Axonal TDP-43 condensates drive neuromuscular junction disruption through inhibition of local synthesis of nuclear encoded mitochondrial proteins

AbstractMislocalization of the predominantly nuclear RNA/DNA binding protein, TDP-43, occurs in motor neurons of ~95% of amyotrophic lateral sclerosis (ALS) patients, but the contribution of axonal TDP-43 to this neurodegenerative disease is unclear. Here, we show TDP-43 accumulation in intra-muscular nerves from ALS patients and in axons of human iPSC-derived motor neurons of ALS patient, as well as in motor neurons and neuromuscular junctions (NMJs) of a TDP-43 mislocalization mouse model. In axons, TDP-43 is hyper-phosphorylated and promotes G3BP1-positive ribonucleoprotein (RNP) condensate assembly, consequently inhibiting local protein synthesis in distal axons and NMJs. Specifically, the axonal and synaptic levels of nuclear-encoded mitochondrial proteins are reduced. Clearance of axonal TDP-43 or dissociation of G3BP1 condensates restored local translation and resolved TDP-43-derived toxicity in both axons and NMJs. These findings support an axonal gain of function of TDP-43 in ALS, which can be targeted for therapeutic development.

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Axonal TDP-43 Drives NMJ Disruption through Inhibition of Local Protein Synthesis

10.21203/rs.3.rs-87662/v1 ◽

2020 ◽

Author(s):

Topaz Altman ◽

Ariel Ionescu ◽

Amjad Ibraheem ◽

Dominik Priesmann ◽

Tal Gradus-Pery ◽

...

Keyword(s):

Protein Synthesis ◽

Motor Neurons ◽

Neuromuscular Junctions ◽

Dna Binding Protein ◽

Mitochondrial Proteins ◽

Local Protein Synthesis ◽

Rna Granules ◽

Nuclear Rna ◽

Als Patients ◽

Local Protein

Abstract Mislocalization of the predominantly nuclear RNA/DNA binding protein, TDP-43, occurs in motor neurons of ~95% of ALS patients, but the contribution of axonal TDP-43 to this fatal neurodegenerative disease is unclear. Here, we find TDP-43 accumulation in the axons of intra-muscular nerves from ALS patients, and in motor neurons and neuromuscular junctions (NMJs) of a mouse model with TDP-43 mislocalization. This leads to the formation of G3BP1- and TDP-43- positive RNA-granules in motor neuron axons, and to inhibition of local protein synthesis in axons and NMJs. Specifically, the axonal and synaptic levels of nuclear-encoded mitochondria proteins are reduced. Clearance of axonal TDP-43 restored local translation of the nuclear-encoded mitochondrial proteins and rescued TDP-43-derived axonal and NMJ toxicity. These findings suggest that targeting TDP-43 axonal gain of function may mediate a therapeutic effect in ALS.

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Human sensorimotor organoids derived from healthy and amyotrophic lateral sclerosis stem cells form neuromuscular junctions

Nature Communications ◽

10.1038/s41467-021-24776-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

João D. Pereira ◽

Daniel M. DuBreuil ◽

Anna-Claire Devlin ◽

Aaron Held ◽

Yechiam Sapir ◽

...

Keyword(s):

Stem Cells ◽

Amyotrophic Lateral Sclerosis ◽

Motor Neurons ◽

Pluripotent Stem Cells ◽

Neuromuscular Junctions ◽

Familial Als ◽

Induced Pluripotent ◽

Human Ipsc ◽

Lateral Sclerosis ◽

Individual Human

AbstractHuman induced pluripotent stem cells (iPSC) hold promise for modeling diseases in individual human genetic backgrounds and thus for developing precision medicine. Here, we generate sensorimotor organoids containing physiologically functional neuromuscular junctions (NMJs) and apply the model to different subgroups of amyotrophic lateral sclerosis (ALS). Using a range of molecular, genomic, and physiological techniques, we identify and characterize motor neurons and skeletal muscle, along with sensory neurons, astrocytes, microglia, and vasculature. Organoid cultures derived from multiple human iPSC lines generated from individuals with ALS and isogenic lines edited to harbor familial ALS mutations show impairment at the level of the NMJ, as detected by both contraction and immunocytochemical measurements. The physiological resolution of the human NMJ synapse, combined with the generation of major cellular cohorts exerting autonomous and non-cell autonomous effects in motor and sensory diseases, may prove valuable to understand the pathophysiological mechanisms of ALS.

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Riluzole Exhibits No Therapeutic Efficacy on a Transgenic Rat model of Amyotrophic Lateral Sclerosis

Current Neurovascular Research ◽

10.2174/1567202617666200409125227 ◽

2020 ◽

Vol 17 (3) ◽

pp. 275-285 ◽

Cited By ~ 5

Author(s):

Si Chen ◽

Qiao Liao ◽

Ke Lu ◽

Jinxia Zhou ◽

Cao Huang ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Motor Neurons ◽

Rat Model ◽

Microglial Activation ◽

Transgenic Rat ◽

Intragastric Administration ◽

Behavioral Deficits ◽

Als Patients ◽

Lateral Sclerosis ◽

Transgenic Rat Model

Background: Amyotrophic lateral sclerosis (ALS) is a neurological disorder clinically characterized by motor system dysfunction, with intraneuronal accumulation of the TAR DNAbinding protein 43 (TDP-43) being a pathological hallmark. Riluzole is a primarily prescribed medicine for ALS patients, while its therapeutical efficacy appears limited. TDP-43 transgenic mice are existing animal models for mechanistic/translational research into ALS. Methods: We developed a transgenic rat model of ALS expressing a mutant human TDP-43 transgene (TDP-43M337V) and evaluated the therapeutic effect of Riluzole on this model. Relative to control, rats with TDP-43M337V expression promoted by the neurofilament heavy subunit (NEF) gene or specifically in motor neurons promoted by the choline acetyltransferase (ChAT) gene showed progressive worsening of mobility and grip strength, along with loss of motor neurons, microglial activation, and intraneuronal accumulation of TDP-43 and ubiquitin aggregations in the spinal cord. Results: Compared to vehicle control, intragastric administration of Riluzole (30 mg/kg/d) did not mitigate the behavioral deficits nor alter the neuropathologies in the transgenics. Conclusion: These findings indicate that transgenic rats recapitulate the basic neurological and neuropathological characteristics of human ALS, while Riluzole treatment can not halt the development of the behavioral and histopathological phenotypes in this new transgenic rodent model of ALS.

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Utility of Transcranial Magnetic Simulation in Studying Upper Motor Neuron Dysfunction in Amyotrophic Lateral Sclerosis

Brain Sciences ◽

10.3390/brainsci11070906 ◽

2021 ◽

Vol 11 (7) ◽

pp. 906

Author(s):

Nimeshan Geevasinga ◽

Mehdi Van den Bos ◽

Parvathi Menon ◽

Steve Vucic

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Motor Neurons ◽

Cortical Excitability ◽

Muscular Atrophy ◽

Close Relationship ◽

Bulbar Onset ◽

Als Patients ◽

Transcranial Magnetic Simulation ◽

Cortical Dysfunction ◽

Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is characterised by progressive dysfunction of the upper and lower motor neurons. The disease can evolve over time from focal limb or bulbar onset to involvement of other regions. There is some clinical heterogeneity in ALS with various phenotypes of the disease described, from primary lateral sclerosis, progressive muscular atrophy and flail arm/leg phenotypes. Whilst the majority of ALS patients are sporadic in nature, recent advances have highlighted genetic forms of the disease. Given the close relationship between ALS and frontotemporal dementia, the importance of cortical dysfunction has gained prominence. Transcranial magnetic stimulation (TMS) is a noninvasive neurophysiological tool to explore the function of the motor cortex and thereby cortical excitability. In this review, we highlight the utility of TMS and explore cortical excitability in ALS diagnosis, pathogenesis and insights gained from genetic and variant forms of the disease.

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Cerebrospinal Fluid Chitinases as Biomarkers for Amyotrophic Lateral Sclerosis

Diagnostics ◽

10.3390/diagnostics11071210 ◽

2021 ◽

Vol 11 (7) ◽

pp. 1210

Author(s):

Júlia Costa ◽

Marta Gromicho ◽

Ana Pronto-Laborinho ◽

Conceição Almeida ◽

Ricardo A. Gomes ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Cerebrospinal Fluid ◽

Motor Neurons ◽

Neurological Diseases ◽

Disease Diagnosis ◽

Enzyme Linked Immunosorbent Assay ◽

Progression Rate ◽

Therapeutic Trials ◽

Als Patients ◽

Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative neuromuscular disease that affects motor neurons controlling voluntary muscles. Survival is usually 2–5 years after onset, and death occurs due to respiratory failure. The identification of biomarkers would be very useful to help in disease diagnosis and for patient stratification based on, e.g., progression rate, with implications in therapeutic trials. Neurofilaments constitute already-promising markers for ALS and, recently, chitinases have emerged as novel marker targets for the disease. Here, we investigated cerebrospinal fluid (CSF) chitinases as potential markers for ALS. Chitotriosidase (CHIT1), chitinase-3-like protein 1 (CHI3L1), chitinase-3-like protein 2 (CHI3L2) and the benchmark marker phosphoneurofilament heavy chain (pNFH) were quantified by an enzyme-linked immunosorbent assay (ELISA) from the CSF of 34 ALS patients and 24 control patients with other neurological diseases. CSF was also analyzed by UHPLC-mass spectrometry. All three chitinases, as well as pNFH, were found to correlate with disease progression rate. Furthermore, CHIT1 was elevated in ALS patients with high diagnostic performance, as was pNFH. On the other hand, CHIT1 correlated with forced vital capacity (FVC). The three chitinases correlated with pNFH, indicating a relation between degeneration and neuroinflammation. In conclusion, our results supported the value of CHIT1 as a diagnostic and progression rate biomarker, and its potential as respiratory function marker. The results opened novel perspectives to explore chitinases as biomarkers and their functional relevance in ALS.

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Local protein synthesis of neuronal MT1-MMP for agrin-induced presynaptic development

Development ◽

10.1242/dev.199000 ◽

2021 ◽

Vol 148 (10) ◽

Author(s):

Jun Yu ◽

Marilyn Janice Oentaryo ◽

Chi Wai Lee

Keyword(s):

Protein Synthesis ◽

Neuronal Development ◽

Neuromuscular Junctions ◽

Neuronal Cell ◽

Time Lapse ◽

Protein Synthesis Inhibitor ◽

Synthesis Inhibitor ◽

Local Protein Synthesis ◽

Presynaptic Differentiation ◽

Local Protein

ABSTRACT Upon the stimulation of extracellular cues, a significant number of proteins are synthesized distally along the axon. Although local protein synthesis is crucial for various stages throughout neuronal development, its involvement in presynaptic differentiation at developing neuromuscular junctions remains unknown. By using axon severing and microfluidic chamber assays, we first showed that treatment of a protein synthesis inhibitor, cycloheximide, inhibits agrin-induced presynaptic differentiation in cultured Xenopus spinal neurons. Newly synthesized proteins are prominently detected, as revealed by the staining of click-reactive cell-permeable puromycin analog O-propargyl-puromycin, at agrin bead-neurite contacts involving the mTOR/4E-BP1 pathway. Next, live-cell time-lapse imaging demonstrated the local capturing and immobilization of ribonucleoprotein granules upon agrin bead stimulation. Given that our recent study reported the roles of membrane-type 1 matrix metalloproteinase (MT1-MMP) in agrin-induced presynaptic differentiation, here we further showed that MT1-MMP mRNA is spatially enriched and locally translated at sites induced by agrin beads. Taken together, this study reveals an essential role for axonal MT1-MMP translation, on top of the well-recognized long-range transport of MT1-MMP proteins synthesized from neuronal cell bodies, in mediating agrin-induced presynaptic differentiation.

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Spatiotemporal Dynamics of Molecular Pathology in Amyotrophic Lateral Sclerosis

10.1101/389270 ◽

2018 ◽

Cited By ~ 2

Author(s):

Silas Maniatis ◽

Tarmo Äijö ◽

Sanja Vickovic ◽

Catherine Braine ◽

Kristy Kang ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Motor Neuron ◽

Motor Neurons ◽

Molecular Mechanisms ◽

Early Time ◽

Spatiotemporal Dynamics ◽

Course Of Disease ◽

Molecular Events ◽

Als Patients ◽

Lateral Sclerosis

AbstractParalysis occurring in amyotrophic lateral sclerosis (ALS) results from denervation of skeletal muscle as a consequence of motor neuron degeneration. Interactions between motor neurons and glia contribute to motor neuron loss, but the spatiotemporal ordering of molecular events that drive these processes in intact spinal tissue remains poorly understood. Here, we use spatial transcriptomics to obtain gene expression measurements of mouse spinal cords over the course of disease, as well as of postmortem tissue from ALS patients, to characterize the underlying molecular mechanisms in ALS. We identify novel pathway dynamics, regional differences between microglia and astrocyte populations at early time-points, and discern perturbations in several transcriptional pathways shared between murine models of ALS and human postmortem spinal cords.One Sentence SummaryAnalysis of the ALS spinal cord using Spatial Transcriptomics reveals spatiotemporal dynamics of disease driven gene regulation.

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Microphysiological 3D model of amyotrophic lateral sclerosis (ALS) from human iPS-derived muscle cells and optogenetic motor neurons

Science Advances ◽

10.1126/sciadv.aat5847 ◽

2018 ◽

Vol 4 (10) ◽

pp. eaat5847 ◽

Cited By ~ 66

Author(s):

Tatsuya Osaki ◽

Sebastien G. M. Uzel ◽

Roger D. Kamm

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Motor Unit ◽

Motor Neurons ◽

Neuromuscular Junctions ◽

Research Effort ◽

Muscle Contractions ◽

Drug Candidates ◽

Chip Technology ◽

Sporadic Als ◽

Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disease involving loss of motor neurons (MNs) and muscle atrophy, still has no effective treatment, despite much research effort. To provide a platform for testing drug candidates and investigating the pathogenesis of ALS, we developed an ALS-on-a-chip technology (i.e., an ALS motor unit) using three-dimensional skeletal muscle bundles along with induced pluripotent stem cell (iPSC)–derived and light-sensitive channelrhodopsin-2–induced MN spheroids from a patient with sporadic ALS. Each tissue was cultured in a different compartment of a microfluidic device. Axon outgrowth formed neuromuscular junctions on the muscle fiber bundles. Light was used to activate muscle contraction, which was measured on the basis of pillar deflections. Compared to a non-ALS motor unit, the ALS motor unit generated fewer muscle contractions, there was MN degradation, and apoptosis increased in the muscle. Furthermore, the muscle contractions were recovered by single treatments and cotreatment with rapamycin (a mechanistic target of rapamycin inhibitor) and bosutinib (an Src/c-Abl inhibitor). This recovery was associated with up-regulation of autophagy and degradation of TAR DNA binding protein–43 in the MNs. Moreover, administering the drugs via an endothelial cell barrier decreased the expression of P-glycoprotein (an efflux pump that transports bosutinib) in the endothelial cells, indicating that rapamycin and bosutinib cotreatment has considerable potential for ALS treatment. This ALS-on-a-chip and optogenetics technology could help to elucidate the pathogenesis of ALS and to screen for drug candidates.

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What is “Hyper” in the ALS Hypermetabolism?

Mediators of Inflammation ◽

10.1155/2017/7821672 ◽

2017 ◽

Vol 2017 ◽

pp. 1-11 ◽

Cited By ~ 21

Author(s):

Alberto Ferri ◽

Roberto Coccurello

Keyword(s):

Motor Neurons ◽

Body Weight Loss ◽

Point Of View ◽

Whole Body ◽

Glucose And Lipid Metabolism ◽

Als Patients ◽

Amp Activated Protein Kinase ◽

Body Energy ◽

Defective Metabolism ◽

Lateral Sclerosis

The progressive and fatal loss of upper (brain) and lower (spinal cord) motor neurons and muscle denervation concisely condenses the clinical picture of amyotrophic lateral sclerosis (ALS). Despite the multiple mechanisms believed to underlie the selective loss of motor neurons, ALS aetiology remains elusive and obscure. Likewise, there is also a cluster of alterations in ALS patients in which muscle wasting, body weight loss, eating dysfunction, and abnormal energy dissipation coexist. Defective energy metabolism characterizes the ALS progression, and such paradox of energy balance stands as a challenge for the understanding of ALS pathogenesis. The hypermetabolism in ALS will be examined from tissue-specific energy imbalance (e.g., skeletal muscle) to major energetic pathways (e.g., AMP-activated protein kinase) and whole-body energy alterations including glucose and lipid metabolism, nutrition, and potential involvement of interorgan communication. From the point of view here expressed, the hypermetabolism in ALS should be evaluated as a magnifying glass through which looking at the ALS pathogenesis is from a different perspective in which defective metabolism can disclose novel mechanistic interpretations and lines of intervention.

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Amyotrophic Lateral Sclerosis

10.1093/med/9780199937837.003.0028 ◽

2017 ◽

Cited By ~ 1

Author(s):

Brian J. Wainger

Keyword(s):

Neurodegenerative Diseases ◽

Motor Neurons ◽

Molecular Processes ◽

Cellular Models ◽

Subtype Specificity ◽

Sporadic Disease ◽

Neuronal Subtype ◽

Human Ipsc ◽

Insight Into ◽

Lateral Sclerosis

Mouse and cellular models of ALS including stem cells have revealed tremendous insight into the molecular processes that lead to ALS. Models of ALS and other neurodegenerative diseases have led to emergent molecular themes that span several diseases. Future models must account for neuronal subtype specificity of different neurodegenerative diseases, particularly between tightly related diseases such as FTD and ALS. Human iPSC-derived motor neurons offer promise both with regard to the use of human cells and in particular the ability to model sporadic disease, which is critically important given the overwhelming abundance of sporadic disease in ALS and other neurodegenerative diseases.

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