Motor Neurons In Spinal Cord Research Articles

Molecular mechanisms by which targeted muscle reinnervation improves the microenvironment of spinal cord motor neurons and target muscles

Targeted muscle reinnervation is a clinically valuable nerve transfers technology used to reconstruct the information sources reconstruct the motor nerve information sources lost because of nerve injury. This study aimed to investigate the effects and underlying molecular mechanisms of hind limb TMR on motor neurons and target muscles in rats after tibial nerve transection (TNT). Immunohistochemistry was performed to detect acetylcholinesterase expression in the target muscles and myelin basic protein, neuregulin-1 (NRG1), and ErbB2 expression in the tibial nerve of rats. Masson's trichrome staining was performed to observe fibrillar collagen expression in the target muscles. Western blot analysis was used to detect the protein expression of NRG1 and its receptor, ErbB2, in the target muscles. TMR significantly enhanced NRG1, ErbB2, and myelin basic protein expression in nerve fibers compared with those in the TNT group and exerted a protective effect on the maintenance of a large number of nerve fibers and myelin sheath thickness. The above results indicated that TMR can regulate NRG1 and ErbB2 expression in residual nerve fibers and protect the integrity of the myelin sheath, thus improving the functional status of the target muscles, which is beneficial for restoring hind limb motor function after TNT.

Functional implication of ubiquitinating and deubiquitinating mechanisms in TDP-43 proteinopathies.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease in which motor neurons in spinal cord and motor cortex are progressively lost. About 15% cases of ALS also develop the frontotemporal dementia (FTD), in which the frontotemporal lobar degeneration (FTLD) occurs in the frontal and temporal lobes of the brain. Among the pathologic commonalities in ALS and FTD is ubiquitin-positive cytoplasmic aggregation of TDP-43 that may reflect both its loss-of-function and gain-of-toxicity from proteostasis impairment. Deep understanding of how protein quality control mechanisms regulate TDP-43 proteinopathies still remains elusive. Recently, a growing body of evidence indicates that ubiquitinating and deubiquitinating pathways are critically engaged in the fate decision of aberrant or pathological TDP-43 proteins. E3 ubiquitin ligases coupled with deubiquitinating enzymes may influence the TDP-43-associated proteotoxicity through diverse events, such as protein stability, translocation, and stress granule or inclusion formation. In this article, we recapitulate our current understanding of how ubiquitinating and deubiquitinating mechanisms can modulate TDP-43 protein quality and its pathogenic nature, thus shedding light on developing targeted therapies for ALS and FTD by harnessing protein degradation machinery.

ROS scavengers decrease γH2ax spots in motor neuronal nuclei of ALS model mice in vitro

Background: Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease characterized by the loss of motor neurons in cerebral cortex, brainstem and spinal cord. Numerous studies have demonstrated signs of oxidative stress in postmortem neuronal tissue, cerebrospinal fluid, plasma and urine of ALS patients, without focusing on the specific processes within motor neurons. Thus, we aimed to investigate the relevance of reactive oxygen species (ROS) detoxification mechanisms and its consequences on the formation of toxic/lethal DNA double strand breaks (DSBs) in the ALS model of the Wobbler mouse.Methods: Live cell imaging in dissociated motor neuronal cultures was used to investigate the production of ROS using Dihydroethidium (DHE). The expression levels of ROS detoxifying molecules were investigated by qPCR as well as Western blots. Furthermore, the expression levels of DNA damage response proteins p53bp1 and H2ax were investigated using qPCR and immunofluorescence staining. Proof-of-principle experiments using ROS scavengers were performed in vitro to decipher the influence of ROS on the formation of DNA double strand breaks quantifying the γH2ax spots formation.Results: Here, we verified an elevated ROS-level in spinal motor neurons of symptomatic Wobbler mice in vitro. As a result, an increased number of DNA damage response proteins p53bp1 and γH2ax in dissociated motor neurons of the spinal cord of Wobbler mice was observed. Furthermore, we found a significantly altered expression of several antioxidant molecules in the spinal cord of Wobbler mice, suggesting a deficit in ROS detoxification mechanisms. This hypothesis could be verified by using ROS scavenger molecules in vitro to reduce the number of γH2ax foci in dissociated motor neurons and thus counteract the harmful effects of ROS.Conclusion: Our data indicate that maintenance of redox homeostasis may play a key role in the therapy of the neurodegenerative disease ALS. Our results underline a necessity for multimodal treatment approaches to prolong the average lifespan of motor neurons and thus slow down the progression of the disease, since a focused intervention in one pathomechanism seems to be insufficient in ALS therapy.

Co-deposition of SOD1, TDP-43 and p62 proteinopathies in ALS: evidence for multifaceted pathways underlying neurodegeneration

Multiple neurotoxic proteinopathies co-exist within vulnerable neuronal populations in all major neurodegenerative diseases. Interactions between these pathologies may modulate disease progression, suggesting they may constitute targets for disease-modifying treatments aiming to slow or halt neurodegeneration. Pairwise interactions between superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TDP-43) and ubiquitin-binding protein 62/sequestosome 1 (p62) proteinopathies have been reported in multiple transgenic cellular and animal models of amyotrophic lateral sclerosis (ALS), however corresponding examination of these relationships in patient tissues is lacking. Further, the coalescence of all three proteinopathies has not been studied in vitro or in vivo to date. These data are essential to guide therapeutic development and enhance the translation of relevant therapies into the clinic. Our group recently profiled SOD1 proteinopathy in post-mortem spinal cord tissues from familial and sporadic ALS cases, demonstrating an abundance of structurally-disordered (dis)SOD1 conformers which become mislocalized within these vulnerable neurons compared with those of aged controls. To explore any relationships between this, and other, ALS-linked proteinopathies, we profiled TDP-43 and p62 within spinal cord motor neurons of the same post-mortem tissue cohort using multiplexed immunofluorescence and immunohistochemistry. We identified distinct patterns of SOD1, TDP43 and p62 co-deposition and subcellular mislocalization between motor neurons of familial and sporadic ALS cases, which we primarily attribute to SOD1 gene status. Our data demonstrate co-deposition of p62 with mutant and wild-type disSOD1 and phosphorylated TDP-43 in familial and sporadic ALS spinal cord motor neurons, consistent with attempts by p62 to mitigate SOD1 and TDP-43 deposition. Wild-type SOD1 and TDP-43 co-deposition was also frequently observed in ALS cases lacking SOD1 mutations. Finally, alterations to the subcellular localization of the three proteins were tightly correlated, suggesting close relationships between the regulatory mechanisms governing the subcellular compartmentalization of these proteins. Our study is the first to profile spatial relationships between SOD1, TDP-43 and p62 pathologies in post-mortem spinal cord motor neurons of ALS patients, previously only studied in vitro. Our findings suggest interactions between these three key ALS-linked proteins are likely to modulate the formation of their respective proteinopathies, and perhaps the rate of motor neuron degeneration, in ALS patients.

Disruption of the VAPB-PTPIP51 ER-mitochondria tethering proteins in post-mortem human amyotrophic lateral sclerosis.

Signaling between the endoplasmic reticulum (ER) and mitochondria regulates many neuronal functions that are perturbed in amyotrophic lateral sclerosis (ALS) and perturbation to ER-mitochondria signaling is seen in cell and transgenic models of ALS. However, there is currently little evidence that ER-mitochondria signaling is altered in human ALS. ER-mitochondria signaling is mediated by interactions between the integral ER protein VAPB and the outer mitochondrial membrane protein PTPIP51 which act to recruit and “tether” regions of ER to the mitochondrial surface. The VAPB-PTPI51 tethers are now known to regulate a number of ER-mitochondria signaling functions. These include delivery of Ca2+ from ER stores to mitochondria, mitochondrial ATP production, autophagy and synaptic activity. Here we investigate the VAPB-PTPIP51 tethers in post-mortem control and ALS spinal cords. We show that VAPB protein levels are reduced in ALS. Proximity ligation assays were then used to quantify the VAPB-PTPIP51 interaction in spinal cord motor neurons in control and ALS cases. These studies revealed that the VAPB-PTPIP51 tethers are disrupted in ALS. Thus, we identify a new pathogenic event in post-mortem ALS.

Dysregulation of Tweak and Fn14 in skeletal muscle of spinal muscular atrophy mice

BackgroundSpinal muscular atrophy (SMA) is a childhood neuromuscular disorder caused by depletion of the survival motor neuron (SMN) protein. SMA is characterized by the selective death of spinal cord motor neurons, leading to progressive muscle wasting. Loss of skeletal muscle in SMA is a combination of denervation-induced muscle atrophy and intrinsic muscle pathologies. Elucidation of the pathways involved is essential to identify the key molecules that contribute to and sustain muscle pathology. The tumor necrosis factor-like weak inducer of apoptosis (TWEAK)/TNF receptor superfamily member fibroblast growth factor-inducible 14 (Fn14) pathway has been shown to play a critical role in the regulation of denervation-induced muscle atrophy as well as muscle proliferation, differentiation, and metabolism in adults. However, it is not clear whether this pathway would be important in highly dynamic and developing muscle.MethodsWe thus investigated the potential role of the TWEAK/Fn14 pathway in SMA muscle pathology, using the severe Taiwanese Smn−/−; SMN2 and the less severe Smn2B/− SMA mice, which undergo a progressive neuromuscular decline in the first three post-natal weeks. We also used experimental models of denervation and muscle injury in pre-weaned wild-type (WT) animals and siRNA-mediated knockdown in C2C12 muscle cells to conduct additional mechanistic investigations.ResultsHere, we report significantly dysregulated expression of Tweak, Fn14, and previously proposed downstream effectors during disease progression in skeletal muscle of the two SMA mouse models. In addition, siRNA-mediated Smn knockdown in C2C12 myoblasts suggests a genetic interaction between Smn and the TWEAK/Fn14 pathway. Further analyses of SMA, Tweak−/−, and Fn14−/− mice revealed dysregulated myopathy, myogenesis, and glucose metabolism pathways as a common skeletal muscle feature, providing further evidence in support of a relationship between the TWEAK/Fn14 pathway and Smn. Finally, administration of the TWEAK/Fn14 agonist Fc-TWEAK improved disease phenotypes in the two SMA mouse models.ConclusionsOur study provides mechanistic insights into potential molecular players that contribute to muscle pathology in SMA and into likely differential responses of the TWEAK/Fn14 pathway in developing muscle.

Mapping of STB/HAP1 Immunoreactivity in the Mouse Brainstem and its Relationships with Choline Acetyltransferase, with Special Emphasis on Cranial Nerve Motor and Preganglionic Autonomic Nuclei

Huntingtin-associated protein 1 (HAP1) is a core component of stigmoid body (STB) and is known as a neuroprotective interactor with causal agents for various neurodegenerative diseases. Brain regions rich in STB/HAP1 immunoreactivity are usually spared from cell death, whereas brain regions with negligible STB/HAP1 immunoreactivity are the major neurodegenerative targets. Recently, we have shown that STB/HAP1 is abundantly expressed in the spinal preganglionic sympathetic/parasympathetic neurons but absent in the motoneurons of spinal cord, indicating that spinal motoneurons are more vulnerable to neurodegenerative diseases. In light of STB/HAP1 neuroprotective effects, it is also essential to clarify the distribution of STB/HAP1 in another major neurodegenerative target, the brainstem. Here, we examined the expression and detailed immunohistochemical distribution of STB/HAP1 and its relationships with choline acetyltransferase (ChAT) in the midbrain, pons, and medulla oblongata of adult mice. Abundant STB/HAP1 immunoreactive neurons were disseminated in the periaqueductal gray, Edinger-Westphal nucleus, raphe nuclei, locus coeruleus, pedunculopontine tegmental nucleus, superior/inferior salivatory nucleus, and dorsal motor nucleus of vagus. Double-label immunohistochemistry of HAP1 with ChAT (or with urocortin-1 for Edinger-Westphal nucleus centrally projecting population) confirmed that STB/HAP1 was highly present in parasympathetic preganglionic neurons but utterly absent in cranial nerve motor nuclei throughout the brainstem. These results suggest that due to deficient putative STB/HAP1-protectivity, cranial nerve motor nuclei might be more vulnerable to certain neurodegenerative stresses than STB/HAP1-expressing brainstem nuclei, including preganglionic parasympathetic nuclei. Our current results also lay a basic foundation for future studies that seek to clarify the physiological/pathological roles of STB/HAP1 in the brainstem.

The Role and Therapeutic Potential of the Integrated Stress Response in Amyotrophic Lateral Sclerosis.

In amyotrophic lateral sclerosis (ALS) patients, loss of cellular homeostasis within cortical and spinal cord motor neurons triggers the activation of the integrated stress response (ISR), an intracellular signaling pathway that remodels translation and promotes a gene expression program aimed at coping with stress. Beyond its neuroprotective role, under regimes of chronic or excessive stress, ISR can also promote cell/neuronal death. Given the two-edged sword nature of ISR, many experimental attempts have tried to establish the therapeutic potential of ISR enhancement or inhibition in ALS. This review discusses the complex interplay between ISR and disease progression in different models of ALS, as well as the opportunities and limitations of ISR modulation in the hard quest to find an effective therapy for ALS.

A method to investigate muscle target-specific transcriptional signatures of single motor neurons.

Motor neurons in the vertebrate spinal cord have long served as a paradigm to study the transcriptional logic of cell type specification and differentiation. At limb levels, pool-specific transcriptional signatures first restrict innervation to only one particular muscle in the periphery, and get refined, once muscle connection has been established. Accordingly, to study the transcriptional dynamics and specificity of the system, a method for establishing muscle target-specific motor neuron transcriptomes would be required. To investigate target-specific transcriptional signatures of single motor neurons, here we combine ex-ovo retrograde axonal labeling in mid-gestation chicken embryos with manual isolation of individual fluorescent cells and Smart-seq2 single-cell RNA-sequencing. We validate our method by injecting the dorsal extensor metacarpi radialis and ventral flexor digiti quarti wing muscles and harvesting a total of 50 fluorescently labeled cells, in which we detect up to 12,000 transcribed genes. Additionally, we present visual cues and cDNA metrics predictive of sequencing success. Our method provides a unique approach to study muscle target-specific motor neuron transcriptomes at a single-cell resolution. We anticipate that our method will provide key insights into the transcriptional logic underlying motor neuron pool specialization and proper neuromuscular circuit assembly and refinement.

R-loop Mediated DNA Damage and Impaired DNA Repair in Spinal Muscular Atrophy.

Defects in DNA repair pathways are a major cause of DNA damage accumulation leading to genomic instability and neurodegeneration. Efficient DNA damage repair is critical to maintain genomicstability and support cell function and viability. DNA damage results in the activation of cell death pathways, causing neuronal death in an expanding spectrum of neurological disorders, such as amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), Alzheimer’s disease (AD), and spinal muscular atrophy (SMA). SMA is a neurodegenerative disorder caused by mutations in the Survival Motor Neuron 1 (SMN1) gene. SMA is characterized by the degeneration of spinal cord motor neurons due to low levels of the SMN protein. The molecular mechanism of selective motor neuron degeneration in SMA was unclear for about 20 years. However, several studies have identified biochemical and molecular mechanisms that may contribute to the predominant degeneration of motor neurons in SMA, including the RhoA/ROCK, the c-Jun NH2-terminal kinase (JNK), and p53-mediated pathways, which are involved in mediating DNA damage-dependent cell death. Recent studies provided insight into selective degeneration of motor neurons, which might be caused by accumulation of R-loop-mediated DNA damage and impaired non-homologous end joining (NHEJ) DNA repair pathway leading to genomic instability. Here, we review the latest findings involving R-loop-mediated DNA damage and defects in neuron-specific DNA repair mechanisms in SMA and discuss these findings in the context of other neurodegenerative disorders linked to DNA damage.

Altered SOD1 maturation and post-translational modification in amyotrophic lateral sclerosis spinal cord.

Aberrant self-assembly and toxicity of wild-type and mutant superoxide dismutase 1 (SOD1) has been widely examined in silico, in vitro and in transgenic animal models of amyotrophic lateral sclerosis. Detailed examination of the protein in disease-affected tissues from amyotrophic lateral sclerosis patients, however, remains scarce.We used histological, biochemical and analytical techniques to profile alterations to SOD1 protein deposition, subcellular localization, maturation and post-translational modification in post-mortem spinal cord tissues from amyotrophic lateral sclerosis cases and controls. Tissues were dissected into ventral and dorsal spinal cord grey matter to assess the specificity of alterations within regions of motor neuron degeneration.We provide evidence of the mislocalization and accumulation of structurally disordered, immature SOD1 protein conformers in spinal cord motor neurons of SOD1-linked and non-SOD1-linked familial amyotrophic lateral sclerosis cases, and sporadic amyotrophic lateral sclerosis cases, compared with control motor neurons. These changes were collectively associated with instability and mismetallation of enzymatically active SOD1 dimers, as well as alterations to SOD1 post-translational modifications and molecular chaperones governing SOD1 maturation. Atypical changes to SOD1 protein were largely restricted to regions of neurodegeneration in amyotrophic lateral sclerosis cases, and clearly differentiated all forms of amyotrophic lateral sclerosis from controls. Substantial heterogeneity in the presence of these changes was also observed between amyotrophic lateral sclerosis cases.Our data demonstrate that varying forms of SOD1 proteinopathy are a common feature of all forms of amyotrophic lateral sclerosis, and support the presence of one or more convergent biochemical pathways leading to SOD1 proteinopathy in amyotrophic lateral sclerosis. Most of these alterations are specific to regions of neurodegeneration, and may therefore constitute valid targets for therapeutic development.

Electrophysiological Recordings from Embryonic Mouse Motoneurons Cultured on Electrospun Poly-Lactic Acid (PLA) and Polypyrrole-Coated PLA Scaffolds.

Background:Biomaterials used as cell growth stimulants should be able to provide adequate cell adhesion with no alteration in cell function. In this work, we developed a 3D model of mouse spinal cord motoneurons on scaffolds composed of electrospun PLA fibers and plasma-polymerized PPy-coated PLA fibers. Methods:The functionality of the cultured motoneurons was assessed by evaluating both the electrophysiological response (i.e., the whole-cell Na+ and K+ currents and the firing of action potentials) and also the expression of the VAChaT by immunostaining techniques. While the expression of the VAChaT was confirmed on motoneurons cultured on the fibrous scaffolds, the electrophysiological responses indicated Na+ and K+ currents with lower amplitude and slower action potentials when compared to the response recorded from spinal cord motoneurons cultured on Poly-DL-Ornithine/Laminin- and plasma-polymerized PPy-coated coverslips.Results:From a morphological viewpoint, motoneurons cultured on PLA and PPy-coated PLA scaffolds did not show the development of dendritic and/or axonal processes, which were satisfactorily observed in the bidimensional cultures.Conclusion:We hypothesize that the apparently limited development of dendritic and/or axonal processes could produce a deleterious effect on the electrophysiological response of the cells, which might be due to the limited growth surface available in the fibrous scaffolds and/or to an undesired effect of the purification process.

Sema6D Regulates Zebrafish Vascular Patterning and Motor Neuronal Axon Growth in Spinal Cord.

Vessels and nerves are closely associated in anatomy as well as functions. Accumulating evidences have demonstrated that axon-guiding signals may affect endothelial cells migration and path finding, which is crucial for the patterning of both the complex vascular network and neural system. However, studies regarding the functional overlap between vascular and neuronal orchestrating are still incomplete. Semaphorin6D (Sema6D) belongs to the Semaphorin family and has been identified as an important regulating factor in diverse biological processes. Its roles in vascular development are still unclear. Here, we confirmed that sema6D is enriched in neural system and blood vessels of zebrafish embryos by in situ hybridization. Then, the deficiency of sema6D caused by specific antisense morpholino-oligonucleotides (MO) led to dramatic path finding defects in both intersegmental vessels (ISVs) and primary motor neurons (PMNs) of spinal cord in zebrafish embryos. Furthermore, these defective phenotypes were confirmed in F0 generation of sema6D knockouts and rescue experiments by overexpression of sema6D mRNA in sema6D morphants. These data collectively indicate that sema6D regulates zebrafish vascular patterning and motor neuronal axon growth in the spinal cord, which might be of great therapeutical use to regulate vessel and nerve guidance in the relevant diseases that affect both systems.

The mechanism of the WNT5A and FZD4 receptor mediated WNT/β–catenin pathway in the degeneration of ALS spinal cord motor neurons

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with unknown etiology, characterized by motor neuron degeneration, and there is no highly effective treatment. The canonical WNT/β-catenin signaling pathway has a critical role in the physiological and pathophysiological processes of the central nervous system. In this study, we investigated the regulatory mechanism of the WNT/β-catenin signaling pathway from the perspective of ligand-receptor binding and its relationship with the degeneration of ALS motor neurons. We used hSOD1-G93A mutant ALS transgenic mice and hSOD1-G93A mutant NSC34 cells combined with morphological and molecular biology techniques to determine the role of the WNT/β–catenin pathway in ALS. Our findings demonstrated that WNT5A regulates the WNT/β-catenin signaling pathway by binding to the FZD4 receptor in the pathogenesis of ALS and affects the proliferation and apoptosis of ALS motor neurons. Therefore, these findings may lead to the development of novel therapies to support the survival of ALS motor neurons.

Vulnerability of the spinal motor neuron presynaptic terminal sub-proteome in ALS

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder, characterised by the loss of motor neurons and subsequent paralysis. Evidence indicates that synaptic alterations are associated with the early stages of ALS pathogenesis. A hallmark of ALS postmortem tissue is the presence of proteinaceous inclusions, indicative of disturbed protein homeostasis, particularly in spinal cord motor neurons. We recently demonstrated that spinal cord motor neurons contain a supersaturated proteome, as they possess proteins at concentrations that exceed their solubility limits, resulting in a metastable proteome conducive to protein misfolding and aggregation. Recent evidence indicates metastable sub-proteomes within neuronal compartments, such as the synapse, may be particularly vulnerable and underlie their involvement in the initial stages of neurodegenerative diseases. To investigate if the motor neuron presynaptic terminal possesses a metastable sub-proteome, we used human and mouse spinal cord motor neuron expression data to calculate supersaturation scores. Here, we found that both the human and mouse presynaptic terminal sub-proteomes have higher supersaturation scores than the entire motor neuron proteome. In addition, we observed that proteins down-regulated in ALS were over-represented in the synapse. These results provide support for the notion that the metastability of the sub-proteome within the motor neuron presynaptic terminal may be particularly susceptible to protein homeostasis disturbances in ALS, and may contribute to explaining the observed synaptic dysfunction in ALS.

Methylmercury Causes Neurodegeneration and Downregulation of Myelin Basic Protein in the Spinal Cord of Offspring Rats after Maternal Exposure.

Methylmercury (MeHg) is one of the most dangerous toxic pollutants spread throughout the earth. Chronic MeHg intoxication by contaminated food ingestion is the most common threat to human health, including impairment to the developing fetus. The present study aims at investigating the effects of maternal exposure to MeHg during gestation and lactation on the spinal cord of offspring. Pregnant rats received oral doses of MeHg (40 μg/kg/day) over a period of 42 days (21 gestation and 21 lactation). Control animals received the vehicle only. Total mercury concentration was measured in blood samples from offspring collected at the 41st postnatal day. Counting of motor neurons and immunoreactivity for myelin basic protein (MBP) were assessed in the spinal cords in both control and MeHg-intoxicated animals. Our results showed that MeHg promoted an increase in blood Hg levels. In addition, it caused a reduction in the number of spinal cord motor neurons as well as decreased MBP immunoreactivity in the cervical, thoracic and lumbar segments. Our present findings suggest that MeHg intoxication during rat pregnancy and lactation is associated with a pattern of motor neuron degeneration and downregulation of myelin basic protein in different segments of a developing spinal cord. Further studies are needed to establish the effect of MeHg intoxication in both young and adult rats.

Key Disease Mechanisms Linked to Amyotrophic Lateral Sclerosis in Spinal Cord Motor Neurons.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no modifying treatments available. The molecular mechanisms underpinning disease pathogenesis are not fully understood. Recent studies have employed co-expression networks to identify key genes, known as “switch genes”, responsible for dramatic transcriptional changes in the blood of ALS patients. In this study, we directly investigate the root cause of ALS by examining the changes in gene expression in motor neurons that degenerate in patients. Co-expression networks identified in ALS patients’ spinal cord motor neurons revealed 610 switch genes in seven independent microarrays. Switch genes were enriched in several pathways, including viral carcinogenesis, PI3K-Akt, focal adhesion, proteoglycans in cancer, colorectal cancer, and thyroid hormone signaling. Transcription factors ELK1 and GATA2 were identified as key master regulators of the switch genes. Protein-chemical network analysis identified valproic acid, cyclosporine, estradiol, acetaminophen, quercetin, and carbamazepine as potential therapeutics for ALS. Furthermore, the chemical analysis identified metals and organic compounds including, arsenic, copper, nickel, and benzo(a)pyrene as possible mediators of neurodegeneration. The identification of switch genes provides insights into previously unknown biological pathways associated with ALS.

Motor neuron TDP-43 proteinopathy in progressive supranuclear palsy and corticobasal degeneration

Abstract TDP-43 is mislocalized from the nucleus and aggregates within the cytoplasm of affected neurons in cases of amyotrophic lateral sclerosis. TDP-43 pathology has also been found in brain tissues under non-amyotrophic lateral sclerosis conditions, suggesting mechanistic links between TDP-43-related amyotrophic lateral sclerosis and various neurological disorders. This study aimed to assess TDP-43 pathology in the spinal cord motor neurons of tauopathies. We examined 106 spinal cords from consecutively autopsied cases with progressive supranuclear palsy (n = 26), corticobasal degeneration (n = 12), globular glial tauopathy (n = 5), Alzheimer’s disease (n = 21) or Pick's disease (n = 6) and neurologically healthy controls (n = 36). Ten of the progressive supranuclear palsy cases (38%) and seven of the corticobasal degeneration cases (58%) showed mislocalization and cytoplasmic aggregation of TDP-43 in spinal cord motor neurons, which was prominent in the cervical cord. TDP-43 aggregates were found to be skein-like, round-shaped, granular or dot-like and contained insoluble C-terminal fragments showing blotting pattern of amyotrophic lateral sclerosis or frontotemporal lobar degeneration. The lower motor neurons also showed cystatin-C aggregates, although Bunina bodies were absent in haematoxylin-eosin staining. The spinal cord TDP-43 pathology was often associated with TDP-43 pathology of the primary motor cortex. Positive correlations were shown between the severities of TDP-43 and four-repeat (4R)-tau aggregates in the cervical cord. TDP-43 and 4R-tau aggregates burdens positively correlated with microglial burden in anterior horn. TDP-43 pathology of spinal cord motor neuron did not develop in an age-dependent manner and was not found in the Alzheimer’s disease, Pick's disease, globular glial tauopathy and control groups. Next, we assessed SFPQ expression in spinal cord motor neurons; SFPQ is a recently identified regulator of amyotrophic lateral sclerosis/frontotemporal lobar degeneration pathogenesis, and it is also reported that interaction between SFPQ and FUS regulates splicing of MAPT exon 10. Immunofluorescent and proximity-ligation assays revealed altered SFPQ/FUS-interactions in the neuronal nuclei of progressive supranuclear palsy, corticobasal degeneration and amyotrophic lateral sclerosis-TDP cases but not in Alzheimer’s disease, Pick's disease and globular glial tauopathy cases. Moreover, SFPQ expression was depleted in neurons containing TDP-43 or 4R-tau aggregates of progressive supranuclear palsy and corticobasal degeneration cases. Our results indicate that progressive supranuclear palsy and corticobasal degeneration may have properties of systematic motor neuron TDP-43 proteinopathy, suggesting mechanistic links with amyotrophic lateral sclerosis-TDP. SFPQ dysfunction, arising from altered interaction with FUS, may be a candidate of the common pathway.

GPX4 deficiency-dependent phospholipid peroxidation drives motor deficits of ALS

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by oxidative stress that triggers motor neurons loss in the brain and spinal cord. However, the mechanisms underlying the exact role of oxidative stress in ALS-associated neural degeneration are not definitively established. Oxidative stress-generated phospholipid peroxides are known to have extensive physiological and pathological consequences to tissues. Here, we discovered that the deficiency of glutathione peroxidase 4 (GPX4), an essential antioxidant peroxidase, led to the accumulation of phospholipid peroxides and resulted in a loss of motor neurons in spinal cords of ALS mice. Mutant human SOD1G93A transgenic mice were intrathecally injected with neuron-targeted adeno-associated virus (AAV) expressing GPX4 (GPX4-AAV) or phospholipid peroxidation inhibitor, ferrostatin-1. The results showed that impaired motor performance and neural loss induced by SOD1G93A toxicity in the lumbar spine were substantially alleviated by ferrostatin-1 treatment and AAV-mediated GPX4 delivery. In addition, the denervation of neuron-muscle junction and spinal atrophy in ALS mice were rescued by neural GPX4 overexpression, suggesting that GPX4 is essential for the motor neural maintenance and function. In comparison, conditional knockdown of Gpx4 in the spinal cords of Gpx4fl/fl mice triggered an obvious increase of phospholipid peroxides and the occurrence of ALS-like motor phenotype. Altogether, our findings underscore the importance of GPX4 in maintaining phospholipid redox homeostasis in the spinal cord and presents GPX4 as an attractive therapeutic target for ALS treatment.

Selective Loss of MATR3 in Spinal Interneurons, Upper Motor Neurons and Hippocampal CA1 Neurons in a MATR3 S85C Knock-In Mouse Model of Amyotrophic Lateral Sclerosis.

Simple SummaryAmyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting the motor neurons in the brain and spinal cord. Mutations in the gene Matr3 have been linked to ALS, including the autosomal dominant missense mutation S85C. We previously created a mouse model containing the S85C mutation in the Matr3 gene to understand how it causes ALS. The S85C mice exhibited MATR3 staining loss in selective populations of degenerating neurons, such as Purkinje cells in the cerebellum and α-motor neurons in the lumbar spinal cord. However, studies have shown that neurons other than motor neurons may be involved in contributing to ALS; therefore, we investigated additional neuronal cell types in the spinal cord and brain. Here, we found that MATR3 staining is selectively reduced in interneurons and α-motor neurons of the cervical and thoracic regions of the spinal cord, as well as in subsets of upper motor neurons and hippocampal neurons. These neurons did not exhibit cell body loss; however, how the MATR3 loss affects neuronal function remains to be determined. Overall, these findings demonstrate that the MATR3 S85C mutation affects other neuronal types of the brain and spinal cord in addition to motor neurons, suggesting that these additional neuronal types are involved in ALS pathogenesis.The neuropathological hallmark of amyotrophic lateral sclerosis (ALS) is motor neuron degeneration in the spinal cord and cortex. Accumulating studies report that other neurons in the central nervous system (CNS) are also affected in ALS. Mutations in Matr3, which encodes a nuclear matrix protein involved in RNA splicing, have been linked to ALS. Previously, we generated a MATR3 S85C knock-in (KI) mouse model that recapitulates early-stage features of ALS. We reported that MATR3 S85C KI mice exhibit defects in lumbar spinal cord motor neurons and in cerebellar Purkinje cells, which are associated with reduced MATR3 immunoreactivity. Here, we show that neurons in various other regions of the CNS are affected in MATR3 S85C KI mice. Using histological analyses, we found selective loss of MATR3 staining in α-motor neurons, but not γ-motor neurons in the cervical and thoracic spinal cord. Loss of MATR3 was also found in parvalbumin-positive interneurons in the cervical, thoracic and lumbar spinal cord. In addition, we found the loss of MATR3 in subsets of upper motor neurons and hippocampal CA1 neurons. Collectively, our findings suggest that these additional neuronal types may contribute to the disease process in MATR3 S85C KI mice.