Novel genome-editing-based approaches to treat motor neuron diseases: Promises and challenges.

Motor neuron diseases are untreatable with common pharmacological approaches. Spinal muscular atrophy (SMA) is caused by SMN1 gene mutations leading to lowered SMN expression. Symptoms are alleviated in infants with a higher copy number of the SMN2 gene, which, however, displays a splicing defect resulting in low SMN levels. Amyotrophic lateral sclerosis (ALS) is caused by a number of mutations, with C9orf72 repeat expansions the most common genetic cause and SOD1 gain-of-function mutations the first genetic cause identified for this disease. Genetic therapies based on oligonucleotides that enhance SMN2 splicing and SMN production or lower SOD1 expression have shown promise in initial clinical trials for individuals with SMA and ALS harboring SOD1 mutations, respectively. Gene addition/silencing approaches using adeno-associated viruses (AAVs) are also currently under clinical investigation in trials for SMA and ALS. Here we provide a brief overview of these efforts and their advantages and challenges. We also review genome editing approaches aimed at correcting the disease-causing mutations or modulating the expression of genetic modifiers, e.g., by repairing SOD1 mutations or the SMN2 splicing defect or deleting C9orf72 expanded repeats. These studies have shown promising results to approach therapeutic trials that should significantly lower the progression of these deadly disorders.

Loss of C9ORF72 impairs autophagy and synergizes with polyQ Ataxin-2 to induce motor neuron dysfunction and cell death.

An intronic expansion of GGGGCC repeats within the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). Ataxin-2 with intermediate length of polyglutamine expansions (Ataxin-2 Q30x) is a genetic modifier of the disease. Here, we found that C9ORF72 forms a complex with the WDR41 and SMCR8 proteins to act as a GDP/GTP exchange factor for RAB8a and RAB39b and to thereby control autophagic flux. Depletion of C9orf72 in neurons partly impairs autophagy and leads to accumulation of aggregates of TDP-43 and P62 proteins, which are histopathological hallmarks of ALS-FTD SMCR8 is phosphorylated by TBK1 and depletion of TBK1 can be rescued by phosphomimetic mutants of SMCR8 or by constitutively active RAB39b, suggesting that TBK1, SMCR8, C9ORF72, and RAB39b belong to a common pathway regulating autophagy. While depletion of C9ORF72 only has a partial deleterious effect on neuron survival, it synergizes with Ataxin-2 Q30x toxicity to induce motor neuron dysfunction and neuronal cell death. These results indicate that partial loss of function of C9ORF72 is not deleterious by itself but synergizes with Ataxin-2 toxicity, suggesting a double-hit pathological mechanism in ALS-FTD.

Loss of VPS13C Function in Autosomal-Recessive Parkinsonism Causes Mitochondrial Dysfunction and Increases PINK1/Parkin-Dependent Mitophagy.

Autosomal-recessive early-onset parkinsonism is clinically and genetically heterogeneous. The genetic causes of approximately 50% of autosomal-recessive early-onset forms of Parkinson disease (PD) remain to be elucidated. Homozygozity mapping and exome sequencing in 62 isolated individuals with early-onset parkinsonism and confirmed consanguinity followed by data mining in the exomes of 1,348 PD-affected individuals identified, in three isolated subjects, homozygous or compound heterozygous truncating mutations in vacuolar protein sorting 13C (VPS13C). VPS13C mutations are associated with a distinct form of early-onset parkinsonism characterized by rapid and severe disease progression and early cognitive decline; the pathological features were striking and reminiscent of diffuse Lewy body disease. In cell models, VPS13C partly localized to the outer membrane of mitochondria. Silencing of VPS13C was associated with lower mitochondrial membrane potential, mitochondrial fragmentation, increased respiration rates, exacerbated PINK1/Parkin-dependent mitophagy, and transcriptional upregulation of PARK2 in response to mitochondrial damage. This work suggests that loss of function of VPS13C is a cause of autosomal-recessive early-onset parkinsonism with a distinctive phenotype of rapid and severe progression.

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

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.

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

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.

Two novel COLVI long chains in zebrafish that are essential for muscle development.

Collagen VI (COLVI), a protein ubiquitously expressed in connective tissues, is crucial for structural integrity, cellular adhesion, migration and survival. Six different genes are recognized in mammalians, encoding six COLVI-chains that assemble as two 'short' (α1, α2) and one 'long' chain (theoretically any one of α3-6). In humans, defects in the most widely expressed heterotrimer (α123), due to mutations in the COL6A1-3 genes, cause a heterogeneous group of neuromuscular disorders, collectively termed COLVI-related muscle disorders. Little is known about the function(s) of the recently described α4-6 chains and no mutations have been detected yet. In this study, we characterized two novel COLVI long chains in zebrafish that are most homologous to the mammalian α4 chain; therefore, we named the corresponding genes col6a4a and col6a4b. These orthologues represent ancestors of the mammalian Col6a4-6 genes. By in situ hybridization and RT-qPCR, we unveiled a distinctive expression kinetics for col6a4b, compared with the other col6a genes. Using morpholino antisense oligonucleotides targeting col6a4a, col6a4b and col6a2, we modelled partial and complete COLVI deficiency, respectively. All morphant embryos presented altered muscle structure and impaired motility. While apoptosis was not drastically increased, autophagy induction was defective in all morphants. Furthermore, motoneuron axon growth was abnormal in these morphants. Importantly, some phenotypical differences emerged between col6a4a and col6a4b morphants, suggesting only partial functional redundancy. Overall, our results further confirm the importance of COLVI in zebrafish muscle development and may provide important clues for potential human phenotypes associated with deficiency of the recently described COLVI-chains.

Loss of function of C9orf72 causes motor deficits in a zebrafish model of amyotrophic lateral sclerosis.

OBJECTIVE: To define the role that repeat expansions of a GGGGCC hexanucleotide sequence of the C9orf72 gene play in the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). A genetic model for ALS was developed to determine whether loss of function of the zebrafish orthologue of C9orf72 (zC9orf72) leads to abnormalities in neuronal development. METHODS: C9orf72 mRNA levels were quantified in brain and lymphoblasts derived from FTLD and ALS/FTLD patients and in zebrafish. Knockdown of the zC9orf72 was performed using 2 specific antisense morpholino oligonucleotides to block transcription. Quantifications of spontaneous swimming and tactile escape response, as well as measurements of axonal projections from the spinal cord, were performed. RESULTS: Significantly decreased expression of C9orf72 transcripts in brain and lymphoblasts was found in sporadic FTLD and ALS/FTLD patients with normal-size or expanded hexanucleotide repeats. The zC9orf72 is selectively expressed in the developing nervous system at developmental stages. Loss of function of the zC9orf72 transcripts causes both behavioral and cellular deficits related to locomotion without major morphological abnormalities. These deficits were rescued upon overexpression of human C9orf72 mRNA transcripts. INTERPRETATION: Our results indicate C9orf72 haploinsufficiency could be a contributing factor in the spectrum of ALS/FTLD neurodegenerative disorders. Loss of function of the zebrafish orthologue of zC9orf72 expression in zebrafish is associated with axonal degeneration of motor neurons that can be rescued by expressing human C9orf72 mRNA, highlighting the specificity of the induced phenotype. These results reveal a pathogenic consequence of decreased C9orf72 levels, supporting a loss of function mechanism of disease.

Microglia Mitigate Neuronal Activation in a Zebrafish Model of Dravet Syndrome.

It has been known for a long time that epileptic seizures provoke brain neuroinflammation involving the activation of microglial cells. However, the role of these cells in this disease context and the consequences of their inflammatory activation on subsequent neuron network activity remain poorly understood so far. To fill this gap of knowledge and gain a better understanding of the role of microglia in the pathophysiology of epilepsy, we used an established zebrafish Dravet syndrome epilepsy model based on Scn1Lab sodium channel loss-of-function, combined with live microglia and neuronal Ca2+ imaging, local field potential (LFP) recording, and genetic microglia ablation. Data showed that microglial cells in scn1Lab-deficient larvae experiencing epileptiform seizures displayed morphological and biochemical changes characteristic of M1-like pro-inflammatory activation; i.e., reduced branching, amoeboid-like morphology, and marked increase in the number of microglia expressing pro-inflammatory cytokine Il1ß. More importantly, LFP recording, Ca2+ imaging, and swimming behavior analysis showed that microglia-depleted scn1Lab-KD larvae displayed an increase in epileptiform seizure-like neuron activation when compared to that seen in scn1Lab-KD individuals with microglia. These findings strongly suggest that despite microglia activation and the synthesis of pro-inflammatory cytokines, these cells provide neuroprotective activities to epileptic neuronal networks, making these cells a promising therapeutic target in epilepsy.

Correction: Association of Rare Genetic Variants in Opioid Receptors with Tourette Syndrome.

[This corrects the article DOI: 10.5334/tohm.464.].

Hypertonicity sensing in organum vasculosum lamina terminalis neurons: a mechanical process involving TRPV1 but not TRPV4.

Primary osmosensory neurons in the mouse organum vasculosum lamina terminalis (OVLT) transduce hypertonicity via the activation of nonselective cation channels that cause membrane depolarization and increased action potential discharge, and this effect is absent in mice lacking expression of the transient receptor potential vanilloid 1 (Trpv1) gene (Ciura and Bourque, 2006). However other experiments have indicated that channels encoded by Trpv4 also contribute to central osmosensation in mice (Liedtke and Friedman, 2003; Mizuno et al., 2003). At present, the mechanism by which hypertonicity modulates cation channels in OVLT neurons is unknown, and it remains unclear whether Trpv1 and Trpv4 both contribute to this process. Here, we show that physical shrinking is necessary and sufficient to mediate hypertonicity sensing in OVLT neurons isolated from adult mice. Steps coupling progressive decreases in cell volume to increased neuronal activity were quantitatively equivalent whether shrinking was evoked by osmotic pressure or mechanical aspiration. Finally, modulation of OVLT neurons by tonicity or mechanical stimulation was unaffected by deletion of trpv4 but was abolished in cells lacking Trpv1 or wild-type neurons treated with the TRPV1 antagonist SB366791. Thus, hypertonicity sensing is a mechanical process requiring Trpv1, but not Trpv4.

Oxytocin-induced analgesia and scratching are mediated by the vasopressin-1A receptor in the mouse.

The neuropeptides oxytocin (OXT) and arginine vasopressin (AVP) contribute to the regulation of diverse cognitive and physiological functions including nociception. Indeed, OXT has been reported to be analgesic when administered directly into the brain, the spinal cord, or systemically. Here, we characterized the phenotype of oxytocin receptor (OTR) and vasopressin-1A receptor (V1AR) null mutant mice in a battery of pain assays. Surprisingly, OTR knock-out mice displayed a pain phenotype identical to their wild-type littermates. Moreover, systemic administration of OXT dose-dependently produced analgesia in both wild-type and OTR knock-out mice in three different assays, the radiant-heat paw withdrawal test, the von Frey test of mechanical sensitivity, and the formalin test of inflammatory nociception. In contrast, OXT-induced analgesia was completely absent in V1AR knock-out mice. In wild-type mice, OXT-induced analgesia could be fully prevented by pretreatment with a V1AR but not an OTR antagonist. Receptor binding studies demonstrated that the distribution of OXT and AVP binding sites in mouse lumbar spinal cord resembles the pattern observed in rat. AVP binding sites diffusely label the lumbar spinal cord, whereas OXT binding sites cluster in the substantia gelatinosa of the dorsal horn. In contrast, quantitative real-time reverse transcription (RT)-PCR revealed that V1AR but not OTR mRNA is abundantly expressed in mouse dorsal root ganglia, where it localizes to small- and medium-diameter cells as shown by single-cell RT-PCR. Hence, V1ARs expressed in dorsal root ganglia might represent a previously unrecognized target for the analgesic action of OXT and AVP.

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

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 have uncovered a large spectrum of genes implicated in various forms of epilepsy, highlighting the heterogeneous nature of this disorder. Appropriate animal models are essential for investigating the pathological mechanisms triggered by genetic mutations implicated in epilepsy and for developing specialized, targeted therapies. In recent years, zebrafish has emerged as a valuable vertebrate organism for modeling epilepsies, with the use of both genetic manipulation and exposure to known epileptogenic drugs, such as pentylenetetrazole (PTZ), to identify novel anti-epileptic therapeutics. Deleterious mutations in the mTOR regulator DEPDC5 have been associated with various forms of focal epilepsies and knock-down of the zebrafish orthologue causes hyperactivity associated with spontaneous seizure-like episodes, as well as enhanced electrographic activity and characteristic turn wheel swimming. Here, we described the method involved in generating the DEPDC5 loss-of-function model and illustrate the protocol for assessing motor activity at 28 and 48 h post fertilization (hpf), as well as a method for recording field activity in the zebrafish optic tectum. An illustration of the effect of the epileptogenic drug PTZ on neuronal activity over time is also provided.

Osmotic and thermal control of magnocellular neurosecretory neurons--role of an N-terminal variant of trpv1.

The release of vasopressin (antidiuretic hormone) plays a key role in the osmoregulatory response of mammals to changes in salt or water intake and in the rate of water loss through evaporation during thermoregulatory cooling. Previous work has shown that the hypothalamus encloses the sensory elements that modulate vasopressin release during systemic changes in fluid osmolality or body temperature. These responses depend in part on a synaptic regulation of vasopressin neurons by afferent inputs arising from osmosensory and thermosensory neurons in the preoptic area. However, recent studies in rats and mice have shown that vasopressin neurons in the supraoptic nucleus also display intrinsic osmosensory and thermosensory properties. Isolated vasopressin neurons exposed to increases in perfusate temperature or osmolality generate increases in non-selective cation channel activity that cause membrane depolarization and increase neuronal excitability. These channels are calcium-permeable and can be blocked by ruthenium red. Moreover, intrinsic responses to osmotic and thermal stimuli are absent in magnocellular neurosecretory cells isolated from mice lacking the transient receptor potential vanilloid-1 (trpv1) gene, which encodes the capsaicin receptor. Immunostaining of vasopressin-releasing neurons with anti-TRPV1 antibodies reveals the presence of amino acids present in the carboxy terminus of the protein, but not those lying in the amino terminal domain. Thus, magnocellular neurosecretory neurons appear to express an N-terminal variant of trpv1 which lacks sensitivity to capsaicin, but which enables osmosensing and thermosensing.

Association of Rare Genetic Variants in Opioid Receptors with Tourette Syndrome.

Background: Genes involved in Tourette syndrome (TS) remain largely unknown. We aimed to identify genetic factors contributing to TS in a French cohort of 120 individuals using a combination of hypothesis-driven and exome-sequencing approaches. Methods: We first sequenced exons of SLITRK1-6 and HDC in the TS cohort and subsequently sequenced the exome of 12 individuals harboring rare variants in these genes to find additional rare variants contributing to the disorder under the hypothesis of oligogenic inheritance. We further screened three candidate genes (OPRK1, PCDH10, and NTSR2) preferentially expressed in the basal ganglia, and three additional genes involved in neurotensin and opioid signaling (OPRM1, NTS, and NTSR1), and compared variant frequencies in TS patients and 788 matched control individuals. We also investigated the impact of altering the expression of Oprk1 in zebrafish. Results: Thirteen ultrarare missense variants of SLITRK1-6 and HDC were identified in 12 patients. Exome sequencing in these patients revealed rare possibly deleterious variants in 3,041 genes, 54 of which were preferentially expressed in the basal ganglia. Comparison of variant frequencies altering selected candidate genes in TS and control individuals revealed an excess of potentially disrupting variants in OPRK1, encoding the opioid kappa receptor, in TS patients. Accordingly, we show that downregulation of the Oprk1 orthologue in zebrafish induces a hyperkinetic phenotype in early development. Discussion: These results support a heterogeneous and complex genetic etiology of TS, possibly involving rare variants altering the opioid pathway in some individuals, which could represent a novel therapeutic target in this disorder.

Trpv4 Mediates Hypotonic Inhibition of Central Osmosensory Neurons via Taurine Gliotransmission.

The maintenance of hydromineral homeostasis requires bidirectional detection of changes in extracellular fluid osmolality by primary osmosensory neurons (ONs) in the organum vasculosum laminae terminalis (OVLT). Hypertonicity excites ONs in part through the mechanical activation of a variant transient receptor potential vanilloid-1 channel (dn-Trpv1). However, the mechanism by which local hypotonicity inhibits ONs in the OVLT remains unknown. Here, we show that hypotonicity can reduce the basal activity of dn-Trpv1 channels and hyperpolarize acutely isolated ONs. Surprisingly, we found that mice lacking dn-Trpv1 maintain normal inhibitory responses to hypotonicity when tested in situ. In the intact setting, hypotonicity inhibits ONs through a non-cell-autonomous mechanism that involves glial release of the glycine receptor agonist taurine through hypotonicity activated anion channels (HAAC) that are activated subsequent to Ca2+ influx through Trpv4 channels. Our study clarifies how Trpv4 channels contribute to the inhibition of OVLT ONs during hypotonicity in situ.

Depdc5 knockdown causes mTOR-dependent motor hyperactivity in zebrafish.

OBJECTIVE: DEPDC5 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 DEPDC5 in the etiology of focal epilepsy. DEPDC5 is a component of the GATOR1 complex, acting as a negative regulator of mTOR signaling. METHODS: Zebrafish represents a vertebrate model suitable for genetic analysis and drug screening in epilepsy-related disorders. In this study, we defined the expression of depdc5 during development and established an epilepsy model with reduced Depdc5 expression. RESULTS: Here we report a zebrafish model of Depdc5 loss-of-function that displays a measurable behavioral phenotype, including hyperkinesia, circular swimming, and increased neuronal activity. These phenotypic features persisted throughout embryonic development and were significantly reduced upon treatment with the mTORC1 inhibitor, rapamycin, as well as overexpression of human WT DEPDC5 transcript. No phenotypic rescue was obtained upon expression of epilepsy-associated DEPDC5 mutations (p.Arg487* and p.Arg485Gln), indicating that these mutations cause a loss of function of the protein. INTERPRETATION: This study demonstrates that Depdc5 knockdown leads to early-onset phenotypic features related to motor and neuronal hyperactivity. Restoration of phenotypic features by WT but not epilepsy-associated Depdc5 mutants, as well as by mTORC1 inhibition confirm the role of Depdc5 in the mTORC1-dependent molecular cascades, defining this pathway as a potential therapeutic target for DEPDC5-inherited forms of focal epilepsy.

Transient receptor potential vanilloid 1 is required for intrinsic osmoreception in organum vasculosum lamina terminalis neurons and for normal thirst responses to systemic hyperosmolality.

Recent studies have indicated that members of the transient receptor potential vanilloid (TRPV) family of cation channels are required for the generation of normal osmoregulatory responses, yet the mechanism of osmosensory transduction in primary osmoreceptor neurons of the CNS remains to be defined. Indeed, despite ample evidence suggesting that the organum vasculosum lamina terminalis (OVLT) serves as the primary locus of the brain for the detection of osmotic stimuli, evidence that neurons in the OVLT are intrinsically osmosensitive has remained elusive. Here we show that murine OVLT neurons are intrinsically sensitive to increases in the osmolality of the extracellular fluid. Hypertonic conditions provoked increases in membrane cation conductance that resulted in the generation of an inward current, depolarizing osmoreceptor potentials, and enhanced action potential discharge. Moreover, we found that this osmosensory signal transduction cascade was absent in OVLT neurons from TRPV1 knock-out (TRPV1-/-) mice and that responses of wild type (WT) OVLT neurons could be blocked by ruthenium red, an inhibitor of TRPV channels. Finally, TRPV1-/- mice showed significantly attenuated water intake in response to systemic hypertonicity compared with WT controls. These findings indicate that OVLT neurons act as primary osmoreceptors and that a product of the trpv1 gene is required for osmosensory transduction.

Hes6 inhibits astrocyte differentiation and promotes neurogenesis through different mechanisms.

The mechanisms regulating the generation of cell diversity in the mammalian cerebral cortex are beginning to be elucidated. In that regard, Hairy/Enhancer of split (Hes) 1 and 5 are basic helix-loop-helix (bHLH) factors that inhibit the differentiation of pluripotent cortical progenitors into neurons. In contrast, a related Hes family member termed Hes6 promotes neurogenesis. It is shown here that knockdown of endogenous Hes6 causes supernumerary cortical progenitors to differentiate into cells that exhibit an astrocytic morphology and express the astrocyte marker protein GFAP. Conversely, exogenous Hes6 expression in cortical progenitors inhibits astrocyte differentiation. The negative effect of Hes6 on astrocyte differentiation is independent of its ability to promote neuronal differentiation. We also show that neither its proneuronal nor its anti-gliogenic functions appear to depend on Hes6 ability to bind to DNA via the basic arm of its bHLH domain. Both of these activities require Hes6 to be localized to nuclei, but only its anti-gliogenic function depends on two short peptides, LNHLL and WRPW, that are conserved in all Hes6 proteins. These findings suggest that Hes6 is an important regulator of the neurogenic phase of cortical development by promoting the neuronal fate while suppressing astrocyte differentiation. They suggest further that separate molecular mechanisms underlie the proneuronal and anti-gliogenic activities of Hes6 in cortical progenitor cells.

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.