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Amyotrophic lateral sclerosis (ALS) is a heterogenous neurodegenerative disorder that affects motor neurons and voluntary muscle control1. ALS heterogeneity includes the age of manifestation, the rate of progression and the anatomical sites of symptom onset. Disease-causing mutations in specific genes have been identified and define different subtypes of ALS1. Although several ALS-associated genes have been shown to affect immune functions2, whether specific immune features account for ALS heterogeneity is poorly understood. Amyotrophic lateral sclerosis-4 (ALS4) is characterized by juvenile onset and slow progression3. Patients with ALS4 show motor difficulties by the time that they are in their thirties, and most of them require devices to assist with walking by their fifties. ALS4 is caused by mutations in the senataxin gene (SETX). Here, using Setx knock-in mice that carry the ALS4-causative L389S mutation, we describe an immunological signature that consists of clonally expanded, terminally differentiated effector memory (TEMRA) CD8 T cells in the central nervous system and the blood of knock-in mice. Increased frequencies of antigen-specific CD8 T cells in knock-in mice mirror the progression of motor neuron disease and correlate with anti-glioma immunity. Furthermore, bone marrow transplantation experiments indicate that the immune system has a key role in ALS4 neurodegeneration. In patients with ALS4, clonally expanded TEMRA CD8 T cells circulate in the peripheral blood. Our results provide evidence of an antigen-specific CD8 T cell response in ALS4, which could be used to unravel disease mechanisms and as a potential biomarker of disease state.

Fused in sarcoma (FUS) is an RNA-binding protein that is genetically and pathologically associated with rare and aggressive forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). To explore the mechanisms by which mutant FUS causes neurodegeneration in ALS-FTD, we generated a series of FUS knock-in mouse lines that express the equivalent of ALS-associated mutant FUSP525L and FUSΔEX14 protein. In FUS mutant mice, we show progressive, age-dependent motor neuron loss as a consequence of a dose-dependent gain of toxic function, associated with the insolubility of FUS and related RNA-binding proteins. In this disease-relevant mouse model of ALS-FUS, we show that ION363, a non-allele-specific FUS antisense oligonucleotide, efficiently silences Fus and reduces postnatal levels of FUS protein in the brain and spinal cord, delaying motor neuron degeneration. In a patient with ALS with a FUSP525L mutation, we provide preliminary evidence that repeated intrathecal infusions of ION363 lower wild-type and mutant FUS levels in the central nervous system, resulting in a marked reduction in the burden of FUS aggregates that are a pathological hallmark of disease. In mouse genetic and human clinical studies, we provide evidence in support of FUS silencing as a therapeutic strategy in FUS-dependent ALS and FTD., An antisense oligonucleotide targeting the RNA-binding protein FUS transcript lowers FUS levels in mice and in the central nervous system of a single patient with FUS-dependent ALS

Neurodegenerative disorders are a family of diseases that remain poorly treated despite their growing global health burden. A shared feature of many neurodegenerative disorders is the accumulation of toxic misfolded proteins. To gain insight into the mechanisms and modulators of protein misfolding, we developed a multiplex reverse genetics platform. Using this novel platform 29 cell-based models expressing proteins that undergo misfolding in neurodegeneration were probed against more than a thousand genetic modifiers. The resulting data provide insight into the nature of modifiers that act on multiple misfolded proteins as compared to those that show activity on only one. To illustrate the utility of this platform, we extensively characterized a potent hit from our screens, the human chaperone DNAJB6. We show that DNAJB6 is a general modifier of the toxicity and solubility of multiple amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD)-linked RNA-binding proteins (RBPs), including FUS, TDP-43, and hnRNPA1. Biophysical examination of DNAJB6 demonstrated that it co-phase separates with, and alters the behavior of FUS containing condensates by locking them into a loose gel-like state which prevents their fibrilization. Domain mapping and a deep mutational scan of DNAJB6 support the critical importance for DNAJB6 phase separation in its effects on multiple RNA-binding proteins. Crucially, these studies also suggest that this property can be further tuned to generate novel variants with enhanced activity that might illuminate potential avenues for clinical translation.

Introduction/Aims. Primary lateral sclerosis (PLS) is exceedingly rare and has been an enigmatic disease. Recent progress has drastically changed this perception, with early biomarkers being investigated and potential medications for PLS emerging at the preclinical stage. The aim of this paper is to describe a study of PLS natural history and discuss the limitations and proposed solutions to the study of a rare and slowly progressive disease. Methods. The PLS Natural History Study is a 30-site, 24-month, prospective study that is supported by multiple funding sources. The study aims to enroll 50 early PLS (disease duration ≤4 years) and 50 definite PLS (disease duration 4 to 15 years) participants using modified PLS Diagnostic Criteria. Smartphone-based assessments including semi-quantitative and quantitative measures and patient-reported outcomes are utilized. In-person quantitative measures are also completed during site visits. The change in the PLS Functional Rating Scale score is the primary outcome. The study utilizes the NeuroBANK® patient-centric data capture and management platform. The biostatistical analysis plan has been developed. Results. In one year, 28 participants have been recruited. Enrollment has been much slower than anticipated due to the COVID-19 pandemic, the rarity of PLS, and potential study competition for internal resources from ALS clinical trials. Discussion. We discuss the need for more innovative methods to enroll and study individuals with such rare diseases and propose a number of mechanisms by which more efficient enrollment could be facilitated.

Amyotrophic Lateral Sclerosis (ALS) is a heterogeneous neurodegenerative disorder that affects motor neurons in the brain and spinal cord, causing progressive loss of voluntary muscle control1,2. ALS heterogeneity includes the age of manifestation, the rate of progression, and the anatomical sites of symptom onset. In addition, disease-causing mutations in specific genes have been identified and are used to catalog different subtypes of ALS3. Interestingly, several ALS-associated genes have been shown to affect immune functions, and a variety of aberrant inflammatory events have been reported in patients and mouse models4-11, suggesting that specific immune features can also account for ALS heterogeneity. ALS4 is characterized by juvenile-onset and slow progression12. After experiencing mild symptoms during their childhood, ALS4 patients show motor difficulties by their 30s, and most of them require walkers or wheelchairs by their 50s. ALS4 is caused by dominant mutations in the gene SETX. Using Setx knock-in (KI) mice carrying the ALS4 causative L389S mutation, we discovered an immunological signature consisting of clonally activated CD8 T cells specifically in the central nervous system and blood of KI animals. Expansion of antigen-specific CD8 T cells mirrors disease progression. Bone marrow transplantation experiments indicate an essential role of the immune system in ALS4 neurodegeneration. Furthermore, we found that clonally expanded CD8 T cells circulate in the peripheral blood of ALS4 patients. Our results provide evidence of an antigen-specific CD8 T cell response linked to ALS4, and can serve not only to unravel specific disease mechanisms, but as a potential biomarker of disease activity.

Background The pathological hallmark in MSA is oligodendrocytic glial cytoplasmic inclusions (GCIs) containing α-synuclein, in addition to neuronal loss and astrogliosis especially involving the striatonigral and olivopontocerebellar systems. Rarely, TAR DNA-binding protein of 43 kDa (TDP-43), a component of ubiquitinated inclusions observed mainly in amyotrophic lateral sclerosis and frontotemporal lobar degeneration has been demonstrated in cases of MSA and, more recently, was shown to colocalize with α-synuclein pathology in GCIs in 2 patients. Methods A 66-year-old woman presented with a syndrome characterized by spasticity, dysautonomia, bulbar dysfunction, and parkinsonism. Symptoms progressed until her death at age 74. Neuropathological evaluation was performed at the New York Brain Bank at Columbia University. Results On gross examination, there was striking severe volume loss of the left striatum compared to mild involvement of the right striatum. Microscopically, neuronal loss and gliosis of the putamen and globus pallidus were severe on the left side, in contrast to mild involvement on the right side. Immunohistochemistry for α-synuclein revealed widespread GCIs. The sections subjected to TDP-43 antibodies showed a few GCIs with definite nucleocytoplasmic translocation of the labeling within the lenticular nucleus and within the paracentral cortex. Conclusions This report adds to the evidence that TDP-43 and α-synuclein colocalize in GCIs. Whether this coexistence contributes to the pathogenesis of a subset of MSA patients or is an age-related process is not known. More cases with these peculiar pathological hallmarks might help determine whether TDP-43 contributes to neurodegeneration in a subset of patients with MSA.

Large-scale sequencing efforts in amyotrophic lateral sclerosis (ALS) have implicated novel genes using gene-based collapsing methods. However, pathogenic mutations may be concentrated in specific genic regions. To address this, we developed two collapsing strategies: One focuses rare variation collapsing on homology-based protein domains as the unit for collapsing, and the other is a gene-level approach that, unlike standard methods, leverages existing evidence of purifying selection against missense variation on said domains. The application of these two collapsing methods to 3093 ALS cases and 8186 controls of European ancestry, and also 3239 cases and 11,808 controls of diversified populations, pinpoints risk regions of ALS genes, including SOD1, NEK1, TARDBP, and FUS. While not clearly implicating novel ALS genes, the new analyses not only pinpoint risk regions in known genes but also highlight candidate genes as well.

Hannu Laaksovirta konsortion jäsenenä. The Genomics England Research Consortium, The International ALS/FTD Genomics Consortium (iAFGC), The International FTD Genetics Consortium (IFGC), The International LBD Genomics Consortium (iLBDGC), The NYGC ALS Consortium, The PROSPECT Consortium,17 James B. Rowe,17 Luisa Benussi,18 Giuliano Binetti,18,19 Roberta Ghidoni,18 Edwin Jabbari,20,21 Coralie Viollet,22 Jonathan D. Glass,23 Andrew B. Singleton,24 Vincenzo Silani,25,26 Owen A. Ross,27 Mina Ryten,8,28,29 Ali Torkamani,30 Toshiko Tanaka,31 Luigi Ferrucci,31 Susan M. Resnick,32 We examined the role of repeat expansions in the pathogenesis of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) by analyzing whole-genome sequence data from 2,442 FTD/ALS patients, 2,599 Lewy body dementia (LBD) patients, and 3,158 neurologically healthy subjects. Pathogenic expansions (range, 40?64 CAG repeats) in the huntingtin (HTT) gene were found in three (0.12%) patients diagnosed with pure FTD/ALS syndromes but were not present in the LBD or healthy cohorts. We replicated our findings in an independent collection of 3,674 FTD/ALS patients. Postmortem evaluations of two patients revealed the classical TDP-43 pathology of FTD/ALS, as well as huntingtin-positive, ubiquitin-positive aggregates in the frontal cortex. The neostriatal atrophy that pathologically defines Huntington?s disease was absent in both cases. Our findings reveal an etiological relationship between HTT repeat expansions and FTD/ALS syndromes and indicate that genetic screening of FTD/ALS patients for HTT repeat expansions should be considered. We examined the role of repeat expansions in the pathogenesis of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) by analyzing whole-genome sequence data from 2,442 FTD/ALS patients, 2,599 Lewy body dementia (LBD) patients, and 3,158 neurologically healthy subjects. Pathogenic expansions (range, 40?64 CAG repeats) in the huntingtin (HTT) gene were found in three (0.12%) patients diagnosed with pure FTD/ALS syndromes but were not present in the LBD or healthy cohorts. We replicated our findings in an independent collection of 3,674 FTD/ALS patients. Postmortem evaluations of two patients revealed the classical TDP-43 pathology of FTD/ALS, as well as huntingtin-positive, ubiquitin-positive aggregates in the frontal cortex. The neostriatal atrophy that pathologically defines Huntington?s disease was absent in both cases. Our findings reveal an etiological relationship between HTT repeat expansions and FTD/ALS syndromes and indicate that genetic screening of FTD/ALS patients for HTT repeat expansions should be considered.

Dysregulation of the DNA/RNA-binding protein FUS causes certain subtypes of ALS/FTD by largely unknown mechanisms. Recent evidence has shown that FUS toxic gain of function due either to mutations or to increased expression can disrupt critical cellular processes, including mitochondrial functions. Here, we demonstrate that in human cells overexpressing wild-type FUS or expressing mutant derivatives, the protein associates with multiple mRNAs, and these are enriched in mRNAs encoding mitochondrial respiratory chain components. Notably, this sequestration leads to reduced levels of the encoded proteins, which is sufficient to bring about disorganized mitochondrial networks, reduced aerobic respiration and increased reactive oxygen species. We further show that mutant FUS associates with mitochondria and with mRNAs encoded by the mitochondrial genome. Importantly, similar results were also observed in fibroblasts derived from ALS patients with FUS mutations. Finally, we demonstrate that FUS loss of function does not underlie the observed mitochondrial dysfunction, and also provides a mechanism for the preferential sequestration of the respiratory chain complex mRNAs by FUS that does not involve sequence-specific binding. Together, our data reveal that respiratory chain complex mRNA sequestration underlies the mitochondrial defects characteristic of ALS/FTD and contributes to the FUS toxic gain of function linked to this disease spectrum.

Reliable and consistent pluripotent stem cell reporter systems for efficient purification and visualization of motor neurons are essential reagents for the study of normal motor neuron biology and for effective disease modeling. To overcome the inherent noisiness of transgene-based reporters, we developed a new series of human induced pluripotent stem cell lines by knocking in tdTomato, Cre, or CreERT2 recombinase into the HB9 (MNX1) or VACHT (SLC18A3) genomic loci. The new lines were validated by directed differentiation into spinal motor neurons and immunostaining for motor neuron markers HB9 and ISL1. To facilitate efficient purification of spinal motor neurons, we further engineered the VACHT-Cre cell line with a validated, conditional CD14-GFP construct that allows for both fluorescence-based identification of motor neurons, as well as magnetic-activated cell sorting (MACS) to isolate differentiated motor neurons at scale. The targeting strategies developed here offer a standardized platform for reproducible comparison of motor neurons across independently derived pluripotent cell lines.

The RNA-binding protein FUS participates in several RNA biosynthetic processes and has been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Here we report that FUS controls back-splicing reactions leading to circular RNA (circRNA) production. We identified circRNAs expressed in in vitro-derived mouse motor neurons (MNs) and determined that the production of a considerable number of these circRNAs is regulated by FUS. Using RNAi and overexpression of wild-type and ALS-associated FUS mutants, we directly correlate the modulation of circRNA biogenesis with alteration of FUS nuclear levels and with putative toxic gain of function activities. We also demonstrate that FUS regulates circRNA biogenesis by binding the introns flanking the back-splicing junctions and that this control can be reproduced with artificial constructs. Most circRNAs are conserved in humans and specific ones are deregulated in human-induced pluripotent stem cell-derived MNs carrying the FUSP525L mutation associated with ALS.

The study of amyotrophic lateral sclerosis (ALS) and potential interventions would be facilitated if motor axon degeneration could be more readily visualized. Here we demonstrate that stimulated Raman scattering (SRS) microscopy could be used to sensitively monitor peripheral nerve degeneration in ALS mouse models and ALS autopsy materials. Three-dimensional imaging of pre-symptomatic SOD1 mouse models and data processing by a correlation-based algorithm revealed that significant degeneration of peripheral nerves could be detected coincidentally with the earliest detectable signs of muscle denervation and preceded physiologically measurable motor function decline. We also found that peripheral degeneration was an early event in FUS as well as C9ORF72 repeat expansion models of ALS, and that serial imaging allowed long-term observation of disease progression and drug effects in living animals. Our study demonstrates that SRS imaging is a sensitive and quantitative means of measuring disease progression, greatly facilitating future studies of disease mechanisms and candidate therapeutics., Sensitive and label-free imaging methods to visualize nerve degeneration are currently lacking. Here authors show that stimulated Raman scattering (SRS) microscopy can be used to monitor peripheral nerve degeneration in mouse models of amyotrophic lateral sclerosis (ALS) and in postmortem tissue from ALS patients.

Motor function in mammalian species depends on the maturation of spinal circuits formed by a large variety of interneurons that regulate motoneuron firing and motor output. Interneuron activity is in turn modulated by the organization of their synaptic inputs, but the principles governing the development of specific synaptic architectures unique to each premotor interneuron are unknown. For example, Renshaw cells receive, at least in the neonate, convergent inputs from sensory afferents (likely Ia) and motor axons, raising the question of whether they interact during Renshaw cell development. In other well-studied neurons, such as Purkinje cells, heterosynaptic competition between inputs from different sources shapes synaptic organization. To examine the possibility that sensory afferents modulate synaptic maturation on developing Renshaw cells, we used three animal models in which afferent inputs in the ventral horn are dramatically reduced (ER81(-/-) knockout), weakened (Egr3(-/-) knockout), or strengthened (mlcNT3(+/-) transgenic). We demonstrate that increasing the strength of sensory inputs on Renshaw cells prevents their deselection and reduces motor axon synaptic density, and, in contrast, absent or diminished sensory afferent inputs correlate with increased densities of motor axons synapses. No effects were observed on other glutamatergic inputs. We conclude that the early strength of Ia synapses influences their maintenance or weakening during later development and that heterosynaptic influences from sensory synapses during early development regulates the density and organization of motor inputs on mature Renshaw cells.

Large-scale sequencing efforts in amyotrophic lateral sclerosis (ALS) have implicated novel genes using gene-based collapsing methods. However, pathogenic mutations may be concentrated in specific genic regions. To address this, we developed two collapsing strategies, one focuses rare variation collapsing on homology-based protein domains as the unit for collapsing and another gene-level approach that, unlike standard methods, leverages existing evidence of purifying selection against missense variation on said domains. The application of these two collapsing methods to 3,093 ALS cases and 8,186 controls of European ancestry, and also 3,239 cases and 11,808 controls of diversified populations, pinpoints risk regions of ALS genes including SOD1, NEK1, TARDBP and FUS. While not clearly implicating novel ALS genes, the new analyses not only pinpoint risk regions in known genes but also highlight candidate genes as well.

International audience; Hyperexcitability has been suggested to contribute to motoneuron degeneration in amyotrophic lateral sclerosis (ALS). If this is so, and given that the physiological type of a motor unit determines the relative susceptibility of its motoneuron in ALS, then one would expect the most vulnerable motoneurons to display the strongest hyperexcitability prior to their degeneration, whereas the less vulnerable should display a moderate hyperexcitability, if any. We tested this hypothesis in vivo in two unrelated ALS mouse models by correlating the electrical properties of motoneurons with their physiological types, identified based on their motor unit contractile properties. We found that, far from being hyperexcitable, the most vulnerable motoneurons become unable to fire repetitively despite the fact that their neuromuscular junctions were still functional. Disease markers confirm that this loss of function is an early sign of degeneration. Our results indicate that intrinsic hyperexcitability is unlikely to be the cause of motoneuron degeneration.

© 2018 Elsevier Inc., To identify novel genes associated with ALS, we undertook two lines of investigation. We carried out a genome-wide association study comparing 20,806 ALS cases and 59,804 controls. Independently, we performed a rare variant burden analysis comparing 1,138 index familial ALS cases and 19,494 controls. Through both approaches, we identified kinesin family member 5A (KIF5A) as a novel gene associated with ALS. Interestingly, mutations predominantly in the N-terminal motor domain of KIF5A are causative for two neurodegenerative diseases: hereditary spastic paraplegia (SPG10) and Charcot-Marie-Tooth type 2 (CMT2). In contrast, ALS-associated mutations are primarily located at the C-terminal cargo-binding tail domain and patients harboring loss-of-function mutations displayed an extended survival relative to typical ALS cases. Taken together, these results broaden the phenotype spectrum resulting from mutations in KIF5A and strengthen the role of cytoskeletal defects in the pathogenesis of ALS.

Mutations in fused in sarcoma (FUS) cause amyotrophic lateral sclerosis (ALS). FUS is a multifunctional protein involved in the biogenesis and activity of several types of RNAs, and its role in the pathogenesis of ALS may involve both direct effects of disease-associated mutations through gain- and loss-of-function mechanisms and indirect effects due to the cross talk between different classes of FUS-dependent RNAs. To explore how FUS mutations impinge on motor neuron-specific RNA-based circuitries, we performed transcriptome profiling of small and long RNAs of motor neurons (MNs) derived from mouse embryonic stem cells carrying a FUS-P517L knock-in mutation, which is equivalent to human FUS-P525L, associated with a severe and juvenile-onset form of ALS. Combining ontological, predictive and molecular analyses, we found an inverse correlation between several classes of deregulated miRNAs and their corresponding mRNA targets in both homozygous and heterozygous P517L MNs. We validated a circuitry in which the upregulation of miR-409-3p and miR-495-3p, belonging to a brain-specific miRNA subcluster implicated in several neurodevelopmental disorders, produced the downregulation of Gria2, a subunit of the glutamate α‐amino‐3‐hydroxy‐5‐methyl-4-isoxazole propionic acid (AMPA) receptor with a significant role in excitatory neurotransmission. Moreover, we found that FUS was involved in mediating such miRNA repression. Gria2 alteration has been proposed to be implicated in MN degeneration, through disturbance of Ca2+ homeostasis, which triggers a cascade of damaging “excitotoxic” events. The molecular cross talk identified highlights a role for FUS in excitotoxicity and in miRNA-dependent regulation of Gria2. This circuitry also proved to be deregulated in heterozygosity, which matches the human condition perfectly. Electronic supplementary material The online version of this article (10.1007/s12035-018-0884-4) contains supplementary material, which is available to authorized users.

Long non-coding RNAs (lncRNAs) are currently recognized as crucial players in nervous system development, function and pathology. In Amyotrophic Lateral Sclerosis (ALS), identification of causative mutations in FUS and TDP-43 or hexanucleotide repeat expansion in C9ORF72 point to the essential role of aberrant RNA metabolism in neurodegeneration. In this study, by taking advantage of an in vitro differentiation system generating mouse motor neurons (MNs) from embryonic stem cells, we identified and characterized the long non-coding transcriptome of MNs. Moreover, by using mutant mouse MNs carrying the equivalent of one of the most severe ALS-associated FUS alleles (P517L), we identified lncRNAs affected by this mutation. Comparative analysis with human MNs derived in vitro from induced pluripotent stem cells indicated that candidate lncRNAs are conserved between mouse and human. Our work provides a global view of the long non-coding transcriptome of MN, as a prerequisite toward the comprehension of the still poorly characterized non-coding side of MN physiopathology. Keywords: LncRNAs, Mouse ESCs, Motor neurons, Differentiation, FUS, ALS

Increasing evidence suggests that necroptosis, a form of programmed cell death (PCD), contributes to neurodegeneration in several disorders, including ALS. Supporting this view, investigations in bothin vitroandin vivomodels of ALS have implicated key molecular determinants of necroptosis in the death of spinal motor neurons (MNs). Consistent with a pathogenic role of necroptosis in ALS, we showed increased mRNA levels for the three main necroptosis effectorsRipk1,Ripk3, andMlklin the spinal cord of mutant superoxide dismutase-1 (SOD1G93A) transgenic mice (Tg), an established model of ALS. In addition, protein levels of receptor-interacting protein kinase 1 (RIPK1; but not of RIPK3, MLKL or activated MLKL) were elevated in spinal cord extracts from these Tg SOD1G93Amice. In postmortem motor cortex samples from sporadic and familial ALS patients, no change in protein levels of RIPK1 were detected. Silencing ofRipk3in cultured MNs protected them from toxicity associated with SOD1G93Aastrocytes. However, constitutive deletion ofRipk3in Tg SOD1G93Amice failed to provide behavioral or neuropathological improvement, demonstrating no similar benefit ofRipk3silencingin vivo. Lastly, we detected no genotype-specific myelin decompaction, proposed to be a proxy of necroptosis in ALS, in either Tg SOD1G93AorOptineurinknock-out mice, another ALS mouse model. These findings argue against a role for RIPK3 in Tg SOD1G93A-induced neurodegeneration and call for further preclinical investigations to determine if necroptosis plays a critical role in the pathogenesis of ALS.

The molecular and cellular basis of selective motor neuron (MN) vulnerability in amyotrophic lateral sclerosis (ALS) is not known. In genetically distinct mouse models of familial ALS expressing mutant superoxide dismutase-1 (SOD1), TAR DNA-binding protein 43 (TDP-43), and fused in sarcoma (FUS), we demonstrate selective degeneration of alpha MNs (α-MNs) and complete sparing of gamma MNs (γ-MNs), which selectively innervate muscle spindles. Resistant γ-MNs are distinct from vulnerable α-MNs in that they lack synaptic contacts from primary afferent (IA) fibers. Elimination of these synapses protects α-MNs in the SOD1 mutant, implicating this excitatory input in MN degeneration. Moreover, reduced IA activation by targeted reduction of γ-MNs in SOD1G93A mutants delays symptom onset and prolongs lifespan, demonstrating a pathogenic role of surviving γ-MNs in ALS. This study establishes the resistance of γ-MNs as a general feature of ALS mouse models and demonstrates that synaptic excitation of MNs within a complex circuit is an important determinant of relative vulnerability in ALS.

An expanded GGGGCC hexanucleotide in C9ORF72 (C9) is the most frequent known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). It has been proposed that expanded transcripts adopt G-quadruplex (G-Q) structures and associate with proteins, but whether this occurs and contributes to disease is unknown. Here we show first that the protein that predominantly associates with GGGGCC repeat RNA in vitro is the splicing factor hnRNP H, and that this interaction is linked to G-Q formation. We then show that G-Q RNA foci are more abundant in C9 ALS patient fibroblasts and astrocytes compared to those without the expansion, and more frequently colocalize with hnRNP H. Importantly, we demonstrate dysregulated splicing of multiple known hnRNP H-target transcripts in C9 patient brains, which correlates with elevated insoluble hnRNP H/G-Q aggregates. Together, our data implicate C9 expansion-mediated sequestration of hnRNP H as a significant contributor to neurodegeneration in C9 ALS/FTD. DOI: http://dx.doi.org/10.7554/eLife.17820.001

Motor pools comprise a heterogeneous population of motor neurons that innervate distinct intramuscular targets. While the organization of motor neurons into motor pools has been well described, the time course and mechanism of motor pool diversification into functionally distinct classes remains unclear. γ-Motor neurons (γ-MNs) and α-motor neurons (α-MNs) differ in size, molecular identity, synaptic input and peripheral target. While α-MNs innervate extrafusal skeletal muscle fibers to mediate muscle contraction, γ-MNs innervate intrafusal fibers of the muscle spindle, and regulate sensitivity of the muscle spindle in response to stretch. In this study, we find that the secreted signaling molecule Wnt7a is selectively expressed in γ-MNs in the mouse spinal cord by embryonic day 17.5 and continues to molecularly distinguish γ;-from α-MNs into the third postnatal week. Our data demonstrate that Wnt7a is the earliest known γ-MN marker, supporting a model of developmental divergence between α- and γ-MNs at embryonic stages. Furthermore, using Wnt7a expression as an early marker of γ-MN identity, we demonstrate a previously unknown dependence of γ-MNs on a muscle spindle-derived, GDNF-independent signal during the first postnatal week.

The fatal adult motor neuron disease amyotrophic lateral sclerosis (ALS) shares some clinical and pathological overlap with frontotemporal dementia (FTD), an early-onset neurodegenerative disorder. The RNA/DNA-binding proteins fused in sarcoma (FUS; also known as TLS) and TAR DNA binding protein-43 (TDP-43) have recently been shown to be genetically and pathologically associated with familial forms of ALS and FTD. It is currently unknown whether perturbation of these proteins results in disease through mechanisms that are independent of normal protein function or via the pathophysiological disruption of molecular processes in which they are both critical. Here, we report that Drosophila mutants in which the homolog of FUS is disrupted exhibit decreased adult viability, diminished locomotor speed, and reduced life span compared with controls. These phenotypes were fully rescued by wild-type human FUS, but not ALS-associated mutant FUS proteins. A mutant of the Drosophila homolog of TDP-43 had similar, but more severe, deficits. Through cross-rescue analysis, we demonstrated that FUS acted together with and downstream of TDP-43 in a common genetic pathway in neurons. Furthermore, we found that these proteins associated with each other in an RNA-dependent complex. Our results establish that FUS and TDP-43 function together in vivo and suggest that molecular pathways requiring the combined activities of both of these proteins may be disrupted in ALS and FTD.

We investigated factors controlling the development of connections between muscle spindle afferents, spinal motor neurons, and inhibitory Renshaw cells. Several mutants were examined to establish the role of muscle spindles, muscle spindle-derived NT3, and excess NT3 in determining the specificity and strength of these connections. The findings suggest that although spindle-derived factors are not necessary for the initial formation and specificity of the synapses, spindle-derived NT3 seems necessary for strengthening homonymous connections between Ia afferents and motor neurons during the second postnatal week. We also found evidence for functional monosynaptic connections between sensory afferents and neonatal Renshaw cells although the density of these synapses decreases at P15. We conclude that muscle spindle synapses are weakened on Renshaw cells while they are strengthened on motor neurons. Interestingly, the loss of sensory synapses on Renshaw cells was reversed in mice overexpresssing NT3 in the periphery, suggesting that different levels of NT3 are required for functional maintenance and strengthening of spindle afferent inputs on motor neurons and Renshaw cells.

Motor function in mammalian species depends on the maturation of spinal circuits formed by a large variety of interneurons that regulate motoneuron firing and motor output. Interneuron activity is in turn modulated by the organization of their synaptic inputs, but the principles governing the development of specific synaptic architectures unique to each premotor interneuron are unknown. For example, Renshaw cells receive, at least in the neonate, convergent inputs from sensory afferents (likely Ia) and motor axons, raising the question of whether they interact during Renshaw cell development. In other well-studied neurons, such as Purkinje cells, heterosynaptic competition between inputs from different sources shapes synaptic organization. To examine the possibility that sensory afferents modulate synaptic maturation on developing Renshaw cells, we used three animal models in which afferent inputs in the ventral horn are dramatically reduced (ER81(-/-) knockout), weakened (Egr3(-/-) knockout), or strengthened (mlcNT3(+/-) transgenic). We demonstrate that increasing the strength of sensory inputs on Renshaw cells prevents their deselection and reduces motor axon synaptic density, and, in contrast, absent or diminished sensory afferent inputs correlate with increased densities of motor axons synapses. No effects were observed on other glutamatergic inputs. We conclude that the early strength of Ia synapses influences their maintenance or weakening during later development and that heterosynaptic influences from sensory synapses during early development regulates the density and organization of motor inputs on mature Renshaw cells.

The maturation of synaptic structures depends on inductive interactions between axons and their prospective targets. One example of such an interaction is the influence of proprioceptive sensory axons on the differentiation of muscle spindles. We have monitored the expression of three transcription factors, Egr3, Pea3, and Erm, that delineate early muscle spindle development in an assay of muscle spindle-inducing signals. We provide genetic evidence that Neuregulin1 (Nrg1) is required for proprioceptive afferent-evoked induction of muscle spindle differentiation in the mouse. Ig-Nrg1 isoforms are preferentially expressed by proprioceptive sensory neurons and are sufficient to induce muscle spindle differentiation in vivo, whereas CRD-Nrg1 isoforms are broadly expressed in sensory and motor neurons but are not required for muscle spindle induction.

SummaryMutations in the RNA binding protein FUS cause amyotrophic lateral sclerosis (ALS), a fatal adult motor neuron disease. Decreased expression of SMN causes the fatal childhood motor neuron disorder spinal muscular atrophy (SMA). The SMN complex localizes in both the cytoplasm and nuclear Gems, and loss of Gems is a cellular hallmark of fibroblasts in patients with SMA. Here, we report that FUS associates with the SMN complex, mediated by U1 snRNP and by direct interactions between FUS and SMN. Functionally, we show that FUS is required for Gem formation in HeLa cells, and expression of FUS containing a severe ALS-causing mutation (R495X) also results in Gem loss. Strikingly, a reduction in Gems is observed in ALS patient fibroblasts expressing either mutant FUS or TDP-43, another ALS-causing protein that interacts with FUS. The physical and functional interactions among SMN, FUS, TDP-43, and Gems indicate that ALS and SMA share a biochemical pathway, providing strong support for the view that these motor neuron diseases are related.

We investigated factors controlling the development of connections between muscle spindle afferents, spinal motor neurons and inhibitory Renshaw cells. Several mutants were examined to establish the role of muscle spindles, muscle spindle-derived NT3 and excess NT3 in determining the specificity and strength of these connections. The findings suggest that although spindle-derived factors are not necessary for the initial formation and specificity of the synapses, spindle-derived NT3 seems necessary for strengthening homonymous connections between Ia afferents and motor neurons during the second postnatal week. We also found evidence for functional monosynaptic connections between sensory afferents and neonatal Renshaw cells although the density of these synapses decreases at P15. We conclude that muscle spindle synapses are weakened on Renshaw cells while they are strengthened on motor neurons. Interestingly, the loss of sensory synapses on Renshaw cells was reversed in mice over-expresssing NT3 in the periphery, suggesting that different levels of NT3 are required for functional maintenance and strengthening of spindle afferent inputs on motor neurons and Renshaw cells.

Background Gamma motor neurons (γ-MNs) selectively innervate muscle spindle intrafusal fibers and regulate their sensitivity to stretch. They constitute a distinct subpopulation that differs in morphology, physiology and connectivity from α-MNs, which innervate extrafusal muscle fibers and exert force. The mechanisms that control the differentiation of functionally distinct fusimotor neurons are unknown. Progress on this question has been limited by the absence of molecular markers to specifically distinguish and manipulate γ-MNs. Recently, it was reported that early embryonic γ-MN precursors are dependent on GDNF. Using this knowledge we characterized genetic strategies to label developing γ-MNs based on GDNF receptor expression, showed their strict dependence for survival on muscle spindle-derived GDNF and generated an animal model in which γ-MNs are selectively lost. Results In mice heterozygous for both the Hb9::GFP transgene and a tau-lacZ-labeled (TLZ) allele of the GDNF receptor Gfrα1, we demonstrated that small motor neurons with high Gfrα1-TLZ expression and lacking Hb9::GFP display structural and synaptic features of γ-MNs and are selectively lost in mutants lacking target muscle spindles. Loss of muscle spindles also results in the downregulation of Gfrα1 expression in some large diameter MNs, suggesting that spindle-derived factors may also influence populations of α-MNs with β-skeletofusimotor collaterals. These molecular markers can be used to identify γ-MNs from birth to the adult and to distinguish γ- from β-motor axons in the periphery. We also found that postnatal γ-MNs are also distinguished by low expression of the neuronal nuclear protein (NeuN). With these markers of γ-MN identity, we show after conditional elimination of GDNF from muscle spindles that the survival of γ-MNs is selectively dependent on spindle-derived GDNF during the first 2 weeks of postnatal development. Conclusion Neonatal γ-MNs display a unique molecular profile characterized by the differential expression of a series of markers - Gfrα1, Hb9::GFP and NeuN - and the selective dependence on muscle spindle-derived GDNF. Deletion of GDNF expression from muscle spindles results in the selective elimination of γ-MNs with preservation of the spindle and its sensory innervation. This provides a mouse model with which to explore the specific role of γ-fusimotor activity in motor behaviors.

Optical imaging methods rely upon visualization of three types of signals: (1) intrinsic optical signals, including light scattering and reflectance, birefringence, and spectroscopic changes of intrinsic molecules, such as NADH or oxyhemoglobin; (2) changes in fluorescence or absorbance of voltage-sensitive membrane dyes; and (3) changes in fluorescence or absorbance of calcium-sensitive indicator dyes. Of these, the most widely used approach is fluorescent microscopy of calcium-sensitive dyes. This unit describes protocols for the use of calcium-sensitive dyes and voltage-dependent dyes for studies of neuronal activity in culture, tissue slices, and en-bloc preparations of the central nervous system.

The mechanisms controlling the formation of synaptic connections between muscle spindle afferents and spinal motor neurons are believed to be regulated by factors originating from muscle spindles. Here, we find that the connections form with appropriate specificity in mice with abnormal spindle development caused by the conditional elimination of the neuregulin1 receptor ErbB2 from muscle precursors. However, despite a modest (∼30%) decrease in the number of afferent terminals on motor neuron somata, the amplitude of afferent-evoked synaptic potentials recorded in motor neurons was reduced by ∼80%, suggesting that many of the connections that form are functionally silent. The selective elimination of neurotrophin 3 (NT3) from muscle spindles had no effect on the amplitude of afferent-evoked ventral root potentials until the second postnatal week, revealing a late role for spindle-derived NT3 in the functional maintenance of the connections. These findings indicate that spindle-derived factors regulate the strength of the connections but not their initial formation or their specificity.

For studies of motor neuron function or for therapeutic purposes, novel pseudotype HIV-1-based vectors were developed that are capable of expressing transgenes in motor neurons following injection into mouse hind limb muscles. To specifically target motor neurons, glycoproteins from two rabies virus (RV) isolates, the mouse-brain adapted challenge virus 24 (CVS-24) variants, CVS-N2c and CVS-B2c were evaluated for pseudotype formation with an HIV-1-based vector. Both RV glycoproteins incorporated into vector envelopes, and both pseudotypes yielded high titers with Hek293T and cortical plate neuron cultures. Increased neuronotropism by the CVS-N2c pseudotype was not observed, suggesting that vector tropism is not solely determined by the fusogenic viral glycoprotein. Vector injection into hind limb muscles resulted in EYFP reporter gene expression in the injected muscle fibers and in spinal cord motor neurons innervating the same muscle, indicating retrograde vector transport. Intramuscular vector injections into the soleus and tibialis anterior muscles transduced 26% and 16% of all motor neurons in each motor nucleus, respectively. These transduction efficiencies may allow novel approaches to functional studies of the motor system and the treatment of neuromuscular disease.

We have used expression cloning in Xenopus oocytes to isolate two different cDNAs encoding functional N-methyl-D-aspartate (NMDA) receptor subunits. The two receptors (NMDA-R1A and -R1B) display different pharmacologic properties as a consequence of alternative exon addition within the putative ligand-binding domain. The splicing choice is regulated such that R1B is the predominant form of receptor in the cerebellum, whereas R1A predominates in other brain regions. Expression of either of the subunits alone in oocytes results in an NMDA-evoked inward current with electrophysiologic properties closely resembling those of the NMDA receptors observed in neurons. Thus, the complex properties exhibited by the NMDA receptor in neurons can be generated by the expression of a single receptor subunit.