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.
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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
Robust neural information transfer relies on a delicate molecular nano-architecture of chemical synapses. Neurotransmitter release is controlled by a specific arrangement of proteins within presynaptic active zones. How the specific presynaptic molecular architecture relates to postsynaptic organization and how synaptic nano-architecture is transsynaptically regulated to enable stable synaptic transmission remain enigmatic. Using time-gated stimulated emission-depletion microscopy at the Drosophila neuromuscular junction, we found that presynaptic nanorings formed by the active-zone scaffold Bruchpilot (Brp) align with postsynaptic glutamate receptor (GluR) rings. Individual rings harbor approximately four transsynaptically aligned Brp-GluR nanocolumns. Similar nanocolumn rings are formed by the presynaptic protein Unc13A and GluRs. Intriguingly, acute GluR impairment triggers transsynaptic nanocolumn formation on the minute timescale during homeostatic plasticity. We reveal distinct phases of structural transsynaptic homeostatic plasticity, with postsynaptic GluR reorganization preceding presynaptic Brp modulation. Finally, homeostatic control of transsynaptic nano-architecture and neurotransmitter release requires the auxiliary GluR subunit Neto. Thus, transsynaptic nanocolumn rings provide a substrate for rapid homeostatic stabilization of synaptic efficacy.
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Proteínas de Drosophila , Unión Neuromuscular , Animales , Unión Neuromuscular/metabolismo , Drosophila/metabolismo , Transmisión Sináptica , Sinapsis/metabolismo , Receptores de Glutamato/metabolismo , Proteínas de Drosophila/metabolismo , Neurotransmisores/metabolismo , Proteínas de la Membrana/metabolismoRESUMEN
Neurons exhibit a striking degree of functional diversity, each one tuned to the needs of the circuitry in which it is embedded. A fundamental functional dichotomy occurs in activity patterns, with some neurons firing at a relatively constant "tonic" rate, while others fire in bursts, a "phasic" pattern. Synapses formed by tonic versus phasic neurons are also functionally differentiated, yet the bases of their distinctive properties remain enigmatic. A major challenge toward illuminating the synaptic differences between tonic and phasic neurons is the difficulty in isolating their physiological properties. At the Drosophila neuromuscular junction, most muscle fibers are coinnervated by two motor neurons: the tonic "MN-Ib" and phasic "MN-Is." Here, we used selective expression of a newly developed botulinum neurotoxin transgene to silence tonic or phasic motor neurons in Drosophila larvae of either sex. This approach highlighted major differences in their neurotransmitter release properties, including probability, short-term plasticity, and vesicle pools. Furthermore, Ca2+ imaging demonstrated â¼2-fold greater Ca2+ influx at phasic neuron release sites relative to tonic, along with an enhanced synaptic vesicle coupling. Finally, confocal and super-resolution imaging revealed that phasic neuron release sites are organized in a more compact arrangement, with enhanced stoichiometry of voltage-gated Ca2+ channels relative to other active zone scaffolds. These data suggest that distinctions in active zone nano-architecture and Ca2+ influx collaborate to differentially tune glutamate release at tonic versus phasic synaptic subtypes.SIGNIFICANCE STATEMENT "Tonic" and "phasic" neuronal subtypes, based on differential firing properties, are common across many nervous systems. Using a recently developed approach to selectively silence transmission from one of these two neurons, we reveal specialized synaptic functional and structural properties that distinguish these specialized neurons. This study provides important insights into how input-specific synaptic diversity is achieved, which could have implications for neurologic disorders that involve changes in synaptic function.
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Unión Neuromuscular , Sinapsis , Animales , Sinapsis/fisiología , Unión Neuromuscular/metabolismo , Vesículas Sinápticas/metabolismo , Neuronas Motoras/fisiología , DrosophilaRESUMEN
Synapses exhibit an astonishing degree of adaptive plasticity in healthy and disease states. We have investigated whether synapses also adjust to life stages imposed by novel developmental programs for which they were never molded by evolution. Under conditions in which Drosophila larvae are terminally arrested, we have characterized synaptic growth, structure and function at the neuromuscular junction (NMJ). Although wild-type larvae transition to pupae after 5â days, arrested third instar (ATI) larvae persist for 35â days, during which time NMJs exhibit extensive overgrowth in muscle size, presynaptic release sites and postsynaptic glutamate receptors. Remarkably, despite this exuberant growth, stable neurotransmission is maintained throughout the ATI lifespan through a potent homeostatic reduction in presynaptic neurotransmitter release. Arrest of the larval stage in stathmin mutants also reveals a degree of progressive instability and neurodegeneration that was not apparent during the typical larval period. Hence, an adaptive form of presynaptic depression stabilizes neurotransmission during an extended developmental period of unconstrained synaptic growth. More generally, the ATI manipulation provides a powerful system for studying neurodegeneration and plasticity across prolonged developmental timescales.
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Drosophila/crecimiento & desarrollo , Drosophila/genética , Larva/crecimiento & desarrollo , Larva/genética , Depresión Sináptica a Largo Plazo/genética , Degeneración Nerviosa/genética , Unión Neuromuscular/crecimiento & desarrollo , Animales , Axones/patología , Proteínas de Drosophila/genética , Femenino , Homeostasis/genética , Masculino , Mutación , Unión Neuromuscular/metabolismo , Interferencia de ARN , Proteínas Smad Reguladas por Receptores/genética , Estatmina/genética , Sinapsis/metabolismo , Transmisión Sináptica/genéticaRESUMEN
Presynaptic glutamate receptors (GluRs) modulate neurotransmitter release and are physiological targets for regulation during various forms of plasticity. Although much is known about the auxiliary subunits associated with postsynaptic GluRs, far less is understood about presynaptic auxiliary GluR subunits and their functions. At the Drosophila neuromuscular junction, a presynaptic GluR, DKaiR1D, localizes near active zones and operates as an autoreceptor to tune baseline transmission and enhance presynaptic neurotransmitter release in response to diminished postsynaptic GluR functionality, a process referred to as presynaptic homeostatic potentiation (PHP). Here, we identify an auxiliary subunit that collaborates with DKaiR1D to promote these synaptic functions. This subunit, dSol-1, is the homolog of the Caenorhabditis elegans CUB (Complement C1r/C1s, Uegf, Bmp1) domain protein Sol-1. We find that dSol-1 functions in neurons to facilitate baseline neurotransmission and to enable PHP expression, properties shared with DKaiR1D Intriguingly, presynaptic overexpression of dSol-1 is sufficient to enhance neurotransmitter release through a DKaiR1D-dependent mechanism. Furthermore, dSol-1 is necessary to rapidly increase the abundance of DKaiR1D receptors near active zones during homeostatic signaling. Together with recent work showing the CUB domain protein Neto2 is necessary for the homeostatic modulation of postsynaptic GluRs in mammals, our data demonstrate that dSol-1 is required for the homeostatic regulation of presynaptic GluRs. Thus, we propose that CUB domain proteins are fundamental homeostatic modulators of GluRs on both sides of the synapse.
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Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Proteínas de la Membrana/metabolismo , Neurotransmisores/metabolismo , Terminales Presinápticos/metabolismo , Animales , Drosophila/genética , Proteínas de Drosophila/genética , Homeostasis , Proteínas de la Membrana/genética , Unión Neuromuscular/metabolismo , Plasticidad Neuronal , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismo , Transmisión SinápticaRESUMEN
Homeostatic signaling systems are fundamental forms of biological regulation that maintain stable functionality in a changing environment. In the nervous system, synapses are crucial substrates for homeostatic modulation, serving to establish, maintain, and modify the balance of excitation and inhibition. Synapses must be sufficiently flexible to enable the plasticity required for learning and memory but also endowed with the stability to last a lifetime. In response to the processes of development, growth, remodeling, aging, and disease that challenge synapses, latent forms of adaptive plasticity become activated to maintain synaptic stability. In recent years, new insights into the homeostatic control of synaptic function have been achieved using the powerful Drosophila neuromuscular junction (NMJ). This review will focus on work over the past 10 years that has illuminated the cellular and molecular mechanisms of five homeostats that operate at the fly NMJ. These homeostats adapt to loss of postsynaptic neurotransmitter receptor functionality, glutamate imbalance, axonal injury, as well as aberrant synaptic growth and target innervation. These diverse homeostats work independently yet can be simultaneously expressed to balance neurotransmission. Growing evidence from this model glutamatergic synapse suggests these ancient homeostatic signaling systems emerged early in evolution and are fundamental forms of plasticity that also function to stabilize mammalian cholinergic NMJs and glutamatergic central synapses.
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Proteínas de Drosophila/metabolismo , Drosophila/fisiología , Homeostasis , Unión Neuromuscular/fisiología , Plasticidad Neuronal , Sinapsis/fisiología , Transmisión Sináptica , Animales , Transducción de SeñalRESUMEN
The ubiquitin ligase Highwire restrains synaptic growth and promotes evoked neurotransmission at NMJ synapses in Drosophila Highwire regulates synaptic morphology by downregulating the MAP3K Wallenda, but excess Wallenda signaling does not account for the decreased presynaptic release observed in highwire mutants. Hence, Highwire likely has a second substrate that inhibits neurotransmission. Highwire targets the NAD+ biosynthetic and axoprotective enzyme dNmnat to regulate axonal injury responses. dNmnat localizes to synapses and interacts with the active zone protein Bruchpilot, leading us to hypothesize that Highwire promotes evoked release by downregulating dNmnat. Here, we show that excess dNmnat is necessary in highwire mutants and sufficient in wild-type larvae to reduce quantal content, likely via disruption of active zone ultrastructure. Catalytically active dNmnat is required to drive defects in evoked release, and depletion of a second NAD+ synthesizing enzyme is sufficient to suppress these defects in highwire mutants, suggesting that excess NAD+ biosynthesis is the mechanism inhibiting neurotransmission. Thus, Highwire downregulates dNmnat to promote evoked synaptic release, suggesting that Highwire balances the axoprotective and synapse-inhibitory functions of dNmnat.
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Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Drosophila melanogaster/fisiología , NAD/biosíntesis , Proteínas del Tejido Nervioso/metabolismo , Nicotinamida-Nucleótido Adenililtransferasa/metabolismo , Transmisión Sináptica , Animales , Biocatálisis , Drosophila melanogaster/ultraestructura , Mutación/genética , Unión Neuromuscular/metabolismo , Terminales Presinápticos/metabolismo , Terminales Presinápticos/ultraestructura , ProbabilidadRESUMEN
Synapses grow, prune, and remodel throughout development, experience, and disease. This structural plasticity can destabilize information transfer in the nervous system. However, neural activity remains stable throughout life, implying that adaptive countermeasures exist that maintain neurotransmission within proper physiological ranges. Aberrant synaptic structure and function have been associated with a variety of neural diseases, including Fragile X syndrome, autism, and intellectual disability. We have screened 300 mutants in Drosophila larvae of both sexes for defects in synaptic growth at the neuromuscular junction, identifying 12 mutants with severe reductions or enhancements in synaptic growth. Remarkably, electrophysiological recordings revealed that synaptic strength was unchanged in all but one of these mutants compared with WT. We used a combination of genetic, anatomical, and electrophysiological analyses to illuminate three mechanisms that stabilize synaptic strength despite major disparities in synaptic growth. These include compensatory changes in (1) postsynaptic neurotransmitter receptor abundance, (2) presynaptic morphology, and (3) active zone structure. Together, this characterization identifies new mutants with defects in synaptic growth and the adaptive strategies used by synapses to homeostatically stabilize neurotransmission in response.SIGNIFICANCE STATEMENT This study reveals compensatory mechanisms used by synapses to ensure stable functionality during severe alterations in synaptic growth using the neuromuscular junction of Drosophila melanogaster as a model system. Through a forward genetic screen, we identify mutants that exhibit dramatic undergrown or overgrown synapses yet express stable levels of synaptic strength, with three specific compensatory mechanisms discovered. Thus, this study reveals novel insights into the adaptive strategies that constrain neurotransmission within narrow physiological ranges while allowing considerable flexibility in overall synapse number. More broadly, these findings provide insights into how stable synaptic function may be maintained in the nervous system during periods of intensive synaptic growth, pruning, and remodeling.
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Plasticidad Neuronal/fisiología , Transmisión Sináptica/fisiología , Animales , Animales Modificados Genéticamente , Drosophila , Femenino , Masculino , Mutación , Unión Neuromuscular/fisiologíaRESUMEN
Neurons communicate through Ca2+-dependent neurotransmitter release at presynaptic active zones (AZs). Neurotransmitter release properties play a key role in defining information flow in circuits and are tuned during multiple forms of plasticity. Despite their central role in determining neurotransmitter release properties, little is known about how Ca2+ channel levels are modulated to calibrate synaptic function. We used CRISPR to tag the Drosophila CaV2 Ca2+ channel Cacophony (Cac) and, in males in which all Cac channels are tagged, investigated the regulation of endogenous Ca2+ channels during homeostatic plasticity. We found that heterogeneously distributed Cac is highly predictive of neurotransmitter release probability at individual AZs and differentially regulated during opposing forms of presynaptic homeostatic plasticity. Specifically, AZ Cac levels are increased during chronic and acute presynaptic homeostatic potentiation (PHP), and live imaging during acute expression of PHP reveals proportional Ca2+ channel accumulation across heterogeneous AZs. In contrast, endogenous Cac levels do not change during presynaptic homeostatic depression (PHD), implying that the reported reduction in Ca2+ influx during PHD is achieved through functional adaptions to pre-existing Ca2+ channels. Thus, distinct mechanisms bidirectionally modulate presynaptic Ca2+ levels to maintain stable synaptic strength in response to diverse challenges, with Ca2+ channel abundance providing a rapidly tunable substrate for potentiating neurotransmitter release over both acute and chronic timescales.SIGNIFICANCE STATEMENT Presynaptic Ca2+ dynamics play an important role in establishing neurotransmitter release properties. Presynaptic Ca2+ influx is modulated during multiple forms of homeostatic plasticity at Drosophila neuromuscular junctions to stabilize synaptic communication. However, it remains unclear how this dynamic regulation is achieved. We used CRISPR gene editing to endogenously tag the sole Drosophila Ca2+ channel responsible for synchronized neurotransmitter release, and found that channel abundance is regulated during homeostatic potentiation, but not homeostatic depression. Through live imaging experiments during the adaptation to acute homeostatic challenge, we visualize the accumulation of endogenous Ca2+ channels at individual active zones within 10 min. We propose that differential regulation of Ca2+ channels confers broad capacity for tuning neurotransmitter release properties to maintain neural communication.
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Canales de Calcio/fisiología , Proteínas de Drosophila/fisiología , Plasticidad Neuronal , Terminales Presinápticos/fisiología , Potenciales Sinápticos , Animales , Drosophila/fisiología , Homeostasis , MasculinoRESUMEN
Recent advances in next-generation sequencing approaches have revolutionized our understanding of transcriptional expression in diverse systems. However, measurements of transcription do not necessarily reflect gene translation, the process of ultimate importance in understanding cellular function. To circumvent this limitation, biochemical tagging of ribosome subunits to isolate ribosome-associated mRNA has been developed. However, this approach, called TRAP, lacks quantitative resolution compared to a superior technology, ribosome profiling. Here, we report the development of an optimized ribosome profiling approach in Drosophila. We first demonstrate successful ribosome profiling from a specific tissue, larval muscle, with enhanced resolution compared to conventional TRAP approaches. We next validate the ability of this technology to define genome-wide translational regulation. This technology is leveraged to test the relative contributions of transcriptional and translational mechanisms in the postsynaptic muscle that orchestrate the retrograde control of presynaptic function at the neuromuscular junction. Surprisingly, we find no evidence that significant changes in the transcription or translation of specific genes are necessary to enable retrograde homeostatic signaling, implying that post-translational mechanisms ultimately gate instructive retrograde communication. Finally, we show that a global increase in translation induces adaptive responses in both transcription and translation of protein chaperones and degradation factors to promote cellular proteostasis. Together, this development and validation of tissue-specific ribosome profiling enables sensitive and specific analysis of translation in Drosophila.
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Drosophila/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Biosíntesis de Proteínas/genética , Subunidades Ribosómicas/genética , Análisis de Secuencia de ARN/métodos , Adaptación Fisiológica/genética , Animales , Drosophila/metabolismo , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Chaperonas Moleculares/genética , Proteostasis/genética , ARN Mensajero/análisis , Sensibilidad y EspecificidadRESUMEN
Amyotrophic lateral sclerosis (ALS) is debilitating neurodegenerative disease characterized by motor neuron dysfunction and progressive weakening of the neuromuscular junction (NMJ). Hereditary ALS is strongly associated with variants in the human C9orf72 gene. We have characterized C9orf72 pathology at the Drosophila NMJ and utilized several approaches to restore synaptic strength in this model. First, we demonstrate a dramatic reduction in synaptic arborization and active zone number at NMJs following C9orf72 transgenic expression in motor neurons. Further, neurotransmission is similarly reduced at these synapses, consistent with severe degradation. However, despite these defects, C9orf72 synapses still retain the ability to express presynaptic homeostatic plasticity, a fundamental and adaptive form of NMJ plasticity in which perturbation to postsynaptic neurotransmitter receptors leads to a retrograde enhancement in presynaptic release. Next, we show that these endogenous but dormant homeostatic mechanisms can be harnessed to restore synaptic strength despite C9orf72 pathogenesis. Finally, activation of regenerative signaling is not neuroprotective in motor neurons undergoing C9orf72 toxicity. Together, these experiments define synaptic dysfunction at NMJs experiencing ALS-related degradation and demonstrate the potential to activate latent plasticity as a novel therapeutic strategy to restore synaptic strength.
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Proteína C9orf72/metabolismo , Unión Neuromuscular/metabolismo , Plasticidad Neuronal/genética , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Animales Modificados Genéticamente , Proteína C9orf72/genética , Modelos Animales de Enfermedad , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Homeostasis , Humanos , Neuronas Motoras/metabolismo , Unión Neuromuscular/genética , Plasticidad Neuronal/fisiología , Transducción de Señal , Sinapsis/metabolismo , Transmisión SinápticaRESUMEN
The protein family of degenerin/epithelial sodium channels (DEG/ENaCs) is composed of diverse animal-specific, non-voltage-gated ion channels that play important roles in regulating cationic gradients across epithelial barriers. Some family members are also enriched in neural tissues in both vertebrates and invertebrates. However, the specific neurophysiological functions of most DEG/ENaC-encoding genes remain poorly understood. The fruit fly Drosophila melanogaster is an excellent model for deciphering the functions of DEG/ENaC genes because its genome encodes an exceptionally large number of DEG/ENaC subunits termed pickpocket (ppk) 1-31 Here we demonstrate that ppk29 contributes specifically to the postsynaptic modulation of excitatory synaptic transmission at the larval neuromuscular junction. Electrophysiological data indicate that the function of ppk29 in muscle is necessary for normal postsynaptic responsivity to neurotransmitter release and for normal coordinated larval movement. The ppk29 mutation does not affect gross synaptic morphology and ultrastructure, which indicates that the observed phenotypes are likely due to defects in glutamate receptor function. Together, our data indicate that DEG/ENaC ion channels play a fundamental role in the postsynaptic regulation of excitatory neurotransmission.SIGNIFICANCE STATEMENT Members of the degenerin/epithelial sodium channel (DEG/ENaC) family are broadly expressed in epithelial and neuronal tissues. To date, the neurophysiological functions of most family members remain unknown. Here, by using the power of Drosophila genetics in combination with electrophysiological and behavioral approaches, we demonstrate that the DEG/ENaC-encoding gene pickpocket 29 contributes to baseline neurotransmission, possibly via the modulation of postsynaptic glutamate receptor functionality.
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Proteínas de Drosophila/fisiología , Drosophila/fisiología , Potenciales Postsinápticos Excitadores/fisiología , Activación del Canal Iónico/fisiología , Canales Iónicos/fisiología , Unión Neuromuscular/fisiología , Sodio/metabolismo , Animales , Células Cultivadas , Canales de Sodio Degenerina/fisiología , Canales Epiteliales de Sodio/fisiología , Transmisión Sináptica/fisiologíaRESUMEN
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.
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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
GCaMP8f is a sensitive genetically encoded Ca2+ indicator that enables imaging of neuronal activity. Here, we present a protocol to perform Ca2+ imaging of the Drosophila neuromuscular junction using GCaMP8f targeted to pre- or postsynaptic compartments. We describe ratiometric Ca2+ imaging using GCaMP8f fused to mScarlet and synaptotagmin that reveals Ca2+ dynamics at presynaptic terminals. We then detail "quantal" imaging of miniature transmission events using GCaMP8f targeted to postsynaptic compartments by fusion to a PDZ-binding motif. For complete details on the use and execution of this protocol, please refer to Li et al.,1 Han et al.,2 Perry et al.,3 and Han et al.4.
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Proteínas de Drosophila , Drosophila , Animales , Drosophila/fisiología , Unión Neuromuscular/fisiología , Proteínas de Drosophila/genética , Terminales Presinápticos/fisiología , NeuronasRESUMEN
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.
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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
Tissue-specific gene knockout by CRISPR/Cas9 is a powerful approach for characterizing gene functions in animal development. However, this approach has been successfully applied in only a small number of Drosophila tissues. The Drosophila motor nervous system is an excellent model system for studying the biology of neuromuscular junction (NMJ). To expand tissue-specific CRISPR to the Drosophila motor system, here we present a CRISPR-mediated tissue-restricted mutagenesis (CRISPR-TRiM) toolkit for knocking out genes in motoneurons, muscles, and glial cells. We validated the efficacy of this toolkit by knocking out known genes in each tissue, demonstrated its orthogonal use with the Gal4/UAS binary expression system, and showed simultaneous knockout of multiple redundant genes. Using these tools, we discovered an essential role for SNARE pathways in NMJ maintenance. Furthermore, we demonstrate that the canonical ESCRT pathway suppresses NMJ bouton growth by downregulating the retrograde Gbb signaling. Lastly, we found that axon termini of motoneurons rely on ESCRT-mediated intra-axonal membrane trafficking to lease extracellular vesicles at the NMJ.
RESUMEN
Ionotropic glutamate receptors (GluRs) are targets for modulation in Hebbian and homeostatic synaptic plasticity and are remodeled by development, experience, and disease. We have probed the impact of synaptic glutamate levels on the two postsynaptic GluR subtypes at the Drosophila neuromuscular junction, GluRA and GluRB. We first demonstrate that GluRA and GluRB compete to establish postsynaptic receptive fields, and that proper GluR abundance and composition can be orchestrated in the absence of any synaptic glutamate release. However, excess glutamate adaptively tunes postsynaptic GluR abundance, echoing GluR scaling observed in mammalian systems. Furthermore, when GluRA vs. GluRB competition is eliminated, GluRB becomes insensitive to glutamate modulation. In contrast, GluRA is now homeostatically regulated by excess glutamate to maintain stable miniature activity, where Ca2+ permeability through GluRA receptors is required. Thus, excess glutamate, GluR competition, and Ca2+ signaling collaborate to selectively target GluR subtypes for homeostatic regulation at postsynaptic compartments.
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Proteínas de Drosophila , Sinapsis , Animales , Sinapsis/fisiología , Ácido Glutámico , Unión Neuromuscular/fisiología , Drosophila , Plasticidad Neuronal/fisiología , MamíferosRESUMEN
Active zones are colossal and complex molecular machines that transform electrical signals into rapid neurotransmitter secretion. In this issue of Neuron, Tan et al. (2022) elegantly distill central functions of this synaptic apparatus by tethering a small domain of the scaffold RIM near Ca2+ channels.