RESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that results from many diverse genetic causes. Although therapeutics specifically targeting known causal mutations may rescue individual types of ALS, these approaches cannot treat most cases since they have unknown genetic etiology. Thus, there is a pressing need for therapeutic strategies that rescue multiple forms of ALS. Here, we show that pharmacological inhibition of PIKFYVE kinase activates an unconventional protein clearance mechanism involving exocytosis of aggregation-prone proteins. Reducing PIKFYVE activity ameliorates ALS pathology and extends survival of animal models and patient-derived motor neurons representing diverse forms of ALS including C9ORF72, TARDBP, FUS, and sporadic. These findings highlight a potential approach for mitigating ALS pathogenesis that does not require stimulating macroautophagy or the ubiquitin-proteosome system.
Asunto(s)
Esclerosis Amiotrófica Lateral , Fosfatidilinositol 3-Quinasas , Animales , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Neuronas Motoras , Mutación , Proteína FUS de Unión a ARN/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Modelos Animales de EnfermedadRESUMEN
Proteome modifications downstream of monogenic or polygenic disorders have the potential to uncover novel molecular mechanisms participating in pathogenesis and/or extragenic modification of phenotypic expression. We tested this idea by determining the proteome sensitive to genetic defects in a locus encoding dysbindin, a protein required for synapse biology and implicated in schizophrenia risk. We applied quantitative mass spectrometry to identify proteins expressed in neuronal cells the abundance of which was altered after downregulation of the schizophrenia susceptibility factor dysbindin (Bloc1s8) or two other dysbindin-interacting polypeptides, which assemble into the octameric biogenesis of lysosome-related organelles complex 1 (BLOC-1). We found 491 proteins sensitive to dysbindin and BLOC-1 loss of function. Gene ontology of these 491 proteins singled out the actin cytoskeleton and the actin polymerization factor, the Arp2/3 complex, as top statistical molecular pathways contained within the BLOC-1-sensitive proteome. Subunits of the Arp2/3 complex were downregulated by BLOC-1 loss of function, thus affecting actin dynamics in early endosomes of BLOC-1-deficient cells. Furthermore, we demonstrated that Arp2/3, dysbindin, and subunits of the BLOC-1 complex biochemically and genetically interact, modulating Drosophila melanogaster synapse morphology and homeostatic synaptic plasticity. Our results indicate that ontologically prioritized proteomics identifies novel pathways that modify synaptic phenotypes associated with neurodevelopmental disorder gene defects. SIGNIFICANCE STATEMENT: The mechanisms associated with schizophrenia are mostly unknown despite the increasing number of genetic loci identified that increase disease risk. We present an experimental strategy that impartially and comprehensively interrogates the proteome of neurons to identify effects of genetic mutations in a schizophrenia risk factor, dysbindin. We find that the expression of the actin polymerization complex Arp2/3 is reduced in dysbindin-deficient cells, thus affecting actin-dependent phenotypes in two cellular compartments where dysbindin resides, endosomes and presynapses. Our studies indicate that a central cellular structure affected by schizophrenia susceptibility loci is the actin cytoskeleton, an organelle necessary for synaptic function in the presynaptic and postsynaptic compartment.
Asunto(s)
Proteína 3 Relacionada con la Actina/genética , Angiopoyetinas/genética , Proteínas Portadoras/genética , Proteínas Asociadas a la Distrofina/genética , Lectinas/genética , Esquizofrenia/genética , Sinapsis , Actinas/genética , Proteína 2 Similar a la Angiopoyetina , Proteínas Similares a la Angiopoyetina , Animales , Células Cultivadas , Citoesqueleto/genética , Drosophila melanogaster , Disbindina , Femenino , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Endogámicos C57BL , Polimerizacion , ProteomaRESUMEN
Neurodevelopmental disorders arise from single or multiple gene defects. However, the way multiple loci interact to modify phenotypic outcomes remains poorly understood. Here, we studied phenotypes associated with mutations in the schizophrenia susceptibility gene dysbindin (dysb), in isolation or in combination with null alleles in the dysb network component Blos1. In humans, the Blos1 ortholog Bloc1s1 encodes a polypeptide that assembles, with dysbindin, into the octameric BLOC-1 complex. We biochemically confirmed BLOC-1 presence in Drosophila neurons, and measured synaptic output and complex adaptive behavior in response to BLOC-1 perturbation. Homozygous loss-of-function alleles of dysb, Blos1, or compound heterozygotes of these alleles impaired neurotransmitter release, synapse morphology, and homeostatic plasticity at the larval neuromuscular junction, and impaired olfactory habituation. This multiparameter assessment indicated that phenotypes were differentially sensitive to genetic dosages of loss-of-function BLOC-1 alleles. Our findings suggest that modification of a second genetic locus in a defined neurodevelopmental regulatory network does not follow a strict additive genetic inheritance, but rather, precise stoichiometry within the network determines phenotypic outcomes.
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Proteínas Portadoras/genética , Proteínas de Drosophila/genética , Dosificación de Gen/fisiología , Red Nerviosa/fisiología , Plasticidad Neuronal/fisiología , Esquizofrenia/genética , Sinapsis/genética , Animales , Animales Modificados Genéticamente , Drosophila , Disbindina , Proteínas Asociadas a la Distrofina , Femenino , Red Nerviosa/ultraestructura , Esquizofrenia/fisiopatología , Sinapsis/ultraestructura , Transmisión Sináptica/fisiologíaRESUMEN
The fruit fly Drosophila melanogaster has been established as a premier experimental model system for neuroscience research. These organisms are genetically tractable, yet their nervous systems are sufficiently complex to study diverse processes that are conserved across metazoans, including neural cell fate determination and migration, axon guidance, synaptogenesis and function, behavioral neurogenetics, and responses to neuronal injury. For several decades, Drosophila neuroscientists have taken advantage of a vast toolkit of genetic and molecular techniques to reveal fundamental principles of neuroscience illuminating to all systems, including the first behavioral mutants from Seymour Benzer's pioneering work in the 1960s and 1970s, the cloning of the first potassium channel in the 1980s, and the identification of the core genes that orchestrate axon guidance and circadian rhythms in the 1990s. Over the past decade, new tools and innovations in genetic, imaging, and electrophysiological technologies have enabled the visualization, in vivo, of dynamic processes in synapses with unprecedented resolution. We will review some of the fresh insights into synaptic development, function, and plasticity that have recently emerged in Drosophila with an emphasis on the unique advantages of this model system.
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Drosophila/fisiología , Plasticidad Neuronal/fisiología , Neuronas/fisiología , Sinapsis/fisiología , Animales , Conducta Animal/fisiología , Neurogénesis/fisiologíaRESUMEN
The molecular mechanisms underlying the homeostatic modulation of presynaptic neurotransmitter release are largely unknown. We have previously used an electrophysiology-based forward genetic screen to assess the function of >400 neuronally expressed genes for a role in the homeostatic control of synaptic transmission at the neuromuscular junction of Drosophila melanogaster. This screen identified a critical function for dysbindin, a gene linked to schizophrenia in humans (Dickman and Davis, 2009). Biochemical studies in other systems have shown that Snapin interacts with Dysbindin, prompting us to test whether Snapin might be involved in the mechanisms of synaptic homeostasis. Here, we demonstrate that loss of snapin blocks the homeostatic modulation of presynaptic vesicle release following inhibition of postsynaptic glutamate receptors. This is true for both the rapid induction of synaptic homeostasis induced by pharmacological inhibition of postsynaptic glutamate receptors, and the long-term expression of synaptic homeostasis induced by the genetic deletion of the muscle-specific GluRIIA glutamate receptor subunit. Loss of snapin does not alter baseline synaptic transmission, synapse morphology, synapse growth, or the number or density of active zones, indicating that the block of synaptic homeostasis is not a secondary consequence of impaired synapse development. Additional genetic evidence suggests that snapin functions in concert with dysbindin to modulate vesicle release and possibly homeostatic plasticity. Finally, we provide genetic evidence that the interaction of Snapin with SNAP25, a component of the SNARE complex, is also involved in synaptic homeostasis.
Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiología , Homeostasis/fisiología , Plasticidad Neuronal/fisiología , Terminales Presinápticos/fisiología , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/fisiología , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Disbindina , Proteínas Asociadas a la Distrofina , Fenómenos Electrofisiológicos , Homeostasis/genética , Inmunohistoquímica , Unión Neuromuscular/fisiología , Unión Neuromuscular/ultraestructura , Plasticidad Neuronal/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas SNARE/genética , Proteínas SNARE/fisiología , Transducción de Señal/genética , Transducción de Señal/fisiología , Transmisión Sináptica/fisiología , Proteína 25 Asociada a Sinaptosomas/genética , Proteína 25 Asociada a Sinaptosomas/metabolismoRESUMEN
Importin proteins act both at the nuclear pore to promote substrate entry and in the cytosol during signal trafficking. Here, we describe mutations in the Drosophila gene importin-beta11, which has not previously been analyzed genetically. Mutants of importin-beta11 died as late pupae from neuronal defects, and neuronal importin-beta11 was present not only at nuclear pores but also in the cytosol and at synapses. Neurons lacking importin-beta11 were viable and properly differentiated but exhibited discrete defects. Synaptic transmission was defective in adult photoreceptors and at larval neuromuscular junctions (NMJs). Mutant photoreceptor axons formed grossly normal projections and synaptic terminals in the brain, but synaptic arbors on larval muscles were smaller while still containing appropriate synaptic components. Bone morphogenic protein (BMP) signaling was the apparent cause of the observed NMJ defects. Importin-beta11 interacted genetically with the BMP pathway, and at mutant synaptic boutons, a key component of this pathway, phosphorylated mothers against decapentaplegic (pMAD), was reduced. Neuronal expression of an importin-beta11 transgene rescued this phenotype as well as the other observed neuromuscular phenotypes. Despite the loss of synaptic pMAD, pMAD persisted in motor neuron nuclei, suggesting a specific impairment in the local function of pMAD. Restoring levels of pMAD to mutant terminals via expression of constitutively active type I BMP receptors or by reducing retrograde transport in motor neurons also restored synaptic strength and morphology. Thus, importin-beta11 function interacts with the BMP pathway to regulate a pool of pMAD that must be present at the presynapse for its proper development and function.
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Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Carioferinas/metabolismo , Unión Neuromuscular/fisiología , Neuronas/fisiología , Sinapsis/fisiología , Factores de Transcripción/metabolismo , Animales , Animales Modificados Genéticamente , Axones/fisiología , Transporte Biológico Activo , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Encéfalo/citología , Encéfalo/crecimiento & desarrollo , Encéfalo/fisiología , Núcleo Celular/fisiología , Citosol/fisiología , Drosophila , Proteínas de Drosophila/genética , Carioferinas/genética , Neuronas Motoras/citología , Neuronas Motoras/fisiología , Mutación , Unión Neuromuscular/citología , Unión Neuromuscular/crecimiento & desarrollo , Neuronas/citología , Células Fotorreceptoras de Invertebrados/citología , Células Fotorreceptoras de Invertebrados/fisiología , Terminales Presinápticos/fisiología , Transducción de Señal , Transmisión Sináptica/fisiologíaRESUMEN
The morphological transition of growth cones to synaptic boutons characterizes synaptogenesis. Here we have isolated mutations in immaculate connections (imac; CG8566), a previously uncharacterized Drosophila gene encoding a member of the Kinesin-3 family. Whereas earlier studies in Drosophila implicated Kinesin-1 in transporting synaptic vesicle precursors, we find that Imac is essential for this transport. An unexpected feature of imac mutants is the failure of synaptic boutons to form. Motor neurons lacking imac properly target to muscles but remain within target fields as thin processes, a structure that is distinct from either growth cones or mature terminals. Few active zones form at these endings. We show that the arrest of synaptogenesis is not a secondary consequence of the absence of transmission. Our data thus indicate that Imac transports components required for synaptic maturation and provide insight into presynaptic maturation as a process that can be differentiated from axon outgrowth and targeting.
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Proteínas de Drosophila/fisiología , Cinesinas/fisiología , Unión Neuromuscular/fisiología , Terminales Presinápticos/fisiología , Vesículas Sinápticas/fisiología , Animales , Animales Modificados Genéticamente , Drosophila , Proteínas de Drosophila/genética , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Cinesinas/genética , Microscopía Electrónica de Transmisión , Neuronas Motoras/citología , Proteínas del Tejido Nervioso/metabolismo , Unión Neuromuscular/citología , Células Fotorreceptoras de Invertebrados/citología , Terminales Presinápticos/ultraestructura , Transporte de Proteínas/fisiología , Vesículas Sinápticas/ultraestructuraRESUMEN
Voltage-dependent calcium channels regulate many aspects of neuronal biology, including synaptic transmission. In addition to their alpha1 subunit, which encodes the essential voltage gate and selective pore, calcium channels also contain auxiliary alpha2delta, beta, and gamma subunits. Despite progress in understanding the biophysical properties of calcium channels, the in vivo functions of these auxiliary subunits remain unclear. We have isolated mutations in the gene encoding an alpha2delta calcium channel subunit (d alpha2delta-3) using a forward genetic screen in Drosophila. Null mutations in this gene are embryonic lethal and can be rescued by expression in the nervous system, demonstrating that the essential function of this subunit is neuronal. The photoreceptor phenotype of d alpha2delta-3 mutants resembles that of the calcium channel alpha1 mutant cacophony (cac), suggesting shared functions. We have examined in detail genotypes that survive to the third-instar stage. Electrophysiological recordings demonstrate that synaptic transmission is severely impaired in these mutants. Thus the alpha2delta calcium channel subunit is critical for calcium-dependent synaptic function. As such, this Drosophila isoform is the likely partner to the presynaptic calcium channel alpha1 subunit encoded by the cac locus. Consistent with this hypothesis, cacGFP fluorescence at the neuromuscular junction is reduced in d alpha2delta-3 mutants. This is the first characterization of an alpha2delta-3 mutant in any organism and indicates a necessary role for alpha2delta-3 in presynaptic vesicle release and calcium channel expression at active zones.
Asunto(s)
Canales de Calcio/genética , Mutación/fisiología , Unión Neuromuscular/fisiología , Subunidades de Proteína/genética , Transmisión Sináptica/genética , Animales , Animales Modificados Genéticamente , Calcio/metabolismo , Drosophila , Proteínas de Drosophila/genética , Electrorretinografía/métodos , Embrión no Mamífero , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Unión Neuromuscular/genética , Terminales Presinápticos/metabolismo , Subunidades de Proteína/metabolismoRESUMEN
Many types of synapses have highly characteristic shapes and tightly regulated distributions of active zones, parameters that are important to the function of neuronal circuits. The development of terminal arborizations must therefore include mechanisms to regulate the spacing of terminals, the frequency of branching, and the distribution and density of release sites. At present, however, the mechanisms that control these features remain obscure. Here, we report the development of supernumerary or "satellite" boutons in a variety of endocytic mutants at the Drosophila neuromuscular junction. Mutants in endophilin, synaptojanin, dynamin, AP180, and synaptotagmin all show increases in supernumerary bouton structures. These satellite boutons contain releasable vesicles and normal complements of synaptic proteins that are correctly localized within terminals. Interestingly, however, synaptojanin terminals have more active zones per unit of surface area and more dense bodies (T-bars) within these active zones, which may in part compensate for reduced transmission per active zone. The altered structural development of the synapse is selectively encountered in endocytosis mutants and is not observed when synaptic transmission is reduced by mutations in glutamate receptors or when synaptic transmission is blocked by tetanus toxin. We propose that endocytosis plays a critical role in sculpting the structure of synapses, perhaps through the endocytosis of unknown regulatory signals that organize morphogenesis at synaptic terminals.
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Proteínas de Drosophila/genética , Drosophila/crecimiento & desarrollo , Endocitosis/fisiología , Unión Neuromuscular/crecimiento & desarrollo , Terminales Presinápticos/ultraestructura , Aciltransferasas/genética , Animales , Drosophila/genética , Drosophila/ultraestructura , Dinaminas/genética , Endocitosis/genética , Procesamiento de Imagen Asistido por Computador , Larva/genética , Larva/crecimiento & desarrollo , Larva/ultraestructura , Microscopía Electrónica de Rastreo , Proteínas de Ensamble de Clatrina Monoméricas/genética , Mutación , Proteínas del Tejido Nervioso/genética , Unión Neuromuscular/ultraestructura , Fenotipo , Monoéster Fosfórico Hidrolasas/genética , Terminales Presinápticos/metabolismo , Terminales Presinápticos/fisiologíaRESUMEN
Research on neuropeptide function has advanced rapidly, yet there is still no spatio-temporally resolved method to measure the release of neuropeptides in vivo. Here we introduce Neuropeptide Release Reporters (NPRRs): novel genetically-encoded sensors with high temporal resolution and genetic specificity. Using the Drosophila larval neuromuscular junction (NMJ) as a model, we provide evidence that NPRRs recapitulate the trafficking and packaging of native neuropeptides, and report stimulation-evoked neuropeptide release events as real-time changes in fluorescence intensity, with sub-second temporal resolution.
Asunto(s)
Genes Reporteros , Ingeniería Genética , Imagen Molecular , Neuropéptidos/metabolismo , Sinapsis/metabolismo , Animales , Drosophila/metabolismo , Drosophila/ultraestructura , Sinapsis/ultraestructuraRESUMEN
Postsynaptic compartments can be specifically modulated during various forms of synaptic plasticity, but it is unclear whether this precision is shared at presynaptic terminals. Presynaptic homeostatic plasticity (PHP) stabilizes neurotransmission at the Drosophila neuromuscular junction, where a retrograde enhancement of presynaptic neurotransmitter release compensates for diminished postsynaptic receptor functionality. To test the specificity of PHP induction and expression, we have developed a genetic manipulation to reduce postsynaptic receptor expression at one of the two muscles innervated by a single motor neuron. We find that PHP can be induced and expressed at a subset of synapses, over both acute and chronic time scales, without influencing transmission at adjacent release sites. Further, homeostatic modulations to CaMKII, vesicle pools, and functional release sites are compartmentalized and do not spread to neighboring pre- or post-synaptic structures. Thus, both PHP induction and expression mechanisms are locally transmitted and restricted to specific synaptic compartments.
Asunto(s)
Drosophila melanogaster/fisiología , Potenciales Postsinápticos Excitadores , Homeostasis , Unión Neuromuscular/fisiología , Terminales Presinápticos/fisiología , Sinapsis/fisiología , Animales , Proteínas de Drosophila/metabolismo , Receptores Ionotrópicos de Glutamato/metabolismo , Transmisión SinápticaRESUMEN
The biogenesis of lysosome-related organelles complex-1 (BLOC-1) and the bloc-one-related complex (BORC) are the cytosolic protein complexes required for specialized membrane protein traffic along the endocytic route and the spatial distribution of endosome-derived compartments, respectively. BLOC-1 and BORC complex subunits and components of their interactomes have been associated with the risk and/or pathomechanisms of neurodevelopmental disorders. Thus, cellular processes requiring BLOC-1 and BORC interactomes have the potential to offer novel insight into mechanisms underlying behavioral defects. We focus on interactions between BLOC-1 or BORC subunits with the actin and microtubule cytoskeleton, membrane tethers, and SNAREs. These interactions highlight requirements for BLOC-1 and BORC in membrane movement by motors, control of actin polymerization, and targeting of membrane proteins to specialized cellular domains such as the nerve terminal and the primary cilium. We propose that the endosome-primary cilia pathway is an underappreciated hub in the genesis and mechanisms of neurodevelopmental disorders. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 311-330, 2018.
Asunto(s)
Cilios/metabolismo , Endosomas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Trastornos del Neurodesarrollo/metabolismo , Animales , HumanosRESUMEN
Homeostatic signaling systems are thought to interface with other forms of plasticity to ensure flexible yet stable levels of neurotransmission. The role of neurotransmitter receptors in this process, beyond mediating neurotransmission itself, is not known. Through a forward genetic screen, we have identified the Drosophila kainate-type ionotropic glutamate receptor subunit DKaiR1D to be required for the retrograde, homeostatic potentiation of synaptic strength. DKaiR1D is necessary in presynaptic motor neurons, localized near active zones, and confers robustness to the calcium sensitivity of baseline synaptic transmission. Acute pharmacological blockade of DKaiR1D disrupts homeostatic plasticity, indicating that this receptor is required for the expression of this process, distinct from developmental roles. Finally, we demonstrate that calcium permeability through DKaiR1D is necessary for baseline synaptic transmission, but not for homeostatic signaling. We propose that DKaiR1D is a glutamate autoreceptor that promotes robustness to synaptic strength and plasticity with active zone specificity.
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Ácido Glutámico/metabolismo , Homeostasis/fisiología , Receptores Presinapticos/metabolismo , Transmisión Sináptica/fisiología , Animales , Humanos , RatonesRESUMEN
The endoplasmic reticulum (ER) is an extensive organelle in neurons with important roles at synapses including the regulation of cytosolic Ca2+, neurotransmission, lipid metabolism, and membrane trafficking. Despite intriguing evidence for these crucial functions, how the presynaptic ER influences synaptic physiology remains enigmatic. To gain insight into this question, we have generated and characterized mutations in the single extended synaptotagmin (Esyt) ortholog in Drosophila melanogaster Esyts are evolutionarily conserved ER proteins with Ca2+-sensing domains that have recently been shown to orchestrate membrane tethering and lipid exchange between the ER and plasma membrane. We first demonstrate that Esyt localizes to presynaptic ER structures at the neuromuscular junction. Next, we show that synaptic growth, structure, and homeostatic plasticity are surprisingly unperturbed at synapses lacking Esyt expression. However, neurotransmission is reduced in Esyt mutants, consistent with a presynaptic role in promoting neurotransmitter release. Finally, neuronal overexpression of Esyt enhances synaptic growth and the sustainment of the vesicle pool during intense activity, suggesting that increased Esyt levels may modulate the membrane trafficking and/or resting Ca2+ pathways that control synapse extension. Thus, we identify Esyt as a presynaptic ER protein that can promote neurotransmission and synaptic growth, revealing the first in vivo neuronal functions of this conserved gene family.
Asunto(s)
Proteínas de Drosophila/metabolismo , Retículo Endoplásmico/metabolismo , Proyección Neuronal , Terminales Presinápticos/metabolismo , Transmisión Sináptica , Sinaptotagminas/metabolismo , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster , Unión Neuromuscular/metabolismo , Unión Neuromuscular/fisiología , Terminales Presinápticos/fisiología , Sinaptotagminas/genéticaRESUMEN
Membrane trafficking pathways must be exquisitely coordinated at synaptic terminals to maintain functionality, particularly during conditions of high activity. We have generated null mutations in the Drosophila homolog of pallidin, a central subunit of the biogenesis of lysosome-related organelles complex-1 (BLOC-1), to determine its role in synaptic development and physiology. We find that Pallidin localizes to presynaptic microtubules and cytoskeletal structures, and that the stability of Pallidin protein is highly dependent on the BLOC-1 components Dysbindin and Blos1. We demonstrate that the rapidly recycling vesicle pool is not sustained during high synaptic activity in pallidin mutants, leading to accelerated rundown and slowed recovery. Following intense activity, we observe a loss of early endosomes and a concomitant increase in tubular endosomal structures in synapses without Pallidin. Together, our data reveal that Pallidin subserves a key role in promoting efficient synaptic vesicle recycling and re-formation through early endosomes during sustained activity.
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Proteínas de Drosophila/metabolismo , Endocitosis/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Transmisión Sináptica/fisiología , Vesículas Sinápticas/metabolismo , Animales , Animales Modificados Genéticamente , Drosophila , Proteínas de Drosophila/genética , Disbindina , Proteínas Asociadas a la Distrofina/metabolismo , Endosomas/metabolismo , Proteínas del Ojo/metabolismo , Homeostasis/fisiología , Immunoblotting , Inmunohistoquímica , Microscopía Electrónica , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/metabolismo , Unión Neuromuscular/metabolismo , Unión Neuromuscular/ultraestructura , Biogénesis de Organelos , Técnicas de Placa-Clamp , Estabilidad ProteicaRESUMEN
Presynaptic homeostatic plasticity (PHP) controls synaptic transmission in organisms from Drosophila to human and is hypothesized to be relevant to the cause of human disease. However, the underlying molecular mechanisms of PHP are just emerging and direct disease associations remain obscure. In a forward genetic screen for mutations that block PHP we identified mctp (Multiple C2 Domain Proteins with Two Transmembrane Regions). Here we show that MCTP localizes to the membranes of the endoplasmic reticulum (ER) that elaborate throughout the soma, dendrites, axon and presynaptic terminal. Then, we demonstrate that MCTP functions downstream of presynaptic calcium influx with separable activities to stabilize baseline transmission, short-term release dynamics and PHP. Notably, PHP specifically requires the calcium coordinating residues in each of the three C2 domains of MCTP. Thus, we propose MCTP as a novel, ER-localized calcium sensor and a source of calcium-dependent feedback for the homeostatic stabilization of neurotransmission.
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Calcio/metabolismo , Plasticidad Neuronal , Neuronas/fisiología , Transmisión Sináptica , Animales , Línea Celular , DrosophilaRESUMEN
It is now appreciated that the brain is immunologically active. Highly conserved innate immune signaling responds to pathogen invasion and injury and promotes structural refinement of neural circuitry. However, it remains generally unknown whether innate immune signaling has a function during the day-to-day regulation of neural function in the absence of pathogens and irrespective of cellular damage or developmental change. Here we show that an innate immune receptor, a member of the peptidoglycan pattern recognition receptor family (PGRP-LC), is required for the induction and sustained expression of homeostatic synaptic plasticity. This receptor functions presynaptically, controlling the homeostatic modulation of the readily releasable pool of synaptic vesicles following inhibition of postsynaptic glutamate receptor function. Thus, PGRP-LC is a candidate receptor for retrograde, trans-synaptic signaling, a novel activity for innate immune signaling and the first known function of a PGRP-type receptor in the nervous system of any organism.
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Proteínas Portadoras/fisiología , Homeostasis/fisiología , Inmunidad Innata/fisiología , Plasticidad Neuronal/fisiología , Terminales Presinápticos/fisiología , Animales , Animales Modificados Genéticamente , Proteínas Portadoras/ultraestructura , Drosophila melanogaster , Terminales Presinápticos/ultraestructuraRESUMEN
At synapses in organisms ranging from fly to human, a decrease in postsynaptic neurotransmitter receptor function elicits a homeostatic increase in presynaptic release that restores baseline synaptic efficacy. This process, termed presynaptic homeostasis, requires a retrograde, trans-synaptic signal of unknown identity. In a forward genetic screen for homeostatic plasticity genes, we identified multiplexin. Multiplexin is the Drosophila homolog of Collagen XV/XVIII, a matrix protein that can be proteolytically cleaved to release Endostatin, an antiangiogenesis signaling factor. Here we demonstrate that Multiplexin is required for normal calcium channel abundance, presynaptic calcium influx, and neurotransmitter release. Remarkably, Endostatin has a specific activity, independent of baseline synapse development, that is required for the homeostatic modulation of presynaptic calcium influx and neurotransmitter release. Our data support a model in which proteolytic release of Endostatin signals trans-synaptically, acting in concert with the presynaptic CaV2.1 calcium channel, to promote presynaptic homeostasis.
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Calcio/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Colágeno/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Endostatinas/metabolismo , Homeostasis/fisiología , Plasticidad Neuronal/fisiología , Neurotransmisores/metabolismo , Animales , Canales de Calcio/metabolismo , Humanos , Transducción de Señal/fisiología , Sinapsis/metabolismoRESUMEN
Phosphorylation has emerged as a crucial regulatory mechanism in the nervous system to integrate the dynamic signalling required for proper synaptic development, function and plasticity, particularly during changes in neuronal activity. Here we present evidence that Minibrain (Mnb; also known as Dyrk1A), a serine/threonine kinase implicated in autism spectrum disorder and Down syndrome, is required presynaptically for normal synaptic growth and rapid synaptic vesicle endocytosis at the Drosophila neuromuscular junction (NMJ). We find that Mnb-dependent phosphorylation of Synaptojanin (Synj) is required, in vivo, for complex endocytic protein interactions and to enhance Synj activity. Neuronal stimulation drives Mnb mobilization to endocytic zones and triggers Mnb-dependent phosphorylation of Synj. Our data identify Mnb as a synaptic kinase that promotes efficient synaptic vesicle recycling by dynamically calibrating Synj function at the Drosophila NMJ, and in turn endocytic capacity, to adapt to conditions of high synaptic activity.