Preservation of miR-9-5p and miR-124-3p in ALS-resistant oculomotor neurons contrasts with their downregulation in vulnerable spinal motor neurons, irrespective of TDP-43 pathology

Preservation of miR-9-5p and miR-124-3p in ALS-resistant oculomotor neurons contrasts with their downregulation in vulnerable spinal motor neurons, irrespective of TDP-43 pathology | 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 Preservation of miR-9-5p and miR-124-3p in ALS-resistant oculomotor neurons contrasts with their downregulation in vulnerable spinal motor neurons, irrespective of TDP-43 pathology Crystal McLellan, Danae Campos-Melo, Robert Hammond, Michael J. Strong This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7537447/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Mar, 2026 Read the published version in Acta Neuropathologica → Version 1 posted 10 You are reading this latest preprint version Abstract Selective vulnerability of motor neurons is a defining feature of amyotrophic lateral sclerosis (ALS) and provides a valuable framework for uncovering mechanisms that distinguish resilient from vulnerable neuronal populations. We investigated whether dysregulation of neuroprotective microRNAs (miRNAs), miR-9-5p and miR-124-3p, contributes to the differential susceptibility of motor neuron subtypes. We focused on spinal motor neurons (SMNs), which undergo drastic degeneration in ALS, and oculomotor neurons (OMNs), which remain functionally intact and rarely degenerate, allowing preservation of eye movement in ALS patients. Using a modified multiplexed fluorescent in situ hybridization protocol combined with immunofluorescence, we quantified the expression of miR-9-5p and miR-124-3p in cervical SMNs and OMNs from ALS and control cases. We observed significant downregulation of both miRNAs in ALS SMNs, while their expression was maintained in ALS OMNs. Stratification of ALS SMNs by TDP-43 pathological status revealed similarly reduced miRNA expression in neurons with and without cytoplasmic inclusions, suggesting that miRNA downregulation occurs independently of visible TDP-43 pathology. To explore a potential mechanism, we assessed the localization of the Dicer cofactor TRBP and found that it was sequestered into TDP-43 inclusions in ALS SMNs. However, TRBP remained normally localized in neurons without cytoplasmic inclusions, indicating that sequestration cannot fully account for miRNA reduction across all ALS motor neurons. These findings support a model in which early or subtle disruptions, preceding visible pathology, also contribute to miRNA downregulation in ALS. Our study also indicates cell-type-specific preservation of miRNA networks as a possible contributor to oculomotor neuron resilience. Amyotrophic lateral sclerosis Selective motor neuron vulnerability miR-9-5p miR-124-3p TDP-43 Fluorescent in situ hybridization Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Amyotrophic lateral sclerosis (ALS) is characterized by progressive loss of motor neurons resulting in paralysis and death, yet some motor neuron subtypes remarkably remain resistant[31]. Spinal motor neurons (SMNs) undergo early and severe degeneration, while other populations, such as oculomotor neurons (OMNs) that control eye movement, are largely spared [17, 23]. Post-mortem analysis shows that ALS OMNs typically retain nuclear expression of transactive response DNA-binding protein 43 kDa (TDP-43), an RNA-binding protein strongly associated with ALS pathology, whereas SMNs commonly exhibit TDP-43 mislocalization to the cytoplasm and subsequent formation of biomolecular condensates[4, 8, 42]. Features such as unique cellular architecture, distinct gene expression, specialized metabolism, and reduced glutamate excitotoxicity are suspected to contribute to the resilience of OMNs[3, 5, 11, 20, 27, 40]. Elucidating these and other novel mechanisms that confer protection could help with the development of targeted neuroprotective approaches in ALS. Dysregulated RNA metabolism is increasingly recognized as an important pathological feature of ALS, and microRNAs (miRNAs) have emerged as both dysregulated molecules and promising biomarkers for disease progression[9, 10, 14, 15, 21, 36]. miRNAs are short (~22 nt) non-coding RNAs that fine-tune gene expression post-transcriptionally by guiding the RNA-induced silencing complex (RISC) to target mRNAs, leading to translational repression or degradation. miRNA production begins in the nucleus, where Drosha and DGCR8 cleave primary transcripts into precursor miRNAs, which are exported to the cytoplasm and further processed by Dicer, aided by TAR RNA-binding protein (TRBP) and PACT. The guide strand is incorporated into RISC, where Argonaute 2 (AGO2) directs it to target mRNAs for translational repression or degradation. Because of their stability and cell-specificity, miRNAs are useful for understanding ALS pathogenesis and providing candidate biomarkers. Several studies have reported a global downregulation of miRNAs in ALS spinal cord tissue homogenates and lumbar motor neurons, as demonstrated by in situ hybridization, qPCR, and RNA sequencing[9, 13–15, 37]. One proposed mechanism for this downregulation involves impaired Drosha/DGCR8 and Dicer/TRBP function downstream of ALS-causing mutations and cellular stress. In cell culture stress assays, AGO2 was also shown to interact with the stress granule (SG) protein TIA-1, linking miRNA machinery to SG dynamics[14]. Alternatively, other evidence from cell culture suggests that TDP-43 mislocalization, triggered by cellular stress, can disrupt a negative feedback loop involving specific miRNAs, where loss of nuclear TDP-43 leads to decreased expression of these miRNAs in the cytoplasm[19]. miR-9-5p and miR-124-3p have been reported to exhibit reduced expression in spinal cords of individuals with ALS[9, 12, 14, 15, 18, 46]. Targets of both miRNAs are implicated in neuronal development and function, including neurogenesis, cell fate specification, synaptic remodeling, and structural maturation of axons and dendrites, indicating their importance in neuronal integrity[18, 22, 38, 45]. This functional relevance may explain why SMNs are particularly susceptible to ALS-related pathology. However, miRNA expression profiling in ALS-resistant motor neurons, such as OMNs, is lacking. Since OMNs usually maintain nuclear TDP-43 throughout the disease course, exhibit resistance to degeneration compared to SMNs, and miR-9-5p and miR-124-3p targets are responsible for neuronal maintenance, we hypothesized that OMNs and SMNs from ALS patients would differ in their expression of miR-9-5p and miR-124-3p compared to control cases. We assessed the expression of miR-9-5p and miR-124-3p in postmortem sporadic ALS and control tissue using fluorescent in situ hybridization, targeting both OMNs and SMNs. Immunofluorescence (IF) for TDP-43 was included to evaluate miRNA expression and cellular pathology. This approach enabled direct comparison of miRNA expression in vulnerable and resistant motor neuron populations. Materials and Methods Patient tissue Fixed tissue sections of cervical spinal cord and superior midbrain were used for fluorescent in situ hybridization and immunofluorescence (FISH-IF) experiments. Formalin-fixed, post-mortem tissue control patients or patients diagnosed with sporadic ALS according to El Escorial criteria (World Federation of Neurology) by MJS were processed, embedded in paraffin, sectioned at 7 um thickness and mounted on glass slides[6]. Basic demographic information of the patients is provided in Supplementary file1: Table S1. The cervical spinal cord was selected for analysis because it is significantly affected in ALS while still retaining a substantial population of observable motor neurons. SMNs of the ventral horn were identified in the anterior region of the spinal cord sections by the larger size of the ventral horn compared to the dorsal horn and the presence of prominent motor neuron cell bodies. The oculomotor nucleus was identified anatomically in midbrain sections at the level of the superior colliculus paired posteromedial nuclei ventral to the periaqueductal gray matter, medial to the median longitudinal fasciculus. and from which ventrally exiting fascicles are present. In several cases, the midbrain was initially cut on the mid-sagittal plane and sectioned into halves prior to cutting and mounting for examination. Fluorescent in situ hybridization and Immunofluorescence Double fluorescent in situ hybridization and immunofluorescence (dFISH-IF) was used to detect target miRNA miR-9-5p or miR-124-3p with candidate normalizers miR-132-3p or U6 small nuclear RNA (U6 snRNA) and TDP-43 in motor neurons, along with the motor neuron marker NeuroTrace435/455 (Nissl-staining substance; does not stain nucleus). U6 snRNA was used previously for normalization in control and ALS lumbar spinal cord in situ experiments[14]. FISH-IF was performed similarly to a published protocol and is briefly outlined here with modifications[34]. Tissue sections were dewaxed with xylenes and rehydrated with a series of ethanol dilutions. Antigen retrieval was performed in 10 mM sodium citrate, pH 6.0, using a pressure cooker for 15 minutes at high pressure at 100°C. dFISH was performed with inventoried digoxigenin (DIG) or 6-carboxyfluorescein (FAM) labelled mirCURY LNA™ miRNA detection probes (GeneGlobe, Qiagen; for probe details see Supplementary file1: Table S2). Two differently labeled LNA probes (DIG or FAM) with similar hybridization temperatures (within 10°C) were added to tissue sections, and each was detected by sequential HRP antibody-tyramide signal amplification (TSA; Cyanine 3 or 5, Akoya). Triple FISH (tFISH) was performed similarly to the above method, followed by sequential hybridization and detection of the third probe. Immunofluorescence of the same slides was performed using an anti-TDP-43 antibody (ProteinTech) at 1:150 in 1xPBS with 2% BSA (bovine serum albumin) after blocking in 1xPBS with 5% BSA and incubated overnight at 4°C (Supplementary file1: Table S2). An Alexa Fluor 488-conjugated secondary antibody (Invitrogen) at 1:250 in 1xPBS with 2% BSA was used to detect the TDP-43 antibody. NeuroTrace 435/455 (Invitrogen) was used to label neurons as directed by the manufacturer, except permeabilization buffer consisted of 0.3% TritonX-100, and NeuroTrace was diluted 1:200 before addition to sections. Tissue sections were mounted with Dako Fluorescence Mounting Medium (Agilent). For immunofluorescence without in situ hybridization, we examined 4 control cases and 5 ALS cases. The same procedure described above was performed, except blocking and antibodies (anti-TDP-43 mouse antibody at 1:200 and anti-TRBP rabbit antibody at 1:100) were added directly after antigen retrieval. NeuroTrace 435/455 staining and mounting were then performed as described above. using immunofluorescence with antibodies to TDP-43 and TRBP. Confocal microscopy Images of dFISH-IF experiments performed for quantification were captured with Stellaris 5 or SP8 confocal microscopes and Leica Application Suite-X (LAS-X) software (Leica Microsystems). Navigator was used to acquire tiled images of the tissue sections with a 20x objective. Fluorescent dyes detected were NeuroTrace435/455 (Nissl-staining substance), Alexa Fluor 488 (TDP-43), Cyanine 3 (target miRNAs), and Cyanine 5 (reference RNA). A narrow detection window was used to detect each fluorophore, and no crosstalk was observed. For each experiment, confocal settings were optimized to the slide containing the highest motor neuron RNA fluorescent signal, such that no saturation was detected in the image for each fluorophore to be quantified (miRNA and U6 snRNA) and applied equally to all other slides during image acquisition. Representative dFISH-IF images for publication were taken with a Leica SP8 confocal microscope using a 63x objective with 1.4 NA and deconvolved by Lightning software (Leica Microsystems). Quantification Tiled images were opened in Fiji-ImageJ and regions of interest were drawn freehand around individual motor neurons[39]. NeuroTrace 435/455 staining, cellular anatomy, and motor neuron size/morphology were used as a combined reference to select motor neurons. The number of cases used per experiment ranged from 3 to 5 for controls and 8-11 for ALS (Supplementary file1: Table S1). The number of quantified motor neurons was typically greater than 50 per case slide. All observable motor neurons that fit the criteria were selected for quantification. Fluorescence representing miRNA expression per cell was measured as integrated fluorescence density. Target miRNA signal was either corrected to background fluorescence or corrected and then normalized to another cellular RNA species for expression[28]. MiR-132-3p in midbrain sections or U6 snRNA in cervical spinal cord sections were evaluated as candidate reference species by comparing corrected expression between control and ALS in two experiments. Cervical SMNs quantified in ALS cases were subdivided into three TDP-43-pathological subgroups based on the TDP-43 immunofluorescence pattern. Cells with an observable nucleus (based on lack of Nissl staining and U6 snRNA staining) were sorted into “ALS nuclear ” (largely nuclear TDP-43), “ALS cyto no inclusions” (largely cytoplasmic TDP-43 without inclusions), or “ALS cyto with inclusions” (largely cytoplasmic TDP-43 with inclusions) subtypes. At this magnification (20x objective) fine or subtle inclusions could not be reliably resolved, therefore a conservative approach was adopted whereby only clearly discernible inclusions were recorded in the “with inclusions” subtype and the remaining motor neurons with nuclear loss were included in “no inclusions”. Data preparation and statistical analysis Separate mixed-effects models were fit to each experiment to compare ALS and control groups for normalized and corrected RNA expression. This analysis compares RNA expression outcomes between ALS and control cases while accounting for patient-level variability, which was apparent from the data and confirmed in the analysis[1, 2]. Pairwise comparisons between ALS motor neuron subtypes were performed using Wald Z-tests on fixed effects from the mixed-effects models. Outliers were removed prior to analysis using GraphPad Prism’s ROUT method (Q = 1%). Because preliminary modeling suggested non-normality and unequal variance for some datasets, all data were log-transformed to stabilize variance; this also enabled interpretation as a percent change in ALS versus controls. A constant offset (|minimum|+1) was added where values were ≤0. Linear mixed-effects models were fitted with disease status as the fixed effect (control as reference) and a random intercept for each patient to account for repeated measures (non-independent observations). Predicted means were back-transformed to compute percent differences, and 95% confidence intervals were obtained by cluster bootstrapping (1,000 iterations). Significance was set at 0.05, and p-values were corrected within each miRNA-tissue family using the Benjamini-Yekutieli procedure to control false discovery rate under correlation (i.e. corrected miRNA expression comparisons are correlated with normalized miRNA comparisons). To visualize distributions of miRNA expression in motor neuron subpopulations by TDP-43 pathology, overlaid histograms and kernel density estimates (KDEs) were generated. Histograms were normalized using density scaling to allow direct comparison despite unequal group sizes, and smoothed KDE curves were plotted to show overall distribution shapes. All data processing and analysis were performed in Python 3.13.3[35]. The pandas package (2.2.3) was used for data manipulation and handling of tabular datasets, while numpy (2.2.4) was employed for numerical operations. Statistical modeling was conducted using the statsmodels package (0.14.4). The scipy library (1.15.2) was used for statistical functions and residual diagnostics. Data visualization was carried out using matplotlib (3.10.1) and seaborn (0.13.2). File path operations were managed using Python's built-in os module. Results Multiplex dFISH-IF allows co-detection of miRNAs, reference RNAs, and TDP-43 We developed a multiplexed fluorescent in situ hybridization method for use on human postmortem tissue to detect multiple low-abundance RNA targets and proteins. This approach allowed simultaneous visualization of up to three RNA species in the same tissue section (Supplementary file1: Fig S1). We applied a modified version of this method to profile miR-9-5p or miR-124-3p expression simultaneously with TDP-43 localization. We successfully detected miRNA, reference RNA, and TDP-43 in all cases analyzed (dFISH-IF). miR-132-3p was selected as a candidate reference RNA for OMNs based on small-scale preliminary testing that showed little difference in its expression between ALS and control cases and because it produced a consistently strong fluorescent signal in this tissue with little background (data not shown). Preliminary testing also indicated that miR-132-3p was reduced in ALS SMNs thus we looked for a substitute reference for spinal cord comparisons (data not shown). Because U6 snRNA has previously been used to normalize these miRNAs in published FISH studies, we adopted this approach for normalization of miRNA expression in spinal cord. Candidate reference RNAs were validated for normalization based on corrected expression showing no significant difference between ALS and control tissues (Supplementary file1: Fig S2). miR-9-5p and miR-124-3p expression are downregulated in ALS cervical spinal motor neurons We used multiplexed dFISH-IF in cervical spinal cord tissue to assess whether the previously reported downregulation of miR-9-5p and miR-124-3p in ALS could be detected with this method. Representative confocal images of motor neurons found and analyzed by dFISH-IF in cervical spinal cord control and ALS cases are shown in Fig. 1. We observed that most ALS cases contained SMNs with TDP-43-positive cytoplasmic inclusions (or aggregates), as well as motor neurons with either nuclear TDP-43 localization or nuclear clearance of TDP-43 (Fig. 1). Subsequent quantification of normalized miRNA expression revealed that both miR-9-5p and miR-124-3p were reduced in the ALS group compared to control for SMNs (Fig. 1 and 4a, c, and e). miR-9-5p was reduced in ALS by 52.3% (95% CI: 39.0, 63.3; p = 0.00222), and miR-124-3p was reduced in ALS by 69.7% (95% CI: 47.1, 83.2; p = 0.00647) (Fig. 4a, c and e). Similar results were obtained with quantification data that was not normalized but background-corrected (Supplementary file1: Fig S3). These findings validate our dFISH-IF protocol as a robust method for detecting disease-associated changes in miRNA expression alongside TDP-43 patterns, and they align with previous studies reporting downregulation of these miRNAs in ALS. miR-9-5p and miR-124-3p expression are preserved in ALS oculomotor neurons We then extended the analysis to midbrain tissue to assess the levels of miR-9-5p and miR-124-3p in ALS OMNs. To assist with anatomical orientation, low-magnification images of the oculomotor nucleus from control and ALS cases illustrating miR-9-5p, miR-124-3p, and TDP-43 expression are shown in Fig. 2. Representative confocal images of individual OMNs analyzed by dFISH-IF from control and ALS cases at higher magnification are shown in Fig. 3. We did not detect cytoplasmic TDP-43 inclusions or loss of nuclear TDP-43 in OMNs from control or ALS cases (Fig. 3). Notably, neither miRNA showed a statistically significant difference between ALS and control OMNs. Quantification revealed that miR-9-5p expression showed a non-significant 90.5% increase compared to control (p = 0.657), while miR-124-3p showed a non-significant 42.9% decrease (p = 0.144) (Fig. 4b, d, and e). This indicates that both miRNAs show altered regulation in OMNs compared to SMNs. Given that downregulation of the target miRNAs was statistically significant in ALS SMNs, the lack of significant differences in OMNs between ALS and control is consistent with the hypothesis of regional differential regulation. TRBP colocalizes with cytoplasmic TDP-43 inclusions in ALS cervical spinal motor neurons We then asked whether the observed downregulation of mature miR-9-5p and miR-124-3p in ALS might result from sequestration of miRNA biogenesis factors, as proposed by Emde et al .[14] To directly assess disruption of the Dicer complex, we focused on TRBP and evaluated its subcellular localization with TDP-43. In all control cases, TDP-43 was detected in the nucleus, whereas TRBP was cytoplasmic, often spatially associated with the Nissl marker at the rough endoplasmic reticulum (Fig. 5). In ALS SMNs with nuclear TDP-43 and no cytoplasmic inclusions (ALS nuclear ), TRBP was also detected in the cytoplasm, with a similar pattern as control motor neurons (Fig. 5). ALS motor neurons with a complete cytoplasmic shift of TDP-43 from the nucleus to the cytoplasm, along with small or diffuse TDP-43-positive cytoplasmic inclusions (ALS cyto, diffuse ), showed TRBP colocalization with these inclusions (white arrows for examples, Fig. 5). The colocalization between TRBP and TDP-43 was most apparent in ALS motor neurons with overt cytoplasmic inclusions, either spheroid or filamentous/skein-like (white arrows for examples in ALS cyto, spheroid or ALS cyto, skein ). We also detected this same pattern of colocalization with a second TDP-43 antibody and the same TRBP antibody using an immunofluorescence method to limit background signal and found the same colocalization pattern (Supplementary file1: Fig. S4). TDP-43-TRBP colocalization was also maintained throughout the motor neurons, as shown by a representative maximum projection of a z-stack acquisition from a SMN with a filamentous inclusion (Supplementary file1: Fig. S4b). These findings suggest that TRBP is found within TDP-43-specific cytoplasmic condensates, while its localization remains normal in ALS motor neurons without overt TDP-43 pathology, similar to representative control motor neurons. miR-9-5p and miR-124-3p downregulation occurs irrespective of overt TDP-43 pathology TDP-43 pathology in ALS exists along a spectrum, ranging from nuclear localization to complete nuclear clearance accompanied by cytoplasmic diffuse, spheroid, skein-like, or filamentous inclusions (Fig. 1). We observed motor neurons displaying these various subtypes of TDP-43 localization in the ALS cervical spinal cord tissues analyzed, but not within OMNs, where TDP-43 was nuclear. Because miRNA expression was preserved in OMNs without TDP-43 pathology but reduced in cervical spinal cord with TDP-43 pathology, we stratified ALS SMNs into subtypes based on TDP-43 localization (nuclear, cytoplasmic, or cytoplasmic with inclusions) to assess its influence on miRNA expression. We compared background-corrected miRNA expression in control cases to three subpopulations of ALS motor neurons: those with 1) largely nuclear TDP-43 or 2) largely cytoplasmic TDP-43, which was then further divided into 2a) those without visible TDP-43 inclusions or 2b) those with visible TDP-43 inclusions. Control data and ALS subgroup data were selected from the same raw data used for Fig. 4a and c, except that background-corrected target miRNA expression was used, as initial observations suggested that ALS motor neurons with cytoplasmic inclusions often contained little reference RNA. miR-9-5p and miR-124-3p were downregulated in all three ALS subpopulations compared to control and all comparisons were statistically significant except for the miR-9-5p “ALS cyto no inclusion” group (uncorrected p = 0.0370, corrected p = 0.188), possibly due to a small sample size (10 cases, 43 motor neurons) (Fig. 6). Downregulation in the total ALS SMN population quantified was 60.4% for miR-9-5p and 69.6% for miR-124-3p. Similarly, miR-9-5p was reduced by 57.7% in nuclear TDP-43, 49.7% in cytoplasmic TDP-43 without inclusions, and 61.6% in cytoplasmic TDP-43 with inclusions, while miR-124-3p was reduced by 69.0% in nuclear TDP-43, 60.7% in cytoplasmic TDP-43 without inclusions, and 67.6% in cytoplasmic TDP-43 with inclusions (Fig. 6a and c). These results indicate that SMNs without TDP-43 pathology show similarly reduced miR-9-5p and miR-124-3p expression as motor neurons with obvious evidence of TDP-43 cytoplasmic inclusions. Additionally, there was a similar distribution of miRNA expression for each pathological subgroup, supporting the statistical non-significance of the results (Fig. 5b and d). Together, these findings suggest that reduced miR-9-5p and miR-124-3p expression in ALS SMNs occurs independently of visible cytoplasmic TDP-43 aggregation. We also compared miRNA expression changes among the ALS TDP-43 subtypes for each miRNA within the context of this analysis. Most subtype comparisons were not statistically significant for either miRNA, however miR-124-3p expression increased by 26.4% in ALS neurons with cytoplasmic TDP-43 mislocalization (no inclusions) compared to ALS motor neurons with nuclear TDP-43 (p = 0.0222) (Fig. 6c). For miR-9-5p, expression was 18.8% higher in the cytoplasmic group (no inclusions) relative to the nuclear group, however this difference was not statistically significant (p = 0.519) (Fig. 6a). Discussion This work identifies two examples of miRNA dysregulation as cell-type-specific events in ALS disease progression, revealing a potential new mechanism of selective neuronal vulnerability. In agreement with previous studies, our profiling method revealed that miR-9-5p and miR-124-3p, two highly enriched neuronal miRNAs, were significantly downregulated in ALS cervical SMNs. Reduced miR-9-5p and miR-124-3p are predicted to derepress targets such as REST, MAP1B, PTBP1, and ROCK1, leading to impaired neuronal transcription, axonal instability, altered splicing, and disrupted mitochondrial function[33, 43, 44]. These pathways are validated in motor neurons and align with ALS phenotypes, including axonal transport defects, mitochondrial impairment, and abnormal neuron-glia interactions. In contrast, both miRNAs were unchanged in ALS OMNs, consistent with their resistance to degeneration and lack of TDP-43 pathology. The preservation of these miRNAs in OMNs likely maintains or enhances regulatory control over these disrupted pathways, conferring resilience against ALS-related stress. Although it is possible that maintained miRNA levels in OMNs reflect a downstream consequence of reduced pathological burden, the functional relevance of these specific miRNAs supports an active protective role. Further experimental validation is needed to confirm this and determine the specific pathways responsible for miRNA-mediated protection in OMNs. miR-132-3p expression was stable between ALS and control OMNs. Although used here as a reference, this brain-enriched miRNA regulates cytoskeletal plasticity, mitochondrial dynamics, and pro-survival signaling, and its maturation is TDP-43-dependent[24]. Because it is predicted to be downregulated in ALS spinal cord, its preserved expression in OMNs may represent a third example of miRNA preservation in this resilient population[9, 16]. Future work will determine whether this preservation is specific to a subset of neuroprotective miRNAs or reflects broader maintenance of miRNA regulatory networks. To explore a mechanism for miRNA downregulation in ALS, we examined TRBP localization with TDP-43. We hypothesized that impaired miRNA biogenesis upstream of RISC contributes to pathology, given prior evidence of AGO2 in SGs in cell models[14]. Because TDP-43 is an SG-associated RNA-binding protein in ALS, it provides a disease-relevant scaffold for assessing TRBP interactions in postmortem neurons. TRBP exhibited cytoplasmic localization in control SMNs and ALS SMNs where TDP-43 was nuclear, however, in ALS SMNs with cytoplasmic TDP-43 inclusions, TRBP colocalized with the inclusions. TRBP is a cytoplasmic double-stranded RNA-binding protein that modulates PKR-dependent SG formation, providing a mechanistic basis for its presence in protein-RNA assemblies that overlap with TDP-43 complexes.[32]. Although evidence from cell culture shows that TDP-43 and TRBP can co-exist in Dicer-containing complexes under normal conditions, TRBP is not recovered when immunoprecipitating TDP-43, indicating no stable association[24]. Thus, the co-localization we observed in ALS SMNs more likely reflects a pathological sequestration into condensates or SG-like assemblies. Together, these results suggest that chronic stress and aggregation may aberrantly incorporate miRNA biogenesis factors into persistent SG-like structures in ALS patients, leading to decreased miRNA expression. The colocalization results implied that dysregulated miRNA biogenesis by sequestration would only apply to neurons with visible TDP-43 inclusions. Furthermore, loss of nuclear TDP-43 is known to impair processing of specific miRNAs and disrupt feedback loops that normally regulate TDP-43 via specific miRNAs, consequently decreasing miRNA expression[19, 24, 30, 46]. These findings provided a rationale for quantifying miR-9-5p and miR-124-3p expression in subgroups of SMNs stratified by TDP-43 pathology. We asked whether nuclear TDP-43-positive motor neurons retain higher miRNA expression compared with neurons showing cytoplasmic or aggregated TDP-43. Contrary to our expectation, we found equivalent downregulation across all TDP-43 pathology subtypes compared to control for both miRNAs, indicating that nuclear TDP-43 alone in ALS is insufficient to maintain normal levels of these miRNAs. This may be caused by early and possibly TDP-43-independent mechanisms affecting RNA regulatory pathways. It also indicates that cytoplasmic TDP-43 inclusions are not necessary for miRNA dysregulation, and that motor neurons without visible aggregates may still be functionally impaired. These findings support a model in which miRNA downregulation occurs early in the cellular disease process, before the formation of visible TDP-43 inclusions. Even in the absence of visible TDP-43/TRBP inclusions, ALS motor neurons may experience early or subtle molecular disruptions that could impair miRNA biogenesis. Soluble mislocalized TDP-43, low-level cellular stress, or transient stress responses, such as early-stage granule formation, could be sufficient to disrupt Dicer complex function or engage alternative pathways that reduce mature miRNA levels, even in the absence of histologically detectable pathology at our current resolution. Retention of nuclear TDP-43 does not preclude SMNs from being actively engaged in the disease process. Identifying the molecular changes that precede or contribute to TDP-43 mislocalization is a critical area of ongoing research. Studies have demonstrated that nuclear TDP-43 can exhibit early conformational pathology coinciding with STMN2 (Stathmin-2) cryptic splicing, preceding clinical onset[41]. Similar loss-of-function effects occur at the UNC13A (Unc-13 Homolog A) locus, where cryptic exon inclusion leads to nonsense-mediated decay in ALS/FTD (frontotemporal dementia) patient tissue[7, 25, 29]. Beyond splicing defects, researchers have reported that nonfibrillar TDP-43 accumulates on the rough endoplasmic reticulum early in disease, potentially disrupting protein synthesis prior to visible aggregation[26]. Together, these observations and our findings support the interpretation that nuclear TDP-43-positive neurons in ALS can already be engaged in pathological processes that impair RNA regulation and cellular homeostasis. They also provide a rationale for further investigation into miRNA targets that are disrupted early in ALS cellular progression and developing therapeutic strategies that preserve RNA regulatory pathways to protect vulnerable neurons. Declarations Acknowledgements The authors acknowledge funding from the Canadian Institutes of Health Research (CIHR) and preliminary work important for this project, but not shown, conducted by Alex Martin. The authors acknowledge the use of ChatGPT (OpenAI, San Francisco, CA, USA) to assist with Python coding. Author contributions Conceptualization: RH, MJS, DCM. Methodology and Design: CM, DCM, RH, MJS. Experimentation/Data Acquisition: CM. Investigation: CM, MJS, DCM. Formal Analysis: CM. Supervision: RH, MJS, DCM. Manuscript (writing): CM. Manuscript (editing): CM, MJS, DCM, RH. All authors read, reviewed, and approved the final manuscript. Funding This work was supported by a Canadian Institutes of Health Research (CIHR) grant 201806SOP-411481 (MJS). Data availability All data generated or analyzed in this study are included in this published article, found in the supplementary files, or can be made available on reasonable request to the corresponding author. Conflict of interest The authors declare that there are no competing interests. Ethics approval and consent to participate Ethics approval for the use of human postmortem tissue was obtained from the University of Western Ontario, Health Sciences Research Ethics Board (HSREB) (protocols #103735 and #124855). Informed consent Informed consent for autopsy and retention of tissues for research purposes was obtained in written format from all patients antemortem or, if not possible, from their spouse postmortem. No minors participated in this study. To ensure anonymization, at the time of utilization for this research project, all cases were assigned a unique identifier with the master list correlating the unique identifier maintained in a locked cabinet in the office of the principal investigator (MJS) as per HSREB requirements. Author details 1 Molecular Medicine Group, Robarts Research Institute, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada. 2 Department of Pathology, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada. 3 Department of Clinical Neurological Sciences, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada. 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Supplementary Files Supplementaryfile1.docx Cite Share Download PDF Status: Published Journal Publication published 26 Mar, 2026 Read the published version in Acta Neuropathologica → Version 1 posted Editorial decision: Revision requested 19 Oct, 2025 Reviews received at journal 29 Sep, 2025 Reviews received at journal 26 Sep, 2025 Reviewers agreed at journal 11 Sep, 2025 Reviewers agreed at journal 10 Sep, 2025 Reviewers agreed at journal 08 Sep, 2025 Reviewers invited by journal 06 Sep, 2025 Editor assigned by journal 06 Sep, 2025 Submission checks completed at journal 06 Sep, 2025 First submitted to journal 04 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7537447","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":513700704,"identity":"73535d63-598d-414a-8ac6-85fe29e5571b","order_by":0,"name":"Crystal McLellan","email":"","orcid":"","institution":"Western University","correspondingAuthor":false,"prefix":"","firstName":"Crystal","middleName":"","lastName":"McLellan","suffix":""},{"id":513700705,"identity":"3de8e97b-f306-410a-be4e-379fdcc66c3b","order_by":1,"name":"Danae Campos-Melo","email":"","orcid":"","institution":"Western University","correspondingAuthor":false,"prefix":"","firstName":"Danae","middleName":"","lastName":"Campos-Melo","suffix":""},{"id":513700706,"identity":"039ef6ec-1dcd-4f61-ac41-b9ce6dc876b2","order_by":2,"name":"Robert Hammond","email":"","orcid":"","institution":"Western University","correspondingAuthor":false,"prefix":"","firstName":"Robert","middleName":"","lastName":"Hammond","suffix":""},{"id":513700707,"identity":"ac59296e-511b-4f8a-96a3-1bc88cf786c3","order_by":3,"name":"Michael J. Strong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAApklEQVRIiWNgGAWjYBADOVIUM4NJY9K1JDYQrUG3//yxDx931KZvuJF8gOFHDRFazG4kM8+ceeZ47oYbaQmMPceI0sLMzMzbdix3w+0cA2YGNmK0nD8M1pJuANbyjxgtB5JBWmoSwFoY24hyWLIx48y2A4Yz7z9LONjbR5TDDj5m+NhWJ8935vDBBz++EaEFCg6DyQPEa2BgqCNF8SgYBaNgFIw0AACYGDfoet4gLAAAAABJRU5ErkJggg==","orcid":"","institution":"Western University","correspondingAuthor":true,"prefix":"","firstName":"Michael","middleName":"J.","lastName":"Strong","suffix":""}],"badges":[],"createdAt":"2025-09-04 15:08:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7537447/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7537447/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00401-026-03005-6","type":"published","date":"2026-03-26T16:09:03+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":91305042,"identity":"dfaa4312-eea5-40d2-bd45-a2932957ae4d","added_by":"auto","created_at":"2025-09-15 06:24:44","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":363279,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of miR-9-5p and miR-124-3p in motor neurons of the cervical spinal cord. Confocal images of representative control and ALS motor neurons are shown, stained with probes to miR-9-5p or miR-124-3p (both shown in yellow) and U6 small nuclear RNA (magenta), along with an antibody to TDP-43 (green) and NeuroTrace 435/455, Nissl-staining substance (blue). dFISH-IF staining for each probe/target is shown by channel, and a merged image is also provided. Each row represents a single motor neuron. ALS motor neurons shown are representative of different TDP-43 pathologies observed in cervical spinal cord cases. These include TDP-43 localization that is largely nuclear (ALS\u003csub\u003enuclear\u003c/sub\u003e), largely cytoplasmic with relatively small diffuse/granulofilamentous inclusions (ALS\u003csub\u003ecyto\u003c/sub\u003e), cytoplasmic with round spheroid inclusions (ALS\u003csub\u003ecyto, spheroid\u003c/sub\u003e), and cytoplasmic with obvious skein/filamentous-like inclusions (ALS\u003csub\u003ecyto, skein\u003c/sub\u003e). Not all motor neurons have an observable nucleus in the image plane. Images were acquired with a 63x objective (1.4 NA) and deconvolved using Leica Lightning software. Scale bars are indicated in white and represent 25 µm\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7537447/v1/d3c6b8f1785da252a16f33e7.jpg"},{"id":91305101,"identity":"f53b2b62-a6d1-4c29-85cf-8837fe023e14","added_by":"auto","created_at":"2025-09-15 06:24:47","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":574965,"visible":true,"origin":"","legend":"\u003cp\u003eOculomotor nucleus in control (a) and ALS (b) postmortem tissue, illustrating miRNA expression and TDP-43 localization. Merged confocal images of representative control and ALS midbrain tissue sections showing dFISH-IF staining using probes to miR-124-3p (magenta), miR-132-3p (cyan), and an antibody to TDP-43 (green) are shown (white indicates merge of magenta and cyan). Tiled images were acquired with a 10x objective at 1.5 zoom and merged using Leica Navigator software. The oculomotor nuclei regions are outlined with red dashed lines. Scale bars are indicated in white and represent 1000 µm\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7537447/v1/4453860eb46c240c7c801ceb.jpg"},{"id":91305045,"identity":"fef047fc-51aa-49e0-a275-4ed0b5f7aedb","added_by":"auto","created_at":"2025-09-15 06:24:45","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":273612,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of miR-9-5p and miR-124-3p in oculomotor neurons. Confocal images of representative control and ALS OMNs are shown, stained with probes to miR-9-5p or miR-124-3p (both shown in yellow) and miR-132-3p (magenta), along with an antibody to TDP-43 (green) and NeuroTrace 435/455, Nissl-staining substance (blue). Double FISH-IF staining for each probe/target is shown individually (by channel), and a merged image is also provided in the last column. Each row represents a single motor neuron. Images were acquired with a 63x objective (1.4 NA) and deconvolved using Leica Lightning software. Scale bars are indicated in white and represent 25 µm\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7537447/v1/2c52039b141e901ed3acaf84.jpg"},{"id":91305102,"identity":"77228366-1fbb-4059-9724-93d798709f6b","added_by":"auto","created_at":"2025-09-15 06:24:48","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":245113,"visible":true,"origin":"","legend":"\u003cp\u003eQuantification of miR-9-5p and miR-124-3p expression levels in motor neurons of the cervical spinal cord and oculomotor nucleus. a-d Levels of miR-9-5p and miR-124-3p were measured in individual motor neurons from the cervical spinal cord and oculomotor nucleus of ALS and control patients from double FISH-IF stained tissue sections. Quantification was based on integrated fluorescence density per neuron from confocal images of tissue sections, followed by background correction and normalization to the indicated reference RNA. Single-cell and summary data are shown, with quantified individual motor neurons represented by grey dots, median and interquartile range by boxplots, and mean by a red diamond. Statistical comparisons were performed using a log-transformed linear mixed-effects model accounting for patient-level variation. Number of cases analyzed for each panel is as follows: a, control = 4, ALS = 10; b, control = 5, ALS = 8; c, control = 4, ALS = 10 ; d, control = 3, ALS = 7. e Forest plot indicating the relative changes in ALS compared to control for miR-9-5p and miR-124-3p normalized expression in each of the specified motor neuron regions. Percent change was calculated from predicted values obtained from the linear mixed-effects model, and 95% confidence intervals (indicated by horizontal bars) were estimated using clustered bootstrapping. P-values reflect multiple testing correction within each miRNA/tissue family as described in the Methods\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7537447/v1/b13d4dc33de093fed08afcba.jpg"},{"id":91305044,"identity":"9cf4a891-aa29-47dd-97aa-93515a7401f0","added_by":"auto","created_at":"2025-09-15 06:24:45","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":341399,"visible":true,"origin":"","legend":"\u003cp\u003eTDP-43 and TRBP expression in control and ALS cervical spinal motor neurons with and without TDP-43-cytoplasmic inclusions. a Confocal images of representative ALS motor neurons are shown, immunostained with a mouse antibody to TDP-43 (green) and a rabbit antibody to TRBP (magenta), including NeuroTrace 435/455, Nissl-staining substance (blue). Individual channels are shown, and two merged images are provided as described. Each row contains a single representative motor neuron, categorized by TDP-43 pathological staining. White arrows indicate examples of TDP-43 and TRBP colocalization, but do not reflect the only examples. Images were acquired with a 63x objective (1.4 NA) and deconvolved using Leica Lightning software. Motor neurons were taken with slightly different zoom settings to better observe the inclusion structures. Scale bars are indicated in white and represent 20 µm\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7537447/v1/5029c329537138adbedc2577.jpg"},{"id":91305128,"identity":"176e0da0-a833-4e1b-966f-bec1f7919afd","added_by":"auto","created_at":"2025-09-15 06:24:48","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":226034,"visible":true,"origin":"","legend":"\u003cp\u003eALS-associated downregulation of miR-9-5p and miR-124-3p is independent of TDP-43 aggregation in cervical spinal motor neurons. a, c Motor neuron TDP-43 pathology was visually assessed with immunofluorescence in the same images used for quantification of miRNA expression shown in Fig 4a and c. ALS motor neurons with observable nuclei based on morphology and Nissl staining were grouped into either “nuclear” (where they contained no obvious cytoplasmic TDP-43 inclusions and were accompanied by largely TDP-43 nuclear localization), “cyto no inclusions” (where they contained no obvious cytoplasmic TDP-43 inclusions but were accompanied by TDP-43 nuclear clearing) or “cyto with inclusions” (where they contained both obvious cytoplasmic TDP-43 inclusions and nuclear clearing). Expression levels reflect corrected total cell fluorescence (CTCF). Single-cell (grey dots) and summary data are shown with median and interquartile range by boxplots (“nuclear”, blue; “cyto no inclusions”, orange; “cyto with inclusions”, purple), and the mean by a red diamond. Statistical comparisons were performed among each TDP-43-pathology subtype and control using a log-transformed linear mixed-effects model. Number of cases analyzed for miR-9-5p: control, n = 4, ALS\u003csub\u003enuclear\u003c/sub\u003e = 11, ALS\u003csub\u003ecyto\u003c/sub\u003e no inclusions = 10, ALS\u003csub\u003ecyto\u003c/sub\u003e with inclusions = 10. Number of cases analyzed for miR-124-3p: control, n = 4, ALS\u003csub\u003enuclear\u003c/sub\u003e = 10, ALS\u003csub\u003ecyto\u003c/sub\u003e no inclusions = 9, ALS\u003csub\u003ecyto\u003c/sub\u003e with inclusions = 9. P-values reflect multiple testing correction within each miRNA/tissue family as described in the Methods. b, d\u003cstrong\u003e \u003c/strong\u003emiR-9-5p and miR-124-3p expression distribution in the three ALS subtypes described above is depicted by histograms. Histograms shown are normalized to probability density to account for sample size differences. Kernel density estimates (KDEs) are shown to better illustrate the distribution of miRNA levels across each subtype.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7537447/v1/917a7260f10b68b843369182.jpg"},{"id":106093158,"identity":"f9139de7-3cf2-445f-b0b5-a354532f7a43","added_by":"auto","created_at":"2026-04-03 11:35:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2745550,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7537447/v1/1feec016-6be7-4f89-a064-8f2915cdb9b8.pdf"},{"id":91305525,"identity":"0c69ce88-15da-4b28-969b-e15bc15c8380","added_by":"auto","created_at":"2025-09-15 06:26:10","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1944070018,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7537447/v1/549863192896db4e00a93871.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Preservation of miR-9-5p and miR-124-3p in ALS-resistant oculomotor neurons contrasts with their downregulation in vulnerable spinal motor neurons, irrespective of TDP-43 pathology","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAmyotrophic lateral sclerosis (ALS) is characterized by progressive loss of motor neurons resulting in paralysis and death, yet some motor neuron subtypes remarkably remain resistant[31]. Spinal motor neurons (SMNs) undergo early and severe degeneration, while other populations, such as oculomotor neurons (OMNs) that control eye movement, are largely spared [17, 23]. Post-mortem analysis shows that ALS OMNs typically retain nuclear expression of transactive response DNA-binding protein 43 kDa (TDP-43), an RNA-binding protein strongly associated with ALS pathology, whereas SMNs commonly exhibit TDP-43 mislocalization to the cytoplasm and subsequent formation of biomolecular condensates[4, 8, 42]. Features such as unique cellular architecture, distinct gene expression, specialized metabolism, and reduced glutamate excitotoxicity are suspected to contribute to the resilience of OMNs[3, 5, 11, 20, 27, 40]. Elucidating these and other novel mechanisms that confer protection could help with the development of targeted neuroprotective approaches in ALS.\u003c/p\u003e\n\u003cp\u003eDysregulated RNA metabolism is increasingly recognized as an important pathological feature of ALS, and microRNAs (miRNAs) have emerged as both dysregulated molecules and promising biomarkers for disease progression[9, 10, 14, 15, 21, 36]. miRNAs are short (~22 nt) non-coding RNAs that fine-tune gene expression post-transcriptionally by guiding the RNA-induced silencing complex (RISC) to target mRNAs, leading to translational repression or degradation. miRNA production begins in the nucleus, where Drosha and DGCR8 cleave primary transcripts into precursor miRNAs, which are exported to the cytoplasm and further processed by Dicer, aided by TAR RNA-binding protein (TRBP) and PACT. The guide strand is incorporated into RISC, where Argonaute 2 (AGO2) directs it to target mRNAs for translational repression or degradation. Because of their stability and cell-specificity, miRNAs are useful for understanding ALS pathogenesis and providing candidate biomarkers.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSeveral studies have reported a global downregulation of miRNAs in ALS spinal cord tissue homogenates and lumbar motor neurons, as demonstrated by \u003cem\u003ein situ\u003c/em\u003e hybridization, qPCR, and RNA sequencing[9, 13–15, 37]. One proposed mechanism for this downregulation involves impaired Drosha/DGCR8 and Dicer/TRBP function downstream of ALS-causing mutations and cellular stress. In cell culture stress assays, AGO2 was also shown to interact with the stress granule (SG) protein TIA-1, linking miRNA machinery to SG dynamics[14]. Alternatively, other evidence from cell culture suggests that TDP-43 mislocalization, triggered by cellular stress, can disrupt a negative feedback loop involving specific miRNAs, where loss of nuclear TDP-43 leads to decreased expression of these miRNAs in the cytoplasm[19].\u003c/p\u003e\n\u003cp\u003emiR-9-5p and miR-124-3p have been reported to exhibit reduced expression in spinal cords of individuals with ALS[9, 12, 14, 15, 18, 46]. Targets of both miRNAs are implicated in neuronal development and function, including neurogenesis, cell fate specification, synaptic remodeling, and structural maturation of axons and dendrites, indicating their importance in neuronal integrity[18, 22, 38, 45]. This functional relevance may explain why SMNs are particularly susceptible to ALS-related pathology. However, miRNA expression profiling in ALS-resistant motor neurons, such as OMNs, is lacking. Since OMNs usually maintain nuclear TDP-43 throughout the disease course, exhibit resistance to degeneration compared to SMNs, and miR-9-5p and miR-124-3p targets are responsible for neuronal maintenance, we hypothesized that OMNs and SMNs from ALS patients would differ in their expression of miR-9-5p and miR-124-3p compared to control cases. We assessed the expression of miR-9-5p and miR-124-3p in postmortem sporadic ALS and control tissue using fluorescent \u003cem\u003ein situ\u003c/em\u003e hybridization, targeting both OMNs and SMNs. Immunofluorescence (IF) for TDP-43 was included to evaluate miRNA expression and cellular pathology.\u0026nbsp;This approach enabled direct comparison of miRNA expression in vulnerable and resistant motor neuron populations.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003ePatient tissue\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFixed tissue sections of cervical spinal cord and superior midbrain were used for fluorescent \u003cem\u003ein situ\u0026nbsp;\u003c/em\u003ehybridization and immunofluorescence (FISH-IF) experiments. Formalin-fixed, post-mortem tissue control patients or patients diagnosed with sporadic ALS according to El Escorial criteria (World Federation of Neurology) by MJS were processed, embedded in paraffin, sectioned at 7 um thickness and mounted on glass slides[6]. Basic demographic information of the patients is provided in Supplementary file1: Table S1. The cervical spinal cord was selected for analysis because it is significantly affected in ALS while still retaining a substantial population of observable motor neurons. SMNs of the ventral horn were identified in the anterior region of the spinal cord sections by the larger size of the ventral horn compared to the dorsal horn and the presence of prominent motor neuron cell bodies. The oculomotor nucleus was identified anatomically in midbrain sections at the level of the superior colliculus paired posteromedial nuclei ventral to the periaqueductal gray matter, medial to the median longitudinal fasciculus. and from which ventrally exiting fascicles are present. In several cases, the midbrain was initially cut on the mid-sagittal plane and sectioned into halves prior to cutting and mounting for examination.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFluorescent \u003cem\u003ein situ\u003c/em\u003e hybridization and Immunofluorescence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDouble fluorescent \u003cem\u003ein situ\u003c/em\u003e hybridization and immunofluorescence (dFISH-IF) was used to detect target miRNA miR-9-5p or miR-124-3p with candidate normalizers miR-132-3p or U6 small nuclear RNA (U6 snRNA) and TDP-43 in motor neurons, along with the motor neuron marker NeuroTrace435/455 (Nissl-staining substance; does not stain nucleus). U6 snRNA was used previously for normalization in control and ALS lumbar spinal cord \u003cem\u003ein situ\u003c/em\u003e experiments[14]. FISH-IF was performed similarly to a published protocol and is briefly outlined here with modifications[34]. Tissue sections were dewaxed with xylenes and rehydrated with a series of ethanol dilutions. Antigen retrieval was performed in 10 mM sodium citrate, pH 6.0, using a pressure cooker for 15 minutes at high pressure at 100°C. dFISH was performed with inventoried digoxigenin (DIG) or 6-carboxyfluorescein (FAM) labelled mirCURY LNA™ miRNA detection probes (GeneGlobe, Qiagen; for probe details see Supplementary file1: Table S2). Two differently labeled LNA probes (DIG or FAM) with similar hybridization temperatures (within 10°C) were added to tissue sections, and each was detected by sequential HRP antibody-tyramide signal amplification (TSA; Cyanine 3 or 5, Akoya). Triple FISH (tFISH) was performed similarly to the above method, followed by sequential hybridization and detection of the third probe. Immunofluorescence of the same slides was performed using an anti-TDP-43 antibody (ProteinTech) at 1:150 in 1xPBS with 2% BSA (bovine serum albumin) after blocking in 1xPBS with 5% BSA and incubated overnight at 4°C (Supplementary file1: Table S2). An Alexa Fluor 488-conjugated secondary antibody (Invitrogen) at 1:250 in 1xPBS with 2% BSA was used to detect the TDP-43 antibody. NeuroTrace 435/455 (Invitrogen) was used to label neurons as directed by the manufacturer, except permeabilization buffer consisted of 0.3% TritonX-100, and NeuroTrace was diluted 1:200 before addition to sections. Tissue sections were mounted with Dako Fluorescence Mounting Medium (Agilent).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor immunofluorescence without \u003cem\u003ein situ\u003c/em\u003e hybridization, we examined 4 control cases and 5 ALS cases. The same procedure described above was performed, except blocking and antibodies (anti-TDP-43 mouse antibody at 1:200 and anti-TRBP rabbit antibody at 1:100) were added directly after antigen retrieval. NeuroTrace 435/455 staining and mounting were then performed as described above. \u0026nbsp;using immunofluorescence with antibodies to TDP-43 and TRBP.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConfocal microscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImages of dFISH-IF experiments performed for quantification were captured with Stellaris 5 or SP8 confocal microscopes and Leica Application Suite-X (LAS-X) software (Leica Microsystems). Navigator was used to acquire tiled images of the tissue sections with a 20x objective. Fluorescent dyes detected were NeuroTrace435/455 (Nissl-staining substance), Alexa Fluor 488 (TDP-43), Cyanine 3 (target miRNAs), and Cyanine 5 (reference RNA). A narrow detection window was used to detect each fluorophore, and no crosstalk was observed. For each experiment, confocal settings were optimized to the slide containing the highest motor neuron RNA fluorescent signal, such that no saturation was detected in the image for each fluorophore to be quantified (miRNA and U6 snRNA) and applied equally to all other slides during image acquisition. Representative dFISH-IF images for publication were taken with a Leica SP8 confocal microscope using a 63x objective with 1.4 NA and deconvolved by Lightning software (Leica Microsystems).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTiled images were opened in Fiji-ImageJ and regions of interest were drawn freehand around individual motor neurons[39]. NeuroTrace 435/455 staining, cellular anatomy, and motor neuron size/morphology were used as a combined reference to select motor neurons. The number of cases used per experiment ranged from 3 to 5 for controls and 8-11 for ALS (Supplementary file1: Table S1). The number of quantified motor neurons was typically greater than 50 per case slide. All observable motor neurons that fit the criteria were selected for quantification. Fluorescence representing miRNA expression per cell was measured as integrated fluorescence density. Target miRNA signal was either corrected to background fluorescence or corrected and then normalized to another cellular RNA species for expression[28]. MiR-132-3p in midbrain sections or U6 snRNA in cervical spinal cord sections were evaluated as candidate reference species by comparing corrected expression between control and ALS in two experiments. Cervical SMNs quantified in ALS cases were subdivided into three TDP-43-pathological subgroups based on the TDP-43 immunofluorescence pattern. Cells with an observable nucleus (based on lack of Nissl staining and U6 snRNA staining) were sorted into “ALS\u003csub\u003enuclear\u003c/sub\u003e” (largely nuclear TDP-43), “ALS\u003csub\u003ecyto\u003c/sub\u003e no inclusions” (largely cytoplasmic TDP-43 without inclusions), or “ALS\u003csub\u003ecyto\u003c/sub\u003e with inclusions” (largely cytoplasmic TDP-43 with inclusions) subtypes. At this magnification (20x objective) fine or subtle inclusions could not be reliably resolved, therefore a conservative approach was adopted whereby only clearly discernible inclusions were recorded in the “with inclusions” subtype and the remaining motor neurons with nuclear loss were included in “no inclusions”.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData preparation and statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeparate mixed-effects models were fit to each experiment to compare ALS and control groups for normalized and corrected RNA expression. This analysis compares RNA expression outcomes between ALS and control cases while accounting for patient-level variability, which was apparent from the data and confirmed in the analysis[1, 2]. Pairwise comparisons between ALS motor neuron subtypes were performed using Wald Z-tests on fixed effects from the mixed-effects models.\u003c/p\u003e\n\u003cp\u003eOutliers were removed prior to analysis using GraphPad Prism’s ROUT method (Q = 1%). Because preliminary modeling suggested non-normality and unequal variance for some datasets, all data were log-transformed to stabilize variance; this also enabled interpretation as a percent change in ALS versus controls. A constant offset (|minimum|+1) was added where values were ≤0. Linear mixed-effects models were fitted with disease status as the fixed effect (control as reference) and a random intercept for each patient to account for repeated measures (non-independent observations). Predicted means were back-transformed to compute percent differences, and 95% confidence intervals were obtained by cluster bootstrapping (1,000 iterations). Significance was set at 0.05, and p-values were corrected within each miRNA-tissue family using the Benjamini-Yekutieli procedure to control false discovery rate under correlation (i.e. corrected miRNA expression comparisons are correlated with normalized miRNA comparisons).\u003c/p\u003e\n\u003cp\u003eTo visualize distributions of miRNA expression in motor neuron subpopulations by TDP-43 pathology, overlaid histograms and kernel density estimates (KDEs) were generated. Histograms were normalized using density scaling to allow direct comparison despite unequal group sizes, and smoothed KDE curves were plotted to show overall distribution shapes.\u003c/p\u003e\n\u003cp\u003eAll data processing and analysis were performed in Python 3.13.3[35]. The pandas package (2.2.3) was used for data manipulation and handling of tabular datasets, while numpy (2.2.4) was employed for numerical operations. Statistical modeling was conducted using the statsmodels package (0.14.4). The scipy library (1.15.2) was used for statistical functions and residual diagnostics. Data visualization was carried out using matplotlib (3.10.1) and seaborn (0.13.2). File path operations were managed using Python's built-in os module.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eMultiplex dFISH-IF allows co-detection of miRNAs, reference RNAs, and TDP-43\u003cbr\u003e\u003c/strong\u003eWe developed a multiplexed fluorescent \u003cem\u003ein situ\u003c/em\u003e hybridization method for use on human postmortem tissue to detect multiple low-abundance RNA targets and proteins. This approach allowed simultaneous visualization of up to three RNA species in the same tissue section (Supplementary file1: Fig S1). We applied a modified version of this method to profile miR-9-5p or miR-124-3p expression simultaneously with TDP-43 localization. We successfully detected miRNA, reference RNA, and TDP-43 in all cases analyzed (dFISH-IF).\u003c/p\u003e\n\u003cp\u003emiR-132-3p was selected as a candidate reference RNA for OMNs based on small-scale preliminary testing that showed little difference in its expression between ALS and control cases and because it produced a consistently strong fluorescent signal in this tissue with little background (data not shown). Preliminary testing also indicated that miR-132-3p was reduced in ALS SMNs thus we looked for a substitute reference for spinal cord comparisons (data not shown). Because U6 snRNA has previously been used to normalize these miRNAs in published FISH studies, we adopted this approach for normalization of miRNA expression in spinal cord. Candidate reference RNAs were validated for normalization based on corrected expression showing no significant difference between ALS and control tissues (Supplementary file1: Fig S2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emiR-9-5p and miR-124-3p expression are downregulated in ALS cervical spinal motor neurons\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe used multiplexed dFISH-IF in cervical spinal cord tissue to assess whether the previously reported downregulation of miR-9-5p and miR-124-3p in ALS could be detected with this method. Representative confocal images of motor neurons found and analyzed by dFISH-IF in cervical spinal cord control and ALS cases are shown in Fig. 1. We observed that most ALS cases contained SMNs with TDP-43-positive cytoplasmic inclusions (or aggregates), as well as motor neurons with either nuclear TDP-43 localization or nuclear clearance of TDP-43 (Fig. 1). Subsequent quantification of normalized miRNA expression revealed that both miR-9-5p and miR-124-3p were reduced in the ALS group compared to control for SMNs (Fig. 1 and 4a, c, and e). miR-9-5p was reduced in ALS by 52.3% (95% CI: 39.0, 63.3; p = 0.00222), and miR-124-3p was reduced in ALS by 69.7% (95% CI: 47.1, 83.2; p = 0.00647) (Fig. 4a, c and e). Similar results were obtained with quantification data that was not normalized but background-corrected (Supplementary file1: Fig S3). These findings validate our dFISH-IF protocol as a robust method for detecting disease-associated changes in miRNA expression alongside TDP-43 patterns, and they align with previous studies reporting downregulation of these miRNAs in ALS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emiR-9-5p and miR-124-3p expression are preserved in ALS oculomotor neurons\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe then extended the analysis to midbrain tissue to assess the levels of miR-9-5p and miR-124-3p in ALS OMNs. To assist with anatomical orientation, low-magnification images of the oculomotor nucleus from control and ALS cases illustrating miR-9-5p, miR-124-3p, and TDP-43 expression are shown in Fig. 2. Representative confocal images of individual OMNs analyzed by dFISH-IF from control and ALS cases at higher magnification are shown in Fig. 3. We did not detect cytoplasmic TDP-43 inclusions or loss of nuclear TDP-43 in OMNs from control or ALS cases (Fig. 3). Notably, neither miRNA showed a statistically significant difference between ALS and control OMNs. Quantification revealed that miR-9-5p expression showed a non-significant 90.5% increase compared to control (p = 0.657), while miR-124-3p showed a non-significant 42.9% decrease (p = 0.144) (Fig. 4b, d, and e). This indicates that both miRNAs show altered regulation in OMNs compared to SMNs. Given that downregulation of the target miRNAs was statistically significant in ALS SMNs, the lack of significant differences in OMNs between ALS and control is consistent with the hypothesis of regional differential regulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTRBP colocalizes with cytoplasmic TDP-43 inclusions in ALS cervical spinal motor neurons\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe then asked whether the observed downregulation of mature miR-9-5p and miR-124-3p in ALS might result from sequestration of miRNA biogenesis factors, as proposed by Emde \u003cem\u003eet al\u003c/em\u003e.[14] To directly assess disruption of the Dicer complex, we focused on TRBP and evaluated its subcellular localization with TDP-43. In all control cases, TDP-43 was detected in the nucleus, whereas TRBP was cytoplasmic, often spatially associated with the Nissl marker at the rough endoplasmic reticulum (Fig. 5). In ALS SMNs with nuclear TDP-43 and no cytoplasmic inclusions (ALS\u003csub\u003enuclear\u003c/sub\u003e), TRBP was also detected in the cytoplasm, with a similar pattern as control motor neurons (Fig. 5). ALS motor neurons with a complete cytoplasmic shift of TDP-43 from the nucleus to the cytoplasm, along with small or diffuse TDP-43-positive cytoplasmic inclusions (ALS\u003csub\u003ecyto, diffuse\u003c/sub\u003e), showed TRBP colocalization with these inclusions (white arrows for examples, Fig. 5). The colocalization between TRBP and TDP-43 was most apparent in ALS motor neurons with overt cytoplasmic inclusions, either spheroid or filamentous/skein-like (white arrows for examples in ALS\u003csub\u003ecyto, spheroid\u003c/sub\u003e or ALS\u003csub\u003ecyto, skein\u003c/sub\u003e). We also detected this same pattern of colocalization with a second TDP-43 antibody and the same TRBP antibody using an immunofluorescence method to limit background signal and found the same colocalization pattern (Supplementary file1: Fig. S4). TDP-43-TRBP colocalization was also maintained throughout the motor neurons, as shown by a representative maximum projection of a z-stack acquisition from a SMN with a filamentous inclusion (Supplementary file1: Fig. S4b). These findings suggest that TRBP is found within TDP-43-specific cytoplasmic condensates, while its localization remains normal in ALS motor neurons without overt TDP-43 pathology, similar to representative control motor neurons.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emiR-9-5p and miR-124-3p downregulation occurs irrespective of overt TDP-43 pathology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTDP-43 pathology in ALS exists along a spectrum, ranging from nuclear localization to complete nuclear clearance accompanied by cytoplasmic diffuse, spheroid, skein-like, or filamentous inclusions (Fig. 1). We observed motor neurons displaying these various subtypes of TDP-43 localization in the ALS cervical spinal cord tissues analyzed, but not within OMNs, where TDP-43 was nuclear. Because miRNA expression was preserved in OMNs without TDP-43 pathology but reduced in cervical spinal cord with TDP-43 pathology, we stratified ALS SMNs into subtypes based on TDP-43 localization (nuclear, cytoplasmic, or cytoplasmic with inclusions) to assess its influence on miRNA expression.\u003c/p\u003e\n\u003cp\u003eWe compared background-corrected miRNA expression in control cases to three subpopulations of ALS motor neurons: those with 1) largely nuclear TDP-43 or 2) largely cytoplasmic TDP-43, which was then further divided into 2a) those without visible TDP-43 inclusions or 2b) those with visible TDP-43 inclusions. Control data and ALS subgroup data were selected from the same raw data used for Fig. 4a and c, except that background-corrected target miRNA expression was used, as initial observations suggested that ALS motor neurons with cytoplasmic inclusions often contained little reference RNA. miR-9-5p and miR-124-3p were downregulated in all three ALS subpopulations compared to control and all comparisons were statistically significant except for the miR-9-5p “ALS\u003csub\u003ecyto\u003c/sub\u003e no inclusion” group (uncorrected p = 0.0370, corrected p = 0.188), possibly due to a small sample size (10 cases, 43 motor neurons) (Fig. 6). Downregulation in the total ALS SMN population quantified was 60.4% for miR-9-5p and 69.6% for miR-124-3p. Similarly, miR-9-5p was reduced by 57.7% in nuclear TDP-43, 49.7% in cytoplasmic TDP-43 without inclusions, and 61.6% in cytoplasmic TDP-43 with inclusions, while miR-124-3p was reduced by 69.0% in nuclear TDP-43, 60.7% in cytoplasmic TDP-43 without inclusions, and 67.6% in cytoplasmic TDP-43 with inclusions (Fig. 6a and c). These results indicate that SMNs without TDP-43 pathology show similarly reduced miR-9-5p and miR-124-3p expression as motor neurons with obvious evidence of TDP-43 cytoplasmic inclusions. Additionally, there was a similar distribution of miRNA expression for each pathological subgroup, supporting the statistical non-significance of the results (Fig. 5b and d). Together, these findings suggest that reduced miR-9-5p and miR-124-3p expression in ALS SMNs occurs independently of visible cytoplasmic TDP-43 aggregation.\u003c/p\u003e\n\u003cp\u003eWe also compared miRNA expression changes among the ALS TDP-43 subtypes for each miRNA within the context of this analysis. Most subtype comparisons were not statistically significant for either miRNA, however miR-124-3p expression increased by 26.4% in ALS neurons with cytoplasmic TDP-43 mislocalization (no inclusions) compared to ALS motor neurons with nuclear TDP-43 (p = 0.0222) (Fig. 6c). For miR-9-5p, expression was 18.8% higher in the cytoplasmic group (no inclusions) relative to the nuclear group, however this difference was not statistically significant (p = 0.519) (Fig. 6a).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis work identifies two examples of miRNA dysregulation as cell-type-specific events in ALS disease progression, revealing a potential new mechanism of selective neuronal vulnerability. In agreement with previous studies, our profiling method revealed that miR-9-5p and miR-124-3p, two highly enriched neuronal miRNAs, were significantly downregulated in ALS cervical SMNs. Reduced miR-9-5p and miR-124-3p are predicted to derepress targets such as REST, MAP1B, PTBP1, and ROCK1, leading to impaired neuronal transcription, axonal instability, altered splicing, and disrupted mitochondrial function[33, 43, 44]. These pathways are validated in motor neurons and align with ALS phenotypes, including axonal transport defects, mitochondrial impairment, and abnormal neuron-glia interactions. In contrast, both miRNAs were unchanged in ALS OMNs, consistent with their resistance to degeneration and lack of TDP-43 pathology. The preservation of these miRNAs in OMNs likely maintains or enhances regulatory control over these disrupted pathways, conferring resilience against ALS-related stress. Although it is possible that maintained miRNA levels in OMNs reflect a downstream consequence of reduced pathological burden, the functional relevance of these specific miRNAs supports an active protective role. Further experimental validation is needed to confirm this and determine the specific pathways responsible for miRNA-mediated protection in OMNs.\u003c/p\u003e\n\u003cp\u003emiR-132-3p expression was stable between ALS and control OMNs. Although used here as a reference, this brain-enriched miRNA regulates cytoskeletal plasticity, mitochondrial dynamics, and pro-survival signaling, and its maturation is TDP-43-dependent[24]. Because it is predicted to be downregulated in ALS spinal cord, its preserved expression in OMNs may represent a third example of miRNA preservation in this resilient population[9, 16]. Future work will determine whether this preservation is specific to a subset of neuroprotective miRNAs or reflects broader maintenance of miRNA regulatory networks.\u003c/p\u003e\n\u003cp\u003eTo explore a mechanism for miRNA downregulation in ALS, we examined TRBP localization with TDP-43. We hypothesized that impaired miRNA biogenesis upstream of RISC contributes to pathology, given prior evidence of AGO2 in SGs in cell models[14]. Because TDP-43 is an SG-associated RNA-binding protein in ALS, it provides a disease-relevant scaffold for assessing TRBP interactions in postmortem neurons. TRBP exhibited cytoplasmic localization in control SMNs and ALS SMNs where TDP-43 was nuclear, however, in ALS SMNs with cytoplasmic TDP-43 inclusions, TRBP colocalized with the inclusions. TRBP is a cytoplasmic double-stranded RNA-binding protein that modulates PKR-dependent SG formation, providing a mechanistic basis for its presence in protein-RNA assemblies that overlap with TDP-43 complexes.[32]. Although evidence from cell culture shows that TDP-43 and TRBP can co-exist in Dicer-containing complexes under normal conditions, TRBP is not recovered when immunoprecipitating TDP-43, indicating no stable association[24]. Thus, the co-localization we observed in ALS SMNs more likely reflects a pathological sequestration into condensates or SG-like assemblies. Together, these results suggest that chronic stress and aggregation may aberrantly incorporate miRNA biogenesis factors into persistent SG-like structures in ALS patients, leading to decreased miRNA expression.\u003c/p\u003e\n\u003cp\u003eThe colocalization results implied that dysregulated miRNA biogenesis by sequestration would only apply to neurons with visible TDP-43 inclusions. Furthermore, loss of nuclear TDP-43 is known to impair processing of specific miRNAs and disrupt feedback loops that normally regulate TDP-43 via specific miRNAs, consequently decreasing miRNA expression[19, 24, 30, 46]. These findings provided a rationale for quantifying miR-9-5p and miR-124-3p expression in subgroups of SMNs stratified by TDP-43 pathology. We asked whether nuclear TDP-43-positive motor neurons retain higher miRNA expression compared with neurons showing cytoplasmic or aggregated TDP-43. \u0026nbsp;Contrary to our expectation, we found equivalent downregulation across all TDP-43 pathology subtypes compared to control for both miRNAs, indicating that nuclear TDP-43 alone in ALS is insufficient to maintain normal levels of these miRNAs. This may be caused by early and possibly TDP-43-independent mechanisms affecting RNA regulatory pathways. It also indicates that cytoplasmic TDP-43 inclusions are not necessary for miRNA dysregulation, and that motor neurons without visible aggregates may still be functionally impaired.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese findings support a model in which miRNA downregulation occurs early in the cellular disease process, before the formation of visible TDP-43 inclusions. Even in the absence of visible TDP-43/TRBP inclusions, ALS motor neurons may experience early or subtle molecular disruptions that could impair miRNA biogenesis. Soluble mislocalized TDP-43, low-level cellular stress, or transient stress responses, such as early-stage granule formation, could be sufficient to disrupt Dicer complex function or engage alternative pathways that reduce mature miRNA levels, even in the absence of histologically detectable pathology at our current resolution.\u003c/p\u003e\n\u003cp\u003eRetention of nuclear TDP-43 does not preclude SMNs from being actively engaged in the disease process. Identifying the molecular changes that precede or contribute to TDP-43 mislocalization is a critical area of ongoing research. Studies have demonstrated that nuclear TDP-43 can exhibit early conformational pathology coinciding with \u003cem\u003eSTMN2\u003c/em\u003e (Stathmin-2) cryptic splicing, preceding clinical onset[41]. Similar loss-of-function effects occur at the \u003cem\u003eUNC13A\u003c/em\u003e (Unc-13 Homolog A) locus, where cryptic exon inclusion leads to nonsense-mediated decay in ALS/FTD (frontotemporal dementia) patient tissue[7, 25, 29]. Beyond splicing defects, researchers have reported that nonfibrillar TDP-43 accumulates on the rough endoplasmic reticulum early in disease, potentially disrupting protein synthesis prior to visible aggregation[26]. Together, these observations and our findings support the interpretation that nuclear TDP-43-positive neurons in ALS can already be engaged in pathological processes that impair RNA regulation and cellular homeostasis. They also provide a rationale for further investigation into miRNA targets that are disrupted early in ALS cellular progression and developing therapeutic strategies that preserve RNA regulatory pathways to protect vulnerable neurons.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge funding from the Canadian Institutes of Health Research (CIHR) and preliminary work important for this project, but not shown, conducted by Alex Martin. The authors acknowledge the use of ChatGPT (OpenAI, San Francisco, CA, USA) to assist with Python coding.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: RH, MJS, DCM. Methodology and Design: CM, DCM, RH, MJS.\u003c/p\u003e\n\u003cp\u003eExperimentation/Data Acquisition: CM. Investigation: CM, MJS, DCM. Formal Analysis: CM. Supervision: RH, MJS, DCM. Manuscript (writing): CM. Manuscript (editing): CM, MJS, DCM, RH. All authors read, reviewed, and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by a Canadian Institutes of Health Research (CIHR) grant 201806SOP-411481 (MJS).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed in this study are included in this published article, found in the supplementary files, or can be made available on reasonable request to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthics approval for the use of human postmortem tissue was obtained from the University of Western Ontario, Health Sciences Research Ethics Board (HSREB) (protocols #103735 and #124855).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed consent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent for autopsy and retention of tissues for research purposes was obtained in written format from all patients antemortem or, if not possible, from their spouse postmortem. No minors participated in this study. To ensure anonymization, at the time of utilization for this research project, all cases were assigned a unique identifier with the master list correlating the unique identifier maintained in a locked cabinet in the office of the principal investigator (MJS) as per HSREB requirements.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eMolecular Medicine Group, Robarts Research Institute, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada. \u003csup\u003e2\u003c/sup\u003eDepartment of Pathology, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada.\u0026nbsp;\u003csup\u003e3\u003c/sup\u003eDepartment of Clinical Neurological Sciences, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAarts E, Dolan CV, Verhage M, van der Sluis S (2015) Multilevel analysis quantifies variation in the experimental effect while optimizing power and preventing false positives. 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PLoS One 8:e76055. https://doi.org/10.1371/journal.pone.0076055\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"acta-neuropathologica","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aneu","sideBox":"Learn more about [Acta Neuropathologica](https://link.springer.com/journal/401)","snPcode":"401","submissionUrl":"https://submission.springernature.com/new-submission/401/3","title":"Acta Neuropathologica","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Amyotrophic lateral sclerosis, Selective motor neuron vulnerability, miR-9-5p, miR-124-3p, TDP-43, Fluorescent in situ hybridization ","lastPublishedDoi":"10.21203/rs.3.rs-7537447/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7537447/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Selective vulnerability of motor neurons is a defining feature of amyotrophic lateral sclerosis (ALS) and provides a valuable framework for uncovering mechanisms that distinguish resilient from vulnerable neuronal populations. We investigated whether dysregulation of neuroprotective microRNAs (miRNAs), miR-9-5p and miR-124-3p, contributes to the differential susceptibility of motor neuron subtypes. We focused on spinal motor neurons (SMNs), which undergo drastic degeneration in ALS, and oculomotor neurons (OMNs), which remain functionally intact and rarely degenerate, allowing preservation of eye movement in ALS patients. Using a modified multiplexed fluorescent in situ hybridization protocol combined with immunofluorescence, we quantified the expression of miR-9-5p and miR-124-3p in cervical SMNs and OMNs from ALS and control cases. We observed significant downregulation of both miRNAs in ALS SMNs, while their expression was maintained in ALS OMNs. Stratification of ALS SMNs by TDP-43 pathological status revealed similarly reduced miRNA expression in neurons with and without cytoplasmic inclusions, suggesting that miRNA downregulation occurs independently of visible TDP-43 pathology. To explore a potential mechanism, we assessed the localization of the Dicer cofactor TRBP and found that it was sequestered into TDP-43 inclusions in ALS SMNs. However, TRBP remained normally localized in neurons without cytoplasmic inclusions, indicating that sequestration cannot fully account for miRNA reduction across all ALS motor neurons. These findings support a model in which early or subtle disruptions, preceding visible pathology, also contribute to miRNA downregulation in ALS. Our study also indicates cell-type-specific preservation of miRNA networks as a possible contributor to oculomotor neuron resilience.","manuscriptTitle":"Preservation of miR-9-5p and miR-124-3p in ALS-resistant oculomotor neurons contrasts with their downregulation in vulnerable spinal motor neurons, irrespective of TDP-43 pathology","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-15 06:23:34","doi":"10.21203/rs.3.rs-7537447/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-19T05:34:21+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-29T10:44:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-26T08:28:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"129595663946782942919586429543093042419","date":"2025-09-11T13:44:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"281961648807821486143569496967456441155","date":"2025-09-10T13:24:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"332171690761991509755943776826237737873","date":"2025-09-08T14:21:31+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-06T11:04:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-06T11:00:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-06T06:11:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"Acta Neuropathologica","date":"2025-09-04T14:59:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"acta-neuropathologica","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aneu","sideBox":"Learn more about [Acta Neuropathologica](https://link.springer.com/journal/401)","snPcode":"401","submissionUrl":"https://submission.springernature.com/new-submission/401/3","title":"Acta Neuropathologica","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e1accb4f-3843-44a8-add5-7a34f0667ce4","owner":[],"postedDate":"September 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-30T16:20:25+00:00","versionOfRecord":{"articleIdentity":"rs-7537447","link":"https://doi.org/10.1007/s00401-026-03005-6","journal":{"identity":"acta-neuropathologica","isVorOnly":false,"title":"Acta Neuropathologica"},"publishedOn":"2026-03-26 16:09:03","publishedOnDateReadable":"March 26th, 2026"},"versionCreatedAt":"2025-09-15 06:23:34","video":"","vorDoi":"10.1007/s00401-026-03005-6","vorDoiUrl":"https://doi.org/10.1007/s00401-026-03005-6","workflowStages":[]},"version":"v1","identity":"rs-7537447","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7537447","identity":"rs-7537447","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}