behavioral-and-physiological-analysis-in-a-zebrafish-model-of-epilepsy

Behavioral And Physiological Analysis In A Zebrafish Model Of Epilepsy

Epilepsy represents one of the most common neurological disorders, affecting an estimated 50 million people worldwide. Recent advances in genetic research ...

Mechanisms of protein homeostasis are crucial for overseeing the clearance of misfolded and toxic proteins over the lifetime of an organism, thereby ensuring the health of neurons and other cells of the central nervous system. The highly conserved pathway of autophagy is particularly necessary for preventing and counteracting pathogenic insults that may lead to neurodegeneration. In line with this, mutations in genes that encode essential autophagy factors result in impaired autophagy and lead to neurodegenerative conditions such as amyotrophic lateral sclerosis (ALS). However, the mechanistic details underlying the neuroprotective role of autophagy, neuronal resistance to autophagy induction, and the neuron-specific effects of autophagy-impairing mutations remain incompletely defined. Further, the manner and extent to which non-cell autonomous effects of autophagy dysfunction contribute to ALS pathogenesis are not fully understood. Here, we review the current understanding of the interplay between autophagy and ALS pathogenesis by providing an overview of critical steps in the autophagy pathway, with special focus on pivotal factors impaired by ALS-causing mutations, their physiologic effects on autophagy in disease models, and the cell type-specific mechanisms regulating autophagy in non-neuronal cells which, when impaired, can contribute to neurodegeneration. This review thereby provides a framework not only to guide further investigations of neuronal autophagy but also to refine therapeutic strategies for ALS and related neurodegenerative diseases.: ALS: amyotrophic lateral sclerosis; Atg: autophagy-related; CHMP2B: charged multivesicular body protein 2B; DPR: dipeptide repeat; FTD: frontotemporal dementia; iPSC: induced pluripotent stem cell; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; PINK1: PTEN induced kinase 1; RNP: ribonuclear protein; sALS: sporadic ALS; SPHK1: sphingosine kinase 1; TARDBP/TDP-43: TAR DNA binding protein; TBK1: TANK-binding kinase 1; TFEB: transcription factor EB; ULK: unc-51 like autophagy activating kinase; UPR: unfolded protein response; UPS: ubiquitin-proteasome system; VCP: valosin containing protein.

Autophagy and ALS: mechanistic insights and therapeutic implications.

Mutations in the SOD1 and TARDBP genes have been commonly identified in Amyotrophic Lateral Sclerosis (ALS). Recently, mutations in the Fused in sarcoma gene (FUS) were identified in familial (FALS) ALS cases and sporadic (SALS) patients. Similarly to TDP-43 (coded by TARDBP gene), FUS is an RNA binding protein. Using the zebrafish (Danio rerio), we examined the consequences of expressing human wild-type (WT) FUS and three ALS-related mutations, as well as their interactions with TARDBP and SOD1. Knockdown of zebrafish Fus yielded a motor phenotype that could be rescued upon co-expression of wild-type human FUS. In contrast, the two most frequent ALS-related FUS mutations, R521H and R521C, unlike S57Î”, failed to rescue the knockdown phenotype, indicating loss of function. The R521H mutation caused a toxic gain of function when expressed alone, similar to the phenotype observed upon knockdown of zebrafish Fus. This phenotype was not aggravated by co-expression of both mutant human TARDBP (G348C) and FUS (R521H) or by knockdown of both zebrafish Tardbp and Fus, consistent with a common pathogenic mechanism. We also observed that WT FUS rescued the Tardbp knockdown phenotype, but not vice versa, suggesting that TARDBP acts upstream of FUS in this pathway. In addition we observed that WT SOD1 failed to rescue the phenotype observed upon overexpression of mutant TARDBP or FUS or upon knockdown of Tardbp or Fus; similarly, WT TARDBP or FUS also failed to rescue the phenotype induced by mutant SOD1 (G93A). Finally, overexpression of mutant SOD1 exacerbated the motor phenotype caused by overexpression of mutant FUS. Together our results indicate that TARDBP and FUS act in a pathogenic pathway that is independent of SOD1.

FUS and TARDBP but not SOD1 interact in genetic models of amyotrophic lateral sclerosis.

Mutationen in den Genen SOD1 und TARDBP wurden h&auml;ufig in die Amyotrophe Lateralsklerose (ALS). Vor kurzem wurden die Fused im Sarkom-gen (FUS) sporadisch (SALS) Patienten und famili&auml;re ALS (FALS) F&auml;lle Mutationen im. Ebenso ist FUS TDP-43 (TARDBP-Gen codiert), ein verbindliches Protein der RNS. Mithilfe der Zebrab&auml;rbling (Danio Rerio), haben wir die Folgen menschlichen Wildtyp (WT) FUS sowie drei ALS im Zusammenhang mit Mutationen und deren Wechselwirkungen mit SOD1, TARDBP zum Ausdruck zu bringen. Niederschlag von Zebrafish Fus ergab einen motor Ph&auml;notyp, der auf Koexpression von Wildtyp menschliche FUS gerettet werden konnte. Im Gegensatz dazu h&auml;ufig die beiden am meisten ALS-bezogene FUS Mutationen, R521H und R521C, im Gegensatz zu S57&Delta;, der Niederschlag Ph&auml;notyp retten konnte, Verlust der Funktion angibt. Die R521H-Mutation verursacht einen giftigen Gewinn Funktion ausgedr&uuml;ckt, allein, &auml;hnlich wie der Ph&auml;notyp auf Niederschlag von Zebrafish Fus beobachtet. Dieser Ph&auml;notyp war keine Verst&auml;rkung durch Koexpression von beiden Mutante menschlichen TARDBP (G348C) und FUS (R521H) oder durch Niederschlag der Zebrafisch Tardbp und Fus, mit einem gemeinsamen pathogenen Mechanismus konsistent. Wir auch beobachtet, dass WT FUS Tardbp Niederschlag Ph&auml;notyps, gerettet, aber nicht umgekehrt, darauf hindeutet, dass TARDBP stromaufw&auml;rts fungiert der FUS in diesem Weg. Zus&auml;tzlich beobachteten wir, dass WT SOD1 fehlgeschlagen ist, der Ph&auml;notyp beobachtet eine &Uuml;berexpression von mutant TARDBP oder FUS oder Niederschlag Tardbp oder Fus zu retten; ebenso WT TARDBP oder FUS konnte auch nicht der Ph&auml;notyp von Mutant SOD1 induziert zu retten (G93A). Schlie&szlig;lich noch eine &Uuml;berexpression von Mutant SOD1 der motor Ph&auml;notyp, verursacht durch eine &Uuml;berexpression von Mutant FUS versch&auml;rft. Zusammen zeigen unsere Ergebnisse, dass TARDBP und FUS pathogenen Weg handeln, die unabh&auml;ngig von SOD1.

FUS und TARDBP aber nicht SOD1 interagieren auf genetische Modelle der amyotrophen Lateralsklerose.

TMEM106B was identified as a risk factor for frontotemporal lobar degeneration (FTD) with TAR DNA-binding protein 43 kDa inclusions. It has been reported that variants in this gene are genetic modifiers of the disease and that this association is stronger in patients carrying a GRN mutation or a pathogenic expansion in chromosome 9 open reading frame 72 (C9orf72) gene. Here, we investigated the contribution of TMEM106B polymorphisms in cohorts of FTD and FTD with amyotrophic lateral sclerosis patients from France and Italy. Patients carrying the C9orf72 expansion (n = 145) and patients with GRN mutations (n = 76) were compared with a group of FTD patients (n = 384) negative for mutations and to a group of healthy controls (n = 552). In our cohorts, the presence of the C9orf72 expansion did not correlate with TMEM106B genotypes but the association was very strong in individuals with pathogenic GRN mutations (p = 9.54 × 10(-6)). Our data suggest that TMEM106B genotypes differ in FTD patient cohorts and strengthen the protective role of TMEM106B in GRN carriers. Further studies are needed to determine whether TMEM106B polymorphisms are associated with other genetic causes for FTD, including C9orf72 repeat expansions.

Defining the association of TMEM106B variants among frontotemporal lobar degeneration patients with GRN mutations and C9orf72 repeat expansions.

The aim of this study was to establish the frequency of ATXN2 polyglutamine (polyQ) expansion in large cohorts of patients with amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and progressive supranuclear palsy (PSP), and to evaluate whether ATXN2 could act as a modifier gene in patients carrying the C9orf72 expansion.

Contribution of ATXN2 intermediary polyQ expansions in a spectrum of neurodegenerative disorders.

Mutations in SQSTM1, encoding for the protein SQSTM1/p62, have been recently reported in 1-3.5% of patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration (ALS/FTLD). Inclusions positive for SQSTM1/p62 have been detected in patients with neurodegenerative disorders, including ALS/FTLD. In order to investigate the pathogenic mechanisms induced by SQSTM1 mutations in ALS/FTLD, we developed a zebrafish model. Knock-down of the sqstm1 zebrafish ortholog, as well as impairment of its splicing, led to a specific phenotype, consisting of behavioral and axonal anomalies. Here, we report swimming deficits associated with shorter motor neuronal axons that could be rescued by the overexpression of wild-type human SQSTM1. Interestingly, no rescue of the loss-of-function phenotype was observed when overexpressing human SQSTM1 constructs carrying ALS/FTLD-related mutations. Consistent with its role in autophagy regulation, we found increased mTOR levels upon knock-down of sqstm1. Furthermore, treatment of zebrafish embryos with rapamycin, a known inhibitor of the mTOR pathway, yielded an amelioration of the locomotor phenotype in the sqstm1 knock-down model. Our results suggest that loss-of-function of SQSTM1 causes phenotypic features characterized by locomotor deficits and motor neuron axonal defects that are associated with a misregulation of autophagic processes. 

Sqstm1 knock-down causes a locomotor phenotype ameliorated by rapamycin in a zebrafish model of ALS/FTLD.

Several genetic causes have been recently described for neurological diseases, increasing our knowledge of the common pathological mechanisms involved in these disorders. Mutation analysis has shown common causative factors for two major neurodegenerative disorders, ALS and FTD. Shared pathological and genetic markers as well as common neurological signs between these diseases have given rise to the notion of an ALS/FTD spectrum. This overlap among genetic factors causing ALS/FTD and the coincidence of mutated alleles (including causative, risk and modifier variants) have given rise to the notion of an oligogenic model of disease. In this review we summarize major advances in the elucidation of novel genetic factors in these diseases which have led to a better understanding of the common pathogenic factors leading to neurodegeneration.

Defining the genetic connection linking amyotrophic lateral sclerosis (ALS) with frontotemporal dementia (FTD).

 was identified as a major genetic cause of focal epilepsy with deleterious mutations found in a wide range of inherited forms of focal epilepsy, associated with malformation of cortical development in certain cases. Identification of frameshift, truncation, and deletion mutations implicates haploinsufficiency of  in the etiology of focal epilepsy. DEPDC5 is a component of the GATOR1 complex, acting as a negative regulator of mTOR signaling.

Depdc5 knockdown causes mTOR-dependent motor hyperactivity in zebrafish.

Amyotrophic lateral sclerosis (ALS) is a late-onset progressive neurodegenerative disorder that affects both upper and lower motor neurons, leading to muscle atrophy with spasticity and eventual death in 3-5 years after the disease onset. More than 50 mutations linked to ALS have been found in the gene , encoding the protein TDP-43 that is the predominant component of neuronal inclusions in ALS. TDP-43 is an RNA binding protein with glycine-rich domains that binds to more than 6,000 RNAs in the human brain. However, ALS-related mutations do not appear to affect the function of these genes, indicating that a toxic gain-of-function may occur. We generated transgenic zebrafish lines expressing human TDP-43, either the wild-type form or the ALS-causative G348C mutation identified in a subset of ALS patients, with the transgene expression driven by an inducible heat shock promoter in order to bypass a potential early mortality. The expression of the mutant but not the wild-type human TDP-43 in zebrafish embryos induced a reduction of the locomotor activity in response to touch compared to controls and moderate axonopathy of the motor neurons of the spinal cord, with premature branching of the main axonal branch, recapitulating previous results obtained by mRNA injections. We used these lines to investigate transcriptomic changes due to the presence of mutant TDP-43 using RNA sequencing and have found 159 genes that are differentially expressed compared to control, with 67 genes up-regulated and 92 genes down-regulated. These transcriptomic changes are in line with recent transcriptomic data obtained in mouse models, indicating that these zebrafish transgenic lines are adequate to further study TDP-43-related ALS.

Transcriptomic Analysis of Zebrafish TDP-43 Transgenic Lines.

Neurofilaments (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 , which encodes two different isoforms via a splicing of the primary transcript ( and ). In vivo imaging showed that  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  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.

Functional Characterization of Neurofilament Light Splicing and Misbalance in Zebrafish.

The "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.

Article	Year
Autophagy and ALS: mechanistic insights and therapeutic implications. Autophagy\| PubMed ID: 34057020	2021
FUS and TARDBP but not SOD1 interact in genetic models of amyotrophic lateral sclerosis. PLoS genetics\| PubMed ID: 21829392	2011
Defining the association of TMEM106B variants among frontotemporal lobar degeneration patients with GRN mutations and C9orf72 repeat expansions. Neurobiology of aging\| PubMed ID: 25085782	2014
Contribution of ATXN2 intermediary polyQ expansions in a spectrum of neurodegenerative disorders. Neurology\| PubMed ID: 25098532	2014
Sqstm1 knock-down causes a locomotor phenotype ameliorated by rapamycin in a zebrafish model of ALS/FTLD. Human molecular genetics\| PubMed ID: 25410659	2015
Defining the genetic connection linking amyotrophic lateral sclerosis (ALS) with frontotemporal dementia (FTD). Trends in genetics : TIG\| PubMed ID: 25869998	2015
Depdc5 knockdown causes mTOR-dependent motor hyperactivity in zebrafish. Annals of clinical and translational neurology\| PubMed ID: 29761115	2018
Transcriptomic Analysis of Zebrafish TDP-43 Transgenic Lines. Frontiers in molecular neuroscience\| PubMed ID: 30618614	2018
Functional Characterization of Neurofilament Light Splicing and Misbalance in Zebrafish. Cells\| PubMed ID: 32429483	2020
TDP-43 Regulation of AChE Expression Can Mediate ALS-Like Phenotype in Zebrafish. Cells\| PubMed ID: 33499374	2021

Edor Kabashi

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