Inhibition of N6-Methyladenosine Accumulation by Targeting METTL3 Mitigates Tau Pathology and Cognitive Decline in Alzheimer’s Disease

Inhibition of N6-Methyladenosine Accumulation by Targeting METTL3 Mitigates Tau Pathology and Cognitive Decline in Alzheimer’s Disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Inhibition of N6-Methyladenosine Accumulation by Targeting METTL3 Mitigates Tau Pathology and Cognitive Decline in Alzheimer’s Disease Lulu Jiang, Allison Tucker, Camron Sepehri, Dev Patel, Qingbo Wang, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8379573/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Dysregulation of N6-methyladenosine (m6A) modification of RNA has emerged as a novel feature of Alzheimer’s disease (AD). Here, we investigate the relationship between m6A modification and AD pathology, and the therapeutic potential of modulating excessive m6A via its “writer” methyltransferase METTL3 in a humanized P301S tau transgenic mouse model of AD (PS19). We observed significantly elevated m6A levels in human post-mortem AD frontal cortex tissue compared to healthy controls, which positively correlated with hyperphosphorylated tau and amyloid-β (Aβ) deposition. These effects were recapitulated in the PS19 tau mice model of AD. Importantly, treatment of PS19 mice with the METTL3 inhibitor STM2457 reduced excessive m6A, alleviated tau pathology, and attenuated neurodegeneration. Behavioral assessments further demonstrated that STM2457-treated PS19 mice exhibited significantly improved learning and memory relative to untreated PS19 mice. Our results identify m6A as a critical contributor to AD pathogenesis and demonstrate that pharmacological inhibition of METTL3 represents a promising therapeutic strategy to improve cognition in AD. Health sciences/Diseases/Neurological disorders/Neurodegeneration Biological sciences/Neuroscience/Epigenetics in the nervous system/Epigenetics and behaviour Alzheimer’s disease tauopathy N6-methyladenosine RNA modification cognition METTL3 inhibitor STM2457 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Highlights N6-methyladenosine (m 6 A) is elevated in human AD brain tissue and correlates with both tau and Aβ pathology. m 6 A elevation is age dependent and closely correlated with pathological progression in a tauopathy mouse model of AD. METTL3 inhibition reduces m 6 A levels and alleviates tau pathology, neurodegeneration, and neuroinflammation in PS19 mice. STM2457 treatment improves learning and memory, supporting METTL3 as a promising therapeutic target for AD. Introduction In the last 20 years, reported deaths from Alzheimer’s disease (AD) have increased over 140%, emphasizing the importance of early detection and new disease-modifying treatment strategies 1 . For decades, AD research has been focused on identifying hallmarks of amyloid-β (Aβ) deposition and aggregation of tau neurofibrillary tangles. Emerging research on the role of RNA modifications in AD pathogenesis is a promising research direction for elucidating AD beyond proteiopathies 2–4 . N6-methyladenosine (m 6 A) RNA modification is a key regulator of RNA metabolism and has been studied extensively in the context of neurodevelopment and oncogenesis, though its role in neurodegeneration remains underexplored and debated. Important roles of m 6 A in RNA metabolism include pre-mRNA processing, splicing, nuclear export, mRNA expression, and RNA degradation 5–14 . Dysregulation of m 6 A machinery, including its writers, readers, and erasers, has been linked to impaired memory formation and cognitive decline 5,15–25 . METTL3 is a core writer protein and catalytic portion of the METTL3-METTL14 methyltransferase complex, which carries out the modification resulting in m 6 A RNA 26–28 . Elevated expression of METTL3 has been implicated in AD and related rodent models 29 . Further investigation into m 6 A’s impact on key AD-related pathways could provide novel therapeutic targets, offering a fresh perspective on disease intervention and potential RNA-based treatments. Our previous research discovered a novel association of m 6 A RNA with oligomeric tau (oTau) in the neuronal cytoplasm through the HNRNPA2B1 RNA binding protein, which behaves as an m 6 A reader. Our finding of a 3-5-fold increase of cytoplasmic m 6 A abundance in disease tissues (P301S tau mice and AD-affected human brain tissue) suggests that m 6 A plays a significant role in the pathogenic pathway of AD. This m 6 A-HNRNPA2B1-oTau interaction contributes to oTau’s integrated stress response and reduced protein synthesis 30 . Given the central role of hyperphosphorylated and oligomeric tau in AD pathology, these findings highlight the potential for modulating excessive m 6 A accumulation as a strategy to control tau aggregation. To comprehensively assess m 6 A RNA dynamics during AD progression and aging, we analyzed the correlation between m 6 A RNA accumulation and pathological Aβ and tau deposition in human post-mortem brain tissue as well as in mouse models of tauopathy across different ages. We found a consistent increase in m 6 A RNA levels that strongly correlated with the accumulation of pathological tau and Aβ in human brains from early to late Braak stages, which was further confirmed in aging and disease-progression of mouse models. To explore the therapeutic potential of targeting excessive m 6 A RNA accumulation, we examined the effects of STM2457, a selective METTL3 inhibitor, on AD-related pathology in P301S PS19 tauopathy mice. Remarkably, STM2457 treatment rescued cognitive function, reduced pathological tau accumulation, prevented neuronal loss, and ameliorated neuroinflammation, as demonstrated by behavioral assessments, immunostaining, and biochemical analyses. Results Characterization of m 6 A modifications associated with tau hyperphosphorylation and Aβ deposition across Alzheimer’s disease progression Previous investigations examining m 6 A RNA levels in human post-mortem brain tissue from AD individuals have yielded conflicting results. Work examining the anterior cingulate gyrus and middle temporal gyrus found reduced levels of m 6 A RNA 19,31 , however, when the anterior frontal gyrus was examined, there were elevated levels of m 6 A 30 . Thus, we sought to reaffirm this cortical increase in m 6 A RNA in post-mortem brain tissue. We examined post-mortem brain tissue collected from Brodmann area 10; this region was selected based on consistent evidence from prior studies indicating a strong positive correlation between altered gene expression and disease severity 32 . The brain tissue in this study was collected from eight AD patients and eight cognitively normal control (NC) individuals. The results shown include brain tissue from NC individuals between Braak stages I-IV and AD individuals between Braak stages V-VI. Tau phosphorylated at threonine 217 (pTau217) is an early AD marker that correlates with disease severity and can be used to evaluate pathogenic tau in tissue 33,34 . Using 3,3’-Diaminobenzidine (DAB) staining, pTau217, Aβ (4G8 antibody), and m 6 A RNA levels were evaluated (Fig. 1a) . As expected, we observed elevated pTau217 and Aβ pathology in AD brains (Fig. 1a-c) . When comparing AD to NC, there was significantly more m 6 A in the AD post-mortem brains than the controls (Fig. 1a, d) . Additionally, pTau217, Aβ, and m 6 A were all more prominent in later Braak Stages (V and VI) ( Fig. 1e ). Correlation analysis between pTau217, Aβ, and m 6 A revealed that pathologies were positively correlated with increased levels of m 6 A. The Pearson correlation coefficient (R) between m 6 A and tau pathology was 1.00 ( p =2.21x10 -5 ), while the correlation coefficient with Aβ was 0.80 ( p =0.055) (Fig. 1e) . This data reaffirms the presence of elevated m 6 A levels in cortical regions of AD post-mortem brain tissue, in alignment with the pathological accumulation of Aβ and tau and shows a particularly strong correlation with pathogenic tau. Age-related accumulation of m 6 A in humanized P301S tau transgenic mouse model of AD Previous work demonstrated an important role for m 6 A RNA methylation in regulating neurodevelopment and aging 5,20,35,36 . Data in Fig. 1 from post-mortem AD brain tissue showed that marked accumulation of m 6 A is highly correlated with tau pathology. Next, we quantified how m 6 A levels change with age in a mouse model of progressive tau pathology. To do this, we utilized PS19 mice overexpressing human P301S mutant tau and examined brain tissue collected from 6- or 9-month-old mice. To confirm the pathological relevance of the PS19 model, we labeled hippocampal and cortical regions using the MC1 antibody to identify misfolded tau. MC1 fluorescence intensity was significantly increased in PS19 compared to C57BL/6 wild-type (WT) mice in the hippocampus and cortex (CTX) at both 6- and 9-months of age (Fig. 2a-f) . Considering advanced tau pathology in AD is associated with robust neurodegeneration, we also probed for MAP2, a marker of neuronal cell bodies and dendrites, to evaluate neuronal integrity. MAP2 fluorescence intensity was significantly decreased in the hippocampus and CTX of PS19 mice in 9-month brains (Fig. 2a-c and j-l) . These results indicate that PS19 mice exhibit both increased tau pathology and neurodegeneration as anticipated. More importantly, staining for m 6 A revealed elevated m 6 A RNA levels in the cortex and hippocampal CA3 region of PS19 mouse brains when compared to WT controls at 9-months of age ( Fig. 2a-c and g-i) . Interestingly, we observed that m 6 A RNA was significantly increased in 9-month-old PS19 mouse hippocampus (CA1 and CA3) and CTX when compared to the 6-month-old PS19 group (Fig. 2g-i) . However, for all brain regions examined, m 6 A levels were comparable at 6 months of age between PS19 and WT mice (Fig. 2g-i) . Additionally, DAB staining of brain tissue from 5-month- and 9-month-old WT and PS19 mice revealed that m 6 A levels were significantly elevated in 9-month-old PS19 compared to age-matched WT samples, and there is a significant increase in m 6 A from the 6-month to 9-month timepoint for PS19 mice ( Extended Data Fig. 1) . Altogether, our data suggests that the age-related increase in tau pathology and neurodegeneration in PS19 mice is accompanied by a parallel increase in m 6 A RNA modification. Inhibition of METTL3 reduces excessive m 6 A RNA levels in tauopathy mice Our results demonstrated that m 6 A levels were elevated in both AD post-mortem brains and tauopathic mouse brains. RNA methylation is a reversible epigenetic modification, which makes it an attractive target for pharmacological intervention. Therefore, we attempted to restore the homeostasis of RNA methylation through pharmacologically removing excessive m 6 A by inhibiting its methyltransferase METTL3. STM2457 is an inhibitor of METTL3 that has been examined as a potential therapeutic for myeloid leukemia; that work demonstrated STM2457 can penetrate the blood brain barrier and infiltrate the brain 37 . STM2457 inhibits METTL3 by binding to its S-adenosyl methionine binding site, thus disrupting its catalytic activity without influencing the activity of other RNA methyltransferases 37 . Given its selective METTL3 inhibition, robust brain permeability, and high potency in IC₅₀ value of 16.9 nM 37 , we utilized STM2457 as a treatment strategy, anticipating that the achievable brain dose would fall within its therapeutically effective range. STM2457 was administered continuously for six weeks at a dose of 0.25 mg/kg/day using an osmotic pump that was subcutaneously implanted in the mice starting at 4 months of age (Fig. 3a) . The concentrations of STM2457 in the hippocampus of 9-month-old STM2457-treated mice were measured by liquid chromatography–mass spectrometry (LC–MS). Our result showed an average of 0.96 ng of STM2457 was detected per 10 mg of hippocampal tissue used for analysis (Fig. 3b ). Next, the efficiency of STM2457 treatment on levels of m 6 A in select brain regions was evaluated. Hippocampal levels of m 6 A in PS19 and WT mice with and without STM2457 treatment were assessed with DAB staining with m 6 A antigen probed by an m 6 A-specific antibody (Fig. 3c) . Notably, PS19 mice exhibited a significant increase in m 6 A intensity in CA3 compared to WT mice. Importantly, STM2457 treatment robustly countered the presence of this excessive m 6 A (Fig. 3d) . A similar pattern was observed in CA1, with STM2457 resulting in a significant reduction of m 6 A in PS19 mice. However, high levels of m 6 A were not apparent in the CA1 of untreated PS19 mice (Fig. 3e) . This consistent reduction of m 6 A levels across both CA3 and CA1 indicates that STM2457 treatment effectively controls hippocampal m 6 A in PS19 mice. Changes in m 6 A levels by STM2457 were also validated through immunohistochemical analysis. We conducted an m 6 A dot blot of RNA extracted from cortical tissue of 9-month-old PS19 and WT mice and found that STM2457 treatment significantly reduced total m 6 A RNA levels in PS19 mice, but not in WT mice (Fig. 3f, g) . Immunofluorescence labeling of brain tissue for m 6 A also revealed a significant reduction of m 6 A in CA1 and CTX of PS19 STM2457-treated mice (Fig. 3h-j) . Together, this data demonstrates that STM2457 counteracts excessive m 6 A RNA modification in the cortex and hippocampus as measured with multiple modalities. To further evaluate the inhibition effect of STM2457 on the expression of main methyltransferase and demethylase in the m 6 A regulatory complex, we examined the protein levels of both METTL3, METTL14, and ALKBH5 in the CTX of mice with and without treatment by western blot. Our results revealed that STM2457 treatment in PS19 mice resulted in decreased levels of METTL3 and METTL14 (Fig. 3k-m) . On its own, METTL14 is a separate methyltransferase, however when METTL14 forms a complex with METTL3, METTL14 becomes catalytically inactive due to an occluded active site, and instead acts as structural support for METTL3, promoting the catalytic activity of METTL3 38 . Interestingly, METTL3 levels were not significantly altered by STM2457 treatment in WT mice, while METTL14 levels are significantly increased (Fig. 3l, m) . Similarly, the demethylase ALKBH5 levels were significantly downregulated in PS19 treated mice but not WT ( Extended Data Fig. 2a, b ). These results indicate a coordinated and complementary regulatory relationship between m 6 A writers and erasers in restoring m 6 A homeostasis. Altogether, the evaluation on the treatment efficacy indicates that STM2457, delivered at a constant and controlled dose via osmotic pump, effectively inhibits METTL3 and reduces excessive m 6 A methylated RNA levels in the hippocampus and cortex of PS19 mice. STM2457-mediated reduction of m 6 A RNA rescues behavioral deficits in mouse tauopathy model. RNA modification via m 6 A methylation has been implicated in homeostatic learning and memory processes and neurodegeneration-related deficits, along with the associated m 6 A writers and erasers 21–25,39 . The therapeutic potential of STM2457 relies heavily on its ability to prevent cognitive deficits, as these are some of the most debilitating AD sequelae. Therefore, we evaluated the effect of sustained STM2457 treatment on locomotion and cognitive behaviors of mice using a series of behavioral assays, including the Morris water maze (MWM), elevated plus maze (EPM), Y maze and open field test. MWM was used to evaluate spatial learning and memory. Spatial learning was assessed over four consecutive days of training by measuring the time taken to locate a hidden platform. Reference memory was evaluated over two subsequent probe trials in which the platform was removed, and preference for the former platform location was measured. On the final test day (Day 7), escape latency and search patterns were analyzed to determine the degree to which mice relied on spatial versus non-spatial strategies 40 . MWM analysis showed that 9-month-old PS19 mice exhibited significant learning and memory deficits compared to WT controls, and that these deficits were dramatically ameliorated in the same cohort treated with STM2457 (Fig. 4a-c) . Specifically, PS19 mice traveled greater distances to reach the platform during the 4 days of spatial learning phase compared to WT control (Fig. 4a, b) . Additionally, untreated PS19 mice took significantly longer to reach the platform on the final day (Day 7). (Fig. 4c) . These results indicate impaired spatial learning and memory in PS19 mice. Treatment with STM2457 improved performance, with PS19 mice showing comparable average distance from the platform on the fourth day of training and spending similar time to reach the platform on Day-7 as WT mice (Fig. 4a-c) . Notably, STM2457-treated mice exhibited a temporary increase in escape latency on days 2 to 3, coinciding with the transition from non-spatial to spatial search strategies. Following this transition, they achieved significantly lower escape latencies and shorter distances to the platform, indicating enhanced spatial memory consolidation rather than impairment. This pattern is consistent with prior studies showing that mice with superior learning performance often exhibit an initial exploratory phase characterized by increased path length or escape latency before adopting a more efficient spatial navigation strategy in the Morris water maze 41,42 . The EPM is typically used to assess anxiety-like behaviors, however we utilized the test to evaluate cognition. Previous work demonstrated that the EPM can reveal whether the mice recognize the danger associated with the open arms, a behavior that is impaired in mice with cognitive deficits. 43 . Our EPM analysis revealed that PS19 mice exhibited impaired cognitive capacity (Fig. 4d) . At baseline, WT mice explored both open and closed arms but showed a clear preference for the closed arm, reflecting their ability to recognize the risks of the open arm. In contrast, untreated PS19 mice spent similar amounts of time in both arms, indicating impaired risk recognition. STM2457-treated PS19 mice, however, demonstrated a preference for the closed arm, exhibiting a behavioral pattern similar to WT mice (Fig. 4d) . Evaluation of spatial working memory using the Y maze revealed that the number of spontaneous entries was significantly elevated in PS19 mice when compared to WT, but with treatment this difference was ameliorated (Fig. 4e). Additionally, the percent spontaneous alteration, a measure that gives insight into working memory capabilities, revealed a deficit in percent spontaneous alteration in untreated PS19 mice relative to untreated WT mice (Fig. 4f) . With STM2457 treatment there was a significant increase in percent spontaneous alteration relative to untreated PS19 mice, indicative of improved spatial working memory (Fig. 4f) . The open field test was used to evaluate locomotor activity and gain insight into motor deficits commonly observed in PS19 mice. These deficits are attributed to the clasping and limb retraction that precede hind limb paralysis that develops at aged time-points with substantial pathological tau burden 44 . In the open field test, PS19 mice traveled significantly less total distance than WT mice, whereas STM2457-treated PS19 mice showed locomotor activity comparable to WT controls ( Fig. 4g ). This improvement indicates that PS19 mice with treatment experienced fewer motor deficits or peripheral symptoms typically preceding hind limb weakness and paralysis. Taken together, these behavioral assays demonstrate that STM2457 treatment markedly improves learning, memory, and overall cognition in PS19 mice, which typically exhibit deficits in these behavioral paradigms. STM2457 treatment mitigates tau pathology and protects against neurodegeneration. After assessing the effects of STM2457 on cognition, learning, and memory in PS19 mice, we examined brain tissue from 9-month-old mice to determine whether pathological tau accumulation or neurodegeneration had been alleviated. Immunofluorescent staining of brain sections revealed depleted levels of excessive MC1-positive misfolded tau in PS19 mice treated with STM2457 (Fig. 5a-d, Extended Data Fig. 3a, b) . Changes in MC1 were also accompanied by changes in m 6 A demonstrated previously (Fig. 3g-i) . This depletion was evident in the CTX, CA3, and CA1 (Fig. 5b-d) . Hyperphosphorylation of tau in PS19 mice was assessed using the phosphorylation-specific antibodies CP13 and AT8, which recognize pathologically relevant tau phosphorylated at Ser202 and Ser202/Thr205, respectively. Western blot analysis showed that levels of both CP13- and AT8-positive tau were elevated in untreated PS19 mice compared to WT. STM2457 treatment significantly reduced tau phosphorylation at the Ser202 and Ser202/Thr205 sites in PS19 mice (Fig. 5e-g) . Together, these data demonstrate that, alongside cognitive improvements, STM2457 treatment can reduce multiple forms of misfolded and hyperphosphorylated tau pathology. Based on the reduction in tau pathology and improvement in cognitive behavior, we continued to investigate whether neurodegeneration was slowed or prevented. To assess neuronal integrity, PS19 brain tissue was stained with the NeuN antibody (a Neuronal Nuclei marker). Untreated PS19 mice showed reduced neuronal integrity in the CA1 and cortex relative to WT ( Fig. 5h-k ). Compared to untreated PS19 mice, STM2457-treated mice exhibited significantly increased neuronal density in the CA3, CA1, and cortex, restoring levels to those observed in WT mice ( Fig. 5i-k ). In summation, these results demonstrate that STM2457 treatment mitigates tau pathology and protect neurons against degeneration in brain regions bearing pathological tau aggregation and excessive m 6 A accumulation. STM2457 treatment attenuates glial inflammatory response in PS19 mice. AD pathology is associated with neuroinflammation involving altered glial cell activation, including neuroprotective acute activation and neurotoxic chronic activation 45,46 . Additionally, m 6 A modification has been implicated in regulating microglial inflammatory responses and microglia-neuron interactions 20,47,48 . Considering the effects STM2457 had on cognition, tau pathology, and neurodegeneration, we next considered how glial activation states change with treatment in PS19 mice. To elucidate the impact of STM2457 on AD-related glial activation, we examined the morphological changes of microglia and astrocytes indicative of the shift into differently activated inflammatory states. We used an IBA1 antibody to mark both resting and activated microglia and used immunofluorescence intensity to assess morphological alteration and activation status. Immunofluorescent staining of hippocampal (CA1 and CA3) tissue from 9-month-old PS19 mice revealed increased ameboid-like morphology of microglia and associated increased fluorescence intensity that was attenuated in mice that received STM2457 treatment (Fig. 6a-d) . Interestingly, STM2457-treated WT mice showed a slight increase in IBA1-positive microglial intensity, despite the absence of detectable pathology or behavioral changes. This may indicate a more proactive microglial state associated with METTL3 inhibition by STM2457, potentially reflecting its role in maintaining m 6 A homeostasis. A similar investigation of astrogliosis-associated morphology revealed increased fluorescence intensity indicative of hypertrophy of the cell body and processes in the CA1, CA3, and CTX of 9-month-old PS19 mice; this reactive morphology was quelled by STM2457 treatment (Fig. 6e-h) . Altogether, these findings indicate that, in addition to reducing tau pathology and restoring neuronal integrity, STM2457 treatment also reduces the neuroinflammatory responses of microglia and astrocytes. Discussion Epigenetic modifications are increasingly recognized as contributors to AD pathogenesis, highlighting new therapeutic opportunities. Among these, m 6 A RNA modification has emerged as a potential regulator of AD-related processes, though prior findings have varied across pathological contexts 19,30,49,50 . Our study supports a link between excessive m 6 A and tau pathology and shows that inhibiting the m 6 A writer METTL3 with small molecule STM2457 reduces tau deposition, mitigates neurodegeneration, and prevents cognitive decline in PS19 mice. Our analysis of post-mortem human brain tissue revealed that increased m 6 A modification is present in the frontal cortex (Brodmann’s area 10) of AD brains, which is consistent with previous findings in the same brain region 30 . We also observed that m 6 A levels rise concurrently with increasing Braak stage, hyperphosphorylated tau accumulation, and Aβ plaque deposition. To be noted, other studies also reported inconsistent m 6 A alterations across cortical regions in human post-mortem brain tissue, including reduced m 6 A in the anterior cingulate gyrus and middle temporal gyrus 19,31 . These discrepancies suggest region-specific regulation of m 6 A and highlight the need for comprehensive spatial profiling of m 6 A in both diseased and non-diseased human brains. Using humanized P301S tau transgenic mouse models of AD, we found that PS19 mice exhibit elevated m 6 A levels in the cortex and hippocampus at 9 months compared to age-matched WT and levels increased further from 6 to 9 months. These findings align with prior reports that m 6 A varies with age, both independently and in the context of aging-related diseases 20,31,36,51 . Given the prominence of tau pathology in the hippocampus and cortex, this increase suggests a potential age-related link between elevated m 6 A RNA and tau accumulation, highlighting the dynamic and transient nature of m 6 A regulation in the brain 5 . Our findings in human AD cases and PS19 mice support the modulation of m 6 A levels by targeting its regulatory proteins, such as methyltransferase METTL3, as a promising therapeutic strategy for AD. STM2457, originally developed as a selective METTL3 inhibitor for hematologic malignancies, demonstrates several pharmacological properties that favor potential clinical development, including its small molecule structure, nanomolar potency, ability to cross the blood brain barrier, and capacity to reduce m 6 A levels in vivo 37 . Treatment of PS19 mice with STM2457, delivered continuously for 6 weeks via a surgically implanted osmotic pump subcutaneously with precise dose control, reduced m 6 A levels to a range more comparable to age-matched WT mice as shown by immunolabeling and RNA dot blot. Assessment of STM2457 treatment on tau deposition revealed a robust reduction in MC1-positive misfolded tau, pTau (Ser202), and pTau (Ser202/Thr205) in the hippocampus and cortex of treated PS19 mice, indicating that removal of excessive m 6 A effectively limits pathological tau accumulation. Protein levels of key m 6 A regulators were assessed to elucidate the regulatory mechanisms and coordination underlying m 6 A homeostasis. METTL3 and METTL14 protein levels were significantly reduced in STM2457-treated PS19 mice. In contrast, STM2457-treated WT mice showed no change in METTL3 and a slight increase in METTL14 relative to untreated WT. This variance in translational effect suggests the presence of a compensatory mechanism that attempts to restore the balance of the m 6 A regulatory axis. Evaluation of neuronal density further revealed that reduced tau pathology in treated PS19 mice is accompanied by protection against neurodegeneration. Together, these findings demonstrated that STM2457 successfully inhibited METTL3 and reduced m 6 A methylation, decreased tau pathology, and mitigated neurodegeneration. Glial activation is a key component of the inflammatory response to pathogenic tau and Aβ in AD 52–56 , and increasing evidence indicates that m 6 A RNA methylation participates in regulating neuroinflammatory cascades 48,57–61 . Recent studies have also shown differential clustering of m 6 A regulators in infiltrating immune cells from AD patients 62 , further suggesting a link between m 6 A signaling and immune activation. In this study we found that STM2457 treatment reduced microglial activation and astrogliosis in PS19 mice, as reflected by decreased inflammatory glial morphology. However, because glial activation in tauopathy may arise secondary to pathological tau aggregation, the reduction in inflammatory morphology could be partly attributable to decreased tau deposition. Even so, our findings also raise the possibility that m 6 A directly modulates glial immune responses. To more comprehensively evaluate this potential role, future studies are planned to assess additional inflammatory endpoints, including reactive oxygen species production and pro-inflammatory cytokine release. Importantly, the therapeutic relevance of STM2457 lies in its ability to reverse cognitive impairment, one of the most debilitating features of AD. In our study, behavioral testing demonstrated that STM2457 fully rescued the learning, memory, and general cognitive deficits observed in PS19 mice. Improvements in locomotion in the open field test indicates that STM2457 mitigates motor impairments associated with this tauopathy model. These findings align with other studies reporting that reducing or eliminating METTL3 activity can benefit cognition in amyloid related AD mouse models 63 , 64 . Notably, unlike previous work that has focused predominantly on amyloid-based models, our study reveals a direct role for m 6 A in modulating tau pathology, which is particularly important because tau burden shows a stronger correlation with cognitive decline in AD 65 . This distinction underscores the unique contribution of our findings in supporting the role of m 6 A as a key regulator of tau-driven neurodegeneration. Taken together with prior studies, our findings support the therapeutic potential of targeting m 6 A in AD. m 6 A RNA modification influences multiple disease-relevant pathways, including tau misfolding, Aβ clearance, neuroinflammation, and synaptic function 19,36,47,49,66 . Regional and stage-dependent variation in m 6 A patterns further indicate that modulating METTL3 could restore disrupted transcriptomic regulation in a context-specific manner. Specifically, the prolonged presence of STM2457 in the hippocampus following sustained subcutaneous delivery via osmotic pumps enhances its potential as a viable therapeutic agent. Nevertheless, m 6 A is regulated by complex enzymatic networks, and their remain questions regarding the consequences of long-term METTL3 inhibition. Our observation that METTL3 and METTL14 respond differently to STM2457 in WT versus PS19 mice also points to distinct compensatory mechanisms that require deeper investigation. Overall, selective METTL3 inhibitors represent a promising therapeutic avenue, and advancing STM2457 and related compounds will require continued evaluation of safety, dosing, and cellular consequences to support future clinical translation. In conclusion, this work suggests that reducing excessive m 6 A RNA levels using STM2457 reduces tau pathology, neurodegeneration, neuroinflammatory glial morphology, and improves learning and memory. This impact of m 6 A methylation on tau pathogenesis presents new avenues for therapeutic intervention. This work highlights the potential of STM2457 as a novel AD therapeutic, whose impact on m 6 A methylation in specific cell types and pathogenic processes needs further investigation. Materials and Methods Human post-mortem brain tissue Anonymous human brain tissue used in this project was obtained from the Goizuetta Alzheimer’s Disease Center and was collected in accordance with IRB protocols of Emory University. Human anterior prefrontal cortex (Brodmann Area 10) was used for immunohistochemical analysis and immunofluorescence labeling in the current study. These brain regions were selected based on consistent evidence from prior studies indicating a strong positive correlation between altered gene expression and disease severity 67 . The samples were de-identified and are described below. The tissue was fixed in periodate-lysine-paraformaldehyde (PLP) fixative for 2 hrs, followed by overnight incubation in 30% sucrose, after which tissue sections were cut at thickness of 30μm. Fixed human brain samples The human brain tissue samples used in this study were all de-identified. All studies included both sexes, and results were integrated by covariate analysis, as described below. Primary Neuropathologic Diagnosis Braak Stage Age at Onset Age at Death/Bx Duration (years) ApoE Race Sex Control I 59 E2/3 b m Control I 70 E3/3 b m Control I 72 E3/3 w m Control II 78 E3/3 w f Control IV 84 E2/3 b f Control III 91 E3/3 w f Control III 92 E3/3 w f Control II 94 E3/3 w m Control/asymptomatic AD II 64 E4/4 w f Control/asymptomatic AD IV 80 E3/4 w m Control/asymptomatic AD II 81 E3/3 w m Control/asymptomatic AD I 87 E3/4 w m Control/asymptomatic AD III 87 E2/3 w f Control/asymptomatic AD IV 89 E3/3 w m Control/asymptomatic AD IV 91 E3/3 w m AD VI 49 59 10 E3/3 w m AD VI 59 69 10 E3/4 w m AD VI 53 71 18 E3/4 w m AD VI 65 80 15 E3/4 b f AD V 77 83 6 NA w f AD V 86 92 6 E3/4 w f AD V 82 92 10 E3/3 w m AD VI 80 93 13 E3/4 w f Animals Use of all animals was approved by the University of Virginia Institutional and Animal Care and Use Committee. All animals were housed in an IACUC-approved vivarium at the University of Virginia School of Medicine. P301S tau PS19 transgenic mice: PS19 mice overexpressing human P301S Tau (B6;C3-Tg(Prnp-MAPT*P301S) PS19Vle/J, stock #008169) were purchased from Jackson Laboratories. Male PS19 P301S tau +/- mice and female C57BL/6 wild type were used as breeding pairs and the F1 generation of P301S tau +/- (PS19) and P301S tau -/- (wild type) were used for the experiment. Littermates of the same sex were randomly assigned to experimental groups. Mice were sacrificed for experimentation at the age of 6- and 9-months-old, respectively. The perfusion and fixation method used for brain tissue was critical for successful immunostaining conducted in this study. Following anesthesia, mice were perfused through the heart with 20mL ice cold PBS at the speed of 4mL/min for 10 mins until the mouse tail became curved and stiff. The mouse brains were dissected and placed in 4% paraformaldehyde (PFA) on ice for 24 hrs before being transferred 0.05% sodium azide/PBS solution. To prepare for brain sectioning, the fixed mice brains were transferred into 30% sucrose/PBS until the brains sank to the bottom of the tube (about 48h) and then sectioned. The fixed brains were cryosectioned into 30µm coronal sections and stored in 0.005% sodium azide/PBS solution at 4°C for up to 3 months. For long-term storage, the sections were transferred into cryoprotectant solution (30% glycerol and 30% ethylene glycol in PBS) and stored at -20 °C. Immunohistochemistry staining Floating 30µm brain sections were placed onto slides. All subsequent steps were performed at room temperature unless otherwise specified. Sections were outlined with a hydrophobic barrier using a PAP pen and air-dried before staining. Sections were washed in PBS for 10 min then treated with 1% H2O2 diluted in dH2O for 15 min. Sections were rinsed twice in PBS for 15 min each, then blocked with 0.4% Triton X-100 with 1% Bovine serum albumin (BSA) and 4% normal goat serum (NGS) in PBS for 30 min. After blocking, sections were incubated overnight in 1° antibody diluted in DAKO antibody diluent (5% BSA in 0.25% TritonX-100/PBS) at 4°C with photobleaching. The following day, sections were rinsed twice in PBS for 15 min then incubated for one hr in BTA solution (0.44% Biotinylated goat anti-mouse IgG diluted in 0.3% Triton X-100/PBS). Sections were rinsed twice in PBS then treated with Avidin Biotin Complex (ABC) (0.88% Avidin, 0.88% Biotin diluted in 0.3% Triton X-100/PBS, prepared 30 min prior to use; Vector Laboratories, Cat# PK-6101). After ABC incubation, sections were washed three times in PBS for 10 min each. DAB solution was prepared by adding 1 tablet DAB (Millipore Sigma, cat# D4293-5SET) and 1 tablet Urea (Sigma-Aldrich, Cat# D4193) to 5mL dH2O and vortexed until fully dissolved. DAB solution was added to sections individually for one to three min until section turned medium brown in color, then were immediately placed in PBS. Sections were washed twice with PBS for five min each, then dried overnight at 37°C. The following day, sections were dehydrated by washing in 70% EtOH, 85% EtOH, 95% EtOH, and 100% EtOH for two min each followed by washing in 100% EtOH for five min. Slides were then cleared by washing twice in Xylene for five min followed by immediately dropping 50µL of Permount Mounting Medium (Thermo Fisher Scientific, Cat# SP15-500) and covering with glass coverslips. Slides were dried overnight in the fume hood and stored at 4°C for long term storage. All washing and incubated steps were done with agitation. Primary antibody used for brain tissue DAB staining was mouse monoclonal purified IgG anti-m 6 A, 1:2000 (Synaptic Systems, Cat# 202 011, RRID: AB_2619890). RNA dot blot RNA dot blot protocol was adapted from Zhao et al. 64 RNA extraction was performed using RNeasy Plus Universal Mini Kit (73404, Qiagen) on ~20-25mg of cortical tissue that had been frozen in RNA later. ~400ng/µL of extracted RNA was heated to 95ºC for 3 min to disrupt secondary structures, then samples were immediately placed on ice. Then 1µL 10mM HEPES buffer and 1µL methanol were added to the RNA, and the volume was brought up to 10µL using molecular biology grade water. Then 10µL of each sample was dotted onto a positively charged nylon membrane (Cytiva, Amersham Hybond -N+, RPN1210B). Following this, the membrane was heated in an oven to 65ºC for 45 min, then immediately placed in a UV crosslinker with the following settings: 125 mJoule/cm2 at 254 nM for 5 min. Then the membrane was washed with PBST (1X PBS, 0.01% Tween-20) with gentle shaking for 10 min. The membrane was then blocked in 5% skimmed milk powder in 1X PBS for 1 hr with gentle shaking at RT. The primary antibody used was mouse IgG3 anti-m6A monoclonal antibody (Proteintech, Cat # 68055-1-Ig, RRID: AB_2918796) in a 1:1000 dilution in 5% BSA in PBST (1X PBS, 0.01% Tween-20) solution. The membrane was submerged in primary antibody and incubated either at RT with gentle shaking for 2 hrs, or at 4ºC with gentle shaking overnight (14-16 hrs). The membrane was then washed 3x in PBST for 10 min. A horseradish peroxidase concentrate was used as a secondary antibody, specifically goat anti-mouse IgG (H+L) horseradish peroxidase-conjugated secondary antibody (Thermo Fisher Scientific, Cat# G-21040, RRID: AB_2536527) was used at a 1:2000 dilution in 5% BSA in PBST. The membrane was incubated for 1 hr at RT with gentle shaking. The membrane was washed 4x for 10 min each in PBST with gentle shaking. Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific, Cat #32106) was used for luminescent development: 4 mL of detection reagent 1 peroxidase solution and 4 mL of detection reagent 2 luminol enhancer were added to the membrane to incubate for 5 min. The membrane was then imaged on a BioRad ChemiDoc imaging system. Afterwards, the membrane was briefly washed with PBST to remove the ECL substrate and was then submerged in Pierce™ Restore Plus Western Blot Stripping Buffer (Thermo Fisher Scientific, Cat# 46428) for 5 min. Then the membrane is placed in a Methylene Blue staining buffer (0.2% methylene blue in 0.4M sodium acetate and 0.4M acetic acid) for 30 min with gentle shaking. The membrane was washed with molecular biology grade water until the solution was clear (~3 4–5-minute washes). Membrane was imaged on BioRad ChemiDoc imaging system using blue and green StarBright B700 and B520 Blot settings, respectively. Western blot At the time of perfusion cortical tissue samples intended for western blot were snap frozen and transferred to a 1.5mL Protein LoBind Eppendorf tube and stored at -80ºC. Samples were subsequently lysed by mechanical homogenization 40µL Hsiao TBS buffer (50mM Tris, pH 8.0, 274mM NaCl, 5mM KCl) supplemented with cOmplete Protease Inhibitor (Roche, Cat# 11836153001) and PhosSTOP Phosphatase Inhibitor (Roche, Cat# 04906845001). 200U of Benzonase (Sigma-Aldrich, cat# E1014-25KU) with 1mM MgCl2 was added for removal of nucleic acids. Protein lysate concentration was quantified by the Pierce BCA assay according to the manufacturer’s protocol (Thermo Fisher Scientific, cat# 23227). 10-20µg of protein lysate were separated by denaturing gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane. The membrane was blocked for an hr in 5% non-fat milk in .01% Tween-20 in 1x PBS (PBST), and incubated in primary antibody (dilution antibody dependent) in 5% BSA PBST overnight at 4°C. The following day, the membrane was washed with PBST for 3 times for 10 min each then incubated with secondary antibody goat anti-mouse IgG (H+L) horseradish peroxidase-conjugated secondary antibody 1:5000 (Thermo Fisher Scientific, Cat# G-21040, RRID: AB_2536527) in 5% BSA PBST for 2 hrs at RT. The membrane was washed with PBST 3 times for 10 min before briefly being washed with PBS prior to visualization. Protein bands were visualized using Pierce™ enhanced chemiluminescence (ECL) Western Blotting Substrate (Thermo Fisher Scientific, Cat# 32106) and imaged by ChemiDoc Imaging System (Bio-Rad). Band intensities were quantified using ImageJ. Protein abundance was normalized to total Βeta Actin with background subtraction. Primary antibodies used for immunoblotting were as follows: : rabbit IgG METTL3 polyclonal antibody 1:2000 (Proteintech, Cat# 15073-1-AP, RRID: AB_2142033), rabbit IgG METTL14 polyclonal antibody 1:2000 (Proteintech, Cat# 26158-1-AP, RRID: AB_2800447), rabbit IgG ALKBH5 polyclonal antibody 1:1000 (Proteintech, Cat# 16837-1-AP, RRID: AB_2242665), mouse IgG Phospho-Tau (Ser202, Thr205) Moloclonal Antibody (AT8) 1:2000 (Invitrogen, Cat# MN1020, RRID: AB_223647); mouse anti-tau p202 (CP13) 1:500 (provided by Feinstein Institute through MTA) and mouse β-Actin Monoclonal Antibody 1:2000 (Proteintech, Cat# 66009-1-Ig, RRID: AB_2687938). Immunofluorescence labeling Coronal sections, including cortex and hippocampus, were washed in PBS for 10 mins and then permeabilized in 0.5mL PBS/0.25% Triton X-100 (PBST). The selected brain tissues were then blocked in blocking solution (5% BSA and 5% normal donkey serum in PBST) for1.5-2 hrs at room temperature (RT). Then sections were incubated in primary antibodies diluted in 5% BSA/PBST overnight at 4°C. On the second day, brain sections were washed three times in PBST, 15 min each. Brain sections were then incubated in 2° antibodies (1:1000 for Alexa fluor-conjugated antibodies made in goat/donkey purchased from Thermo Fisher Scientific) diluted in 5% BSA/PBST for 2 hrs at room temperature. For DAPI nuclei stain, DAPI (1:10,000of 1mg/mL stock) was diluted in PBST and incubated with brain sections for 15 min followed by two washes with PBST then one wash with PBS, 10 min each. The brain sections were mounted onto microscope glass slides using Prolong gold antifade reagent. Images were captured by Leica Stellaris 5 confocal microscope. Primary antibodies used for brain tissue staining were as follows: mouse monoclonal purified IgG anti-m 6 A, 1:2000 (Synaptic Systems, Cat# 202 011, RRID: AB_2619890); mouse anti-MC1, 1:300 (provided by Feinstein Institute through MTA); chicken anti-microtubule-associated protein antibody, 1:1000 (Aves Labs, Cat# MAP, RRID: AB_2313549); rabbit anti-Iba1, 1:1000 (Fuji Film Wako Pure Chemical Corporation, Cat# 019-19741, RRID: AB_839504); rat monoclonal antibody anti-GFAP, 1:1000 (Thermo Fisher Scientific, Cat# 13-0300, RRID: AB_2532994); guinea pig polyclonal anti-NeuN antibody (Synaptic Systems, Cat# 266 004, RRID: AB_2619988). STM2457 treatment via osmotic pump delivery STM2457 (MedChem Express, Cat#HY-134836), a potent and selective METTL3 inhibitor (IC 50 = 16.9nM), is delivered via Alzet osmotic pump model 2006 for continuous administration (0.15µL/hr, 42-day duration, 200µL reservoir volume). Osmotic pumps are miniature infusion devices designed for controlled and continuous agent delivery in laboratory animals, commonly used for systemic administration when implanted subcutaneously. In our experiment, the working solution of STM2457 at 2.08mg/mL (4.6mM) was prepared and immediately followed by 200µL infused into the osmotic pump to maintain accurate dosing at 0.25mg/kg/day in vivo . The osmotic pumps were then implanted subcutaneously on the neck of the 4-month-old mice (~30mg), including wild type control, and PS19 (N= 10 mice per condition). This ensured precise and sustained release of STM2457 at a rate of 0.15µL/h (0.312µg/h) for 42 days. For preparation of STM2457 working solution, it was first dissolved in DMSO at 50mg/mL (112.48mM) with ultrasonic treatment and heated to 60°C to ensure complete dissolution while avoiding repeated freeze-thaw cycles. The stock solution is stored at -80°C for up to 6 months or at -20°C for up to 1 month. The final working solution is prepared by sequentially adding 10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline, yielding a clear solution with solubility ≥ 2.08mg/mL. This formulation ensures stability and consistent STM2457 delivery via subcutaneously implanted osmotic pump . Liquid chromatography–mass spectrometry (LC-MS) 10mg of Hippocampal frozen tissues were pestled, followed by addition of 200µL of ethyl alcohol water mixture (50% ethyl alcohol:50%water). The Tissue + solvent samples were vortexed for 10 min under ambient temperature. After vortex, Tissue + solvent samples were centrifuged at 10000 RPM for 10 mins. 170µL of supernatants were collected and aliquoted. The whole extraction process was repeated three times. 80µL of supernatants were used for non-spiked LC-MS analysis and 90µL of supernatants were used for spiked LC-MS analysis. 10µL of 116µM STM2457 standard solution were spiked into 90µL supernatants to reach a rough final concentration of 11.6µM. Data-dependent acquisition (DDA) analyses were performed on Orbitrap Exploris coupled to Vanquish system (Thermo Fisher Scientific). 10µL of supernatants were separated on a HALO 90 Å AQ-C18 column (4.6 x 150 mm, 5µm, C18, Advanced Materials Technology, Delaware, Cat# USEQT001307) at 250µL per min. Mobile phase A consisted of 50% LC/MS grade H 2 O (W6-4, Fisher Scientific) and 50% LC/MS grade methanol (34860-4L-R, Sigma-Aldrich), mobile phase B consisted of LC/MS grade ethyl alcohol (459829-4L, Sigma-Aldrich), and both mobile phases contained 0.1% FA. The LC gradient was: 0% B to 50% B in 5 min, 98% B in 10 min until 12 min, and 0% B in 15 min, with a total gradient length of 15 min. A targeted MS1 scan and a ddMS2 scan were paired for quantification and qualification purpose. Targeted MS1spectra were collected at 120,000 resolution and ddMS2 spectra at 90,000 resolution. 70% HCD collision energy was used for ddMS2 fragmentation. Elevated plus maze The elevated plus maze (EPM) protocol was adapted from Marco et al 68 . The EPM was used to assess cognition similar to the application in Jürgensen et al 43 . The apparatus consisted of two opposing closed arms covered by high walls (20 cm tall opaque black Plexiglass, 35 x 6 cm 2 ), while the other two arms remained open to the air (uncovered, 35 x 6 cm 2 ) extended from a common center square, elevated 121cm above the floor. Mice were placed onto the opaque white Plexiglass floor in the center facing a closed arm and allowed to explore the entire raised platform for 5 min. Data was recorded using an overhead camera with the EthoVision XT automated tracking system (Nodulus Information Technology), which provided the time spent in the open and closed arms. Between trials, the maze was thoroughly cleaned with 70% ethanol. Open field The open field test protocol was adapted from Lammert et al. 69 and was used to evaluate spontaneous locomotor activity. The open field consists of a square area (40 x 40 cm 2 ) with white Plexiglass walls and floor, evenly illuminated with dim lighting. All mice were individually placed in the upper left corner of the open field and allowed to explore the area for 10 min. Movements were recorded using an overhead camera and data was collected using the EthoVision XT automated tracking system (Nodulus Information Technology). Locomotor activity was quantified as total distance traveled (cm). Between trials, the maze was thoroughly cleaned with 70% ethanol. Morris water maze The Morris water maze (MWM) protocol was adapted from March et al 68 and Vorhees & Williams 40 . The MWM was used to evaluate spatial learning and memory in 9-month-old mice WT and PS19 mice. The mice were placed in a 1m diameter pool of opaque water. The water was made opaque by adding a nontoxic tempera white paint and the water was kept at room temperature. A hidden 10-cm diameter platform was placed at approximately 1cm underneath the surface of the water on which the mice can stand on. The maze was divided into four quadrants. Four trials were performed each day across a four-day learning period. The mice were placed in the pool at various starting positions throughout the three quadrants that did not contain the platform. Four large markers (a square, circle, triangle, and X) were placed on the tub walls which provide visual cues to allow the mice to determine platform location. On Day-1 the mice were placed in their starting positions and allowed to search for the platform for 60s. Each mouse was allowed to remain on the platform for 2 min for the first trial, 1 minute the second trial, 30s the third trial, and 5s the last trial before being removed from the maze. If the mouse did not find the platform within 60s, the time to find the platform was recorded as 60s. Days 2-4 were all the exact same where the mice underwent four trials and had 60s to locate the platform. On these days the mice had to remain on the platform for 5s before being removed from the maze. Days 5 and 6 were “probe” days used to assess memory function, these days only contained single trials where the platform had been removed. The mice were once again allowed to search for the platform for 60s and the time spent in the quadrant where the platform was previously located was recorded. Day 7 of the test included four trials and had the platform returned to the same location as Days 1-4 and the mice were given 60s to locate the platform and had to remain on the platform for at least 5s before being removed from the maze. During the test data was recorded using an overhead camera and with the EthoVision XT automated tracking system (Nodulus Information Technology) which provided the average distance from the platform (cm), total swimming time, swimming time in the target vs. opposing quadrants, and the path taken to reach the platform. The time for the mice to reach the platform on Day 7 was considered escape latency. Between trials, the maze was thoroughly cleaned with 70% ethanol. Y-maze The Y-maze protocol was adapted from Kraeuter et al 70 . The Y-maze was used to evaluate spatial working memory. The apparatus consisted of three identical opaque arms (35 cm long × 8 cm wide × 15 cm high) positioned at 120° angles, these arms were labeled A, B, and C. For this protocol we designated the arm labeled “A” as the starting arm, with the arms labeled “B” and “C” designated as non-starting arms. Each mouse was placed in the distal part of the starting arm of the maze, facing the center of the maze, and allowed to explore all arms freely for 8 min. An arm entry was defined as the center point of the mouse entering the arm. The number of spontaneous entries included the sum of the entries the mice made into the non-starting arms. The number of alterations included how many times a mouse entered all three arms consecutively (e.g., ABC, ACB). The total number of arm entries included the sum of all entries into any arm. The percent spontaneous alternation was calculated as: % Spontaneous Alteration = (Number of alterations)/ [Total number of arm entries -2]) *100 Data was recorded using an overhead camera and with the EthoVision XT automated tracking system (Nodulus Information Technology). Between trials, the maze was thoroughly cleaned with 70% ethanol. Schematics Schematics were created with BioRender.com Brightfield image analysis Bright images (DAB) were captured by Keyence BZ-X810 Microscope (Keyence, BZ-X810) using BZ-X800 Viewer Software (Keyence). The staining intensities in DAB labeled brain sections were measured by ImageJ. Raw integrated density (RawIntDen) was recorded and normalized by area measured when appropriate. Fluorescent Image analysis Fluorescent images were captured by STELLARIS 5 Confocal Microscope (Leica Microsystems, Stellaris 5) using LAS X Life Science Microscope Software Platform (Leica Microsystems). The staining intensities in immunofluorescence-labeled brain sections were measured by ImageJ. Either integrated density (IntDen) or raw integrated density (RawIntDen) were recorded and normalized by area measured when appropriate. Statistical analysis Statistical analyses and figures artwork were performed using GraphPad Prism version 10.2.0 for Windows. Pairwise data was compared using unpaired student’s t-test. All group data are expressed as mean ± SEM. Column means were compared using one-way ANOVA. Group means were compared using two-way ANOVA with factors of genotype, age time course, or treatment status. When two-way ANOVA showed a significant difference, pairwise comparisons between group means were examined by Tukey’s or uncorrected Fisher’s LSD multiple comparisons test. Significance was defined at p < 0.05. Declarations Availability of data and materials All data reported in this paper will be shared by the lead contact upon request. The raw IF, DAB and WB images, and the animal behavior test videos and analysis will be made publicly available at Mendeley Data (Reserved DOI:10.17632/pynkg2hdcf.1). ACKNOWLEDGEMENTS We acknowledge the late Dr. Peter Davies (Northwell/Hofstra) and the Feinstein Institutes for Medical Research for providing the CP13 and MC1 antibodies. We would like to thank the following funding agencies for their support to L.J.: NIH/NIA (R01AG091577); The Grand Challenges Research Investments in Brain and Neuroscience at UVA; Virginia Alzheimer’s Disease Center 2023 ‘Pitch and Catch’ funding; UVA Brain Institute Transformative Neuroscience Pilot Grants; Strang Neuroscience Research Award; Jim and Bruce Eck Fund; Commonwealth Health Research Board Award; Cure Alzheimer’s Fund, and the Bill Howard’s Golf Tournament for Alzheimer’s Disease Research Grant. We thank Professor Daniel Engel for his thoughtful review, constructive edits, and valuable feedback on the manuscript. Author contributions L.J. conceived the study. A.T., C.S., D.P., Q.W., S.Y., E.S., Y.C., A.D., D.G., W.G., and L.J. performed the investigations and generated the visualizations. Data analysis was conducted by A.T., C.S., D.P., Q.W., S.Y., E.S., Y.C., A.D., D.G., X.M.; L.J. A.T., C.S., D.P., E.S., A.D., D.G., and L.J. contributed to writing and editing the manuscript. The study was supervised by L.J.. Declaration of Interests L.J. and A.T. have filed a patent application related to the therapeutic approach described in this manuscript (U.S. Provisional Patent Application No. 63/933,846). All other authors declare no competing interests. References Alzheimer’s disease facts and figures. Alzheimers Dement 20 , 3708–3821 (2024). Hu, B. et al. Rewired m6A of promoter antisense RNAs in Alzheimer’s disease regulates neuronal genes in 3D nucleome. Nat Commun 16 , 5251 (2025). Kobayashi, A. et al. Emerging Roles and Mechanisms of RNA Modifications in Neurodegenerative Diseases and Glioma. Cells 13 , 457 (2024). Martinez-Feduchi, P., Jin, P. & Yao, B. Epigenetic modifications of DNA and RNA in Alzheimer’s disease. Front Mol Neurosci 17 , 1398026 (2024). Shafik, A. M. et al. N6-methyladenosine dynamics in neurodevelopment and aging, and its potential role in Alzheimer’s disease. Genome Biol 22 , 17 (2021). Murakami, S. & Jaffrey, S. R. Hidden codes in mRNA: Control of gene expression by m6A. Mol Cell 82 , 2236–2251 (2022). Hong, J., Xu, K. & Lee, J. H. Biological roles of the RNA m6A modification and its implications in cancer. Exp Mol Med 54 , 1822–1832 (2022). Zaccara, S. & Jaffrey, S. R. A Unified Model for the Function of YTHDF Proteins in Regulating m6A-Modified mRNA. Cell 181 , 1582-1595.e18 (2020). Dominissini, D. et al. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 485 , 201–206 (2012). Yang, Y. et al. Dynamic m6A modification and its emerging regulatory role in mRNA splicing. Sci. Bull. 60 , 21–32 (2015). Roundtree, I. A. et al. YTHDC1 mediates nuclear export of N6-methyladenosine methylated mRNAs. eLife 6 , e31311 (2017). Lesbirel, S. et al. The m6A-methylase complex recruits TREX and regulates mRNA export. Sci Rep 8 , 13827 (2018). Edens, B. M. et al. FMRP Modulates Neural Differentiation through m6A-Dependent mRNA Nuclear Export. Cell Rep 28 , 845-854.e5 (2019). Meyer, K. D. et al. 5′ UTR m6A Promotes Cap-Independent Translation. Cell 163 , 999–1010 (2015). Du, B. et al. N6-methyladenosine (m6A) modification and its clinical relevance in cognitive dysfunctions. Aging (Albany NY) 13 , 20716–20737 (2021). Zhang, R., Zhang, Y., Guo, F., Li, S. & Cui, H. RNA N6-Methyladenosine Modifications and Its Roles in Alzheimer’s Disease. Front. Cell. Neurosci. 16 , (2022). Jiang, X. et al. The role of m6A modification in the biological functions and diseases. Sig Transduct Target Ther 6 , 74 (2021). Han, M. et al. Abnormality of m6A mRNA Methylation Is Involved in Alzheimer’s Disease. Front Neurosci 14 , 98 (2020). Zhao, F. et al. METTL3-dependent RNA m6A dysregulation contributes to neurodegeneration in Alzheimer’s disease through aberrant cell cycle events. Molecular Neurodegeneration 16 , 70 (2021). Sun, J. et al. The Potential Role of m6A RNA Methylation in the Aging Process and Aging-Associated Diseases. Front Genet 13 , 869950 (2022). Leonetti, A. M., Chu, M. Y., Ramnaraign, F. O., Holm, S. & Walters, B. J. An Emerging Role of m6A in Memory: A Case for Translational Priming. International Journal of Molecular Sciences 21 , 7447 (2020). Lv, Z. et al. Downregulation of m6A Methyltransferase in the Hippocampus of Tyrobp–/– Mice and Implications for Learning and Memory Deficits. Front. Neurosci. 16 , (2022). Pupak, A. et al. Altered m6A RNA methylation contributes to hippocampal memory deficits in Huntington’s disease mice. Cell Mol Life Sci 79 , 416 (2022). Deng, J. et al. N6-methyladenosine demethylase FTO regulates synaptic and cognitive impairment by destabilizing PTEN mRNA in hypoxic-ischemic neonatal rats. Cell Death Dis 14 , 1–14 (2023). Walters, B. J. et al. The Role of The RNA Demethylase FTO (Fat Mass and Obesity-Associated) and mRNA Methylation in Hippocampal Memory Formation. Neuropsychopharmacol 42 , 1502–1510 (2017). Bokar, J. A., Shambaugh, M. E., Polayes, D., Matera, A. G. & Rottman, F. M. Purification and cDNA cloning of the AdoMet-binding subunit of the human mRNA (N6-adenosine)-methyltransferase. RNA 3 , 1233–1247 (1997). Liu, J. et al. A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation. Nat Chem Biol 10 , 93–95 (2014). Oerum, S., Meynier, V., Catala, M. & Tisné, C. A comprehensive review of m6A/m6Am RNA methyltransferase structures. Nucleic Acids Res 49 , 7239–7255 (2021). F, Z. et al. METTL3-dependent RNA m 6 A dysregulation contributes to neurodegeneration in Alzheimer’s disease through aberrant cell cycle events. Mol Neurodegener 16 , 70–70 (2021). Jiang, L. et al. Interaction of tau with HNRNPA2B1 and N6-methyladenosine RNA mediates the progression of tauopathy. Mol Cell 81 , 4209-4227.e12 (2021). Castro-Hernández, R. et al. Conserved reduction of m6A RNA modifications during aging and neurodegeneration is linked to changes in synaptic transcripts. Proc Natl Acad Sci U S A 120 , e2204933120. Haroutunian, V., Katsel, P. & Schmeidler, J. Transcriptional vulnerability of brain regions in Alzheimer’s disease and dementia. Neurobiol Aging 30 , 561–573 (2009). Palmqvist, S. et al. Discriminative Accuracy of Plasma Phospho-tau217 for Alzheimer Disease vs Other Neurodegenerative Disorders. JAMA 324 , 772–781 (2020). Ashton, N. J. et al. Diagnostic Accuracy of a Plasma Phosphorylated Tau 217 Immunoassay for Alzheimer Disease Pathology. JAMA Neurol 81 , 255–263 (2024). Huang, H., Song, R., Wong, J. J.-L., Anggono, V. & Widagdo, J. The N6-methyladenosine RNA landscape in the aged mouse hippocampus. https://doi.org/10.1111/acel.13755 doi:10.1111/acel.13755. Fan, Y., Lv, X., Chen, Z., Peng, Y. & Zhang, M. m6A methylation: Critical roles in aging and neurological diseases. Front. Mol. Neurosci. 16 , (2023). Yankova, E. et al. Small molecule inhibition of METTL3 as a strategy against myeloid leukaemia. Nature 593 , 597–601 (2021). Wang, P., Doxtader, K. A. & Nam, Y. Structural basis for cooperative function of Mettl3 and Mettl14 methyltransferases. Mol Cell 63 , 306–317 (2016). Koranda, J. L. et al. Mettl14 Is Essential for Epitranscriptomic Regulation of Striatal Function and Learning. Neuron 99 , 283-292.e5 (2018). Vorhees, C. V. & Williams, M. T. Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat Protoc 1 , 848–858 (2006). D’Hooge, R. & De Deyn, P. P. Applications of the Morris water maze in the study of learning and memory. Brain Res Brain Res Rev 36 , 60–90 (2001). Garthe, A., Behr, J. & Kempermann, G. Adult-generated hippocampal neurons allow the flexible use of spatially precise learning strategies. PLoS One 4 , e5464 (2009). Jürgenson, M., Aonurm-Helm, A. & Zharkovsky, A. Behavioral profile of mice with impaired cognition in the elevated plus-maze due to a deficiency in neural cell adhesion molecule. Pharmacol Biochem Behav 96 , 461–468 (2010). Merchán-Rubira, J., Sebastián-Serrano, Á., Díaz-Hernández, M., Avila, J. & Hernández, F. Peripheral nervous system effects in the PS19 tau transgenic mouse model of tauopathy. Neurosci Lett 698 , 204–208 (2019). Al-Ghraiybah, N. F. et al. Glial Cell-Mediated Neuroinflammation in Alzheimer’s Disease. Int J Mol Sci 23 , 10572 (2022). Kwon, H. S. & Koh, S.-H. Neuroinflammation in neurodegenerative disorders: the roles of microglia and astrocytes. Transl Neurodegener 9 , 42 (2020). Li, Q., Wen, S., Ye, W., Zhao, S. & Liu, X. The potential roles of m6A modification in regulating the inflammatory response in microglia. Journal of Neuroinflammation 18 , 149 (2021). Ding, L. et al. m6A Reader Igf2bp1 Regulates the Inflammatory Responses of Microglia by Stabilizing Gbp11 and Cp mRNAs. Front Immunol 13 , 872252 (2022). Huang, H., Camats-Perna, J., Medeiros, R., Anggono, V. & Widagdo, J. Altered Expression of the m6A Methyltransferase METTL3 in Alzheimer’s Disease. eNeuro 7 , (2020). Zhang, X. et al. Differential methylation of circRNA m6A in an APP/PS1 Alzheimer’s disease mouse model. Mol Med Rep 27 , 55 (2023). Wu, Z. et al. m6A epitranscriptomic regulation of tissue homeostasis during primate aging. Nat Aging 3 , 705–721 (2023). Zhang, W., Xiao, D., Mao, Q. & Xia, H. Role of neuroinflammation in neurodegeneration development. Sig Transduct Target Ther 8 , 267 (2023). Pascoal, T. A. et al. Microglial activation and tau propagate jointly across Braak stages. Nat Med 27 , 1592–1599 (2021). d’Errico, P. et al. Microglia contribute to the propagation of Aβ into unaffected brain tissue. Nat Neurosci 25 , 20–25 (2022). Bellaver, B. et al. Astrocyte reactivity influences amyloid-β effects on tau pathology in preclinical Alzheimer’s disease. Nat Med 29 , 1775–1781 (2023). Dai, D. L., Li, M. & Lee, E. B. Human Alzheimer’s disease reactive astrocytes exhibit a loss of homeostastic gene expression. Acta Neuropathologica Communications 11 , 127 (2023). Wu, X. et al. The m6A methyltransferase METTL3 drives neuroinflammation and neurotoxicity through stabilizing BATF mRNA in microglia. Cell Death Differ 32 , 100–117 (2025). Zhang, F. et al. Regulation of N6-methyladenosine (m6A) RNA methylation in microglia-mediated inflammation and ischemic stroke. Front. Cell. Neurosci. 16 , (2022). Wen, Y.-L., Guo, F., Gu, T.-T., Zeng, Y.-P. & Cao, X. Transcriptomic Regulation by Astrocytic m6A Methylation in the mPFC. Genes Cells 30 , e70003 (2025). Li, Y., Li, J., Yu, Q., Ji, L. & Peng, B. METTL14 regulates microglia/macrophage polarization and NLRP3 inflammasome activation after ischemic stroke by the KAT3B-STING axis. Neurobiology of Disease 185 , 106253 (2023). Zhu, L. et al. METTL3/IGF2BP2/IκBα axis participates in neuroinflammation in Alzheimer’s disease by regulating M1/M2 polarization of microglia. Neurochemistry International 186 , 105964 (2025). Hui, Y. et al. Immunological characterization and diagnostic models of RNA N6-methyladenosine regulators in Alzheimer’s disease. Sci Rep 13 , 14588 (2023). Yin, H. et al. Loss of the m6A methyltransferase METTL3 in monocyte-derived macrophages ameliorates Alzheimer’s disease pathology in mice. PLOS Biology 21 , e3002017 (2023). Zhao, X. et al. Mettl3 regulates the pathogenesis of Alzheimer’s disease via fine-tuning Lingo2. Mol Psychiatry 30 , 4047–4063 (2025). Long, J. M. & Holtzman, D. M. Alzheimer Disease: An Update on Pathobiology and Treatment Strategies. Cell 179 , 312–339 (2019). Xia, L. et al. A new perspective on Alzheimer’s disease: m6A modification. Front Genet 14 , 1166831 (2023). Haroutunian, V., Katsel, P. & Schmeidler, J. Transcriptional vulnerability of brain regions in Alzheimer’s disease and dementia. Neurobiol Aging 30 , 561–573 (2009). Marco, A. R. et al. Therapeutic VEGFC treatment provides protection against traumatic-brain-injury-driven tauopathy pathogenesis. Cell Reports 44 , (2025). Lammert, C. R. et al. AIM2 inflammasome surveillance of DNA damage shapes neurodevelopment. Nature 580 , 647–652 (2020). Kraeuter, A.-K., Guest, P. C. & Sarnyai, Z. The Y-Maze for Assessment of Spatial Working and Reference Memory in Mice. in Pre-Clinical Models: Techniques and Protocols (ed. Guest, P. C.) 105–111 (Springer, New York, NY, 2019). doi:10.1007/978-1-4939-8994-2_10. Additional Declarations Yes there is potential Competing Interest. L.J. and A.T. have filed a patent application related to the therapeutic approach described in this manuscript (U.S. Provisional Patent Application No. 63/933,846). All other authors declare no competing interests. Supplementary Files m6AMSSupplemental20251216.docx Extended Figures Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8379573","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":562597728,"identity":"ff348a69-3583-4c0d-841c-262b0eb915d4","order_by":0,"name":"Lulu 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20:20:59","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8379573/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8379573/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":98627561,"identity":"b8d7182a-74fe-4e09-940e-2a4222a36400","added_by":"auto","created_at":"2025-12-19 17:10:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":749187,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelation between pathological progress of tau hyperphosphorylation, Aβ plaques deposition, and excessive m\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eA accumulation. (a)\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eRepresentative images of 3,3’-Diaminobenzidine (DAB) staining of tau (pTau217 antibody) and Aβ (4G8 antibody) pathology in parallel with m\u003csup\u003e6\u003c/sup\u003eA labeling in post-mortem brain tissue of cognitively normal control (NC) and AD brain sections of Brodmann’s area 10. Scale bar = 100µm. \u003cstrong\u003e(b)\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eQuantification of pTau217 intensity by integrated density (IntDen) in post-mortem brain tissue. \u003cstrong\u003e(c)\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eQuantification of Aβ plaques (4G8 antibody) intensity by IntDen in post-mortem brain. \u003cstrong\u003e(d)\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eQuantification of m\u003csup\u003e6\u003c/sup\u003eA intensity by IntDen in post-mortem brain tissue. \u003cstrong\u003e(e)\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eCorrelation analysis demonstrating relationship between pTau217, Aβ, and m\u003csup\u003e6\u003c/sup\u003eA pixels per section across Braak stages. The Pearson correlation coefficient between m\u003csup\u003e6\u003c/sup\u003eA and tau pathology was r = 0.97 (\u003cem\u003ep\u003c/em\u003e=0.001), correlation coefficient with Aβ was r = 0.89 (\u003cem\u003ep \u003c/em\u003e=\u003cem\u003e \u003c/em\u003e0.018). **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.005, ****\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001. Error bars = SEM. Statistical differences were analyzed using unpaired Student’s t-test. \u003cem\u003eN \u003c/em\u003e= 8 brains per condition. \u003cem\u003en\u003c/em\u003e = 16 sections per condition \u003cstrong\u003e(b-e)\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8379573/v1/0f7e9798ad6af0484fb2dba7.png"},{"id":98577679,"identity":"b2542782-306b-45dd-a64d-07b6e6f12302","added_by":"auto","created_at":"2025-12-19 07:45:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":816565,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAge-related accumulation of m\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eA in humanized tauopathy mouse model of AD. (a-c)\u003c/strong\u003e, Images showing DAPI (cyan), m\u003csup\u003e6\u003c/sup\u003eA (red), MC1 positive misfolded tau (green), and MAP2 (magenta) immunofluorescence staining in \u003cstrong\u003e(a)\u003c/strong\u003e, CA3, \u003cstrong\u003e(b)\u003c/strong\u003e, CA1, \u003cstrong\u003e(c)\u003c/strong\u003e, and cortex (CTX), regions of P301S tau (PS19) transgenic mice and C57BL/6 (WT) mice at 6-months and 9-months of age. Scale bar = 50µm. \u003cstrong\u003e(d-f)\u003c/strong\u003e, Quantification of MC1 immunofluorescence intensity by integrated density (IntDen) normalized to area in \u003cstrong\u003e(d)\u003c/strong\u003e, CA3, \u003cstrong\u003e(e), \u003c/strong\u003eCA1, and \u003cstrong\u003e(f), \u003c/strong\u003eCTX. \u003cstrong\u003eg-i\u003c/strong\u003e, Quantification of m\u003csup\u003e6\u003c/sup\u003eA immunofluorescence intensity by IntDen normalized to area as shown in \u003cstrong\u003e(g)\u003c/strong\u003e, CA3, \u003cstrong\u003e(h)\u003c/strong\u003e, CA1, and \u003cstrong\u003e(i)\u003c/strong\u003e, CTX. \u003cstrong\u003e(j-l)\u003c/strong\u003e, Quantification of neurodegeneration by MAP2 immunofluorescent intensity by IntDen normalized to area as shown in \u003cstrong\u003e(j)\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eCA3, \u003cstrong\u003e(k)\u003c/strong\u003e, CA1, and \u003cstrong\u003e(l)\u003c/strong\u003e, CTX. Statistical analysis was performed using two-way ANOVA with Tukey’s multiple comparisons test. n. Error bars = SEM. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.005, ****\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001. \u003cem\u003eN\u003c/em\u003e = 5 brains per condition. \u003cem\u003en\u003c/em\u003e = 10 sections per condition.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8379573/v1/20bb3bc26d476c08a9a6b6bf.png"},{"id":98577674,"identity":"43018f15-2433-4cd2-8ab3-5afe9c211e52","added_by":"auto","created_at":"2025-12-19 07:45:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":771130,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of METTL3 reduces excessive m\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eA in mice of tauopathy. \u003c/strong\u003e\u0026nbsp;\u003cstrong\u003e(a)\u003c/strong\u003e, Schematic of timeline of STM2457 treatment in C57BL/6 mice (WT) and P301S tau transgenic mice (PS19). \u003cstrong\u003e(b), \u003c/strong\u003eQuantification of STM2457 in 10mg hippocampal tissue from WT mice with and without STM2457 treatment by LC-MS. \u003cstrong\u003e(c),\u003c/strong\u003e Images showing DAB staining of m\u003csup\u003e6\u003c/sup\u003eA in the CA1 and CA3 regions of 9-month-old WT and PS19 mice with and without STM2457 treatment. Scale bar = 100µm. \u003cstrong\u003ed\u003c/strong\u003e, \u003cstrong\u003ee\u003c/strong\u003e, Quantification of intensity of DAB stained m\u003csup\u003e6\u003c/sup\u003eA by raw integrated density (RawIntDen) in \u003cstrong\u003e(d\u003c/strong\u003e, CA3 and \u003cstrong\u003e(e)\u003c/strong\u003e, CA1. \u003cem\u003eN \u003c/em\u003e= 5 brains per condition, \u003cem\u003en \u003c/em\u003e= 10 sections per condition. \u003cstrong\u003e(f)\u003c/strong\u003e, m\u003csup\u003e6\u003c/sup\u003eA RNA dot blot for PS19 and WT mice with or without STM2457 treatment. \u003cstrong\u003e(g)\u003c/strong\u003e, Quantification of m\u003csup\u003e6\u003c/sup\u003eA RNA dot blot signals, expressed as fold changes in m\u003csup\u003e6\u003c/sup\u003eA intensity (RawIntDen), in PS19 and WT mice with or without STM2457 treatment. \u003cem\u003eN\u003c/em\u003e = 12 brains per condition. \u003cstrong\u003e(h)\u003c/strong\u003e, Immunofluorescence analysis of m\u003csup\u003e6\u003c/sup\u003eA in CA1 and CTX regions of PS19 and WT mice with or without STM2457 treatment. Scale bar = 50µm. \u003cstrong\u003ei\u003c/strong\u003e, \u003cstrong\u003ej\u003c/strong\u003e, Quantification of m\u003csup\u003e6\u003c/sup\u003eA immunofluorescence intensity by RawIntDen in the \u003cstrong\u003e(i), \u003c/strong\u003eCA1 and \u003cstrong\u003e(j), \u003c/strong\u003eCTX. \u003cstrong\u003e(k), \u003c/strong\u003eWestern blot of PS19 and WT mouse CTX\u003cstrong\u003e \u003c/strong\u003eprobed for METTL3, METTL14, and β-actin control. \u003cstrong\u003el\u003c/strong\u003e, \u003cstrong\u003em\u003c/strong\u003e, Quantification of relative protein expression level of \u003cstrong\u003e(l),\u003c/strong\u003e METTL3 and \u003cstrong\u003e(m)\u003c/strong\u003e, METTL14. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.005, ****\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001.\u003cem\u003e N\u003c/em\u003e = 5 brains per condition,\u003cem\u003e n\u003c/em\u003e = 10 sections per condition \u003cstrong\u003e(d-j)\u003c/strong\u003e. \u003cem\u003eN\u003c/em\u003e = 3\u003cstrong\u003e (k-m).\u003c/strong\u003e Error bars = SEM. Statistical analysis was done using two-way ANOVA with Tukey’s multiple comparisons test \u003cstrong\u003e(d, e, i, j).\u003c/strong\u003e Statistical analysis was done using two-way ANOVA with Fisher’s LSD \u003cstrong\u003e(g, l, m)\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8379573/v1/07f2b758de719fe84efcdde6.png"},{"id":98577678,"identity":"dc38bfd5-8076-4416-8d9e-d62070d78add","added_by":"auto","created_at":"2025-12-19 07:45:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":296027,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTreatment of PS19 mice with STM2457 ameliorates behavioral deficits. (a)\u003c/strong\u003e, Traces from video recordings of mouse movement in the MWM on Day 7. Platform indicated with red circle. \u003cstrong\u003e(b)\u003c/strong\u003e, Average distance from platform (cm) represents mean distance from the platform in each quadrant across days 1 to 4 for PS19 and WT mice, with or without STM2457 treatment.\u003cstrong\u003e (c)\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eEscape latency (s) indicates time spent finding the platform on Day 7. \u003cstrong\u003e(d)\u003c/strong\u003e, Time (s) spent in the open and closed arms of the EPM. \u003cstrong\u003e(e), \u003c/strong\u003eNumber of spontaneous entries into the non-starting arms of the Y maze. \u003cstrong\u003e(f)\u003c/strong\u003e, Percent spontaneous alteration of mice in the Y maze.\u003cstrong\u003e (g)\u003c/strong\u003e, Total distance (cm) traveled in the OF test. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.005, ****\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001. \u003cem\u003eN\u003c/em\u003e= 10 mice per condition, \u003cem\u003en\u003c/em\u003e = 4 trials per mouse \u003cstrong\u003e(a-c)\u003c/strong\u003e and \u003cem\u003eN = \u003c/em\u003e10 mice per condition \u003cstrong\u003e(d-g)\u003c/strong\u003e. Error bars = SEM. Statistical analysis was performed using Fisher’s LSD \u003cstrong\u003e(b, d, e-g)\u003c/strong\u003e. Statistical analysis was done using two-way ANOVA with Tukey’s multiple comparisons test\u003cstrong\u003e (c)\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8379573/v1/8b585e73db5ec4c29ce2aec8.png"},{"id":98627439,"identity":"fd067e58-f1a6-460d-8c1a-cf611fad4a7d","added_by":"auto","created_at":"2025-12-19 17:10:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":927463,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSTM2457 treatment mitigates tau pathology and neurodegeneration.\u003c/strong\u003e \u003cstrong\u003e(a)\u003c/strong\u003e, Representative immunofluorescence images of CA1 of WT and PS19 control and STM2457 treated mice with DAPI (cyan), misfolded tau (green, MC1 antibody), and m\u003csup\u003e6\u003c/sup\u003eA (red) Scale bar = 50µm. \u003cstrong\u003eb-d, \u003c/strong\u003eQuantification of immunofluorescence intensity of misfolded tau by integrated density (IntDen) normalized to area in the \u003cstrong\u003e(b)\u003c/strong\u003e, CA3, \u003cstrong\u003e(c)\u003c/strong\u003e, CA1, and \u003cstrong\u003e(d)\u003c/strong\u003e, CTX. \u003cem\u003eN \u003c/em\u003e= 10 mice per condition, \u003cem\u003en\u003c/em\u003e = 20 sections per condition. \u003cstrong\u003e(e)\u003c/strong\u003e, Western blot analysis of phosphorylated tau at Ser202 and Ser202/Thr205, detected using CP13 and AT8 antibodies, respectively, with β-actin as the internal loading control. \u003cem\u003eN\u003c/em\u003e = 3, \u003cem\u003en\u003c/em\u003e = 12. \u003cstrong\u003e(f)\u003c/strong\u003e, Quantification of hyperphosphorylated tau by western blot of Ser202 phosphorylated tau (CP13 antibody) with background subtracted and normalized to β-actin. \u003cstrong\u003e(g)\u003c/strong\u003e, Quantification of hyperphosphorylated tau expression by western blot of Ser202/Thr205 phosphorylated tau isoforms (AT8 antibody) with background subtracted and normalized to β-actin. \u003cstrong\u003e(h)\u003c/strong\u003e, Representative immunofluorescence images of the CA3, CA1, and CTX with NeuN (magenta) stain. Scale bar = 50µm. \u003cstrong\u003ei-k\u003c/strong\u003e, Quantification of NeuN by raw integrated density in the \u003cstrong\u003e(i)\u003c/strong\u003e, CA3, \u003cstrong\u003e(j)\u003c/strong\u003e, CA1, and \u003cstrong\u003e(k)\u003c/strong\u003e, CTX. \u003cem\u003eN\u003c/em\u003e = 5, \u003cem\u003en\u003c/em\u003e = 10. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.005, ****\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001. Error bars = SEM. Statistical analysis was performed using two-way ANOVA with Fisher’s LSD \u003cstrong\u003e(b-d, f, g)\u003c/strong\u003e. Statistical analysis done using two-way ANOVA with Tukey’s multiple comparisons test. \u003cstrong\u003e(i-k)\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8379573/v1/ef401275bcb2b0523709ac82.png"},{"id":98577676,"identity":"a1b73afd-d854-4107-86b2-1677eba5bdec","added_by":"auto","created_at":"2025-12-19 07:45:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":836220,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTreatment with STM2457 attenuates glial inflammatory response in PS19 mice. \u0026nbsp;(a), \u003c/strong\u003eRepresentative immunofluorescence images of CA1 and CA3 of treated and control PS19 and WT mice with IBA1 (green). Scale bar = 50µm. \u003cstrong\u003e(b)\u003c/strong\u003e, Representative immunofluorescence image magnification illustrating resting state microglial morphology in WT CA1 (left) and ameboid-like morphology of inflammatory microglia cell body (red arrow) in PS19 CA1 (right). Scale bar = 17.5µm. \u003cstrong\u003ec, d\u003c/strong\u003e, Quantification of immunofluorescence intensity of IBA1 by raw integrated density (RawIntDen) in the \u003cstrong\u003e(c)\u003c/strong\u003e, CA1, and \u003cstrong\u003e(d)\u003c/strong\u003e, CA3. \u003cstrong\u003e(e)\u003c/strong\u003e, Representative immunofluorescence images of the CA1, CA3, and CTX of treated and control PS19 and WT mice with GFAP (cyan). Scale bar = 50µm. \u003cstrong\u003ef-h\u003c/strong\u003e, Quantification of GFAP immunofluorescent intensity by RawIntDen in the \u003cstrong\u003e(f)\u003c/strong\u003e, CA1, \u003cstrong\u003e(g)\u003c/strong\u003e, CA3, and \u003cstrong\u003e(h)\u003c/strong\u003e, CTX. \u003cem\u003eN\u003c/em\u003e = 5 brains per condition, \u003cem\u003en\u003c/em\u003e= 10 sections per condition. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.005, ****\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001. Error bars = SEM. Statistical analysis was done using two-way ANOVA with Tukey’s multiple comparisons test.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8379573/v1/3f18b71a3cc90578b40b647b.png"},{"id":98632086,"identity":"3247a089-f3b8-447a-a09c-fdf78af014de","added_by":"auto","created_at":"2025-12-19 17:21:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5941543,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8379573/v1/9411b2d2-660e-4597-aa10-048d22378d0b.pdf"},{"id":98577680,"identity":"c04cc41c-233b-42b8-9df7-ab93ca66a258","added_by":"auto","created_at":"2025-12-19 07:45:07","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":25367775,"visible":true,"origin":"","legend":"Extended Figures","description":"","filename":"m6AMSSupplemental20251216.docx","url":"https://assets-eu.researchsquare.com/files/rs-8379573/v1/03257a931b77c8060a410f64.docx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nL.J. and A.T. have filed a patent application related to the therapeutic approach described in this manuscript (U.S. Provisional Patent Application No. 63/933,846). All other authors declare no competing interests.","formattedTitle":"Inhibition of N6-Methyladenosine Accumulation by Targeting METTL3 Mitigates Tau Pathology and Cognitive Decline in Alzheimer’s Disease","fulltext":[{"header":"Highlights","content":"\u003cul\u003e\n \u003cli\u003eN6-methyladenosine (m\u003csup\u003e6\u003c/sup\u003eA) is elevated in human AD brain tissue and correlates with both tau and A\u0026beta; pathology.\u003c/li\u003e\n \u003cli\u003em\u003csup\u003e6\u003c/sup\u003eA elevation is age dependent and closely correlated with pathological progression in a tauopathy mouse model of AD.\u003c/li\u003e\n \u003cli\u003eMETTL3 inhibition reduces m\u003csup\u003e6\u003c/sup\u003eA levels and alleviates tau pathology, neurodegeneration, and neuroinflammation in PS19 mice.\u003c/li\u003e\n \u003cli\u003eSTM2457 treatment improves learning and memory, supporting METTL3 as a promising therapeutic target for AD.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eIn the last 20 years, reported deaths from Alzheimer\u0026rsquo;s disease (AD) have increased over 140%, emphasizing the importance of early detection and new disease-modifying treatment strategies\u003csup\u003e1\u003c/sup\u003e. For decades, AD research has been focused on identifying hallmarks of amyloid-\u0026beta; (A\u0026beta;) deposition and aggregation of tau neurofibrillary tangles. Emerging research on the role of RNA modifications in AD pathogenesis is a promising research direction for elucidating AD beyond proteiopathies\u003csup\u003e2\u0026ndash;4\u003c/sup\u003e. N6-methyladenosine (m\u003csup\u003e6\u003c/sup\u003eA) RNA modification is a key regulator of RNA metabolism and has been studied extensively in the context of neurodevelopment and oncogenesis, though its role in neurodegeneration remains underexplored and debated. Important roles of m\u003csup\u003e6\u003c/sup\u003eA in RNA metabolism include pre-mRNA processing, splicing, nuclear export, mRNA expression, and RNA degradation\u003csup\u003e5\u0026ndash;14\u003c/sup\u003e. Dysregulation of m\u003csup\u003e6\u003c/sup\u003eA machinery, including its writers, readers, and erasers, has been linked to impaired memory formation and cognitive decline\u003csup\u003e5,15\u0026ndash;25\u003c/sup\u003e. METTL3 is a core writer protein and catalytic portion of the METTL3-METTL14 methyltransferase complex, which carries out the modification resulting in m\u003csup\u003e6\u003c/sup\u003eA RNA\u003csup\u003e26\u0026ndash;28\u003c/sup\u003e. Elevated expression of METTL3 has been implicated in AD and related rodent models\u003csup\u003e29\u003c/sup\u003e. Further investigation into m\u003csup\u003e6\u003c/sup\u003eA\u0026rsquo;s impact on key AD-related pathways could provide novel therapeutic targets, offering a fresh perspective on disease intervention and potential RNA-based treatments.\u003c/p\u003e\n\u003cp\u003eOur previous research discovered a novel association of m\u003csup\u003e6\u003c/sup\u003eA RNA with oligomeric tau (oTau) in the neuronal cytoplasm through the HNRNPA2B1 RNA binding protein, which behaves as an m\u003csup\u003e6\u003c/sup\u003eA reader. Our finding of a 3-5-fold increase of cytoplasmic m\u003csup\u003e6\u003c/sup\u003eA abundance in disease tissues (P301S tau mice and AD-affected human brain tissue) suggests that m\u003csup\u003e6\u003c/sup\u003eA plays a significant role in the pathogenic pathway of AD. This m\u003csup\u003e6\u003c/sup\u003eA-HNRNPA2B1-oTau interaction contributes to oTau\u0026rsquo;s integrated stress response and reduced protein synthesis\u003csup\u003e30\u003c/sup\u003e. Given the central role of hyperphosphorylated and oligomeric tau in AD pathology, these findings highlight the potential for modulating excessive m\u003csup\u003e6\u003c/sup\u003eA accumulation as a strategy to control tau aggregation.\u003c/p\u003e\n\u003cp\u003eTo comprehensively assess m\u003csup\u003e6\u003c/sup\u003eA RNA dynamics during AD progression and aging, we analyzed the correlation between m\u003csup\u003e6\u003c/sup\u003eA RNA accumulation and pathological A\u0026beta; and tau deposition in human post-mortem brain tissue as well as in mouse models of tauopathy across different ages. We found a consistent increase in m\u003csup\u003e6\u003c/sup\u003eA RNA levels that strongly correlated with the accumulation of pathological tau and A\u0026beta; in human brains from early to late Braak stages, which was further confirmed in aging and disease-progression of mouse models. To explore the therapeutic potential of targeting excessive m\u003csup\u003e6\u003c/sup\u003eA RNA accumulation, we examined the effects of STM2457, a selective METTL3 inhibitor, on AD-related pathology in P301S PS19 tauopathy mice. Remarkably, STM2457 treatment rescued cognitive function, reduced pathological tau accumulation, prevented neuronal loss, and ameliorated neuroinflammation, as demonstrated by behavioral assessments, immunostaining, and biochemical analyses.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCharacterization of m\u003csup\u003e6\u003c/sup\u003eA modifications associated with tau hyperphosphorylation and A\u0026beta; deposition across Alzheimer\u0026rsquo;s disease progression\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrevious investigations examining m\u003csup\u003e6\u003c/sup\u003eA RNA levels in human post-mortem brain tissue from AD individuals have yielded conflicting results. Work examining the anterior cingulate gyrus and middle temporal gyrus found reduced levels of m\u003csup\u003e6\u003c/sup\u003eA RNA\u003csup\u003e19,31\u003c/sup\u003e, however, when the anterior frontal gyrus was examined, there were elevated levels of m\u003csup\u003e6\u003c/sup\u003eA\u003csup\u003e30\u003c/sup\u003e. Thus, we sought to reaffirm this cortical increase in m\u003csup\u003e6\u003c/sup\u003eA RNA in post-mortem brain tissue. We examined post-mortem brain tissue collected from Brodmann area 10; this region was selected based on consistent evidence from prior studies indicating a strong positive correlation between altered gene expression and disease severity\u003csup\u003e32\u003c/sup\u003e. The brain tissue in this study was collected from eight AD patients and eight cognitively normal control (NC) individuals. The results shown include brain tissue from NC individuals between Braak stages I-IV and AD individuals between Braak stages V-VI. Tau phosphorylated at threonine 217 (pTau217) is an early AD marker that correlates with disease severity and can be used to evaluate pathogenic tau in tissue\u003csup\u003e33,34\u003c/sup\u003e. Using 3,3\u0026rsquo;-Diaminobenzidine (DAB) staining, pTau217, A\u0026beta; (4G8 antibody), and m\u003csup\u003e6\u003c/sup\u003eA RNA levels were evaluated \u003cstrong\u003e(Fig. 1a)\u003c/strong\u003e. As expected, we observed elevated pTau217 and A\u0026beta; pathology in AD brains \u003cstrong\u003e(Fig. 1a-c)\u003c/strong\u003e. When comparing AD to NC, there was significantly more m\u003csup\u003e6\u003c/sup\u003eA in the AD post-mortem brains than the controls \u003cstrong\u003e(Fig. 1a, d)\u003c/strong\u003e. Additionally, pTau217, A\u0026beta;, and m\u003csup\u003e6\u003c/sup\u003eA were all more prominent in later Braak Stages (V and VI) (\u003cstrong\u003eFig. 1e\u003c/strong\u003e). Correlation analysis between pTau217, A\u0026beta;, and m\u003csup\u003e6\u003c/sup\u003eA revealed that pathologies were positively correlated with increased levels of m\u003csup\u003e6\u003c/sup\u003eA. The Pearson correlation coefficient (R) between\u0026nbsp;m\u003csup\u003e6\u003c/sup\u003eA\u0026nbsp;and tau pathology was 1.00 (\u003cem\u003ep\u003c/em\u003e=2.21x10\u003csup\u003e-5\u003c/sup\u003e), while the correlation coefficient with A\u0026beta; was 0.80 (\u003cem\u003ep\u003c/em\u003e=0.055) \u003cstrong\u003e(Fig. 1e)\u003c/strong\u003e. This data reaffirms the presence of elevated m\u003csup\u003e6\u003c/sup\u003eA levels in cortical regions of AD post-mortem brain tissue, in alignment with the pathological accumulation of A\u0026beta; and tau and shows a particularly strong correlation with pathogenic tau.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAge-related accumulation of m\u003csup\u003e6\u003c/sup\u003eA\u0026nbsp;in humanized P301S tau transgenic mouse model of AD\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrevious work demonstrated an important role for m\u003csup\u003e6\u003c/sup\u003eA RNA methylation in regulating neurodevelopment and aging \u003csup\u003e5,20,35,36\u003c/sup\u003e. Data in \u003cstrong\u003eFig. 1\u003c/strong\u003e from post-mortem AD brain tissue showed that marked accumulation of m\u003csup\u003e6\u003c/sup\u003eA is highly correlated with tau pathology. Next, we quantified how m\u003csup\u003e6\u003c/sup\u003eA levels change with age in a mouse model of progressive tau pathology. To do this, we utilized PS19 mice overexpressing human P301S mutant tau and examined brain tissue collected from 6- or 9-month-old mice. To confirm the pathological relevance of the PS19 model, we labeled hippocampal and cortical regions using the MC1 antibody to identify misfolded tau. MC1 fluorescence intensity was significantly increased in PS19 compared to C57BL/6 wild-type (WT) mice in the hippocampus and cortex (CTX) at both 6- and 9-months of age \u003cstrong\u003e(Fig. 2a-f)\u003c/strong\u003e. Considering advanced tau pathology in AD is associated with robust neurodegeneration, we also probed for MAP2, a marker of neuronal cell bodies and dendrites, to evaluate neuronal integrity. MAP2 fluorescence intensity was significantly decreased in the hippocampus and CTX of PS19 mice in 9-month brains \u003cstrong\u003e(Fig. 2a-c and j-l)\u003c/strong\u003e. These results indicate that PS19 mice exhibit both increased tau pathology and neurodegeneration as anticipated. More importantly, staining for m\u003csup\u003e6\u003c/sup\u003eA revealed elevated m\u003csup\u003e6\u003c/sup\u003eA RNA levels in the cortex and hippocampal CA3 region of PS19 mouse brains when compared to WT controls at 9-months of age (\u003cstrong\u003eFig. 2a-c and g-i)\u003c/strong\u003e. Interestingly, we observed that m\u003csup\u003e6\u003c/sup\u003eA RNA was significantly increased in 9-month-old PS19 mouse hippocampus (CA1 and CA3) and CTX when compared to the 6-month-old PS19 group \u003cstrong\u003e(Fig. 2g-i)\u003c/strong\u003e. However, for all brain regions examined, m\u003csup\u003e6\u003c/sup\u003eA levels were comparable at 6 months of age between PS19 and WT mice \u003cstrong\u003e(Fig. 2g-i)\u003c/strong\u003e. Additionally, DAB staining of brain tissue from 5-month- and 9-month-old WT and PS19 mice revealed that m\u003csup\u003e6\u003c/sup\u003eA levels were significantly elevated in 9-month-old PS19 compared to age-matched WT samples, and there is a significant increase in m\u003csup\u003e6\u003c/sup\u003eA from the 6-month to 9-month timepoint for PS19 mice (\u003cstrong\u003eExtended Data Fig. 1)\u003c/strong\u003e. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAltogether, our data suggests that the age-related increase in tau pathology and neurodegeneration in PS19 mice is accompanied by a parallel increase in m\u003csup\u003e6\u003c/sup\u003eA RNA modification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eInhibition of METTL3 reduces excessive m\u003csup\u003e6\u003c/sup\u003eA RNA levels\u0026nbsp;in tauopathy mice\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur results demonstrated that m\u003csup\u003e6\u003c/sup\u003eA levels were elevated in both AD post-mortem brains and tauopathic mouse brains. RNA methylation is a reversible epigenetic modification, which makes it an attractive target for pharmacological intervention. Therefore, we attempted to restore the homeostasis of RNA methylation through pharmacologically removing excessive m\u003csup\u003e6\u003c/sup\u003eA by inhibiting its methyltransferase METTL3. STM2457 is an inhibitor of METTL3 that has been examined as a potential therapeutic for myeloid leukemia; that work demonstrated STM2457 can penetrate the blood brain barrier and infiltrate the brain\u003csup\u003e37\u003c/sup\u003e. STM2457 inhibits METTL3 by binding to its S-adenosyl methionine binding site, thus disrupting its catalytic activity without influencing the activity of other RNA methyltransferases\u003csup\u003e37\u003c/sup\u003e. Given its selective METTL3 inhibition, robust brain permeability, and high potency in IC₅₀ value of 16.9 nM\u003csup\u003e37\u003c/sup\u003e, we utilized STM2457 as a treatment strategy, anticipating that the achievable brain dose would fall within its therapeutically effective range. STM2457 was administered continuously for six weeks at a dose of 0.25 mg/kg/day using an osmotic pump that was subcutaneously implanted in the mice starting at 4 months of age \u003cstrong\u003e(Fig. 3a)\u003c/strong\u003e. The concentrations of STM2457 in the hippocampus of 9-month-old STM2457-treated mice were measured by liquid chromatography\u0026ndash;mass spectrometry (LC\u0026ndash;MS). Our result showed an average of 0.96 ng of STM2457 was detected per 10 mg of hippocampal tissue used for analysis \u003cstrong\u003e(Fig. 3b\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eNext, the efficiency of STM2457 treatment on levels of m\u003csup\u003e6\u003c/sup\u003eA in select brain regions was evaluated. Hippocampal levels of m\u003csup\u003e6\u003c/sup\u003eA in PS19 and WT mice with and without STM2457 treatment were assessed with DAB staining with m\u003csup\u003e6\u003c/sup\u003eA antigen probed by an m\u003csup\u003e6\u003c/sup\u003eA-specific antibody \u003cstrong\u003e(Fig. 3c)\u003c/strong\u003e. Notably, PS19 mice exhibited a significant increase in m\u003csup\u003e6\u003c/sup\u003eA intensity in CA3 compared to WT mice. Importantly, STM2457 treatment robustly countered the presence of this excessive m\u003csup\u003e6\u003c/sup\u003eA \u003cstrong\u003e(Fig. 3d)\u003c/strong\u003e. A similar pattern was observed in CA1, with STM2457 resulting in a significant reduction of m\u003csup\u003e6\u003c/sup\u003eA in PS19 mice. However, high levels of m\u003csup\u003e6\u003c/sup\u003eA were not apparent in the CA1 of untreated PS19 mice \u003cstrong\u003e(Fig. 3e)\u003c/strong\u003e. This consistent reduction of m\u003csup\u003e6\u003c/sup\u003eA levels across both CA3 and CA1 indicates that STM2457 treatment effectively controls\u003csup\u003e\u0026nbsp;\u003c/sup\u003ehippocampal m\u003csup\u003e6\u003c/sup\u003eA\u003csup\u003e\u0026nbsp;\u003c/sup\u003ein PS19 mice. Changes in m\u003csup\u003e6\u003c/sup\u003eA levels by STM2457 were also validated through immunohistochemical analysis. We conducted an m\u003csup\u003e6\u003c/sup\u003eA dot blot of RNA extracted from cortical tissue of 9-month-old PS19 and WT mice and found that STM2457 treatment significantly reduced total m\u003csup\u003e6\u003c/sup\u003eA RNA levels in PS19 mice, but not in WT mice \u003cstrong\u003e(Fig. 3f, g)\u003c/strong\u003e. \u0026nbsp;Immunofluorescence labeling of brain tissue for m\u003csup\u003e6\u003c/sup\u003eA also revealed a significant reduction of m\u003csup\u003e6\u003c/sup\u003eA in CA1 and CTX of PS19 STM2457-treated mice \u003cstrong\u003e(Fig. 3h-j)\u003c/strong\u003e. Together, this data demonstrates that STM2457 counteracts excessive m\u003csup\u003e6\u003c/sup\u003eA RNA modification in the cortex and hippocampus as measured with multiple modalities.\u003c/p\u003e\n\u003cp\u003eTo further evaluate the inhibition effect of STM2457 on the expression of main methyltransferase and demethylase in the m\u003csup\u003e6\u003c/sup\u003eA regulatory complex, we examined the protein levels of both METTL3, METTL14, and ALKBH5 in the CTX of mice with and without treatment by western blot. Our results revealed that STM2457 treatment in PS19 mice resulted in decreased levels of METTL3 and METTL14 \u003cstrong\u003e(Fig. 3k-m)\u003c/strong\u003e. On its own, METTL14 is a separate methyltransferase, however when METTL14 forms a complex with METTL3, METTL14 becomes catalytically inactive due to an occluded active site, and instead acts as structural support for METTL3, promoting the catalytic activity of METTL3\u003csup\u003e38\u003c/sup\u003e. Interestingly, METTL3 levels were not significantly altered by STM2457 treatment in WT mice, while METTL14 levels are significantly increased \u003cstrong\u003e(Fig. 3l, m)\u003c/strong\u003e. Similarly, the demethylase ALKBH5 levels were significantly downregulated in PS19 treated mice but not WT (\u003cstrong\u003eExtended Data Fig. 2a, b\u003c/strong\u003e). These results indicate a coordinated and complementary regulatory relationship between m\u003csup\u003e6\u003c/sup\u003eA writers and erasers in restoring m\u003csup\u003e6\u003c/sup\u003eA homeostasis.\u003c/p\u003e\n\u003cp\u003eAltogether, the evaluation on the treatment efficacy indicates that STM2457, delivered at a constant and controlled dose via osmotic pump, effectively inhibits METTL3 and reduces excessive m\u003csup\u003e6\u003c/sup\u003eA methylated RNA levels in the hippocampus and cortex of PS19 mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSTM2457-mediated reduction of m\u003csup\u003e6\u003c/sup\u003eA RNA\u0026nbsp;rescues behavioral deficits in mouse\u0026nbsp;tauopathy model.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA modification via m\u003csup\u003e6\u003c/sup\u003eA methylation has been implicated in homeostatic learning and memory processes and neurodegeneration-related deficits, along with the associated m\u003csup\u003e6\u003c/sup\u003eA writers and erasers\u003csup\u003e21\u0026ndash;25,39\u003c/sup\u003e. The therapeutic potential of STM2457 relies heavily on its ability to prevent cognitive deficits, as these are some of the most debilitating AD sequelae. Therefore, we evaluated the effect of sustained STM2457 treatment on locomotion and cognitive behaviors of mice using a series of behavioral assays, including the Morris water maze (MWM), elevated plus maze (EPM), Y maze and open field test.\u003c/p\u003e\n\u003cp\u003eMWM was used to evaluate spatial learning and memory. Spatial learning was assessed over four consecutive days of training by measuring the time taken to locate a hidden platform. Reference memory was evaluated over two subsequent probe trials in which the platform was removed, and preference for the former platform location was measured. On the final test day (Day 7), escape latency and search patterns were analyzed to determine the degree to which mice relied on spatial versus non-spatial strategies\u003csup\u003e40\u003c/sup\u003e. MWM analysis showed that 9-month-old PS19 mice exhibited significant learning and memory deficits compared to WT controls, and that these deficits were dramatically ameliorated in the same cohort treated with STM2457 \u003cstrong\u003e(Fig. 4a-c)\u003c/strong\u003e. Specifically, PS19 mice traveled greater distances to reach the platform during the 4 days of spatial learning phase compared to WT control \u003cstrong\u003e(Fig. 4a, b)\u003c/strong\u003e. Additionally, untreated PS19 mice took significantly longer to reach the platform on the final day (Day 7). \u003cstrong\u003e(Fig. 4c)\u003c/strong\u003e. These results indicate impaired spatial learning and memory in PS19 mice. Treatment with STM2457 improved performance, with PS19 mice showing comparable average distance from the platform on the fourth day of training and spending similar time to reach the platform on Day-7 as WT mice \u003cstrong\u003e(Fig. 4a-c)\u003c/strong\u003e. Notably, STM2457-treated mice exhibited a temporary increase in escape latency on days 2 to 3, coinciding with the transition from non-spatial to spatial search strategies. Following this transition, they achieved significantly lower escape latencies and shorter distances to the platform, indicating enhanced spatial memory consolidation rather than impairment. This pattern is consistent with prior studies showing that mice with superior learning performance often exhibit an initial exploratory phase characterized by increased path length or escape latency before adopting a more efficient spatial navigation strategy in the Morris water maze \u003csup\u003e41,42\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe EPM is typically used to assess anxiety-like behaviors, however we utilized the test to evaluate cognition. Previous work demonstrated that the EPM can reveal whether the mice recognize the danger associated with the open arms, a behavior that is impaired in mice with cognitive deficits.\u003csup\u003e43\u003c/sup\u003e. Our EPM analysis revealed that PS19 mice exhibited impaired cognitive capacity \u003cstrong\u003e(Fig. 4d)\u003c/strong\u003e. At baseline, WT mice explored both open and closed arms but showed a clear preference for the closed arm, reflecting their ability to recognize the risks of the open arm. In contrast, untreated PS19 mice spent similar amounts of time in both arms, indicating impaired risk recognition. STM2457-treated PS19 mice, however, demonstrated a preference for the closed arm, exhibiting a behavioral pattern similar to WT mice \u003cstrong\u003e(Fig. 4d)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eEvaluation of spatial working memory using the Y maze revealed that the number of spontaneous entries was significantly elevated in PS19 mice when compared to WT, but with treatment this difference was ameliorated \u003cstrong\u003e(Fig. 4e).\u0026nbsp;\u003c/strong\u003eAdditionally, the percent spontaneous alteration, a measure that gives insight into working memory capabilities, revealed a deficit in percent spontaneous alteration in untreated PS19 mice relative to untreated WT mice \u003cstrong\u003e(Fig. 4f)\u003c/strong\u003e. With STM2457 treatment there was a significant increase in percent spontaneous alteration relative to untreated PS19 mice, indicative of improved spatial working memory \u003cstrong\u003e(Fig. 4f)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eThe open field test was used to evaluate locomotor activity and gain insight into motor deficits commonly observed in PS19 mice. These deficits are attributed to the clasping and limb retraction that precede hind limb paralysis that develops at aged time-points with substantial pathological tau burden\u003csup\u003e44\u003c/sup\u003e. In the open field test, PS19 mice traveled significantly less total distance than WT mice, whereas STM2457-treated PS19 mice showed locomotor activity comparable to WT controls (\u003cstrong\u003eFig. 4g\u003c/strong\u003e). This improvement indicates that PS19 mice with treatment experienced fewer motor deficits or peripheral symptoms typically preceding hind limb weakness and paralysis.\u003c/p\u003e\n\u003cp\u003eTaken together, these behavioral assays demonstrate that STM2457 treatment markedly improves learning, memory, and overall cognition in PS19 mice, which typically exhibit deficits in these behavioral paradigms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSTM2457 treatment mitigates tau pathology and protects against neurodegeneration.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;After assessing the effects of STM2457 on cognition, learning, and memory in PS19 mice, we examined brain tissue from 9-month-old mice to determine whether pathological tau accumulation or neurodegeneration had been alleviated. Immunofluorescent staining of brain sections revealed depleted levels of excessive MC1-positive misfolded tau in PS19 mice treated with STM2457 \u003cstrong\u003e(Fig. 5a-d, Extended Data Fig. 3a, b)\u003c/strong\u003e. Changes in MC1 were also accompanied by changes in m\u003csup\u003e6\u003c/sup\u003eA demonstrated previously \u003cstrong\u003e(Fig. 3g-i)\u003c/strong\u003e. This depletion was evident in the CTX, CA3, and CA1 \u003cstrong\u003e(Fig. 5b-d)\u003c/strong\u003e. Hyperphosphorylation of tau in PS19 mice was assessed using the phosphorylation-specific antibodies CP13 and AT8, which recognize pathologically relevant tau phosphorylated at Ser202 and Ser202/Thr205, respectively. Western blot analysis showed that levels of both CP13- and AT8-positive tau were elevated in untreated PS19 mice compared to WT. STM2457 treatment significantly reduced tau phosphorylation at the Ser202 and Ser202/Thr205 sites in PS19 mice \u003cstrong\u003e(Fig. 5e-g)\u003c/strong\u003e. Together, these data demonstrate that, alongside cognitive improvements, STM2457 treatment can reduce multiple forms of misfolded and hyperphosphorylated tau pathology.\u003c/p\u003e\n\u003cp\u003eBased on the reduction in tau pathology and improvement in cognitive behavior, we continued to investigate whether neurodegeneration was slowed or prevented. To assess neuronal integrity, PS19 brain tissue was stained with the NeuN antibody (a Neuronal Nuclei marker). Untreated PS19 mice showed reduced neuronal integrity in the CA1 and cortex relative to WT (\u003cstrong\u003eFig. 5h-k\u003c/strong\u003e). Compared to untreated PS19 mice, STM2457-treated mice exhibited significantly increased neuronal density in the CA3, CA1, and cortex, restoring levels to those observed in WT mice (\u003cstrong\u003eFig. 5i-k\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eIn summation, these results demonstrate that STM2457 treatment mitigates tau pathology and protect neurons against degeneration in brain regions bearing pathological tau aggregation and excessive m\u003csup\u003e6\u003c/sup\u003eA accumulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSTM2457 treatment\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eattenuates glial inflammatory response in PS19\u0026nbsp;mice.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAD pathology is associated with neuroinflammation involving altered glial cell activation, including neuroprotective acute activation and neurotoxic chronic activation\u003csup\u003e45,46\u003c/sup\u003e. Additionally, m\u003csup\u003e6\u003c/sup\u003eA modification has been implicated in regulating microglial inflammatory responses and microglia-neuron interactions\u003csup\u003e20,47,48\u003c/sup\u003e. Considering the effects STM2457 had on cognition, tau pathology, and neurodegeneration, we next considered how glial activation states change with treatment in PS19 mice.\u003c/p\u003e\n\u003cp\u003eTo elucidate the impact of STM2457 on AD-related glial activation, we examined the morphological changes of microglia and astrocytes indicative of the shift into differently activated inflammatory states. We used an IBA1 antibody to mark both resting and activated microglia and used immunofluorescence intensity to assess morphological alteration and activation status. Immunofluorescent staining of hippocampal (CA1 and CA3) tissue from 9-month-old PS19 mice revealed increased ameboid-like morphology of microglia and associated increased fluorescence intensity that was attenuated in mice that received STM2457 treatment \u003cstrong\u003e(Fig. 6a-d)\u003c/strong\u003e. Interestingly, STM2457-treated WT mice showed a slight increase in IBA1-positive microglial intensity, despite the absence of detectable pathology or behavioral changes. This may indicate a more proactive microglial state associated with METTL3 inhibition by STM2457, potentially reflecting its role in maintaining\u0026nbsp;m\u003csup\u003e6\u003c/sup\u003eA homeostasis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA similar investigation of astrogliosis-associated morphology revealed increased fluorescence intensity indicative of hypertrophy of the cell body and processes in the CA1, CA3, and CTX of 9-month-old PS19 mice; this reactive morphology was quelled by STM2457 treatment \u003cstrong\u003e(Fig. 6e-h)\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAltogether, these findings indicate that, in addition to reducing tau pathology and restoring neuronal integrity, STM2457 treatment also reduces the neuroinflammatory responses of microglia and astrocytes.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eEpigenetic modifications are increasingly recognized as contributors to AD pathogenesis, highlighting new therapeutic opportunities. Among these, m\u003csup\u003e6\u003c/sup\u003eA RNA modification has emerged as a potential regulator of AD-related processes, though prior findings have varied across pathological contexts\u003csup\u003e19,30,49,50\u003c/sup\u003e. Our study supports a link between excessive m\u003csup\u003e6\u003c/sup\u003eA and tau pathology and shows that inhibiting the m\u003csup\u003e6\u003c/sup\u003eA writer METTL3 with small molecule STM2457 reduces tau deposition, mitigates neurodegeneration, and prevents cognitive decline in PS19 mice.\u003c/p\u003e\n\u003cp\u003eOur analysis of post-mortem human brain tissue revealed that increased m\u003csup\u003e6\u003c/sup\u003eA modification is present in the frontal cortex (Brodmann\u0026rsquo;s area 10) of \u0026nbsp;AD brains, which is consistent with previous findings in the same brain region\u003csup\u003e30\u003c/sup\u003e. We also observed that m\u003csup\u003e6\u003c/sup\u003eA levels rise concurrently with increasing Braak stage, hyperphosphorylated tau accumulation, and A\u0026beta; plaque deposition. To be noted, other studies also reported inconsistent m\u003csup\u003e6\u003c/sup\u003eA alterations across cortical regions in human post-mortem brain tissue, including reduced m\u003csup\u003e6\u003c/sup\u003eA in the anterior cingulate gyrus and middle temporal gyrus\u003csup\u003e19,31\u003c/sup\u003e. These discrepancies suggest region-specific regulation of m\u003csup\u003e6\u003c/sup\u003eA and highlight the need for comprehensive spatial profiling of m\u003csup\u003e6\u003c/sup\u003eA in both diseased and non-diseased human brains.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUsing humanized P301S tau transgenic mouse models of AD, we found that PS19 mice exhibit elevated m\u003csup\u003e6\u003c/sup\u003eA levels in the cortex and hippocampus at 9 months compared to age-matched WT and levels increased further from 6 to 9 months. These findings align with prior reports that m\u003csup\u003e6\u003c/sup\u003eA varies with age, both independently and in the context of aging-related diseases\u003csup\u003e20,31,36,51\u003c/sup\u003e. Given the prominence of tau pathology in the hippocampus and cortex, this increase suggests a potential age-related link between elevated m\u003csup\u003e6\u003c/sup\u003eA RNA and tau accumulation, highlighting the dynamic and transient nature of m\u003csup\u003e6\u003c/sup\u003eA regulation in the brain\u003csup\u003e5\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eOur findings in human AD cases and PS19 mice support the modulation of m\u003csup\u003e6\u003c/sup\u003eA levels by targeting its regulatory proteins, such as methyltransferase METTL3, as a promising therapeutic strategy for AD. STM2457, originally developed as a selective METTL3 inhibitor for hematologic malignancies, demonstrates several pharmacological properties that favor potential clinical development, including its small molecule structure, nanomolar potency, ability to cross the blood brain barrier, and capacity to reduce m\u003csup\u003e6\u003c/sup\u003eA levels \u003cem\u003ein vivo\u003c/em\u003e\u003csup\u003e37\u003c/sup\u003e. Treatment of PS19 mice with STM2457, delivered continuously for 6 weeks via a surgically implanted osmotic pump subcutaneously with precise dose control, reduced m\u003csup\u003e6\u003c/sup\u003eA levels to a range more comparable to age-matched WT mice as shown by immunolabeling and RNA dot blot. Assessment of STM2457 treatment on tau deposition revealed a robust reduction in MC1-positive misfolded tau, pTau (Ser202), and pTau (Ser202/Thr205) in the hippocampus and cortex of treated PS19 mice, indicating that removal of excessive m\u003csup\u003e6\u003c/sup\u003eA effectively limits pathological tau accumulation. Protein levels of key m\u003csup\u003e6\u003c/sup\u003eA regulators were assessed to elucidate the regulatory mechanisms and coordination underlying m\u003csup\u003e6\u003c/sup\u003eA homeostasis. METTL3 and METTL14 protein levels were significantly reduced in STM2457-treated PS19 mice. In contrast, STM2457-treated WT mice showed no change in METTL3 and a slight increase in METTL14 relative to untreated WT. This variance in translational effect suggests the presence of a compensatory mechanism that attempts to restore the balance of the m\u003csup\u003e6\u003c/sup\u003eA regulatory axis. Evaluation of neuronal density further revealed that reduced tau pathology in treated PS19 mice is accompanied by protection against neurodegeneration. Together, these findings demonstrated that STM2457 successfully inhibited METTL3 and reduced m\u003csup\u003e6\u003c/sup\u003eA methylation, decreased tau pathology, and mitigated neurodegeneration.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGlial activation is a key component of the inflammatory response to pathogenic tau and A\u0026beta; in AD\u003csup\u003e52\u0026ndash;56\u003c/sup\u003e, and increasing evidence indicates that m\u003csup\u003e6\u003c/sup\u003eA RNA methylation participates in regulating neuroinflammatory cascades\u003csup\u003e48,57\u0026ndash;61\u003c/sup\u003e. Recent studies have also shown differential clustering of m\u003csup\u003e6\u003c/sup\u003eA regulators in infiltrating immune cells from AD patients\u003csup\u003e62\u003c/sup\u003e, further suggesting a link between m\u003csup\u003e6\u003c/sup\u003eA signaling and immune activation. In this study we found that STM2457 treatment reduced microglial activation and astrogliosis in PS19 mice, as reflected by decreased inflammatory glial morphology. However, because glial activation in tauopathy may arise secondary to pathological tau aggregation, the reduction in inflammatory morphology could be partly attributable to decreased tau deposition. Even so, our findings also raise the possibility that m\u003csup\u003e6\u003c/sup\u003eA directly modulates glial immune responses. To more comprehensively evaluate this potential role, future studies are planned to assess additional inflammatory endpoints, including reactive oxygen species production and pro-inflammatory cytokine release.\u003c/p\u003e\n\u003cp\u003eImportantly, the therapeutic relevance of STM2457 lies in its ability to reverse cognitive impairment, one of the most debilitating features of AD. In our study, behavioral testing demonstrated that STM2457 fully rescued the learning, memory, and general cognitive deficits observed in PS19 mice. Improvements in locomotion in the open field test indicates that STM2457 mitigates motor impairments associated with this tauopathy model. These findings align with other studies reporting that reducing or eliminating METTL3 activity can benefit cognition in amyloid related AD mouse models\u0026nbsp;\u003csup\u003e63\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e64\u003c/sup\u003e. Notably, unlike previous work that has focused predominantly on amyloid-based models, our study reveals a direct role for m\u003csup\u003e6\u003c/sup\u003eA in modulating tau pathology, which is particularly important because tau burden shows a stronger correlation with cognitive decline in AD\u003csup\u003e65\u003c/sup\u003e. This distinction underscores the unique contribution of our findings in supporting the role of m\u003csup\u003e6\u003c/sup\u003eA as a key regulator of tau-driven neurodegeneration.\u003c/p\u003e\n\u003cp\u003eTaken together with prior studies, our findings support the therapeutic potential of targeting m\u003csup\u003e6\u003c/sup\u003eA in AD. m\u003csup\u003e6\u003c/sup\u003eA RNA modification influences multiple disease-relevant pathways, including tau misfolding, A\u0026beta; clearance, neuroinflammation, and synaptic function\u003csup\u003e19,36,47,49,66\u003c/sup\u003e. Regional and stage-dependent variation in m\u003csup\u003e6\u003c/sup\u003eA patterns further indicate that modulating METTL3 could restore disrupted transcriptomic regulation in a context-specific manner. Specifically, the prolonged presence of STM2457 in the hippocampus following sustained subcutaneous delivery via osmotic pumps enhances its potential as a viable therapeutic agent. Nevertheless, m\u003csup\u003e6\u003c/sup\u003eA is regulated by complex enzymatic networks, and their remain questions regarding the consequences of long-term METTL3 inhibition. Our observation that METTL3 and METTL14 respond differently to STM2457 in WT versus PS19 mice also points to distinct compensatory mechanisms that require deeper investigation. Overall, selective METTL3 inhibitors represent a promising therapeutic avenue, and advancing STM2457 and related compounds will require continued evaluation of safety, dosing, and cellular consequences to support future clinical translation.\u003c/p\u003e\n\u003cp\u003eIn conclusion, this work suggests that reducing excessive m\u003csup\u003e6\u003c/sup\u003eA RNA levels using STM2457 reduces tau pathology, neurodegeneration, neuroinflammatory glial morphology, and improves learning and memory. This impact of m\u003csup\u003e6\u003c/sup\u003eA methylation on tau pathogenesis presents new avenues for therapeutic intervention. This work highlights the potential of STM2457 as a novel AD therapeutic, whose impact on m\u003csup\u003e6\u003c/sup\u003eA methylation in specific cell types and pathogenic processes needs further investigation.\u0026nbsp;\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHuman post-mortem brain tissue\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAnonymous human brain tissue used in this project was obtained from the Goizuetta Alzheimer\u0026rsquo;s Disease Center and was collected in accordance with IRB protocols of Emory University. Human anterior prefrontal cortex (Brodmann Area 10) was used for immunohistochemical analysis and immunofluorescence labeling in the current study. These brain regions were selected based on consistent evidence from prior studies indicating a strong positive correlation between altered gene expression and disease severity\u003csup\u003e67\u003c/sup\u003e. The samples were de-identified and are described below. The tissue was fixed in periodate-lysine-paraformaldehyde (PLP) fixative for 2 hrs, followed by overnight incubation in 30% sucrose, after which tissue sections were cut at thickness of 30\u0026mu;m.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFixed human brain samples\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human brain tissue samples used in this study were all de-identified. All studies included both sexes, and results were integrated by covariate analysis, as described below.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 31.129%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003ePrimary Neuropathologic Diagnosis\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003eBraak Stage\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.67742%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003eAge at Onset\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.5484%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003eAge at Death/Bx\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.2258%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003eDuration (years)\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.54839%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003eApoE\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.74194%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003eRace\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6.12903%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003eSex\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 31.129%;\"\u003e\n \u003cp\u003e\u003cu\u003eControl\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10%;\"\u003e\n \u003cp\u003e\u003cu\u003eI\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.67742%;\"\u003e\n \u003cp\u003e\u003cu\u003e \u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.5484%;\"\u003e\n \u003cp\u003e\u003cu\u003e59\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.2258%;\"\u003e\n \u003cp\u003e\u003cu\u003e \u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.54839%;\"\u003e\n \u003cp\u003e\u003cu\u003eE2/3\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.74194%;\"\u003e\n \u003cp\u003e\u003cu\u003eb\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6.12903%;\"\u003e\n \u003cp\u003e\u003cu\u003em\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 31.129%;\"\u003e\n \u003cp\u003e\u003cu\u003eControl\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10%;\"\u003e\n \u003cp\u003e\u003cu\u003eI\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.67742%;\"\u003e\n \u003cp\u003e\u003cu\u003e \u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.5484%;\"\u003e\n \u003cp\u003e\u003cu\u003e70\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.2258%;\"\u003e\n \u003cp\u003e\u003cu\u003e \u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.54839%;\"\u003e\n \u003cp\u003e\u003cu\u003eE3/3\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.74194%;\"\u003e\n \u003cp\u003e\u003cu\u003eb\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6.12903%;\"\u003e\n \u003cp\u003e\u003cu\u003em\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 31.129%;\"\u003e\n \u003cp\u003e\u003cu\u003eControl\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10%;\"\u003e\n \u003cp\u003e\u003cu\u003eI\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.67742%;\"\u003e\n \u003cp\u003e\u003cu\u003e \u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.5484%;\"\u003e\n \u003cp\u003e\u003cu\u003e72\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.2258%;\"\u003e\n \u003cp\u003e\u003cu\u003e \u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.54839%;\"\u003e\n \u003cp\u003e\u003cu\u003eE3/3\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.74194%;\"\u003e\n \u003cp\u003e\u003cu\u003ew\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6.12903%;\"\u003e\n \u003cp\u003e\u003cu\u003em\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 31.129%;\"\u003e\n \u003cp\u003e\u003cu\u003eControl\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10%;\"\u003e\n \u003cp\u003e\u003cu\u003eII\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.67742%;\"\u003e\n \u003cp\u003e\u003cu\u003e \u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.5484%;\"\u003e\n \u003cp\u003e\u003cu\u003e78\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.2258%;\"\u003e\n \u003cp\u003e\u003cu\u003e \u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.54839%;\"\u003e\n \u003cp\u003e\u003cu\u003eE3/3\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.74194%;\"\u003e\n \u003cp\u003e\u003cu\u003ew\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6.12903%;\"\u003e\n \u003cp\u003e\u003cu\u003ef\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 31.129%;\"\u003e\n \u003cp\u003e\u003cu\u003eControl\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10%;\"\u003e\n \u003cp\u003e\u003cu\u003eIV\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.67742%;\"\u003e\n \u003cp\u003e\u003cu\u003e \u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.5484%;\"\u003e\n \u003cp\u003e\u003cu\u003e84\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.2258%;\"\u003e\n \u003cp\u003e\u003cu\u003e \u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.54839%;\"\u003e\n \u003cp\u003e\u003cu\u003eE2/3\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.74194%;\"\u003e\n \u003cp\u003e\u003cu\u003eb\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6.12903%;\"\u003e\n \u003cp\u003e\u003cu\u003ef\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 31.129%;\"\u003e\n \u003cp\u003e\u003cu\u003eControl\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10%;\"\u003e\n \u003cp\u003e\u003cu\u003eIII\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.67742%;\"\u003e\n \u003cp\u003e\u003cu\u003e \u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.5484%;\"\u003e\n \u003cp\u003e\u003cu\u003e91\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.2258%;\"\u003e\n \u003cp\u003e\u003cu\u003e \u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.54839%;\"\u003e\n \u003cp\u003e\u003cu\u003eE3/3\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.74194%;\"\u003e\n \u003cp\u003e\u003cu\u003ew\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6.12903%;\"\u003e\n \u003cp\u003e\u003cu\u003ef\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 31.129%;\"\u003e\n \u003cp\u003e\u003cu\u003eControl\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10%;\"\u003e\n \u003cp\u003e\u003cu\u003eIII\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.67742%;\"\u003e\n \u003cp\u003e\u003cu\u003e \u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.5484%;\"\u003e\n \u003cp\u003e\u003cu\u003e92\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.2258%;\"\u003e\n \u003cp\u003e\u003cu\u003e \u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.54839%;\"\u003e\n \u003cp\u003e\u003cu\u003eE3/3\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.74194%;\"\u003e\n \u003cp\u003e\u003cu\u003ew\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6.12903%;\"\u003e\n \u003cp\u003e\u003cu\u003ef\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 31.129%;\"\u003e\n \u003cp\u003e\u003cu\u003eControl\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10%;\"\u003e\n \u003cp\u003e\u003cu\u003eII\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.67742%;\"\u003e\n \u003cp\u003e\u003cu\u003e \u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.5484%;\"\u003e\n \u003cp\u003e\u003cu\u003e94\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.2258%;\"\u003e\n \u003cp\u003e\u003cu\u003e \u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.54839%;\"\u003e\n \u003cp\u003e\u003cu\u003eE3/3\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.74194%;\"\u003e\n \u003cp\u003e\u003cu\u003ew\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6.12903%;\"\u003e\n \u003cp\u003e\u003cu\u003em\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 31.129%;\"\u003e\n \u003cp\u003e\u003cu\u003eControl/asymptomatic AD\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10%;\"\u003e\n \u003cp\u003e\u003cu\u003eII\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.67742%;\"\u003e\n \u003cp\u003e\u003cu\u003e \u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.5484%;\"\u003e\n \u003cp\u003e\u003cu\u003e64\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.2258%;\"\u003e\n \u003cp\u003e\u003cu\u003e \u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.54839%;\"\u003e\n \u003cp\u003e\u003cu\u003eE4/4\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.74194%;\"\u003e\n \u003cp\u003e\u003cu\u003ew\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6.12903%;\"\u003e\n \u003cp\u003e\u003cu\u003ef\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 31.129%;\"\u003e\n \u003cp\u003e\u003cu\u003eControl/asymptomatic AD\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10%;\"\u003e\n \u003cp\u003e\u003cu\u003eIV\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.67742%;\"\u003e\n \u003cp\u003e\u003cu\u003e \u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.5484%;\"\u003e\n \u003cp\u003e\u003cu\u003e80\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.2258%;\"\u003e\n \u003cp\u003e\u003cu\u003e \u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.54839%;\"\u003e\n \u003cp\u003e\u003cu\u003eE3/4\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.74194%;\"\u003e\n \u003cp\u003e\u003cu\u003ew\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6.12903%;\"\u003e\n \u003cp\u003e\u003cu\u003em\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 31.129%;\"\u003e\n \u003cp\u003e\u003cu\u003eControl/asymptomatic AD\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10%;\"\u003e\n \u003cp\u003e\u003cu\u003eII\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.67742%;\"\u003e\n \u003cp\u003e\u003cu\u003e \u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.5484%;\"\u003e\n \u003cp\u003e\u003cu\u003e81\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.2258%;\"\u003e\n \u003cp\u003e\u003cu\u003e \u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.54839%;\"\u003e\n \u003cp\u003e\u003cu\u003eE3/3\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.74194%;\"\u003e\n \u003cp\u003e\u003cu\u003ew\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6.12903%;\"\u003e\n \u003cp\u003e\u003cu\u003em\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 31.129%;\"\u003e\n \u003cp\u003e\u003cu\u003eControl/asymptomatic AD\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10%;\"\u003e\n \u003cp\u003e\u003cu\u003eI\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.67742%;\"\u003e\n \u003cp\u003e\u003cu\u003e \u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.5484%;\"\u003e\n \u003cp\u003e\u003cu\u003e87\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.2258%;\"\u003e\n \u003cp\u003e\u003cu\u003e \u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.54839%;\"\u003e\n \u003cp\u003e\u003cu\u003eE3/4\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.74194%;\"\u003e\n \u003cp\u003e\u003cu\u003ew\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6.12903%;\"\u003e\n \u003cp\u003e\u003cu\u003em\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 31.129%;\"\u003e\n \u003cp\u003e\u003cu\u003eControl/asymptomatic AD\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10%;\"\u003e\n \u003cp\u003e\u003cu\u003eIII\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.67742%;\"\u003e\n \u003cp\u003e\u003cu\u003e \u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.5484%;\"\u003e\n \u003cp\u003e\u003cu\u003e87\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.2258%;\"\u003e\n \u003cp\u003e\u003cu\u003e \u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.54839%;\"\u003e\n \u003cp\u003e\u003cu\u003eE2/3\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.74194%;\"\u003e\n \u003cp\u003e\u003cu\u003ew\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6.12903%;\"\u003e\n \u003cp\u003e\u003cu\u003ef\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 31.129%;\"\u003e\n \u003cp\u003e\u003cu\u003eControl/asymptomatic AD\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10%;\"\u003e\n \u003cp\u003e\u003cu\u003eIV\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.67742%;\"\u003e\n \u003cp\u003e\u003cu\u003e \u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.5484%;\"\u003e\n \u003cp\u003e\u003cu\u003e89\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.2258%;\"\u003e\n \u003cp\u003e\u003cu\u003e \u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.54839%;\"\u003e\n \u003cp\u003e\u003cu\u003eE3/3\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.74194%;\"\u003e\n \u003cp\u003e\u003cu\u003ew\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6.12903%;\"\u003e\n \u003cp\u003e\u003cu\u003em\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 31.129%;\"\u003e\n \u003cp\u003e\u003cu\u003eControl/asymptomatic AD\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10%;\"\u003e\n \u003cp\u003e\u003cu\u003eIV\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.67742%;\"\u003e\n \u003cp\u003e\u003cu\u003e \u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.5484%;\"\u003e\n \u003cp\u003e\u003cu\u003e91\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.2258%;\"\u003e\n \u003cp\u003e\u003cu\u003e \u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.54839%;\"\u003e\n \u003cp\u003e\u003cu\u003eE3/3\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.74194%;\"\u003e\n \u003cp\u003e\u003cu\u003ew\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6.12903%;\"\u003e\n \u003cp\u003e\u003cu\u003em\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 31.129%;\"\u003e\n \u003cp\u003e\u003cu\u003eAD\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10%;\"\u003e\n \u003cp\u003e\u003cu\u003eVI\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.67742%;\"\u003e\n \u003cp\u003e\u003cu\u003e49\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.5484%;\"\u003e\n \u003cp\u003e\u003cu\u003e59\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.2258%;\"\u003e\n \u003cp\u003e\u003cu\u003e10\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.54839%;\"\u003e\n \u003cp\u003e\u003cu\u003eE3/3\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.74194%;\"\u003e\n \u003cp\u003e\u003cu\u003ew\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6.12903%;\"\u003e\n \u003cp\u003e\u003cu\u003em\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 31.129%;\"\u003e\n \u003cp\u003e\u003cu\u003eAD\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10%;\"\u003e\n \u003cp\u003e\u003cu\u003eVI\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.67742%;\"\u003e\n \u003cp\u003e\u003cu\u003e59\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.5484%;\"\u003e\n \u003cp\u003e\u003cu\u003e69\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.2258%;\"\u003e\n \u003cp\u003e\u003cu\u003e10\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.54839%;\"\u003e\n \u003cp\u003e\u003cu\u003eE3/4\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.74194%;\"\u003e\n \u003cp\u003e\u003cu\u003ew\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6.12903%;\"\u003e\n \u003cp\u003e\u003cu\u003em\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 31.129%;\"\u003e\n \u003cp\u003e\u003cu\u003eAD\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10%;\"\u003e\n \u003cp\u003e\u003cu\u003eVI\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.67742%;\"\u003e\n \u003cp\u003e\u003cu\u003e53\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.5484%;\"\u003e\n \u003cp\u003e\u003cu\u003e71\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.2258%;\"\u003e\n \u003cp\u003e\u003cu\u003e18\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.54839%;\"\u003e\n \u003cp\u003e\u003cu\u003eE3/4\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.74194%;\"\u003e\n \u003cp\u003e\u003cu\u003ew\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6.12903%;\"\u003e\n \u003cp\u003e\u003cu\u003em\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 31.129%;\"\u003e\n \u003cp\u003e\u003cu\u003eAD\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10%;\"\u003e\n \u003cp\u003e\u003cu\u003eVI\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.67742%;\"\u003e\n \u003cp\u003e\u003cu\u003e65\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.5484%;\"\u003e\n \u003cp\u003e\u003cu\u003e80\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.2258%;\"\u003e\n \u003cp\u003e\u003cu\u003e15\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.54839%;\"\u003e\n \u003cp\u003e\u003cu\u003eE3/4\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.74194%;\"\u003e\n \u003cp\u003e\u003cu\u003eb\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6.12903%;\"\u003e\n \u003cp\u003e\u003cu\u003ef\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 31.129%;\"\u003e\n \u003cp\u003e\u003cu\u003eAD\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10%;\"\u003e\n \u003cp\u003e\u003cu\u003eV\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.67742%;\"\u003e\n \u003cp\u003e\u003cu\u003e77\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.5484%;\"\u003e\n \u003cp\u003e\u003cu\u003e83\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.2258%;\"\u003e\n \u003cp\u003e\u003cu\u003e6\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.54839%;\"\u003e\n \u003cp\u003e\u003cu\u003eNA \u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.74194%;\"\u003e\n \u003cp\u003e\u003cu\u003ew\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6.12903%;\"\u003e\n \u003cp\u003e\u003cu\u003ef\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 31.129%;\"\u003e\n \u003cp\u003e\u003cu\u003eAD\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10%;\"\u003e\n \u003cp\u003e\u003cu\u003eV\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.67742%;\"\u003e\n \u003cp\u003e\u003cu\u003e86\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.5484%;\"\u003e\n \u003cp\u003e\u003cu\u003e92\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.2258%;\"\u003e\n \u003cp\u003e\u003cu\u003e6\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.54839%;\"\u003e\n \u003cp\u003e\u003cu\u003eE3/4\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.74194%;\"\u003e\n \u003cp\u003e\u003cu\u003ew\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6.12903%;\"\u003e\n \u003cp\u003e\u003cu\u003ef\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 31.129%;\"\u003e\n \u003cp\u003e\u003cu\u003eAD\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10%;\"\u003e\n \u003cp\u003e\u003cu\u003eV\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.67742%;\"\u003e\n \u003cp\u003e\u003cu\u003e82\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.5484%;\"\u003e\n \u003cp\u003e\u003cu\u003e92\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.2258%;\"\u003e\n \u003cp\u003e\u003cu\u003e10\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.54839%;\"\u003e\n \u003cp\u003e\u003cu\u003eE3/3\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.74194%;\"\u003e\n \u003cp\u003e\u003cu\u003ew\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6.12903%;\"\u003e\n \u003cp\u003e\u003cu\u003em\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 31.129%;\"\u003e\n \u003cp\u003e\u003cu\u003eAD\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10%;\"\u003e\n \u003cp\u003e\u003cu\u003eVI\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.67742%;\"\u003e\n \u003cp\u003e\u003cu\u003e80\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.5484%;\"\u003e\n \u003cp\u003e\u003cu\u003e93\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13.2258%;\"\u003e\n \u003cp\u003e\u003cu\u003e13\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.54839%;\"\u003e\n \u003cp\u003e\u003cu\u003eE3/4\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.74194%;\"\u003e\n \u003cp\u003e\u003cu\u003ew\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6.12903%;\"\u003e\n \u003cp\u003e\u003cu\u003ef\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAnimals\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUse of all animals was approved by the University of Virginia Institutional and Animal Care and Use Committee. All animals were housed in an IACUC-approved vivarium at the University of Virginia School of Medicine. P301S tau PS19 transgenic mice: PS19 mice overexpressing human P301S Tau (B6;C3-Tg(Prnp-MAPT*P301S) PS19Vle/J, stock #008169) were purchased from Jackson Laboratories. Male PS19 P301S tau\u003csup\u003e+/-\u0026nbsp;\u003c/sup\u003emice and female C57BL/6 wild type were used as breeding pairs and the F1 generation of P301S tau\u003csup\u003e+/-\u003c/sup\u003e(PS19) and P301S tau\u003csup\u003e-/-\u0026nbsp;\u003c/sup\u003e(wild type) were used for the experiment. Littermates of the same sex were randomly assigned to experimental groups. Mice were sacrificed for experimentation at the age of 6- and 9-months-old, respectively.\u003c/p\u003e\n\u003cp\u003eThe perfusion and fixation method used for brain tissue was critical for successful immunostaining conducted in this study. Following anesthesia, mice were perfused through the heart with 20mL ice cold PBS at the speed of 4mL/min for 10 mins until the mouse tail became curved and stiff. The mouse brains were dissected and placed in 4% paraformaldehyde (PFA) on ice for 24 hrs before being transferred 0.05% sodium azide/PBS solution. To prepare for brain sectioning, the fixed mice brains were transferred into 30% sucrose/PBS until the brains sank to the bottom of the tube (about 48h) and then sectioned. The fixed brains were cryosectioned into 30\u0026micro;m coronal sections and stored in 0.005% sodium azide/PBS solution at 4\u0026deg;C for up to 3 months. For long-term storage, the sections were transferred into cryoprotectant solution (30% glycerol and 30% ethylene glycol in PBS) and stored at -20 \u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eImmunohistochemistry staining\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFloating 30\u0026micro;m brain sections were placed onto slides. All subsequent steps were performed at room temperature unless otherwise specified. Sections were outlined with a hydrophobic barrier using a PAP pen and air-dried before staining. Sections were washed in PBS for 10 min then treated with 1% H2O2 diluted in dH2O for 15 min. Sections were rinsed twice in PBS for 15 min each, then blocked with 0.4% Triton X-100 with 1% Bovine serum albumin (BSA) and 4% normal goat serum (NGS) in PBS for 30 min. After blocking, sections were incubated overnight in 1\u0026deg; antibody diluted in DAKO antibody diluent (5% BSA in 0.25% TritonX-100/PBS) at 4\u0026deg;C with photobleaching. The following day, sections were rinsed twice in PBS for 15 min then incubated for one hr in BTA solution (0.44% Biotinylated goat anti-mouse IgG diluted in 0.3% Triton X-100/PBS). Sections were rinsed twice in PBS then treated with Avidin Biotin Complex (ABC) (0.88% Avidin, 0.88% Biotin diluted in 0.3% Triton X-100/PBS, prepared 30 min prior to use; Vector Laboratories, Cat# PK-6101). After ABC incubation, sections were washed three times in PBS for 10 min each. DAB solution was prepared by adding 1 tablet DAB (Millipore Sigma, cat# D4293-5SET) and 1 tablet Urea (Sigma-Aldrich, Cat# D4193) to 5mL dH2O and vortexed until fully dissolved. DAB solution was added to sections individually for one to three min until section turned medium brown in color, then were immediately placed in PBS. Sections were washed twice with PBS for five min each, then dried overnight at 37\u0026deg;C. The following day, sections were dehydrated by washing in 70% EtOH, 85% EtOH, 95% EtOH, and 100% EtOH for two min each followed by washing in 100% EtOH for five min. Slides were then cleared by washing twice in Xylene for five min followed by immediately dropping 50\u0026micro;L of Permount Mounting Medium (Thermo Fisher Scientific, Cat# SP15-500) and covering with glass coverslips. Slides were dried overnight in the fume hood and stored at 4\u0026deg;C for long term storage. All washing and incubated steps were done with agitation. Primary antibody used for brain tissue DAB staining was mouse monoclonal purified IgG anti-m\u003csup\u003e6\u003c/sup\u003eA, 1:2000 (Synaptic Systems, Cat# 202 011, RRID: AB_2619890).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eRNA dot blot\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA dot blot protocol was adapted from Zhao et al.\u003csup\u003e64\u003c/sup\u003e RNA extraction was performed using RNeasy Plus Universal Mini Kit (73404, Qiagen) on ~20-25mg of cortical tissue that had been frozen in RNA later. ~400ng/\u0026micro;L of extracted RNA was heated to 95\u0026ordm;C for 3 min to disrupt secondary structures, then samples were immediately placed on ice. Then 1\u0026micro;L 10mM HEPES buffer and 1\u0026micro;L methanol were added to the RNA, and the volume was brought up to 10\u0026micro;L using molecular biology grade water. Then 10\u0026micro;L of each sample was dotted onto a positively charged nylon membrane (Cytiva, Amersham Hybond -N+, RPN1210B). Following this, the membrane was heated in an oven to 65\u0026ordm;C for 45 min, then immediately placed in a UV crosslinker with the following settings: 125 mJoule/cm2 at 254 nM for 5 min. Then the membrane was washed with PBST (1X PBS, 0.01% Tween-20) with gentle shaking for 10 min. The membrane was then blocked in 5% skimmed milk powder in 1X PBS for 1 hr with gentle shaking at RT. The primary antibody used was mouse IgG3 anti-m6A monoclonal antibody (Proteintech, Cat # 68055-1-Ig, RRID: AB_2918796) in a 1:1000 dilution in 5% BSA in PBST (1X PBS, 0.01% Tween-20) solution. The membrane was submerged in primary antibody and incubated either at RT with gentle shaking for 2 hrs, or at 4\u0026ordm;C with gentle shaking overnight (14-16 hrs). The membrane was then washed 3x in PBST for 10 min. A horseradish peroxidase concentrate was used as a secondary antibody, specifically goat anti-mouse IgG (H+L) horseradish peroxidase-conjugated secondary antibody (Thermo Fisher Scientific, Cat# G-21040, RRID: AB_2536527) was used at a 1:2000 dilution in 5% BSA in PBST. The membrane was incubated for 1 hr at RT with gentle shaking. \u0026nbsp;The membrane was washed 4x for 10 min each in PBST with gentle shaking. Pierce\u0026trade; ECL Western Blotting Substrate (Thermo Fisher Scientific, Cat #32106) was used for luminescent development: 4 mL of detection reagent 1 peroxidase solution and 4 mL of detection reagent 2 luminol enhancer were added to the membrane to incubate for 5 min. The membrane was then imaged on a BioRad ChemiDoc imaging system. Afterwards, the membrane was briefly washed with PBST to remove the ECL substrate and was then submerged in Pierce\u0026trade; Restore Plus Western Blot Stripping Buffer (Thermo Fisher Scientific, Cat# 46428) for 5 min. Then the membrane is placed in a Methylene Blue staining buffer (0.2% methylene blue in 0.4M sodium acetate and 0.4M acetic acid) for 30 min with gentle shaking. The membrane was washed with molecular biology grade water until the solution was clear (~3 4\u0026ndash;5-minute washes). Membrane was imaged on BioRad ChemiDoc imaging system using blue and green StarBright B700 and B520 Blot settings, respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eWestern blot\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the time of perfusion cortical tissue samples intended for western blot were snap frozen and transferred to a 1.5mL Protein LoBind Eppendorf tube and stored at -80\u0026ordm;C. Samples were subsequently lysed by mechanical homogenization 40\u0026micro;L Hsiao TBS buffer (50mM\u0026nbsp;Tris, pH 8.0,\u0026nbsp;274mM\u0026nbsp;NaCl,\u0026nbsp;5mM\u0026nbsp;KCl) supplemented with cOmplete Protease Inhibitor (Roche, Cat# 11836153001) and PhosSTOP Phosphatase Inhibitor (Roche, Cat# 04906845001). 200U of Benzonase (Sigma-Aldrich, cat# E1014-25KU) with 1mM MgCl2 was added for removal of nucleic acids. Protein lysate concentration was quantified by the Pierce BCA assay according to the manufacturer\u0026rsquo;s protocol (Thermo Fisher Scientific, cat#\u0026nbsp;23227).\u0026nbsp; 10-20\u0026micro;g of protein lysate were separated by denaturing gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane. The membrane was blocked for an hr in 5% non-fat milk in .01% Tween-20 in 1x PBS (PBST), and incubated in primary antibody (dilution antibody dependent) in 5% BSA PBST overnight at 4\u0026deg;C. The following day, the membrane was washed with PBST for 3 times for 10 min each then incubated with secondary antibody\u0026nbsp;goat anti-mouse IgG (H+L) horseradish peroxidase-conjugated secondary antibody 1:5000 (Thermo Fisher Scientific, Cat# G-21040, RRID: AB_2536527)\u0026nbsp;in 5% BSA PBST for 2 hrs at RT. The membrane was washed with PBST 3 times for 10 min before briefly being washed with PBS prior to visualization.\u0026nbsp;Protein bands were visualized using Pierce\u0026trade; enhanced chemiluminescence (ECL) Western Blotting Substrate (Thermo Fisher Scientific, Cat# 32106) and imaged by ChemiDoc Imaging System (Bio-Rad). Band intensities were quantified using ImageJ. Protein abundance was normalized to total \u0026Beta;eta Actin with background subtraction. Primary antibodies used for immunoblotting were as follows:\u0026nbsp;: rabbit IgG METTL3 polyclonal antibody 1:2000 (Proteintech, Cat# 15073-1-AP, RRID: AB_2142033), rabbit IgG METTL14 polyclonal antibody 1:2000 (Proteintech, Cat# 26158-1-AP, RRID: AB_2800447), rabbit IgG ALKBH5 polyclonal antibody 1:1000 (Proteintech, Cat# 16837-1-AP, RRID: AB_2242665), mouse IgG Phospho-Tau (Ser202, Thr205) Moloclonal Antibody (AT8) 1:2000 (Invitrogen, Cat# MN1020, RRID: AB_223647); mouse anti-tau p202 (CP13) 1:500 (provided by Feinstein Institute through MTA) and mouse \u0026beta;-Actin Monoclonal Antibody 1:2000 (Proteintech, Cat# 66009-1-Ig, RRID: AB_2687938).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eImmunofluorescence labeling\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCoronal sections, including cortex and hippocampus, were washed in PBS for 10 mins and then permeabilized in 0.5mL PBS/0.25% Triton X-100 (PBST). The selected brain tissues were then blocked in blocking solution (5% BSA and 5% normal donkey serum in PBST) for1.5-2 hrs at room temperature (RT). Then sections were incubated in primary antibodies diluted in 5% BSA/PBST overnight at 4\u0026deg;C. On the second day, brain sections were washed three times in PBST, 15 min each. Brain sections were then incubated in 2\u0026deg; antibodies (1:1000 for Alexa fluor-conjugated antibodies made in goat/donkey purchased from Thermo Fisher Scientific) diluted in 5% BSA/PBST for 2 hrs at room temperature. For DAPI nuclei stain, DAPI (1:10,000of 1mg/mL stock) was diluted in PBST and incubated with brain sections for 15 min followed by two washes with PBST then one wash with PBS, 10 min each. The brain sections were mounted onto microscope glass slides using Prolong gold antifade reagent. Images were captured by Leica Stellaris 5 confocal microscope. Primary antibodies used for brain tissue staining were as follows: mouse\u0026nbsp;monoclonal purified IgG anti-m\u003csup\u003e6\u003c/sup\u003eA, 1:2000 (Synaptic Systems, Cat# 202 011, RRID: AB_2619890); mouse anti-MC1, 1:300 (provided by Feinstein Institute through MTA); chicken anti-microtubule-associated protein antibody, 1:1000 (Aves Labs, Cat# MAP, RRID: AB_2313549); rabbit anti-Iba1, 1:1000 (Fuji Film Wako Pure Chemical Corporation, Cat# 019-19741, RRID: AB_839504); rat monoclonal antibody anti-GFAP, 1:1000 (Thermo Fisher Scientific, Cat# 13-0300, RRID: AB_2532994); guinea pig polyclonal anti-NeuN antibody (Synaptic Systems, Cat# 266 004, RRID: AB_2619988).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSTM2457 treatment via osmotic pump delivery\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSTM2457 (MedChem Express, Cat#HY-134836), a potent and selective METTL3 inhibitor (IC\u003csub\u003e50\u003c/sub\u003e = 16.9nM), is delivered via Alzet osmotic pump model 2006 for continuous administration (0.15\u0026micro;L/hr, 42-day duration, 200\u0026micro;L reservoir volume). Osmotic pumps are miniature infusion devices designed for controlled and continuous agent delivery in laboratory animals, commonly used for systemic administration when implanted subcutaneously.\u003c/p\u003e\n\u003cp\u003eIn our experiment, the working solution of STM2457 at 2.08mg/mL (4.6mM) was prepared and immediately followed by 200\u0026micro;L infused into the osmotic pump to maintain accurate dosing at 0.25mg/kg/day \u003cem\u003ein vivo\u003c/em\u003e. The osmotic pumps were then implanted subcutaneously on the neck of the 4-month-old mice (~30mg), including wild type control, and PS19 \u003cem\u003e(N=\u003c/em\u003e10 mice per condition). This ensured precise and sustained release of STM2457 at a rate of 0.15\u0026micro;L/h (0.312\u0026micro;g/h) for 42 days.\u003c/p\u003e\n\u003cp\u003eFor preparation of STM2457 working solution, it was first dissolved in DMSO at 50mg/mL (112.48mM) with ultrasonic treatment and heated to 60\u0026deg;C to ensure complete dissolution while avoiding repeated freeze-thaw cycles. The stock solution is stored at -80\u0026deg;C for up to 6 months or at -20\u0026deg;C for up to 1 month. The final working solution is prepared by sequentially adding 10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline, yielding a clear solution with solubility \u0026ge; 2.08mg/mL. This formulation ensures stability and consistent STM2457 delivery via subcutaneously implanted osmotic pump\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eLiquid chromatography\u0026ndash;mass spectrometry (LC-MS)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e10mg of Hippocampal frozen tissues were pestled, followed by addition of 200\u0026micro;L of ethyl alcohol water mixture (50% ethyl alcohol:50%water). The Tissue + solvent samples were vortexed for 10 min under ambient temperature. After vortex, Tissue + solvent samples were centrifuged at 10000 RPM for 10 mins. 170\u0026micro;L of supernatants were collected and aliquoted. The whole extraction process was repeated three times. 80\u0026micro;L of supernatants were used for non-spiked LC-MS analysis and 90\u0026micro;L of supernatants were used for spiked LC-MS analysis. 10\u0026micro;L of 116\u0026micro;M STM2457 standard solution were spiked into 90\u0026micro;L supernatants to reach a rough final concentration of 11.6\u0026micro;M.\u003c/p\u003e\n\u003cp\u003eData-dependent acquisition (DDA) analyses were performed on Orbitrap Exploris coupled to Vanquish system (Thermo Fisher Scientific). 10\u0026micro;L of supernatants were separated on a HALO 90 \u0026Aring; AQ-C18 column (4.6 x 150 mm, 5\u0026micro;m, C18, Advanced Materials Technology, Delaware, Cat# USEQT001307) at 250\u0026micro;L per min. Mobile phase A consisted of 50% LC/MS grade H\u003csub\u003e2\u003c/sub\u003eO (W6-4, Fisher Scientific) and 50% LC/MS grade methanol (34860-4L-R, Sigma-Aldrich), mobile phase B consisted of LC/MS grade ethyl alcohol (459829-4L, Sigma-Aldrich), and both mobile phases contained 0.1% FA. The LC gradient was: 0% B to 50% B in 5\u0026thinsp;min, 98% B in 10\u0026thinsp;min until 12 min, and 0% B in 15\u0026thinsp;min, with a total gradient length of 15\u0026thinsp;min. A targeted MS1 scan and a ddMS2 scan were paired for quantification and qualification purpose. Targeted MS1spectra were collected at 120,000 resolution and ddMS2 spectra at 90,000 resolution. 70% HCD collision energy was used for ddMS2 fragmentation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eElevated plus maze\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe elevated plus maze (EPM) protocol was adapted from Marco et al\u003csup\u003e68\u003c/sup\u003e. The EPM was used to assess cognition similar to the application in J\u0026uuml;rgensen et al\u003csup\u003e43\u003c/sup\u003e. The apparatus consisted of two opposing closed arms covered by high walls (20 cm tall opaque black Plexiglass, 35 x 6 cm\u003csup\u003e2\u003c/sup\u003e), while the other two arms remained open to the air (uncovered, 35 x 6 cm\u003csup\u003e2\u003c/sup\u003e) extended from a common center square, elevated 121cm above the floor. Mice were placed onto the opaque white Plexiglass floor in the center facing a closed arm and allowed to explore the entire raised platform for 5 min. Data was recorded using an overhead camera with the EthoVision XT automated tracking system (Nodulus Information Technology), which provided the time spent in the open and closed arms. Between trials, the maze was thoroughly cleaned with 70% ethanol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eOpen field\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe open field test protocol was adapted from Lammert et al.\u003csup\u003e69\u003c/sup\u003e and was used to evaluate spontaneous locomotor activity. The open field consists of a square area (40 x 40 cm\u003csup\u003e2\u003c/sup\u003e) with white Plexiglass walls and floor, evenly illuminated with dim lighting. All mice were individually placed in the upper left corner of the open field and allowed to explore the area for 10 min. Movements were recorded using an overhead camera and data was collected using the EthoVision XT automated tracking system (Nodulus Information Technology). Locomotor activity was quantified as total distance traveled (cm).\u0026nbsp;Between trials, the maze was thoroughly cleaned with 70% ethanol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMorris water maze\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Morris water maze (MWM) protocol was adapted from March et al\u003csup\u003e68\u003c/sup\u003e and Vorhees \u0026amp; Williams\u003csup\u003e40\u003c/sup\u003e. The MWM was used to evaluate spatial learning and memory in 9-month-old mice WT and PS19 mice. The mice were placed in a 1m diameter pool of opaque water. The water was made opaque by adding a nontoxic tempera white paint and the water was kept at room temperature. A hidden 10-cm diameter platform was placed at approximately 1cm underneath the surface of the water on which the mice can stand on. The maze was divided into four quadrants. Four trials were performed each day across a four-day learning period. The mice were placed in the pool at various starting positions throughout the three quadrants that did not contain the platform. Four large markers (a square, circle, triangle, and X) were placed on the tub walls which provide visual cues to allow the mice to determine platform location. On Day-1 the mice were placed in their starting positions and allowed to search for the platform for 60s. Each mouse was allowed to remain on the platform for 2 min for the first trial, 1 minute the second trial, 30s the third trial, and 5s the last trial before being removed from the maze. If the mouse did not find the platform within 60s, the time to find the platform was recorded as 60s. Days 2-4 were all the exact same where the mice underwent four trials and had 60s to locate the platform. On these days the mice had to remain on the platform for 5s before being removed from the maze. Days 5 and 6 were \u0026ldquo;probe\u0026rdquo; days used to assess memory function, these days only contained single trials where the platform had been removed. The mice were once again allowed to search for the platform for 60s and the time spent in the quadrant where the platform was previously located was recorded. Day 7 of the test included four trials and had the platform returned to the same location as Days 1-4 and the mice were given 60s to locate the platform and had to remain on the platform for at least 5s before being removed from the maze. During the test data was recorded using an overhead camera and with the EthoVision XT automated tracking system (Nodulus Information Technology) which provided the average distance from the platform (cm), total swimming time, swimming time in the target vs. opposing quadrants, and the path taken to reach the platform. The time for the mice to reach the platform on Day 7 was considered escape latency. Between trials, the maze was thoroughly cleaned with 70% ethanol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eY-maze\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Y-maze protocol was adapted from Kraeuter et al\u003csup\u003e70\u003c/sup\u003e. The Y-maze was used to evaluate spatial working memory. The apparatus consisted of three identical opaque arms (35 cm long \u0026times; 8 cm wide \u0026times; 15 cm high) positioned at 120\u0026deg; angles, these arms were labeled A, B, and C. For this protocol we designated the arm labeled \u0026ldquo;A\u0026rdquo; as the starting arm, with the arms labeled \u0026ldquo;B\u0026rdquo; and \u0026ldquo;C\u0026rdquo; designated as non-starting arms. Each mouse was placed in the distal part of the starting arm of the maze, facing the center of the maze, and allowed to explore all arms freely for 8 min. An arm entry was defined as the center point of the mouse entering the arm. The number of spontaneous entries included the sum of the entries the mice made into the non-starting arms. The number of alterations included how many times a mouse entered all three arms consecutively (e.g., ABC, ACB). The total number of arm entries included the sum of all entries into any arm. The percent spontaneous alternation was calculated as:\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e% Spontaneous Alteration = (Number of alterations)/ [Total number of arm entries -2]) *100\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eData was recorded using an overhead camera and with the EthoVision XT automated tracking system (Nodulus Information Technology).\u0026nbsp;Between trials, the maze was thoroughly cleaned with 70% ethanol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSchematics\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSchematics were created with BioRender.com\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eBrightfield image analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBright images (DAB) were captured by Keyence BZ-X810 Microscope (Keyence, BZ-X810) using BZ-X800 Viewer Software (Keyence). The staining intensities in DAB labeled brain sections were measured by ImageJ. Raw integrated density (RawIntDen) was recorded and normalized by area measured when appropriate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFluorescent Image analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFluorescent images were captured by STELLARIS 5 Confocal Microscope (Leica Microsystems, Stellaris 5) using LAS X Life Science Microscope Software Platform (Leica Microsystems). The staining intensities in immunofluorescence-labeled brain sections were measured by ImageJ. Either integrated density (IntDen) or raw integrated density (RawIntDen) were recorded and normalized by area measured when appropriate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses and figures artwork were performed using GraphPad Prism version 10.2.0 for Windows. Pairwise data was compared using unpaired student\u0026rsquo;s t-test. All group data are expressed as mean \u0026plusmn; SEM. Column means were compared using one-way ANOVA. Group means were compared using two-way ANOVA with factors of genotype, age time course, or treatment status. When two-way ANOVA showed a significant difference, pairwise comparisons between group means were examined by Tukey\u0026rsquo;s or uncorrected Fisher\u0026rsquo;s LSD multiple comparisons test. Significance was defined at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data reported in this paper will be shared by the lead contact upon request. The raw IF, DAB and WB images, and the animal behavior test videos and analysis will be made publicly available at Mendeley Data (Reserved DOI:10.17632/pynkg2hdcf.1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge the late Dr. Peter Davies (Northwell/Hofstra) and the Feinstein Institutes for Medical Research for providing the CP13 and MC1 antibodies. We would like to thank the following funding agencies for their support to L.J.: NIH/NIA (R01AG091577); The Grand Challenges Research Investments in Brain and Neuroscience at UVA; Virginia Alzheimer\u0026rsquo;s Disease Center 2023 \u0026lsquo;Pitch and Catch\u0026rsquo; funding; UVA Brain Institute Transformative Neuroscience Pilot Grants; Strang Neuroscience Research Award; Jim and Bruce Eck Fund; Commonwealth Health Research Board Award; Cure Alzheimer\u0026rsquo;s\u0026nbsp;Fund, and the\u0026nbsp;Bill Howard\u0026rsquo;s Golf Tournament for Alzheimer\u0026rsquo;s Disease Research Grant. We thank Professor Daniel Engel for his thoughtful review, constructive edits, and valuable feedback on the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL.J. conceived the study. A.T., C.S., D.P., Q.W., S.Y., E.S., Y.C., A.D., D.G., W.G., and L.J. performed the investigations and generated the visualizations. Data analysis was conducted by A.T., C.S., D.P., Q.W., S.Y., E.S., Y.C., A.D., D.G., X.M.; L.J. A.T., C.S., D.P., E.S., A.D., D.G., and L.J. contributed to writing and editing the manuscript. The study was supervised by L.J..\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL.J. and A.T. have filed a patent application related to the therapeutic approach described in this manuscript (U.S. Provisional Patent Application No. 63/933,846). All other authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlzheimer\u0026rsquo;s disease facts and figures. \u003cem\u003eAlzheimers Dement\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 3708\u0026ndash;3821 (2024).\u003c/li\u003e\n\u003cli\u003eHu, B. \u003cem\u003eet al.\u003c/em\u003e Rewired m6A of promoter antisense RNAs in Alzheimer\u0026rsquo;s disease regulates neuronal genes in 3D nucleome. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 5251 (2025).\u003c/li\u003e\n\u003cli\u003eKobayashi, A. \u003cem\u003eet al.\u003c/em\u003e Emerging Roles and Mechanisms of RNA Modifications in Neurodegenerative Diseases and Glioma. \u003cem\u003eCells\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 457 (2024).\u003c/li\u003e\n\u003cli\u003eMartinez-Feduchi, P., Jin, P. \u0026amp; Yao, B. Epigenetic modifications of DNA and RNA in Alzheimer\u0026rsquo;s disease. \u003cem\u003eFront Mol Neurosci\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 1398026 (2024).\u003c/li\u003e\n\u003cli\u003eShafik, A. M. \u003cem\u003eet al.\u003c/em\u003e N6-methyladenosine dynamics in neurodevelopment and aging, and its potential role in Alzheimer\u0026rsquo;s disease. \u003cem\u003eGenome Biol\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 17 (2021).\u003c/li\u003e\n\u003cli\u003eMurakami, S. \u0026amp; Jaffrey, S. R. Hidden codes in mRNA: Control of gene expression by m6A. \u003cem\u003eMol Cell\u003c/em\u003e \u003cstrong\u003e82\u003c/strong\u003e, 2236\u0026ndash;2251 (2022).\u003c/li\u003e\n\u003cli\u003eHong, J., Xu, K. \u0026amp; Lee, J. H. Biological roles of the RNA m6A modification and its implications in cancer. \u003cem\u003eExp Mol Med\u003c/em\u003e \u003cstrong\u003e54\u003c/strong\u003e, 1822\u0026ndash;1832 (2022).\u003c/li\u003e\n\u003cli\u003eZaccara, S. \u0026amp; Jaffrey, S. R. A Unified Model for the Function of YTHDF Proteins in Regulating m6A-Modified mRNA. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e181\u003c/strong\u003e, 1582-1595.e18 (2020).\u003c/li\u003e\n\u003cli\u003eDominissini, D. \u003cem\u003eet al.\u003c/em\u003e Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e485\u003c/strong\u003e, 201\u0026ndash;206 (2012).\u003c/li\u003e\n\u003cli\u003eYang, Y. \u003cem\u003eet al.\u003c/em\u003e Dynamic m6A modification and its emerging regulatory role in mRNA splicing. \u003cem\u003eSci. Bull.\u003c/em\u003e \u003cstrong\u003e60\u003c/strong\u003e, 21\u0026ndash;32 (2015).\u003c/li\u003e\n\u003cli\u003eRoundtree, I. A. \u003cem\u003eet al.\u003c/em\u003e YTHDC1 mediates nuclear export of N6-methyladenosine methylated mRNAs. \u003cem\u003eeLife\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, e31311 (2017).\u003c/li\u003e\n\u003cli\u003eLesbirel, S. \u003cem\u003eet al.\u003c/em\u003e The m6A-methylase complex recruits TREX and regulates mRNA export. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 13827 (2018).\u003c/li\u003e\n\u003cli\u003eEdens, B. M. \u003cem\u003eet al.\u003c/em\u003e FMRP Modulates Neural Differentiation through m6A-Dependent mRNA Nuclear Export. \u003cem\u003eCell Rep\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e, 845-854.e5 (2019).\u003c/li\u003e\n\u003cli\u003eMeyer, K. D. \u003cem\u003eet al.\u003c/em\u003e 5\u0026prime; UTR m6A Promotes Cap-Independent Translation. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e163\u003c/strong\u003e, 999\u0026ndash;1010 (2015).\u003c/li\u003e\n\u003cli\u003eDu, B. \u003cem\u003eet al.\u003c/em\u003e N6-methyladenosine (m6A) modification and its clinical relevance in cognitive dysfunctions. \u003cem\u003eAging (Albany NY)\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 20716\u0026ndash;20737 (2021).\u003c/li\u003e\n\u003cli\u003eZhang, R., Zhang, Y., Guo, F., Li, S. \u0026amp; Cui, H. RNA N6-Methyladenosine Modifications and Its Roles in Alzheimer\u0026rsquo;s Disease. \u003cem\u003eFront. Cell. Neurosci.\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, (2022).\u003c/li\u003e\n\u003cli\u003eJiang, X. \u003cem\u003eet al.\u003c/em\u003e The role of m6A modification in the biological functions and diseases. \u003cem\u003eSig Transduct Target Ther\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 74 (2021).\u003c/li\u003e\n\u003cli\u003eHan, M. \u003cem\u003eet al.\u003c/em\u003e Abnormality of m6A mRNA Methylation Is Involved in Alzheimer\u0026rsquo;s Disease. \u003cem\u003eFront Neurosci\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 98 (2020).\u003c/li\u003e\n\u003cli\u003eZhao, F. \u003cem\u003eet al.\u003c/em\u003e METTL3-dependent RNA m6A dysregulation contributes to neurodegeneration in Alzheimer\u0026rsquo;s disease through aberrant cell cycle events. \u003cem\u003eMolecular Neurodegeneration\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 70 (2021).\u003c/li\u003e\n\u003cli\u003eSun, J. \u003cem\u003eet al.\u003c/em\u003e The Potential Role of m6A RNA Methylation in the Aging Process and Aging-Associated Diseases. \u003cem\u003eFront Genet\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 869950 (2022).\u003c/li\u003e\n\u003cli\u003eLeonetti, A. M., Chu, M. Y., Ramnaraign, F. O., Holm, S. \u0026amp; Walters, B. J. An Emerging Role of m6A in Memory: A Case for Translational Priming. \u003cem\u003eInternational Journal of Molecular Sciences\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 7447 (2020).\u003c/li\u003e\n\u003cli\u003eLv, Z. \u003cem\u003eet al.\u003c/em\u003e Downregulation of m6A Methyltransferase in the Hippocampus of Tyrobp\u0026ndash;/\u0026ndash; Mice and Implications for Learning and Memory Deficits. \u003cem\u003eFront. Neurosci.\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, (2022).\u003c/li\u003e\n\u003cli\u003ePupak, A. \u003cem\u003eet al.\u003c/em\u003e Altered m6A RNA methylation contributes to hippocampal memory deficits in Huntington\u0026rsquo;s disease mice. \u003cem\u003eCell Mol Life Sci\u003c/em\u003e \u003cstrong\u003e79\u003c/strong\u003e, 416 (2022).\u003c/li\u003e\n\u003cli\u003eDeng, J. \u003cem\u003eet al.\u003c/em\u003e N6-methyladenosine demethylase FTO regulates synaptic and cognitive impairment by destabilizing PTEN mRNA in hypoxic-ischemic neonatal rats. \u003cem\u003eCell Death Dis\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 1\u0026ndash;14 (2023).\u003c/li\u003e\n\u003cli\u003eWalters, B. J. \u003cem\u003eet al.\u003c/em\u003e The Role of The RNA Demethylase FTO (Fat Mass and Obesity-Associated) and mRNA Methylation in Hippocampal Memory Formation. \u003cem\u003eNeuropsychopharmacol\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e, 1502\u0026ndash;1510 (2017).\u003c/li\u003e\n\u003cli\u003eBokar, J. A., Shambaugh, M. E., Polayes, D., Matera, A. G. \u0026amp; Rottman, F. M. Purification and cDNA cloning of the AdoMet-binding subunit of the human mRNA (N6-adenosine)-methyltransferase. \u003cem\u003eRNA\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 1233\u0026ndash;1247 (1997).\u003c/li\u003e\n\u003cli\u003eLiu, J. \u003cem\u003eet al.\u003c/em\u003e A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation. \u003cem\u003eNat Chem Biol\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 93\u0026ndash;95 (2014).\u003c/li\u003e\n\u003cli\u003eOerum, S., Meynier, V., Catala, M. \u0026amp; Tisn\u0026eacute;, C. A comprehensive review of m6A/m6Am RNA methyltransferase structures. \u003cem\u003eNucleic Acids Res\u003c/em\u003e \u003cstrong\u003e49\u003c/strong\u003e, 7239\u0026ndash;7255 (2021).\u003c/li\u003e\n\u003cli\u003eF, Z. \u003cem\u003eet al.\u003c/em\u003e METTL3-dependent RNA m\u003csup\u003e6\u003c/sup\u003eA dysregulation contributes to neurodegeneration in Alzheimer\u0026rsquo;s disease through aberrant cell cycle events. \u003cem\u003eMol Neurodegener\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 70\u0026ndash;70 (2021).\u003c/li\u003e\n\u003cli\u003eJiang, L. \u003cem\u003eet al.\u003c/em\u003e Interaction of tau with HNRNPA2B1 and N6-methyladenosine RNA mediates the progression of tauopathy. \u003cem\u003eMol Cell\u003c/em\u003e \u003cstrong\u003e81\u003c/strong\u003e, 4209-4227.e12 (2021).\u003c/li\u003e\n\u003cli\u003eCastro-Hern\u0026aacute;ndez, R. \u003cem\u003eet al.\u003c/em\u003e Conserved reduction of m6A RNA modifications during aging and neurodegeneration is linked to changes in synaptic transcripts. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e120\u003c/strong\u003e, e2204933120.\u003c/li\u003e\n\u003cli\u003eHaroutunian, V., Katsel, P. \u0026amp; Schmeidler, J. Transcriptional vulnerability of brain regions in Alzheimer\u0026rsquo;s disease and dementia. \u003cem\u003eNeurobiol Aging\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 561\u0026ndash;573 (2009).\u003c/li\u003e\n\u003cli\u003ePalmqvist, S. \u003cem\u003eet al.\u003c/em\u003e Discriminative Accuracy of Plasma Phospho-tau217 for Alzheimer Disease vs Other Neurodegenerative Disorders. \u003cem\u003eJAMA\u003c/em\u003e \u003cstrong\u003e324\u003c/strong\u003e, 772\u0026ndash;781 (2020).\u003c/li\u003e\n\u003cli\u003eAshton, N. J. \u003cem\u003eet al.\u003c/em\u003e Diagnostic Accuracy of a Plasma Phosphorylated Tau 217 Immunoassay for Alzheimer Disease Pathology. \u003cem\u003eJAMA Neurol\u003c/em\u003e \u003cstrong\u003e81\u003c/strong\u003e, 255\u0026ndash;263 (2024).\u003c/li\u003e\n\u003cli\u003eHuang, H., Song, R., Wong, J. J.-L., Anggono, V. \u0026amp; Widagdo, J. The N6-methyladenosine RNA landscape in the aged mouse hippocampus. https://doi.org/10.1111/acel.13755 doi:10.1111/acel.13755.\u003c/li\u003e\n\u003cli\u003eFan, Y., Lv, X., Chen, Z., Peng, Y. \u0026amp; Zhang, M. m6A methylation: Critical roles in aging and neurological diseases. \u003cem\u003eFront. Mol. Neurosci.\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, (2023).\u003c/li\u003e\n\u003cli\u003eYankova, E. \u003cem\u003eet al.\u003c/em\u003e Small molecule inhibition of METTL3 as a strategy against myeloid leukaemia. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e593\u003c/strong\u003e, 597\u0026ndash;601 (2021).\u003c/li\u003e\n\u003cli\u003eWang, P., Doxtader, K. A. \u0026amp; Nam, Y. Structural basis for cooperative function of Mettl3 and Mettl14 methyltransferases. \u003cem\u003eMol Cell\u003c/em\u003e \u003cstrong\u003e63\u003c/strong\u003e, 306\u0026ndash;317 (2016).\u003c/li\u003e\n\u003cli\u003eKoranda, J. L. \u003cem\u003eet al.\u003c/em\u003e Mettl14 Is Essential for Epitranscriptomic Regulation of Striatal Function and Learning. \u003cem\u003eNeuron\u003c/em\u003e \u003cstrong\u003e99\u003c/strong\u003e, 283-292.e5 (2018).\u003c/li\u003e\n\u003cli\u003eVorhees, C. V. \u0026amp; Williams, M. T. Morris water maze: procedures for assessing spatial and related forms of learning and memory. \u003cem\u003eNat Protoc\u003c/em\u003e \u003cstrong\u003e1\u003c/strong\u003e, 848\u0026ndash;858 (2006).\u003c/li\u003e\n\u003cli\u003eD\u0026rsquo;Hooge, R. \u0026amp; De Deyn, P. P. Applications of the Morris water maze in the study of learning and memory. \u003cem\u003eBrain Res Brain Res Rev\u003c/em\u003e \u003cstrong\u003e36\u003c/strong\u003e, 60\u0026ndash;90 (2001).\u003c/li\u003e\n\u003cli\u003eGarthe, A., Behr, J. \u0026amp; Kempermann, G. Adult-generated hippocampal neurons allow the flexible use of spatially precise learning strategies. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, e5464 (2009).\u003c/li\u003e\n\u003cli\u003eJ\u0026uuml;rgenson, M., Aonurm-Helm, A. \u0026amp; Zharkovsky, A. Behavioral profile of mice with impaired cognition in the elevated plus-maze due to a deficiency in neural cell adhesion molecule. \u003cem\u003ePharmacol Biochem Behav\u003c/em\u003e \u003cstrong\u003e96\u003c/strong\u003e, 461\u0026ndash;468 (2010).\u003c/li\u003e\n\u003cli\u003eMerch\u0026aacute;n-Rubira, J., Sebasti\u0026aacute;n-Serrano, \u0026Aacute;., D\u0026iacute;az-Hern\u0026aacute;ndez, M., Avila, J. \u0026amp; Hern\u0026aacute;ndez, F. Peripheral nervous system effects in the PS19 tau transgenic mouse model of tauopathy. \u003cem\u003eNeurosci Lett\u003c/em\u003e \u003cstrong\u003e698\u003c/strong\u003e, 204\u0026ndash;208 (2019).\u003c/li\u003e\n\u003cli\u003eAl-Ghraiybah, N. F. \u003cem\u003eet al.\u003c/em\u003e Glial Cell-Mediated Neuroinflammation in Alzheimer\u0026rsquo;s Disease. \u003cem\u003eInt J Mol Sci\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 10572 (2022).\u003c/li\u003e\n\u003cli\u003eKwon, H. S. \u0026amp; Koh, S.-H. Neuroinflammation in neurodegenerative disorders: the roles of microglia and astrocytes. \u003cem\u003eTransl Neurodegener\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 42 (2020).\u003c/li\u003e\n\u003cli\u003eLi, Q., Wen, S., Ye, W., Zhao, S. \u0026amp; Liu, X. The potential roles of m6A modification in regulating the inflammatory response in microglia. \u003cem\u003eJournal of Neuroinflammation\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 149 (2021).\u003c/li\u003e\n\u003cli\u003eDing, L. \u003cem\u003eet al.\u003c/em\u003e m6A Reader Igf2bp1 Regulates the Inflammatory Responses of Microglia by Stabilizing Gbp11 and Cp mRNAs. \u003cem\u003eFront Immunol\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 872252 (2022).\u003c/li\u003e\n\u003cli\u003eHuang, H., Camats-Perna, J., Medeiros, R., Anggono, V. \u0026amp; Widagdo, J. Altered Expression of the m6A Methyltransferase METTL3 in Alzheimer\u0026rsquo;s Disease. \u003cem\u003eeNeuro\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, (2020).\u003c/li\u003e\n\u003cli\u003eZhang, X. \u003cem\u003eet al.\u003c/em\u003e Differential methylation of circRNA m6A in an APP/PS1 Alzheimer\u0026rsquo;s disease mouse model. \u003cem\u003eMol Med Rep\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 55 (2023).\u003c/li\u003e\n\u003cli\u003eWu, Z. \u003cem\u003eet al.\u003c/em\u003e m6A epitranscriptomic regulation of tissue homeostasis during primate aging. \u003cem\u003eNat Aging\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 705\u0026ndash;721 (2023).\u003c/li\u003e\n\u003cli\u003eZhang, W., Xiao, D., Mao, Q. \u0026amp; Xia, H. Role of neuroinflammation in neurodegeneration development. \u003cem\u003eSig Transduct Target Ther\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 267 (2023).\u003c/li\u003e\n\u003cli\u003ePascoal, T. A. \u003cem\u003eet al.\u003c/em\u003e Microglial activation and tau propagate jointly across Braak stages. \u003cem\u003eNat Med\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 1592\u0026ndash;1599 (2021).\u003c/li\u003e\n\u003cli\u003ed\u0026rsquo;Errico, P. \u003cem\u003eet al.\u003c/em\u003e Microglia contribute to the propagation of A\u0026beta; into unaffected brain tissue. \u003cem\u003eNat Neurosci\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 20\u0026ndash;25 (2022).\u003c/li\u003e\n\u003cli\u003eBellaver, B. \u003cem\u003eet al.\u003c/em\u003e Astrocyte reactivity influences amyloid-\u0026beta; effects on tau pathology in preclinical Alzheimer\u0026rsquo;s disease. \u003cem\u003eNat Med\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 1775\u0026ndash;1781 (2023).\u003c/li\u003e\n\u003cli\u003eDai, D. L., Li, M. \u0026amp; Lee, E. B. Human Alzheimer\u0026rsquo;s disease reactive astrocytes exhibit a loss of homeostastic gene expression. \u003cem\u003eActa Neuropathologica Communications\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 127 (2023).\u003c/li\u003e\n\u003cli\u003eWu, X. \u003cem\u003eet al.\u003c/em\u003e The m6A methyltransferase METTL3 drives neuroinflammation and neurotoxicity through stabilizing BATF mRNA in microglia. \u003cem\u003eCell Death Differ\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e, 100\u0026ndash;117 (2025).\u003c/li\u003e\n\u003cli\u003eZhang, F. \u003cem\u003eet al.\u003c/em\u003e Regulation of N6-methyladenosine (m6A) RNA methylation in microglia-mediated inflammation and ischemic stroke. \u003cem\u003eFront. Cell. Neurosci.\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, (2022).\u003c/li\u003e\n\u003cli\u003eWen, Y.-L., Guo, F., Gu, T.-T., Zeng, Y.-P. \u0026amp; Cao, X. Transcriptomic Regulation by Astrocytic m6A Methylation in the mPFC. \u003cem\u003eGenes Cells\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, e70003 (2025).\u003c/li\u003e\n\u003cli\u003eLi, Y., Li, J., Yu, Q., Ji, L. \u0026amp; Peng, B. METTL14 regulates microglia/macrophage polarization and NLRP3 inflammasome activation after ischemic stroke by the KAT3B-STING axis. \u003cem\u003eNeurobiology of Disease\u003c/em\u003e \u003cstrong\u003e185\u003c/strong\u003e, 106253 (2023).\u003c/li\u003e\n\u003cli\u003eZhu, L. \u003cem\u003eet al.\u003c/em\u003e METTL3/IGF2BP2/I\u0026kappa;B\u0026alpha; axis participates in neuroinflammation in Alzheimer\u0026rsquo;s disease by regulating M1/M2 polarization of microglia. \u003cem\u003eNeurochemistry International\u003c/em\u003e \u003cstrong\u003e186\u003c/strong\u003e, 105964 (2025).\u003c/li\u003e\n\u003cli\u003eHui, Y. \u003cem\u003eet al.\u003c/em\u003e Immunological characterization and diagnostic models of RNA N6-methyladenosine regulators in Alzheimer\u0026rsquo;s disease. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 14588 (2023).\u003c/li\u003e\n\u003cli\u003eYin, H. \u003cem\u003eet al.\u003c/em\u003e Loss of the m6A methyltransferase METTL3 in monocyte-derived macrophages ameliorates Alzheimer\u0026rsquo;s disease pathology in mice. \u003cem\u003ePLOS Biology\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, e3002017 (2023).\u003c/li\u003e\n\u003cli\u003eZhao, X. \u003cem\u003eet al.\u003c/em\u003e Mettl3 regulates the pathogenesis of Alzheimer\u0026rsquo;s disease via fine-tuning Lingo2. \u003cem\u003eMol Psychiatry\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 4047\u0026ndash;4063 (2025).\u003c/li\u003e\n\u003cli\u003eLong, J. M. \u0026amp; Holtzman, D. M. Alzheimer Disease: An Update on Pathobiology and Treatment Strategies. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e179\u003c/strong\u003e, 312\u0026ndash;339 (2019).\u003c/li\u003e\n\u003cli\u003eXia, L. \u003cem\u003eet al.\u003c/em\u003e A new perspective on Alzheimer\u0026rsquo;s disease: m6A modification. \u003cem\u003eFront Genet\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 1166831 (2023).\u003c/li\u003e\n\u003cli\u003eHaroutunian, V., Katsel, P. \u0026amp; Schmeidler, J. Transcriptional vulnerability of brain regions in Alzheimer\u0026rsquo;s disease and dementia. \u003cem\u003eNeurobiol Aging\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 561\u0026ndash;573 (2009).\u003c/li\u003e\n\u003cli\u003eMarco, A. R. \u003cem\u003eet al.\u003c/em\u003e Therapeutic VEGFC treatment provides protection against traumatic-brain-injury-driven tauopathy pathogenesis. \u003cem\u003eCell Reports\u003c/em\u003e \u003cstrong\u003e44\u003c/strong\u003e, (2025).\u003c/li\u003e\n\u003cli\u003eLammert, C. R. \u003cem\u003eet al.\u003c/em\u003e AIM2 inflammasome surveillance of DNA damage shapes neurodevelopment. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e580\u003c/strong\u003e, 647\u0026ndash;652 (2020).\u003c/li\u003e\n\u003cli\u003eKraeuter, A.-K., Guest, P. C. \u0026amp; Sarnyai, Z. The Y-Maze for Assessment of Spatial Working and Reference Memory in Mice. in \u003cem\u003ePre-Clinical Models: Techniques and Protocols\u003c/em\u003e (ed. Guest, P. C.) 105\u0026ndash;111 (Springer, New York, NY, 2019). doi:10.1007/978-1-4939-8994-2_10.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Alzheimer’s disease, tauopathy, N6-methyladenosine, RNA modification, cognition, METTL3 inhibitor, STM2457","lastPublishedDoi":"10.21203/rs.3.rs-8379573/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8379573/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Dysregulation of N6-methyladenosine (m6A) modification of RNA has emerged as a novel feature of Alzheimer’s disease (AD). Here, we investigate the relationship between m6A modification and AD pathology, and the therapeutic potential of modulating excessive m6A via its “writer” methyltransferase METTL3 in a humanized P301S tau transgenic mouse model of AD (PS19). We observed significantly elevated m6A levels in human post-mortem AD frontal cortex tissue compared to healthy controls, which positively correlated with hyperphosphorylated tau and amyloid-β (Aβ) deposition. These effects were recapitulated in the PS19 tau mice model of AD. Importantly, treatment of PS19 mice with the METTL3 inhibitor STM2457 reduced excessive m6A, alleviated tau pathology, and attenuated neurodegeneration. Behavioral assessments further demonstrated that STM2457-treated PS19 mice exhibited significantly improved learning and memory relative to untreated PS19 mice. Our results identify m6A as a critical contributor to AD pathogenesis and demonstrate that pharmacological inhibition of METTL3 represents a promising therapeutic strategy to improve cognition in AD.","manuscriptTitle":"Inhibition of N6-Methyladenosine Accumulation by Targeting METTL3 Mitigates Tau Pathology and Cognitive Decline in Alzheimer’s Disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-19 07:45:02","doi":"10.21203/rs.3.rs-8379573/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aebeca7a-8a4d-468c-a69e-06f6f212aabf","owner":[],"postedDate":"December 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":59913618,"name":"Health sciences/Diseases/Neurological disorders/Neurodegeneration"},{"id":59913619,"name":"Biological sciences/Neuroscience/Epigenetics in the nervous system/Epigenetics and behaviour"}],"tags":[],"updatedAt":"2025-12-19T07:45:04+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-19 07:45:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8379573","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8379573","identity":"rs-8379573","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}