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• W2983140136 abstract "•Aged microglia have an activated phenotype that affects motor neuron (MN) health•MN damage leads to age-related defects at the neuromuscular junction•An age-related decline in motor units (MUs) results in loss of muscle innervation•Modulation of microglia by exercise or CSF1R inhibition preserves MUs in aged mice Age-related loss of skeletal muscle innervation by motor neurons leads to impaired neuromuscular function and is a well-established clinical phenomenon. However, the underlying pathogenesis remains unclear. Studying mice, we find that the number of motor units (MUs) can be maintained by counteracting neurotoxic microglia in the aged spinal cord. We observe that marked innervation changes, detected by motor unit number estimation (MUNE), occur prior to loss of muscle function in aged mice. This coincides with gene expression changes indicative of neuronal remodeling and microglial activation in aged spinal cord. Voluntary exercise prevents loss of MUs and reverses microglia activation. Depleting microglia by CSF1R inhibition also prevents the age-related decline in MUNE and neuromuscular junction disruption, implying a causal link. Our results suggest that age-related changes in spinal cord microglia contribute to neuromuscular decline in aged mice and demonstrate that removal of aged neurotoxic microglia can prevent or reverse MU loss. Age-related loss of skeletal muscle innervation by motor neurons leads to impaired neuromuscular function and is a well-established clinical phenomenon. However, the underlying pathogenesis remains unclear. Studying mice, we find that the number of motor units (MUs) can be maintained by counteracting neurotoxic microglia in the aged spinal cord. We observe that marked innervation changes, detected by motor unit number estimation (MUNE), occur prior to loss of muscle function in aged mice. This coincides with gene expression changes indicative of neuronal remodeling and microglial activation in aged spinal cord. Voluntary exercise prevents loss of MUs and reverses microglia activation. Depleting microglia by CSF1R inhibition also prevents the age-related decline in MUNE and neuromuscular junction disruption, implying a causal link. Our results suggest that age-related changes in spinal cord microglia contribute to neuromuscular decline in aged mice and demonstrate that removal of aged neurotoxic microglia can prevent or reverse MU loss. The motor unit (MU), representing a single alpha motor neuron and the skeletal muscle fibers it innervates at the neuromuscular junction (NMJ), is the fundamental unit responsible for control of movement. A slow, progressive decline in the number of motor neurons innervating skeletal muscle occurs in the elderly and manifests as a decrease in MU numbers, detected by nerve conduction studies (NCSs). The decline in MUs has been considered to be a major factor in the development of sarcopenia, the age-related loss of muscle mass, and the decline in mobility and fine motor control in the elderly (Piasecki et al., 2016Piasecki M. Ireland A. Jones D.A. McPhee J.S. Age-dependent motor unit remodelling in human limb muscles.Biogerontology. 2016; 17: 485-496Crossref PubMed Scopus (55) Google Scholar). These changes are among the earliest signs of deterioration in motor neuron health and are thought to result from initial retraction of the motor axon from the muscle, a process that has been termed “dying back degeneration” (Chung et al., 2017Chung T. Park J.S. Kim S. Montes N. Walston J. Höke A. Evidence for dying-back axonal degeneration in age-associated skeletal muscle decline.Muscle Nerve. 2017; 55: 894-901Crossref PubMed Scopus (21) Google Scholar). Despite being first reported nearly 50 years ago (McComas, 1995McComas A.J. Motor-unit estimation: the beginning.J. Clin. Neurophysiol. 1995; 12: 560-564Crossref PubMed Scopus (17) Google Scholar, McComas et al., 1971McComas A.J. Fawcett P.R.W. Campbell M.J. Sica R.E.P. Electrophysiological estimation of the number of motor units within a human muscle.J. Neurol. Neurosurg. Psychiatry. 1971; 34: 121-131Crossref PubMed Scopus (505) Google Scholar), the mechanisms underlying this age-associated change in innervation with aging are still unclear. Loss of skeletal muscle innervation has been shown to start in humans at the age of approximately 50 years and gradually continue, so that, by the age of 80, roughly 50% of total MUs are lost (Faulkner et al., 2007Faulkner J.A. Larkin L.M. Claflin D.R. Brooks S.V. Age-related changes in the structure and function of skeletal muscles.Clin. Exp. Pharmacol. Physiol. 2007; 34: 1091-1096Crossref PubMed Scopus (404) Google Scholar). It has also been shown that the number of MUs is maintained in old master runners, suggesting that exercise supports the preservation of skeletal muscle innervation during aging (Power et al., 2010Power G.A. Dalton B.H. Behm D.G. Vandervoort A.A. Doherty T.J. Rice C.L. Motor unit number estimates in masters runners: use it or lose it?.Med. Sci. Sports Exerc. 2010; 42: 1644-1650Crossref PubMed Scopus (104) Google Scholar). Pre-clinical research has further demonstrated pronounced structural changes at the NMJ in aged mice, including evidence of increased denervation that could be prevented by caloric restriction and chronic exercise (Cheng et al., 2013Cheng A. Morsch M. Murata Y. Ghazanfari N. Reddel S.W. Phillips W.D. Sequence of age-associated changes to the mouse neuromuscular junction and the protective effects of voluntary exercise.PLoS ONE. 2013; 8: e67970Crossref PubMed Scopus (53) Google Scholar, Valdez et al., 2010Valdez G. Tapia J.C. Kang H. Clemenson Jr., G.D.J. Gage F.H. Lichtman J.W. Sanes J.R. Attenuation of age-related changes in mouse neuromuscular synapses by caloric restriction and exercise.Proc. Natl. Acad. Sci. USA. 2010; 107: 14863-14868Crossref PubMed Scopus (308) Google Scholar, Valdez et al., 2012Valdez G. Tapia J.C. Lichtman J.W. Fox M.A. Sanes J.R. Shared resistance to aging and ALS in neuromuscular junctions of specific muscles.PLoS ONE. 2012; 7: e34640Crossref PubMed Scopus (142) Google Scholar). These studies hypothesized that the NMJ structural changes represent an age-related decline in neuromuscular function. However, recent work has suggested that the age-related structural changes at the NMJ (e.g., fragmentation) may, in part, represent a compensatory adaptation to maintain effective neurotransmission (Willadt et al., 2016Willadt S. Nash M. Slater C.R. Age-related fragmentation of the motor endplate is not associated with impaired neuromuscular transmission in the mouse diaphragm.Sci. Rep. 2016; 6: 24849Crossref PubMed Scopus (57) Google Scholar, Willadt et al., 2018Willadt S. Nash M. Slater C. Age-related changes in the structure and function of mammalian neuromuscular junctions.Ann. N Y Acad. Sci. 2018; 1412: 41-53Crossref Scopus (26) Google Scholar). During the aging process, a chronic and gradual increase in inflammation occurs in the CNS that is mediated by, among other long-lived cells, microglia (Koellhoffer et al., 2017Koellhoffer E.C. McCullough L.D. Ritzel R.M. Old Maids: Aging and Its Impact on Microglia Function.Int. J. Mol. Sci. 2017; 18: 769Crossref PubMed Scopus (83) Google Scholar). In the healthy adult CNS, microglia comprise 10%–15% of all cells and are distributed in both the brain (with differences per brain area) and spinal cord to survey the surrounding environment. As the resident innate immune cells of the CNS, microglia in their “homeostatic state,” with long and ramified processes, act as sentinels of pathogen infection and injury and participate in innate and adaptive immune responses. With aging, increased microglial density and decreased regularity of distribution have been observed together with morphological changes that are generally associated with functional alterations (Kierdorf and Prinz, 2013Kierdorf K. Prinz M. Factors regulating microglia activation.Front. Cell. Neurosci. 2013; 7: 44Crossref PubMed Scopus (205) Google Scholar). However, the mechanisms that trigger these changes during aging, which may be referred to as microglial activation and are often considered neurotoxic, are poorly understood. Microglial activation during aging may be driven in part by repetitive activation by various stimuli followed by failure to return to the homeostatic phenotype (Wong, 2013Wong W.T. Microglial aging in the healthy CNS: phenotypes, drivers, and rejuvenation.Front. Cell. Neurosci. 2013; 7: 22Crossref PubMed Scopus (150) Google Scholar). Increased expression of surface markers (such as major histocompatibility complex [MHC] class II) and production of neurotoxic factors (such as pro-inflammatory cytokines, reactive oxygen species [ROS], and nitric oxide [NO]) have been described extensively as indicative of the activated phenotype and may contribute to neuronal damage (von Bernhardi et al., 2015von Bernhardi R. Eugenín-von Bernhardi L. Eugenín J. Microglial cell dysregulation in brain aging and neurodegeneration.Front. Aging Neurosci. 2015; 7: 124Crossref PubMed Scopus (290) Google Scholar). Loss of trophic support may contribute further. Neuronal damage may then drive sustained microglial activation (so-called “reactive microgliosis”), resulting in a neurotoxic positive feedback loop and a continuous cycle of neuronal death. Neuronal loss in several neurodegenerative diseases may be explained by this proposed mechanism (Block et al., 2007Block M.L. Zecca L. Hong J.S. Microglia-mediated neurotoxicity: uncovering the molecular mechanisms.Nat. Rev. Neurosci. 2007; 8: 57-69Crossref PubMed Scopus (2741) Google Scholar). Our work uncovered enrichment in microglial and neuroinflammatory markers in the murine spinal cord transcriptome at 18 months of age. This was concurrent with a significant decline in the number of functional MUs, as measured by in vivo NCSs, suggesting a link between upregulation of toxic microglia markers in aged spinal cord and loss of MUs. Surprisingly, limited changes in skeletal muscle gene expression and no change in muscle mass or compound muscle action potential (CMAP) were detected at the same age, confirming that deterioration of motor neurons precedes age-dependent loss of muscle mass. Further, using two strategies to modulate microglial function in vivo, chronic exercise and selective depletion with a colony-stimulating factor 1 receptor (CSF1R) inhibitor, we demonstrated that age-dependent loss of MUs can be rescued. Altogether, our findings support the hypothesis that aging is associated with microglia-mediated neurotoxicity in the spinal cord that is harmful to motor neurons, promotes retraction of the axon from skeletal muscle, and underlies MU loss. Therefore, reducing microglial-mediated neuroinflammation may represent a targeted intervention to preserve motor neurons and prevent MU loss in aging. To investigate the effect of age on muscle innervation, we performed NCSs on wild-type C57BL/6 mice as a function of increasing age (Figure 1). This analysis revealed a significant decline in motor unit number estimation (MUNE) between the ages of 10 and 18 months (Figure 1A), indicating profound loss of innervation by vulnerable motor neurons upon aging. Stabilization of MUNE beyond 18 months was observed and suggests preservation of less vulnerable MUs that are not lost during the aging process (Pun et al., 2006Pun S. Santos A.F. Saxena S. Xu L. Caroni P. Selective vulnerability and pruning of phasic motoneuron axons in motoneuron disease alleviated by CNTF.Nat. Neurosci. 2006; 9: 408-419Crossref PubMed Scopus (432) Google Scholar). In contrast, the CMAP was stable over time, indicating that the total number of innervated muscle fibers was unaffected (Figure 1B). This is consistent with collateral sprouting of new nerve terminals from healthy motor neuron axons to re-innervate vacated NMJs and a consequent increase in size of the remaining MUs, as reported by others (Chung et al., 2018Chung T. Tian Y. Walston J. Hoke A. Increased Single-fiber jitter level is associated with reduction in motor function with aging.Am. J. Phys. Med. Rehabil. 2018; 97: 551-556Crossref Scopus (4) Google Scholar, Hepple and Rice, 2016Hepple R.T. Rice C.L. Innervation and neuromuscular control in ageing skeletal muscle.J. Physiol. 2016; 594: 1965-1978Crossref PubMed Scopus (139) Google Scholar). To explore the molecular changes associated with age, we performed high-throughput RNA sequencing (RNA-seq) on spinal cord and skeletal muscle from mice at the extremes of the MUNE decline (Figure 1A), which we defined as adult (10 months) compared with aged adult (18 months, hereafter referred to as aged). Sequencing was performed on 10 biological replicates per tissue collected (lumbar spinal cord, quadriceps, and gastrocnemius skeletal muscle) for each age group. Applying the criteria for significance (adjusted p ≤ 0.01 and absolute log2 fold change of ≥ 1), we identified 17 differentially regulated genes in spinal cord, all of which were upregulated with age. Interestingly, many of these genes are known to be expressed by the resident immune cells in the CNS, the microglia (Figure 1C; Table 1).Table 1List of 17 Differentially Expressed Genes between Aged and Adult Spinal Cord, Including Relative Expression Changes and Their Microglial Enrichment in the “Brain RNAseq” DatasetGene IDGene SymbolDescriptionSpinal Cord, Aged versus Adult“Brain RNAseq” DataEffect Size (Log2 Fold Change)Significance (BH Adjusted p Value)Microglia Expression (FPKM)Microglia Enrichment Log2 (Microglia/Other Compartments)Microglia Enrichment Z Score (Microglia/Other Compartments)12483Cd22CD22 antigen1.161.8 10−51.7154.1002.26813011Cst7cystatin F (leukocystatin)2.249 10−710.3916.0922.26714727Lilr4bleukocyte immunoglobulin-like receptor, subfamily B, member 4B1.031.510−3N/AN/AN/A14728Lilrb4aleukocyte immunoglobulin-like receptor, subfamily B, member 4A1.132.310−6N/AN/AN/A16411Itgaxintegrin alpha X1.881.8 10−54.1795.2442.26816854Lgals3lectin, galactose binding, soluble 31.671.1 10−78.8723.4362.18317105Lyz2lysozyme 21.097.3 10−5216.2386.4172.26617381Mmp12matrix metallopeptidase 123.171.7 10−40.5072.3412.26818566Pdcd1programmed cell death 11.537.7 10−52.0084.3282.26820302Ccl3chemokine (C-C motif) ligand 31.71.7 10−52,453.8998.0992.26721857Timp1tissue inhibitor of metalloproteinase 11.37.4 10−40.240−3.192−0.40150706Postnperiostin, osteoblast-specific factor11.6 10−31.1880.3890.24656644Clec7aC-type lectin domain family 7, member a1.411.1 10−71.2453.3932.26193695Gpnmbglycoprotein (transmembrane) nmb2.023.4 10−63.2482.0131.411109225Ms4a7membrane-spanning 4-domains, subfamily A, member 71.422.5 10−326.1645.9702.264242341Atp6v0d2ATPase, H+ transporting, lysosomal V0 subunit D22.363.9 10−30.2231.1592.268386463Cdsncorneodesmosin1.078.4 10−30.1000.0000.926 Open table in a new tab A similar analysis of the two skeletal muscles revealed no or very limited effects of aging concomitant with MUNE reduction; only 3 genes (Erfe, Lep, and Nnat) were identified as differentially expressed in quadriceps and none in gastrocnemius with aging (Figure 1D). Targeted qPCR further confirmed the lack of regulation of known muscle atrophy and denervation markers (Figure 1E). Similarly, gene expression markers of muscle fiber types showed no statistically significant differences between the two age groups (Figure 1F). Using MRI to assess muscle atrophy, we did not observe significant differences between adult and aged mice in either calf muscle volume (CMV) or calf muscle cross-sectional area (CSA) (Figures 1G and 1H). This is consistent with an absence of muscle loss in our animals, as determined by normalized muscle mass measurements at the end of the study (Figure 1I). Taken together, these data demonstrate that loss of functional MUs with age is associated with early molecular changes in the spinal cord but no detectable changes in muscle mass or in muscle markers for atrophy and denervation and suggests that spinal cord health alteration precedes muscle loss. To gain further insight into the mechanisms associated with the age-related expression changes observed in the spinal cord, we first performed a gene set enrichment analysis (GSEA) with Gene Ontology (GO) term gene sets (Subramanian et al., 2005Subramanian A. Tamayo P. Mootha V.K. Mukherjee S. Ebert B.L. Gillette M.A. Paulovich A. Pomeroy S.L. Golub T.R. Lander E.S. Mesirov J.P. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles.Proc. Natl. Acad. Sci. USA. 2005; 102: 15545-15550Crossref PubMed Scopus (20168) Google Scholar). GSEA is a powerful method for uncovering meaningful and significant coordinated expression changes of biologically and functionally associated genes. Downregulated pathways suggested the occurrence of altered neuronal remodeling, involving impairment of key biological processes such as synaptic assembly, axon extension, and ion transport (Figure 2A; Table S1). Importantly, the analysis also uncovered highly statistically significant enrichment toward upregulation of transcripts related to the innate immune response, immune system processes, and the inflammatory response (Figure 2B; Table S2), suggesting that neuro-inflammation may play a role in loss of MUs associated with aging. Thus, both individual gene expression (Table 1) and pathway enrichment (Figure 2B) suggest a role for microglia in the aging spinal cord. To further characterize these findings, we created CNS cell type-specific signatures (microglia, neurons, and endothelial cells) based on the brain tissue atlas “Brain RNAseq” published by Zhang et al., 2014Zhang Y. Chen K. Sloan S.A. Bennett M.L. Scholze A.R. O’Keeffe S. Phatnani H.P. Guarnieri P.C. Caneda C. Ruderisch N. et al.an RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex.J. Neurosci. 2014; 34: 11929-11947Crossref PubMed Scopus (2296) Google Scholar (Table S3) and the microglial sensome established by Hickman et al., 2013Hickman S.E. Kingery N.D. Ohsumi T.K. Borowsky M.L. Wang L.C. Means T.K. El Khoury J. The microglial sensome revealed by direct RNA sequencing.Nat. Neurosci. 2013; 16: 1896-1905Crossref PubMed Scopus (780) Google Scholar (Table S4). We then re-interrogated our spinal cord aging transcriptome using GSEA on a compendium of more than 6,000 internal Novartis signatures, and both microglial signatures were identified among the top most enriched (top 5), with a false discovery rate equal to 10−86 and 10−40, respectively (Figures 2C and 2D). Although the neuron signature genes showed a significant coordinated decrease in expression, endothelial cells did not appear to be modulated (Figure 2C). Microglia gene upregulation was further confirmed by qPCR of a selected set of genes related to activated microglia, most of which are members of the 17 significantly regulated genes and include few disease-associated microglia (DAM) genes (Cst7 and Lpl) (Keren-Shaul et al., 2017Keren-Shaul H. Spinrad A. Weiner A. Matcovitch-Natan O. Dvir-Szternfeld R. Ulland T.K. David E. Baruch K. Lara-Astaiso D. Toth B. et al.A Unique Microglia Type Associated with Restricting Development of Alzheimer’s Disease.Cell. 2017; 169: 1276-1290.e17Abstract Full Text Full Text PDF PubMed Scopus (1241) Google Scholar; Figure 2E). Histological analysis of IBA1, a microglial marker, showed increased immunostaining in the spinal cord of aged animals. This was predominantly due to morphological changes in the microglia, as confirmed by an increased number of cellular processes, with a non-significant trend to increased cell numbers (Figure 2F). Brain microglia cells from adult and aged mice were analyzed further following isolation by fluorescence-activated cell sorting (FACS). Here, markers of activation (CD86, MHC class IIa, and CD68) were notably increased in aged compared with younger microglia (Figure 2G). To address whether the increased innate immune response observed in the CNS was also present in the periphery, we examined expression of the macrophage markers Adgre1 and Itgam in skeletal muscle and the sciatic nerve of adult and aged mice but found no changes (Figure 2H). In contrast, gene expression of common microglia markers, such as Aif1 and Itgam, in spinal cord of the same set of mice was increased significantly (Figure 2I). This confirms that an activated immune response occurs in the spinal cord but not in peripheral tissues, such as skeletal muscle and the sciatic nerve, upon aging. To further support the link between aging and microglia, we examined PolG mice, which have an accelerated aging phenotype because of the presence of a D257A mutation in the “proofreading” domain of the DNA polymerase gamma gene in mitochondria (Kujoth et al., 2005Kujoth G.C. Hiona A. Pugh T.D. Someya S. Panzer K. Wohlgemuth S.E. Hofer T. Seo A.Y. Sullivan R. Jobling W.A. et al.Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammalian aging.Science. 2005; 309: 481-484Crossref PubMed Scopus (1453) Google Scholar). This analysis revealed broadly similar changes in expression patterns of microglia genes in PolG mice at the age of 10 months compared with age-matched control mice (Figure S1). Because the age-related increase in the microglia gene signature in the spinal cord occurred concomitant with loss of MUs, we hypothesized that the two events may be causally related. To begin to address this, we asked whether physical exercise could reverse these impairments in a coordinated manner. Exercise has been reported to delay age-dependent MU loss in master athletes (Power et al., 2010Power G.A. Dalton B.H. Behm D.G. Vandervoort A.A. Doherty T.J. Rice C.L. Motor unit number estimates in masters runners: use it or lose it?.Med. Sci. Sports Exerc. 2010; 42: 1644-1650Crossref PubMed Scopus (104) Google Scholar) and is well established to provide beneficial effects on neuromuscular performance even in the elderly (Hunter et al., 2016Hunter S.K. Pereira H.M. Keenan K.G. The aging neuromuscular system and motor performance.J. Appl. Physiol. 2016; 121: 982-995Crossref PubMed Scopus (129) Google Scholar). Moreover, Valdez et al., 2010Valdez G. Tapia J.C. Kang H. Clemenson Jr., G.D.J. Gage F.H. Lichtman J.W. Sanes J.R. Attenuation of age-related changes in mouse neuromuscular synapses by caloric restriction and exercise.Proc. Natl. Acad. Sci. USA. 2010; 107: 14863-14868Crossref PubMed Scopus (308) Google Scholar showed that exercise attenuates age-related changes at the NMJ by reducing the frequency of denervated postsynaptic sites in skeletal muscle. Nevertheless, an association between the beneficial effect of exercise on MU loss and changes in the spinal cord, such as the altered microglia gene expression profile, has not yet been established. We thus enrolled a cohort of animals into a voluntary exercise trial. Mouse cages were fitted with running wheels, and 16-month-old mice were allowed to run freely for 2 months. The distance run per mouse was carefully recorded on an hourly basis. Substantial differences among animals in terms of distance run and daily running pattern (Figures S2A and S2B) were observed, with some animals running up to 10 km/day in the course of the study. NCS electromyography (EMG) recordings to obtain MUNE were performed before and at the end of the trial (Figure 3A) and compared with control adult and aged sedentary groups. As expected, the aged mice in both groups displayed a significantly reduced MU number at baseline compared with adult mice. After 2 months, the aged sedentary group showed a small, non-significant decline in MU number. Remarkably, however, the exercised aged mice not only maintained but significantly restored MU numbers to levels similar to those found in the younger sedentary cohort (Figures 3B and 3C). This strongly suggests that exercise can mediate both neuronal remodeling and strengthen innervation in aged mice. Further supporting this, we also observed a significant positive correlation between the amount of exercise (total distance run in kilometers) and MUNE change (percent) (Figure 3D). Two animals (mouse IDs 5.4 and 8.4, red dots in Figure 3D and Figures S2A and S2B) in the exercise group did not engage in voluntary running during the trial and exhibited no improvement in MUNE. These animals were excluded from subsequent group-level analyses. RNA-seq profiling of spinal cord samples of the exercised cohort showed that only a handful of genes (Calca, Uts2, and Dao) were marginally modulated by this exercise regimen compared with age-matched mice. The changes in these genes were, however, confirmed by qPCR (Figure 3E). Interestingly, one of these genes, Calca, encodes the neuropeptide Calcitonin gene-related peptide (CGRP) in motor neurons and is known to have a role in regeneration and reinnervation of skeletal muscle (Chung, 2018Chung A.M. Calcitonin gene-related peptide (CGRP): role in peripheral nerve regeneration.Rev. Neurosci. 2018; 29: 369-376Crossref Scopus (14) Google Scholar). Consistent with published work (Rom and Reznick, 2016Rom O. Reznick A.Z. The role of E3 ubiquitin-ligases MuRF-1 and MAFbx in loss of skeletal muscle mass.Free Radic. Biol. Med. 2016; 98: 218-230Crossref Scopus (72) Google Scholar, Yan et al., 2011Yan Z. Okutsu M. Akhtar Y.N. Lira V.A. Regulation of exercise-induced fiber type transformation, mitochondrial biogenesis, and angiogenesis in skeletal muscle.J. Appl. Physiol. 2011; 110: 264-274Crossref PubMed Scopus (189) Google Scholar), we identified significant changes in the two muscles, including upregulation of sarcolipin (Sln), myomesin (Myom3), and ATP citrate lyase (Acly). In addition, we performed qPCR on known exercise-responsive genes (Figure S2C) as well as histological assessments of the muscle tissues (Figures S2D and S2E) in quadriceps: the common atrophy markers Murf1 and Mafbx were downregulated, as shown by qPCR analysis following exercise, and a reduction in type IIb muscle fibers was mirrored by an increase in type IIa fibers, as shown by histology and qPCR analysis, suggesting the expected shift toward a slow oxidative phenotype of muscle fibers with exercise. Microglial gene signatures, found previously to be significantly upregulated with age in the spinal cord, were downregulated significantly following the chronic exercise regimen (“Brain RNAseq” microglia false discovery rate (FDR), 10−22; microglial sensome FDR, 10−5.4; Figure 3F). The neuron signature was, on the other hand, slightly but significantly upregulated (FDR, 10−4) (Figure 3F). Thus, the beneficial effects of exercise on age-associated neuromuscular impairment in this study were concomitant with changes in microglial activation. Full RNA-seq data are publicly available in the GEO: GSE122116. To determine whether the beneficial effect on reinnervation could also be achieved at older ages, we repeated the exercise trial with mice at 21 months of age. Here, MUNE was again measured before and after the 2 months of study. Voluntary wheel running in these very old mice also restored the number of MUs to the level of adult animals (Figures S3A and S3B). However, this was not associated with substantial changes in gene expression in gastrocnemius muscle, suggesting somewhat reduced muscular plasticity at this advanced age, when MUNE reduction is already maximal (Figure S3C). Because exercise attenuated the activated microglial phenotype concomitant with reversal of age-dependent MU reduction (Figure 3F), we hypothesized that aged microglia may engender a neurotoxic environment in the spinal cord that is detrimental to motor neuron health and results in compromised muscle innervation by spinal motor neurons (Figures 1A and 2). Therefore, we asked whether removing microglia from the aged CNS could reduce or prevent MU loss. To deplete microglia, we used a highly specific and potent CSF1R kinase inhibitor, BLZ945 (Krauser et al., 2015Krauser J.A. Jin Y. Walles M. Pfaar U. Sutton J. Wiesmann M. Graf D. Pflimlin-Fritschy V. Wolf T. Camenisch G. Swart P. Phenotypic and metabolic investigation of a CSF-1R kinase receptor inhibitor (BLZ945) and its pharmacologically active metabolite.Xenobiotica. 2015; 45: 107-123Crossref Scopus (14) Google Scholar, Pyonteck et al., 2013Pyonteck S.M. Akkari L. Schuhmacher A.J. Bowman R.L. Sevenich L. Quail D.F. Olson O.C. Quick M.L. Huse J.T. Teijeiro V. et al.CSF-1R inhibition alters macrophage polarization and blocks glioma progression.Nat. Med. 2013; 19: 1264-1272Crossref PubMed Scopus (1081) Google Scholar). CSF1R is a tyrosine kinase receptor activated by the ligands CSF1 and interleukin-34 (IL-34) and plays an essential role in the regulation of survival, proliferation, and differentiation of microglia and related cells of the myeloid lineage, such as macrophages and monocytes. Pharmacological inhibition of CSF1R has been well established as an efficient means to deplete CNS microglia (Beckmann et al., 2018Beckmann N. Giorgetti E. Neuhaus A. Zurbruegg S. Accart N. Smith P. Perdoux J. Perrot L. Nash M. Desrayaud S. et al.Brain region-specific enhancement of remyelination and prevention of demyelination by the CSF1R kinase inhibitor BLZ945.Acta Neuropathol. Commun. 2018; 6: 9Crossref Scopus (42) Google Scholar, Elmore et al., 2018Elmore M.R.P. Hohsfield L.A. Kramár E.A. Soreq L. Lee R.J. Pham S.T. Najafi A.R. Spangenberg E.E. Wood M.A. West B.L. Green K.N. Replacement of microglia in the aged brain reverses cognitive, synaptic, and neuronal deficits in mice.Aging Cell. 2018; 17: e12832Crossref PubMed Scopus (100) Google Scholar). We validated this in vitro on human induced pluripotent stem cell (iPSC)-derived macrophages (iMACS) by showing a concentration-dependent cell reduction over 5 days (Figures S4A and S4B). Previous work showed that, following drug-mediated depletion, microglia fully repo" @default.
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• W2983140136 title "Modulation of Microglia by Voluntary Exercise or CSF1R Inhibition Prevents Age-Related Loss of Functional Motor Units" @default.
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