Planar cell polarity zebrafish models of congenital scoliosis reveal novel underlying defects in notochord morphogenesis.

Congenital scoliosis (CS) is a type of vertebral malformation whose etiology remains elusive. The notochord is pivotal for vertebrae development but its role in CS is still understudied. Zebrafish knockout of ptk7a, a planar cell polarity (PCP) gene that is essential for convergence and extension (C&E) of the notochord, developed congenital scoliosis-like vertebral malformations (CVM). Maternal zygotic ptk7a mutants displayed severe C&E defects of the notochord. Excessive apoptosis occurred in the malformed notochord, causing a significantly reduced number of vacuolated cells, and compromising the mechanical properties of the notochord. The latter manifested as a less stiff extracellular matrix along with a significant reduction in the number of the caveolae and severely loosened intercellular junctions in the vacuolated region. These defects led to focal kinks, abnormal mineralization, and CVM exclusively at the anterior spine. Loss of function of another PCP gene, vangl2, also revealed excessive apoptosis in the notochord associated with CVM. This study suggests a new model for CS pathogenesis that is associated with defects in notochord C&E and highlights an essential role of PCP signaling in vertebrae development.

Expression of C9orf72-related dipeptides impairs motor function in a vertebrate model.

Large expansions of hexanucleotide GGGGCC (G4C2) repeats (hundreds to thousands) in the first intron of the chromosome 9 open reading frame 72 (C9orf72) locus are the strongest known genetic factor associated with amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Different hypotheses exist about the underlying disease mechanism including loss of function by haploinsufficiency, toxicity arising as a result of RNA or dipeptide repeats (DPRs). Five different DPRs are produced by repeat-associated non-ATG-initiated translation of the G4C2 repeats. Though earlier studies have indicated toxicity of the DPRs in worms, flies, primary cultured cells and cell lines, the effect of expressing DPRs of amyotrophic lateral sclerosis-relevant length has not been tested on motor behaviour in vertebrate models. In this study, by expressing constructs with alternate codons encoding different lengths of each DPR (40, 200 and 1000) in the vertebrate zebrafish model, the GR DPR was found to lead to the greatest developmental lethality and morphological defects, and GA, the least. However, expressing 1000 repeats of any DPR, including the 'non-toxic' GA DPR led to locomotor defects. Based on these observations, a transgenic line stably expressing 100 GR repeats was generated to allow specific regional and temporal expression of GR repeats in vivo. Expression of GR DPRs ubiquitously resulted in severe morphological defects and reduced swimming. However, when expressed specifically in motor neurons, the developmental defects were significantly reduced, but the swimming phenotype persisted, suggesting that GR DPRs have a toxic effect on motor neuron function. This was validated by the reduction in motor neuron length even in already formed motor neurons when GR was expressed in these. Hence, the expression of C9orf72-associated DPRs can cause significant motor deficits in vertebrates.

Mutations in CAPN1 Cause Autosomal-Recessive Hereditary Spastic Paraplegia.

Hereditary spastic paraplegia (HSP) is a genetically and clinically heterogeneous disease characterized by spasticity and weakness of the lower limbs with or without additional neurological symptoms. Although more than 70 genes and genetic loci have been implicated in HSP, many families remain genetically undiagnosed, suggesting that other genetic causes of HSP are still to be identified. HSP can be inherited in an autosomal-dominant, autosomal-recessive, or X-linked manner. In the current study, we performed whole-exome sequencing to analyze a total of nine affected individuals in three families with autosomal-recessive HSP. Rare homozygous and compound-heterozygous nonsense, missense, frameshift, and splice-site mutations in CAPN1 were identified in all affected individuals, and sequencing in additional family members confirmed the segregation of these mutations with the disease (spastic paraplegia 76 [SPG76]). CAPN1 encodes calpain 1, a protease that is widely present in the CNS. Calpain 1 is involved in synaptic plasticity, synaptic restructuring, and axon maturation and maintenance. Three models of calpain 1 deficiency were further studied. In Caenorhabditis elegans, loss of calpain 1 function resulted in neuronal and axonal dysfunction and degeneration. Similarly, loss-of-function of the Drosophila melanogaster ortholog calpain B caused locomotor defects and axonal anomalies. Knockdown of calpain 1a, a CAPN1 ortholog in Danio rerio, resulted in abnormal branchiomotor neuron migration and disorganized acetylated-tubulin axonal networks in the brain. The identification of mutations in CAPN1 in HSP expands our understanding of the disease causes and potential mechanisms.

γ-Aminobutyric acid receptor alpha 1 subunit loss of function causes genetic generalized epilepsy by impairing inhibitory network neurodevelopment.

OBJECTIVE: In humans, mutations of the γ-aminobutyric acid receptor subunit 1 (GABRA1) cause either mild or severe generalized epilepsy. Although these epilepsy-causing mutations have been shown to disrupt the receptor activity in vitro, their in vivo consequences on brain development and activity are not known. Here, we aim at unraveling the epileptogenesis mechanisms of GABRA1 loss of function. METHODS: We generated a gabra1-/- zebrafish mutant line displaying highly penetrant epileptic seizures. We sought to identify the underlying molecular mechanisms through unbiased whole transcriptomic assay of gabra1-/- larval brains. RESULTS: Interestingly, mutant fish show fully penetrant seizures at juvenile stages that accurately mimic tonic-clonic generalized seizures observed in patients. Moreover, highly penetrant seizures can be induced by light stimulation, thus providing us with the first zebrafish model in which evident epileptic seizures can be induced by nonchemical agents. Our transcriptomic assay identified misregulated genes in several pathways essential for correct brain development. More specifically, we show that the early development of the brain inhibitory network is specifically affected. Although the number of GABAergic neurons is not altered, we observed a drastic reduction in the number of inhibitory synapses and a decreased complexity of the GABAergic network. This is consistent with the disruption in expression of many genes involved in axon guidance and synapse formation. SIGNIFICANCE: Together with the role of GABA in neurodevelopment, our data identify a novel aspect of epileptogenesis, suggesting that the substratum of GABRA1-deficiency epilepsy is a consequence of early brain neurodevelopmental defects, in particular at the level of inhibitory network wiring.

Disruption of CLPB is associated with congenital microcephaly, severe encephalopathy and 3-methylglutaconic aciduria.

BACKGROUND: The heterogeneous group of 3-methylglutaconic aciduria disorders includes several inborn errors of metabolism that affect mitochondrial function through poorly understood mechanisms. We describe four newborn siblings, from a consanguineous family, who showed microcephaly, small birth weight, severe encephalopathy and 3-methylglutaconic aciduria. Their neurological examination was characterised by severe hypertonia and the induction of prolonged clonic movements of the four limbs upon minimal tactile stimulation. METHODS AND RESULTS: Using homozygosity mapping and exome sequencing, we identified a homozygous truncating mutation (p.I562Tfs*23) in CLPB segregating with the disease in this family. CLPB codes for a member of the family of ATPases associated with various cellular activities (AAA(+) proteins) whose function remains unknown. We found that CLPB expression is abolished in fibroblasts from the patients. To investigate the function of this gene, we interfered with the translation of the zebrafish clpb orthologue using an antisense morpholino. The clpb morphants showed an abnormal touch-evoked response with increased swim velocity and tail beat frequency. This motor phenotype is reminiscent of that observed in the patients and is suggestive of increased excitability in neuronal circuits. Interestingly, knocking down clpb reduced the number of inhibitory glycinergic interneurons and increased a population of excitatory glutamatergic neurons in the spinal cord. CONCLUSIONS: Altogether, our study suggests that disruption of CLPB causes a novel form of neonatal encephalopathy associated with 3-methylglutaconic aciduria.

WNK1/HSN2 mutation in human peripheral neuropathy deregulates KCC2 expression and posterior lateral line development in zebrafish (Danio rerio).

Hereditary sensory and autonomic neuropathy type 2 (HSNAII) is a rare pathology characterized by an early onset of severe sensory loss (all modalities) in the distal limbs. It is due to autosomal recessive mutations confined to exon "HSN2" of the WNK1 (with-no-lysine protein kinase 1) serine-threonine kinase. While this kinase is well studied in the kidneys, little is known about its role in the nervous system. We hypothesized that the truncating mutations present in the neural-specific HSN2 exon lead to a loss-of-function of the WNK1 kinase, impairing development of the peripheral sensory system. To investigate the mechanisms by which the loss of WNK1/HSN2 isoform function causes HSANII, we used the embryonic zebrafish model and observed strong expression of WNK1/HSN2 in neuromasts of the peripheral lateral line (PLL) system by immunohistochemistry. Knocking down wnk1/hsn2 in embryos using antisense morpholino oligonucleotides led to improper PLL development. We then investigated the reported interaction between the WNK1 kinase and neuronal potassium chloride cotransporter KCC2, as this transporter is a target of WNK1 phosphorylation. In situ hybridization revealed kcc2 expression in mature neuromasts of the PLL and semi-quantitative RT-PCR of wnk1/hsn2 knockdown embryos showed an increased expression of kcc2 mRNA. Furthermore, overexpression of human KCC2 mRNA in embryos replicated the wnk1/hsn2 knockdown phenotype. We validated these results by obtaining double knockdown embryos, both for wnk1/hsn2 and kcc2, which alleviated the PLL defects. Interestingly, overexpression of inactive mutant KCC2-C568A, which does not extrude ions, allowed a phenocopy of the PLL defects. These results suggest a pathway in which WNK1/HSN2 interacts with KCC2, producing a novel regulation of its transcription independent of KCC2's activation, where a loss-of-function mutation in WNK1 induces an overexpression of KCC2 and hinders proper peripheral sensory nerve development, a hallmark of HSANII.

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

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 S57Δ, 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.

Synaptic scaling and the development of a motor network.

Neurons respond homeostatically to chronic changes in network activity with compensatory changes such as a uniform alteration in the size of miniature postsynaptic current (mPSC) amplitudes termed synaptic scaling. However, little is known about the impact of synaptic scaling on the function of neural networks in vivo. We used the embryonic zebrafish to address the effect of synaptic scaling on the neural network underlying locomotion. Activity was decreased during development by TTX injection to block action potentials or CNQX injection to block glutamatergic transmission. Alternatively TNFalpha was chronically applied. Recordings from spinal neurons showed that glutamatergic mPSCs scaled up approximately 25% after activity reduction and fortuitously scaled down approximately 20% after TNFalpha treatment, and were unchanged following blockade of neuromuscular activity alone with alpha-bungarotoxin. Regardless of the direction of scaling, immediately following reversal of treatment no chronic effect was distinguishable in motoneuron activity patterns or in swimming behavior. We also acutely induced a similar increase of glutamatergic mPSC amplitudes using cyclothiazide to reduce AMPA receptor desensitization or decrease of glutamatergic mPSC amplitudes using a low concentration of CNQX to partially block AMPA receptors. Though the strength of the motor output was altered, neither chronic nor acute treatments disrupted the patterning of synaptic activity or swimming. Our results show, for the first time, that scaling of glutamatergic synapses can be induced in vivo in the zebrafish and that synaptic patterning is less plastic than synaptic strength during development.

A novel conserved evx1 enhancer links spinal interneuron morphology and cis-regulation from fish to mammals.

Spinal interneurons are key components of locomotor circuits, driving such diverse behaviors as swimming in fish and walking in mammals. Recent work has linked the expression of evolutionarily conserved transcription factors to key features of interneurons in diverse species, raising the possibility that these interneurons are functionally related. Consequently, the determinants of interneuron subtypes are predicted to share conserved cis-regulation in vertebrates with very different spinal cords. Here, we establish a link between cis-regulation and morphology of spinal interneurons that express the Evx1 homeodomain transcription factor from fish to mammals. Using comparative genomics, and complementary transgenic approaches, we have identified a novel enhancer of evx1, that includes two non-coding elements conserved in vertebrates. We show that pufferfish evx1 transgenes containing this enhancer direct reporter expression to a subset of spinal commissural interneurons in zebrafish embryos. Pufferfish, zebrafish and mouse evx1 downstream genomic enhancers label selectively Evx1(+) V0 commissural interneurons in chick and rat embryos. By dissecting the zebrafish evx1 enhancer, we identify a role for a 25 bp conserved cis-element in V0-specific gene expression. Our findings support the notion that spinal interneurons shared between distantly related vertebrates, have been maintained in part via the preservation of highly conserved cis-regulatory modules.

Neurogenic role of the depolarizing chloride gradient revealed by global overexpression of KCC2 from the onset of development.

GABA- and glycine-induced depolarization is thought to provide important developmental signals, but the role of the underlying chloride gradient has not been examined from the onset of development. We therefore overexpressed globally the potassium-chloride cotransporter 2 (KCC2) in newly fertilized zebrafish embryos to reverse the chloride gradient. This rendered glycine hyperpolarizing in all neurons, tested at the time that motor behaviors (but not native KCC2) first appear. KCC2 overexpression resulted in fewer mature spontaneously active spinal neurons, more immature silent neurons, and disrupted motor activity. We observed fewer motoneurons and interneurons, a reduction in the elaboration of axonal tracts, and smaller brains and spinal cords. However, we observed no increased apoptosis and a normal complement of sensory neurons, glia, and progenitors. These results suggest that chloride-mediated excitation plays a crucial role in promoting neurogenesis from the earliest stages of embryonic development.

Glycine Regulates Neural Stem Cell Proliferation During Development via Lnx1-Dependent Notch Signaling.

During development of the zebrafish embryo, glycine signaling promotes the differentiation of neural stem cells (NSCs). We found that glycine signaling suppresses the expression of Ligand of Numb X1 (lnx1, Ligand of numb protein-x1), a gene of unknown function during NSC differentiation that is selectively expressed in the embryonic central nervous system (CNS). As a consequence, Numb levels were stabilized and Notch activity (measured as her4.1 expression) was reduced, promoting NSC differentiation. These consequent actions were blocked by knockdown of lnx1. In contrast, lnx1 overexpression increased NSC proliferation and led to defects of neural tube closure at the early stages of development. Thus, our data provide evidence that glycine/lnx1 signaling modulates NSC proliferation by regulation of Notch signaling.

Individual knock out of glycine receptor alpha subunits identifies a specific requirement of glra1 for motor function in zebrafish.

Glycine receptors (GlyRs) are ligand-gated chloride channels mediating inhibitory neurotransmission in the brain stem and spinal cord. They function as pentamers composed of alpha and beta subunits for which 5 genes have been identified in human (GLRA1, GLRA2, GLRA3, GLRA4, GLRB). Several in vitro studies showed that the pentameric subtype composition as well as its stoichiometry influence the distribution and the molecular function of the receptor. Moreover, mutations in some of these genes are involved in different human conditions ranging from tinnitus to epilepsy and hyperekplexia, suggesting distinct functions of the different subunits. Although the beta subunit is essential for synaptic clustering of the receptor, the specific role of each alpha subtype is still puzzling in vivo. The zebrafish genome encodes for five glycine receptor alpha subunits (glra1, glra2, glra3, glra4a, glra4b) thus offering a model of choice to investigate the respective role of each subtype on general motor behaviour. After establishing a phylogeny of GlyR subunit evolution between human and zebrafish, we checked the temporal expression pattern of these transcripts during embryo development. Interestingly, we found that glra1 is the only maternally transmitted alpha subunit. We also showed that the expression of the different GlyR subunits starts at different time points during development. Lastly, in order to decipher the role of each alpha subunit on the general motor behaviour of the fish, we knocked out individually each alpha subunit by CRISPR/Cas9-targeted mutagenesis. Surprisingly, we found that knocking out any of the alpha2, 3, a4a or a4b subunit did not lead to any obvious developmental or motor phenotype. However, glra1-/- (hitch) embryos depicted a strong motor dysfunction from 3 days, making them incapable to swim and thus leading to their premature death. Our results infer a strong functional redundancy between alpha subunits and confirm the central role played by glra1 for proper inhibitory neurotransmission controlling locomotion. The genetic tools we developed here will be of general interest for further studies aiming at dissecting the role of GlyRs in glycinergic transmission in vivo and the hitch mutant (hic) is of specific relevance as a new model of hyperekplexia.

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.

Targeted knockout of GABA-A receptor gamma 2 subunit provokes transient light-induced reflex seizures in zebrafish larvae.

Epilepsy is a common primary neurological disorder characterized by the chronic tendency of a patient to experience epileptic seizures, which are abnormal body movements or cognitive states that result from excessive, hypersynchronous brain activity. Epilepsy has been found to have numerous etiologies and, although about two-thirds of epilepsies were classically considered idiopathic, the majority of those are now believed to be of genetic origin. Mutations in genes involved in gamma-aminobutyric acid (GABA)-mediated inhibitory neurotransmission have been associated with a broad range of epilepsy syndromes. Mutations in the GABA-A receptor gamma 2 subunit gene (GABRG2), for example, have been associated with absence epilepsy and febrile seizures in humans. Several rodent models of GABRG2 loss of function depict clinical features of the disease; however, alternative genetic models more amenable for the study of ictogenesis and for high-throughput screening purposes are still needed. In this context, we generated a gabrg2 knockout (KO) zebrafish model (which we called R23X) that displayed light/dark-induced reflex seizures. Through high-resolution in vivo calcium imaging of the brain, we showed that this phenotype is associated with widespread increases in neuronal activity that can be effectively alleviated by the anti-epileptic drug valproic acid. Moreover, these seizures only occur at the larval stages but disappear after 1 week of age. Interestingly, our whole-transcriptome analysis showed that gabrg2 KO does not alter the expression of genes in the larval brain. As a result, the gabrg2-/- zebrafish is a novel in vivo genetic model of early epilepsies that opens new doors to investigate ictogenesis and for further drug-screening assays.

Transcriptomic Analysis of Zebrafish TDP-43 Transgenic Lines.

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 TARDBP, 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.

Glycine decarboxylase deficiency-induced motor dysfunction in zebrafish is rescued by counterbalancing glycine synaptic level.

Glycine encephalopathy (GE), or nonketotic hyperglycinemia (NKH), is a rare recessive genetic disease caused by defective glycine cleavage and characterized by increased accumulation of glycine in all tissues. Here, based on new case reports of GLDC loss-of-function mutations in GE patients, we aimed to generate a zebrafish model of severe GE in order to unravel the molecular mechanism of the disease. Using CRISPR/Cas9, we knocked out the gldc gene and showed that gldc-/- fish recapitulate GE on a molecular level and present a motor phenotype reminiscent of severe GE symptoms. The molecular characterization of gldc-/- mutants showed a broad metabolic disturbance affecting amino acids and neurotransmitters other than glycine, with lactic acidosis at stages preceding death. Although a transient imbalance was found in cell proliferation in the brain of gldc-/- zebrafish, the main brain networks were not affected, thus suggesting that GE pathogenicity is mainly due to metabolic defects. We confirmed that the gldc-/- hypotonic phenotype is due to NMDA and glycine receptor overactivation, and demonstrated that gldc-/- larvae depict exacerbated hyperglycinemia at these synapses. Remarkably, we were able to rescue the motor dysfunction of gldc-/- larvae by counterbalancing pharmacologically or genetically the level of glycine at the synapse.

Non-canonical mTOR-Independent Role of DEPDC5 in Regulating GABAergic Network Development.

Mutations in DEPDC5 are causal factors for a broad spectrum of focal epilepsies, but the underlying pathogenic mechanisms are still largely unknown. To address this question, a zebrafish depdc5 knockout model showing spontaneous epileptiform events in the brain, increased drug-induced seizure susceptibility, general hypoactivity, premature death at 2-3 weeks post-fertilization, as well as the expected hyperactivation of mTOR signaling was developed. Using this model, the role of DEPDC5 in brain development was investigated using an unbiased whole-transcriptomic approach. Surprisingly, in addition to mTOR-associated genes, many genes involved in synaptic function, neurogenesis, axonogenesis, and GABA network activity were found to be dysregulated in larval brains. Although no gross defects in brain morphology or neuron loss were observed, immunostaining of depdc5-/- brains for several GABAergic markers revealed specific defects in the fine branching of the GABAergic network. Consistently, some defects in depdc5-/- could be compensated for by treatment with GABA, corroborating that GABA signaling is indeed involved in DEPDC5 pathogenicity. Further, the mTOR-independent nature of these neurodevelopmental defects was demonstrated by the inability of rapamycin to rescue the GABAergic network defects observed in depdc5-/- brains and, conversely, the inability of GABA to rescue the hypoactivity in another genetic model showing mTOR hyperactivation. This study hence provides the first in vivo evidence that DEPDC5 plays previously unknown roles apart from its canonical function as an mTOR inhibitor. Moreover, these results propose that defective neurodevelopment of GABAergic networks could be a key factor in epileptogenesis when DEPDC5 is mutated.

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.

Homology Directed Knockin of Point Mutations in the Zebrafish tardbp and fus Genes in ALS Using the CRISPR/Cas9 System.

The methodology for site-directed editing of single nucleotides in the vertebrate genome is of considerable interest for research in biology and medicine. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 type II (Cas9) system has emerged as a simple and inexpensive tool for editing genomic loci of interest in a variety of animal models. In zebrafish, error-prone non-homologous end joining (NHEJ) has been used as a simple method to disrupt gene function. We sought to develop a method to easily create site-specific SNPs in the zebrafish genome. Here, we report simple methodologies for using CRISPR/Cas9-mediated homology directed repair using single-stranded oligodeoxynucleotide donor templates (ssODN) for site-directed single nucleotide editing, for the first time in two disease-related genes, tardbp and fus.