Molecular mechanisms by which mitochondrial dysfunction drives neuromuscular junction degeneration in amyotrophic lateral sclerosis.

Mutations in copper-zinc superoxide dismutase (SOD1) have been linked to a subset of familial amytrophic lateral sclerosis (fALS), a fatal neurodegenerative disease characterized by progressive motor neuron death. An increasing amount of evidence supports that mitochondrial dysfunction and apoptosis activation play a critical role in the fALS etiology, but little is known about the mechanisms by which SOD1 mutants cause the mitochondrial dysfunction and apoptosis. In this study, we use proteomic approaches to identify the mitochondrial proteins that are altered in the presence of a fALS-causing mutant G93A-SOD1. A comprehensive characterization of mitochondrial proteins from NSC34 cells, a motor neuron-like cell line, was achieved by two independent proteomic approaches. Four hundred seventy unique proteins were identified in the mitochondrial fraction collectively, 75 of which are newly discovered proteins that previously had only been reported at the cDNA level. Two-dimensional gel electrophoresis was subsequently used to analyze the differences between the mitochondrial proteomes of NSC34 cells expressing wild-type and G93A-SOD1. Nine and 36 protein spots displayed elevated and suppressed abundance respectively in G93A-SOD1-expressing cells. The 45 spots were identified by MS, and they include proteins involved in mitochondrial membrane transport, apoptosis, the respiratory chain, and molecular chaperones. In particular, alterations in the post-translational modifications of voltage-dependent anion channel 2 (VDAC2) were found, and its relevance to regulating mitochondrial membrane permeability and activation of apoptotic pathways is discussed. The potential role of other proteins in the mutant SOD1-mediated fALS is also discussed. This study has produced a short list of mitochondrial proteins that may hold the key to the mechanisms by which SOD1 mutants cause mitochondrial dysfunction and neuronal death. It has laid the foundation for further detailed functional studies to elucidate the role of particular mitochondrial proteins, such as VDAC2, in the pathogenesis of familial ALS.

Amyotrophic lateral sclerosis (ALS) is a neuromuscular disease characterized by specific loss of motor neurons in the spinal cord and brain stem. Although ALS has historically been characterized as a motor neuron disease, there is evidence that motor neurons degenerate in a retrograde manner, beginning in the periphery at the neuromuscular junctions (NMJs) and skeletal muscle. We recently reported a vesicle trafficking protein Bet1L (Bet1 Golgi Vesicular Membrane Trafficking Protein Like) as a new molecule possibly linked to NMJ degeneration in ALS. In this study, we tested the hypothesis that Bet1L gene silencing in skeletal muscle could influence NMJ integrity, motor neuron function, and survival in a rat model of familial ALS (SOD1G93A transgenic). Small interfering RNA (siRNA) targeting the Bet1L gene was injected on a weekly basis into the hindlimb muscle of pre-symptomatic ALS and wild-type (WT) rats. After 3 weeks, intramuscular Bet1L siRNA injection significantly increased the number of denervated NMJs in the injected muscle. Bet1L knockdown decreased motor neuron size in the lumbar spinal cord, which innervated the siRNA-injected hindlimb. Impaired motor function was identified in the hindlimbs of Bet1L siRNA-injected rats. Notably, the effects of Bet1L knockdown on NMJ and motor neuron degeneration were more significant in ALS rats when compared to WT rats. Together, Bet1L knockdown induces denervation of NMJs, but also this knockdown accelerates the disease progression in ALS. Our results provide new evidence to support the potential roles of Bet1L as a key molecule in NMJ maintenance and ALS pathogenesis.

Calcium, mitochondria, and the pathogenesis of ALS: the good, the bad, and the ugly

Glial-Derived Prodegenerative Signaling in the Drosophila Neuromuscular System

Mitochondrial dysfunction in amyotrophic lateral sclerosis

Muscling In on PGC-1α for Improved Quality of Life in ALS

Neuroprotective effects of the mitochondria-targeted antioxidant MitoQ in a model of inherited amyotrophic lateral sclerosis

Axon degeneration and Neuromuscular Junction (NMJ) disruption are key pathologies in the fatal neurodegenerative disease Amyotrophic Lateral Sclerosis (ALS). Despite accumulating evidence that axons and NMJs are impacted at a very early stage of the disease, current knowledge about the mechanisms leading to their degeneration remains elusive. Cytoplasmic mislocalization and accumulation of the protein TDP-43 are considered key pathological hallmarks of ALS, as they occur in ~ 97% of ALS patients, both sporadic and familial. Recent studies have identified pathological accumulation of TDP-43 in intramuscular nerves of muscle biopsies collected from pre-diagnosed, early symptomatic ALS patients. These findings suggest a gain of function for TDP-43 in axons, which might facilitate early NMJ disruption. In this review, we dissect the process leading to axonal TDP-43 accumulation and phosphorylation, discuss the known and hypothesized roles TDP-43 plays in healthy axons, and review possible mechanisms that connect TDP-43 pathology to the axon and NMJ degeneration in ALS.

Amyotrophic lateral sclerosis (ALS) has been traditionally regarded as a progressive neurodegenerative disorder of the motor system. Clinically, ALS is characterised by a combination upper motor neuron [UMN] (brisk deep tendon reflexes, spasticity and extensor plantar response) and lower motor neuron [LMN] signs (fasciculations, muscle wasting and weakness), which form the basis of the ALS diagnostic criteria. This article is protected by copyright. All rights reserved.

Excitation BolsTORs Motor Neurons in ALS Mice

Complex Genetics of Amyotrophic Lateral Sclerosis

The etiological underpinnings of amyotrophic lateral sclerosis (ALS) are complex and incompletely understood, although contributions to pathogenesis by regulators of proteolytic pathways have become increasingly apparent. Here, we present a novel variant in UBQLN4 that is associated with ALS and show that its expression compromises motor axon morphogenesis in mouse motor neurons and in zebrafish. We further demonstrate that the ALS-associated UBQLN4 variant impairs proteasomal function, and identify the Wnt signaling pathway effector beta-catenin as a UBQLN4 substrate. Inhibition of beta-catenin function rescues the UBQLN4 variant-induced motor axon phenotypes. These findings provide a strong link between the regulation of axonal morphogenesis and a new ALS-associated gene variant mediated by protein degradation pathways.DOI: http://dx.doi.org/10.7554/eLife.25453.001

Transcriptome analysis using patient iPSC-derived skeletal myocytes: Bet1L as a new molecule possibly linked to neuromuscular junction degeneration in ALS

<h3>Objective:</h3> To investigate the role of astrocytes in apolipoprotein B-100 (ApoB)-mediated motor neuron degeneration in sporadic amyotrophic lateral sclerosis (ALS). <h3>Background:</h3> ALS is a devastating neurodegenerative disease which results in motor neuron degeneration in the brain and spinal cord. Our previous research identified ApoB as a neurotoxic factor as it is upregulated in the cerebrospinal fluid (CSF) of ALS patients and can induce motor disability and motor neuron degeneration <i>in vivo</i> in mice, as well as death of human motor neurons <i>in vitro</i>. Here, we investigate whether astrocytes play a role in exacerbating or protecting against ApoB-induced motor neuron death. <h3>Design/Methods:</h3> Primary human astrocytes were plated on matrigel/DMEM-F12-coated chamber slides and grown in astrocyte growth media (AGM) overnight. The next day, the AGM was replaced with motor neuron maintenance media (MNMM) and iPSC-derived human motor neurons were plated directly on top of the astrocytes or on coated slides without astrocytes. On day 9, cells were either treated with 1 ng/μL ApoB diluted in MNMM or 50% ALS CSF/MNMM. Twenty-four hours later cells were fixed in 4% PFA for ChAT and ApoB immunocytochemistry. <h3>Results:</h3> ALS CSF and ApoB induce human motor neuron degeneration, as indicated by significantly smaller ChAT⁺ clusters compared to the media cultures. In contrast, ALS CSF and ApoB have no effect on the size of ChAT⁺ motor neuron clusters co-cultured with human astrocytes. ApoB staining intensity on ApoB-treated motor neurons is significantly reduced in the co-cultures vs. monocultures. <h3>Conclusions:</h3> Healthy astrocytes appear to protect motor neurons from the neurotoxic effects of ApoB by reducing uptake of ApoB into motor neurons. This suggests that intrinsic defects in ALS astrocytes disrupt this neuroprotective function, leaving motor neurons susceptible to ApoB-mediated degeneration. Future experiments will seek to elucidate how astrocyte dysfunction contributes to motor neuron degeneration in ALS. <b>Disclosure:</b> Ms. Griffin has nothing to disclose. Miss Gao has nothing to disclose. Dr. Wong has nothing to disclose. Dr. Sadiq has nothing to disclose.

Suppressed Autophagy Flux in Skeletal Muscle of an Amyotrophic Lateral Sclerosis Mouse Model