Reversal of cognitive deficits in FUSR521G amyotrophic lateral sclerosis mice by arimoclomol and a class I histone deacetylase inhibitor independent of heat shock protein induction.

Protein misfolding and mislocalization are common to both familial and sporadic forms of amyotrophic lateral sclerosis (ALS). Maintaining proteostasis through induction of heat shock proteins (HSP) to increase chaperoning capacity is a rational therapeutic strategy in the treatment of ALS. However, the threshold for upregulating stress-inducible HSPs remains high in neurons, presenting a therapeutic obstacle. This study used mouse models expressing the ALS variants FUSR521G or SOD1G93A to follow up on previous work in cultured motor neurons showing varied effects of the HSP co-inducer, arimoclomol, and class I histone deacetylase (HDAC) inhibitors on HSP expression depending on the ALS variant being expressed. As in cultured neurons, neither expression of the transgene nor drug treatments induced expression of HSPs in cortex, spinal cord or muscle of FUSR521G mice, indicating suppression of the heat shock response. Nonetheless, arimoclomol, and RGFP963, restored performance on cognitive tests and improved cortical dendritic spine densities. In SOD1G93A mice, multiple HSPs were upregulated in hindlimb skeletal muscle, but not in lumbar spinal cord with the exception of HSPB1 associated with astrocytosis. Drug treatments improved contractile force but reduced the increase in HSPs in muscle rather than facilitating their expression. The data point to mechanisms other than amplification of the heat shock response underlying recovery of cognitive function in ALS-FUS mice by arimoclomol and class I HDAC inhibition and suggest potential benefits in counteracting cognitive impairment in ALS, frontotemporal dementia and related disorders.

Perisynaptic Schwann Cells: Guardians of Neuromuscular Junction Integrity and Function in Health and Disease.

The neuromuscular junction (NMJ) is a highly reliable synapse to carry the control of the motor commands of the nervous system over the muscles. Its development, organization, and synaptic properties are highly structured and regulated to support such reliability and efficacy. Yet, the NMJ is also highly plastic, able to react to injury, and able to adapt to changes. This balance between structural stability and synaptic efficacy on one hand and structural plasticity and repair on another hand is made possible by perisynaptic Schwann cells (PSCs), glial cells at this synapse. They regulate synaptic efficacy and structural plasticity of the NMJ in a dynamic, bidirectional manner owing to their ability to decode synaptic transmission and by their interactions with trophic-related factors. Alteration of these fundamental roles of PSCs is also important in the maladapted response of NMJs in various diseases and in aging.

Impact of histone deacetylase inhibition and arimoclomol on heat shock protein expression and disease biomarkers in primary culture models of familial ALS.

Protein misfolding and mislocalization are common themes in neurodegenerative disorders, including motor neuron disease, and amyotrophic lateral sclerosis (ALS). Maintaining proteostasis is a crosscutting therapeutic target, including the upregulation of heat shock proteins (HSP) to increase chaperoning capacity. Motor neurons have a high threshold for upregulating stress-inducible HSPA1A, but constitutively express high levels of HSPA8. This study compared the expression of these HSPs in cultured motor neurons expressing three variants linked to familial ALS: TAR DNA binding protein 43 kDa (TDP-43)G348C, fused in sarcoma (FUS)R521G, or superoxide dismutase I (SOD1)G93A. All variants were poor inducers of Hspa1a, and reduced levels of Hspa8 mRNA and protein, indicating multiple compromises in chaperoning capacity. To promote HSP expression, cultures were treated with the putative HSP coinducer, arimoclomol, and class I histone deacetylase inhibitors, to promote active chromatin for transcription, and with the combination. Treatments had variable, often different effects on the expression of Hspa1a and Hspa8, depending on the ALS variant expressed, mRNA distribution (somata and dendrites), and biomarker of toxicity measured (histone acetylation, maintaining nuclear TDP-43 and the neuronal Brm/Brg-associated factor chromatin remodeling complex component Brg1, mitochondrial transport, FUS aggregation). Overall, histone deacetylase inhibition alone was effective on more measures than arimoclomol. As in the FUS model, arimoclomol failed to induce HSPA1A or preserve Hspa8 mRNA in the TDP-43 model, despite preserving nuclear TDP-43 and Brg1, indicating neuroprotective properties other than HSP induction. The data speak to the complexity of drug mechanisms against multiple biomarkers of ALS pathogenesis, as well as to the importance of HSPA8 for neuronal proteostasis in both somata and dendrites.

mTORC1-mediated acquisition of reward-related representations by hippocampal somatostatin interneurons.

Plasticity of principal cells and inhibitory interneurons underlies hippocampal memory. Bidirectional modulation of somatostatin cell mTORC1 activity, a crucial translational control mechanism in synaptic plasticity, causes parallel changes in hippocampal CA1 somatostatin interneuron (SOM-IN) long-term potentiation and hippocampus-dependent memory, indicating a key role in learning. However, SOM-IN activity changes and behavioral correlates during learning, and the role of mTORC1 in these processes, remain ill-defined. To address these questions, we used two-photon Ca2+ imaging from SOM-INs during a virtual reality goal-directed spatial memory task in head-fixed control mice (SOM-IRES-Cre mice) or in mice with conditional knockout of Rptor (SOM-Rptor-KO mice) to block mTORC1 activity in SOM-INs. We found that control mice learn the task, but SOM-Raptor-KO mice exhibit a deficit. Also, SOM-IN Ca2+ activity became increasingly related to reward during learning in control mice but not in SOM-Rptor-KO mice. Four types of SOM-IN activity patterns related to reward location were observed, "reward off sustained", "reward off transient", "reward on sustained" and "reward on transient", and these responses showed reorganization after reward relocation in control but not SOM-Rptor-KO mice. Thus, SOM-INs develop mTORC1-dependent reward- related activity during learning. This coding may bi-directionally interact with pyramidal cells and other structures to represent and consolidate reward location.

Skeletal muscle in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS), the major adult-onset motor neuron disease, has been viewed almost exclusively as a disease of upper and lower motor neurons, with muscle changes interpreted as a consequence of the progressive loss of motor neurons and neuromuscular junctions. This has led to the prevailing view that the involvement of muscle in ALS is only secondary to motor neuron loss. Skeletal muscle and motor neurons reciprocally influence their respective development and constitute a single functional unit. In ALS, multiple studies indicate that skeletal muscle dysfunction might contribute to progressive muscle weakness, as well as to the final demise of neuromuscular junctions and motor neurons. Furthermore, skeletal muscle has been shown to participate in disease pathogenesis of several monogenic diseases closely related to ALS. Here, we move the narrative towards a better appreciation of muscle as a contributor of disease in ALS. We review the various potential roles of skeletal muscle cells in ALS, from passive bystanders to active players in ALS pathophysiology. We also compare ALS to other motor neuron diseases and draw perspectives for future research and treatment.

Functional adaptation of glial cells at neuromuscular junctions in response to injury.

Synaptic elements from neuromuscular junctions (NMJs) undergo massive morphological and functional changes upon nerve injury. While morphological changes of NMJ-associated glia in response to injury has been investigated, their functional properties remain elusive. Perisynaptic Schwann cells (PSCs), glial cells at the NMJ, are essential for NMJ maintenance and repair, and are involved in synaptic efficacy and plasticity. Importantly, these functions are regulated by PSCs ability to detect synaptic transmission through, notably, muscarinic (mAChRs) and purinergic receptors' activation. Using Ca2+ imaging and electrophysiological recordings of synaptic transmission at the mouse NMJ, we investigated PSC receptors activation following denervation and during reinnervation in adults and at denervated NMJs in an ALS mouse model (SOD1G37R ). We observed reduced PSCs mAChR-mediated Ca2+ responses at denervated and reinnervating NMJs. Importantly, PSC phenotypes during denervation and reinnervation were distinct than the one observed during NMJ maturation. At denervated NMJs, exogenous activation of mAChRs greatly diminished galectin-3 expression, a glial marker of phagocytosis. PSCs Ca2+ responses at reinnervating NMJs did not correlate with the number of innervating axons or process extensions. Interestingly, we observed an extended period of reduced PSC mAChRs activation after the injury (up to 60 days), suggesting a glial memory of injury. PSCs associated with denervated NMJs in an ALS model (SOD1G37R mice) did not show any muscarinic adaptation, a phenotype incompatible with NMJ repair. Understanding functional mechanisms that underlie this glial response to injury may contribute to favor complete NMJ and motor recovery.

Expression and Roles of Lynx1, a Modulator of Cholinergic Transmission, in Skeletal Muscles and Neuromuscular Junctions in Mice.

Lynx1 is a glycosylphosphatidylinositol (GPI)-linked protein shown to affect synaptic plasticity through modulation of nicotinic acetylcholine receptor (nAChR) subtypes in the brain. Because of this function and structural similarity to α-bungarotoxin, which binds muscle-specific nAChRs with high affinity, Lynx1 is a promising candidate for modulating nAChRs in skeletal muscles. However, little is known about the expression and roles of Lynx1 in skeletal muscles and neuromuscular junctions (NMJs). Here, we show that Lynx1 is expressed in skeletal muscles, increases during development, and concentrates at NMJs. We also demonstrate that Lynx1 interacts with muscle-specific nAChR subunits. Additionally, we present data indicating that Lynx1 deletion alters the response of skeletal muscles to cholinergic transmission and their contractile properties. Based on these findings, we asked if Lynx1 deletion affects developing and adult NMJs. Loss of Lynx1 had no effect on NMJs at postnatal day 9 (P9) and moderately increased their size at P21. Thus, Lynx1 plays a minor role in the structural development of NMJs. In 7- and 12-month-old mice lacking Lynx1, there is a marked increase in the incidence of NMJs with age- and disease-associated morphological alterations. The loss of Lynx1 also reduced the size of adult muscle fibers. Despite these effects, Lynx1 deletion did not alter the rate of NMJ reinnervation and stability following motor axon injury. These findings suggest that Lynx1 is not required during fast remodeling of the NMJ, as is the case during reformation following crushing of motor axons and development. Instead, these data indicate that the primary role of Lynx1 may be to maintain the structure and function of adult and aging NMJs.

hnRNP A1B, a Splice Variant of HNRNPA1, Is Spatially and Temporally Regulated.

RNA binding proteins (RBPs) play a key role in cellular growth, homoeostasis and survival and are tightly regulated. A deep understanding of their spatiotemporal regulation is needed to understand their contribution to physiology and pathology. Here, we have characterized the spatiotemporal expression pattern of hnRNP A1 and its splice variant hnRNP A1B in mice. We have found that hnRNP A1B expression is more restricted to the CNS compared to hnRNP A1, and that it can form an SDS-resistant dimer in the CNS. Also, hnRNP A1B expression becomes progressively restricted to motor neurons in the ventral horn of the spinal cord, compared to hnRNP A1 which is more broadly expressed. We also demonstrate that hnRNP A1B is present in neuronal processes, while hnRNP A1 is absent. This finding supports a hypothesis that hnRNP A1B may have a cytosolic function in neurons that is not shared with hnRNP A1. Our results demonstrate that both isoforms are differentially expressed across tissues and have distinct localization profiles, suggesting that the two isoforms may have specific subcellular functions that can uniquely contribute to disease progression.

Properties of Glial Cell at the Neuromuscular Junction Are Incompatible with Synaptic Repair in the SOD1G37R ALS Mouse Model.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motoneurons (MNs) in a motor-unit (MU)-dependent manner. Glial dysfunction contributes to numerous aspects of the disease. At the neuromuscular junction (NMJ), early alterations in perisynaptic Schwann cell (PSC), glial cells at this synapse, may impact their ability to regulate NMJ stability and repair. Indeed, muscarinic receptors (mAChRs) regulate the repair phenotype of PSCs and are overactivated at disease-resistant NMJs [soleus muscle (SOL)] in SOD1G37R mice. However, it remains unknown whether this is the case at disease-vulnerable NMJs and whether it translates into an impairment of PSC-dependent repair mechanisms. We used SOL and sternomastoid (STM) muscles from SOD1G37R mice and performed Ca2+-imaging to monitor PSC activity and used immunohistochemistry to analyze their repair and phagocytic properties. We show that PSC mAChR-dependent activity was transiently increased at disease-vulnerable NMJs (STM muscle). Furthermore, PSCs from both muscles extended disorganized processes from denervated NMJs and failed to initiate or guide nerve terminal sprouts at disease-vulnerable NMJs, a phenomenon essential for compensatory reinnervation. This was accompanied by a failure of numerous PSCs to upregulate galectin-3 (MAC-2), a marker of glial axonal debris phagocytosis, on NMJ denervation in SOD1 mice. Finally, differences in these PSC-dependent NMJ repair mechanisms were MU type dependent, thus reflecting MU vulnerability in ALS. Together, these results reveal that neuron-glia communication is ubiquitously altered at the NMJ in ALS. This appears to prevent PSCs from adopting a repair phenotype, resulting in a maladapted response to denervation at the NMJ in ALS.SIGNIFICANCE STATEMENT Understanding how the complex interplay between neurons and glial cells ultimately lead to the degeneration of motor neurons and loss of motor function is a fundamental question to comprehend amyotrophic lateral sclerosis (ALS). An early and persistent alteration of glial cell activity takes place at the neuromuscular junction (NMJ), the output of motor neurons, but its impact on NMJ repair remains unknown. Here, we reveal that glial cells at disease-vulnerable NMJs often fail to guide compensatory nerve terminal sprouts and to adopt a phagocytic phenotype on denervated NMJs in SOD1G37R mice. These results show that glial cells at the NMJ elaborate an inappropriate response to NMJ degeneration in a manner that reflects motor-unit (MU) vulnerability and potentially impairs compensatory reinnervation.

Sex-Specific Differences in Motor-Unit Remodeling in a Mouse Model of ALS.

Progressive loss of neuromuscular junctions (NMJs) is an early event in amyotrophic lateral sclerosis (ALS), preceding the global degeneration of motor axons and being accompanied by new axonal sprouting within the same axonal arbor. Some aspects of ALS onset and progression seem to be affected by sex in animal models of the disease. However, whether there are sex-specific differences in the pattern or time course of NMJ loss and repair within single motor axons remains unknown. We performed further analysis of a previously published in vivo dataset, obtained from male and female SOD1G37R mice. We found that NMJ losses are as frequent in male and female motor axons but, intriguingly, axonal sprouting is more frequent in female than male mice, resulting in a net increase of axonal arborization. Interestingly, these numerous new axonal branches in female mice are associated with a slightly faster decline in grip strength, increased NMJ denervation, and reduced α-motor neuron survival. Collectively, these results suggest that excessive axonal sprouting and motor-unit (MU) expansion in female SOD1G37R mice are maladaptive during ALS progression.

Improved Human Muscle Biopsy Method To Study Neuromuscular Junction Structure and Functions with Aging.

Reduced mobility and physical independence of elders has emerged as a major clinical and public health priority with extended life expectancy. The impact of the neuromuscular function on muscle activity and properties has emerged as a critical factor influencing the progress and outcome of muscle changes with aging. However, very little is known about the neuromuscular junctions (NMJs) in humans, in part due to technical constraints limiting the access to healthy, fresh neuromuscular tissue. Here, we describe a method, called Biopsy using Electrostimulation for Enhanced NMJ Sampling (BeeNMJs) that improves the outcome of muscle biopsies. We used local cutaneous stimulation to identify the area enriched with NMJs for each participant at the right Vastus lateralis (VL). The needle biopsy was then performed in proximity of that point. The BeeNMJs procedure was safe for the participants. We observed NMJs in 53.3% of biopsies in comparison with only 16.7% using the traditional method. Furthermore, we observed an average of 30.13 NMJs per sample compared to only 2.33 for the traditional method. Importantly, high-quality neuromuscular material was obtained whereby pre-, postsynaptic, and glial elements were routinely labeled, simultaneously with myosin heavy chain type I. The BeeNMJs approach will facilitate studies of NMJs, particularly in human disease or aging process.

The Novel Small Molecule TRVA242 Stabilizes Neuromuscular Junction Defects in Multiple Animal Models of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disorder in which the neuromuscular junction progressively degenerates, leading to movement difficulties, paralysis, and eventually death. ALS is currently being treated by only two FDA-approved drugs with modest efficacy in slowing disease progression. Often, the translation of preclinical findings to bedside terminates prematurely as the evaluation of potential therapeutic compounds focuses on a single study or a single animal model. To circumscribe these issues, we screened 3,765 novel small molecule derivatives of pimozide, a recently identified repurposed neuroleptic for ALS, in Caenorhabditis elegans, confirmed the hits in zebrafish and validated the most active compounds in mouse genetic models. Out of the 27 small molecules identified from the high-throughput screen in worms, 4 were found to recover locomotor defects in C. elegans and genetic zebrafish models of ALS. TRVA242 was identified as the most potent compound as it significantly improved efficiency in rescuing locomotor, motorneuron, and neuromuscular junction synaptic deficits in a C. elegans TDP-43 model and in multiple zebrafish genetic (TDP-43, SOD1, and C9ORF72) models of ALS. The actions of TRVA242 were also conserved in a mammalian model as it also stabilized neuromuscular junction deficits in a mouse SOD1 model of ALS. Compounds such as TRVA242 therefore represent new potential therapeutics for the treatment of ALS.

Purinergic-Dependent Glial Regulation of Synaptic Plasticity of Competing Terminals and Synapse Elimination at the Neuromuscular Junction.

The precise wiring of synaptic connections requires the elimination of supernumerary inputs competing for innervation of the same target cell. This competition is activity-dependent, strengthening some inputs whereas others are eliminated. Although glial cells are required for the elimination and clearance of terminals, their involvement in activity-dependent synaptic competition remains ill-defined. Here, we used the developing neuromuscular junctions of mice to show that perisynaptic glial cells, through 2Y1 purinergic receptors (P2Y1Rs), decode synaptic efficacy of competing terminals in a Ca2+-dependent manner. This glial activity induces long-lasting synaptic potentiation of strong but not weak terminals via presynaptic adenosine 2A receptors. Blockade of glial activity by intracellular Ca2+ chelation or blockade of P2Y1Rs prevents this plasticity. In addition, blockade of P2Y1Rs delays synapse elimination in vivo. Hence, P2Y1Rs drive glial cell regulation of strong synaptic inputs and influence synapse competition and elimination.

GABAergic modulation of olfactomotor transmission in lampreys.

Odor-guided behaviors, including homing, predator avoidance, or food and mate searching, are ubiquitous in animals. It is only recently that the neural substrate underlying olfactomotor behaviors in vertebrates was uncovered in lampreys. It consists of a neural pathway extending from the medial part of the olfactory bulb (medOB) to locomotor control centers in the brainstem via a single relay in the caudal diencephalon. This hardwired olfactomotor pathway is present throughout life and may be responsible for the olfactory-induced motor behaviors seen at all life stages. We investigated modulatory mechanisms acting on this pathway by conducting anatomical (tract tracing and immunohistochemistry) and physiological (intracellular recordings and calcium imaging) experiments on lamprey brain preparations. We show that the GABAergic circuitry of the olfactory bulb (OB) acts as a gatekeeper of this hardwired sensorimotor pathway. We also demonstrate the presence of a novel olfactomotor pathway that originates in the non-medOB and consists of a projection to the lateral pallium (LPal) that, in turn, projects to the caudal diencephalon and to the mesencephalic locomotor region (MLR). Our results indicate that olfactory inputs can induce behavioral responses by activating brain locomotor centers via two distinct pathways that are strongly modulated by GABA in the OB. The existence of segregated olfactory subsystems in lampreys suggests that the organization of the olfactory system in functional clusters may be a common ancestral trait of vertebrates.

Dynamic neuromuscular remodeling precedes motor-unit loss in a mouse model of ALS.

Despite being an early event in ALS, it remains unclear whether the denervation of neuromuscular junctions (NMJ) is simply the first manifestation of a globally degenerating motor neuron. Using in vivo imaging of single axons and their NMJs over a three-month period, we identify that single motor-units are dismantled asynchronously in SOD1G37R mice. We reveal that weeks prior to complete axonal degeneration, the dismantling of axonal branches is accompanied by contemporaneous new axonal sprouting resulting in synapse formation onto nearby NMJs. Denervation events tend to propagate from the first lost NMJ, consistent with a contribution of neuromuscular factors extrinsic to motor neurons, with distal branches being more susceptible. These results show that NMJ denervation in ALS is a complex and dynamic process of continuous denervation and new innervation rather than a manifestation of sudden global motor neuron degeneration.

Astrocytes detect and upregulate transmission at inhibitory synapses of somatostatin interneurons onto pyramidal cells.

Astrocytes are important regulators of excitatory synaptic networks. However, astrocytes regulation of inhibitory synaptic systems remains ill defined. This is particularly relevant since GABAergic interneurons regulate the activity of excitatory cells and shape network function. To address this issue, we combined optogenetics and pharmacological approaches, two-photon confocal imaging and whole-cell recordings to specifically activate hippocampal somatostatin or paravalbumin-expressing interneurons (SOM-INs or PV-INs), while monitoring inhibitory synaptic currents in pyramidal cells and Ca2+ responses in astrocytes. We found that astrocytes detect SOM-IN synaptic activity via GABABR and GAT-3-dependent Ca2+ signaling mechanisms, the latter triggering the release of ATP. In turn, ATP is converted into adenosine, activating A1Rs and upregulating SOM-IN synaptic inhibition of pyramidal cells, but not PV-IN inhibition. Our findings uncover functional interactions between a specific subpopulation of interneurons, astrocytes and pyramidal cells, involved in positive feedback autoregulation of dendritic inhibition of pyramidal cells.

Neuroleptics as therapeutic compounds stabilizing neuromuscular transmission in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressing, fatal disorder with no effective treatment. We used simple genetic models of ALS to screen phenotypically for potential therapeutic compounds. We screened libraries of compounds in C. elegans, validated hits in zebrafish, and tested the most potent molecule in mice and in a small clinical trial. We identified a class of neuroleptics that restored motility in C. elegans and in zebrafish, and the most potent was pimozide, which blocked T-type Ca2+ channels in these simple models and stabilized neuromuscular transmission in zebrafish and enhanced it in mice. Finally, a short randomized controlled trial of sporadic ALS subjects demonstrated stabilization of motility and evidence of target engagement at the neuromuscular junction. Simple genetic models are, thus, useful in identifying promising compounds for the treatment of ALS, such as neuroleptics, which may stabilize neuromuscular transmission and prolong survival in this disease.

A Novel Egr-1-Agrin Pathway and Potential Implications for Regulation of Synaptic Physiology and Homeostasis at the Neuromuscular Junction.

Synaptic transmission requires intricate coordination of the components involved in processing of incoming signals, formation and stabilization of synaptic machinery, neurotransmission and in all related signaling pathways. Changes to any of these components cause synaptic imbalance and disruption of neuronal circuitry. Extensive studies at the neuromuscular junction (NMJ) have greatly aided in the current understanding of synapses and served to elucidate the underlying physiology as well as associated adaptive and homeostatic processes. The heparan sulfate proteoglycan agrin is a vital component of the NMJ, mediating synaptic formation and maintenance in both brain and muscle, but very little is known about direct control of its expression. Here, we investigated the relationship between agrin and transcription factor early growth response-1 (Egr-1), as Egr-1 regulates the expression of many genes involved in synaptic homeostasis and plasticity. Using chromatin immunoprecipitation (ChIP), cell culture with cell lines derived from brain and muscle, and animal models, we show that Egr-1 binds to the AGRN gene locus and suppresses its expression. When compared with wild type (WT), mice deficient in Egr-1 (Egr-1-/-) display a marked increase in AGRN mRNA and agrin full-length and cleavage fragment protein levels, including the 22 kDa, C-terminal fragment in brain and muscle tissue homogenate. Because agrin is a crucial component of the NMJ, we explored possible physiological implications of the Egr-1-agrin relationship. In the diaphragm, Egr-1-/- mice display increased NMJ motor endplate density, individual area and area of innervation. In addition to increased density, soleus NMJs also display an increase in fragmented and faint endplates in Egr-1-/- vs. WT mice. Moreover, the soleus NMJ electrophysiology of Egr-1-/- mice revealed increased quantal content and motor testing showed decreased movement and limb muscle strength compared with WT. This study provides evidence for the potential involvement of a novel Egr-1-agrin pathway in synaptic homeostatic and compensatory mechanisms at the NMJ. Synaptic homeostasis is greatly affected by the process of aging. These and other data suggest that changes in Egr-1 expression may directly or indirectly promote age-related pathologies.

Opposite Synaptic Alterations at the Neuromuscular Junction in an ALS Mouse Model: When Motor Units Matter.

Denervation of the neuromuscular junction (NMJ) precedes the loss of motor neurons (MNs) in amyotrophic lateral sclerosis (ALS). ALS is characterized by a motor unit (MU)-dependent vulnerability where MNs with fast-fatigable (FF) characteristics are lost first, followed by fast fatigue-resistant (FR) and slow (S) MNs. However, changes in NMJ properties as a function of MU types remain debated. We hypothesized that NMJ synaptic functions would be altered precociously in an MU-specific manner, before structural alterations of the NMJ. Synaptic transmission and morphological changes of NMJs have been explored in two nerve-muscle preparations of male SOD1G37R mice and their wild-type (WT) littermates: the soleus (S and FR MU); and the extensor digitorum longus (FF MU). S, FR, and FF NMJs of WT mice showed distinct synaptic properties from which we build an MU synaptic profile (MUSP) that reports MU-dependent NMJ synaptic properties. At postnatal day 180 (P180), FF and S NMJs of SOD1 already showed, respectively, lower and higher quantal content compared with WT mice, before signs of MN death and before NMJ morphological alterations. Changes persisted in both muscles until preonset (P380), while denervation was frequent in the mutant mouse. MN death was evident at this stage. Additional changes occurred at clinical disease onset (P450) for S and FR MU. As a whole, our results reveal a reversed MUSP in SOD1 mutants and highlight MU-specific synaptic changes occurring in a precise temporal sequence. Importantly, changes in synaptic properties appear to be good predictors of vulnerability to neurodegeneration.SIGNIFICANCE STATEMENT The inadequate excitability of motor neurons and their output, the neuromuscular junctions (NMJs), has been considered a key factor in the detrimental outcome of the motor function in amyotrophic lateral sclerosis. However, a conundrum persists at the NMJ whereby persistent but incoherent opposite neurotransmission changes have been reported to take place. This article untangles this conundrum by systematically analyzing the changes in synaptic properties over the course of the disease progression as a function of the motor unit type. This temporal analysis reveals that early synaptic alterations evolve with disease progression but precede NMJ neurodegeneration. These data provide a novel framework of analysis and comparison of synaptic transmission alterations in neurodegenerative disorders.