Harnessing mIR-145 deficiency to modulate inflammation and prevent degeneration in a mouse model of multiple sclerosis

Harnessing mIR-145 deficiency to modulate inflammation and prevent degeneration in a mouse model of multiple sclerosis | 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 Harnessing mIR-145 deficiency to modulate inflammation and prevent degeneration in a mouse model of multiple sclerosis Monique Marylin Alves Almeida, Samantha F. Kornfeld, Yves De Repentigny, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5462410/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Multiple sclerosis (MS) is a progressive inflammatory disease of the central nervous system (CNS) marked by myelin loss, which impairs nerve function. Current therapies fail to halt disease progression or prevent myelin and axonal degeneration. In this study, we explored the impact of miR-145 knockout in a murine model of experimental autoimmune encephalomyelitis (EAE), which mimics MS pathology. Loss of miR-145 reduced clinical severity and significantly decreased immune cell infiltration in the lumbar spinal cord during both the onset and chronic stages of the disease. Additionally, miR-145 loss altered the expression of key inflammatory genes and modulated astrocytic activity throughout EAE. Of significant interest, acute treatment with an antisense oligonucleotide (ASO) targeting miR-145 decreased miR-145 levels and led to reduced disease severity, decreased immune cell infiltration, and an increase in regulatory T cells in EAE mice. Moreover, miR-145 deficiency mitigated axon and myelin degeneration. Our findings suggest that ASOs targeting miR-145 may offer a promising therapeutic strategy, addressing both inflammatory and degenerative components of MS. Myelin oligodendrocytes demyelination neuroinflammation neurodegeneration microRNA Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Background Multiple sclerosis (MS) is the most prevalent immune-mediated inflammatory demyelinating disease of the central nervous system (CNS), significantly impacting society due to its high incidence among young adults( 1 ). The hallmark of MS is the destruction of myelin – a vital lipid-rich sheath that encases and safeguards nerve fibres, which is essential for maintaining axonal health and facilitating effective neurotransmission( 2 , 3 ). Demyelination is driven by myelin-specific, self-reactive T and B lymphocytes that aberrantly infiltrate the CNS and target oligodendrocytes (OLs), the cells responsible for myelin production( 4 ). While recent studies have identified Epstein-Barr virus (EBV) as a risk factor for MS( 5 ), EBV alone is insufficient to trigger the disease, underscoring the multifaceted and multifactorial nature of MS( 5 , 6 ). The classification of MS is broad, with the most recognized categories being relapsing-remitting MS (RRMS), primary progressive MS (PPMS), and secondary progressive MS (SPMS)( 7 ). RRMS is the most common form, characterized by acute relapses, during which immune attacks result in focal inflammatory demyelination and subsequent neurological decline, followed by periods of remission. RRMS patients often experience cumulative disease burden over time, eventually transitioning to SPMS, where remissions are rare, and disability is pronounced. In contrast, PPMS is marked by continuous neurological deterioration from the onset, resulting in a greater overall disease burden despite its slow progression( 7 , 8 ). Current disease-modifying therapies effectively mitigate relapses in RRMS but are less efficient in preventing the transition to SPMS and show limited efficacy in treating established progressive forms of MS( 9 ). Furthermore, while many treatments focus on modulating immune responses to address active inflammation, they do not adequately prevent myelin loss or axonal degeneration, nor do they enhance remyelination – processes that are critical for halting disease progression and promoting regeneration( 10 – 14 ). This underscores a critical gap in therapy and highlights the urgent need for novel, combinatorial approaches that simultaneously target both the inflammatory and degenerative processes of the disease. In previous work, we identified microRNA-145-5p (herein referred to as miR-145) as a negative regulator of OL differentiation in primary OL cultures, where its expression is crucial for maintaining OL precursor cells (OPCs) in a proliferative and undifferentiated state( 15 ). Notably, miR-145 is overexpressed in chronic inactive lesions of progressive MS patients compared to both healthy controls and active lesions from RRMS patients( 16 ). Additionally, our recent findings indicate that miR-145 is particularly abundant in chronic lesions from secondary progressive MS patients( 17 ). In miR-145 knockout mice subjected to chronic cuprizone-induced demyelination, we observed enhanced functional recovery and increased numbers of myelinated axons. Consistent with this, there is a pathological upregulation of miR-145 in wild-type mice exposed to chronic cuprizone treatment( 17 ). Furthermore, miR-145 is elevated in peripheral blood mononuclear cells (PBMCs), serum, and plasma from treatment-naïve RRMS and early SPMS patients( 18 , 19 ). Given the inflammatory nature of MS and the role of peripheral immune cells in disease pathophysiology, further research is necessary to elucidate the beneficial effects of targeting miR-145 in autoimmune-driven inflammatory demyelination. Here, we investigated the impact of miR-145 loss in recovery from experimental autoimmune encephalitis (EAE) in mice. The EAE mouse model of multiple sclerosis closely mimics the immune characteristics of the disease( 20 ). Our results revealed that miR-145-deficient mice exhibited reduced disease burden and decreased immune cell infiltration. This prompted us to assess the therapeutic benefit of an antisense oligonucleotide (ASO) specifically targeting miR-145. We tested whether its administration could improve recovery and prevent degeneration in EAE, while also elucidating the cellular mechanisms by which miR-145 deficiency enhances clinical outcomes. Notably, we observed a significant reduction in clinical severity in the EAE animals, correlated with decreased levels of CD3 + and CD45 + leukocytes. Building on our prior research highlighting the role of miR-145 in OL differentiation( 15 , 17 ), these results suggest a dual function for miR-145 in both modulating inflammatory responses and alleviating degenerative processes in EAE. This dual action positions miR-145 as a promising therapeutic target that addresses both inflammatory and neurodegenerative aspects of MS. Results Loss of miR-145 results in reduced clinical severity throughout the course of EAE We previously demonstrated enhanced remyelination and recovery from cuprizone-induced toxic demyelination using the constitutive miR-145 knockout mouse model ( miR-145 −/− mice)( 17 ). In the cuprizone model, inflammation is restricted to the CNS and leads to OL cell death and subsequent demyelination( 21 ). Thus, this model does not fully depict the pathophysiology of MS, as it lacks the peripheral immune components that are critical to the disease. Given that miR-145 is dysregulated in RRMS and is expressed widely both within and outside the CNS( 18 , 19 ), we employed EAE, a murine model of RRMS, to assess the impact of miR-145 absence on autoimmune-driven demyelination. miR-145 +/+ and miR-145 −/− mice were subjected to EAE induced by myelin oligodendrocyte glycoprotein peptide fragment 35–55 ( MOG 35 − 55 ) antigen. Disease progression was assessed by monitoring clinical scores, which were based on the degree of ascending hind limb paralysis and quantified using a standard clinical score system (0–5 scale)( 22 ), along with body weight changes over the 30-day course of disease (Fig. 1 A). All induced animals developed EAE irrespective of genotype. Notably, the severity of disease was diminished in miR-145 −/− animals, as evidenced by reduced clinical scores and improved weight retention (Fig. 1 B, C). The onset of disease, marked by the first observable clinical symptom, was slightly delayed in miR-145 −/− mice, with perceptible symptoms emerging approximately 1.5 days later (p < 0.0001) than in miR-145 +/+ animals (Fig. 1 D, E). Additionally, miR-145 −/− mice exhibited a reduced mean clinical score at onset (Fig. 1 F, p = 0.0199) and a delayed first instance of paralysis (Fig. 1 G, p 0) to paralysis was similar in miR-145 +/+ and miR-145 −/− animals (Fig. 1 H, p > 0.05). At the peak of the disease, the average maximum clinical score attained was lower in miR-145 −/− animals (Fig. 1 I, p = 0.0043). Furthermore, among animals that experienced hind limb paralysis (score ≥3), the duration of paralysis was significantly shorter in miR-145 −/− mice (Fig. 1 J, p < 0.0001). When analyzing the entire cohort of EAE animals, the proportion that experienced a relapse - defined as more than one instance of paralysis - was 52.7% in miR-145 +/+ mice compared to 17.7% in miR-145 −/− mice. Moreover, only 6.3% of miR-145 +/+ animals did not reach a state of paralysis, compared to 23.1% of miR-145 −/− animals (Fig. 1 K). Lastly, at the chronic stage, miR-145 −/− animals exhibited a lower clinical score than miR-145 +/+ counterparts (Fig. 1 L, p = 0.0066), underscoring the impact of miR-145 deficiency in reducing disease burden in EAE. Myelin retention is improved in miR-145 deficient spinal cord To evaluate the pathological changes associated with the altered clinical course of EAE in miR-145 deficient mice, we assessed myelin content in the lumbar spinal cord – a region primarily affected in EAE( 20 ) – in miR-145 +/+ and miR-145 −/− mice. Tissue samples were collected from naïve animals, as well as at the onset, peak, and chronic stages of EAE. Onset samples were obtained on the day the first clinical symptom was observed, peak samples were taken on the first day animals achieved a clinical score of 3.0 or higher (indicating hind limb paralysis), and chronic samples were collected arbitrarily on day 30 post-induction. Cross-sections of the lumbar spinal cord were immunostained for myelin basic protein (MBP), and the MBP-immunopositive area was quantified as a proportion of the total spinal cord section area (Fig. 2 A-B). No significant differences were noted between naïve miR-145 +/+ and miR-145 −/− animals. As anticipated, miR-145 +/+ animals demonstrated a progressive loss of myelinated area throughout the different phases of EAE. In contrast, consistent with the reduced clinical scores, miR-145 −/− animals retained 1.25-fold more (p = 0.0171) myelinated area over miR-145 +/+ mice at onset, with notable MBP retention in the grey matter (Fig. 2 A). At the chronic stage, miR-145 −/− mice exhibited a trending, yet non-significant, 1.28-fold greater myelinated area (p = 0.0534), with more pronounced MBP retention in the white matter compared to their miR-145 +/+ counterparts (Fig. 2 A-B). However, similar levels of myelin loss were observed at the peak of EAE between the two genotypes, as all animals assessed at this stage were selectively collected while experiencing hind limb paralysis with a clinical score of 3.0 or higher (Fig. 2 B). Loss of miR-145 reduces immune cell infiltration during different phases of EAE with minor alterations in cytokine and chemokine expression in peripheral immune tissues Immune cell infiltration in the lumbar spinal cord is a hallmark of EAE( 20 ), and this was assessed using histological H&E staining. As expected, naïve animals showed no subpial or parenchymal dense nuclei accumulations (Fig. 3 A). At the onset of the disease, miR-145 +/+ animals exhibited significant infiltration characterized by accumulation of dense nuclei, particularly in the ventral horn as well as at the dorsal root-spinal cord junction (Fig. 3 A, B). In contrast, miR-145 −/− mice displayed a 12.70-fold reduction (p = 0.0024) in cell infiltration compared to their miR-145 +/+ counterparts, with infiltration primarily localized to the ventral horn (Fig. 3 A, B). Infiltration was extensive in both genotypes at the peak of EAE, as expected, given that mice were selectively collected while experiencing hindlimb paralysis (Fig. 3 A, C). At the chronic stage, the spinal cords from miR-145 +/+ mice exhibited extensive infiltration, while those from miR-145 −/− mice showed a 2.20-fold reduction in cell infiltration (Fig. 3 A, D, p = 0.0010). These findings align with the improved myelination status observed in miR-145 −/− animals at both the onset and chronic stages of EAE, suggesting that the absence of miR-145 may delay immune cell infiltration into the CNS. We further evaluated the expression of key cytokines and chemokines in the spinal cord across all stages of EAE (Fig. 3 E-J). mRNA expression of several genes was altered at disease onset in miR-145 −/− animals, including significant downregulation of Il1b (Fig. 3 E, 9.62-fold decrease), Ifng (Fig. 3 F, 6.65-fold decrease), Il6 (Fig. 3 G, 6.44-fold decrease), Tnfa (Fig. 3 H, 8.90-fold decrease) and Ccl5 (Fig. 3 J, 30.11-fold decrease), alongside upregulation of Cxcl1 (Fig. 3 I, 2.92-fold increase). Additional changes in gene expression were observed at peak, with Il1b showing a 1.74-fold increase (Fig. 3 E), and during the chronic stage, both Il6 (Fig. 3 G, 72.50-fold decrease) and Ccl5 (Fig. 3 J, 1.80-fold decrease) were downregulated. miR-145 is implicated in peripheral immune responses( 18 , 19 ) and is abundantly expressed in the spleen and thymus (Supp. Figure 1 A). Additionally, miR-145-5p levels were higher in the spinal cord and spleen at the onset of EAE (Supp. Figure 1 B, C), while levels in the thymus remained unaltered throughout the different phases of EAE (Supp. Figure 1 D). Consequently, we investigated whether the reduced immune cell infiltration observed in miR-145 −/− EAE-subjected spinal cords at both the onset and chronic stages could be attributed to alterations in cytokine and chemokine expression, potentially influencing peripheral immune cell activation in EAE. The thymus and spleen (Supp. Figure 2 A-L) were assessed for cytokines and chemokines known to play roles in EAE development in the periphery. Significant downregulations were detected in thymus (Supp. Figure 2 A-F), including reduced expression of Tnfa (Supp. Figure 2 D, 1.45-fold decrease) and Cxcl1 (Supp. Figure 2 E, 1.86-fold decrease, p < 0.0001) at disease onset, as well as reduced expression of Ccl5 in naïve miR-145 −/− animals (Supp. Figure 2 F, 2.28-fold decrease). Conversely, significant upregulation was observed in miR-145 −/− spleen, including Ifng (Supp. Figure 2 H, 1.89-fold increase) at the peak of the disease and Cxcl1 during the chronic stage (Supp. Figure 2 L, 4.33-fold increase). No other significant changes in gene expression were noted in any assessed factors at any time point in either the thymus or spleen. Neuroinflammatory responses are altered in miR-145 deficient animals During autoimmunity-driven inflammatory demyelination, cytokine/chemokine expression reflects not only peripheral immune cell activity but also the responses of glial cells, such as microglia and astrocytes( 23 , 24 ). To determine whether changes in the neuroinflammatory landscape correspond with the improved clinical outcome and differing pathological findings observed in miR-145 −/− animals, we evaluated the activation of microglia and astrocytes. Sections of lumbar spinal cords from miR-145 +/+ and miR-145 −/− mice were stained for Iba1, a marker of microglia/macrophages, at various stages of EAE (Fig. 4 A-E). Total microglia/macrophage numbers were quantified in the parenchyma, excluding submeningeal areas of dense cellular infiltration. Mild alterations in microglial activation were observed during EAE in miR-145 −/− animals compared to miR-145 +/+ (Fig. 4 A-B), with microglial numbers showing a trending yet non-significant reduction at onset (~ 55% reduction, p = 0.123) and during the chronic stage (~ 38% reduction, p = 0.084), along with a trending increase at peak (~ 53% increase, p = 0.084). We further assessed the expression of Ym1, expressed in alternatively activated microglia/macrophages (M2-like cells)( 25 ), iNos, an important pro-inflammatory mediator( 26 ), and Nurr1, an anti-inflammatory factor regulated by miR-145( 27 ). Notably, expression changes were observed across all stages of EAE in Ym1 (Fig. 4 C) and iNos (Fig. 4 D). Ym1 was downregulated at all phases of EAE in miR-145 −/− mice, showing a 61.72-fold decrease at onset, a 2.39-fold decrease at peak, and 6.89-fold decrease at the chronic stage. Conversely, iNos was downregulated at onset (6.05-fold decrease) and chronic phases (17.02-fold decrease) but upregulated at the peak (3.38-fold increase). These findings correlate with the altered numbers of microglia at these time points. Nurr1, a negative regulator of TNFa, was increased 4.21-fold at onset (p < 0.0001), but no significant changes were observed at peak or chronic phases (Fig. 4 E). Astrocytes, which play crucial roles in neuroinflammation( 23 ), were evaluated by immunostaining spinal cord sections with glial fibrillary acid protein (GFAP) antibodies, a well-known astrocyte marker. We quantified both the total numbers of GFAP + astrocytes and the number of hypertrophic reactive astrocytes, defined by GFAP high signal and thickened processes (Fig. 5 A-E). Naïve miR-145 −/− animals showed similar astrocyte numbers and reactivity (Fig. 5 A-B). At both onset (Fig. 5 A, C) and peak (Fig. 5 A, D), miR-145 −/− mice exhibited higher total astrocyte numbers (~ 37% increase, p = 0.049 at onset and ~ 39%, p = 0.038 at peak) and reactive astrocytes (~ 159% increase, p = 0.049 at onset and ~ 187%, p = 0.038 at peak). Interestingly, reactive astrocytes at onset were diffusely distributed throughout the parenchyma in miR-145 +/+ spinal cords, whereas in miR-145 −/− , they were primarily localized to submeningeal areas (Fig. 5 A). In contrast, at the chronic stage (Fig. 5 A, E), miR-145 −/− animals displayed fewer total astrocytes (45% reduction, p = 0.039) and fewer reactive astrocytes (~ 68% reduction, p = 0.039). This suggests that astrocyte-driven inflammation occurs more rapidly and with greater intensity but subsides earlier in miR-145 −/− animals when compared to their wild-type counterparts. To confirm the role of astrocytes in the modulation of inflammation driven by miR-145 deficiency, we assessed the relative expression of GFAP and aquaporin 4 (AQP4), a component of astrocytic end feet that is crucial for maintaining blood-brain barrier integrity and is downregulated when miR-145-5p is overexpressed( 28 , 29 ). We assessed their expression at the mRNA level in the spinal cords of naïve animals as well as throughout EAE. GFAP mRNA expression was altered at EAE onset (5.34-fold increase) and chronic phases (Fig. 5 F, 1.35-fold decrease), correlating with observed changes in astrocyte reactivity at these time points. Surprisingly, AQP4 mRNA expression was downregulated at disease onset (1.33-fold decrease), with no significant changes detected at other time points (Fig. 5 G). Together, these findings indicate broad modifications to the CNS resident immune response resulting from the loss of miR-145, although the underlying mechanisms for these observations remain to be elucidated. Acute miR-145 knockdown with an antisense oligonucleotide (ASO) reduces disease severity To translate our findings into a therapeutic setting, we developed an ASO specifically designed to acutely and transiently inhibit miR-145. WT mice were injected with 20 µg/mouse of custom in vivo miRCURY negative control and miR-145-5p ASOs via subcutaneous injections. Initially, we aimed to determine whether transient antagonism of miR-145 at the peak of EAE would lead to reduced clinical severity later in the disease. A single dose of either control or miR-145 ASOs was administered at the peak of EAE (Supp. Figure 3 A). Given that ASOs do not penetrate the CNS, this timing was chosen to exploit the compromised blood-spinal cord barrier (BSCB) during EAE, thereby enhancing ASO bioavailability within the CNS. Mice were scored daily from day 7 to day 30 post-EAE induction. However, although there was a trend to improved response, treatment with the miR-145 ASO did not result in significant changes in clinical outcomes, as measured by mean clinical score (Supp. Figure 3 B) and percentage weight loss (Supp. Figure 3 C) throughout the course of EAE. Similarly, the mean disease score at the chronic phase was not significantly altered (Supp. Figure 3 D, p = 0.1682), despite observing a 75% (p < 0.0001) reduction in miR-145 levels in the spleen (Supp. Figure 3 E) and a 67% (p < 0.0001) reduction in the spinal cord (Supp. Figure 3 F). In miR-145 −/− animals, a marked reduction in immune cell infiltration was observed at both the onset and chronic phases of EAE (Fig. 3 A-D). To further explore this, we modified our approach by administering ASOs on days 1 and 3 post-EAE induction (Fig. 6 A), aiming to mimic the conditions in miR-145 −/− EAE-subjected animals. This strategy allowed us to evaluate the prophylactic benefits of miR-145 antagonism following myelin antigen immune cell priming and to investigate the cellular mechanisms associated with miR-145 knockdown that may lead to improved clinical outcomes in EAE. Overall, disease severity was notably attenuated in miR-145 ASO-treated animals during the peak of the disease, as indicated by a reduced clinical score at 16 days post-induction (Fig. 6 B, p = 0.0113). However, weight retention did not significantly differ between control and miR-145 ASO-treated groups (Fig. 6 C). The onset of disease was delayed in miR-145 ASO-treated animals, with the first symptoms emerging approximately 2 days earlier in controls compared to miR-145 ASO-treated mice (Fig. 6 D, p = 0.0013). Although the mean clinical score at onset did not significantly differ (Fig. 6 E), the mean clinical score at peak was lower in miR-145 ASO-treated mice (p = 0.0469) (Fig. 6 F). At the chronic phase, miR-145 ASO-treated mice eventually exhibited a disease score comparable to that of their control counterparts (Fig. 6 G), although the average maximum clinical score was only slightly reduced (p = 0.0535) in these animals (Fig. 6 H). miR145 ASO reaches the CNS and efficiently reduces miR-145 levels We further evaluated the efficacy of miR-145 knockdown in lymphoid and spinal cord tissues at the chronic phase of EAE. Administration of the miR-145 ASO resulted in a significant reduction of miR-145-5p levels: an 84% decrease (p < 0.0001) in the spleen (Fig. 6 I), a 62% decrease (p = 0.0050) in the thymus (Fig. 6 J), and a 32% decrease (p = 0.0018) in the spinal cord (Fig. 6 K). Importantly, EAE-subjected mice exhibited no observable signs of toxicity following subcutaneous administration of ASOs. To gain further insights into the distribution and cell-specific targeting of the miR-145 ASO, we developed a custom fluorescent dye attachment (5' FAM labelled) for both control and miR-145 ASOs. A single injection at half the dosage was administered on day 3 post-induction, and tissues were harvested on day 11 (corresponding to EAE disease onset) to assess ASO distribution across various tissues (Supp. Figure 4 A). We harvested and sectioned the liver, spleen, and spinal cord tissues – predicted major sites of ASO accumulation in our study – to evaluate tissue-specific distribution. Tissue exposure times were standardized across conditions based on non-treated controls. Our data revealed more intense fluorescent signal in the liver and spleen, with only mild fluorescence detected in the spinal cord compared to non-treated controls. In the spinal cord, the signal appeared to be more localized within blood vessels (Supp. Figure 4 B). In a secondary approach, we examined the cell-specific accumulation of ASOs administered directly into the CNS. We utilized spinal cord organotypic cultures whereby cultures were kept for 10 days in vitro , followed by a 24 h treatment with FAM-labeled ASOs (Supp. Figure 4 C). Subsequent co-staining with specific markers for various CNS cell types revealed that, although ASO controls exhibited a positive signal, co-localization with CNS cells was lower than compared to miR-145 FAM-labeled ASO-treated animals. This difference may be attributed to the degradation of ASOs by nucleases, as control ASOs are missense oligonucleotides without known binding to microRNAs. Conversely, miR-145-expressing cells displayed more frequent accumulation of the miR-145 FAM-labeled ASO in CNS cells (Supp. Figure 4 D-I). Specific accumulation of ASOs was observed within CC1 + Olig2 + oligodendrocytes (Supp. Figure 4 E), IBA1 + microglia/macrophages (Supp. Figure 4 F), NeuN + neurons (Supp. Figure 4 G), and Nestin + neural precursor cells (Supp. Figure 4 H), with microglia/macrophages demonstrating the highest levels of accumulated miR-145 ASO signal. Notably, no positive signal was detected within GFAP + astrocytes (Supp. Figure 4 I). Collectively, these findings suggest that the miR-145 ASO may also exert effects in CNS cells to promote myelin regeneration. Acute miR-145 knockdown reduces specific immune cell infiltration in the lumbar SC at the peak of EAE To further explore the immune cell landscape in the CNS and its association with improved clinical severity following miR-145 ASO administration, we collected lumbar spinal cord tissue on day 16 post-EAE induction (Fig. 7 A), coinciding with the peak of hindlimb paralysis in most animals. The lumbar spinal cord was stained for various immune cell markers, including clusters of differentiation 3 (CD3), 4 (CD4), 45 (CD45), and 19 (CD19), as well as forkhead box P3 (FoxP3). In miR-145 ASO-treated animals, we observed a ~ 3-fold decrease (p = 0.0018) in the number of CD3 + T cells (Fig. 7 B, C). While no significant differences were noted in the proportion of CD4 + helper T cells (Fig. 7 B, D), the proportion of regulatory FoxP3 + T cells increased by ~ 2-fold (p = 0.0286) in these animals (Fig. 7 B, E). Additionally, there was a 1.6-fold decrease (p = 0.0199) in the number of CD45 + cells of hematopoietic origin (Fig. 7 F, G), while the numbers of CD19 + B cells remained unchanged (Fig. 7 F, H). Collectively, these findings underscore the role of miR-145 in regulating the migration and polarization of peripheral immune cells. Myelin and axon degeneration is attenuated in miR-145 deficient animals We next assessed myelin at the ultrastructural level using transmission electron microscopy (TEM) in miR-145 +/+ , miR-145 −/− , and ASO-treated animals during different phases of EAE. At disease onset (Fig. 8 ), miR-145 −/− mice exhibited no significant differences in myelin thickness compared to miR-145 +/+ (Fig. 8 A-D). In contrast, acute miR-145 ASO knockdown resulted in a significant reduction in g-ratio (Fig. 8 L-N, p < 0.0001), although this did not reach statistical significance when averaged (Fig. 8 O, p = 0.0899). Frequency distribution of g-ratios revealed that both miR-145 −/− (Fig. 8 E) and miR-145 ASO-treated mice (Fig. 8 P) displayed a shift toward a smaller g-ratio (indicating thicker myelin). When g-ratios were averaged and binned into three axon diameter groups, no significant changes were observed for axons up to 2 µm or within the 2–3 µm and > 3 µm diameter categories (Fig. 8 F, Q). The relative abundance of axons across these bins did not change in either group (Fig. 8 G, R), nor did axonal density, although a clear increasing trend was noted (Fig. 8 H, S). Notably, in areas enriched with myelinated axons, there were no changes in the proportion of total myelinated axons (Fig. 8 I, T), axons with uncompacted myelin (Fig. 8 J, U), or those undergoing degeneration (Fig. 8 K, V), all important indicators of myelin health and neurodegeneration( 30 – 32 ). At the peak of the disease (Fig. 9 ), differences emerged, particularly between miR-145 −/− and miR-145 ASO-treated mice. In miR-145 −/− mice, no changes were seen in myelin thickness distribution (Fig. 9 B-C), while the average g-ratio was increased compared to miR-145 +/+ controls, indicating thinner myelin in miR-145 −/− (Fig. 9 D) and ongoing remyelination. Conversely, miR-145 ASO knockdown exhibited a trend towards thicker myelin thickness (p = 0.0717, Fig. 9 M-O). Similarly, frequency distribution of g-ratios indicated that miR-145 −/− mice (Fig. 9 E) showed a distribution toward thinner myelin, whereas miR-145 ASO-treated mice (Fig. 9 P) trended toward thicker myelin. When g-ratios were averaged and binned into three axon diameter groups, no significant changes were observed for axons up to 2 µm or within the 2–3 µm and > 3 µm diameter categories in both miR-145 −/− compared to miR-145 +/+ (Fig. 9 F) and in miR-145 ASO-treated compared to control ASO-treated animals (Fig. 9 Q). In miR-145 −/− , however, a trending increase in g-ratio was observed for axons within the 2–3 µm and > 3 µm diameter axons (p = 0.0684). The relative abundance of axons across these bins did not change (Fig. 9 G, R). miR-145 −/− mice did not show changes in axonal density, whereas miR-145 ASO-treated animals displayed a trend towards increased axon density (Fig. 9 H, S, p = 0.0827). In myelinated axon-rich areas, the proportion of myelinated axons remained unchanged in miR-145 −/− animals (Fig. 9 I), while a slight yet significant increase was observed in miR-145 ASO-treated animals (Fig. 9 T, p = 0.0361). Interestingly, the proportion of axons with uncompacted myelin (Fig. 9 J, U) remained unchanged across both groups, but miR-145 −/− animals displayed a reduced proportion of degenerating axons (Fig. 9 K, p = 0.0011), while no changes were observed in miR-145 ASO-treated animals (Fig. 9 V). At the chronic phase of EAE (Fig. 10 ), axons that remained myelinated in miR-145 +/+ controls trended toward thicker myelin, but no differences were found in g-ratios (distribution, frequency, or average) in miR-145 −/− or miR-145 ASO-treated animals compared to controls (Fig. 10 B-E). A significant increase was noted in the g-ratio (p = 0.0323) for axons up to 2 µm in diameter in miR-145 ASO-treated mice (Fig. 10 F), with no changes found in miR-145 −/− or other diameter bins. The relative abundance of axons across these bins did not change in either group (Fig. 10 G). Notably, a 1.7-fold increase in axonal density was found in miR-145 −/− compared to miR-145 +/+ controls (p = 0.0494), while no statistical significance was observed in miR-145 ASO-treated animals (Fig. 10 H). In areas enriched with myelinated axons, an increased proportion of myelinated axons was found in both miR-145 −/− (p = 0.0062) and miR-145 ASO-treated animals (p = 0.0172). The proportion of axons with uncompacted myelin (Fig. 10 J) was reduced only in miR-145 ASO-treated animals (p = 0.0327). Finally, the proportion of degenerating axons (Fig. 10 K) was reduced by 88% in miR-145 −/− and by 63% in miR-145 ASO-treated animals (p = 0.0101 and p = 0.0425, respectively). Overall, these data highlight that targeting miR-145 may prevent axonal and myelin degeneration, potentially leading to improved clinical outcomes in EAE. Discussion In RRMS, miR-145 has emerged as a significant circulating biomarker, distinguishing between RRMS and SPMS in comparison to healthy controls( 18 , 33 ). Furthermore, its dysregulation is normalized in patients exhibiting successful treatment responses to interferon-beta (IFN-β)( 34 ). However, the role of miR-145 in the pathogenesis and progression of MS has yet to be elucidated. To investigate this, we utilized a miR-145 knockout mouse model in conjunction with the EAE paradigm, a widely accepted mouse model for RRMS. Our findings demonstrate that the absence of miR-145 yields substantial clinical benefits in EAE, characterized by a delayed onset of symptoms and a marked reduction in clinical severity. Notably, a significant proportion of miR-145-deficient animals did not progress to hindlimb paralysis, and those that did exhibited a notably faster recovery. Furthermore, during the chronic phase of EAE, miR-145-deficient mice displayed improved clinical scores, indicating a greater recovery trajectory. The loss of miR-145 significantly impacted immune cell infiltration, leading to notable reductions at both the onset and during the chronic stages of the disease. However, at the peak of the disease, animals exhibited similar levels of infiltration. Research indicates that miR-145 plays dual roles in pro-inflammatory and anti-inflammatory responses across various contexts in the periphery( 35 – 37 ). Additionally, dysregulation of miR-145 has been observed in PBMCs, serum and plasma of individuals with RRMS and early SPMS( 18 , 19 ). In our study, we identified differential expression of miR-145 in the spinal cord and spleen at disease onset. Notably, we found that miR-145 expression in naïve wild-type animals is approximately 25-fold higher in the spleen and about 625-fold higher in the thymus compared to the lumbar spinal cord, indicating that this microRNA may play a crucial role in peripheral lymphoid organs. We also observed that reduced cellular infiltration coincided with decreased expression of several pro-inflammatory cytokines and chemokines, including Ifnγ, Tnfα, Il1β, Il6, and Ccl5 at onset, and Il6 and Ccl5 during the chronic stage. These factors are secreted by infiltrating T cells, monocytes/macrophages, and various CNS cell types( 38 , 39 ). Their diminished expression aligns with the reduction in both infiltrating populations observed with miR-145 loss. This reduction is critical to the inflammatory cascade in EAE, underscoring the potential regulatory role of miR-145 in modulating immune responses. Neuroinflammatory responses, particularly involving microglia and astrocytes, were evident at all stages of EAE in miR-145-deficient mice. In our model, reduced microglial activation was mild at both the onset and chronic stages. Previous studies indicate that microglial activation occurs before the onset of EAE symptoms and the infiltration of immune cells into the CNS( 40 ). This has significant implications for the blood-brain barrier (BBB), as activated microglia can alter the expression of factors critical for maintaining tight junctions among endothelial cells lining the neurovasculature, thus facilitating immune cell entry into the CNS( 41 ). The relationship between alterations in microglial behavior and the loss of miR-145 remains somewhat unclear, as few miR-145-regulated pathways in microglia have been identified to date( 27 , 42 ). One known target of miR-145 in microglia is Nurr1. Previous studies have shown that downregulation of Nurr1 via miR-145 promotes microglial inflammation by negatively regulating TNF-α( 27 ). In our findings, Nurr1 expression was notably upregulated in miR-145 −/− mice, particularly at the onset of EAE, which coincided with TNF-α downregulation in the spinal cord and spleen, as well as with miR-145 upregulation in wild-type mice. This suggests that the Nurr1 pathway may play a role in reducing disease severity in EAE. Additionally, miR-145 has been associated with the polarization of activated microglia/macrophages, promoting a shift towards an anti-inflammatory M2-like phenotype rather than a pro-inflammatory M1-like phenotype, as shown in ischemia models( 43 ). Consequently, we aimed to determine whether the loss of miR-145 leads to diminished pro-inflammatory response in EAE. To explore this, we assessed the expression of the well-known inflammation-associated gene iNos and the anti-inflammatory gene Ym1( 44 ) throughout the course of EAE. Both markers were reduced at the onset and chronic stages in miR-145 −/− mice, likely reflecting an overall decrease in the number of activated microglia /macrophages and cell infiltrates during these time points. At the peak of the disease, iNos was upregulated, while Ym1 remained downregulated in miR-145 −/− mice. This suggests that microglia/macrophage activation during the disease peak favoured an inflammatory phenotype. Nonetheless, recent studies using single-cell RNA sequencing indicate that activated microglia/macrophages represent a far more diverse population than the traditional M1/M2 dichotomy( 45 ). Interpreting innate immune activation solely through this lens may oversimplify the complex heterogeneity present, particularly following demyelination( 46 – 48 ). Astrocyte activation (astrogliosis), characterized by an increased number of astrocytes and morphological changes( 49 , 50 ), was altered in miR-145-deficient animals compared to wild-type throughout the course of EAE. Specifically, astrocyte activation was heightened during the onset and peak stages but decreased in the chronic stage in miR-145-deficient mice. Previous research suggests that early and robust astrocyte activation is beneficial, while late activation can worsen EAE outcomes. In contrast, during the chronic stage, excessive astrocyte activation may be detrimental, whereas reduced activation can improve clinical outcomes( 51 – 53 ). The early loss of astrocyte activation in these studies has been associated with compromised BBB integrity, increased leukocyte infiltration, and more severe demyelination. Conversely, reduced activation during the chronic stage has been linked to enhanced remyelination( 51 – 53 ). Therefore, the loss of miR-145 appears to promote a more favourable pattern of astrocyte activation throughout EAE, likely contributing to the observed improvements in clinical outcomes and underlying pathologies in our model. To elucidate the cellular mechanisms by which miR-145 enhances clinical outcomes and to evaluate its potential as a prophylactic therapy, we developed an ASO targeting miR-145 to reduce its levels following immune priming. While ASO administration did attenuate disease severity, the effects were less pronounced than those observed in the miR-145 −/− mice. This difference may be due to suboptimal delivery to the CNS, highlighting the need for improved CNS-targeting strategies. Axon damage is closely linked to crosstalk between lymphocytes and microglia. Previous work has shown that in progressive MS, meningeal T-cell infiltration in the spinal cord is prominent, outnumbering parenchymal T cells, and exhibits close interactions with meningeal macrophages( 54 ). This suggests that the spinal cord meninges may function as an immunological niche where T lymphocytes are activated through antigen presentation, potentially instructing parenchymal macrophages and microglia to engage in neurotoxic activation( 54 ). Here, acute knockdown of miR-145 reduced CD3 + lymphocytes and CD45 + leukocytes while increasing the proportion of FoxP3 + regulatory T cells. These findings align with observations in miR-145 −/− mice, where immune cell infiltration was reduced in EAE. This suggests that miR-145 may play a role in regulating leukocyte recruitment into the spinal cord during EAE. However, whether this regulation occurs through the prevention of lymphocyte activation or by inhibiting their trafficking from lymphoid organs into the CNS remains to be explored. Interestingly, we found that the chemokine Ccl5 was downregulated in the naïve thymus of miR-145 −/− mice, a primary lymphoid organ essential for the development and maturation of T lymphocytes( 55 ). This downregulation may help explain the differences in disease severity observed between constitutive knockout animals and ASO-treated animals. In our study, ASO treatment, administered after immune priming, resulted in a 62% reduction in miR-145 levels in the thymus. Moreover, secreted CCL5 plays a crucial role in cell migration and leukocyte adhesion within the CNS vasculature during EAE( 56 ). We also observed that Ccl5 was downregulated during both the onset and chronic phases of EAE in miR-145 −/− mice, which could further diminish immune cell infiltration into the parenchyma. This suggests that Ccl5 is likely critical to the reduction of immune cell infiltration in miR-145 −/− EAE-subjected animals. Lastly, we previously demonstrated that the pathological upregulation of miR-145 inhibits OL differentiation and that the loss of miR-145 in this context is sufficient to promote remyelination in a chronic demyelination model( 15 , 17 ). In this study, we assessed myelin and axon health as indicators of overall pathological outcomes in EAE. Due to the acute nature of EAE, evaluating remyelination is challenging, although we still assessed myelin thickness in this investigation. In both miR-145 −/− mice and miR-145 ASO-treated animals, we observed alterations in several parameters of myelin and axon health throughout the course of EAE, with more significant changes noted in the chronic phase. Notably, miR-145 −/− mice exhibited nearly twice as many myelinated axons compared to EAE-subjected ASO-treated controls. Additionally, both miR-145-deficient and miR-145 ASO-treated mice showed reduced myelin decompaction and axon degeneration, further underscoring the role of miR-145 in the CNS. The observed improvements in myelin and axon health following miR-145 loss in EAE may indicate reduced demyelination resulting from decreased inflammatory attacks or suggest a neuroprotective role that warrants further investigation. In the lumbar spinal cord, miR-145 was uniquely upregulated at the onset of EAE in wild-type animals, which may contribute to the inhibition of initial remyelination efforts early in the disease. Interestingly, we also found that the chemokine Cxcl1 was upregulated in the spinal cords of miR-145-deficient mice at the onset of EAE. CXCL1 is secreted by astrocytes in the CNS and promotes the recruitment of OPCs, brain progenitors scattered throughout the CNS that give rise to OLs( 57 ). Conclusions In conclusion, our study reveals that the loss of miR-145 significantly enhances clinical outcomes in murine EAE, driving widespread changes in neuroinflammation and reducing myelin and axon degeneration across all disease stages. Future research should focus on improving the bioavailability of miR-145 ASOs to ensure they effectively cross the blood-brain spinal cord barrier and achieve efficient knockdown, allowing for a comprehensive exploration of the broader implications of miR-145 in the CNS and the therapeutic potential of targeting it. Our findings suggest a dual function for miR-145 in mitigating key pathological features of progressive MS. These insights could pave the way for innovative miR-145-targeted therapies aimed at slowing disease progression and enhancing recovery in MS, offering new hope for effective treatment strategies that address both neurodegeneration and inflammation. Methods Animals Constitutive miR-145 knock-out mice (herein referred to as miR-145 −/− ) were generated on the C57BL/6 background as described in Xin et al( 58 ) and generously provided by Dr. Eric Olson. miR-145 +/+ mice were utilized as controls and for the ASO-associated experiments. Animals were subjected to EAE and euthanized at different time points for subsequent analyses, as indicated. The animals had unrestricted access to water and chow and were kept on a 12:12-hour light-dark cycle. EAE induction EAE was induced using the Hooke Laboratories kit, which contains myelin oligodendrocyte glycoprotein peptide fragment 35–55 (MOG 35 − 55 ) in Complete Freund’s Adjuvant (CFA) emulsion (cat. no. EK-2110). Specifically, female mice aged 9–13 weeks received 0.1 mL of the prepared MOG 35 − 55 in CFA emulsion subcutaneously in the upper and lower back at the cervical and lumbar spinal column levels on day 0, in accordance with the manufacturer’s protocol. This was followed by intraperitoneal administration of 100 ng pertussis toxin in filtered PBS. A second intraperitoneal injection of the same dosage was administered on day 1. Mice were left undisturbed until day 7 post-induction, at which point daily assessments were conducted, and body weight was recorded on alternate days until day 30. Scoring was performed according to Hooke Laboratories guidelines. Animals were euthanized for tissue collection at the onset, peak, and chronic phases of EAE. Control animals consisted of non-induced, age-matched, naïve female mice. Antisense oligonucleotide (ASO) in vivo treatment The miRCURY LNA custom inhibitor (Qiagen, 339204), including negative control (YCI0201821-FZA) and miR-145-5p (YCI0201822-FZA) non-labelled ASOs, were administered at a dosage of 20 µg/mouse via subcutaneous injections. Initially, a single dose of either the control or miR-145 ASOs was administered at the peak of EAE. Subsequently, we modified our approach to perform ASO injections on days 1 and 3 post-EAE induction. For biodistribution assessment, 5’ FAM-labeled ASOs (Qiagen, 339146) were administered subcutaneously on day 3 post-induction at a dosage of 10 µg/mouse. Spinal cord organotypic cultures C57BL/6 pups aged P0-P2 were euthanized, and their spinal cords were dissected and sliced to a thickness of 300 µm, following the protocol previously described with slight modifications( 59 ). The slices were placed onto a semi-porous membrane insert (Millipore, 30 mm diameter, pore size 0.4 µm) and maintained using an interface culture method with 1 mL of serum-based medium. The medium consisted of 25% DMEM (low glucose), 25% F12, 21% Hanks’ balanced salt solution (HBSS), 25% horse serum, 25 mM HEPES, 0.4 mM ascorbic acid, 25 mM glucose, and 1% penicillin-streptomycin. Cultures were maintained for 10 days in vitro (DIV), with media changes every 2–3 days. Treatment with either 50 nM of FAM-labeled control or miR-145 ASOs was applied to the wells for 24 h. After treatment, the wells were washed, and 4% paraformaldehyde (PFA) was added for 1 h at room temperature on a shaker. Three washes with PBS, each lasting 10 min, were performed. Slices were then stored in PBS until immunohistochemical experiments were conducted. RNA isolation and qRT-PCR Total RNA was extracted from all samples using the RNeasy Mini Kit (Qiagen), following the manufacturer’s instructions. Spinal cord, spleen, and thymus were flash-frozen immediately upon collection. A maximum of 30 mg of tissue was used per sample. For RNA isolation, 350 µL or 600 µL of RLT buffer was added directly to the frozen tissues, which were then homogenized. The lysate was mixed 1:1 with 70% ethanol in RNase-free H 2 O, transferred to the RNeasy spin column, and centrifuged at maximal speed for 15 sec. The eluate was discarded, and the column was washed with 700 µL RW1 buffer by spinning at maximal speed for 15 sec. The eluate was again discarded, and the column was washed with 500 µL RPE buffer at maximal speed for 15 sec. The columns were transferred to clean collection tubes and washed a second time with 500 µL RPE for 2 min. Finally, the columns were placed in RNase-free microfuge tubes, and RNA was eluted in 30–50 µL RNase-free H2O by spinning at maximal speed for 1 min. Reverse transcription of mature miR-145-5p and SnU6 was conducted according to Biggar et al.( 60 ) with modifications. Briefly, 300 ng of total RNA was incubated with 5 µL of 250 nM stem-loop primer in a total volume of 10 µL. The annealing reaction was performed at 95°C for 5 min, followed by 60°C for 5 min. Samples were then centrifuged and kept on ice for 1 min. Reverse transcription was performed using 1 µL of M-MLV Reverse Transcriptase (Invitrogen), 4 µL of 5x First Strand Buffer (Invitrogen), 2 µL of 100 mM dithiothreitol (Invitrogen), and 1 µL of premixed dNTPs (final concentration 25 µM each). Each reaction was brought to a total volume of 25 µL using RNase-free water (Qiagen). The reverse transcription protocol was as follows: 16°C for 30 min, followed by 60 cycles of 20°C for 30 sec, 42°C for 30 sec, and 50°C for 1 sec, culminating with a final step at 85°C for 5 min, using an Eppendorf Mastercycler. Primer sequences for miRNA qRT-PCR are provided in Supplementary Table 1. A universal reverse primer was used for the amplification of both miR-145-5p and SnU6. Amplification of miR-145-5p and snU6 cDNA was performed using specific forward primers and a universal reverse primer complementary to the stem-loop portion of the cDNA (Supp. Table 1). Each qRT-PCR reaction contained 12.5 µL of 2x SsoFast EvaGreen Supermix (Bio-Rad), 0.8 µL each of 25 µM forward and universal primers, and 4 µL of cDNA, with the total volume adjusted to 25 µL using RNase-free water (Qiagen). Samples were amplified using the following protocol: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min, using a Bio-Rad CFX Connect. All samples were analyzed in technical triplicate. Primer validation was conducted through standard curve efficiency analysis, melt curve analysis, and electrophoresis of qPCR products on a 5% agarose gel to confirm product size. Primers for miRNA qRT-PCR were sourced from AlphaDNA. Relative expression analysis was performed using CFX Manager™ or CFX Maestro software, utilizing the ΔΔCt method. For mRNA analysis, total cDNA was synthesized using the RT2 First Strand Kit (Qiagen) according to the manufacturer’s protocol. Briefly, 2 µL of Buffer GE (Qiagen) was used to eliminate genomic DNA from 150–200 ng of total RNA in RNase-free water, in a total volume of 10 µL per sample. Samples were incubated at 42°C for 5 min and then placed on ice for at least 1 min. Reverse transcription was performed by adding 4 µL of 5x Buffer BC3 (Qiagen), 1 µL of Control P2 (Qiagen), 2 µL of RE3 Reverse Transcriptase Mix (Qiagen), and 3 µL of RNase-free water (Qiagen), followed by incubation at 42°C for 15 min and 95°C for 5 min. Samples were then diluted with 91 µL of RNase-free water (Qiagen) and stored at -20°C until further use. For mRNA qRT-PCR, PrimePCR pre-optimized primers (Bio-Rad) were employed according to the manufacturer’s protocol. Each reaction contained 4 µL of total cDNA, 10 µL of SsoFast EvaGreen master mix (Bio-Rad), and 1 µL of forward/reverse primer mix, in a total volume of 20 µL. Amplification was performed at 95°C for 5 min, followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec using a Bio-Rad CFX Connect. Relative expression analysis was conducted using CFX Manager™ or CFX Maestro software, employing the ΔΔCt method. Immunohistochemistry and histology (paraffin-embedded tissues) Mice were anesthetized via intraperitoneal injection of tribromoethanol (avertin) and perfused transcardially with 5 mL of phosphate-buffered saline (PBS), followed by 10–20 mL of 4% paraformaldehyde (PFA, EMS Cedarlane). Lumbar spinal cords were then dissected and fixed in 4% PFA for 24–48 h before being transferred to 70% ethanol in water. Lumbar spinal cord samples were processed at the Louise Pelletier Histology Core Facility, Department of Pathology and Laboratory Medicine at the University of Ottawa. The samples were initially embedded in paraffin wax using a LOGOS microwave hybrid tissue processor. Paraffin-embedded samples were cut into 20 µm sections and mounted on slides. Prior to immunohistochemistry and staining, sections were deparaffinized and rehydrated as follows: slides were incubated at 59°C for 30–60 min, followed by immersion in 100% Hemo-D (Histo-Clear, National Diagnostics) for three 5-min washes, 50% Hemo-D/50% ethanol for two 3-min washes, 100% ethanol for two 3-min washes, 95% ethanol for 3 min, 70% ethanol for 3 min, 50% ethanol for 3 min, and finally rinsed twice in water. Antigen retrieval for Iba1 immunostaining was performed using Tris-EDTA buffer (pH 9) at 95–100°C for 20 min in a steamer. After antigen retrieval or rehydration, slides were rinsed three times for 5 min in PBS, permeabilized with 0.5% Triton-X for 20 min, and rinsed again three times in PBS. Sections were then blocked for 1 h in a blocking solution containing 1% bovine serum albumin (BSA), 10% goat serum (GS), and 0.2% Triton-X in PBS at room temperature (RT). Primary and secondary antibodies were diluted in a solution containing 2% BSA, 1% goat serum, 0.2% Triton-X, and incubated at 4°C overnight. The following antibody dilutions were used: rat anti-MBP (1:100), rabbit anti-GFAP (1:1000), and rabbit anti-Iba1 (1:100) (Supp. Table 2). Following the incubation with primary antibodies, sections were washed three times with PBS and incubated for 1 h with fluorophore-labeled secondary antibodies (Alexa Fluor, Invitrogen) at a dilution of 1:500. Sections were subsequently washed once with PBS, counterstained with 4′,6-diamidino-2-phenylindole (DAPI) at a dilution of 1:1000 in PBS for 5 min, and finally washed three times for 5 min in PBS. Dako fluorescent mounting medium was applied sparingly to each section, followed by the placement of a coverslip. For histological analysis, lumbar spinal cord cross-sections were deparaffinized and stained with either Cresyl violet or hematoxylin and eosin using a Leica ST5010 Autostainer XL, in conjunction with a Leica CV5030 Glass Coverslipper. Immunohistochemistry (cryopreserved spinal cords) Mice were anesthetized and transcardially perfused with 10–20 mL of Hank’s Balanced Salt Solution (HBSS, Gibco). Following dissection, lumbar spinal columns were post-fixed in 4% PFA for 24 h at 4°C. The spinal cords were then carefully dissected and stored in 30% sucrose (Fisher) in PBS. Two additional changes of 30% sucrose were performed before embedding the tissues in optimal cutting temperature (OCT) compound and flash-freezing. Cryopreserved spinal cords were sectioned at 16 µm and stored at -80 o C until further staining. Briefly, sections were rehydrated in 1X PBS for 5 min, followed by permeabilization and blocking with 5% BSA and 0.3% Triton-X100 in 1X PBS for 1 h at RT. Tissue sections were then incubated overnight at 4°C with appropriate primary antibodies (listed in Supp. Table 2), diluted in a solution containing 1% goat serum, 1% BSA, and 0.2% Triton-X100 in 1X PBS. After washing three times with 1X PBS, secondary antibodies (1:500) diluted in 1X PBS were applied to the tissue sections for 1 h at RT. For nuclear staining, sections were incubated with DAPI diluted in 1X PBS for 5 min. Sections were then washed three times in PBS and mounted with Fluoromount G (Invitrogen). Immunohistochemistry (organotypic cultures) Cultured spinal cord slices were quickly washed once with 1X PBS and subsequently fixed with 4% PFA for 1 h, as previously described( 61 ). Following fixation, slices were washed three times with 1X PBS and incubated in a blocking buffer containing 5% BSA and 1% Triton-X100 in 1X PBS for 3 h at RT. Primary antibodies (listed in Supp. Table 2) were then added to a solution containing 6% normal goat serum, 0.5% BSA, and 1% Triton-X100 in 1X PBS, and incubated overnight at 4°C on a shaker. Afterward, slices were washed three times with 1X PBS, and secondary antibodies (1:500) along with DAPI (1:1000) diluted in PBS-T (1%) were incubated for 2 h at RT on a shaker. Slices were then washed three times for 10 min each and mounted with Fluoromount G. Transmission Electron Microscopy (TEM) Mice were anesthetized and perfused transcardially with 10–20 mL of HBSS, followed by 10–20 mL of Karnovsky’s fixative (4% PFA, 2% glutaraldehyde, and 0.1 M sodium cacodylate in PBS, pH 7.4). Whole spinal columns were extracted and fixed overnight (or until processed) at 4°C in the same fixative. Following fixation, the ventral white matter of the lumbar spinal cords was dissected and cut into straight segments of 1 mm in length under a stereomicroscope. Specimens were subsequently washed twice in 0.1 M sodium cacodylate buffer for 1 h each and once overnight at room temperature. Segments were post-fixed with 1% osmium tetroxide in 0.1 M sodium cacodylate buffer for 1 h at room temperature and then washed three times in distilled water for 5 min each. Specimens were dehydrated in a graded series of ethanol, including 30%, 50%, 70%, 85%, and 95% ethanol, followed by two 30-min washes in 100% ethanol. This was followed by two 15-min washes in 50% ethanol/50% acetone and two 15-min washes in 100% acetone. Segments were then infiltrated in 30% Spurr resin/acetone for 20 min and once for 15 h (overnight), followed by 50% Spurr resin/acetone for 6 h, and then in fresh 100% Spurr resin overnight. The Spurr resin was changed twice daily for three days at room temperature. All infiltration steps were performed on a rotator at low speed. Finally, specimens were embedded in fresh liquid Spurr resin, oriented inside molds, and polymerized overnight at 70°C. Ultrathin sections (80 nm) were collected onto 200-mesh copper grids and stained with 2% aqueous uranyl acetate and Reynolds’ lead citrate. Imaging and quantification Fluorescence images were captured using an Axio Imager M1 microscope with an AxioCam HRm Rev.2 camera and Axiovision 4.8.2 software. Confocal images were obtained with a Zeiss LSM 510 Meta DuoScan microscope using Zen 8.0 software. Electron micrographs of the lumbar spinal cord were taken with a JEM-1400Plus electron microscope at ×4000 magnification. Histology and immunohistochemistry samples from paraffin-embedded tissues were imaged using a Zeiss AxioScan slide scanner with a Colibri 7 camera and Zen 2.6 slidescan software. All images were analyzed using ImageJ, with treatment conditions blinded during image analyses. For paraffin-embedded samples, images were taken at 20x magnification, with 20–25 images quantified per sample; for those taken at 10x, 5–10 images were quantified per sample. For flash-frozen tissues, images were taken at 5x magnification, and 5–10 images were quantified per animal. For qualitative assessment of spinal cord organotypic cultures, 20x objective images from 5–7 slices per condition were taken. Histological and immunohistochemical analyses of the lumbar spinal cord involved cross-sections taken from the proximal end, mid-lumbar spine, and distal end. To calculate percent infiltrated area and percent myelinated area, the total spinal cord area and nucleus-dense area (based on H&E staining) or myelinated area (based on MBP + staining) were measured using ImageJ from every third 20 µm section across 2–3 sections from each of the three sampled areas, resulting in a total of 6–9 sections per animal. For MBP area analysis, a uniform threshold value was used for all images, and the “Measure Particles” plugin in ImageJ was employed to produce MBP traces. GFAP + and Iba1 + cell quantifications involved counting individual cells across the entire section, with Iba1 + counts excluding areas of dense cell infiltration. For immune cell labelling, FIJI automated cell counting based on outlines applied from the binary image was performed on whole spinal cord sections. A total of 5–8 sections per animal were quantified and averaged for each marker. Electron micrographs were captured using the Hitachi 7100 transmission electron microscope at a magnification of x4000. G-ratios were calculated by measuring the axon and the total fibre (axon plus myelin), converting these measurements to diameters, and dividing the axon diameter by the fibre. Areas enriched in myelinated axons were preferentially chosen for g-ratio and morphometric analyses. The g-ratio was measured for myelinated axons with normal morphology while excluding axons exhibiting abnormal myelin morphology (e.g., uncompacted myelin, splitting, empty sheaths) or signs of degradation or swelling, as previously described( 32 , 62 , 63 ). On average, 150 axons per sample were analyzed. Axonal and myelin abnormalities were assessed through morphometric analysis, determining the proportion of uncompacted myelin and degenerating axons characterized by swelling, darkening of the axoplasm, and organelle accumulation via manual counting. Morphometric analyses were evaluated from 10 images per animal. All images were counted in Fiji software in a blinded manner, with representative images processed in Photoshop and GraphPad Prism 10.0. Biorender was utilized for generating schematics. Statistical analysis Statistical analyses were performed using GraphPad Prism software (versions 6 or 10.3.0), except qRT-PCR analyses. Normal distribution and equal variance were assumed for all datasets, though not formally tested. Sample sizes were determined based on prior publications( 64 ). Pairwise comparisons were made using a two-tailed Student’s t-test. For comparisons involving more than two conditions, one-way or two-way analysis of variance (ANOVA), or Multiple unpaired t-tests of two-way ANOVA were employed, followed by the appropriate post-hoc tests. Linear regression analyses were performed for g-ratio assessments, and Kaplan-Meier curves were analyzed using the Mantel-Cox test. Errors on mean values are expressed as ± SEM, unless otherwise specified. The number of experiments and statistical details are included in the corresponding figure legends. P-values of < 0.05 were deemed significant, with significance levels indicated as follows: * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. Abbreviations AQP4: Aquaporin 4 ASO: Antisense oligonucleotide BBB: Blood-brain barrier BSCB: Blood-spinal cord barrier Ccl5: Chemokine (C-C motif) ligand 5 CD: Cluster of differentiation CNS: Central nervous system Cxcl1: Chemokine (C-X-C motif) ligand 1 EAE: Experimental autoimmune encephalomyelitis EBV: Epstein-Barr virus FAM: Fluorescein amidite FoxP3: Forkhead box P3 GFAP: Glial Fibrillary Acidic Protein H&E: Hematoxylin and eosin Iba1: Ionized calcium-binding adaptor molecule 1 Ifng: Interferon-gamma Il1b: Interleukin-1 beta Il6: Interleukin 6 iNos: Inducible nitric oxide synthase MBP: Myelin basic protein MOG 35-55 : Myelin Oligodendrocyte Glycoprotein Peptide Fragment 35-55 MS: Multiple sclerosis Nurr1: Nuclear receptor 4A2 OLs: Oligodendrocytes OPCs: Oligodendrocyte precursor cells PBMCs: peripheral blood mononuclear cells PPMS: Primary progressive multiple sclerosis RRMS: Relapsing-remitting multiple sclerosis SPMS: Secondary progressive multiple sclerosis TEM: Transmission electron microscopy Tnfa: Tumor necrosis factor alpha Ym1: Chitinase-like protein 3 (Chil3) Declarations Ethics approval and consent to participate All animal-related experimental protocols were approved by the University of Ottawa Animal Care Committee and adhered to the standards outlined in the Canadian Council on Animal Care’s Guide to the Care and Use of Experimental Animals, as well as the Animals for Research Act. We ensured compliance with all relevant ethical regulations regarding the use of animals. Consent for publication Not applicable Availability of data and materials All authors had access to the study data and reviewed and approved the final manuscript. All data associated with this study are available in the main text or supplementary materials. Raw data can be provided in full upon request. Competing interests The authors disclose no conflicts of interest. The funders mentioned above were not involved in the study design, data collection and analysis, decision to publish, or manuscript preparation. Funding This work was supported by grants from the Canadian Institutes of Health Research [grant number PJT-162121]; the Multiple Sclerosis Society of Canada [grant number MSSC-3779]. MMAA was supported by the University of Ottawa Brain and Mind Institute TRIMS Award. SFK was supported by a CIHR Banting and Best CGS-D award and an MS Canada Doctoral Fellowship. SEC was supported by a CIHR Banting and Best CGS-D award and a CNMD Star award. ERS was supported by a CNMD Star award. RY was supported by a QEII Graduate Scholarship in Science and Technology Studentship. Authors' contributions M.M.A.A. and S.F.K. conceived and designed the study, with supervision and support from R.K. The methodology was developed by M.M.A.A., S.F.K., S.E.C., Y.D.R., R.Y., S.G., and A.B. Formal analysis was conducted by M.M.A.A., S.F.K., M.A.A., I.G, E.R.S. The investigation was carried out by M.M.A.A. and S.F.K. Graphical abstract was made by E.R.S. The manuscript was primarily written by M.M.A.A., with significant contributions from S.F.K., whose thesis provided key textual elements. R.K. critically edited it. Funding acquisition was managed by R.K. Acknowledgements We thank Dr. Eric Olson for the kind provision of transgenic mice. 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Pharmacol Res. 2020;159:104997. de Almeida MMA, Watson AES, Bibi S, Dittmann NL, Goodkey K, Sharafodinzadeh P, et al. Fractalkine enhances oligodendrocyte regeneration and remyelination in a demyelination mouse model. Stem Cell Reports. 2023;18(2):519-33. Bando Y, Nomura T, Bochimoto H, Murakami K, Tanaka T, Watanabe T, et al. Abnormal morphology of myelin and axon pathology in murine models of multiple sclerosis. Neurochem Int. 2015;81:16-27. Madsen PM, Desu HL, Vaccari JPdR, Florimon Y, Ellman DG, Keane RW, et al. Oligodendrocytes modulate the immune-inflammatory response in EAE via TNFR2 signaling. Brain, Behavior, and Immunity. 2020;84(July 2019):132-46. Additional Declarations No competing interests reported. Supplementary Files Supp1MAandSK2024.tiff Supplementary Figure 1. miR-145-5p is differentially expressed in lumbar spinal cord, spleen and thymus. A. Relative expression of miR-145-5p in the lumbar spinal cord, spleen, and thymus of miR-145 +/+ age-matched naïve animals. mRNA expression from the spleen and thymus was normalized to the spinal cord. B-D. Relative expression of miR-145-5p in miR-145 +/+ lumbar spinal cord (B), spleen (C) and thymus (D) at onset, peak and chronic stages of EAE, normalized to naïve controls. N=3, **=p<0.01, ***=p<0.001, ordinary one-way ANOVA followed by Tukey’s test. Analyzed by DDCt, normalized snU6. Values represent mean ± SEM. SupplTable1MASK2024.docx SupplTable2MASK2024.docx Supp2MAandSK2024.tiff Supplementary Figure 2. Cytokine and chemokine expression show alterations in miR-145 deficient thymus and spleen throughout EAE. A-L. Relative expression of cytokines (Il1b, Ifng, Il6, and Tnfa) and chemokines (Cxcl1 and Ccl5) in the thymus (A-F) and spleen (G-L) from miR-145 +/+ and miR-145 -/- naïve animals, and at onset, peak and during the chronic stages of EAE. N=3, *=p<0.05, **=p<0.01, ****=p<0.0001, multiple unpaired t-tests. Normalized to Actb and Ppia. Analyzed by DDCt method. Values represent mean ± SEM. Supp3MAandSK2024.tiff Supplementary Figure 3. miR-145 ASO injection at the peak of EAE does not alter disease severity, although it efficiently knocked down miR-145 in the spleen and spinal cord. A. Schematic representation of EAE induction and ASO treatment. Female C57BL/6 mice aged 9-13-week-old were subjected to EAE. Animals were followed daily from day 7 post-EAE induction, and at day 16 post-induction (~peak of disease), were subcutaneously injected with the negative control or miR-145 ASOs at a single dose of 20 μg/mouse. B. Mean clinical scores over the course of EAE. C. Mean weights expressed as a percentage of starting weight during EAE. N=4, multiple unpaired t-tests. Values represent mean ± SEM. D. Mean score at the chronic phase of EAE (Day 30). N=4 for each treatment, two-tailed unpaired Student’s t-tests. E-F. Relative expression of miR-145-5p in the spleen (E) and SC (F) at the chronic stage of EAE from control and miR-145 ASOs. Values represent mean ± SEM, ****=p<0.0001. Analyzed by DDCt, normalized snU6. Values represent mean ± SEM. Supp4MAandSK2024.tiff Supplementary Figure 4. FAM-ASO distributes across different tissues and accumulates in various CNS cell types. A. Schematic representation of EAE induction and FAM-ASO treatment. Female C57BL/6 mice aged 9-13-week-old were subjected to EAE and injected subcutaneously with FAM-labelled control or miR-145 ASOs on day 3 post-EAE induction. Animals were monitored daily from day 7 to day 11 post-EAE induction. On day 11, tissues were harvested. B. Qualitative images of the liver, spleen, and spinal cord from negative controls and ASO-treated animals. Scale bar = 50 mm. N=2 for each treatment. C. Schematic representation of organotypic spinal cord cultures. Cultures were maintained for 10 days in vitro , followed by transfection with either 50 nM of FAM-labeled control or miR-145 ASOs for 24 h. D. Schematic illustrating that cells lacking miR-145 expression exhibit minimal fluorescence due to specific binding of the FAM-labeled ASO to its complementary target RNA. Conversely, the mismatched control ASO also shows minimal fluorescence, indicating successful transfection with limited microRNA interaction. In contrast, miR-145-expressing cells are expected to show a specific accumulation of FAM-labeled miR-145 ASO. E-I. Spinal cord organotypic cultures were fixed and stained for Olig2 (E, blue), CC1 (E, pseudo-green), IBA1 (F, magenta), NeuN (G, red), Nestin (H, red), and GFAP (I, magenta). FAM-ASO is shown as pseudo-red in E and as green in F-I. N=2. Scale bar = 20 mm. 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Schematic representation of EAE induction paradigm. \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e were injected with myelin oligodendrocyte antigen (MOG\u003csub\u003e35-55\u003c/sub\u003e) with complete Freund’s adjuvant on day 0, followed by pertussis toxin (PTX) injections on days 0 and 1. Animals were monitored daily from days 7-30. B. Mean clinical scores throughout the course of EAE. C. Mean weights expressed as percentage of starting weight during EAE. (B-C) \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e N=20, \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eN=16, *=p\u0026lt;0.05, **=p\u0026lt;0.01, ***=p\u0026lt;0.001, analyzed by multiple unpaired t-tests. Values represent mean ± SEM. D. Kaplan Meier curve illustrating time to onset; p\u0026lt;0.0001 by log-rank test. E. Mean onset day. F. Mean score at onset. (D-F) \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e N=41, \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eN=31, *=p\u0026lt;0.05, ****=p\u0026lt;0.0001, analyzed by two-tailed unpaired Student’s t-tests. Values represent mean ± SEM. G. Kaplan Meier curve showing time to the first instance of paralysis. \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eN=38, \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e N=28, p\u0026lt;0.0001 by log-rank test. H. Mean time from onset to paralysis. I. Mean maximum clinical score. J. Mean total days paralyzed. K. Proportions of all EAE-induced animals monitored throughout disease course divided by those exhibiting no paralysis, 1 instance of paralysis, or \u0026gt;1 instance of paralysis during the 30-day disease course. L. Mean score at the chronic stage (day 30). (H-L) \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e N=20, \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e N=16, **=p\u0026lt;0.01, ****=p\u0026lt;0.0001, analyzed by two-tailed unpaired Student’s t-tests. Values represent mean ± SEM.\u003c/p\u003e","description":"","filename":"Figure1MAandSK2024.png","url":"https://assets-eu.researchsquare.com/files/rs-5462410/v1/56ebb332ccd33af13e479a3c.png"},{"id":71807221,"identity":"c4b4b3bb-0dea-46eb-a4d7-236b59a3d08d","added_by":"auto","created_at":"2024-12-18 17:42:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":7814332,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMBP loss is reduced in the lumbar spinal cord at onset and during the chronic stage of EAE in miR-145 deficient animals.\u003c/strong\u003e A. Fluorescence micrographs and MBP area traces in the lumbar spinal cord of naïve animals, at onset, peak and during the chronic stage of EAE in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e (left panels) and \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e (right panels). Spinal cords were stained for MBP (green) and counterstained with DAPI (blue). Scale bar = 200 µm. B. Quantifications of MBP area as a percentage of total spinal cord area in naïve animals, and at onset, peak and during the chronic stages of EAE. N=3, *=p\u0026lt;0.05, **=p\u0026lt;0.01, ***=p\u0026lt;0.0001; analyzed by multiple unpaired t-tests. Values represent mean ± SEM.\u003c/p\u003e","description":"","filename":"Figure2MAandSK2024.png","url":"https://assets-eu.researchsquare.com/files/rs-5462410/v1/923ae18d6cff256dbba01351.png"},{"id":71806083,"identity":"524102d7-1750-42f1-8f32-06f71a18499e","added_by":"auto","created_at":"2024-12-18 17:34:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":7196515,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmune cell infiltration of the lumbar spinal cord and chemokine expression are altered during EAE in miR-145 deficient mice. \u003c/strong\u003eA. Hematoxylin and eosin staining of the lumbar spinal cord in naïve animals, at onset, peak and during chronic stages of EAE in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e (left panels) and \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e(right panels). Scale bar = 200 µm. A dashed rectangle highlights a zoomed-in image showing submeningeal and parenchymal infiltration (black arrows) in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eat peak. Scale bar = 200 µm. B-D. Quantifications of infiltrated area as a percentage of total spinal cord area at onset (B), peak (C) and chronic stage (D) of EAE. N=3, **=p\u0026lt;0.01, ***=p\u0026lt;0.001, two-tailed unpaired Student’s t-tests. Values represent mean ± SEM. E-J. Relative expression of cytokines (Il1b, Ifng, Il6, and Tnfa) and chemokines (Cxcl1 and Ccl5) in the lumbar spinal cord from \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e naïve animals, and at onset, peak and during the chronic stage of EAE. N=3, *=p\u0026lt;0.05, ****=p\u0026lt;0.0001, multiple unpaired t-tests. Data normalized to Actb and Ppia. Analyzed by DDCt method. Values represent mean ± SEM.\u003c/p\u003e","description":"","filename":"Figure3MAandSK2024.png","url":"https://assets-eu.researchsquare.com/files/rs-5462410/v1/6979c5cfd679ff016c3acf99.png"},{"id":71807223,"identity":"2e7c2d3e-24e0-44b4-b5c8-a5ff4ccb0299","added_by":"auto","created_at":"2024-12-18 17:42:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4712164,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-145 loss results in altered microglial numbers throughout EAE.\u003c/strong\u003e A. Fluorescence micrographs of whole lumbar spinal cord cross-sections from naïve, EAE onset, peak and chronic stages in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003e(left panels) and \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e animals (right panels) stained for Iba1 (white) and counterstained with DAPI (blue). Scale bar = 200 µm. B. Quantification of Iba+ cells per mm\u003csup\u003e2\u003c/sup\u003e in the parenchyma, excluding areas of dense cellular infiltration. N=3,\u003cstrong\u003e \u003c/strong\u003e*=p\u0026lt;0.05, **=p\u0026lt;0.01, ****=p\u0026lt;0.0001, multiple unpaired t-tests. C-E. Relative\u003cstrong\u003e \u003c/strong\u003eexpression of Ym1 (C), iNos (D) and Nurr1 (E) in the lumbar spinal cord from \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e naïve animals, and at onset, peak and during chronic stage of EAE. N=3, *=p\u0026lt;0.05,\u003cstrong\u003e \u003c/strong\u003e**=p\u0026lt;0.01, ***=p\u0026lt;0.001, ****=p\u0026lt;0.0001, multiple unpaired t-tests. Data normalized to Actb and Ppia, analyzed by the DDCt method. Values represent mean ±SEM.\u003c/p\u003e","description":"","filename":"Figure4MAandSK2024.png","url":"https://assets-eu.researchsquare.com/files/rs-5462410/v1/5f67858e45308844ca7b7e02.png"},{"id":71806098,"identity":"bf62fff0-8bc2-4795-904e-9782b3541614","added_by":"auto","created_at":"2024-12-18 17:34:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3226318,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-145 loss affects astrocyte numbers and reactivity throughout EAE.\u003c/strong\u003e A. Fluorescence micrographs of whole lumbar spinal cord cross-sections from naïve, EAE onset, peak, and chronic stages in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eanimals. Right panels show magnified regions indicated by dashed rectangles on the left. Sections were stained for GFAP (white) and counterstained with DAPI (blue). Scale bars = 200 µm. B-E. Quantification of astrocytes (GFAP+) and hypertrophic astrocytes (GFAP\u003csup\u003ehigh\u003c/sup\u003e/thickened processes) in the spinal cord at naïve (B), onset (C), peak (D) and chronic (E) stages of EAE in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e animals. N=3, *=p\u0026lt;0.05, multiple unpaired t-tests. Values represent mean ± SEM. F-G. Relative expression of GFAP (F) and AQP4 (G) in the spinal cord at naïve, EAE onset, peak and chronic stages of EAE in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e animals. N=3, *=p\u0026lt;0.05, ****=p\u0026lt;0.0001, multiple unpaired t-tests. Data normalized to Actb and Ppia, analyzed by the DDCt method. Values represent mean ±SEM.\u003c/p\u003e","description":"","filename":"Figure5MAandSK2024.png","url":"https://assets-eu.researchsquare.com/files/rs-5462410/v1/5ba59885727010ca6681b124.png"},{"id":71807224,"identity":"3015f00e-e2e9-445d-85eb-b3313a921328","added_by":"auto","created_at":"2024-12-18 17:42:55","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2212742,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-145 ASO injection on days 1 and 3 post-EAE induction slightly reduces disease severity and efficiently knocks down miR-145 in the lymphoid tissues and spinal cord. \u003c/strong\u003eA. Schematic representation of EAE induction and ASO treatment.\u0026nbsp;\u0026nbsp; Female C57BL/6 mice aged 9-13-week-old were subjected to EAE and injected subcutaneously with negative control or miR-145 ASOs on days 1 and 3 post-EAE induction. Animals were monitored daily from day 7 to day 30 post-EAE induction. B. Mean clinical scores over the course of EAE. C. Mean weights expressed as a percentage of starting weight during EAE. (B-C) Control ASO n=14, miR-145 ASO\u003csup\u003e \u003c/sup\u003en=10, *=p\u0026lt;0.05, multiple unpaired t-tests. Values represent mean ± SEM. D. Mean onset day; Control ASO n=14, miR-145 ASO n=11. E. Mean score at onset; Control ASO n=17, miR-145 ASO n=17. F. Mean score at peak; Control ASO n=15, miR-145 ASO\u003csup\u003e \u003c/sup\u003en=13. G. Mean score at the chronic phase (day 30); Control ASO n=7, miR-145 ASO\u003csup\u003e \u003c/sup\u003en=5. H. Mean maximum clinical score; Control ASO n=14, miR-145 ASO n=10. I-K. Relative expression of miR-145-5p in the spleen (I), thymus (J) and spinal cord (K) at the chronic stage of EAE from control and miR-145 ASOs. Analyzed by DDCt, normalized to snU6. (D-K) *=p\u0026lt;0.05, **=p\u0026lt;0.01, ****=p\u0026lt;0.0001, two-tailed unpaired Student’s t-tests. Values represent mean ± SEM.\u003c/p\u003e","description":"","filename":"Figure6MAandSK2024.png","url":"https://assets-eu.researchsquare.com/files/rs-5462410/v1/9842292058ca98609ca7ed43.png"},{"id":71806093,"identity":"6a4cd094-ec9f-48b7-848e-def80bc6b9b5","added_by":"auto","created_at":"2024-12-18 17:34:56","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2803226,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmune cell infiltration of the lumbar spinal cord and chemokine expression are altered during EAE in miR-145 deficient mice. \u003c/strong\u003eA. Schematic representation of EAE induction and ASO treatment. B. Representative images of the lumbar spinal cord stained for FoxP3, CD3 and CD4 in controls and miR-145 ASO-treated mice at the peak of EAE. Scale bar = 50 µm. The inset shows a cropped region to highlight the immunostaining in the spinal cord. Scale bar = 50 µm. C-D. Quantifications of CD3+ cells (C), the proportion of CD4+ cells (D), and FoxP3+ cells (E) among CD3+ lymphocytes. N=3, *=p\u0026lt;0.05, **=p\u0026lt;0.01, two-tailed unpaired Student’s t-tests. Values represent mean ± SEM.\u003cstrong\u003e \u003c/strong\u003eF. Representative images of the lumbar spinal cord stained for CD45 and CD19, counterstained for DAPI in controls and miR-145 ASO-treated at the peak of EAE. Scale bar = 50 µm. The inset shows a cropped region to highlight the immunostaining in the spinal cord. Scale bar = 50 µm. G-H. Quantifications of CD45+ cells (G) and CD19+ cells (H). N=3, *=p\u0026lt;0.05, two-tailed unpaired Student’s t-tests. Values represent mean ± SEM.\u003c/p\u003e","description":"","filename":"Figure7MAandSK2024.png","url":"https://assets-eu.researchsquare.com/files/rs-5462410/v1/b07ee749c406e3c771266d41.png"},{"id":71806095,"identity":"7123d298-1d97-4443-a200-029a2d6a1966","added_by":"auto","created_at":"2024-12-18 17:34:56","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2890316,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-145 deficiency results in slightly thicker myelin at disease onset.\u003c/strong\u003e A, L. Schematic of EAE induction paradigm. A. \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice were injected with myelin oligodendrocyte antigen (MOG\u003csub\u003e35-55\u003c/sub\u003e) with complete Freund’s adjuvant on day 0 along with pertussis toxin (PTX), followed by a second dose of PTX on day 1. L. \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e animals received control and miR-145 ASOs subcutaneous injections on days 1 and 3 post-induction. Animals were followed daily from day 7 to day 11, and at day 11 (approximate disease onset), spinal cords were harvested for TEM. B, M. Representative TEM images of ventral lumbar spinal cord sections from \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice (B) and \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eASO-treated animals (M). Scale bars: 2 mm. C-K, N-V. Analysis of g-ratio vs. axon diameter correlation (C, N), average g-ratio for all axons (D, O), g-ratio frequency distribution (E, P), and average g-ratio for binned axons (F, Q). G, R. Analysis of binned relative frequency (G, R) and total (H, S) myelinated axon density, and proportion (I, T). J-K, U-V. Morphometric analysis of the proportion of uncompacted myelin (J, U) and degenerating axons (K, V) over total myelinated axons. Error bars represent SEM. Each data point in C, N represents an axon. Data in D, H-K and O, S-V were analyzed using two-tailed unpaired Student’s t-tests; C and N were analyzed with linear regression, and data in F-G and Q-R with multiple t-tests. N=3 mice per group.\u003c/p\u003e","description":"","filename":"Figure8MAandSK2024.png","url":"https://assets-eu.researchsquare.com/files/rs-5462410/v1/d0d138a214f2c6183f2acffd.png"},{"id":71806088,"identity":"72c8b2aa-769e-4573-8b3e-27fbba5f1416","added_by":"auto","created_at":"2024-12-18 17:34:55","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":2920582,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-145 deficiency results in thinner myelin and reduced axonal degeneration, while acute knockdown slightly increases myelinated axons proportion at peak.\u003c/strong\u003e A, L. Schematic of EAE induction paradigm. A. \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e were injected with myelin oligodendrocyte antigen (MOG\u003csub\u003e35-55\u003c/sub\u003e) with complete Freund’s adjuvant on day 0 along with pertussis toxin (PTX), followed by a second dose of PTX on day 1. L. \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e animals received control and miR-145 ASOs subcutaneous injections on days 1 and 3 post-induction. Animals were followed daily from day 7 to day 16, and at day 16 (approximate disease peak), spinal cords were harvested for TEM. B, M. Representative TEM images of ventral lumbar spinal cord sections from \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice (B) and \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eASO-treated animals (M). Scale bars: 2 mm. C-K, N-V. Analysis of g-ratio vs. axon diameter correlation (C, N), average g-ratio for all axons (D, O), g-ratio frequency distribution (E, P) or average g-ratio for binned axons (F, Q). G, R. Analysis of binned relative frequency (G, R) and total (H, S) myelinated axon density, and proportion (I, T). J-K, U-V. Morphometric analysis of the proportion of uncompacted myelin (J, U) and degenerating axons (K, V) over total myelinated axons. Error bars represent SEM. Each data point in C, N represents an axon. Data in D, H-K and O, S-V were analyzed using two-tailed unpaired Student’s t-tests; C and N were analyzed with linear regression, and data in F-G and Q-R with multiple t-tests. N=4 mice per group, ** p\u0026lt;0.01, * p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure9MAandSK2024.png","url":"https://assets-eu.researchsquare.com/files/rs-5462410/v1/b18d81645738b315a3ebfaf2.png"},{"id":71806097,"identity":"af53739e-8c58-4960-b8c2-a2fc401b4694","added_by":"auto","created_at":"2024-12-18 17:34:56","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":2585371,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMyelin and axon degeneration are attenuated in miR-145 deficient mice during the chronic phase of EAE.\u003c/strong\u003e A. Schematic of EAE induction paradigm. A. \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eand miR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e were injected with myelin oligodendrocyte antigen (MOG\u003csub\u003e35-55\u003c/sub\u003e) with complete Freund’s adjuvant on day 0, along with pertussis toxin (PTX), followed by a second dose of PTX on day 1. \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e animals were subcutaneously injected with control and miR-145 ASOs on days 1 and 3 post-induction. Animals were monitored daily from day 7 to day 30 (chronic phase), and spinal cords were harvested for TEM. B. Representative TEM images of ventral lumbar spinal cord sections from control ASO, \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e and miR-145 ASO-treated mice (B). Scale bars: 2 mm. C-F. Analysis of g-ratio vs. axon diameter correlation (C), g-ratio frequency distribution (D), average g-ratio for all axons (E) or average g-ratio for binned axons (F). G. Analysis of binned relative frequency (G) and total (H) myelinated axon density, and proportion (I). J-K. Morphometric analysis of the proportion of uncompacted myelin (J) and degenerating axons (K) over total myelinated axons. Error bars represent SEM. Each data point in C represents an axon. Data in E, H-K were analyzed using ordinary one-way ANOVA followed by Tukey’s test; C were analyzed with linear regression and data in F-G with two-way ANOVA with Tukey’s post hoc. ** p\u0026lt;0.01, * p\u0026lt;0.05, n=3 mice per group.\u003c/p\u003e","description":"","filename":"Figure10MAandSK2024.png","url":"https://assets-eu.researchsquare.com/files/rs-5462410/v1/9f71f856f84d91e612daa618.png"},{"id":72033231,"identity":"dd52b20a-2898-4bad-b8c7-92a321fc59ff","added_by":"auto","created_at":"2024-12-20 22:01:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":47065029,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5462410/v1/3059cbee-1062-43e7-9f27-d60ee4a3be38.pdf"},{"id":71807219,"identity":"a1776193-3ae7-4fca-95b5-0886c4ac6791","added_by":"auto","created_at":"2024-12-18 17:42:55","extension":"tiff","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":278068,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1. miR-145-5p\u003c/strong\u003e \u003cstrong\u003eis\u003c/strong\u003e \u003cstrong\u003edifferentially expressed in lumbar spinal cord, spleen and thymus.\u003c/strong\u003e A. Relative expression of miR-145-5p in the lumbar spinal cord, spleen, and thymus of \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e age-matched naïve animals. mRNA expression from the spleen and thymus was normalized to the spinal cord. B-D. Relative expression of miR-145-5p in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e lumbar spinal cord (B), spleen (C) and thymus (D) at onset, peak and chronic stages of EAE, normalized to naïve controls. N=3, **=p\u0026lt;0.01, ***=p\u0026lt;0.001, ordinary one-way ANOVA followed by Tukey’s test. Analyzed by DDCt, normalized snU6. Values represent mean ± SEM.\u003c/p\u003e","description":"","filename":"Supp1MAandSK2024.tiff","url":"https://assets-eu.researchsquare.com/files/rs-5462410/v1/cc6a19ac27852a742d87f6f4.tiff"},{"id":71806084,"identity":"bf132c78-3013-4c8b-aa3b-0fd02b679f50","added_by":"auto","created_at":"2024-12-18 17:34:55","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":17560,"visible":true,"origin":"","legend":"","description":"","filename":"SupplTable1MASK2024.docx","url":"https://assets-eu.researchsquare.com/files/rs-5462410/v1/8cf45ee412b2a3a76111cf96.docx"},{"id":71807218,"identity":"801efbbd-90d7-4e24-b0b3-13e73c8cba08","added_by":"auto","created_at":"2024-12-18 17:42:55","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":17989,"visible":true,"origin":"","legend":"","description":"","filename":"SupplTable2MASK2024.docx","url":"https://assets-eu.researchsquare.com/files/rs-5462410/v1/a5a2ddff4de207f4749b40a4.docx"},{"id":71807222,"identity":"4838c885-5335-4032-bf62-0e58a4578773","added_by":"auto","created_at":"2024-12-18 17:42:55","extension":"tiff","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":721446,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2. Cytokine and chemokine expression show alterations in miR-145 deficient thymus and spleen throughout EAE.\u003c/strong\u003e\u0026nbsp; A-L. Relative expression of cytokines (Il1b, Ifng, Il6, and Tnfa) and chemokines (Cxcl1 and Ccl5) in the thymus (A-F) and spleen (G-L) from \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e naïve animals, and at onset, peak and during the chronic stages of EAE. N=3, *=p\u0026lt;0.05, **=p\u0026lt;0.01, ****=p\u0026lt;0.0001, multiple unpaired t-tests. Normalized to Actb and Ppia. Analyzed by DDCt method. Values represent mean ± SEM.\u003c/p\u003e","description":"","filename":"Supp2MAandSK2024.tiff","url":"https://assets-eu.researchsquare.com/files/rs-5462410/v1/1ad242b04d267c0ccdfbf495.tiff"},{"id":71806090,"identity":"0591ddc4-f8c3-473c-8f55-fce39f21cc12","added_by":"auto","created_at":"2024-12-18 17:34:55","extension":"tiff","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":760568,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 3. miR-145 ASO injection at the peak of EAE does not alter disease severity, although it efficiently knocked down miR-145 in the spleen and spinal cord. \u003c/strong\u003eA. Schematic representation of EAE induction and ASO treatment. Female C57BL/6 mice aged 9-13-week-old were subjected to EAE. Animals were followed daily from day 7 post-EAE induction, and at day 16 post-induction (~peak of disease), were subcutaneously injected with the negative control or miR-145 ASOs at a single dose of 20 μg/mouse. B. Mean clinical scores over the course of EAE. C. Mean weights expressed as a percentage of starting weight during EAE. N=4, multiple unpaired t-tests. Values represent mean ± SEM. \u0026nbsp;D. Mean score at the chronic phase of EAE (Day 30). N=4 for each treatment, two-tailed unpaired Student’s t-tests. E-F. Relative expression of miR-145-5p in the spleen (E) and SC (F) at the chronic stage of EAE from control and miR-145 ASOs. Values represent mean ± SEM, ****=p\u0026lt;0.0001. Analyzed by DDCt, normalized snU6. Values represent mean ± SEM.\u003c/p\u003e","description":"","filename":"Supp3MAandSK2024.tiff","url":"https://assets-eu.researchsquare.com/files/rs-5462410/v1/7bbc14a75752e4ed7fdd9875.tiff"},{"id":71806096,"identity":"2a713c83-004d-4396-8391-ca501397f298","added_by":"auto","created_at":"2024-12-18 17:34:56","extension":"tiff","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":3822510,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 4. FAM-ASO distributes across different tissues and accumulates in various CNS cell types. \u003c/strong\u003eA. Schematic representation of EAE induction and FAM-ASO treatment. \u0026nbsp;Female C57BL/6 mice aged 9-13-week-old were subjected to EAE and injected subcutaneously with FAM-labelled control or miR-145 ASOs on day 3 post-EAE induction. Animals were monitored daily from day 7 to day 11 post-EAE induction. On day 11, tissues were harvested. B. Qualitative images of the liver, spleen, and spinal cord from negative controls and ASO-treated animals. Scale bar = 50 mm. N=2 for each treatment. C. Schematic representation of organotypic spinal cord cultures. Cultures were maintained for 10 days \u003cem\u003ein vitro\u003c/em\u003e, followed by transfection with either 50 nM of FAM-labeled control or miR-145 ASOs for 24 h. D. Schematic illustrating that cells lacking miR-145 expression exhibit minimal fluorescence due to specific binding of the FAM-labeled ASO to its complementary target RNA. Conversely, the mismatched control ASO also shows minimal fluorescence, indicating successful transfection with limited microRNA interaction. In contrast, miR-145-expressing cells are expected to show a specific accumulation of FAM-labeled miR-145 ASO. E-I. Spinal cord organotypic cultures were fixed and stained for Olig2 (E, blue), CC1 (E, pseudo-green), IBA1 (F, magenta), NeuN (G, red), Nestin (H, red), and GFAP (I, magenta). FAM-ASO is shown as pseudo-red in E and as green in F-I. N=2. Scale bar = 20 mm.\u003c/p\u003e","description":"","filename":"Supp4MAandSK2024.tiff","url":"https://assets-eu.researchsquare.com/files/rs-5462410/v1/fca6c082906ec5f9e18aaa49.tiff"}],"financialInterests":"No competing interests reported.","formattedTitle":"Harnessing mIR-145 deficiency to modulate inflammation and prevent degeneration in a mouse model of multiple sclerosis","fulltext":[{"header":"Background","content":"\u003cp\u003eMultiple sclerosis (MS) is the most prevalent immune-mediated inflammatory demyelinating disease of the central nervous system (CNS), significantly impacting society due to its high incidence among young adults(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). The hallmark of MS is the destruction of myelin \u0026ndash; a vital lipid-rich sheath that encases and safeguards nerve fibres, which is essential for maintaining axonal health and facilitating effective neurotransmission(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Demyelination is driven by myelin-specific, self-reactive T and B lymphocytes that aberrantly infiltrate the CNS and target oligodendrocytes (OLs), the cells responsible for myelin production(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). While recent studies have identified Epstein-Barr virus (EBV) as a risk factor for MS(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e), EBV alone is insufficient to trigger the disease, underscoring the multifaceted and multifactorial nature of MS(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe classification of MS is broad, with the most recognized categories being relapsing-remitting MS (RRMS), primary progressive MS (PPMS), and secondary progressive MS (SPMS)(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). RRMS is the most common form, characterized by acute relapses, during which immune attacks result in focal inflammatory demyelination and subsequent neurological decline, followed by periods of remission. RRMS patients often experience cumulative disease burden over time, eventually transitioning to SPMS, where remissions are rare, and disability is pronounced. In contrast, PPMS is marked by continuous neurological deterioration from the onset, resulting in a greater overall disease burden despite its slow progression(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCurrent disease-modifying therapies effectively mitigate relapses in RRMS but are less efficient in preventing the transition to SPMS and show limited efficacy in treating established progressive forms of MS(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Furthermore, while many treatments focus on modulating immune responses to address active inflammation, they do not adequately prevent myelin loss or axonal degeneration, nor do they enhance remyelination \u0026ndash; processes that are critical for halting disease progression and promoting regeneration(\u003cspan additionalcitationids=\"CR11 CR12 CR13\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). This underscores a critical gap in therapy and highlights the urgent need for novel, combinatorial approaches that simultaneously target both the inflammatory and degenerative processes of the disease.\u003c/p\u003e \u003cp\u003eIn previous work, we identified microRNA-145-5p (herein referred to as miR-145) as a negative regulator of OL differentiation in primary OL cultures, where its expression is crucial for maintaining OL precursor cells (OPCs) in a proliferative and undifferentiated state(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Notably, miR-145 is overexpressed in chronic inactive lesions of progressive MS patients compared to both healthy controls and active lesions from RRMS patients(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Additionally, our recent findings indicate that miR-145 is particularly abundant in chronic lesions from secondary progressive MS patients(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). In miR-145 knockout mice subjected to chronic cuprizone-induced demyelination, we observed enhanced functional recovery and increased numbers of myelinated axons. Consistent with this, there is a pathological upregulation of miR-145 in wild-type mice exposed to chronic cuprizone treatment(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Furthermore, miR-145 is elevated in peripheral blood mononuclear cells (PBMCs), serum, and plasma from treatment-na\u0026iuml;ve RRMS and early SPMS patients(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Given the inflammatory nature of MS and the role of peripheral immune cells in disease pathophysiology, further research is necessary to elucidate the beneficial effects of targeting miR-145 in autoimmune-driven inflammatory demyelination.\u003c/p\u003e \u003cp\u003eHere, we investigated the impact of miR-145 loss in recovery from experimental autoimmune encephalitis (EAE) in mice. The EAE mouse model of multiple sclerosis closely mimics the immune characteristics of the disease(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Our results revealed that miR-145-deficient mice exhibited reduced disease burden and decreased immune cell infiltration. This prompted us to assess the therapeutic benefit of an antisense oligonucleotide (ASO) specifically targeting miR-145. We tested whether its administration could improve recovery and prevent degeneration in EAE, while also elucidating the cellular mechanisms by which miR-145 deficiency enhances clinical outcomes. Notably, we observed a significant reduction in clinical severity in the EAE animals, correlated with decreased levels of CD3\u0026thinsp;+\u0026thinsp;and CD45\u0026thinsp;+\u0026thinsp;leukocytes. Building on our prior research highlighting the role of miR-145 in OL differentiation(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), these results suggest a dual function for miR-145 in both modulating inflammatory responses and alleviating degenerative processes in EAE. This dual action positions miR-145 as a promising therapeutic target that addresses both inflammatory and neurodegenerative aspects of MS.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eLoss of miR-145 results in reduced clinical severity throughout the course of EAE\u003c/h2\u003e \u003cp\u003eWe previously demonstrated enhanced remyelination and recovery from cuprizone-induced toxic demyelination using the constitutive miR-145 knockout mouse model (\u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice)(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). In the cuprizone model, inflammation is restricted to the CNS and leads to OL cell death and subsequent demyelination(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Thus, this model does not fully depict the pathophysiology of MS, as it lacks the peripheral immune components that are critical to the disease. Given that miR-145 is dysregulated in RRMS and is expressed widely both within and outside the CNS(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e), we employed EAE, a murine model of RRMS, to assess the impact of miR-145 absence on autoimmune-driven demyelination.\u003c/p\u003e \u003cp\u003e \u003cem\u003emiR-145\u003c/em\u003e \u003csup\u003e \u003cem\u003e+/+\u003c/em\u003e \u003c/sup\u003e and \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice were subjected to EAE induced by \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003emyelin oligodendrocyte glycoprotein peptide fragment 35\u0026ndash;55 (\u003c/span\u003eMOG\u003csub\u003e35\u0026thinsp;\u0026minus;\u0026thinsp;55\u003c/sub\u003e) antigen. Disease progression was assessed by monitoring clinical scores, which were based on the degree of ascending hind limb paralysis and quantified using a standard clinical score system (0\u0026ndash;5 scale)(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e), along with body weight changes over the 30-day course of disease (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). All induced animals developed EAE irrespective of genotype. Notably, the severity of disease was diminished in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e animals, as evidenced by reduced clinical scores and improved weight retention (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C). The onset of disease, marked by the first observable clinical symptom, was slightly delayed in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, with perceptible symptoms emerging approximately 1.5 days later (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) than in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, E). Additionally, \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice exhibited a reduced mean clinical score at onset (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, p\u0026thinsp;=\u0026thinsp;0.0199) and a delayed first instance of paralysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) relative to the time of EAE induction (i.e., day 0). However, the time from symptom onset (score\u0026thinsp;\u0026gt;\u0026thinsp;0) to paralysis was similar in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). At the peak of the disease, the average maximum clinical score attained was lower in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI, p\u0026thinsp;=\u0026thinsp;0.0043). Furthermore, among animals that experienced hind limb paralysis (score \u0026ge;3), the duration of paralysis was significantly shorter in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). When analyzing the entire cohort of EAE animals, the proportion that experienced a relapse - defined as more than one instance of paralysis - was 52.7% in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e mice compared to 17.7% in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice. Moreover, only 6.3% of \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e animals did not reach a state of paralysis, compared to 23.1% of \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK). Lastly, at the chronic stage, \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e animals exhibited a lower clinical score than \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL, p\u0026thinsp;=\u0026thinsp;0.0066), underscoring the impact of miR-145 deficiency in reducing disease burden in EAE.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMyelin retention is improved in miR-145 deficient spinal cord\u003c/h3\u003e\n\u003cp\u003eTo evaluate the pathological changes associated with the altered clinical course of EAE in miR-145 deficient mice, we assessed myelin content in the lumbar spinal cord \u0026ndash; a region primarily affected in EAE(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e) \u0026ndash; in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice. Tissue samples were collected from na\u0026iuml;ve animals, as well as at the onset, peak, and chronic stages of EAE. Onset samples were obtained on the day the first clinical symptom was observed, peak samples were taken on the first day animals achieved a clinical score of 3.0 or higher (indicating hind limb paralysis), and chronic samples were collected arbitrarily on day 30 post-induction.\u003c/p\u003e \u003cp\u003eCross-sections of the lumbar spinal cord were immunostained for myelin basic protein (MBP), and the MBP-immunopositive area was quantified as a proportion of the total spinal cord section area (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). No significant differences were noted between na\u0026iuml;ve \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e animals. As anticipated, \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e animals demonstrated a progressive loss of myelinated area throughout the different phases of EAE. In contrast, consistent with the reduced clinical scores, \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e animals retained 1.25-fold more (p\u0026thinsp;=\u0026thinsp;0.0171) myelinated area over \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e mice at onset, with notable MBP retention in the grey matter (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). At the chronic stage, \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice exhibited a trending, yet non-significant, 1.28-fold greater myelinated area (p\u0026thinsp;=\u0026thinsp;0.0534), with more pronounced MBP retention in the white matter compared to their \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). However, similar levels of myelin loss were observed at the peak of EAE between the two genotypes, as all animals assessed at this stage were selectively collected while experiencing hind limb paralysis with a clinical score of 3.0 or higher (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eLoss of miR-145 reduces immune cell infiltration during different phases of EAE with minor alterations in cytokine and chemokine expression in peripheral immune tissues\u003c/b\u003e \u003c/p\u003e \u003cp\u003eImmune cell infiltration in the lumbar spinal cord is a hallmark of EAE(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e), and this was assessed using histological H\u0026amp;E staining. As expected, na\u0026iuml;ve animals showed no subpial or parenchymal dense nuclei accumulations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). At the onset of the disease, \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e animals exhibited significant infiltration characterized by accumulation of dense nuclei, particularly in the ventral horn as well as at the dorsal root-spinal cord junction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). In contrast, \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice displayed a 12.70-fold reduction (p\u0026thinsp;=\u0026thinsp;0.0024) in cell infiltration compared to their \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e counterparts, with infiltration primarily localized to the ventral horn (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). Infiltration was extensive in both genotypes at the peak of EAE, as expected, given that mice were selectively collected while experiencing hindlimb paralysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, C). At the chronic stage, the spinal cords from \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e mice exhibited extensive infiltration, while those from \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice showed a 2.20-fold reduction in cell infiltration (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, D, p\u0026thinsp;=\u0026thinsp;0.0010). These findings align with the improved myelination status observed in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e animals at both the onset and chronic stages of EAE, suggesting that the absence of miR-145 may delay immune cell infiltration into the CNS.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe further evaluated the expression of key cytokines and chemokines in the spinal cord across all stages of EAE (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-J). mRNA expression of several genes was altered at disease onset in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e animals, including significant downregulation of Il1b (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, 9.62-fold decrease), Ifng (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, 6.65-fold decrease), Il6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, 6.44-fold decrease), Tnfa (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH, 8.90-fold decrease) and Ccl5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ, 30.11-fold decrease), alongside upregulation of Cxcl1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI, 2.92-fold increase). Additional changes in gene expression were observed at peak, with Il1b showing a 1.74-fold increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), and during the chronic stage, both Il6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, 72.50-fold decrease) and Ccl5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ, 1.80-fold decrease) were downregulated.\u003c/p\u003e \u003cp\u003emiR-145 is implicated in peripheral immune responses(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e) and is abundantly expressed in the spleen and thymus (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Additionally, miR-145-5p levels were higher in the spinal cord and spleen at the onset of EAE (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C), while levels in the thymus remained unaltered throughout the different phases of EAE (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Consequently, we investigated whether the reduced immune cell infiltration observed in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e EAE-subjected spinal cords at both the onset and chronic stages could be attributed to alterations in cytokine and chemokine expression, potentially influencing peripheral immune cell activation in EAE. The thymus and spleen (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-L) were assessed for cytokines and chemokines known to play roles in EAE development in the periphery. Significant downregulations were detected in thymus (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-F), including reduced expression of Tnfa (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, 1.45-fold decrease) and Cxcl1 (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, 1.86-fold decrease, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) at disease onset, as well as reduced expression of Ccl5 in na\u0026iuml;ve \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e animals (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, 2.28-fold decrease). Conversely, significant upregulation was observed in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e spleen, including Ifng (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH, 1.89-fold increase) at the peak of the disease and Cxcl1 during the chronic stage (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL, 4.33-fold increase). No other significant changes in gene expression were noted in any assessed factors at any time point in either the thymus or spleen.\u003c/p\u003e\n\u003ch3\u003eNeuroinflammatory responses are altered in miR-145 deficient animals\u003c/h3\u003e\n\u003cp\u003eDuring autoimmunity-driven inflammatory demyelination, cytokine/chemokine expression reflects not only peripheral immune cell activity but also the responses of glial cells, such as microglia and astrocytes(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). To determine whether changes in the neuroinflammatory landscape correspond with the improved clinical outcome and differing pathological findings observed in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e animals, we evaluated the activation of microglia and astrocytes. Sections of lumbar spinal cords from \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice were stained for Iba1, a marker of microglia/macrophages, at various stages of EAE (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-E). Total microglia/macrophage numbers were quantified in the parenchyma, excluding submeningeal areas of dense cellular infiltration. Mild alterations in microglial activation were observed during EAE in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e animals compared to \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B), with microglial numbers showing a trending yet non-significant reduction at onset (~\u0026thinsp;55% reduction, p\u0026thinsp;=\u0026thinsp;0.123) and during the chronic stage (~\u0026thinsp;38% reduction, p\u0026thinsp;=\u0026thinsp;0.084), along with a trending increase at peak (~\u0026thinsp;53% increase, p\u0026thinsp;=\u0026thinsp;0.084).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe further assessed the expression of Ym1, expressed in alternatively activated microglia/macrophages (M2-like cells)(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), iNos, an important pro-inflammatory mediator(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e), and Nurr1, an anti-inflammatory factor regulated by miR-145(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Notably, expression changes were observed across all stages of EAE in Ym1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) and iNos (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Ym1 was downregulated at all phases of EAE in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, showing a 61.72-fold decrease at onset, a 2.39-fold decrease at peak, and 6.89-fold decrease at the chronic stage. Conversely, iNos was downregulated at onset (6.05-fold decrease) and chronic phases (17.02-fold decrease) but upregulated at the peak (3.38-fold increase). These findings correlate with the altered numbers of microglia at these time points. Nurr1, a negative regulator of TNFa, was increased 4.21-fold at onset (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), but no significant changes were observed at peak or chronic phases (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eAstrocytes, which play crucial roles in neuroinflammation(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), were evaluated by immunostaining spinal cord sections with glial fibrillary acid protein (GFAP) antibodies, a well-known astrocyte marker. We quantified both the total numbers of GFAP\u0026thinsp;+\u0026thinsp;astrocytes and the number of hypertrophic reactive astrocytes, defined by GFAP high signal and thickened processes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-E). Na\u0026iuml;ve \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e animals showed similar astrocyte numbers and reactivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B). At both onset (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, C) and peak (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, D), \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice exhibited higher total astrocyte numbers (~\u0026thinsp;37% increase, p\u0026thinsp;=\u0026thinsp;0.049 at onset and ~\u0026thinsp;39%, p\u0026thinsp;=\u0026thinsp;0.038 at peak) and reactive astrocytes (~\u0026thinsp;159% increase, p\u0026thinsp;=\u0026thinsp;0.049 at onset and ~\u0026thinsp;187%, p\u0026thinsp;=\u0026thinsp;0.038 at peak). Interestingly, reactive astrocytes at onset were diffusely distributed throughout the parenchyma in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e spinal cords, whereas in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e, they were primarily localized to submeningeal areas (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In contrast, at the chronic stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, E), \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e animals displayed fewer total astrocytes (45% reduction, p\u0026thinsp;=\u0026thinsp;0.039) and fewer reactive astrocytes (~\u0026thinsp;68% reduction, p\u0026thinsp;=\u0026thinsp;0.039). This suggests that astrocyte-driven inflammation occurs more rapidly and with greater intensity but subsides earlier in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e animals when compared to their wild-type counterparts.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo confirm the role of astrocytes in the modulation of inflammation driven by miR-145 deficiency, we assessed the relative expression of GFAP and aquaporin 4 (AQP4), a component of astrocytic end feet that is crucial for maintaining blood-brain barrier integrity and is downregulated when miR-145-5p is overexpressed(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). We assessed their expression at the mRNA level in the spinal cords of na\u0026iuml;ve animals as well as throughout EAE. GFAP mRNA expression was altered at EAE onset (5.34-fold increase) and chronic phases (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF, 1.35-fold decrease), correlating with observed changes in astrocyte reactivity at these time points. Surprisingly, AQP4 mRNA expression was downregulated at disease onset (1.33-fold decrease), with no significant changes detected at other time points (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). Together, these findings indicate broad modifications to the CNS resident immune response resulting from the loss of miR-145, although the underlying mechanisms for these observations remain to be elucidated.\u003c/p\u003e\n\u003ch3\u003eAcute miR-145 knockdown with an antisense oligonucleotide (ASO) reduces disease severity\u003c/h3\u003e\n\u003cp\u003eTo translate our findings into a therapeutic setting, we developed an ASO specifically designed to acutely and transiently inhibit miR-145. WT mice were injected with 20 \u0026micro;g/mouse of custom \u003cem\u003ein vivo\u003c/em\u003e miRCURY negative control and miR-145-5p ASOs via subcutaneous injections.\u003c/p\u003e \u003cp\u003eInitially, we aimed to determine whether transient antagonism of miR-145 at the peak of EAE would lead to reduced clinical severity later in the disease. A single dose of either control or miR-145 ASOs was administered at the peak of EAE (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Given that ASOs do not penetrate the CNS, this timing was chosen to exploit the compromised blood-spinal cord barrier (BSCB) during EAE, thereby enhancing ASO bioavailability within the CNS. Mice were scored daily from day 7 to day 30 post-EAE induction. However, although there was a trend to improved response, treatment with the miR-145 ASO did not result in significant changes in clinical outcomes, as measured by mean clinical score (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) and percentage weight loss (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) throughout the course of EAE. Similarly, the mean disease score at the chronic phase was not significantly altered (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, p\u0026thinsp;=\u0026thinsp;0.1682), despite observing a 75% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) reduction in miR-145 levels in the spleen (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE) and a 67% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) reduction in the spinal cord (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eIn \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e animals, a marked reduction in immune cell infiltration was observed at both the onset and chronic phases of EAE (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-D). To further explore this, we modified our approach by administering ASOs on days 1 and 3 post-EAE induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), aiming to mimic the conditions in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e EAE-subjected animals. This strategy allowed us to evaluate the prophylactic benefits of miR-145 antagonism following myelin antigen immune cell priming and to investigate the cellular mechanisms associated with miR-145 knockdown that may lead to improved clinical outcomes in EAE.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOverall, disease severity was notably attenuated in miR-145 ASO-treated animals during the peak of the disease, as indicated by a reduced clinical score at 16 days post-induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, p\u0026thinsp;=\u0026thinsp;0.0113). However, weight retention did not significantly differ between control and miR-145 ASO-treated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). The onset of disease was delayed in miR-145 ASO-treated animals, with the first symptoms emerging approximately 2 days earlier in controls compared to miR-145 ASO-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, p\u0026thinsp;=\u0026thinsp;0.0013). Although the mean clinical score at onset did not significantly differ (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE), the mean clinical score at peak was lower in miR-145 ASO-treated mice (p\u0026thinsp;=\u0026thinsp;0.0469) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). At the chronic phase, miR-145 ASO-treated mice eventually exhibited a disease score comparable to that of their control counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG), although the average maximum clinical score was only slightly reduced (p\u0026thinsp;=\u0026thinsp;0.0535) in these animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH).\u003c/p\u003e\n\u003ch3\u003emiR145 ASO reaches the CNS and efficiently reduces miR-145 levels\u003c/h3\u003e\n\u003cp\u003eWe further evaluated the efficacy of miR-145 knockdown in lymphoid and spinal cord tissues at the chronic phase of EAE. Administration of the miR-145 ASO resulted in a significant reduction of miR-145-5p levels: an 84% decrease (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) in the spleen (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI), a 62% decrease (p\u0026thinsp;=\u0026thinsp;0.0050) in the thymus (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ), and a 32% decrease (p\u0026thinsp;=\u0026thinsp;0.0018) in the spinal cord (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK). Importantly, EAE-subjected mice exhibited no observable signs of toxicity following subcutaneous administration of ASOs.\u003c/p\u003e \u003cp\u003eTo gain further insights into the distribution and cell-specific targeting of the miR-145 ASO, we developed a custom fluorescent dye attachment (5' FAM labelled) for both control and miR-145 ASOs. A single injection at half the dosage was administered on day 3 post-induction, and tissues were harvested on day 11 (corresponding to EAE disease onset) to assess ASO distribution across various tissues (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eWe harvested and sectioned the liver, spleen, and spinal cord tissues \u0026ndash; predicted major sites of ASO accumulation in our study \u0026ndash; to evaluate tissue-specific distribution. Tissue exposure times were standardized across conditions based on non-treated controls. Our data revealed more intense fluorescent signal in the liver and spleen, with only mild fluorescence detected in the spinal cord compared to non-treated controls. In the spinal cord, the signal appeared to be more localized within blood vessels (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eIn a secondary approach, we examined the cell-specific accumulation of ASOs administered directly into the CNS. We utilized spinal cord organotypic cultures whereby cultures were kept for 10 days \u003cem\u003ein vitro\u003c/em\u003e, followed by a 24 h treatment with FAM-labeled ASOs (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Subsequent co-staining with specific markers for various CNS cell types revealed that, although ASO controls exhibited a positive signal, co-localization with CNS cells was lower than compared to miR-145 FAM-labeled ASO-treated animals. This difference may be attributed to the degradation of ASOs by nucleases, as control ASOs are missense oligonucleotides without known binding to microRNAs. Conversely, miR-145-expressing cells displayed more frequent accumulation of the miR-145 FAM-labeled ASO in CNS cells (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-I).\u003c/p\u003e \u003cp\u003eSpecific accumulation of ASOs was observed within CC1\u0026thinsp;+\u0026thinsp;Olig2\u0026thinsp;+\u0026thinsp;oligodendrocytes (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), IBA1\u0026thinsp;+\u0026thinsp;microglia/macrophages (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF), NeuN\u0026thinsp;+\u0026thinsp;neurons (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG), and Nestin\u0026thinsp;+\u0026thinsp;neural precursor cells (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH), with microglia/macrophages demonstrating the highest levels of accumulated miR-145 ASO signal. Notably, no positive signal was detected within GFAP\u0026thinsp;+\u0026thinsp;astrocytes (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). Collectively, these findings suggest that the miR-145 ASO may also exert effects in CNS cells to promote myelin regeneration.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAcute miR-145 knockdown reduces specific immune cell infiltration in the lumbar SC at the peak of EAE\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further explore the immune cell landscape in the CNS and its association with improved clinical severity following miR-145 ASO administration, we collected lumbar spinal cord tissue on day 16 post-EAE induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), coinciding with the peak of hindlimb paralysis in most animals. The lumbar spinal cord was stained for various immune cell markers, including clusters of differentiation 3 (CD3), 4 (CD4), 45 (CD45), and 19 (CD19), as well as forkhead box P3 (FoxP3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn miR-145 ASO-treated animals, we observed a\u0026thinsp;~\u0026thinsp;3-fold decrease (p\u0026thinsp;=\u0026thinsp;0.0018) in the number of CD3\u0026thinsp;+\u0026thinsp;T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, C). While no significant differences were noted in the proportion of CD4\u0026thinsp;+\u0026thinsp;helper T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, D), the proportion of regulatory FoxP3\u0026thinsp;+\u0026thinsp;T cells increased by ~\u0026thinsp;2-fold (p\u0026thinsp;=\u0026thinsp;0.0286) in these animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, E). Additionally, there was a 1.6-fold decrease (p\u0026thinsp;=\u0026thinsp;0.0199) in the number of CD45\u0026thinsp;+\u0026thinsp;cells of hematopoietic origin (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF, G), while the numbers of CD19\u0026thinsp;+\u0026thinsp;B cells remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF, H). Collectively, these findings underscore the role of miR-145 in regulating the migration and polarization of peripheral immune cells.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMyelin and axon degeneration is attenuated in miR-145 deficient animals\u003c/h2\u003e \u003cp\u003eWe next assessed myelin at the ultrastructural level using transmission electron microscopy (TEM) in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e, and ASO-treated animals during different phases of EAE. At disease onset (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e), \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice exhibited no significant differences in myelin thickness compared to \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-D). In contrast, acute miR-145 ASO knockdown resulted in a significant reduction in g-ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eL-N, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), although this did not reach statistical significance when averaged (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eO, p\u0026thinsp;=\u0026thinsp;0.0899). Frequency distribution of g-ratios revealed that both \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE) and miR-145 ASO-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eP) displayed a shift toward a smaller g-ratio (indicating thicker myelin).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhen g-ratios were averaged and binned into three axon diameter groups, no significant changes were observed for axons up to 2 \u0026micro;m or within the 2\u0026ndash;3 \u0026micro;m and \u0026gt;\u0026thinsp;3 \u0026micro;m diameter categories (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF, Q). The relative abundance of axons across these bins did not change in either group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG, R), nor did axonal density, although a clear increasing trend was noted (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eH, S). Notably, in areas enriched with myelinated axons, there were no changes in the proportion of total myelinated axons (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eI, T), axons with uncompacted myelin (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eJ, U), or those undergoing degeneration (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eK, V), all important indicators of myelin health and neurodegeneration(\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAt the peak of the disease (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e), differences emerged, particularly between \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and miR-145 ASO-treated mice. In \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, no changes were seen in myelin thickness distribution (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB-C), while the average g-ratio was increased compared to \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e controls, indicating thinner myelin in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eD) and ongoing remyelination. Conversely, miR-145 ASO knockdown exhibited a trend towards thicker myelin thickness (p\u0026thinsp;=\u0026thinsp;0.0717, Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eM-O). Similarly, frequency distribution of g-ratios indicated that \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eE) showed a distribution toward thinner myelin, whereas miR-145 ASO-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eP) trended toward thicker myelin.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhen g-ratios were averaged and binned into three axon diameter groups, no significant changes were observed for axons up to 2 \u0026micro;m or within the 2\u0026ndash;3 \u0026micro;m and \u0026gt;\u0026thinsp;3 \u0026micro;m diameter categories in both \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e compared to \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eF) and in miR-145 ASO-treated compared to control ASO-treated animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eQ). In \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e, however, a trending increase in g-ratio was observed for axons within the 2\u0026ndash;3 \u0026micro;m and \u0026gt;\u0026thinsp;3 \u0026micro;m diameter axons (p\u0026thinsp;=\u0026thinsp;0.0684). The relative abundance of axons across these bins did not change (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eG, R). \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice did not show changes in axonal density, whereas miR-145 ASO-treated animals displayed a trend towards increased axon density (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eH, S, p\u0026thinsp;=\u0026thinsp;0.0827).\u003c/p\u003e \u003cp\u003eIn myelinated axon-rich areas, the proportion of myelinated axons remained unchanged in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eI), while a slight yet significant increase was observed in miR-145 ASO-treated animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eT, p\u0026thinsp;=\u0026thinsp;0.0361). Interestingly, the proportion of axons with uncompacted myelin (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eJ, U) remained unchanged across both groups, but \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e animals displayed a reduced proportion of degenerating axons (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eK, p\u0026thinsp;=\u0026thinsp;0.0011), while no changes were observed in miR-145 ASO-treated animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eV).\u003c/p\u003e \u003cp\u003eAt the chronic phase of EAE (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e), axons that remained myelinated in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e controls trended toward thicker myelin, but no differences were found in g-ratios (distribution, frequency, or average) in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e or miR-145 ASO-treated animals compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB-E). A significant increase was noted in the g-ratio (p\u0026thinsp;=\u0026thinsp;0.0323) for axons up to 2 \u0026micro;m in diameter in miR-145 ASO-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eF), with no changes found in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e or other diameter bins. The relative abundance of axons across these bins did not change in either group (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eG). Notably, a 1.7-fold increase in axonal density was found in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e compared to \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e controls (p\u0026thinsp;=\u0026thinsp;0.0494), while no statistical significance was observed in miR-145 ASO-treated animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn areas enriched with myelinated axons, an increased proportion of myelinated axons was found in both \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e (p\u0026thinsp;=\u0026thinsp;0.0062) and miR-145 ASO-treated animals (p\u0026thinsp;=\u0026thinsp;0.0172). The proportion of axons with uncompacted myelin (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eJ) was reduced only in miR-145 ASO-treated animals (p\u0026thinsp;=\u0026thinsp;0.0327). Finally, the proportion of degenerating axons (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eK) was reduced by 88% in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and by 63% in miR-145 ASO-treated animals (p\u0026thinsp;=\u0026thinsp;0.0101 and p\u0026thinsp;=\u0026thinsp;0.0425, respectively).\u003c/p\u003e \u003cp\u003eOverall, these data highlight that targeting miR-145 may prevent axonal and myelin degeneration, potentially leading to improved clinical outcomes in EAE.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn RRMS, miR-145 has emerged as a significant circulating biomarker, distinguishing between RRMS and SPMS in comparison to healthy controls(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Furthermore, its dysregulation is normalized in patients exhibiting successful treatment responses to interferon-beta (IFN-β)(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). However, the role of miR-145 in the pathogenesis and progression of MS has yet to be elucidated. To investigate this, we utilized a miR-145 knockout mouse model in conjunction with the EAE paradigm, a widely accepted mouse model for RRMS.\u003c/p\u003e \u003cp\u003eOur findings demonstrate that the absence of miR-145 yields substantial clinical benefits in EAE, characterized by a delayed onset of symptoms and a marked reduction in clinical severity. Notably, a significant proportion of miR-145-deficient animals did not progress to hindlimb paralysis, and those that did exhibited a notably faster recovery. Furthermore, during the chronic phase of EAE, miR-145-deficient mice displayed improved clinical scores, indicating a greater recovery trajectory.\u003c/p\u003e \u003cp\u003eThe loss of miR-145 significantly impacted immune cell infiltration, leading to notable reductions at both the onset and during the chronic stages of the disease. However, at the peak of the disease, animals exhibited similar levels of infiltration. Research indicates that miR-145 plays dual roles in pro-inflammatory and anti-inflammatory responses across various contexts in the periphery(\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Additionally, dysregulation of miR-145 has been observed in PBMCs, serum and plasma of individuals with RRMS and early SPMS(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). In our study, we identified differential expression of miR-145 in the spinal cord and spleen at disease onset. Notably, we found that miR-145 expression in na\u0026iuml;ve wild-type animals is approximately 25-fold higher in the spleen and about 625-fold higher in the thymus compared to the lumbar spinal cord, indicating that this microRNA may play a crucial role in peripheral lymphoid organs.\u003c/p\u003e \u003cp\u003eWe also observed that reduced cellular infiltration coincided with decreased expression of several pro-inflammatory cytokines and chemokines, including Ifnγ, Tnfα, Il1β, Il6, and Ccl5 at onset, and Il6 and Ccl5 during the chronic stage. These factors are secreted by infiltrating T cells, monocytes/macrophages, and various CNS cell types(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Their diminished expression aligns with the reduction in both infiltrating populations observed with miR-145 loss. This reduction is critical to the inflammatory cascade in EAE, underscoring the potential regulatory role of miR-145 in modulating immune responses.\u003c/p\u003e \u003cp\u003eNeuroinflammatory responses, particularly involving microglia and astrocytes, were evident at all stages of EAE in miR-145-deficient mice. In our model, reduced microglial activation was mild at both the onset and chronic stages. Previous studies indicate that microglial activation occurs before the onset of EAE symptoms and the infiltration of immune cells into the CNS(\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). This has significant implications for the blood-brain barrier (BBB), as activated microglia can alter the expression of factors critical for maintaining tight junctions among endothelial cells lining the neurovasculature, thus facilitating immune cell entry into the CNS(\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe relationship between alterations in microglial behavior and the loss of miR-145 remains somewhat unclear, as few miR-145-regulated pathways in microglia have been identified to date(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). One known target of miR-145 in microglia is Nurr1. Previous studies have shown that downregulation of Nurr1 via miR-145 promotes microglial inflammation by negatively regulating TNF-α(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). In our findings, Nurr1 expression was notably upregulated in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, particularly at the onset of EAE, which coincided with TNF-α downregulation in the spinal cord and spleen, as well as with miR-145 upregulation in wild-type mice. This suggests that the Nurr1 pathway may play a role in reducing disease severity in EAE.\u003c/p\u003e \u003cp\u003eAdditionally, miR-145 has been associated with the polarization of activated microglia/macrophages, promoting a shift towards an anti-inflammatory M2-like phenotype rather than a pro-inflammatory M1-like phenotype, as shown in ischemia models(\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Consequently, we aimed to determine whether the loss of miR-145 leads to diminished pro-inflammatory response in EAE. To explore this, we assessed the expression of the well-known inflammation-associated gene iNos and the anti-inflammatory gene Ym1(\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e) throughout the course of EAE. Both markers were reduced at the onset and chronic stages in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, likely reflecting an overall decrease in the number of activated microglia /macrophages and cell infiltrates during these time points.\u003c/p\u003e \u003cp\u003eAt the peak of the disease, iNos was upregulated, while Ym1 remained downregulated in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice. This suggests that microglia/macrophage activation during the disease peak favoured an inflammatory phenotype. Nonetheless, recent studies using single-cell RNA sequencing indicate that activated microglia/macrophages represent a far more diverse population than the traditional M1/M2 dichotomy(\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Interpreting innate immune activation solely through this lens may oversimplify the complex heterogeneity present, particularly following demyelination(\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAstrocyte activation (astrogliosis), characterized by an increased number of astrocytes and morphological changes(\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e), was altered in miR-145-deficient animals compared to wild-type throughout the course of EAE. Specifically, astrocyte activation was heightened during the onset and peak stages but decreased in the chronic stage in miR-145-deficient mice. Previous research suggests that early and robust astrocyte activation is beneficial, while late activation can worsen EAE outcomes. In contrast, during the chronic stage, excessive astrocyte activation may be detrimental, whereas reduced activation can improve clinical outcomes(\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). The early loss of astrocyte activation in these studies has been associated with compromised BBB integrity, increased leukocyte infiltration, and more severe demyelination. Conversely, reduced activation during the chronic stage has been linked to enhanced remyelination(\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). Therefore, the loss of miR-145 appears to promote a more favourable pattern of astrocyte activation throughout EAE, likely contributing to the observed improvements in clinical outcomes and underlying pathologies in our model.\u003c/p\u003e \u003cp\u003eTo elucidate the cellular mechanisms by which miR-145 enhances clinical outcomes and to evaluate its potential as a prophylactic therapy, we developed an ASO targeting miR-145 to reduce its levels following immune priming. While ASO administration did attenuate disease severity, the effects were less pronounced than those observed in the \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice. This difference may be due to suboptimal delivery to the CNS, highlighting the need for improved CNS-targeting strategies.\u003c/p\u003e \u003cp\u003eAxon damage is closely linked to crosstalk between lymphocytes and microglia. Previous work has shown that in progressive MS, meningeal T-cell infiltration in the spinal cord is prominent, outnumbering parenchymal T cells, and exhibits close interactions with meningeal macrophages(\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). This suggests that the spinal cord meninges may function as an immunological niche where T lymphocytes are activated through antigen presentation, potentially instructing parenchymal macrophages and microglia to engage in neurotoxic activation(\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHere, acute knockdown of miR-145 reduced CD3\u0026thinsp;+\u0026thinsp;lymphocytes and CD45\u0026thinsp;+\u0026thinsp;leukocytes while increasing the proportion of FoxP3\u0026thinsp;+\u0026thinsp;regulatory T cells. These findings align with observations in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, where immune cell infiltration was reduced in EAE. This suggests that miR-145 may play a role in regulating leukocyte recruitment into the spinal cord during EAE. However, whether this regulation occurs through the prevention of lymphocyte activation or by inhibiting their trafficking from lymphoid organs into the CNS remains to be explored.\u003c/p\u003e \u003cp\u003eInterestingly, we found that the chemokine Ccl5 was downregulated in the na\u0026iuml;ve thymus of \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, a primary lymphoid organ essential for the development and maturation of T lymphocytes(\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). This downregulation may help explain the differences in disease severity observed between constitutive knockout animals and ASO-treated animals. In our study, ASO treatment, administered after immune priming, resulted in a 62% reduction in miR-145 levels in the thymus. Moreover, secreted CCL5 plays a crucial role in cell migration and leukocyte adhesion within the CNS vasculature during EAE(\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). We also observed that Ccl5 was downregulated during both the onset and chronic phases of EAE in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, which could further diminish immune cell infiltration into the parenchyma. This suggests that Ccl5 is likely critical to the reduction of immune cell infiltration in \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e EAE-subjected animals.\u003c/p\u003e \u003cp\u003eLastly, we previously demonstrated that the pathological upregulation of miR-145 inhibits OL differentiation and that the loss of miR-145 in this context is sufficient to promote remyelination in a chronic demyelination model(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). In this study, we assessed myelin and axon health as indicators of overall pathological outcomes in EAE. Due to the acute nature of EAE, evaluating remyelination is challenging, although we still assessed myelin thickness in this investigation.\u003c/p\u003e \u003cp\u003eIn both \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice and miR-145 ASO-treated animals, we observed alterations in several parameters of myelin and axon health throughout the course of EAE, with more significant changes noted in the chronic phase. Notably, \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice exhibited nearly twice as many myelinated axons compared to EAE-subjected ASO-treated controls. Additionally, both miR-145-deficient and miR-145 ASO-treated mice showed reduced myelin decompaction and axon degeneration, further underscoring the role of miR-145 in the CNS. The observed improvements in myelin and axon health following miR-145 loss in EAE may indicate reduced demyelination resulting from decreased inflammatory attacks or suggest a neuroprotective role that warrants further investigation.\u003c/p\u003e \u003cp\u003eIn the lumbar spinal cord, miR-145 was uniquely upregulated at the onset of EAE in wild-type animals, which may contribute to the inhibition of initial remyelination efforts early in the disease. Interestingly, we also found that the chemokine Cxcl1 was upregulated in the spinal cords of miR-145-deficient mice at the onset of EAE. CXCL1 is secreted by astrocytes in the CNS and promotes the recruitment of OPCs, brain progenitors scattered throughout the CNS that give rise to OLs(\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, our study reveals that the loss of miR-145 significantly enhances clinical outcomes in murine EAE, driving widespread changes in neuroinflammation and reducing myelin and axon degeneration across all disease stages. Future research should focus on improving the bioavailability of miR-145 ASOs to ensure they effectively cross the blood-brain spinal cord barrier and achieve efficient knockdown, allowing for a comprehensive exploration of the broader implications of miR-145 in the CNS and the therapeutic potential of targeting it.\u003c/p\u003e \u003cp\u003eOur findings suggest a dual function for miR-145 in mitigating key pathological features of progressive MS. These insights could pave the way for innovative miR-145-targeted therapies aimed at slowing disease progression and enhancing recovery in MS, offering new hope for effective treatment strategies that address both neurodegeneration and inflammation.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003c/div\u003e \u003c/div\u003e "},{"header":"Methods","content":"\u003ch2\u003eAnimals\u003c/h2\u003e\u003cp\u003eConstitutive miR-145 knock-out mice (herein referred to as \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e−/−\u003c/em\u003e\u003c/sup\u003e) were generated on the C57BL/6 background as described in Xin et al(\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e) and generously provided by Dr. Eric Olson. \u003cem\u003emiR-145\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e mice were utilized as controls and for the ASO-associated experiments. Animals were subjected to EAE and euthanized at different time points for subsequent analyses, as indicated. The animals had unrestricted access to water and chow and were kept on a 12:12-hour light-dark cycle.\u003c/p\u003e\u003ch2\u003eEAE induction\u003c/h2\u003e\u003cp\u003eEAE was induced using the Hooke Laboratories kit, which contains myelin oligodendrocyte glycoprotein peptide fragment 35–55 (MOG\u003csub\u003e35 − 55\u003c/sub\u003e) in Complete Freund’s Adjuvant (CFA) emulsion (cat. no. EK-2110). Specifically, female mice aged 9–13 weeks received 0.1 mL of the prepared MOG\u003csub\u003e35 − 55\u003c/sub\u003e in CFA emulsion subcutaneously in the upper and lower back at the cervical and lumbar spinal column levels on day 0, in accordance with the manufacturer’s protocol. This was followed by intraperitoneal administration of 100 ng pertussis toxin in filtered PBS. A second intraperitoneal injection of the same dosage was administered on day 1. Mice were left undisturbed until day 7 post-induction, at which point daily assessments were conducted, and body weight was recorded on alternate days until day 30. Scoring was performed according to Hooke Laboratories guidelines. Animals were euthanized for tissue collection at the onset, peak, and chronic phases of EAE. Control animals consisted of non-induced, age-matched, naïve female mice.\u003c/p\u003e\u003ch2\u003eAntisense oligonucleotide (ASO) in vivo treatment\u003c/h2\u003e\u003cp\u003eThe miRCURY LNA custom inhibitor (Qiagen, 339204), including negative control (YCI0201821-FZA) and miR-145-5p (YCI0201822-FZA) non-labelled ASOs, were administered at a dosage of 20 µg/mouse via subcutaneous injections. Initially, a single dose of either the control or miR-145 ASOs was administered at the peak of EAE. Subsequently, we modified our approach to perform ASO injections on days 1 and 3 post-EAE induction. For biodistribution assessment, 5’ FAM-labeled ASOs (Qiagen, 339146) were administered subcutaneously on day 3 post-induction at a dosage of 10 µg/mouse.\u003c/p\u003e\u003ch2\u003eSpinal cord organotypic cultures\u003c/h2\u003e\u003cp\u003eC57BL/6 pups aged P0-P2 were euthanized, and their spinal cords were dissected and sliced to a thickness of 300 µm, following the protocol previously described with slight modifications(\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e). The slices were placed onto a semi-porous membrane insert (Millipore, 30 mm diameter, pore size 0.4 µm) and maintained using an interface culture method with 1 mL of serum-based medium. The medium consisted of 25% DMEM (low glucose), 25% F12, 21% Hanks’ balanced salt solution (HBSS), 25% horse serum, 25 mM HEPES, 0.4 mM ascorbic acid, 25 mM glucose, and 1% penicillin-streptomycin. Cultures were maintained for 10 days in vitro (DIV), with media changes every 2–3 days. Treatment with either 50 nM of FAM-labeled control or miR-145 ASOs was applied to the wells for 24 h. After treatment, the wells were washed, and 4% paraformaldehyde (PFA) was added for 1 h at room temperature on a shaker. Three washes with PBS, each lasting 10 min, were performed. Slices were then stored in PBS until immunohistochemical experiments were conducted.\u003c/p\u003e\u003ch2\u003eRNA isolation and qRT-PCR\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted from all samples using the RNeasy Mini Kit (Qiagen), following the manufacturer’s instructions. Spinal cord, spleen, and thymus were flash-frozen immediately upon collection. A maximum of 30 mg of tissue was used per sample. For RNA isolation, 350 µL or 600 µL of RLT buffer was added directly to the frozen tissues, which were then homogenized. The lysate was mixed 1:1 with 70% ethanol in RNase-free H\u003csub\u003e2\u003c/sub\u003eO, transferred to the RNeasy spin column, and centrifuged at maximal speed for 15 sec. The eluate was discarded, and the column was washed with 700 µL RW1 buffer by spinning at maximal speed for 15 sec. The eluate was again discarded, and the column was washed with 500 µL RPE buffer at maximal speed for 15 sec. The columns were transferred to clean collection tubes and washed a second time with 500 µL RPE for 2 min. Finally, the columns were placed in RNase-free microfuge tubes, and RNA was eluted in 30–50 µL RNase-free H2O by spinning at maximal speed for 1 min.\u003c/p\u003e\u003cp\u003eReverse transcription of mature miR-145-5p and SnU6 was conducted according to Biggar et al.(\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e) with modifications. Briefly, 300 ng of total RNA was incubated with 5 µL of 250 nM stem-loop primer in a total volume of 10 µL. The annealing reaction was performed at 95°C for 5 min, followed by 60°C for 5 min. Samples were then centrifuged and kept on ice for 1 min. Reverse transcription was performed using 1 µL of M-MLV Reverse Transcriptase (Invitrogen), 4 µL of 5x First Strand Buffer (Invitrogen), 2 µL of 100 mM dithiothreitol (Invitrogen), and 1 µL of premixed dNTPs (final concentration 25 µM each). Each reaction was brought to a total volume of 25 µL using RNase-free water (Qiagen). The reverse transcription protocol was as follows: 16°C for 30 min, followed by 60 cycles of 20°C for 30 sec, 42°C for 30 sec, and 50°C for 1 sec, culminating with a final step at 85°C for 5 min, using an Eppendorf Mastercycler. Primer sequences for miRNA qRT-PCR are provided in Supplementary Table\u0026nbsp;1.\u003c/p\u003e\u003cp\u003eA universal reverse primer was used for the amplification of both miR-145-5p and SnU6. Amplification of miR-145-5p and snU6 cDNA was performed using specific forward primers and a universal reverse primer complementary to the stem-loop portion of the cDNA (Supp. Table\u0026nbsp;1). Each qRT-PCR reaction contained 12.5 µL of 2x SsoFast EvaGreen Supermix (Bio-Rad), 0.8 µL each of 25 µM forward and universal primers, and 4 µL of cDNA, with the total volume adjusted to 25 µL using RNase-free water (Qiagen). Samples were amplified using the following protocol: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min, using a Bio-Rad CFX Connect. All samples were analyzed in technical triplicate. Primer validation was conducted through standard curve efficiency analysis, melt curve analysis, and electrophoresis of qPCR products on a 5% agarose gel to confirm product size. Primers for miRNA qRT-PCR were sourced from AlphaDNA. Relative expression analysis was performed using CFX Manager™ or CFX Maestro software, utilizing the ΔΔCt method.\u003c/p\u003e\u003cp\u003eFor mRNA analysis, total cDNA was synthesized using the RT2 First Strand Kit (Qiagen) according to the manufacturer’s protocol. Briefly, 2 µL of Buffer GE (Qiagen) was used to eliminate genomic DNA from 150–200 ng of total RNA in RNase-free water, in a total volume of 10 µL per sample. Samples were incubated at 42°C for 5 min and then placed on ice for at least 1 min. Reverse transcription was performed by adding 4 µL of 5x Buffer BC3 (Qiagen), 1 µL of Control P2 (Qiagen), 2 µL of RE3 Reverse Transcriptase Mix (Qiagen), and 3 µL of RNase-free water (Qiagen), followed by incubation at 42°C for 15 min and 95°C for 5 min. Samples were then diluted with 91 µL of RNase-free water (Qiagen) and stored at -20°C until further use. For mRNA qRT-PCR, PrimePCR pre-optimized primers (Bio-Rad) were employed according to the manufacturer’s protocol. Each reaction contained 4 µL of total cDNA, 10 µL of SsoFast EvaGreen master mix (Bio-Rad), and 1 µL of forward/reverse primer mix, in a total volume of 20 µL. Amplification was performed at 95°C for 5 min, followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec using a Bio-Rad CFX Connect. Relative expression analysis was conducted using CFX Manager™ or CFX Maestro software, employing the ΔΔCt method.\u003c/p\u003e\u003ch2\u003eImmunohistochemistry and histology (paraffin-embedded tissues)\u003c/h2\u003e\u003cp\u003eMice were anesthetized via intraperitoneal injection of tribromoethanol (avertin) and perfused transcardially with 5 mL of phosphate-buffered saline (PBS), followed by 10–20 mL of 4% paraformaldehyde (PFA, EMS Cedarlane). Lumbar spinal cords were then dissected and fixed in 4% PFA for 24–48 h before being transferred to 70% ethanol in water. Lumbar spinal cord samples were processed at the Louise Pelletier Histology Core Facility, Department of Pathology and Laboratory Medicine at the University of Ottawa. The samples were initially embedded in paraffin wax using a LOGOS microwave hybrid tissue processor. Paraffin-embedded samples were cut into 20 µm sections and mounted on slides.\u003c/p\u003e\u003cp\u003ePrior to immunohistochemistry and staining, sections were deparaffinized and rehydrated as follows: slides were incubated at 59°C for 30–60 min, followed by immersion in 100% Hemo-D (Histo-Clear, National Diagnostics) for three 5-min washes, 50% Hemo-D/50% ethanol for two 3-min washes, 100% ethanol for two 3-min washes, 95% ethanol for 3 min, 70% ethanol for 3 min, 50% ethanol for 3 min, and finally rinsed twice in water. Antigen retrieval for Iba1 immunostaining was performed using Tris-EDTA buffer (pH 9) at 95–100°C for 20 min in a steamer. After antigen retrieval or rehydration, slides were rinsed three times for 5 min in PBS, permeabilized with 0.5% Triton-X for 20 min, and rinsed again three times in PBS.\u003c/p\u003e\u003cp\u003eSections were then blocked for 1 h in a blocking solution containing 1% bovine serum albumin (BSA), 10% goat serum (GS), and 0.2% Triton-X in PBS at room temperature (RT). Primary and secondary antibodies were diluted in a solution containing 2% BSA, 1% goat serum, 0.2% Triton-X, and incubated at 4°C overnight. The following antibody dilutions were used: rat anti-MBP (1:100), rabbit anti-GFAP (1:1000), and rabbit anti-Iba1 (1:100) (Supp. Table\u0026nbsp;2).\u003c/p\u003e\u003cp\u003eFollowing the incubation with primary antibodies, sections were washed three times with PBS and incubated for 1 h with fluorophore-labeled secondary antibodies (Alexa Fluor, Invitrogen) at a dilution of 1:500. Sections were subsequently washed once with PBS, counterstained with 4′,6-diamidino-2-phenylindole (DAPI) at a dilution of 1:1000 in PBS for 5 min, and finally washed three times for 5 min in PBS. Dako fluorescent mounting medium was applied sparingly to each section, followed by the placement of a coverslip.\u003c/p\u003e\u003cp\u003eFor histological analysis, lumbar spinal cord cross-sections were deparaffinized and stained with either Cresyl violet or hematoxylin and eosin using a Leica ST5010 Autostainer XL, in conjunction with a Leica CV5030 Glass Coverslipper.\u003c/p\u003e\u003ch2\u003eImmunohistochemistry (cryopreserved spinal cords)\u003c/h2\u003e\u003cp\u003eMice were anesthetized and transcardially perfused with 10–20 mL of Hank’s Balanced Salt Solution (HBSS, Gibco). Following dissection, lumbar spinal columns were post-fixed in 4% PFA for 24 h at 4°C. The spinal cords were then carefully dissected and stored in 30% sucrose (Fisher) in PBS. Two additional changes of 30% sucrose were performed before embedding the tissues in optimal cutting temperature (OCT) compound and flash-freezing. Cryopreserved spinal cords were sectioned at 16 µm and stored at -80\u003csup\u003eo\u003c/sup\u003eC until further staining.\u003c/p\u003e\u003cp\u003eBriefly, sections were rehydrated in 1X PBS for 5 min, followed by permeabilization and blocking with 5% BSA and 0.3% Triton-X100 in 1X PBS for 1 h at RT. Tissue sections were then incubated overnight at 4°C with appropriate primary antibodies (listed in Supp. Table\u0026nbsp;2), diluted in a solution containing 1% goat serum, 1% BSA, and 0.2% Triton-X100 in 1X PBS. After washing three times with 1X PBS, secondary antibodies (1:500) diluted in 1X PBS were applied to the tissue sections for 1 h at RT. For nuclear staining, sections were incubated with DAPI diluted in 1X PBS for 5 min. Sections were then washed three times in PBS and mounted with Fluoromount G (Invitrogen).\u003c/p\u003e\u003ch2\u003eImmunohistochemistry (organotypic cultures)\u003c/h2\u003e\u003cp\u003eCultured spinal cord slices were quickly washed once with 1X PBS and subsequently fixed with 4% PFA for 1 h, as previously described(\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e). Following fixation, slices were washed three times with 1X PBS and incubated in a blocking buffer containing 5% BSA and 1% Triton-X100 in 1X PBS for 3 h at RT. Primary antibodies (listed in Supp. Table\u0026nbsp;2) were then added to a solution containing 6% normal goat serum, 0.5% BSA, and 1% Triton-X100 in 1X PBS, and incubated overnight at 4°C on a shaker. Afterward, slices were washed three times with 1X PBS, and secondary antibodies (1:500) along with DAPI (1:1000) diluted in PBS-T (1%) were incubated for 2 h at RT on a shaker. Slices were then washed three times for 10 min each and mounted with Fluoromount G.\u003c/p\u003e\u003ch2\u003eTransmission Electron Microscopy (TEM)\u003c/h2\u003e\u003cp\u003eMice were anesthetized and perfused transcardially with 10–20 mL of HBSS, followed by 10–20 mL of Karnovsky’s fixative (4% PFA, 2% glutaraldehyde, and 0.1 M sodium cacodylate in PBS, pH 7.4). Whole spinal columns were extracted and fixed overnight (or until processed) at 4°C in the same fixative. Following fixation, the ventral white matter of the lumbar spinal cords was dissected and cut into straight segments of 1 mm in length under a stereomicroscope.\u003c/p\u003e\u003cp\u003eSpecimens were subsequently washed twice in 0.1 M sodium cacodylate buffer for 1 h each and once overnight at room temperature. Segments were post-fixed with 1% osmium tetroxide in 0.1 M sodium cacodylate buffer for 1 h at room temperature and then washed three times in distilled water for 5 min each. Specimens were dehydrated in a graded series of ethanol, including 30%, 50%, 70%, 85%, and 95% ethanol, followed by two 30-min washes in 100% ethanol. This was followed by two 15-min washes in 50% ethanol/50% acetone and two 15-min washes in 100% acetone.\u003c/p\u003e\u003cp\u003eSegments were then infiltrated in 30% Spurr resin/acetone for 20 min and once for 15 h (overnight), followed by 50% Spurr resin/acetone for 6 h, and then in fresh 100% Spurr resin overnight. The Spurr resin was changed twice daily for three days at room temperature. All infiltration steps were performed on a rotator at low speed. Finally, specimens were embedded in fresh liquid Spurr resin, oriented inside molds, and polymerized overnight at 70°C. Ultrathin sections (80 nm) were collected onto 200-mesh copper grids and stained with 2% aqueous uranyl acetate and Reynolds’ lead citrate.\u003c/p\u003e\u003ch2\u003eImaging and quantification\u003c/h2\u003e\u003cp\u003eFluorescence images were captured using an Axio Imager M1 microscope with an AxioCam HRm Rev.2 camera and Axiovision 4.8.2 software. Confocal images were obtained with a Zeiss LSM 510 Meta DuoScan microscope using Zen 8.0 software. Electron micrographs of the lumbar spinal cord were taken with a JEM-1400Plus electron microscope at ×4000 magnification. Histology and immunohistochemistry samples from paraffin-embedded tissues were imaged using a Zeiss AxioScan slide scanner with a Colibri 7 camera and Zen 2.6 slidescan software.\u003c/p\u003e\u003cp\u003eAll images were analyzed using ImageJ, with treatment conditions blinded during image analyses. For paraffin-embedded samples, images were taken at 20x magnification, with 20–25 images quantified per sample; for those taken at 10x, 5–10 images were quantified per sample. For flash-frozen tissues, images were taken at 5x magnification, and 5–10 images were quantified per animal. For qualitative assessment of spinal cord organotypic cultures, 20x objective images from 5–7 slices per condition were taken.\u003c/p\u003e\u003cp\u003eHistological and immunohistochemical analyses of the lumbar spinal cord involved cross-sections taken from the proximal end, mid-lumbar spine, and distal end. To calculate percent infiltrated area and percent myelinated area, the total spinal cord area and nucleus-dense area (based on H\u0026amp;E staining) or myelinated area (based on MBP + staining) were measured using ImageJ from every third 20 µm section across 2–3 sections from each of the three sampled areas, resulting in a total of 6–9 sections per animal. For MBP area analysis, a uniform threshold value was used for all images, and the “Measure Particles” plugin in ImageJ was employed to produce MBP traces. GFAP + and Iba1 + cell quantifications involved counting individual cells across the entire section, with Iba1 + counts excluding areas of dense cell infiltration. For immune cell labelling, FIJI automated cell counting based on outlines applied from the binary image was performed on whole spinal cord sections. A total of 5–8 sections per animal were quantified and averaged for each marker.\u003c/p\u003e\u003cp\u003eElectron micrographs were captured using the Hitachi 7100 transmission electron microscope at a magnification of x4000. G-ratios were calculated by measuring the axon and the total fibre (axon plus myelin), converting these measurements to diameters, and dividing the axon diameter by the fibre. Areas enriched in myelinated axons were preferentially chosen for g-ratio and morphometric analyses. The g-ratio was measured for myelinated axons with normal morphology while excluding axons exhibiting abnormal myelin morphology (e.g., uncompacted myelin, splitting, empty sheaths) or signs of degradation or swelling, as previously described(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e). On average, 150 axons per sample were analyzed. Axonal and myelin abnormalities were assessed through morphometric analysis, determining the proportion of uncompacted myelin and degenerating axons characterized by swelling, darkening of the axoplasm, and organelle accumulation via manual counting. Morphometric analyses were evaluated from 10 images per animal.\u003c/p\u003e\u003cp\u003eAll images were counted in Fiji software in a blinded manner, with representative images processed in Photoshop and GraphPad Prism 10.0. Biorender was utilized for generating schematics.\u003c/p\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using GraphPad Prism software (versions 6 or 10.3.0), except qRT-PCR analyses. Normal distribution and equal variance were assumed for all datasets, though not formally tested. Sample sizes were determined based on prior publications(\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e). Pairwise comparisons were made using a two-tailed Student’s t-test. For comparisons involving more than two conditions, one-way or two-way analysis of variance (ANOVA), or Multiple unpaired t-tests of two-way ANOVA were employed, followed by the appropriate post-hoc tests. Linear regression analyses were performed for g-ratio assessments, and Kaplan-Meier curves were analyzed using the Mantel-Cox test. Errors on mean values are expressed as ± SEM, unless otherwise specified. The number of experiments and statistical details are included in the corresponding figure legends. P-values of \u0026lt; 0.05 were deemed significant, with significance levels indicated as follows: * = p \u0026lt; 0.05, ** = p \u0026lt; 0.01, *** = p \u0026lt; 0.001, **** = p \u0026lt; 0.0001.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAQP4: Aquaporin 4\u003c/p\u003e\n\u003cp\u003eASO: Antisense oligonucleotide\u003c/p\u003e\n\u003cp\u003eBBB: Blood-brain barrier\u003c/p\u003e\n\u003cp\u003eBSCB: Blood-spinal cord barrier\u003c/p\u003e\n\u003cp\u003eCcl5: Chemokine (C-C motif) ligand 5\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCD: Cluster of differentiation\u003c/p\u003e\n\u003cp\u003eCNS: Central nervous system\u003c/p\u003e\n\u003cp\u003eCxcl1: Chemokine (C-X-C motif) ligand 1\u003c/p\u003e\n\u003cp\u003eEAE: Experimental autoimmune encephalomyelitis\u003c/p\u003e\n\u003cp\u003eEBV: Epstein-Barr virus\u003c/p\u003e\n\u003cp\u003eFAM: Fluorescein amidite\u003c/p\u003e\n\u003cp\u003eFoxP3: Forkhead box P3\u003c/p\u003e\n\u003cp\u003eGFAP: Glial Fibrillary Acidic Protein\u003c/p\u003e\n\u003cp\u003eH\u0026amp;E: Hematoxylin and eosin\u003c/p\u003e\n\u003cp\u003eIba1: Ionized calcium-binding adaptor molecule 1\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIfng: Interferon-gamma\u003c/p\u003e\n\u003cp\u003eIl1b: Interleukin-1 beta\u003c/p\u003e\n\u003cp\u003eIl6: Interleukin 6\u003c/p\u003e\n\u003cp\u003eiNos: Inducible nitric oxide synthase\u003c/p\u003e\n\u003cp\u003eMBP: Myelin basic protein\u003c/p\u003e\n\u003cp\u003eMOG\u003csub\u003e35-55\u003c/sub\u003e: Myelin Oligodendrocyte Glycoprotein Peptide Fragment 35-55\u003c/p\u003e\n\u003cp\u003eMS: Multiple sclerosis\u003c/p\u003e\n\u003cp\u003eNurr1: Nuclear receptor 4A2\u003c/p\u003e\n\u003cp\u003eOLs: Oligodendrocytes\u003c/p\u003e\n\u003cp\u003eOPCs: Oligodendrocyte precursor cells\u003c/p\u003e\n\u003cp\u003ePBMCs: peripheral blood mononuclear cells\u003c/p\u003e\n\u003cp\u003ePPMS: Primary progressive multiple sclerosis\u003c/p\u003e\n\u003cp\u003eRRMS: Relapsing-remitting multiple sclerosis\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSPMS: Secondary progressive multiple sclerosis\u003c/p\u003e\n\u003cp\u003eTEM: Transmission electron microscopy\u003c/p\u003e\n\u003cp\u003eTnfa: Tumor necrosis factor alpha\u003c/p\u003e\n\u003cp\u003eYm1:\u0026nbsp;Chitinase-like protein 3 (Chil3)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cem\u003eEthics approval and consent to participate\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll animal-related experimental protocols were approved by the University of Ottawa Animal Care Committee and adhered to the standards outlined in the Canadian Council on Animal Care’s Guide to the Care and Use of Experimental Animals, as well as the Animals for Research Act. We ensured compliance with all relevant ethical regulations regarding the use of animals.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAvailability of data and materials\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll authors had access to the study data and reviewed and approved the final manuscript. All data associated with this study are available in the main text or supplementary materials. Raw data can be provided in full upon request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe authors disclose no conflicts of interest. The funders mentioned above were not involved in the study design, data collection and analysis, decision to publish, or manuscript preparation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the Canadian Institutes of Health Research [grant number PJT-162121]; the Multiple Sclerosis Society of Canada [grant number MSSC-3779]. MMAA was supported by the University of Ottawa Brain and Mind Institute TRIMS Award. SFK was supported by a CIHR Banting and Best CGS-D award and an MS Canada Doctoral Fellowship. SEC was supported by a CIHR Banting and Best CGS-D award and a CNMD Star award. ERS was supported by a CNMD Star award. RY was supported by a QEII Graduate Scholarship in Science and Technology Studentship.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAuthors' contributions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eM.M.A.A. and S.F.K. conceived and designed the study, with supervision and support from R.K. The methodology was developed by M.M.A.A., S.F.K., S.E.C., Y.D.R., R.Y., S.G., and A.B. Formal analysis was conducted by M.M.A.A., S.F.K., M.A.A., I.G, E.R.S. The investigation was carried out by M.M.A.A. and S.F.K. Graphical abstract was made by E.R.S. The manuscript was primarily written by M.M.A.A., with significant contributions from S.F.K., whose thesis provided key textual elements. R.K. critically edited it. Funding acquisition was managed by R.K.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAcknowledgements\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. Eric Olson for the kind provision of transgenic mice. We also extend our sincere thanks to the Animal Care and Veterinary Service at the University of Ottawa for their invaluable support and assistance throughout this project.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWalton C, King R, Rechtman L, Kaye W, Leray E, Marrie RA, et al. Rising prevalence of multiple sclerosis worldwide: Insights from the Atlas of MS, third edition. Mult Scler. 2020;26(14):1816-21.\u003c/li\u003e\n\u003cli\u003eBae HG, Kim TK, Suk HY, Jung S, Jo DG. White matter and neurological disorders. Arch Pharm Res. 2020;43(9):920-31.\u003c/li\u003e\n\u003cli\u003eReich DS, Lucchinetti CF, Calabresi PA. Multiple Sclerosis. N Engl J Med. 2018;378(2):169-80.\u003c/li\u003e\n\u003cli\u003eCorreale J, Marrodan M, Ysrraelit M. Mechanisms of Neurodegeneration and Axonal Dysfunction in Progressive Multiple Sclerosis. Biomedicines. 2019;7(1):14-.\u003c/li\u003e\n\u003cli\u003eBjornevik K, Cortese M, Healy BC, Kuhle J, Mina MJ, Leng Y, et al. 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Brain, Behavior, and Immunity. 2020;84(July 2019):132-46.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Myelin, oligodendrocytes, demyelination, neuroinflammation, neurodegeneration, microRNA","lastPublishedDoi":"10.21203/rs.3.rs-5462410/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5462410/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMultiple sclerosis (MS) is a progressive inflammatory disease of the central nervous system (CNS) marked by myelin loss, which impairs nerve function. Current therapies fail to halt disease progression or prevent myelin and axonal degeneration. In this study, we explored the impact of miR-145 knockout in a murine model of experimental autoimmune encephalomyelitis (EAE), which mimics MS pathology. Loss of miR-145 reduced clinical severity and significantly decreased immune cell infiltration in the lumbar spinal cord during both the onset and chronic stages of the disease. Additionally, miR-145 loss altered the expression of key inflammatory genes and modulated astrocytic activity throughout EAE. Of significant interest, acute treatment with an antisense oligonucleotide (ASO) targeting miR-145 decreased miR-145 levels and led to reduced disease severity, decreased immune cell infiltration, and an increase in regulatory T cells in EAE mice. Moreover, miR-145 deficiency mitigated axon and myelin degeneration. Our findings suggest that ASOs targeting miR-145 may offer a promising therapeutic strategy, addressing both inflammatory and degenerative components of MS.\u003c/p\u003e","manuscriptTitle":"Harnessing mIR-145 deficiency to modulate inflammation and prevent degeneration in a mouse model of multiple sclerosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-18 17:34:48","doi":"10.21203/rs.3.rs-5462410/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d90b5e61-5269-4ace-96b7-b85d59b8cfb3","owner":[],"postedDate":"December 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-20T21:53:12+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-18 17:34:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5462410","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5462410","identity":"rs-5462410","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}