RESUMEN
Identifying genetic modifiers of familial amyotrophic lateral sclerosis (ALS) may reveal targets for therapeutic modulation with potential application to sporadic ALS. GGGGCC (G4C2) repeat expansions in the C9orf72 gene underlie the most common form of familial ALS, and generate toxic arginine-containing dipeptide repeats (DPRs), which interfere with membraneless organelles, such as the nucleolus. Here we considered senataxin (SETX), the genetic cause of ALS4, as a modifier of C9orf72 ALS, because SETX is a nuclear helicase that may regulate RNA-protein interactions involved in ALS dysfunction. After documenting that decreased SETX expression enhances arginine-containing DPR toxicity and C9orf72 repeat expansion toxicity in HEK293 cells and primary neurons, we generated SETX fly lines and evaluated the effect of SETX in flies expressing either (G4C2)58 repeats or glycine-arginine-50 [GR(50)] DPRs. We observed dramatic suppression of disease phenotypes in (G4C2)58 and GR(50) Drosophila models, and detected a striking relocalization of GR(50) out of the nucleolus in flies co-expressing SETX. Next-generation GR(1000) fly models, that show age-related motor deficits in climbing and movement assays, were similarly rescued with SETX co-expression. We noted that the physical interaction between SETX and arginine-containing DPRs is partially RNA-dependent. Finally, we directly assessed the nucleolus in cells expressing GR-DPRs, confirmed reduced mobility of proteins trafficking to the nucleolus upon GR-DPR expression, and found that SETX dosage modulated nucleolus liquidity in GR-DPR-expressing cells and motor neurons. These findings reveal a hitherto unknown connection between SETX function and cellular processes contributing to neuron demise in the most common form of familial ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Humanos , Animales , Esclerosis Amiotrófica Lateral/metabolismo , Dipéptidos/genética , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Arginina/genética , Arginina/metabolismo , Células HEK293 , Neuronas Motoras/metabolismo , Drosophila/metabolismo , ARN/metabolismo , Demencia Frontotemporal/genética , Expansión de las Repeticiones de ADN/genética , ADN Helicasas/genética , ARN Helicasas/genética , Enzimas Multifuncionales/genéticaRESUMEN
Sleep is thought to be controlled by two main processes: a circadian clock that primarily regulates sleep timing and a homeostatic mechanism that detects and responds to sleep need. Whereas abundant experimental evidence suggests that sleep need increases with time spent awake, the contributions of different brain arousal systems have not been assessed independently of each other to determine whether certain neural circuits, rather than waking per se, selectively contribute to sleep homeostasis. Using the fruit fly, Drosophila melanogaster, we found that sustained thermogenetic activation of three independent neurotransmitter systems promoted nighttime wakefulness. However, only sleep deprivation resulting from activation of cholinergic neurons was sufficient to elicit subsequent homeostatic recovery sleep, as assessed by multiple behavioral criteria. In contrast, sleep deprivation resulting from activation of octopaminergic neurons suppressed homeostatic recovery sleep, indicating that wakefulness can be dissociated from accrual of sleep need. Neurons that promote sleep homeostasis were found to innervate the central brain and motor control regions of the thoracic ganglion. Blocking activity of these neurons suppressed recovery sleep but did not alter baseline sleep, further differentiating between neural control of sleep homeostasis and daily fluctuations in the sleep/wake cycle. Importantly, selective activation of wake-promoting neurons without engaging the sleep homeostat impaired subsequent short-term memory, thus providing evidence that neural circuits that regulate sleep homeostasis are important for behavioral plasticity. Together, our data suggest a neural circuit model involving distinct populations of wake-promoting neurons, some of which are involved in homeostatic control of sleep and cognition.
Asunto(s)
Drosophila melanogaster/fisiología , Sueño/fisiología , Animales , Nivel de Alerta/fisiología , Encéfalo/fisiología , Ritmo Circadiano , Homeostasis/fisiología , Memoria a Corto Plazo/fisiología , Modelos Animales , Neuronas/fisiología , Receptores de Amina Biogénica/fisiología , Vigilia/fisiologíaRESUMEN
Many protein-coding genes identified by genome sequencing remain without functional annotation or biological context. Here we define a novel protein-coding gene, Nmf9, based on a forward genetic screen for neurological function. ENU-induced and genome-edited null mutations in mice produce deficits in vestibular function, fear learning and circadian behavior, which correlated with Nmf9 expression in inner ear, amygdala, and suprachiasmatic nuclei. Homologous genes from unicellular organisms and invertebrate animals predict interactions with small GTPases, but the corresponding domains are absent in mammalian Nmf9. Intriguingly, homozygotes for null mutations in the Drosophila homolog, CG45058, show profound locomotor defects and premature death, while heterozygotes show striking effects on sleep and activity phenotypes. These results link a novel gene orthology group to discrete neurological functions, and show conserved requirement across wide phylogenetic distance and domain level structural changes.
Asunto(s)
Péptidos y Proteínas de Señalización del Ritmo Circadiano/genética , Ritmo Circadiano/fisiología , Proteínas de Drosophila/genética , Miedo/fisiología , Proteínas del Tejido Nervioso/genética , Vestíbulo del Laberinto/patología , Amígdala del Cerebelo/metabolismo , Animales , Secuencia de Bases , Conducta Animal/fisiología , Drosophila melanogaster/genética , Femenino , Eliminación de Gen , Locomoción/genética , Masculino , Ratones , Ratones Endogámicos AKR , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Análisis de Secuencia de ADN , Factores Sexuales , Sueño/genética , Sueño/fisiología , Núcleo Supraquiasmático/metabolismo , Pruebas de Función Vestibular , Vestíbulo del Laberinto/fisiologíaRESUMEN
To assess the potential of Drosophila to analyze clinically graded aspects of human disease, we developed a transgenic fly model to characterize Presenilin (PS) gene mutations that cause early-onset familial Alzheimer's disease (FAD). FAD exhibits a wide range in severity defined by ages of onset from 24 to 65 years . PS FAD mutants have been analyzed in mammalian cell culture, but conflicting data emerged concerning correlations between age of onset and PS biochemical activity . Choosing from over 130 FAD mutations in Presenilin-1, we introduced 14 corresponding mutations at conserved residues in Drosophila Presenilin (Psn) and assessed their biological activity in transgenic flies by using genetic, molecular, and statistical methods. Psn FAD mutant activities were tightly linked to their age-of-onset values, providing evidence that disease severity in humans primarily reflects differences in PS mutant lesions rather than contributions from unlinked genetic or environmental modifiers. Our study establishes a precedent for using transgenic Drosophila to study clinical heterogeneity in human disease.
Asunto(s)
Enfermedad de Alzheimer/genética , Proteínas de Drosophila/fisiología , Drosophila/fisiología , Proteínas de la Membrana/fisiología , Adulto , Edad de Inicio , Anciano , Animales , Animales Modificados Genéticamente , Modelos Animales de Enfermedad , Drosophila/genética , Proteínas de Drosophila/genética , Heterogeneidad Genética , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Persona de Mediana Edad , Mutación , Presenilina-1 , PresenilinasRESUMEN
Before establishing terminal synapses with their final muscle targets, migrating motor axons form en passant synaptic contacts with myotomal muscle. Whereas signaling through terminal synapses has been shown to play important roles in pre- and postsynaptic development, little is known about the function of these early en passant synaptic contacts. Here, we show that increased neuromuscular activity through en passant synaptic contacts affects pre- and postsynaptic development. We demonstrate that in zebrafish twister mutants, prolonged neuromuscular transmission causes motor axonal extension and muscular degeneration in a dose-dependent manner. Cloning of twister reveals a novel, dominant gain-of-function mutation in the muscle-specific nicotinic acetylcholine receptor alpha-subunit, CHRNA1. Moreover, electrophysiological analysis demonstrates that the mutant subunit increases synaptic decay times, thereby prolonging postsynaptic activity. We show that as the first en passant synaptic contacts form, excessive postsynaptic activity in homozygous embryos severely impedes pre- and postsynaptic development, leading to degenerative defects characteristic of the human slow-channel congenital myasthenic syndrome. By contrast, in heterozygous embryos, transient and mild increase in postsynaptic activity does not overtly affect postsynaptic morphology but causes transient axonal defects, suggesting bi-directional communication between motor axons and myotomal muscle. Together, our results provide compelling evidence that during pathfinding, myotomal muscle cells communicate extensively with extending motor axons through en passant synaptic contacts.