Diagnostic Challenge and Neuromuscular Junction Contribution to ALS Pathogenesis

International audienceAmyotrophic lateral sclerosis (ALS) represents the major adult-onset motor neuron disease. Both human and animal studies reveal the critical implication of muscle and neuromuscular junctions (NMJs) in the initial phase of this disease. Despite the common efforts, ALS diagnosis remains particularly challenging since many other disorders can overlap yielding similar clinical phenotypic features. A combination of further research on the NMJ parameters that are specific for this disease and laboratory tests are crucial for the early determination of specific changes in the muscle, as well as in motor neuron and the prediction of ALS progression. Also, it could provide a powerful tool in the discrimination of particular ALS and ALS-mimic cases and increase the efficacy of therapeutic treatments

TDP-43 Regulation of AChE Expression Can Mediate ALS-Like Phenotype in Zebrafish

International audienceThe “distal axonopathy” hypothesis in amyotrophic lateral sclerosis (ALS) proposes that pathological changes occur at the neuromuscular junction (NMJ) early in the disease. While acetylcholinesterase (AChE) plays an important role in the functionality of the NMJ, its potential role in ALS remains unexplored. Here, we identified AChE as a limiting factor regulating muscle/motor neuron connection in a vertebrate model of ALS. Knockdown of the TAR DNA-binding protein 43 (TDP-43) orthologue in zebrafish resulted in early defects of motor functions coupled with NMJ disassembly. We found that a partially depleted tdp-43 caused a decrease of ache expression. Importantly, human AChE overexpression reduced the phenotypic defects in the tdp-43 loss of function model, with amelioration of post- and pre-synaptic deficits at the NMJ. In conclusion, our results provide a better understanding of the role of TDP-43 in the NMJ organization and indicate AChE as a contributing factor in the pathology of ALS. In particular, it may be implicated in the early defects that characterize NMJs in this major neurodegenerative disorder. View Full-Tex

Freezing activity brief data from a new FUS mutant zebrafish line

International audienceThe data presented in this paper are related to the research article "Functional characterization of a FUS mutant zebrafish line as a novel genetic model for ALS". In this model the lack of fus causes reduced lifespan as well as impaired motor abilities associated with a decrease of motor neurons axons lenght and an increase of neuromuscular junctions fragmentation. Data in this article describes the global locomotor activity data at 3, 4 and 5 days post fertilization in WT, fus heterozygous (fus+/-) and fus homozygous (fus-/-) zebrafish embryos as a response to visual light stimulation, with particular attention on the freezing respose

Journal Pre-proof Functional characterization of a FUS mutant zebrafish line as a novel genetic model for ALS

International audienceMutations in Fused in sarcoma (FUS), an RNA-binding protein, are known to cause Amyotrophic Lateral Sclerosis (ALS). However, molecular mechanisms due to loss of FUS function remain unclear and controversial. Here, we report the characterization and phenotypic analysis of a deletion mutant of the unique FUS orthologue in zebrafish where Fus protein levels are depleted. The homozygous mutants displayed a reduced lifespan as well as impaired motor abilities associated with specific cellular deficits, including decreased motor neurons length and neuromuscular junctions (NMJ) fragmentation. Furthermore, we demonstrate that these cellular impairments are linked to the misregulation of mRNA expression of acetylcholine receptor (AChR) subunits and histone deacetylase 4, markers of denervation and reinnervation processes observed in ALS patients. In addition, fus loss of function alters tau transcripts favoring the expression of small tau isoforms. Overall, this new animal model extends our knowledge on FUS and supports the relevance of FUS loss of function in ALS physiopathology

The most prevalent genetic cause of ALS-FTD, C9orf72 synergizes the toxicity of ATXN2 intermediate polyglutamine repeats through the autophagy pathway

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Neuromuscular Junction Impairment in Amyotrophic Lateral Sclerosis: Reassessing the Role of Acetylcholinesterase

International audienceAmyotrophic Lateral Sclerosis (ALS) is a highly debilitating disease caused by progressive degeneration of motorneurons (MNs). Due to the wide variety of genes and mutations identified in ALS, a highly varied etiology could ultimately converge to produce similar clinical symptoms. A major hypothesis in ALS research is the “distal axonopathy” with pathological changes occurring at the neuromuscular junction (NMJ), at very early stages of the disease, prior to MNs degeneration and onset of clinical symptoms. The NMJ is a highly specialized cholinergic synapse, allowing signaling between muscle and nerve necessary for skeletal muscle function. This nerve-muscle contact is characterized by the clustering of the collagen-tailed form of acetylcholinesterase (ColQ-AChE), together with other components of the extracellular matrix (ECM) and specific key molecules in the NMJ formation. Interestingly, in addition to their cholinergic role AChE is thought to play several “non-classical” roles that do not require catalytic function, most prominent among these is the facilitation of neurite growth, NMJ formation and survival. In all this context, abnormalities of AChE content have been found in plasma of ALS patients, in which AChE changes may reflect the neuromuscular disruption. We review these findings and particularly the evidences of changes of AChE at neuromuscular synapse in the pre-symptomatic stages of ALS

Functional Characterization of Neurofilament Light Splicing and Misbalance in Zebrafish

International audienceNeurofilaments (NFs), a major cytoskeletal component of motor neurons, play a key role in the differentiation, establishment and maintenance of their morphology and mechanical strength. The de novo assembly of these neuronal intermediate filaments requires the presence of the neurofilament light subunit (NEFL), whose expression is reduced in motor neurons in amyotrophic lateral sclerosis (ALS). This study used zebrafish as a model to characterize the NEFL homologue neflb, which encodes two different isoforms via a splicing of the primary transcript (neflbE4 and neflbE3). In vivo imaging showed that neflb is crucial for proper neuronal development, and that disrupting the balance between its two isoforms specifically affects the NF assembly and motor axon growth, with resultant motor deficits. This equilibrium is also disrupted upon the partial depletion of TDP-43 (TAR DNA-binding protein 43), an RNA-binding protein encoded by the gene TARDBP that is mislocalized into cytoplasmic inclusions in ALS. The study supports the interaction of the NEFL expression and splicing with TDP-43 in a common pathway, both biologically and pathogenetically

Loss of C9 ORF 72 impairs autophagy and synergizes with polyQ Ataxin‐2 to induce motor neuron dysfunction and cell death

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Quantification of the Transcripts Encoding Different Forms of AChE in Various Cell Types: Real-Time PCR Coupled with Standards in Revealing the Copy Number

Acetylcholinesterase (AChE) is encoded by a single gene, and the alternative splicing at the 3' end produces different isoforms, including tailed (AChE(T)), read-through (AChE(R)), and hydrophobic (AChE(H)). Different forms of this enzyme exist in different cell types. Each AChE form has been proposed to have unique function, and all of them could be found in same cell type. Thus, the splicing process of different AChE forms remains unclear. Here, we aimed to establish a quantification method in measuring the absolute amount of each AChE splicing variants within a cell type. By using real-time PCR coupled with standard curves of defined copy of AChE variants, the copies of AChE(T) transcript per 100 ng of total RNA were 5.7 x 10(4) in PC12 (rat neuronal cell), 1.3 x 10(4) in Caco-2 (human intestinal cell), 0.67 x 10(4) in TF-1 (human erythropoietic precursor), 133.3 in SH-SY5Y (human neuronal cell), and 56.7 in human umbilical vein endothelial cells (human endothelial cells). The copies of AChE(H) in these cell types were 0.3 x 10(4), 3.3 x 10(4), 2.7 x 10(4), 133.3, and 46.7, respectively, and AChE(R) were 0.07 x 10(4), 0.13 x 10(4), 890, 3.3, and 2.7, respectively. Furthermore, PC12 and TF-1 cells were chosen for the analysis of AChE splicing pattern during differentiation. The results demonstrated a selective increase in AChE(T) mRNA but not AChE(R) or AChE(H) mRNAs in PC12 upon nerve growth factor-induced neuronal differentiation. PC12 cells could therefore act as a good cell model for the study on alternative splicing mechanism and regulation of AChE(T)