MiR-144 regulates cognitive dysfunction via NLRP3 inflammasome and FoxO1/AdipoR pathway in T2DM mice

MiR-144 regulates cognitive dysfunction via NLRP3 inflammasome and FoxO1/AdipoR pathway in T2DM mice | 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 Research Article MiR-144 regulates cognitive dysfunction via NLRP3 inflammasome and FoxO1/AdipoR pathway in T2DM mice Jinying Zhao, Yuliang Zhou, Shi Cheng, Jia Shen, Yahong Li, Zhipeng Xu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8592557/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Background Type 2 diabetes mellitus (T2DM) is closely associated with cognitive impairment, with underlying pathological mechanisms including chronic inflammation, insulin resistance, and neuronal injury. Recent research indicates that microRNA-144 (miR-144) plays a critical role in these processes, though its exact mechanism remains unclear. Given the critical role of microglia in neuroinflammation and synaptic homeostasis, we investigated whether miR-144 mediates T2DM-related cognitive impairment by modulating microglial function through the Fork-head Box O1 (FoxO1)/AdipoR signaling pathway and activation of NLR family pyrin domain containing 3 (NLRP3) inflammasome. Methods Microglia-specific T2DM mouse models were established using Cx3cr1-Cre mice subjected to a high-fat diet combined with low-dose streptozotocin administration. Microglia-selective overexpression or knockdown of miR-144 was achieved via stereotactic hippocampal injection of adeno-associated virus (AAV). Cognitive function was evaluated using the Morris water maze and novel object recognition tests. Synaptic function and plasticity were assessed by electrophysiological recordings (mEPSCs/mIPSCs and AMPAR/NMDAR-EPSCs), ultrastructural analyses (Golgi staining and transmission electron microscopy), and molecular assays including Western blotting and immunofluorescence. Expression levels of synaptic proteins, Tau phosphorylation, FoxO1, AdipoR1/2, NLRP3 inflammasome components, and inflammatory cytokines were systematically analyzed. Results T2DM mice exhibited significant cognitive deficits accompanied by synaptic dysfunction, increased Tau phosphorylation, and enhanced neuroinflammatory responses. Notably, microglial overexpression of miR-144 recapitulated key pathological features of T2DM, including impaired learning and memory, disrupted synaptic transmission, reduced synaptic protein expression, decreased dendritic spine density, and elevated Tau phosphorylation. Additionally, miR-144 overexpression significantly suppressed FoxO1 and AdipoR1/AdipoR2 expression, leading to activation of the NLRP3 inflammasome and subsequent amplification of neuroinflammation. In contrast, microglial knockdown of miR-144 markedly alleviated cognitive impairment, restored synaptic integrity, suppressed Tau hyperphosphorylation, and attenuated neuroinflammatory signaling, thereby exerting robust neuroprotective effects. Conclusion This study identifies miR-144 as a pivotal regulator of T2DM-related cognitive dysfunction. miR-144 mediates microglial-driven neuroinflammation and synaptic impairment through suppression of the FoxO1/AdipoR signaling pathway and activation of the NLRP3 inflammasome. These findings highlight miR-144 as a potential biomarker and therapeutic target for preventing or treating cognitive impairment associated with T2DM. type 2 diabetes mellitus cognitive impairment synaptic plasticity biomarkers Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Cognitive impairment is a severe and increasingly recognized complication of type 2 diabetes mellitus (T2DM), imposing a substantial burden on patients and healthcare systems. Epidemiological studies indicate that individuals with T2DM have approximately a 1.5-fold higher risk of developing dementia compared with non-diabetic populations[ 1 ]. The pathogenesis of T2DM-related cognitive impairment involves multiple mechanisms, including chronic hyperglycemia, insulin resistance, cerebrovascular dysfunction, neuroinflammation, disrupted cerebral energy metabolism, and abnormal amyloid-beta (Aβ) accumulation. As the resident immune cells of the central nervous system, microglia play a pivotal role in these pathological processes. Dysregulated microglial activation leads to excessive cytotoxicity, synaptic dysfunction, and sustained neuroinflammatory signaling, ultimately accelerating cognitive decline[ 2 ]. NLR family pyrin domain containing 3 (NLRP3) is a protein complex composed of sensor NLRP3, adaptor apoptosis-associated spot-like protein (ASC) and effector caspase-1. This inflammasome is a central mediator of microglia-driven neuroinflammation and has been implicated in the pathogenesis of numerous inflammatory disorders, including neurodegenerative, cardiovascular, and autoimmune diseases[ 3 , 4 ]. In patients with cognitive impairment, NLRP3 expression in microglia is markedly upregulated, facilitating the assembly of ASC and pro–caspase-1. Subsequent caspase-1 activation promotes the maturation and release of Interleukins (IL)-1β and IL-18, thereby triggering an inflammatory cascade[ 5 ]. Notably, the inhibiting the NLRP3 inflammasome has shown promise in improving cognitive decline caused by T2DM, underscoring NLRP3 as a promising therapeutic target T2DM-related cognitive impairment[ 6 ]. However, the upstream regulatory mechanisms governing NLRP3 activation in T2DM-related cognitive impairment remain incompletely understood. MicroRNA-144 (miR-144) is a small non-coding RNA highly enriched in erythrocytes and exists as a mature duplex comprising the guide strand miR-144-3p and the passenger strand miR-144-5p. Among these, miR-144-3p exhibits greater biological stability and exerts predominant regulatory functions in gene expression[ 7 ]. MiR-144 has emerged as a potential diagnostic and prognostic biomarker and is frequently dysregulated in cancer and inflammatory diseases, where it modulates pathological processes by directly or indirectly targeting key signaling molecules[ 8 ]. Importantly, miR-144 expression is significantly elevated in the peripheral blood of patients with T2DM[ 9 ]. Parallel increases in miR-144 levels have also been observed in the hippocampus and medial prefrontal cortex in Alzheimer’s disease (AD) mouse models[ 10 ]. Functionally, miR-144 has been shown to exacerbate cognitive impairment by promoting Aβ deposition[ 11 ]. Conversely, knockdown of miR-144 has been shown to suppress NLRP3 inflammasome activation and restore cognitive function, suggesting that miR-144 may serve as a critical contributor to T2DM-related cognitive impairment[ 12 ]. Adiponectin (APN) is an endogenous adipokine with well-established protective effects in T2DM. Upon binding to its specific receptors, AdipoR1 and AdipoR2, APN activates downstream AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor-α (PPARα) signaling pathways[ 13 ]. Previous studies have demonstrated that AdipoRon, a synthetic adiponectin receptor agonist, reduces Aβ deposition and improves cognitive performance in APP/PS1 mice through activation of the AdipoR1/AMPK pathway[ 14 ]. Consistently, AdipoRon has been shown to alleviate cognitive deficits and synaptic dysfunction in T2DM mouse models, concomitant with reduced hippocampal Tau hyperphosphorylation[ 15 ]. Furthermore, AdipoRon suppresses the expression of NLRP3, ASC, and IL-1β in the hippocampus and prefrontal cortex, thereby attenuating neuroinflammatory responses[ 16 ]. Although these findings highlight AdipoR signaling as a promising therapeutic avenue for T2DM-related cognitive impairment, the precise regulatory mechanisms governing AdipoR expression and function remain unclear. Forkhead box O1 (FoxO1) is a key transcription factor involved in insulin signaling and the pathogenesis of diabetes and has been shown to positively regulate AdipoR expression[ 17 ]. In ob/ob diabetic mice, the expression of AdipoR is downregulated via the Phosphoinositide 3-Kinase (PI3K)/FoxO1 signaling pathway, resulting in adiponectin resistance[ 18 ]. Notably, miR-144 directly targets FoxO1, thereby attenuating adiponectin-mediated protective effects[ 19 ]. These findings suggest that miR-144 may contribute to T2DM-related cognitive impairment through suppression of the FoxO1/AdipoR signaling axis. Our study aims to investigate the pathogenesis of T2DM-related cognitive impairment by focusing on the regulatory role of miR-144 in synaptic plasticity and cognitive function within the hippocampus (Fig. 1 A). By integrating microglia–neuron interactions within the neuroimmune network, we systematically investigated how miR-144 modulates NLRP3 inflammasome activation via the FoxO1/AdipoR signaling pathway. Additionally, given the critical role of the parietal cortex (PC) in higher cognitive function and its extensive neural circuit connections with the hippocampus, we will assess the pathophysiological consistency between these two regions[ 20 ]. Collectively, our findings are expected to provide novel mechanistic insights into T2DM-related cognitive decline and identify potential molecular targets for future therapeutic intervention. Materials and methods Animals Eight-week-old adult male Cx3cr1-Cre mice (SPF grade) were obtained from Shulaibao (Wuhan) Biotechnology Co., Ltd. All animals were housed under specific pathogen-free conditions in a controlled barrier environment with a stable temperature of 22 ± 2°C and humidity of 55 ± 10%, under a 12-hour light/dark cycle. All mice had free access to food and water. After one week of acclimatization, experimental procedures were initiated. T2DM mice model Mice were randomly assigned to either a normal diet (ND) group or a high-fat diet (HFD) group. The ND group received a standard laboratory diet, while the HFD group was fed a diet containing 60% kcal from fat for six weeks. Following the dietary intervention, HFD-fed mice received intraperitoneal injections of streptozotocin (STZ; 40 mg/kg) dissolved in 0.1 mol/L sodium citrate buffer (pH 4.5) once daily for three consecutive days to induce T2DM model. ND-fed mice received an equivalent volume of citrate buffer on the same schedule. Fasting blood glucose (FBG) levels were measured on the fourth day after the initial STZ injection. If FBG did not exceed 11.1 mmol/L, an additional STZ injection (40 mg/kg) was administered. Successful establishment of the T2DM model was confirmed when FBG levels remained above 11.1 mmol/L for at least one week. Intracerebral stereotactic injection After successful induction of the T2DM model, mice underwent stereotaxic injection of adeno-associated virus (AAV) into the hippocampus. The viral vectors rAVV-SFFV-DIO-EGFP-premmu-miR-144-pA, rAVV-SFFV-DIO-[EGFP-WPRE-4XmicroRNA(mmu-miR-144)]-pA,and the empty control vector rAVV-SFFV-EGFP-WPRs were purchased from Wuhan Shumi Brain Science and Technology Co., LTD. To investigate the effects of miR-144 upregulation and downregulation under diabetic and non-diabetic conditions, mice were divided into 7 groups (n = 12 per group; total N = 84): two Control + siRNA groups, blank control group receiving control siRNA; two T2DM + siRNA groups, diabetic control group receiving control siRNA; Control + miR-144(+): non-diabetic group with miR-144 overexpression; Control + miR-144 (-): non-diabetic group with miR-144 knockdown; T2DM + miR-144 (-): diabetic group with miR-144 knockdown. Mice were anesthetized via intraperitoneal injection of sodium pentobarbital (1% solution, 50 mg/kg), and then secured on a stereotactic frame. Four small holes were drilled bilaterally above the skull, and AVV was injected using a microsyringe (ML: ±1.6mm, AP: -1.6mm, DV: 1.5mm; ML: ±2mm, AP: -1.8mm, DV: 2.0mm, Fig. 1 B). AVV was injected at each site with a virus injection dose of 300nL and a rate of 50 nL/min (titer 5×10 12 vg/mL). Behavioral tests were conducted four weeks after viral injection. Behavioral tests Novel object recognition (NOR) test was performed to evaluate learning and memory in mice, which consisted of three phases: habituation, training, and testing. On the first day, mice were allowed to freely explore an empty open-field arena (40 × 40 × 40 cm) for 5 min. On the following day, two identical objects were placed symmetrically in the arena, and mice were allowed to explore for 5 min. After a 24h interval, one familiar object was replaced with a novel object of similar size and material but a different shape. Mice were allowed to explore freely for 5 min. Exploration time was recorded using a video tracking system. The discrimination index (DI) was calculated as follows: DI= [Time spent exploring the novel object / (Time spent exploring the novel object + Time spent exploring the familiar object)] ×100%. The arena and objects were cleaned with 75% ethanol between trials to eliminate olfactory cues. Spatial learning and memory were evaluated using the Morris water maze (MWM) test. The experiment consisted of five days of training followed by a probe trial on day 6. A circular pool was divided into four quadrants, with a hidden platform (10 cm diameter) submerged 1 cm below the water surface in the target quadrant. The water was rendered opaque by the addition of titanium dioxide to ensure the platform was not visible. During training, mice underwent four trials per day from different start positions. Escape latency was recorded, with a maximum trial duration of 60 s. If it failed to find the platform within the period, mice were guided to the platform and allowed to remain there for 10s to reinforce memory. On the sixth day, a probe trial was conducted with the platform was removed, and mice were allowed to swim freely for 60 s. Platform crossings, time spent in the target quadrant, and percentage of distance traveled in the target quadrant were analyzed. Golgi staining Golgi staining was performed using the Hito Golgi box OptimStain™ Prekit (Hitobiotec Corp., USA). Mice were anesthetized via intraperitoneal injection of 1% pentobarbital sodium (50 mg/kg) and subsequently decapitated for brain extraction. Brains immersed in impregnation solution (A : B = 1 : 1) at room temperature in the dark for 14 days, followed by incubation in solution C at 4°C for 2 days. Coronal sections (60µm thick) were prepared using a vibratome, mounted on glass slides, and subjected to staining, dehydration, and sealing. After scanning and collecting the images, dendritic spine length and number in segments were quantified using Fiji software, and density of dendritic spines was calculated. Transmission electron microscopy Hippocampal and parietal cortex tissues were cut into approximately 1mm³ samples, fixed in 2.5% glutaraldehyde at 4°C for 4 h, and rinsed with PBS. After fixation with osmium acid, samples were dehydrated using gradient ethanol and embedded in epoxy resin. The tissue blocks were placed on the microtome for slicing with a thickness of 60nm. Ultrastructural (Talos L120C, Germany) observations were performed using a transmission electron microscope. Western blotting Hippocampal and parietal cortex tissues were homogenized in ice-cold lysis buffer and centrifuged at 12,000 g for 5 min at 4°C. Protein concentration was determined using a BCA assay. Subsequently, the samples were then separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 3% nonfat dried milk for 1h at RT and incubated with primary antibodies overnight at 4°C, followed by secondary antibodies. Signals were detected using ECL and quantified with AlphaEaseFC software. Primary antibodies included: ADIPOR1 (Abcam, ab70362,1:2000), ADIPOR2 (Thermofisher, PA5-100376, 1:1000), FOXO1 (CST, #2880, 1:1000), Synapsin I (Proteintech, 20258-1-AP, 1:2000), PSD95(CST, #2507,1:1000), and β-Actin (Tiandeyue, TDY05, 1:10000). Immunofluorescent staining Brain sections were fixed, cryoprotected, embedded in paraffin. After blocking, sections were incubated with primary antibodies overnight at 4°C, followed by fluorescent secondary antibodies at RT for 1h. Cell nuclei were counterstained with DAPI at RT in the dark, and images were captured using fluorescence microscopy. For the negative control, the primary antibody was omitted from the blocking buffer. Electrophysiology Acute hippocampal slices (300 µm) were prepared in ice-cold oxygenated cutting solution. Slices were immediately incubated in artificial cerebrospinal fluid at 34 ° C for 30min, and then equilibrated at RT for another 30min. For electrophysiological recordings, whole-cell patch-clamp recordings were obtained from CA1 pyramidal neurons in mouse hippocampal slices. Action potentials were blocked by adding 1µM tetrodotoxin (TTX) to the extracellular solution. Miniature excitatory postsynaptic current (mEPSC) and miniature inhibitory postsynaptic current (mIPSC) were recorded at holding potentials of − 70 mV and + 10 mV, respectively. To isolate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR)-EPSCs and N-methyl-D-aspartate receptors (NMDAR)-EPSCs, 50µM picrotoxinin was applied to inhibit GABAergic transmission. These responses were recorded at holding potentials of − 70 mV and + 40 mV, respectively. All experiments were performed at constant temperature of (30 ± 1) °C. Current signals were recorded in voltage-clamp mode using an Axon 700B amplifier and filtered through a 3 kHz filter. Data analysis used Clampfit 11.2 software. Statistical analysis Data are presented as mean ± SD. Statistical analyses were performed using IBM SPSS Statistics 27.0 and GraphPad Prism 10.0. One-way or two-way ANOVA followed by Tukey’s post hoc test was applied as appropriate. A value of P < 0.05 was considered statistically significant. Results Overexpression of miR-144 induces T2DM-like cognitive impairment. In the NOR test (Fig. 1 C-E), DI was significantly reduced in T2DM mice compared with the CON + siRNA group ( P < 0.001). Notably, mice with miR-144 overexpression also exhibited a marked decrease in DI, reaching a level comparable to that observed in the T2DM + siRNA group ( P < 0.05), indicating impaired recognition memory induced by miR-144 upregulation. In the MWM training phase, escape latency progressively decreased from day 1 to day 5 across all groups, and the descent rate of T2DM mice was even slower (Fig. 1 F). On the fifth day, both the T2DM + siRNA group and the CON + miR-144(+) group showed significantly prolonged escape latencies compared with the CON + siRNA mice. However, the impairment was more severe in the T2DM + siRNA group ( P < 0.001) than in the CON + miR-144(+) group ( P < 0.05). During the probe trial following platform removal, T2DM mice demonstrated a significant reduction in both the total distance traveled ( P < 0.01) and the percentage of time spent ( P < 0.01) in the target quadrant compared with CON + siRNA mice (Fig. 1 G–J). Similarly, miR-144 overexpression resulted in decreased exploration distance ( P < 0.05) and reduced time spent in the target quadrant ( P 0.05). Collectively, these results indicate that miR-144 overexpression induces cognitive deficits resembling those observed in T2DM. Overexpression of miR-144 induces T2DM-like synaptic injury. Synaptic dysfunction is a critical early pathological feature underlying cognitive impairment in T2DM. To investigate whether miR-144-induced cognitive deficits were associated with synaptic alterations, the expression of synapse-related proteins and synaptic structural integrity were examined in the hippocampus and parietal cortex. Western blot analysis revealed that, compared with the CON + siRNA group, the expression levels of postsynaptic density protein 95 (PSD95) and Synapsin I were significantly reduced in the hippocampus of both the T2DM + siRNA group (PSD95: P < 0.001; Synapsin I: P < 0.001) and the CON + miR-144(+) group (PSD95: P < 0.001; Synapsin I: P < 0.001) (Figure S1 A–B). Similar reductions were observed in the PC (Figure S1 A, C), with significant decreases in PSD95 [T2DM + siRNA: P < 0.01; CON + miR-144(+): P < 0.01] and Synapsin I [T2DM + siRNA: P < 0.001; CON + miR-144(+): P < 0.001]. Consistent with these findings, immunofluorescence staining demonstrated significantly reduced PSD95 and Synapsin I expression in multiple hippocampal subregions, including CA1(Figure S1 D-F) [PSD95:T2DM + siRNA: P < 0.01; CON + miR-144(+): P < 0.01,Synapsin I: T2DM + siRNA: P < 0.01; CON + miR-144(+): P < 0.05], CA3 [PSD95: T2DM + siRNA: P < 0.001; CON + miR-144(+): P < 0.05,Synapsin I:T2DM + siRNA: P < 0.001; CON + miR-144(+): P < 0.001], DG(Fig. 2 A-C) [PSD95: T2DM + siRNA: P < 0.01, CON + miR-144(+): P < 0.05; Synapsin I: T2DM + siRNA: P < 0.001; CON + miR-144(+): P < 0.01] and PC [PSD95: T2DM + siRNA: P < 0.05; CON + miR-144(+): P < 0.01,Synapsin I: T2DM + siRNA: P < 0.01; CON + miR-144(+): P < 0.05]. Ultrastructural analysis using transmission electron microscopy further revealed a significant reduction in synapse number in the hippocampus ( P < 0.001) and PC ( P < 0.001) of T2DM mice compared with CON + siRNA mice (Fig. 2 D–F). Importantly, miR-144 overexpression induced comparable decreases in synaptic density in both regions (hippocampus: P < 0.001; PC: P < 0.001). Dendritic spines, which serve as key structural substrates for synaptic transmission and plasticity[ 21 ], were further examined using Golgi staining. Spine density was markedly reduced in the CA1 ( P < 0.05), CA3 ( P < 0.05), DG ( P < 0.001), and PC ( P < 0.001) of T2DM mice (Fig. 2 G–H). Similarly, miR-144 overexpression resulted in significant decreases in dendritic spine density across all examined regions (CA1: P < 0.01; CA3: P < 0.05; DG: P < 0.001; PC: P < 0.001). These findings indicate that miR-144 overexpression impairs synaptic structure and plasticity, closely mimicking T2DM-associated synaptic injury. Overexpression of miR-144 induces T2DM-like synaptic transmission dysfunction. Miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) were recorded from CA1 pyramidal neurons in hippocampal slices in the presence of tetrodotoxin (TTX) (Fig. 3 A-H). Quantitative analysis revealed no significant differences in the amplitudes of either mEPSCs or mIPSCs between T2DM mice and CON + siRNA controls ( P > 0.05). In contrast, the frequency of mEPSCs was significantly reduced in T2DM mice ( P < 0.001), while the frequency of mIPSCs was markedly increased ( P < 0.001). Notably, overexpression of miR-144 recapitulated this synaptic transmission phenotype, resulting in a significant decrease in mEPSC frequency ( P < 0.001) and a concomitant increase in mIPSC frequency (P < 0.001). These findings suggest that miR-144 overexpression disrupts synaptic transmission primarily through presynaptic mechanisms, leading to excitation–inhibition imbalance similar to that observed in T2DM. To further assess glutamatergic synaptic function, the ratio of AMPA receptor- to NMDA receptor-mediated excitatory postsynaptic currents (AMPAR/NMDAR ratio) was examined in CA1 pyramidal neurons (Fig. 3 I-J), which was a widely used index of synaptic strength and plasticity[ 22 ]. The AMPAR/NMDAR ratio was significantly reduced in T2DM mice compared with control mice ( P < 0.001). Importantly, miR-144 overexpression closely mimicked the diabetic phenotype, inducing a similarly significant reduction in the AMPAR/NMDAR ratio ( P < 0.001), further indicating impaired excitatory synaptic transmission. Overexpression of miR-144 induces T2DM-like Tau hyperphosphorylation. Tau is the most abundant microtubule-binding protein in the central nervous system and plays a key role in the regulation of synaptic plasticity through phosphorylation-dependent regulation. In particular, phosphorylation at serine 396 (Ser396) has been shown to significantly impair its biological functions[ 23 , 24 ]. Immunofluorescence staining was performed to evaluate the expression of total Tau and phosphorylated Tau at Ser396 (p-Tau396) in hippocampal subregions and the parietal cortex (PC) of T2DM mice, as well as in response to miR-144 overexpression (Fig. 4 A; Figure S2A). Quantitative analysis revealed no significant differences in total Tau expression among the experimental groups (Fig. 4 C; Figure S2C, P > 0.05). In contrast, p-Tau396 levels were significantly elevated in T2DM mice across multiple brain regions, including CA1 ( P < 0.05), CA3 ( P < 0.01) (Figure S2B), DG ( P < 0.001) and PC ( P < 0.001) (Fig. 4 B). Importantly, overexpression of miR-144 also induced a significant increase in p-Tau396 levels in the same regions (CA1: P < 0.01, CA3: P < 0.05, DG: P < 0.01, PC: P < 0.01), closely mirroring the Tau hyperphosphorylation pattern observed in T2DM mice. These results indicate that miR-144 upregulation promotes Tau pathological phosphorylation without altering total Tau expression, thereby contributing to synaptic transmission dysfunction and T2DM-like neurodegenerative changes. Overexpression of miR-144 suppresses the FoxO1/AdipoR pathway and induces T2DM-like neural injury. To determine whether miR-144 contributes to T2DM-associated neural injury through modulation of the FoxO1/AdipoR signaling pathway, protein expression levels of FoxO1, AdipoR1, and AdipoR2 were examined by western blotting (Fig. 4 D-F). Compared with the control group, T2DM mice exhibited a marked reduction in FoxO1 ( P < 0.001), AdipoR1( P < 0.001) and AdipoR2( P < 0.001) expression in the hippocampus. Notably, miR-144 overexpression produced a comparable suppressive effect, significantly downregulating FoxO1 ( P < 0.001), AdipoR1( P < 0.001) and AdipoR2( P < 0.001). Consistent with the hippocampal findings, similar reductions in FoxO1, AdipoR1, and AdipoR2 expression were observed in the PC of both T2DM and miR-144-overexpressing mice when compared with controls (FoxO1: P < 0.01, AdipoR1: P < 0.001, AdipoR2: P < 0.001). These findings indicate that miR-144 overexpression suppresses FoxO1/AdipoR signaling, thereby mimicking the molecular alterations associated with T2DM-related cognitive impairment. miR-144 overexpression and T2DM activate the NLRP3 inflammasome and downstream inflammatory cascades. Given the close association between metabolic dysfunction, neuroinflammation, and cognitive decline, we next investigated whether miR-144 regulates neuroinflammatory responses via the NLRP3 inflammasome. Western blot analysis revealed that the protein levels of NLRP3, the adaptor protein ASC, and cleaved caspase-1 were significantly elevated in both the hippocampus and PC of T2DM mice (Fig. 4 G-I). Specifically, NLRP3 expression was increased in the hippocampus ( P < 0.05) and PC ( P < 0.05), accompanied by marked upregulation of ASC (hippocampus: P < 0.001; PC: P < 0.01) and cleaved caspase-1 (hippocampus: P < 0.001; PC: P < 0.05). Correspondingly, the downstream pro-inflammatory cytokines IL-1β and IL-18 were significantly increased in both regions (IL-1β: hippocampus P < 0.001, PC P < 0.01; IL-18: hippocampus P < 0.001, PC P < 0.05). Importantly, overexpression of miR-144 recapitulated the inflammatory phenotype observed in T2DM mice. miR-144 upregulation significantly increased the protein levels of NLRP3 (hippocampus: P < 0.05; PC: P < 0.05), ASC (hippocampus: P < 0.001; PC: P < 0.01), and cleaved caspase-1 (hippocampus: P < 0.001; PC: P < 0.05), along with corresponding elevations in IL-1β and IL-18 in both the hippocampus and PC. Collectively, these findings demonstrate that miR-144 overexpression mimics the T2DM state by activating the NLRP3 inflammasome and amplifying downstream neuroinflammatory responses. Knockdown of miR-144 ameliorates cognitive dysfunction in T2DM mice. In the NOR test (Fig. 5 A-B), DI was significantly reduced in the T2DM + siRNA group compared with the CON + siRNA group ( P < 0.05), indicating impaired recognition memory. Knockdown of miR-144 significantly increased the DI in T2DM mice ( P 0.05), suggesting that miR-144 suppression does not affect baseline cognitive performance. In the MWM training phase, escape latency progressively decreased from day 1 to day 5 in all experimental groups, with T2DM + siRNA group exhibiting a slower rate of decline (Fig. 5 C). On the fifth day of the training period, the T2DM + siRNA group showed longer escape latency compared with the CON + siRNA group ( P < 0.05). The escape latency of T2DM + miR-144(−) mice began to decrease steadily from the third day onward, with a progressively widening gap compared to T2DM + siRNA mice, culminating in a statistically significant difference by the fifth day( P < 0.05). During the exploration period (Fig. 5 D-G), compared with the control group, T2DM mice exhibited significant reductions in both the distance traveled in the target quadrant ( P < 0.01) and the percentage of time spent in that quadrant ( P < 0.05). These changes were reversed by knockdown of miR-144 ( P < 0.05, P 0.05). Knocking down miR-144 did not markedly improve this reduction( P > 0.05). Consistent with the NOR results, miR-144 knockdown alone did not significantly alter spatial learning or memory performance ( P > 0.05). Collectively, these findings indicate that miR-144 knockdown selectively ameliorates T2DM-associated cognitive deficits without affecting normal cognitive function. Knockdown of miR-144 ameliorates synaptic impairment in T2DM mice. Western blot analysis demonstrated that the expression levels of synaptic proteins PSD95 and Synapsin I were significantly reduced in the hippocampus of T2DM mice compared with controls (PSD95: P < 0.001; Synapsin I: P < 0.001) (Figure S3A–F). Knockdown of miR-144 significantly restored the expression of these synaptic proteins in T2DM mice (PSD95: P < 0.05; Synapsin I: P < 0.05). Similar reductions in PSD95 and Synapsin I were observed in the PC of T2DM mice ( P < 0.001 for both), and miR-144 knockdown significantly increased their expression levels (PSD95: P < 0.001; Synapsin I: P 0.05). Immunofluorescence staining further confirmed these findings. In T2DM mice, PSD95 and Synapsin I expression was significantly decreased in hippocampal subregions, including CA1 (PSD95: P < 0.001; Synapsin I: P < 0.01), CA3 (PSD95: P < 0.01; Synapsin I: P < 0.01), DG (PSD95: P < 0.05; Synapsin I: P < 0.05), as well as in the PC (PSD95: P < 0.05; Synapsin I: P < 0.01) (Figure S3G–I; Fig. 6 A–C). Knockdown of miR-144 significantly increased the expression of both synaptic proteins across these regions, including CA1, CA3, DG, and PC, whereas miR-144 knockdown alone did not induce significant changes ( P > 0.05). Ultrastructural analysis revealed that synapse number was significantly reduced in both the hippocampus ( P < 0.05) and PC ( P < 0.01) of T2DM mice compared with CON + siRNA mice (Fig. 7A, C). These reductions were significantly reversed by miR-144 knockdown (hippocampus: P < 0.05; PC: P < 0.05). Consistently, Golgi staining showed a marked decrease in dendritic spine density in the hippocampal CA1 ( P < 0.001), CA3 ( P < 0.01), DG ( P < 0.001), and PC ( P < 0.01) of T2DM mice compared with controls (Fig. 7B, D). Knockdown of miR-144 significantly increased dendritic spine density across all examined regions (CA1: P < 0.05; CA3: P < 0.001; DG: P < 0.05; PC: P 0.05). Figure 7 Knockdown of miR-144 increases the number of synapses and dendritic spine density in T2DM mice. A Representative electron microscope images of the number of synapses in the hippocampus and PC (Scale bar = 1µm). B Representative Golgi staining images of the hippocampal regions and PC (Scale bar = 5µm). C The number of synapses in the hippocampus and PC. D Relative dendritic spine density in each region of the hippocampus and PC. (n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. Control + siRNA; # P < 0.05, ## P < 0.01, ### P < 0.001 vs. T2DM + siRNA) Knockdown of miR-144 ameliorates the synaptic transmission dysfunction of T2DM mice. In T2DM mice (Fig. 8 A-H), the amplitude of mEPSCs showed no significant difference compared with control mice ( P > 0.05), whereas the frequency of mEPSCs was significantly reduced ( P < 0.05), indicating impaired excitatory synaptic transmission. Knockdown of miR-144 significantly restored the decreased mEPSC frequency ( P 0.05), but the frequency of mIPSC was markedly increased ( P < 0.001). miR-144 knockdown effectively reversed this increase in mIPSC frequency ( P 0.05). Furthermore, the AMPAR/NMDAR ratio of T2DM mice was significantly lower than that in control mice ( P < 0.01), reflecting disrupted excitatory synaptic balance. miR-144 knockdown significantly increased the AMPAR/NMDAR ratio ( P < 0.01), thereby reversing the synaptic transmission deficits induced by T2DM (Fig. 8 I and J). Knockdown of miR-144 alleviates Tau hyperphosphorylation in T2DM mice. Immunofluorescence staining showed that total Tau expression did not differ significantly among the experimental groups ( P > 0.05), and the level of phosphorylated Tau at Ser396 (p-Tau396) was significantly elevated in the hippocampus of T2DM mice (Fig. 9 A-C; Figure S4A-C). Specifically, p-Tau396 levels were markedly increased in the CA1 and CA3 regions of the hippocampus ( P < 0.01), the DG ( P < 0.001), and the PC ( P < 0.001). Importantly, knockdown of miR-144 significantly reduced Tau phosphorylation levels in these brain regions (CA1, DG, PC: P < 0.001; CA3: P 0.05). Knockdown of miR-144 exerts neuroprotective effects in T2DM by activating the FoxO1/AdipoR pathway. The effects of miR-144 knockdown on the expression of FoxO1 and adiponectin receptors were assessed by Western blot analysis. In the hippocampus of T2DM mice, the protein expression levels of FoxO1, AdipoR1, and AdipoR2 were significantly reduced compared with those in control mice (all P < 0.001). Notably, knockdown of miR-144 markedly restored the expression of FoxO1 ( P < 0.01), AdipoR1 ( P < 0.05), and AdipoR2 ( P < 0.001) (Fig. 9 D–G). Consistent with the hippocampal findings, similar expression patterns were observed in the PC (Fig. 9 H–K). The protein levels of FoxO1, AdipoR1, and AdipoR2 were significantly decreased in T2DM mice (all P < 0.001), whereas miR-144 knockdown significantly reversed these alterations, restoring the expression of FoxO1 ( P < 0.001), AdipoR1 ( P < 0.001), and AdipoR2 ( P 0.05). Knockdown of miR-144 suppresses NLRP3 inflammasome activation and downstream inflammatory responses in T2DM mice. The expression levels of key components of the NLRP3 inflammasome—including NLRP3 (hippocampus: P < 0.001; PC: P < 0.001), ASC (hippocampus: P < 0.001; PC: P < 0.001), and cleaved caspase-1 (hippocampus: P < 0.001; PC: P < 0.001)—were significantly elevated in T2DM mice. Correspondingly, the downstream proinflammatory cytokines IL-1β (hippocampus: P < 0.001; PC: P < 0.001) and IL-18 (hippocampus: P < 0.001; PC: P < 0.001) were also markedly increased (Fig. 10 ). Following miR-144 knockdown, the activation of the NLRP3 inflammasome was significantly attenuated, as evidenced by reduced expression of NLRP3 (hippocampus: P < 0.01; PC: P < 0.01), ASC (hippocampus: P < 0.001; PC: P < 0.001), and cleaved caspase-1 (hippocampus: P < 0.001; PC: P < 0.001). In parallel, the expression levels of IL-1β (hippocampus: P < 0.01; PC: P < 0.001) and IL-18 (hippocampus: P < 0.001; PC: P 0.05). Collectively, these findings demonstrate that miR-144 knockdown effectively suppresses NLRP3 inflammasome activation and its downstream inflammatory signaling, thereby exerting potent anti-inflammatory and neuroprotective effects in T2DM mice. Discussion T2DM accounts for approximately 90% of the 537 million diabetes cases worldwide, among which cognitive impairment represents a severe and increasingly recognized complication. Cognitive impairment in T2DM patients substantially impairs quality of life and imposes a significant burden on global healthcare systems[ 25 , 26 ]. Therefore, elucidating the pathogenic mechanisms underlying T2DM-related cognitive impairment and identifying effective therapeutic targets have become major priorities in both basic and clinical research. Accumulating evidence indicates that chronic neuroinflammation, synaptic damage, deposition of Aβ, and hyperphosphorylation of Tau, which are mediated by microglial activation and polarization, collectively constitute the core pathological mechanisms underlying T2DM-related cognitive impairment[ 2 ]. Our previous studies further demonstrated that these pathological processes are closely linked to alterations in hippocampal synaptic structure and function[ 27 ]. To further clarify the underlying mechanisms, we established a T2DM mouse model induced by high-fat diet combined with streptozotocin injection and selectively manipulated miR-144 expression in hippocampal microglia. We systematically evaluated the effects of miR-144 on synaptic integrity and neuroinflammatory responses in both the hippocampus and PC. Our results demonstrated that miR-144 overexpression recapitulated T2DM-like cognitive dysfunction, whereas targeted knockdown of miR-144 significantly ameliorated cognitive impairment in T2DM mice. These findings identify miR-144 as a critical regulator of synaptic structural and functional integrity and suggest that its pathological effects are mediated through NLRP3-dependent neuroinflammation regulated by the miR-144/FoxO1/AdipoR signaling axis. The interconnection between T2DM and neurodegenerative diseases is underscored by shared pathogenic mechanisms, including mitochondrial dysfunction, oxidative stress, and chronic inflammation[ 28 ]. Insulin resistance plays a central role in T2DM by impairing glucose utilization through inhibition of key signaling pathways, such as AMPK/PGC-1α and PI3K/Akt/FoxO1[ 29 , 30 ]. Importantly, reduced PI3K activity in the central nervous system of T2DM patients may lead to hyperphosphorylation of tau, thereby contributing to cognitive impairment[ 31 ]. Previous studies have confirmed that AdipoRon promotes AMPK expression and improves the mobility and cognitive function of T2DM mice[ 15 , 16 ]. Adiponectin has also been reported to inhibit Tau hyperphosphorylation via the PI3K/Akt/GSK-3β pathway[ 32 ]. Consistent with these findings, our behavioral and immunofluorescence analyses confirmed the presence of cognitive deficits and Tau hyperphosphorylation in T2DM mice. Mechanistically, miR-144 overexpression suppressed the FoxO1/AdipoR pathway, thereby inducing T2DM-like cognitive impairment, whereas miR-144 knockdown restored FoxO1 and AdipoR signaling and reversed these pathological alterations. These results align with previously reported neuroprotective effects mediated by AdipoR-AMPK and PI3K/Akt pathways. Synaptic plasticity plays a fundamental role in regulating cognitive function by enabling dynamic changes in synaptic molecular composition, structure, and electrophysiological properties, such as synapse formation, elimination, and functional remodeling[ 33 ]. This mechanism is particularly evident in brain regions critical for learning and memory, such as the hippocampus and prefrontal cortex, where alterations in synaptic structure, number, and function are closely linked to cognitive impairment[ 34 ]. At the molecular level, PSD95, a postsynaptic scaffold protein located within the postsynaptic density, is essential for maintaining synaptic function by regulating synaptic strength, signal transduction, and neuronal survival. Synapsin I, expressed specifically at presynaptic terminals, regulates synaptic vesicle transport, anchoring, and release through phosphorylation-dependent mechanisms[ 35 , 36 ]. In the present study, T2DM mice and miR-144-overexpressing mice exhibited marked reductions in dendritic spine density and synapse number, accompanied by abnormal expression of PSD95 and Synapsin I. Notably, miR-144 knockdown effectively reversed these synaptic structural deficits and normalized synaptic protein expression, ultimately leading to improved cognitive performance. Given the critical role of synaptic transmission in cognitive impairment, we further assessed synaptic efficacy using whole-cell patch-clamp recordings in the hippocampus. NMDARs are key excitatory neurotransmitter receptors in the central nervous system and are critically involved in synaptic transmission, plasticity, and neurodevelopment. AMPARs are the primary ionotropic glutamate receptors on the postsynaptic membrane and are responsible for mediating fast excitatory synaptic transmission in the central nervous system. Following glutamate binding, AMPARs undergo conformational changes that lead to the opening of their ion channels and permit an influx of Na⁺ into postsynaptic neurons. This influx generates an EPSC, which subsequently facilitates the activation of NMDARs[ 37 ]. Studies have confirmed that the overexpression of the NLRP3 inflammasome in the hippocampus induces neuroinflammation, which leads to neuronal damage and subsequent synaptic dysfunction. Specifically, these effects may manifest as follows: inflammatory responses suppress AMPAR expression, decrease the density of AMPAR within synapses, and enhance receptor desensitization. Meanwhile, extrasynaptic NMDAR signaling remains highly active. Ultimately, this imbalance ultimately elevates the AMPAR/NMDAR ratio, disrupts synaptic signaling homeostasis, and consequently contributes to cognitive impairment[ 38 , 39 ]. Wang et al. found that the frequency of mEPSC and mIPSC in pyramidal neurons in CA1 of rat brain was significantly reduced, with no significant change in amplitude[ 40 ]. Moreover, Jiang et al.'s research showed that under high-glucose conditions, the inter-event interval of mEPSCs in hippocampal dentate gyrus granule cells decreased, while the amplitude remains unaltered[ 41 ]. Our results are consistent with existing findings: the hyperglycemic environment in T2DM impairs synaptic transmission, as reflected by reduced frequencies of mEPSCs and mIPSCs in hippocampal CA1 pyramidal neurons, as well as weakened AMPAR/NMDAR-mediated synaptic currents. Modulating miR-144 expression produced similar electrophysiological abnormalities and led to dysfunction of synaptic transmission. Conversely, knockdown of miR-144 restored synaptic plasticity and reversed hyperglycemia-induced synaptic dysfunction. These findings provide new experimental evidence underlying the synaptic injury mechanism in T2DM-related cognitive impairment. FoxO1, a member of the Forkhead box O transcription factor family, plays a crucial role in glucose metabolism and energy homeostasis. Activation of FoxO1 can alleviate diabetes-related symptoms and has emerged as a potential therapeutic target[ 42 ]. Through the PI3K/Akt signaling pathway, FoxO1 inhibits the expression of glycolytic enzymes to regulate glucose homeostasis. Concurrently, it upregulates the expression of specific genes, including APN, AdipoR1, and AdipoR2, thereby modulating energy balance in adipose tissue[ 18 , 43 ]. Previous studies demonstrated that AdipoRon activates the pAMPK/FoxO1 pathway and induces Akt phosphorylation in diabetic cardiomyocytes[ 44 ]. Similarly, our results indicate an activation of the FoxO1/AdipoR pathway in the hippocampus and PC of T2DM models. Furthermore, upregulation of miR-144 expression enhances the expression of FoxO1, AdipoR1, and AdipoR2, whereas its downregulation may suppress the FoxO1/AdipoR pathway and the subsequent inflammatory responses. Neuroinflammation, driven primarily by aberrant microglial activation, is a critical contributor to cognitive dysfunction. As an inflammatory amplifier in microglia, triggering receptor expressed on myeloid cells 1 (TREM1) promotes the release of proinflammatory cytokines, such as IL-1β and IL-18, through activation of the NLRP3 inflammasome, thereby aggravating insulin resistance and cognitive impairment[ 3 , 45 ]. Based on this, targeted regulation of NLRP3 inflammasome activity is considered a potential therapeutic strategy for T2DM and its complications. Emerging evidence highlights the role of miRNAs in fine-tuning NLRP3 signaling. For example, downregulation of miR-138-5p may activate the NLRP3/caspase-1 pathway, leading to hippocampal neuroinflammation and cognitive impairment in rats[ 46 ], and overexpression of miR-223–3p improves cognitive deficits[ 47 ]. Specific miRNA molecules, such as miR-223-3p and miR-133b, have been demonstrated to effectively suppress the activation of the NLRP3 inflammasome. This inhibition can ameliorate abnormal glucose metabolism and its associated pathological processes, highlighting significant therapeutic potential[ 48 , 49 ]. In this study, miR-144 overexpression markedly promoted NLRP3 inflammasome activation and downstream inflammatory cytokine release, whereas miR-144 inhibition effectively suppressed these neuroinflammatory responses in T2DM mice. These findings provide novel mechanistic insights into the regulation of neuroinflammation in T2DM-related cognitive impairment. Several limitations of this study should be acknowledged. Although molecular alterations were analyzed in both the hippocampus and PC, electrophysiological recordings were confined to the hippocampus due to its well-defined architecture and suitability for stable patch-clamp recordings [ 50 ]. Given the consistency of molecular changes between the two regions, future studies will aim to characterize synaptic electrophysiological properties in PC neurons [ 51 ]. Moreover, whether FoxO1 represents the sole or dominant downstream effector of miR-144 remains to be determined. Future investigations involving combined genetic manipulation of miR-144 and FoxO1 will be essential to further elucidate the neuroprotective mechanisms of miR-144. Conclusion In summary, this study elucidates a bidirectional regulatory mechanism mediated by miR-144 in T2DM-related cognitive impairment. We demonstrate that upregulation of miR-144 in hippocampal microglia suppresses the FoxO1/AdipoR signaling pathway in both the hippocampus and PC, thereby activating the NLRP3 inflammasome, promoting Tau hyperphosphorylation, impairing synaptic plasticity, and ultimately inducing cognitive dysfunction. Conversely, targeted knockdown of miR-144 reverses these pathological processes and exerts robust neuroprotective effects. Collectively, these findings identify miR-144 as a promising biomarker and therapeutic target for T2DM-associated cognitive impairment and provide a novel framework for future clinical prediction and intervention strategies. Abbreviations T2DM Type 2 diabetes mellitus Aβ Amyloid-beta miR MicroRNA FoxO1 Fork-head Box O1 ASC Apoptosis-associated spot-like protein AdipoR Adiponectin receptor IL Interleukins AD Alzheimer’ s disease APN Adiponectin AMPK AMP-activated protein kinase PPAR Peroxisome proliferator-activated receptor PI3K Phosphoinositide 3-Kinase STZ Streptozotocin NLRP3 NLR family pyrin domain containing 3 PC Parietal cortex ND Normal diet HFD High-fat diet STZ Streptozotocin FBG Fasting blood glucose AVV Adeno-associated virus NOR Novel object recognition DI Discrimination index MWM Morris water maze RT Room temperature TTX Tetrodotoxin mEPSC Miniature Excitatory Postsynaptic Current mIPSC Miniature Inhibitory Postsynaptic Current NMDARs N-methyl-D-aspartate receptors AMPARs α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors PSD95 Postsynaptic density protein 95 Ser396 Serine 396 Akt Protein kinase B TREM1 Triggering receptor expressed on myeloid cells 1 Declarations Supplementary Information The article contains supplemental figures available at Supplementary Material.docx. 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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-8592557","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":586668881,"identity":"39bc19d2-46d5-4f6e-9686-5482345c2379","order_by":0,"name":"Jinying Zhao","email":"","orcid":"","institution":"Zhongnan Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Jinying","middleName":"","lastName":"Zhao","suffix":""},{"id":586668882,"identity":"e76e55ad-46c9-4228-9c47-a292f666acdd","order_by":1,"name":"Yuliang Zhou","email":"","orcid":"","institution":"Zhongnan Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Yuliang","middleName":"","lastName":"Zhou","suffix":""},{"id":586668883,"identity":"75ebf830-e499-433c-bb37-77c1c9480417","order_by":2,"name":"Shi Cheng","email":"","orcid":"","institution":"Zhongnan Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Shi","middleName":"","lastName":"Cheng","suffix":""},{"id":586668884,"identity":"9a7b2e29-31c9-4e2c-9d94-4030d208acd1","order_by":3,"name":"Jia Shen","email":"","orcid":"","institution":"Zhongnan Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Jia","middleName":"","lastName":"Shen","suffix":""},{"id":586668885,"identity":"fa73230a-61b2-49c9-91dd-2a8db80cf75e","order_by":4,"name":"Yahong Li","email":"","orcid":"","institution":"South-Central Minzu University","correspondingAuthor":false,"prefix":"","firstName":"Yahong","middleName":"","lastName":"Li","suffix":""},{"id":586668886,"identity":"f0b9683e-c30b-4fb9-b3b3-7d9d44abcc6a","order_by":5,"name":"Zhipeng Xu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAs0lEQVRIiWNgGAWjYFACHgaGBB4bBjbStDyQSSNRC+MDm8MkaDA4f/bYg4Sc8/Z80s0PGH5UbCOsRbLhXLpBwpnbiW0yxwwYe87cJqyFn7HHTCKx53YCm0SCATNjGxFa2Jh5gFr+nbNnk0j/QJwWfjaglgSeA4xtEjlE2iLZw2NukMCTnAjUUnCQKL8YnD9j9vAHj529/Iz0jQ9+VBChBQgQkXiAKPUoWkbBKBgFo2AUYAUAKWM1M8YnlEIAAAAASUVORK5CYII=","orcid":"","institution":"Zhongnan Hospital of Wuhan University","correspondingAuthor":true,"prefix":"","firstName":"Zhipeng","middleName":"","lastName":"Xu","suffix":""}],"badges":[],"createdAt":"2026-01-13 13:24:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8592557/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8592557/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102065785,"identity":"5f3f05c1-6fbb-4d50-b397-ec809820f2a5","added_by":"auto","created_at":"2026-02-06 18:11:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":89141,"visible":true,"origin":"","legend":"\u003cp\u003eOverexpression of miR-144 induces T2DM-like recognition, spatial learning and memory dysfunction. \u003cstrong\u003eA\u003c/strong\u003e Experimental flowchart. Brain silhouette, mouse, injection syringes, and food pellets were sourced from https://scidraw.io/ and adapted from (Ann Kennedy, Annie Park, Federico Claudi, Jimmy Dooley, and Izumi Fukunaga) respectively. \u003cstrong\u003eB \u003c/strong\u003eSchematic diagram of the stereotaxic injection of AAV into the mouse bilateral hippocampus and its subsequent diffusion observed by fluorescence microscopy. Injection syringe was sourced from https://scidraw.io/ and adapted from Jimmy Dooley. \u003cstrong\u003eC \u003c/strong\u003eSchematic diagram of the NOR experiment process in mice. Mouse silhouette was sourced from https://scidraw.io/ and adapted from Gil Costa. \u003cstrong\u003eD \u003c/strong\u003eRepresentative trajectory of NOR in mice. \u003cstrong\u003eE \u003c/strong\u003eDI of mice in each group. \u003cstrong\u003eF\u003c/strong\u003e Changes in the escape latency time of mice during training. \u003cstrong\u003eG \u003c/strong\u003eRepresentative trajectory of MWM. \u003cstrong\u003eH \u003c/strong\u003ePercentage of distances of mice in the fourth quadrant during the experimental period. \u003cstrong\u003eI \u003c/strong\u003ePercentage of time when mice were in the fourth quadrant during the experimental period. \u003cstrong\u003eJ \u003c/strong\u003eThe number of times the mice traveled through the platform during the exploration experiment. (n=10. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 vs. Control+siRNA)\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8592557/v1/bfe282464e64765be739a7bb.png"},{"id":102295849,"identity":"7531d723-ecad-491c-a72d-56d602b8ba1a","added_by":"auto","created_at":"2026-02-10 10:15:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":339610,"visible":true,"origin":"","legend":"\u003cp\u003eBy inhibiting synaptic proteins, miR-144 overexpression and T2DM cause synaptic and spine loss. \u003cstrong\u003eA \u003c/strong\u003eRepresentative immunofluorescence images of synaptic proteins in the hippocampus and PC (Scale bar =50μm). \u003cstrong\u003eB-C\u003c/strong\u003eRelative fluorescence intensity of PSD95 and Synapsin I in the DG of the hippocampus and PC. \u003cstrong\u003eD \u003c/strong\u003eRepresentative electron microscope images of the number of synapses in the hippocampus and PC (Scale bar =1μm).\u003cstrong\u003eE-F \u003c/strong\u003eThe number of synapses in the hippocampus and PC. \u003cstrong\u003eG \u003c/strong\u003eRepresentative Golgi staining images of the hippocampal regions and PC (Scale bar =5μm). \u003cstrong\u003eH \u003c/strong\u003eRelative dendritic spine density in each region of the hippocampus and PC. (n=3. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 vs. Control+siRNA)\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8592557/v1/0a16e7504135838916ad75f8.png"},{"id":102404056,"identity":"becb734f-ae7e-411c-81c7-0e1bb9665a4d","added_by":"auto","created_at":"2026-02-11 10:57:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":53885,"visible":true,"origin":"","legend":"\u003cp\u003eOverexpression of miR-144 induces T2DM-like synaptic transmission dysfunction. \u003cstrong\u003eA, E \u003c/strong\u003eRepresentative traces of mEPSCs and mIPSCs recorded from hippocampal CA1 pyramidal neurons in the presence of TTX. \u003cstrong\u003eB, F\u003c/strong\u003e mEPSC and mIPSC amplitudes in each group. \u003cstrong\u003eC, G \u003c/strong\u003eThe frequency of distribution of mEPSC and mIPSC in each group.\u003cstrong\u003e D, H \u003c/strong\u003eCumulative frequency distribution of mEPSC and mIPSC inter-event intervals. The distributions in both the T2DM and miR-144 overexpression groups show a significant shift compared to the control.\u003cstrong\u003e I \u003c/strong\u003eSchematic diagram of AMPAR and NMDAR mediated EPSCs recorded from hippocampal CA1 pyramidal neurons in the presence of picrotoxinin. \u003cstrong\u003eJ \u003c/strong\u003eThe AMPAR/NMDAR-mediated EPSC ratio in each group. (n=3. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 vs. Control+siRNA)\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8592557/v1/f3829c9220271b20bc2ef7c3.png"},{"id":102295663,"identity":"db6634d4-73ba-44f4-8acd-01dd6ec18b5b","added_by":"auto","created_at":"2026-02-10 10:13:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":260598,"visible":true,"origin":"","legend":"\u003cp\u003eHyperphosphorylation of Tau in miR-144 and T2DM mice. miR-144 overexpression and T2DM activate NLRP3 inflammasome pathway. \u003cstrong\u003eA \u003c/strong\u003eRepresentative immunofluorescence images of Tau and p-Tau in the hippocampus and PC (Scale bar =50μm). \u003cstrong\u003eB-C \u003c/strong\u003eRelative fluorescence intensity of Tau and p-Tau in the DG of the hippocampus and PC. \u003cstrong\u003eD\u003c/strong\u003e Representative western blotting of FoxO1 and AdipoR in the hippocampus and PC.\u003cstrong\u003e E-F\u003c/strong\u003e Relative expression levels of FoxO1, AdipoR1, and AdipoR2 in the hippocampus and PC.\u003cstrong\u003e G\u003c/strong\u003e Representative western blotting of NLRP3 inflammasome and downstream inflammatory factors in the hippocampus and PC. \u003cstrong\u003eH-I \u003c/strong\u003eRelative expression levels of NLRP3, ACS, Cleaved Caspase-1, IL-1β, and IL-18 in the hippocampal and PC. (n=3. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 vs. Control+siRNA)\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8592557/v1/42b1227e963ecaf0f6234d3b.png"},{"id":102397238,"identity":"112039d3-625d-431a-a6ae-ee64509c8294","added_by":"auto","created_at":"2026-02-11 10:12:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":86942,"visible":true,"origin":"","legend":"\u003cp\u003eKnockdown of miR-144 improves the recognition, spatial learning, and memory abilities of T2DM mice. \u003cstrong\u003eA \u003c/strong\u003eRepresentative trajectory of NOR in mice (n=10). \u003cstrong\u003eB\u003c/strong\u003e DI in each group. \u003cstrong\u003eC \u003c/strong\u003eChanges in the escape latency time of mice during training. \u003cstrong\u003eD \u003c/strong\u003eRepresentative trajectory of MWM.\u003cstrong\u003e E \u003c/strong\u003ePercentage of distances of mice in the fourth quadrant during the experimental period. \u003cstrong\u003eF \u003c/strong\u003ePercentage of time when mice were in the fourth quadrant during the experimental period. \u003cstrong\u003eG \u003c/strong\u003eThe number of times the mice traveled through the platform during the exploration experiment. (*\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 vs. Control+siRNA; #\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ##\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ###\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 vs. T2DM+siRNA)\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8592557/v1/c526c68bb17e2e05fdfe87d4.png"},{"id":102295756,"identity":"c821a842-d985-4b68-8a63-4bbe79b2fdb1","added_by":"auto","created_at":"2026-02-10 10:14:42","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":172501,"visible":true,"origin":"","legend":"\u003cp\u003eKnockdown of miR-144 improves synaptic protein expression. \u003cstrong\u003eA \u003c/strong\u003eRepresentative immunofluorescence images of synaptic proteins in the hippocampus and PC (Scale bar =50μm, n=3). \u003cstrong\u003eB-C\u003c/strong\u003eRelative fluorescence intensity of PSD95 and SynapsinI in the hippocampus and PC. (*\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 vs. Control+siRNA; #\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ##\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ###\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 vs. T2DM+siRNA)\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8592557/v1/ad26bc8b1d5121a5a15b6a1d.png"},{"id":102295925,"identity":"5902870c-96a7-4a18-b3f2-0f314b4a9326","added_by":"auto","created_at":"2026-02-10 10:16:08","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":331044,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of miR-144 increases the number of synapses and dendritic spine density in T2DM mice. A \u003c/strong\u003eRepresentative electron microscope images of the number of synapses in the hippocampus and PC (Scale bar =1μm). \u003cstrong\u003eB \u003c/strong\u003eRepresentative Golgi staining images of the hippocampal regions and PC (Scale bar =5μm). \u003cstrong\u003eC \u003c/strong\u003eThe number of synapses in the hippocampus and PC. \u003cstrong\u003eD \u003c/strong\u003eRelative dendritic spine density in each region of the hippocampus and PC. (n=3. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 vs. Control+siRNA; #\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ##\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ###\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 vs. T2DM+siRNA)\u003c/p\u003e","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8592557/v1/9075fc5fa83de8e206c9604b.png"},{"id":102295901,"identity":"6a91edd9-b5c7-4de9-8cc1-6ffab8e60bc6","added_by":"auto","created_at":"2026-02-10 10:15:55","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":68064,"visible":true,"origin":"","legend":"\u003cp\u003eKnockdown of miR-144 increases mEPSC frequency and decreases mIPSC frequency in T2DM mice. \u003cstrong\u003eA, E \u003c/strong\u003eRepresentative traces of mEPSCs and mIPSCs recorded from hippocampal CA1 pyramidal neurons in the presence of TTX. \u003cstrong\u003eB, F \u003c/strong\u003emEPSC and mIPSC amplitudes in each group. \u003cstrong\u003eC, G \u003c/strong\u003eThe frequency of distribution of mEPSC and mIPSC in each group. \u003cstrong\u003eD, H \u003c/strong\u003eCumulative frequency distribution of mEPSC and mIPSC inter-event intervals. The distributions in both the T2DM+siRNA and T2DM+miR-144(-) showed a significant shift compared to the control.\u003cstrong\u003e I \u003c/strong\u003eSchematic diagram of AMPAR and NMDAR mediated EPSCs recorded from hippocampal CA1 pyramidal neurons in the presence of picrotoxinin. \u003cstrong\u003eJ \u003c/strong\u003eThe AMPAR/NMDAR-mediated EPSC ratio in each group. (n=3. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 vs. Control+siRNA; #\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ##\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ###\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 vs. T2DM+siRNA)\u003c/p\u003e","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8592557/v1/8dd609e9d713d269ea25ad4a.png"},{"id":102295845,"identity":"8c557541-05bf-4dcb-a867-3c7df882de02","added_by":"auto","created_at":"2026-02-10 10:15:29","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":270953,"visible":true,"origin":"","legend":"\u003cp\u003eKnockdown of miR-144 ameliorates Tau hyperphosphorylation, and affects the expression levels of FoxO1 and AdipoR. \u003cstrong\u003eA \u003c/strong\u003eRepresentative immunofluorescence images of Tau and p-Tau in the hippocampus and PC (Scale bar =50μm).\u003cstrong\u003e B-C \u003c/strong\u003eRelative fluorescence intensity of Tau and p-Tau in the DG of the hippocampus and PC. \u003cstrong\u003eD, H \u003c/strong\u003eRepresentative western blotting of FoxO1 and AdipoR in the hippocampus and PC. \u003cstrong\u003eE-G, I-K\u003c/strong\u003eRelative expression levels of FoxO1, AdipoR1, and AdipoR2 in the hippocampus and PC. (n=3. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 vs. Control+siRNA; #\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ##\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ###\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 vs. T2DM+siRNA)\u003c/p\u003e","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8592557/v1/89327ec06c0770a8c94cbd87.png"},{"id":102295870,"identity":"d8dfb08e-bc51-4097-a6d8-62d0b9db7913","added_by":"auto","created_at":"2026-02-10 10:15:42","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":75733,"visible":true,"origin":"","legend":"\u003cp\u003eKnockdown of miR-144 inhibits the NLRP3 inflammasome and its downstream inflammatory factors in T2DM mice. \u003cstrong\u003eA, G \u003c/strong\u003eRepresentative western blotting of NLRP3 inflammasome and downstream inflammatory factors in the hippocampus and PC. \u003cstrong\u003eB-F \u003c/strong\u003eRelative expression levels of NLRP3, ACS, Cleaved Caspase-1, IL-1β, and IL-18 in the hippocampus.\u003cstrong\u003eH-L \u003c/strong\u003eRelative expression levels of NLRP3, ACS, Cleaved Caspase-1, IL-1β, and IL-18 in the PC. (n=3. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 vs. Control+siRNA; #\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ##\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ###\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 vs. T2DM+siRNA)\u003c/p\u003e","description":"","filename":"Onlinefloatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-8592557/v1/eb21c015d206d68d9c18201b.png"},{"id":102404649,"identity":"6bdf5cd9-233b-4fe7-bc32-3fef04eca8de","added_by":"auto","created_at":"2026-02-11 11:07:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3565403,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8592557/v1/db275705-9449-42b7-b449-5602467426b7.pdf"},{"id":102296094,"identity":"0b8d62fd-e40c-4908-a7d1-2853ad628374","added_by":"auto","created_at":"2026-02-10 10:17:24","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":4475557,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8592557/v1/be02d97a7de2aa327fa60987.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eMiR-144 regulates cognitive dysfunction via NLRP3 inflammasome and FoxO1/AdipoR pathway in T2DM mice\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCognitive impairment is a severe and increasingly recognized complication of type 2 diabetes mellitus (T2DM), imposing a substantial burden on patients and healthcare systems. Epidemiological studies indicate that individuals with T2DM have approximately a 1.5-fold higher risk of developing dementia compared with non-diabetic populations[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The pathogenesis of T2DM-related cognitive impairment involves multiple mechanisms, including chronic hyperglycemia, insulin resistance, cerebrovascular dysfunction, neuroinflammation, disrupted cerebral energy metabolism, and abnormal amyloid-beta (Aβ) accumulation. As the resident immune cells of the central nervous system, microglia play a pivotal role in these pathological processes. Dysregulated microglial activation leads to excessive cytotoxicity, synaptic dysfunction, and sustained neuroinflammatory signaling, ultimately accelerating cognitive decline[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNLR family pyrin domain containing 3 (NLRP3) is a protein complex composed of sensor NLRP3, adaptor apoptosis-associated spot-like protein (ASC) and effector caspase-1. This inflammasome is a central mediator of microglia-driven neuroinflammation and has been implicated in the pathogenesis of numerous inflammatory disorders, including neurodegenerative, cardiovascular, and autoimmune diseases[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In patients with cognitive impairment, NLRP3 expression in microglia is markedly upregulated, facilitating the assembly of ASC and pro\u0026ndash;caspase-1. Subsequent caspase-1 activation promotes the maturation and release of Interleukins (IL)-1β and IL-18, thereby triggering an inflammatory cascade[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Notably, the inhibiting the NLRP3 inflammasome has shown promise in improving cognitive decline caused by T2DM, underscoring NLRP3 as a promising therapeutic target T2DM-related cognitive impairment[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, the upstream regulatory mechanisms governing NLRP3 activation in T2DM-related cognitive impairment remain incompletely understood.\u003c/p\u003e \u003cp\u003eMicroRNA-144 (miR-144) is a small non-coding RNA highly enriched in erythrocytes and exists as a mature duplex comprising the guide strand miR-144-3p and the passenger strand miR-144-5p. Among these, miR-144-3p exhibits greater biological stability and exerts predominant regulatory functions in gene expression[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. MiR-144 has emerged as a potential diagnostic and prognostic biomarker and is frequently dysregulated in cancer and inflammatory diseases, where it modulates pathological processes by directly or indirectly targeting key signaling molecules[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Importantly, miR-144 expression is significantly elevated in the peripheral blood of patients with T2DM[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Parallel increases in miR-144 levels have also been observed in the hippocampus and medial prefrontal cortex in Alzheimer\u0026rsquo;s disease (AD) mouse models[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Functionally, miR-144 has been shown to exacerbate cognitive impairment by promoting Aβ deposition[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Conversely, knockdown of miR-144 has been shown to suppress NLRP3 inflammasome activation and restore cognitive function, suggesting that miR-144 may serve as a critical contributor to T2DM-related cognitive impairment[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAdiponectin (APN) is an endogenous adipokine with well-established protective effects in T2DM. Upon binding to its specific receptors, AdipoR1 and AdipoR2, APN activates downstream AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor-α (PPARα) signaling pathways[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Previous studies have demonstrated that AdipoRon, a synthetic adiponectin receptor agonist, reduces Aβ deposition and improves cognitive performance in APP/PS1 mice through activation of the AdipoR1/AMPK pathway[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Consistently, AdipoRon has been shown to alleviate cognitive deficits and synaptic dysfunction in T2DM mouse models, concomitant with reduced hippocampal Tau hyperphosphorylation[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Furthermore, AdipoRon suppresses the expression of NLRP3, ASC, and IL-1β in the hippocampus and prefrontal cortex, thereby attenuating neuroinflammatory responses[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Although these findings highlight AdipoR signaling as a promising therapeutic avenue for T2DM-related cognitive impairment, the precise regulatory mechanisms governing AdipoR expression and function remain unclear.\u003c/p\u003e \u003cp\u003eForkhead box O1 (FoxO1) is a key transcription factor involved in insulin signaling and the pathogenesis of diabetes and has been shown to positively regulate AdipoR expression[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In ob/ob diabetic mice, the expression of AdipoR is downregulated via the Phosphoinositide 3-Kinase (PI3K)/FoxO1 signaling pathway, resulting in adiponectin resistance[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Notably, miR-144 directly targets FoxO1, thereby attenuating adiponectin-mediated protective effects[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. These findings suggest that miR-144 may contribute to T2DM-related cognitive impairment through suppression of the FoxO1/AdipoR signaling axis.\u003c/p\u003e \u003cp\u003eOur study aims to investigate the pathogenesis of T2DM-related cognitive impairment by focusing on the regulatory role of miR-144 in synaptic plasticity and cognitive function within the hippocampus (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). By integrating microglia\u0026ndash;neuron interactions within the neuroimmune network, we systematically investigated how miR-144 modulates NLRP3 inflammasome activation via the FoxO1/AdipoR signaling pathway. Additionally, given the critical role of the parietal cortex (PC) in higher cognitive function and its extensive neural circuit connections with the hippocampus, we will assess the pathophysiological consistency between these two regions[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Collectively, our findings are expected to provide novel mechanistic insights into T2DM-related cognitive decline and identify potential molecular targets for future therapeutic intervention.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eEight-week-old adult male Cx3cr1-Cre mice (SPF grade) were obtained from Shulaibao (Wuhan) Biotechnology Co., Ltd. All animals were housed under specific pathogen-free conditions in a controlled barrier environment with a stable temperature of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and humidity of 55\u0026thinsp;\u0026plusmn;\u0026thinsp;10%, under a 12-hour light/dark cycle. All mice had free access to food and water. After one week of acclimatization, experimental procedures were initiated.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eT2DM mice model\u003c/h3\u003e\n\u003cp\u003eMice were randomly assigned to either a normal diet (ND) group or a high-fat diet (HFD) group. The ND group received a standard laboratory diet, while the HFD group was fed a diet containing 60% kcal from fat for six weeks. Following the dietary intervention, HFD-fed mice received intraperitoneal injections of streptozotocin (STZ; 40 mg/kg) dissolved in 0.1 mol/L sodium citrate buffer (pH 4.5) once daily for three consecutive days to induce T2DM model. ND-fed mice received an equivalent volume of citrate buffer on the same schedule. Fasting blood glucose (FBG) levels were measured on the fourth day after the initial STZ injection. If FBG did not exceed 11.1 mmol/L, an additional STZ injection (40 mg/kg) was administered. Successful establishment of the T2DM model was confirmed when FBG levels remained above 11.1 mmol/L for at least one week.\u003c/p\u003e\n\u003ch3\u003eIntracerebral stereotactic injection\u003c/h3\u003e\n\u003cp\u003eAfter successful induction of the T2DM model, mice underwent stereotaxic injection of adeno-associated virus (AAV) into the hippocampus. The viral vectors rAVV-SFFV-DIO-EGFP-premmu-miR-144-pA, rAVV-SFFV-DIO-[EGFP-WPRE-4XmicroRNA(mmu-miR-144)]-pA,and the empty control vector rAVV-SFFV-EGFP-WPRs were purchased from Wuhan Shumi Brain Science and Technology Co., LTD. To investigate the effects of miR-144 upregulation and downregulation under diabetic and non-diabetic conditions, mice were divided into 7 groups (n\u0026thinsp;=\u0026thinsp;12 per group; total N\u0026thinsp;=\u0026thinsp;84): two Control\u0026thinsp;+\u0026thinsp;siRNA groups, blank control group receiving control siRNA; two T2DM\u0026thinsp;+\u0026thinsp;siRNA groups, diabetic control group receiving control siRNA; Control\u0026thinsp;+\u0026thinsp;miR-144(+): non-diabetic group with miR-144 overexpression; Control\u0026thinsp;+\u0026thinsp;miR-144 (-): non-diabetic group with miR-144 knockdown; T2DM\u0026thinsp;+\u0026thinsp;miR-144 (-): diabetic group with miR-144 knockdown. Mice were anesthetized via intraperitoneal injection of sodium pentobarbital (1% solution, 50 mg/kg), and then secured on a stereotactic frame. Four small holes were drilled bilaterally above the skull, and AVV was injected using a microsyringe (ML: \u0026plusmn;1.6mm, AP: -1.6mm, DV: 1.5mm; ML: \u0026plusmn;2mm, AP: -1.8mm, DV: 2.0mm, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). AVV was injected at each site with a virus injection dose of 300nL and a rate of 50 nL/min (titer 5\u0026times;10\u003csup\u003e12\u003c/sup\u003evg/mL). Behavioral tests were conducted four weeks after viral injection.\u003c/p\u003e\n\u003ch3\u003eBehavioral tests\u003c/h3\u003e\n\u003cp\u003eNovel object recognition (NOR) test was performed to evaluate learning and memory in mice, which consisted of three phases: habituation, training, and testing. On the first day, mice were allowed to freely explore an empty open-field arena (40 \u0026times; 40 \u0026times; 40 cm) for 5 min. On the following day, two identical objects were placed symmetrically in the arena, and mice were allowed to explore for 5 min. After a 24h interval, one familiar object was replaced with a novel object of similar size and material but a different shape. Mice were allowed to explore freely for 5 min. Exploration time was recorded using a video tracking system. The discrimination index (DI) was calculated as follows: DI= [Time spent exploring the novel object / (Time spent exploring the novel object\u0026thinsp;+\u0026thinsp;Time spent exploring the familiar object)] \u0026times;100%. The arena and objects were cleaned with 75% ethanol between trials to eliminate olfactory cues.\u003c/p\u003e \u003cp\u003eSpatial learning and memory were evaluated using the Morris water maze (MWM) test. The experiment consisted of five days of training followed by a probe trial on day 6. A circular pool was divided into four quadrants, with a hidden platform (10 cm diameter) submerged 1 cm below the water surface in the target quadrant. The water was rendered opaque by the addition of titanium dioxide to ensure the platform was not visible. During training, mice underwent four trials per day from different start positions. Escape latency was recorded, with a maximum trial duration of 60 s. If it failed to find the platform within the period, mice were guided to the platform and allowed to remain there for 10s to reinforce memory. On the sixth day, a probe trial was conducted with the platform was removed, and mice were allowed to swim freely for 60 s. Platform crossings, time spent in the target quadrant, and percentage of distance traveled in the target quadrant were analyzed.\u003c/p\u003e\n\u003ch3\u003eGolgi staining\u003c/h3\u003e\n\u003cp\u003eGolgi staining was performed using the Hito Golgi box OptimStain\u0026trade; Prekit (Hitobiotec Corp., USA). Mice were anesthetized via intraperitoneal injection of 1% pentobarbital sodium (50 mg/kg) and subsequently decapitated for brain extraction. Brains immersed in impregnation solution (A : B\u0026thinsp;=\u0026thinsp;1 : 1) at room temperature in the dark for 14 days, followed by incubation in solution C at 4\u0026deg;C for 2 days. Coronal sections (60\u0026micro;m thick) were prepared using a vibratome, mounted on glass slides, and subjected to staining, dehydration, and sealing. After scanning and collecting the images, dendritic spine length and number in segments were quantified using Fiji software, and density of dendritic spines was calculated.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTransmission electron microscopy\u003c/h2\u003e \u003cp\u003eHippocampal and parietal cortex tissues were cut into approximately 1mm\u0026sup3; samples, fixed in 2.5% glutaraldehyde at 4\u0026deg;C for 4 h, and rinsed with PBS. After fixation with osmium acid, samples were dehydrated using gradient ethanol and embedded in epoxy resin. The tissue blocks were placed on the microtome for slicing with a thickness of 60nm. Ultrastructural (Talos L120C, Germany) observations were performed using a transmission electron microscope.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWestern blotting\u003c/h3\u003e\n\u003cp\u003eHippocampal and parietal cortex tissues were homogenized in ice-cold lysis buffer and centrifuged at 12,000 g for 5 min at 4\u0026deg;C. Protein concentration was determined using a BCA assay. Subsequently, the samples were then separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 3% nonfat dried milk for 1h at RT and incubated with primary antibodies overnight at 4\u0026deg;C, followed by secondary antibodies. Signals were detected using ECL and quantified with AlphaEaseFC software. Primary antibodies included: ADIPOR1 (Abcam, ab70362,1:2000), ADIPOR2 (Thermofisher, PA5-100376, 1:1000), FOXO1 (CST, #2880, 1:1000), Synapsin I (Proteintech, 20258-1-AP, 1:2000), PSD95(CST, #2507,1:1000), and β-Actin (Tiandeyue, TDY05, 1:10000).\u003c/p\u003e\n\u003ch3\u003eImmunofluorescent staining\u003c/h3\u003e\n\u003cp\u003eBrain sections were fixed, cryoprotected, embedded in paraffin. After blocking, sections were incubated with primary antibodies overnight at 4\u0026deg;C, followed by fluorescent secondary antibodies at RT for 1h. Cell nuclei were counterstained with DAPI at RT in the dark, and images were captured using fluorescence microscopy. For the negative control, the primary antibody was omitted from the blocking buffer.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eElectrophysiology\u003c/h2\u003e \u003cp\u003eAcute hippocampal slices (300 \u0026micro;m) were prepared in ice-cold oxygenated cutting solution. Slices were immediately incubated in artificial cerebrospinal fluid at 34 \u0026deg; C for 30min, and then equilibrated at RT for another 30min. For electrophysiological recordings, whole-cell patch-clamp recordings were obtained from CA1 pyramidal neurons in mouse hippocampal slices. Action potentials were blocked by adding 1\u0026micro;M tetrodotoxin (TTX) to the extracellular solution. Miniature excitatory postsynaptic current (mEPSC) and miniature inhibitory postsynaptic current (mIPSC) were recorded at holding potentials of \u0026minus;\u0026thinsp;70 mV and +\u0026thinsp;10 mV, respectively. To isolate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR)-EPSCs and N-methyl-D-aspartate receptors (NMDAR)-EPSCs, 50\u0026micro;M picrotoxinin was applied to inhibit GABAergic transmission. These responses were recorded at holding potentials of \u0026minus;\u0026thinsp;70 mV and +\u0026thinsp;40 mV, respectively. All experiments were performed at constant temperature of (30\u0026thinsp;\u0026plusmn;\u0026thinsp;1) \u0026deg;C. Current signals were recorded in voltage-clamp mode using an Axon 700B amplifier and filtered through a 3 kHz filter. Data analysis used Clampfit 11.2 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Statistical analyses were performed using IBM SPSS Statistics 27.0 and GraphPad Prism 10.0. One-way or two-way ANOVA followed by Tukey\u0026rsquo;s post hoc test was applied as appropriate. A value of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eOverexpression of miR-144 induces T2DM-like cognitive impairment.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn the NOR test (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-E), DI was significantly reduced in T2DM mice compared with the CON\u0026thinsp;+\u0026thinsp;siRNA group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Notably, mice with miR-144 overexpression also exhibited a marked decrease in DI, reaching a level comparable to that observed in the T2DM\u0026thinsp;+\u0026thinsp;siRNA group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating impaired recognition memory induced by miR-144 upregulation. In the MWM training phase, escape latency progressively decreased from day 1 to day 5 across all groups, and the descent rate of T2DM mice was even slower (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). On the fifth day, both the T2DM\u0026thinsp;+\u0026thinsp;siRNA group and the CON\u0026thinsp;+\u0026thinsp;miR-144(+) group showed significantly prolonged escape latencies compared with the CON\u0026thinsp;+\u0026thinsp;siRNA mice. However, the impairment was more severe in the T2DM\u0026thinsp;+\u0026thinsp;siRNA group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) than in the CON\u0026thinsp;+\u0026thinsp;miR-144(+) group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). During the probe trial following platform removal, T2DM mice demonstrated a significant reduction in both the total distance traveled (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and the percentage of time spent (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in the target quadrant compared with CON\u0026thinsp;+\u0026thinsp;siRNA mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG\u0026ndash;J). Similarly, miR-144 overexpression resulted in decreased exploration distance (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and reduced time spent in the target quadrant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Although the number of platform crossings tended to decrease in both groups, the differences did not reach statistical significance (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Collectively, these results indicate that miR-144 overexpression induces cognitive deficits resembling those observed in T2DM.\u003c/p\u003e \u003cp\u003e \u003cb\u003eOverexpression of miR-144 induces T2DM-like synaptic injury.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSynaptic dysfunction is a critical early pathological feature underlying cognitive impairment in T2DM. To investigate whether miR-144-induced cognitive deficits were associated with synaptic alterations, the expression of synapse-related proteins and synaptic structural integrity were examined in the hippocampus and parietal cortex.\u003c/p\u003e \u003cp\u003eWestern blot analysis revealed that, compared with the CON\u0026thinsp;+\u0026thinsp;siRNA group, the expression levels of postsynaptic density protein 95 (PSD95) and Synapsin I were significantly reduced in the hippocampus of both the T2DM\u0026thinsp;+\u0026thinsp;siRNA group (PSD95: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Synapsin I: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and the CON\u0026thinsp;+\u0026thinsp;miR-144(+) group (PSD95: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Synapsin I: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA\u0026ndash;B). Similar reductions were observed in the PC (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA, C), with significant decreases in PSD95 [T2DM\u0026thinsp;+\u0026thinsp;siRNA: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; CON\u0026thinsp;+\u0026thinsp;miR-144(+): \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01] and Synapsin I [T2DM\u0026thinsp;+\u0026thinsp;siRNA: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; CON\u0026thinsp;+\u0026thinsp;miR-144(+): \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001].\u003c/p\u003e \u003cp\u003eConsistent with these findings, immunofluorescence staining demonstrated significantly reduced PSD95 and Synapsin I expression in multiple hippocampal subregions, including CA1(Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD-F) [PSD95:T2DM\u0026thinsp;+\u0026thinsp;siRNA: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; CON\u0026thinsp;+\u0026thinsp;miR-144(+): \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01,Synapsin I: T2DM\u0026thinsp;+\u0026thinsp;siRNA: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; CON\u0026thinsp;+\u0026thinsp;miR-144(+): \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05], CA3 [PSD95: T2DM\u0026thinsp;+\u0026thinsp;siRNA: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; CON\u0026thinsp;+\u0026thinsp;miR-144(+): \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05,Synapsin I:T2DM\u0026thinsp;+\u0026thinsp;siRNA: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; CON\u0026thinsp;+\u0026thinsp;miR-144(+): \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001], DG(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C) [PSD95: T2DM\u0026thinsp;+\u0026thinsp;siRNA: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, CON\u0026thinsp;+\u0026thinsp;miR-144(+): \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Synapsin I: T2DM\u0026thinsp;+\u0026thinsp;siRNA: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; CON\u0026thinsp;+\u0026thinsp;miR-144(+): \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01] and PC [PSD95: T2DM\u0026thinsp;+\u0026thinsp;siRNA: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; CON\u0026thinsp;+\u0026thinsp;miR-144(+): \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01,Synapsin I: T2DM\u0026thinsp;+\u0026thinsp;siRNA:\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; CON\u0026thinsp;+\u0026thinsp;miR-144(+): \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05].\u003c/p\u003e \u003cp\u003eUltrastructural analysis using transmission electron microscopy further revealed a significant reduction in synapse number in the hippocampus (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and PC (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) of T2DM mice compared with CON\u0026thinsp;+\u0026thinsp;siRNA mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD\u0026ndash;F). Importantly, miR-144 overexpression induced comparable decreases in synaptic density in both regions (hippocampus: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; PC: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eDendritic spines, which serve as key structural substrates for synaptic transmission and plasticity[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], were further examined using Golgi staining. Spine density was markedly reduced in the CA1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), CA3 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), DG (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and PC (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) of T2DM mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG\u0026ndash;H). Similarly, miR-144 overexpression resulted in significant decreases in dendritic spine density across all examined regions (CA1: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; CA3: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; DG: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; PC: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). These findings indicate that miR-144 overexpression impairs synaptic structure and plasticity, closely mimicking T2DM-associated synaptic injury.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eOverexpression of miR-144 induces T2DM-like synaptic transmission dysfunction.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMiniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) were recorded from CA1 pyramidal neurons in hippocampal slices in the presence of tetrodotoxin (TTX) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-H). Quantitative analysis revealed no significant differences in the amplitudes of either mEPSCs or mIPSCs between T2DM mice and CON\u0026thinsp;+\u0026thinsp;siRNA controls (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In contrast, the frequency of mEPSCs was significantly reduced in T2DM mice (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while the frequency of mIPSCs was markedly increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Notably, overexpression of miR-144 recapitulated this synaptic transmission phenotype, resulting in a significant decrease in mEPSC frequency (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and a concomitant increase in mIPSC frequency \u003cem\u003e(P\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). These findings suggest that miR-144 overexpression disrupts synaptic transmission primarily through presynaptic mechanisms, leading to excitation\u0026ndash;inhibition imbalance similar to that observed in T2DM.\u003c/p\u003e \u003cp\u003eTo further assess glutamatergic synaptic function, the ratio of AMPA receptor- to NMDA receptor-mediated excitatory postsynaptic currents (AMPAR/NMDAR ratio) was examined in CA1 pyramidal neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI-J), which was a widely used index of synaptic strength and plasticity[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The AMPAR/NMDAR ratio was significantly reduced in T2DM mice compared with control mice (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Importantly, miR-144 overexpression closely mimicked the diabetic phenotype, inducing a similarly significant reduction in the AMPAR/NMDAR ratio (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), further indicating impaired excitatory synaptic transmission.\u003c/p\u003e \u003cp\u003e \u003cb\u003eOverexpression of miR-144 induces T2DM-like Tau hyperphosphorylation.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTau is the most abundant microtubule-binding protein in the central nervous system and plays a key role in the regulation of synaptic plasticity through phosphorylation-dependent regulation. In particular, phosphorylation at serine 396 (Ser396) has been shown to significantly impair its biological functions[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Immunofluorescence staining was performed to evaluate the expression of total Tau and phosphorylated Tau at Ser396 (p-Tau396) in hippocampal subregions and the parietal cortex (PC) of T2DM mice, as well as in response to miR-144 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA; Figure S2A). Quantitative analysis revealed no significant differences in total Tau expression among the experimental groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC; Figure S2C, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In contrast, p-Tau396 levels were significantly elevated in T2DM mice across multiple brain regions, including CA1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), CA3 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Figure S2B), DG (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and PC (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Importantly, overexpression of miR-144 also induced a significant increase in p-Tau396 levels in the same regions (CA1: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, CA3: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, DG: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, PC:\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), closely mirroring the Tau hyperphosphorylation pattern observed in T2DM mice. These results indicate that miR-144 upregulation promotes Tau pathological phosphorylation without altering total Tau expression, thereby contributing to synaptic transmission dysfunction and T2DM-like neurodegenerative changes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eOverexpression of miR-144 suppresses the FoxO1/AdipoR pathway and induces T2DM-like neural injury.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo determine whether miR-144 contributes to T2DM-associated neural injury through modulation of the FoxO1/AdipoR signaling pathway, protein expression levels of FoxO1, AdipoR1, and AdipoR2 were examined by western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-F). Compared with the control group, T2DM mice exhibited a marked reduction in FoxO1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), AdipoR1(\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and AdipoR2(\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) expression in the hippocampus. Notably, miR-144 overexpression produced a comparable suppressive effect, significantly downregulating FoxO1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), AdipoR1(\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and AdipoR2(\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Consistent with the hippocampal findings, similar reductions in FoxO1, AdipoR1, and AdipoR2 expression were observed in the PC of both T2DM and miR-144-overexpressing mice when compared with controls (FoxO1:\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, AdipoR1:\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, AdipoR2:\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). These findings indicate that miR-144 overexpression suppresses FoxO1/AdipoR signaling, thereby mimicking the molecular alterations associated with T2DM-related cognitive impairment.\u003c/p\u003e \u003cp\u003e \u003cb\u003emiR-144 overexpression and T2DM activate the NLRP3 inflammasome and downstream inflammatory cascades.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGiven the close association between metabolic dysfunction, neuroinflammation, and cognitive decline, we next investigated whether miR-144 regulates neuroinflammatory responses via the NLRP3 inflammasome. Western blot analysis revealed that the protein levels of NLRP3, the adaptor protein ASC, and cleaved caspase-1 were significantly elevated in both the hippocampus and PC of T2DM mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG-I). Specifically, NLRP3 expression was increased in the hippocampus (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and PC (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), accompanied by marked upregulation of ASC (hippocampus: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; PC: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and cleaved caspase-1 (hippocampus: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; PC: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Correspondingly, the downstream pro-inflammatory cytokines IL-1β and IL-18 were significantly increased in both regions (IL-1β: hippocampus \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, PC \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; IL-18: hippocampus \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, PC \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Importantly, overexpression of miR-144 recapitulated the inflammatory phenotype observed in T2DM mice. miR-144 upregulation significantly increased the protein levels of NLRP3 (hippocampus: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; PC: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), ASC (hippocampus: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; PC: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and cleaved caspase-1 (hippocampus: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; PC: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), along with corresponding elevations in IL-1β and IL-18 in both the hippocampus and PC. Collectively, these findings demonstrate that miR-144 overexpression mimics the T2DM state by activating the NLRP3 inflammasome and amplifying downstream neuroinflammatory responses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eKnockdown of miR-144 ameliorates cognitive dysfunction in T2DM mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn the NOR test (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B), DI was significantly reduced in the T2DM\u0026thinsp;+\u0026thinsp;siRNA group compared with the CON\u0026thinsp;+\u0026thinsp;siRNA group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating impaired recognition memory. Knockdown of miR-144 significantly increased the DI in T2DM mice (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), reflecting an improvement in cognitive function. Importantly, miR-144 knockdown alone did not induce a significant change in DI compared with control mice (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), suggesting that miR-144 suppression does not affect baseline cognitive performance. In the MWM training phase, escape latency progressively decreased from day 1 to day 5 in all experimental groups, with T2DM\u0026thinsp;+\u0026thinsp;siRNA group exhibiting a slower rate of decline (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). On the fifth day of the training period, the T2DM\u0026thinsp;+\u0026thinsp;siRNA group showed longer escape latency compared with the CON\u0026thinsp;+\u0026thinsp;siRNA group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The escape latency of T2DM\u0026thinsp;+\u0026thinsp;miR-144(\u0026minus;) mice began to decrease steadily from the third day onward, with a progressively widening gap compared to T2DM\u0026thinsp;+\u0026thinsp;siRNA mice, culminating in a statistically significant difference by the fifth day(\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). During the exploration period (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-G), compared with the control group, T2DM mice exhibited significant reductions in both the distance traveled in the target quadrant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and the percentage of time spent in that quadrant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These changes were reversed by knockdown of miR-144 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The number of platform crossings by T2DM mice was reduced compared with the control group, but there was no significant difference(\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Knocking down miR-144 did not markedly improve this reduction(\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Consistent with the NOR results, miR-144 knockdown alone did not significantly alter spatial learning or memory performance (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Collectively, these findings indicate that miR-144 knockdown selectively ameliorates T2DM-associated cognitive deficits without affecting normal cognitive function.\u003c/p\u003e \u003cp\u003e \u003cb\u003eKnockdown of miR-144 ameliorates synaptic impairment in T2DM mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWestern blot analysis demonstrated that the expression levels of synaptic proteins PSD95 and Synapsin I were significantly reduced in the hippocampus of T2DM mice compared with controls (PSD95: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Synapsin I: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Figure S3A\u0026ndash;F). Knockdown of miR-144 significantly restored the expression of these synaptic proteins in T2DM mice (PSD95: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Synapsin I: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Similar reductions in PSD95 and Synapsin I were observed in the PC of T2DM mice (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for both), and miR-144 knockdown significantly increased their expression levels (PSD95: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Synapsin I: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In contrast, miR-144 knockdown alone did not significantly alter synaptic protein expression in either the hippocampus or PC (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eImmunofluorescence staining further confirmed these findings. In T2DM mice, PSD95 and Synapsin I expression was significantly decreased in hippocampal subregions, including CA1 (PSD95: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Synapsin I: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), CA3 (PSD95: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Synapsin I: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), DG (PSD95: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Synapsin I: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), as well as in the PC (PSD95: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Synapsin I: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Figure S3G\u0026ndash;I; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u0026ndash;C). Knockdown of miR-144 significantly increased the expression of both synaptic proteins across these regions, including CA1, CA3, DG, and PC, whereas miR-144 knockdown alone did not induce significant changes (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eUltrastructural analysis revealed that synapse number was significantly reduced in both the hippocampus (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and PC (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) of T2DM mice compared with CON\u0026thinsp;+\u0026thinsp;siRNA mice (Fig.\u0026nbsp;7A, C). These reductions were significantly reversed by miR-144 knockdown (hippocampus: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; PC: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eConsistently, Golgi staining showed a marked decrease in dendritic spine density in the hippocampal CA1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), CA3 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), DG (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and PC (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) of T2DM mice compared with controls (Fig.\u0026nbsp;7B, D). Knockdown of miR-144 significantly increased dendritic spine density across all examined regions (CA1: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; CA3: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; DG: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; PC: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating improved synaptic plasticity. In contrast, miR-144 knockdown alone did not significantly affect dendritic spine density (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;7 Knockdown of miR-144 increases the number of synapses and dendritic spine density in T2DM mice. A\u003c/b\u003e Representative electron microscope images of the number of synapses in the hippocampus and PC (Scale bar =\u0026thinsp;1\u0026micro;m). \u003cb\u003eB\u003c/b\u003e Representative Golgi staining images of the hippocampal regions and PC (Scale bar =\u0026thinsp;5\u0026micro;m). \u003cb\u003eC\u003c/b\u003e The number of synapses in the hippocampus and PC. \u003cb\u003eD\u003c/b\u003e Relative dendritic spine density in each region of the hippocampus and PC. (n\u0026thinsp;=\u0026thinsp;3. *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs. Control\u0026thinsp;+\u0026thinsp;siRNA; #\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ##\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ###\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs. T2DM\u0026thinsp;+\u0026thinsp;siRNA)\u003c/p\u003e \u003cp\u003e \u003cb\u003eKnockdown of miR-144 ameliorates the synaptic transmission dysfunction of T2DM mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn T2DM mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-H), the amplitude of mEPSCs showed no significant difference compared with control mice (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), whereas the frequency of mEPSCs was significantly reduced (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating impaired excitatory synaptic transmission. Knockdown of miR-144 significantly restored the decreased mEPSC frequency (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while miR-144 knockdown alone had no significant effect on mEPSC frequency. Similarly, the amplitude of mIPSCs in T2DM mice did not differ significantly from that in control mice (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), but the frequency of mIPSC was markedly increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). miR-144 knockdown effectively reversed this increase in mIPSC frequency (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), whereas knockdown of miR-144 alone did not alter mIPSC frequency (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Furthermore, the AMPAR/NMDAR ratio of T2DM mice was significantly lower than that in control mice (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), reflecting disrupted excitatory synaptic balance. miR-144 knockdown significantly increased the AMPAR/NMDAR ratio (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), thereby reversing the synaptic transmission deficits induced by T2DM (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eI and J).\u003c/p\u003e \u003cp\u003e \u003cb\u003eKnockdown of miR-144 alleviates Tau hyperphosphorylation in T2DM mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eImmunofluorescence staining showed that total Tau expression did not differ significantly among the experimental groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), and the level of phosphorylated Tau at Ser396 (p-Tau396) was significantly elevated in the hippocampus of T2DM mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003eA-C; Figure S4A-C). Specifically, p-Tau396 levels were markedly increased in the CA1 and CA3 regions of the hippocampus (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), the DG (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and the PC (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Importantly, knockdown of miR-144 significantly reduced Tau phosphorylation levels in these brain regions (CA1, DG, PC: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; CA3: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In contrast, miR-144 knockdown alone did not induce significant changes in p-Tau levels (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eKnockdown of miR-144 exerts neuroprotective effects in T2DM by activating the FoxO1/AdipoR pathway.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe effects of miR-144 knockdown on the expression of FoxO1 and adiponectin receptors were assessed by Western blot analysis. In the hippocampus of T2DM mice, the protein expression levels of FoxO1, AdipoR1, and AdipoR2 were significantly reduced compared with those in control mice (all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Notably, knockdown of miR-144 markedly restored the expression of FoxO1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), AdipoR1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and AdipoR2 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003eD\u0026ndash;G). Consistent with the hippocampal findings, similar expression patterns were observed in the PC (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003eH\u0026ndash;K). The protein levels of FoxO1, AdipoR1, and AdipoR2 were significantly decreased in T2DM mice (all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), whereas miR-144 knockdown significantly reversed these alterations, restoring the expression of FoxO1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), AdipoR1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and AdipoR2 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Importantly, knockdown of miR-144 alone did not significantly affect the basal expression levels of FoxO1, AdipoR1, or AdipoR2 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cb\u003eKnockdown of miR-144 suppresses NLRP3 inflammasome activation and downstream inflammatory responses in T2DM mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe expression levels of key components of the NLRP3 inflammasome\u0026mdash;including NLRP3 (hippocampus: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; PC: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), ASC (hippocampus: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; PC: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and cleaved caspase-1 (hippocampus: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; PC: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001)\u0026mdash;were significantly elevated in T2DM mice. Correspondingly, the downstream proinflammatory cytokines IL-1β (hippocampus: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; PC: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and IL-18 (hippocampus: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; PC: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) were also markedly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e10\u003c/span\u003e). Following miR-144 knockdown, the activation of the NLRP3 inflammasome was significantly attenuated, as evidenced by reduced expression of NLRP3 (hippocampus: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; PC: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), ASC (hippocampus: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; PC: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and cleaved caspase-1 (hippocampus: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; PC: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In parallel, the expression levels of IL-1β (hippocampus: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; PC: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and IL-18 (hippocampus: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; PC: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) were significantly reduced. In contrast, miR-144 knockdown alone did not produce significant effects on inflammasome-related proteins or inflammatory cytokines (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Collectively, these findings demonstrate that miR-144 knockdown effectively suppresses NLRP3 inflammasome activation and its downstream inflammatory signaling, thereby exerting potent anti-inflammatory and neuroprotective effects in T2DM mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eT2DM accounts for approximately 90% of the 537\u0026nbsp;million diabetes cases worldwide, among which cognitive impairment represents a severe and increasingly recognized complication. Cognitive impairment in T2DM patients substantially impairs quality of life and imposes a significant burden on global healthcare systems[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Therefore, elucidating the pathogenic mechanisms underlying T2DM-related cognitive impairment and identifying effective therapeutic targets have become major priorities in both basic and clinical research. Accumulating evidence indicates that chronic neuroinflammation, synaptic damage, deposition of Aβ, and hyperphosphorylation of Tau, which are mediated by microglial activation and polarization, collectively constitute the core pathological mechanisms underlying T2DM-related cognitive impairment[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Our previous studies further demonstrated that these pathological processes are closely linked to alterations in hippocampal synaptic structure and function[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. To further clarify the underlying mechanisms, we established a T2DM mouse model induced by high-fat diet combined with streptozotocin injection and selectively manipulated miR-144 expression in hippocampal microglia. We systematically evaluated the effects of miR-144 on synaptic integrity and neuroinflammatory responses in both the hippocampus and PC. Our results demonstrated that miR-144 overexpression recapitulated T2DM-like cognitive dysfunction, whereas targeted knockdown of miR-144 significantly ameliorated cognitive impairment in T2DM mice. These findings identify miR-144 as a critical regulator of synaptic structural and functional integrity and suggest that its pathological effects are mediated through NLRP3-dependent neuroinflammation regulated by the miR-144/FoxO1/AdipoR signaling axis.\u003c/p\u003e \u003cp\u003eThe interconnection between T2DM and neurodegenerative diseases is underscored by shared pathogenic mechanisms, including mitochondrial dysfunction, oxidative stress, and chronic inflammation[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Insulin resistance plays a central role in T2DM by impairing glucose utilization through inhibition of key signaling pathways, such as AMPK/PGC-1α and PI3K/Akt/FoxO1[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Importantly, reduced PI3K activity in the central nervous system of T2DM patients may lead to hyperphosphorylation of tau, thereby contributing to cognitive impairment[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Previous studies have confirmed that AdipoRon promotes AMPK expression and improves the mobility and cognitive function of T2DM mice[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Adiponectin has also been reported to inhibit Tau hyperphosphorylation via the PI3K/Akt/GSK-3β pathway[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Consistent with these findings, our behavioral and immunofluorescence analyses confirmed the presence of cognitive deficits and Tau hyperphosphorylation in T2DM mice. Mechanistically, miR-144 overexpression suppressed the FoxO1/AdipoR pathway, thereby inducing T2DM-like cognitive impairment, whereas miR-144 knockdown restored FoxO1 and AdipoR signaling and reversed these pathological alterations. These results align with previously reported neuroprotective effects mediated by AdipoR-AMPK and PI3K/Akt pathways.\u003c/p\u003e \u003cp\u003eSynaptic plasticity plays a fundamental role in regulating cognitive function by enabling dynamic changes in synaptic molecular composition, structure, and electrophysiological properties, such as synapse formation, elimination, and functional remodeling[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. This mechanism is particularly evident in brain regions critical for learning and memory, such as the hippocampus and prefrontal cortex, where alterations in synaptic structure, number, and function are closely linked to cognitive impairment[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. At the molecular level, PSD95, a postsynaptic scaffold protein located within the postsynaptic density, is essential for maintaining synaptic function by regulating synaptic strength, signal transduction, and neuronal survival. Synapsin I, expressed specifically at presynaptic terminals, regulates synaptic vesicle transport, anchoring, and release through phosphorylation-dependent mechanisms[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In the present study, T2DM mice and miR-144-overexpressing mice exhibited marked reductions in dendritic spine density and synapse number, accompanied by abnormal expression of PSD95 and Synapsin I. Notably, miR-144 knockdown effectively reversed these synaptic structural deficits and normalized synaptic protein expression, ultimately leading to improved cognitive performance.\u003c/p\u003e \u003cp\u003eGiven the critical role of synaptic transmission in cognitive impairment, we further assessed synaptic efficacy using whole-cell patch-clamp recordings in the hippocampus. NMDARs are key excitatory neurotransmitter receptors in the central nervous system and are critically involved in synaptic transmission, plasticity, and neurodevelopment. AMPARs are the primary ionotropic glutamate receptors on the postsynaptic membrane and are responsible for mediating fast excitatory synaptic transmission in the central nervous system. Following glutamate binding, AMPARs undergo conformational changes that lead to the opening of their ion channels and permit an influx of Na⁺ into postsynaptic neurons. This influx generates an EPSC, which subsequently facilitates the activation of NMDARs[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Studies have confirmed that the overexpression of the NLRP3 inflammasome in the hippocampus induces neuroinflammation, which leads to neuronal damage and subsequent synaptic dysfunction. Specifically, these effects may manifest as follows: inflammatory responses suppress AMPAR expression, decrease the density of AMPAR within synapses, and enhance receptor desensitization. Meanwhile, extrasynaptic NMDAR signaling remains highly active. Ultimately, this imbalance ultimately elevates the AMPAR/NMDAR ratio, disrupts synaptic signaling homeostasis, and consequently contributes to cognitive impairment[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Wang et al. found that the frequency of mEPSC and mIPSC in pyramidal neurons in CA1 of rat brain was significantly reduced, with no significant change in amplitude[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Moreover, Jiang et al.'s research showed that under high-glucose conditions, the inter-event interval of mEPSCs in hippocampal dentate gyrus granule cells decreased, while the amplitude remains unaltered[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Our results are consistent with existing findings: the hyperglycemic environment in T2DM impairs synaptic transmission, as reflected by reduced frequencies of mEPSCs and mIPSCs in hippocampal CA1 pyramidal neurons, as well as weakened AMPAR/NMDAR-mediated synaptic currents. Modulating miR-144 expression produced similar electrophysiological abnormalities and led to dysfunction of synaptic transmission. Conversely, knockdown of miR-144 restored synaptic plasticity and reversed hyperglycemia-induced synaptic dysfunction. These findings provide new experimental evidence underlying the synaptic injury mechanism in T2DM-related cognitive impairment.\u003c/p\u003e \u003cp\u003eFoxO1, a member of the Forkhead box O transcription factor family, plays a crucial role in glucose metabolism and energy homeostasis. Activation of FoxO1 can alleviate diabetes-related symptoms and has emerged as a potential therapeutic target[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Through the PI3K/Akt signaling pathway, FoxO1 inhibits the expression of glycolytic enzymes to regulate glucose homeostasis. Concurrently, it upregulates the expression of specific genes, including APN, AdipoR1, and AdipoR2, thereby modulating energy balance in adipose tissue[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Previous studies demonstrated that AdipoRon activates the pAMPK/FoxO1 pathway and induces Akt phosphorylation in diabetic cardiomyocytes[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Similarly, our results indicate an activation of the FoxO1/AdipoR pathway in the hippocampus and PC of T2DM models. Furthermore, upregulation of miR-144 expression enhances the expression of FoxO1, AdipoR1, and AdipoR2, whereas its downregulation may suppress the FoxO1/AdipoR pathway and the subsequent inflammatory responses.\u003c/p\u003e \u003cp\u003eNeuroinflammation, driven primarily by aberrant microglial activation, is a critical contributor to cognitive dysfunction. As an inflammatory amplifier in microglia, triggering receptor expressed on myeloid cells 1 (TREM1) promotes the release of proinflammatory cytokines, such as IL-1β and IL-18, through activation of the NLRP3 inflammasome, thereby aggravating insulin resistance and cognitive impairment[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Based on this, targeted regulation of NLRP3 inflammasome activity is considered a potential therapeutic strategy for T2DM and its complications. Emerging evidence highlights the role of miRNAs in fine-tuning NLRP3 signaling. For example, downregulation of miR-138-5p may activate the NLRP3/caspase-1 pathway, leading to hippocampal neuroinflammation and cognitive impairment in rats[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], and overexpression of miR-223\u0026ndash;3p improves cognitive deficits[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Specific miRNA molecules, such as miR-223-3p and miR-133b, have been demonstrated to effectively suppress the activation of the NLRP3 inflammasome. This inhibition can ameliorate abnormal glucose metabolism and its associated pathological processes, highlighting significant therapeutic potential[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In this study, miR-144 overexpression markedly promoted NLRP3 inflammasome activation and downstream inflammatory cytokine release, whereas miR-144 inhibition effectively suppressed these neuroinflammatory responses in T2DM mice. These findings provide novel mechanistic insights into the regulation of neuroinflammation in T2DM-related cognitive impairment.\u003c/p\u003e \u003cp\u003eSeveral limitations of this study should be acknowledged. Although molecular alterations were analyzed in both the hippocampus and PC, electrophysiological recordings were confined to the hippocampus due to its well-defined architecture and suitability for stable patch-clamp recordings [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Given the consistency of molecular changes between the two regions, future studies will aim to characterize synaptic electrophysiological properties in PC neurons [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Moreover, whether FoxO1 represents the sole or dominant downstream effector of miR-144 remains to be determined. Future investigations involving combined genetic manipulation of miR-144 and FoxO1 will be essential to further elucidate the neuroprotective mechanisms of miR-144.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, this study elucidates a bidirectional regulatory mechanism mediated by miR-144 in T2DM-related cognitive impairment. We demonstrate that upregulation of miR-144 in hippocampal microglia suppresses the FoxO1/AdipoR signaling pathway in both the hippocampus and PC, thereby activating the NLRP3 inflammasome, promoting Tau hyperphosphorylation, impairing synaptic plasticity, and ultimately inducing cognitive dysfunction. Conversely, targeted knockdown of miR-144 reverses these pathological processes and exerts robust neuroprotective effects. Collectively, these findings identify miR-144 as a promising biomarker and therapeutic target for T2DM-associated cognitive impairment and provide a novel framework for future clinical prediction and intervention strategies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"518\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eT2DM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 426px;\"\u003e\n \u003cp\u003eType 2 diabetes mellitus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eA\u0026beta;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 426px;\"\u003e\n \u003cp\u003eAmyloid-beta\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003emiR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 426px;\"\u003e\n \u003cp\u003eMicroRNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eFoxO1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 426px;\"\u003e\n \u003cp\u003eFork-head Box O1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eASC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 426px;\"\u003e\n \u003cp\u003eApoptosis-associated spot-like protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eAdipoR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 426px;\"\u003e\n \u003cp\u003eAdiponectin receptor\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eIL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 426px;\"\u003e\n \u003cp\u003eInterleukins\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 426px;\"\u003e\n \u003cp\u003eAlzheimer\u0026rsquo; s disease\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eAPN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 426px;\"\u003e\n \u003cp\u003eAdiponectin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eAMPK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 426px;\"\u003e\n \u003cp\u003eAMP-activated protein kinase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003ePPAR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 426px;\"\u003e\n \u003cp\u003ePeroxisome proliferator-activated receptor\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003ePI3K\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 426px;\"\u003e\n \u003cp\u003ePhosphoinositide 3-Kinase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eSTZ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 426px;\"\u003e\n \u003cp\u003eStreptozotocin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eNLRP3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 426px;\"\u003e\n \u003cp\u003eNLR family pyrin domain containing 3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003ePC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 426px;\"\u003e\n \u003cp\u003eParietal cortex\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 426px;\"\u003e\n \u003cp\u003eNormal diet\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eHFD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 426px;\"\u003e\n \u003cp\u003eHigh-fat diet\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eSTZ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 426px;\"\u003e\n \u003cp\u003eStreptozotocin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eFBG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 426px;\"\u003e\n \u003cp\u003eFasting blood glucose\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eAVV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 426px;\"\u003e\n \u003cp\u003eAdeno-associated virus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eNOR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 426px;\"\u003e\n \u003cp\u003eNovel object recognition\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eDI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 426px;\"\u003e\n \u003cp\u003eDiscrimination index\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eMWM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 426px;\"\u003e\n \u003cp\u003eMorris water maze\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eRT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 426px;\"\u003e\n \u003cp\u003eRoom temperature\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eTTX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 426px;\"\u003e\n \u003cp\u003eTetrodotoxin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003emEPSC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 426px;\"\u003e\n \u003cp\u003eMiniature Excitatory Postsynaptic Current\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003emIPSC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 426px;\"\u003e\n \u003cp\u003eMiniature Inhibitory Postsynaptic Current\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eNMDARs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 426px;\"\u003e\n \u003cp\u003eN-methyl-D-aspartate receptors\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eAMPARs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 426px;\"\u003e\n \u003cp\u003e\u0026alpha;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003ePSD95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 426px;\"\u003e\n \u003cp\u003ePostsynaptic density protein 95\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eSer396\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 426px;\"\u003e\n \u003cp\u003eSerine 396\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eAkt\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 426px;\"\u003e\n \u003cp\u003eProtein kinase B\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eTREM1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 426px;\"\u003e\n \u003cp\u003eTriggering receptor expressed on myeloid cells 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe article contains supplemental figures available at Supplementary Material.docx.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cartoon images used in figures were obtained from Scidraw.io.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZ.X. designed the research. J.Z., Y.Z., and S.C. performed the research. J.S. analyzed the data and Y.L., J.Z. and Y.Z. wrote the paper. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (NO. 82271232).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is provided within the manuscript or supplementary information files.\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll the animal experiments were approved by the ethics review committee of Zhongnan Hospital of Wuhan University (ZN2023216).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003evan Sloten TT, Sedaghat S, Carnethon MR, Launer LJ, Stehouwer CDA. 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Adiponectin Attenuates Streptozotocin-Induced Tau Hyperphosphorylation and Cognitive Deficits by Rescuing PI3K/Akt/GSK-3beta Pathway. Neurochem Res. 2018;43(2):316-23.\u003c/li\u003e\n\u003cli\u003eChelini G, Pantazopoulos H, Durning P, Berretta S. The tetrapartite synapse: a key concept in the pathophysiology of schizophrenia. Eur Psychiatry. 2018;50:60-9.\u003c/li\u003e\n\u003cli\u003eWhitlock JR, Sutherland RJ, Witter MP, Moser MB, Moser EI. Navigating from hippocampus to parietal cortex. Proc Natl Acad Sci U S A. 2008;105(39):14755-62.\u003c/li\u003e\n\u003cli\u003eLevy AM, Gomez-Puertas P, Tumer Z. Neurodevelopmental Disorders Associated with PSD-95 and Its Interaction Partners. Int J Mol Sci. 2022;23(8).\u003c/li\u003e\n\u003cli\u003eLonghena F, Faustini G, Brembati V, Pizzi M, Benfenati F, Bellucci A. An updated reappraisal of synapsins: structure, function and role in neurological and psychiatric disorders. 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J Neurosci. 2003;23(7):2961-70.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"type 2 diabetes mellitus, cognitive impairment, synaptic plasticity, biomarkers","lastPublishedDoi":"10.21203/rs.3.rs-8592557/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8592557/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eType 2 diabetes mellitus (T2DM) is closely associated with cognitive impairment, with underlying pathological mechanisms including chronic inflammation, insulin resistance, and neuronal injury. Recent research indicates that microRNA-144 (miR-144) plays a critical role in these processes, though its exact mechanism remains unclear. Given the critical role of microglia in neuroinflammation and synaptic homeostasis, we investigated whether miR-144 mediates T2DM-related cognitive impairment by modulating microglial function through the Fork-head Box O1 (FoxO1)/AdipoR signaling pathway and activation of NLR family pyrin domain containing 3 (NLRP3) inflammasome.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eMicroglia-specific T2DM mouse models were established using Cx3cr1-Cre mice subjected to a high-fat diet combined with low-dose streptozotocin administration. Microglia-selective overexpression or knockdown of miR-144 was achieved via stereotactic hippocampal injection of adeno-associated virus (AAV). Cognitive function was evaluated using the Morris water maze and novel object recognition tests. Synaptic function and plasticity were assessed by electrophysiological recordings (mEPSCs/mIPSCs and AMPAR/NMDAR-EPSCs), ultrastructural analyses (Golgi staining and transmission electron microscopy), and molecular assays including Western blotting and immunofluorescence. Expression levels of synaptic proteins, Tau phosphorylation, FoxO1, AdipoR1/2, NLRP3 inflammasome components, and inflammatory cytokines were systematically analyzed.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eT2DM mice exhibited significant cognitive deficits accompanied by synaptic dysfunction, increased Tau phosphorylation, and enhanced neuroinflammatory responses. Notably, microglial overexpression of miR-144 recapitulated key pathological features of T2DM, including impaired learning and memory, disrupted synaptic transmission, reduced synaptic protein expression, decreased dendritic spine density, and elevated Tau phosphorylation. Additionally, miR-144 overexpression significantly suppressed FoxO1 and AdipoR1/AdipoR2 expression, leading to activation of the NLRP3 inflammasome and subsequent amplification of neuroinflammation. In contrast, microglial knockdown of miR-144 markedly alleviated cognitive impairment, restored synaptic integrity, suppressed Tau hyperphosphorylation, and attenuated neuroinflammatory signaling, thereby exerting robust neuroprotective effects.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study identifies miR-144 as a pivotal regulator of T2DM-related cognitive dysfunction. miR-144 mediates microglial-driven neuroinflammation and synaptic impairment through suppression of the FoxO1/AdipoR signaling pathway and activation of the NLRP3 inflammasome. These findings highlight miR-144 as a potential biomarker and therapeutic target for preventing or treating cognitive impairment associated with T2DM.\u003c/p\u003e","manuscriptTitle":"MiR-144 regulates cognitive dysfunction via NLRP3 inflammasome and FoxO1/AdipoR pathway in T2DM mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-06 18:11:47","doi":"10.21203/rs.3.rs-8592557/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-11T13:02:23+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-11T12:32:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-08T11:14:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"216161593717786803649564832742975710390","date":"2026-02-07T05:04:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"332680011923705447287783325889432759412","date":"2026-02-06T02:22:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"62551794466542790091400166405671892235","date":"2026-02-04T02:02:34+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-04T00:33:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-16T08:52:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-16T08:50:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Neurobiology","date":"2026-01-13T12:56:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c78d1514-9a53-44b5-ac4d-8db719118128","owner":[],"postedDate":"February 6th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-26T10:53:57+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-06 18:11:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8592557","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8592557","identity":"rs-8592557","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}