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Focal Cortical Dysplasia, Hemimegalencephaly and Cortical Tuber are pediatric epileptogenic malformations of cortical development (MCDs) frequently pharmaco-resistant and mostly surgically treated by the resection of epileptic cortex. Availability of cortical resection samples allowed significant mechanistic discoveries directly from human material. Causal brain somatic or germline mutations in the AKT/PI3K/DEPDC5/MTOR genes were identified. GABAa mediated paradoxical depolarization, related to altered chloride (Cl-) homeostasis, was shown to participate to ictogenesis in human pediatric MCDs. However, the link between genomic alterations and neuronal hyperexcitability is still unclear. Here we studied the post translational interactions between the mTOR pathway and the regulation of cation-chloride cotransporters (CCC), KCC2 and NKCC1, that are largely responsible for controlling intracellular Cl- and ultimately GABAergic transmission. For this study, 35 children (25 MTORopathies and 10 pseudo controls, diagnosed by histology plus genetic profiling) were operated for drug resistant epilepsy. Postoperative cortical tissues were recorded on multielectrode array (MEA) to map epileptic activities. CCC expression level and phosphorylation status of the WNK1/SPAK-OSR1 pathway was measured during basal conditions and after pharmacological modulation. Direct interactions between mTOR and WNK1 pathway components were investigated by immunoprecipitation. Membranous incorporation of MCD samples in Xenopus laevis oocytes enabled Cl- conductance and equilibrium potential (EGABA) for GABA measurement. Of the 25 clinical cases, half harbored a somatic mutation in the mTOR pathway, while pS6 expression was increased in all MCD samples. Spontaneous interictal discharges were recorded in 65% of the slices. CCC expression was altered in MCDs, with a reduced KCC2/NKCC1 ratio and decreased KCC2 membranous expression. CCC expression was regulated by the WNK1/SPAK-OSR1 kinases through direct phosphorylation of Thr906 on KCC2, that was reversed by WNK1 and SPAK antagonists (NEM and Staurosporine). mSIN1 subunit of MTORC2 was found to interact with SPAK-OSR1 and WNK1. Interactions between these key epileptogenic pathways could be reversed by the mTOR specific antagonist Rapamycin, leading to a dephosphorylation of CCCs and recovery of the KCC2/NKCC1 ratio. The functional effect of such recovery was validated by the restoration of the depolarizing shift in EGABA by rapamycin, measured after incorporation of MCD membranes to X. laevis oocytes, in line with a reestablishment of normal ECl-. Our study deciphers a protein interaction network through a phosphorylation cascade between MTOR and WNK1/SPAK-OSR1 leading to chloride cotransporters deregulation, increased neuronal chloride levels and GABAa dysfunction in malformations of Cortical Development, linking genomic defects and functional effects and paving the way to target epilepsy therapy.
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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.
Assuntos
Modelos Animais de Doenças , Epilepsias Mioclônicas , Microglia , Neurônios , Peixe-Zebra , Animais , Microglia/metabolismo , Microglia/patologia , Epilepsias Mioclônicas/patologia , Epilepsias Mioclônicas/genética , Epilepsias Mioclônicas/metabolismo , Epilepsias Mioclônicas/fisiopatologia , Neurônios/metabolismo , Neurônios/patologia , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Canal de Sódio Disparado por Voltagem NAV1.1/metabolismo , Interleucina-1beta/metabolismo , Larva , Cálcio/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
[This corrects the article DOI: 10.5334/tohm.464.].
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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.
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Esclerose Lateral Amiotrófica , Atrofia Muscular Espinal , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/terapia , Edição de Genes , Humanos , Lactente , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/terapia , Oligonucleotídeos/metabolismo , Splicing de RNA , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismoRESUMO
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.
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Epilepsia , Peixe-Zebra , Animais , Modelos Animais de Doenças , Epilepsia/induzido quimicamente , Epilepsia/tratamento farmacológico , Epilepsia/genética , Humanos , Pentilenotetrazol/uso terapêutico , Convulsões/patologia , Peixe-Zebra/genéticaRESUMO
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.
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Acetilcolinesterase/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Epistasia Genética , Técnicas de Silenciamento de Genes , Junção Neuromuscular/patologia , Fenótipo , Ligação ProteicaRESUMO
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.
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Estudos de Associação Genética/métodos , Variação Genética/genética , Mutação de Sentido Incorreto/genética , Receptores Opioides/genética , Síndrome de Tourette/diagnóstico , Síndrome de Tourette/genética , Animais , Estudos de Coortes , Feminino , Humanos , Masculino , Peixe-ZebraRESUMO
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.
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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.
Assuntos
Soluções Hipotônicas/farmacologia , Inibição Neural/efeitos dos fármacos , Neurônios/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Canais de Cátion TRPV/metabolismo , Taurina/farmacologia , Animais , Cálcio/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neurônios/efeitos dos fármacos , Concentração OsmolarRESUMO
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.
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Esclerose Lateral Amiotrófica/tratamento farmacológico , Antipsicóticos/farmacocinética , Antipsicóticos/uso terapêutico , Doenças da Junção Neuromuscular/tratamento farmacológico , Animais , Caenorhabditis elegans , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio Tipo T/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Tolerância a Medicamentos , Feminino , Camundongos , Junção Neuromuscular/efeitos dos fármacos , Pimozida/farmacologia , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
The most common genetic cause for amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD) is repeat expansion of a hexanucleotide sequence (GGGGCC) within the C9orf72 genomic sequence. To elucidate the functional role of C9orf72 in disease pathogenesis, we identified certain molecular interactors of this factor. We determined that C9orf72 exists in a complex with SMCR8 and WDR41 and that this complex acts as a GDP/GTP exchange factor for RAB8 and RAB39, 2 RAB GTPases involved in macroautophagy/autophagy. Consequently, C9orf72 depletion in neuronal cultures leads to accumulation of unresolved aggregates of SQSTM1/p62 and phosphorylated TARDBP/TDP-43. However, C9orf72 reduction does not lead to major neuronal toxicity, suggesting that a second stress may be required to induce neuronal cell death. An intermediate size of polyglutamine repeats within ATXN2 is an important genetic modifier of ALS-FTD. We found that coexpression of intermediate polyglutamine repeats (30Q) of ATXN2 combined with C9orf72 depletion increases the aggregation of ATXN2 and neuronal toxicity. These results were confirmed in zebrafish embryos where partial C9orf72 knockdown along with intermediate (but not normal) repeat expansions in ATXN2 causes locomotion deficits and abnormal axonal projections from spinal motor neurons. These results demonstrate that C9orf72 plays an important role in the autophagy pathway while genetically interacting with another major genetic risk factor, ATXN2, to contribute to ALS-FTD pathogenesis.
Assuntos
Esclerose Lateral Amiotrófica/genética , Ataxina-2/metabolismo , Autofagia , Proteína C9orf72/genética , Demência Frontotemporal/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Proteína C9orf72/metabolismo , Caenorhabditis elegans , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Demência Frontotemporal/patologia , Técnicas de Silenciamento de Genes , Predisposição Genética para Doença , Humanos , Camundongos , Neurônios Motores/patologia , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Peptídeos/química , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Sequestossoma-1/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
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.
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Esclerose Lateral Amiotrófica/patologia , Ataxina-2/metabolismo , Autofagia , Demência Frontotemporal/patologia , Neurônios Motores/fisiologia , Peptídeos/metabolismo , Proteínas/metabolismo , Proteína C9orf72 , Morte Celular , Humanos , Neurônios Motores/metabolismoRESUMO
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.
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Mitofagia/genética , Transtornos Parkinsonianos/genética , Proteínas Quinases/genética , Proteínas/genética , Ubiquitina-Proteína Ligases/genética , Adulto , Idoso , Animais , Células COS , Estudos de Casos e Controles , Consanguinidade , Feminino , Inativação Gênica , Heterogeneidade Genética , Células HEK293 , Heterozigoto , Homozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Transtornos Parkinsonianos/diagnóstico , Linhagem , Fenótipo , Proteínas Quinases/metabolismo , Proteínas/metabolismo , Reprodutibilidade dos Testes , Turquia , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Amyotrophic Lateral Sclerosis (ALS) is a highly debilitating disease caused by progressive degeneration of motorneurons (MNs). Due to the wide variety of genes and mutations identified in ALS, a highly varied etiology could ultimately converge to produce similar clinical symptoms. A major hypothesis in ALS research is the "distal axonopathy" with pathological changes occurring at the neuromuscular junction (NMJ), at very early stages of the disease, prior to MNs degeneration and onset of clinical symptoms. The NMJ is a highly specialized cholinergic synapse, allowing signaling between muscle and nerve necessary for skeletal muscle function. This nerve-muscle contact is characterized by the clustering of the collagen-tailed form of acetylcholinesterase (ColQ-AChE), together with other components of the extracellular matrix (ECM) and specific key molecules in the NMJ formation. Interestingly, in addition to their cholinergic role AChE is thought to play several "non-classical" roles that do not require catalytic function, most prominent among these is the facilitation of neurite growth, NMJ formation and survival. In all this context, abnormalities of AChE content have been found in plasma of ALS patients, in which AChE changes may reflect the neuromuscular disruption. We review these findings and particularly the evidences of changes of AChE at neuromuscular synapse in the pre-symptomatic stages of ALS.
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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.
Assuntos
Colágeno Tipo VI/metabolismo , Desenvolvimento Muscular , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Colágeno Tipo VI/genética , Expressão Gênica , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Thirst and antidiuretic hormone secretion occur during hyperthermia or hypertonicity to preserve body hydration. These vital responses are triggered when hypothalamic osmoregulatory neurons become depolarized by ion channels encoded by an unknown product of the transient receptor potential vanilloid-1 gene (Trpv1). Here, we show that rodent osmoregulatory neurons express a transcript of Trpv1 that mediates the selective translation of a TRPV1 variant that lacks a significant portion of the channel's amino terminus (ΔN-TRPV1). The mRNA transcript encoding this variant (Trpv1dn) is widely expressed in the brains of osmoregulating vertebrates, including the human hypothalamus. Transfection of Trpv1dn into heterologous cells induced the expression of ion channels that could be activated by either hypertonicity or by heating in the physiological range. Moreover, expression of Trpv1dn rescued the osmosensory and thermosensory responses of single hypothalamic neurons obtained from Trpv1 knockout mice. ΔN-TRPV1 is therefore a co-detector of core body temperature and fluid tonicity.
Assuntos
Hipotálamo/metabolismo , Neurônios/metabolismo , Osmorregulação/fisiologia , Pressão Osmótica/fisiologia , Canais de Cátion TRPV/genética , Potenciais de Ação/fisiologia , Sequência de Aminoácidos , Animais , Temperatura Corporal , Tamanho Celular , Feto , Regulação da Expressão Gênica , Células HEK293 , Temperatura Alta , Humanos , Hipotálamo/citologia , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Neurônios/citologia , Técnicas de Patch-Clamp , Cultura Primária de Células , Biossíntese de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Ratos Long-Evans , Alinhamento de Sequência , Transdução de Sinais , Canais de Cátion TRPV/metabolismoRESUMO
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.
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Esclerose Lateral Amiotrófica/genética , Demência Frontotemporal/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Animais , Modelos Animais de Doenças , Variação Genética , Humanos , Padrões de Herança , Fatores de Risco , Pesquisa Translacional BiomédicaRESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Esclerose Lateral Amiotrófica/genética , Degeneração Lobar Frontotemporal/genética , Locomoção/genética , Sirolimo/farmacologia , Proteínas de Peixe-Zebra/genética , Esclerose Lateral Amiotrófica/tratamento farmacológico , Animais , Modelos Animais de Doenças , Degeneração Lobar Frontotemporal/tratamento farmacológico , Técnicas de Silenciamento de Genes , Locomoção/efeitos dos fármacos , Fenótipo , Proteína Sequestossoma-1 , Serina-Treonina Quinases TOR/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Homozygous mutations in TREM2 have been recently identified by exome sequencing in families presenting with frontotemporal dementia (FTD)-like phenotype. No study has evaluated the exact frequency of TREM2 mutations in cohorts of FTD patients so far. We sequenced TREM2 in 175 patients with pure FTD, mostly French, to test whether mutations could be implicated in the pathogenesis of the disease. No disease-causing mutation was identified in 175 individuals from the French cohort of FTD patients. We did not identify the polymorphism p.R47H (rs75932628), strongly associated with an increased risk of developing Alzheimer's disease. We conclude that TREM2 mutations are extremely rare in patients with pure FTD, although further investigation in larger populations is needed.
Assuntos
Demência Frontotemporal/etnologia , Demência Frontotemporal/genética , Glicoproteínas de Membrana/genética , Taxa de Mutação , Mutação , Receptores Imunológicos/genética , Estudos de Coortes , Exoma/genética , França/etnologia , Humanos , Pessoa de Meia-Idade , População Branca/genéticaRESUMO
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.