Structured and disordered regions of Ataxin-2 contribute differently to the specificity and efficiency of mRNP granule formation.

Ataxin-2 (ATXN2) is a gene implicated in spinocerebellar ataxia type II (SCA2), amyotrophic lateral sclerosis (ALS) and Parkinsonism. The encoded protein is a therapeutic target for ALS and related conditions. ATXN2 (or Atx2 in insects) can function in translational activation, translational repression, mRNA stability and in the assembly of mRNP-granules, a process mediated by intrinsically disordered regions (IDRs). Previous work has shown that the LSm (Like-Sm) domain of Atx2, which can help stimulate mRNA translation, antagonizes mRNP-granule assembly. Here we advance these findings through a series of experiments on Drosophila and human Ataxin-2 proteins. Results of Targets of RNA Binding Proteins Identified by Editing (TRIBE), co-localization and immunoprecipitation experiments indicate that a polyA-binding protein (PABP) interacting, PAM2 motif of Ataxin-2 may be a major determinant of the mRNA and protein content of Ataxin-2 mRNP granules. Experiments with transgenic Drosophila indicate that while the Atx2-LSm domain may protect against neurodegeneration, structured PAM2- and unstructured IDR- interactions both support Atx2-induced cytotoxicity. Taken together, the data lead to a proposal for how Ataxin-2 interactions are remodelled during translational control and how structured and non-structured interactions contribute differently to the specificity and efficiency of RNP granule condensation as well as to neurodegeneration.

Habituation of Sugar-Induced Proboscis Extension Reflex and Yeast-Induced Habituation Override in Drosophila melanogaster.

Habituation, the process by which animals learn to ignore insignificant stimuli, facilitates engagement with salient features of the environment. However, neural mechanisms underlying habituation also allow responses to familiar stimuli to be reinstated when such stimuli become potentially significant. Thus, the habituated state must allow a mechanism for habituation override. The remarkably precise knowledge of cell identity, connectivity, and information coding in Drosophila sensory circuits, as well as the availability of tools to genetically target these cells, makes Drosophila a valuable and important organism for analysis of habituation and habituation-override mechanisms. Studies of olfactory and gustatory habituation in Drosophila suggest that potentiation of GABAergic neurons underlies certain timescales of habituation and have specified some elements of a gustatory habituation-override pathway. More detailed understanding of gustatory habituation and habituation-override mechanisms will benefit from access to robust behavioral assays for (a) the proboscis extension reflex (PER) elicited by a sweet stimulus, (b) exposure paradigms that result in PER habituation, and, most critically, (c) manipulations that result in PER-habituation override. Here, we describe simple protocols for persistent sucrose exposure of tarsal hairs that lead to habituation of proboscis extension and for presentation of a novel appetitive stimuli that reinstate robust PER to habituated flies. This detailed protocol of gustatory habituation provides (a) a simple method to induce habituation by continuous exposure of the flies to sucrose for 10 min without leading to ingestion and (b) a novel method to override habituation by presenting yeast to the proboscis. Key features • A protocol for stimulation of Drosophila's taste (sugar) sensory neurons that induces gustatory habituation without satiation due to ingestion. • A chemical (yeast) stimulation protocol that rapidly induces habituation override/dishabituation in sugar-habituated Drosophila.

Emotional states: Sweet relief for depressed flies.

Mammals and insects appear to have emotional states with features characteristic of human depression. A new study has defined a neural circuit including serotonergic neurons that drive sugar-induced relief from a depression-like-state in Drosophila.

Local translation provides the asymmetric distribution of CaMKII required for associative memory formation.

How compartment-specific local proteomes are generated and maintained is inadequately understood, particularly in neurons, which display extreme asymmetries. Here we show that local enrichment of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in axons of Drosophila mushroom body neurons is necessary for cellular plasticity and associative memory formation. Enrichment is achieved via enhanced axoplasmic translation of CaMKII mRNA, through a mechanism requiring the RNA-binding protein Mub and a 23-base Mub-recognition element in the CaMKII 3' UTR. Perturbation of either dramatically reduces axonal, but not somatic, CaMKII protein without altering the distribution or amount of mRNA in vivo, and both are necessary and sufficient to enhance axonal translation of reporter mRNA. Together, these data identify elevated levels of translation of an evenly distributed mRNA as a novel strategy for generating subcellular biochemical asymmetries. They further demonstrate the importance of distributional asymmetry in the computational and biological functions of neurons.

A Drosophila Circuit for Habituation Override.

Habituated animals retain a latent capacity for robust engagement with familiar stimuli. In most instances, the ability to override habituation is best explained by postulating that habituation arises from the potentiation of inhibitory inputs onto stimulus-encoding assemblies and that habituation override occurs through disinhibition. Previous work has shown that inhibitory plasticity contributes to specific forms of olfactory and gustatory habituation in Drosophila Here, we analyze how exposure to a novel stimulus causes override of gustatory (proboscis extension reflex; PER) habituation. While brief sucrose contact with tarsal hairs causes naive Drosophila to extend their proboscis, persistent exposure reduces PER to subsequent sucrose stimuli. We show that in so habituated animals, either brief exposure of the proboscis to yeast or direct thermogenetic activation of sensory neurons restores PER response to tarsal sucrose stimulation. Similar override of PER habituation can also be induced by brief thermogenetic activation of a population of tyrosine hydroxylase (TH)-positive neurons, a subset of which send projections to the subesophageal zone (SEZ). Significantly, sensory-neuron induced habituation override requires transmitter release from these TH-positive cells. Treatments that cause override specifically influence the habituated state, with no effect on the naive sucrose response across a range of concentrations. Taken together with other findings, these observations in female flies are consistent with a model in which novel taste stimuli trigger activity in dopaminergic neurons which, directly or indirectly, inhibit GABAergic cells that drive PER habituation. The implications of these findings for general mechanisms of attentional and sensory override of habituation are discussed.SIGNIFICANCE STATEMENT Habituation can be overcome when a new context requires an enhanced response to a familiar stimulus. However, the underlying mechanisms remain incompletely understood. Previous studies have provided evidence that habituation of the sucrose-induced proboscis extension reflex (PER) in Drosophila occurs through potentiation of inhibition onto the PER pathway. This work defines controlled protocols for override of PER habituation and uses them to outline the underlying circuit mechanisms. The results presented support a model in which novel taste stimuli cause dishabituation by activating a subset of tyrosine hydroxylase (TH)-expressing neurons that inhibit GABAergic neurons whose potentiation underlies PER habituation. At a general level, these findings further highlight a central role for inhibition and disinhibition in the control of behavioral flexibility.

The transcriptional response to oxidative stress is independent of stress-granule formation.

Cells respond to stress with translational arrest, robust transcriptional changes, and transcription-independent formation of mRNP assemblies termed stress granules (SGs). Despite considerable interest in the role of SGs in oxidative, unfolded protein and viral stress responses, whether and how SGs contribute to stress-induced transcription have not been rigorously examined. To address this, we characterized transcriptional changes in Drosophila S2 cells induced by acute oxidative-stress and assessed how these were altered under conditions that disrupted SG assembly. Oxidative stress for 3 h predominantly resulted in induction or up-regulation of stress-responsive mRNAs whose levels peaked during recovery after stress cessation. The stress transcriptome is enriched in mRNAs coding for chaperones including HSP70s, small heat shock proteins, glutathione transferases, and several noncoding RNAs. Oxidative stress also induced cytoplasmic SGs that disassembled 3 h after stress cessation. As expected, RNAi-mediated knockdown of the conserved G3BP1/Rasputin protein inhibited SG assembly. However, this disruption had no significant effect on the stress-induced transcriptional response or stress-induced translational arrest. Thus SG assembly and stress-induced gene expression alterations appear to be driven by distinctive signaling processes. We suggest that while SG assembly represents a fast, transient mechanism, the transcriptional response enables a slower, longer-lasting mechanism for adaptation to and recovery from cell stress.

A C-terminal ataxin-2 disordered region promotes Huntingtin protein aggregation and neurodegeneration in Drosophila models of Huntington's disease.

The Ataxin-2 (Atx2) protein contributes to the progression of neurodegenerative phenotypes in animal models of amyotrophic lateral sclerosis (ALS), type 2 spinocerebellar ataxia (SCA-2), Parkinson's disease, and Huntington's disease (HD). However, because the Atx2 protein contains multiple separable activities, deeper understanding requires experiments to address the exact mechanisms by which Atx2 modulates neurodegeneration (ND) progression. Recent work on two ALS models, C9ORF72 and FUS, in Drosophila has shown that a C-terminal intrinsically disordered region (cIDR) of Atx2 protein, required for assembly of ribonucleoprotein (RNP) granules, is essential for the progression of neurodegenerative phenotypes as well as for accumulation of protein inclusions associated with these ALS models. Here, we show that the Atx2-cIDR also similarly contributes to the progression of degenerative phenotypes and accumulation of Huntingtin protein aggregates in Drosophila models of HD. Because Huntingtin is not an established component of RNP granules, these observations support a recently hypothesized, unexpected protein-handling function for RNP granules, which could contribute to the progression of Huntington's disease and, potentially, other proteinopathies.

Learning and memory: Clashing engrams in the fly brain.

Prior experience is known to deeply influence new learning. A recent study describes a neural mechanism where initial sensory experience promotes the establishment of a competing and/or cooperative memory trace that shapes behavioral expression of subsequent learning.

The Making of Long-Lasting Memories: A Fruit Fly Perspective.

Understanding the nature of the molecular mechanisms underlying memory formation, consolidation, and forgetting are some of the fascinating questions in modern neuroscience. The encoding, stabilization and elimination of memories, rely on the structural reorganization of synapses. These changes will enable the facilitation or depression of neural activity in response to the acquisition of new information. In other words, these changes affect the weight of specific nodes within a neural network. We know that these plastic reorganizations require de novo protein synthesis in the context of Long-term memory (LTM). This process depends on neural activity triggered by the learned experience. The use of model organisms like Drosophila melanogaster has been proven essential for advancing our knowledge in the field of neuroscience. Flies offer an optimal combination of a more straightforward nervous system, composed of a limited number of cells, and while still displaying complex behaviors. Studies in Drosophila neuroscience, which expanded over several decades, have been critical for understanding the cellular and molecular mechanisms leading to the synaptic and behavioral plasticity occurring in the context of learning and memory. This is possible thanks to sophisticated technical approaches that enable precise control of gene expression in the fruit fly as well as neural manipulation, like chemogenetics, thermogenetics, or optogenetics. The search for the identity of genes expressed as a result of memory acquisition has been an active interest since the origins of behavioral genetics. From screenings of more or less specific candidates to broader studies based on transcriptome analysis, our understanding of the genetic control behind LTM has expanded exponentially in the past years. Here we review recent literature regarding how the formation of memories induces a rapid, extensive and, in many cases, transient wave of transcriptional activity. After a consolidation period, transcriptome changes seem more stable and likely represent the synthesis of new proteins. The complexity of the circuitry involved in memory formation and consolidation is such that there are localized changes in neural activity, both regarding temporal dynamics and the nature of neurons and subcellular locations affected, hence inducing specific temporal and localized changes in protein expression. Different types of neurons are recruited at different times into memory traces. In LTM, the synthesis of new proteins is required in specific subsets of cells. This de novo translation can take place in the somatic cytoplasm and/or locally in distinct zones of compartmentalized synaptic activity, depending on the nature of the proteins and the plasticity-inducing processes that occur. We will also review recent advances in understanding how localized changes are confined to the relevant synapse. These recent studies have led to exciting discoveries regarding proteins that were not previously involved in learning and memory processes. This invaluable information will lead to future functional studies on the roles that hundreds of new molecular actors play in modulating neural activity.

Antagonistic roles for Ataxin-2 structured and disordered domains in RNP condensation.

Ataxin-2 (Atx2) is a translational control molecule mutated in spinocerebellar ataxia type II and amyotrophic lateral sclerosis. While intrinsically disordered domains (IDRs) of Atx2 facilitate mRNP condensation into granules, how IDRs work with structured domains to enable positive and negative regulation of target mRNAs remains unclear. Using the Targets of RNA-Binding Proteins Identified by Editing technology, we identified an extensive data set of Atx2-target mRNAs in the Drosophila brain and S2 cells. Atx2 interactions with AU-rich elements in 3'UTRs appear to modulate stability/turnover of a large fraction of these target mRNAs. Further genomic and cell biological analyses of Atx2 domain deletions demonstrate that Atx2 (1) interacts closely with target mRNAs within mRNP granules, (2) contains distinct protein domains that drive or oppose RNP-granule assembly, and (3) has additional essential roles outside of mRNP granules. These findings increase the understanding of neuronal translational control mechanisms and inform strategies for Atx2-based interventions under development for neurodegenerative disease.

Impaired inhibitory processing: a new therapeutic target for autism and psychosis?

In the healthy brain, homeostatic balance between excitation and inhibition maintains neural stability. Reduced inhibition may explain shared symptoms observed in autism and psychosis. Here we review evidence suggesting that altered levels of gamma-aminobutyric acid (GABA) may underlie both disorders, providing a potential cross-diagnostic therapeutic target.

The Neurohumanities: An Emerging Partnership for Exploring the Human Experience.

Neuroscience has an extraordinary opportunity to investigate issues historically addressed by the arts, humanities, and social sciences. As a guide, we suggest three features of meaningful progress in the collaborative field, the neurohumanities, which we illustrate through a discussion of "neural schemas."

Glomerulus-Selective Regulation of a Critical Period for Interneuron Plasticity in the Drosophila Antennal Lobe.

Several features of the adult nervous systems develop in a "critical period" (CP), during which high levels of plasticity allow neural circuits to be tuned for optimal performance. Through an analysis of long-term olfactory habituation (LTH) in female Drosophila, we provide new insight into mechanisms by which CPs are regulated in vivo LTH manifests as a persistently reduced behavioral response to an odorant encountered for 4 continuous days and occurs together with the growth of specific, odorant-responsive glomeruli in the antennal lobe. We show that the CP for behavioral and structural plasticity induced by ethyl butyrate (EB) or carbon dioxide (CO2) closes within 48 h after eclosion. The elaboration of excitatory projection neuron (PN) processes likely contribute to glomerular volume increases, as follows: both occur together and similarly require cAMP signaling in the antennal lobe inhibitory local interneurons. Further, the CP for structural plasticity could be extended beyond 48 h if EB- or CO2-responsive olfactory sensory neurons (OSNs) are silenced after eclosion; thus, OSN activity is required for closing the CP. Strikingly, silencing of glomerulus-selective OSNs extends the CP for structural plasticity only in respective target glomeruli. This indicates the existence of a local, short-range mechanism for regulating CP closure. Such a local mechanism for CP regulation can explain why plasticity induced by the odorant geranyl acetate (which is attractive) shows no CP although it involves the same core plasticity mechanisms as CO2 and EB. Local control of closure mechanisms during the critical period can potentially impart evolutionarily adaptive, odorant-specific features to behavioral plasticity.SIGNIFICANCE STATEMENT The critical period for plasticity represents a stage of life at which animals learn specific tasks or features with particular facility. This work provides fresh evidence that mechanisms for regulating critical periods are broadly conserved across evolution. Thus, a critical period for long-term olfactory habituation in Drosophila, which closes early in adulthood can, like the critical period for ocular dominance plasticity in mammals, be extended by blocking sensory neurons early in life. Further observations show that critical periods for plasticity can be regulated by spatially restricted mechanisms, potentially allowing varied critical periods for plasticity to stimuli of different ethological relevance.

Implications of the Sap47 null mutation for synapsin phosphorylation, longevity, climbing proficiency and behavioural plasticity in adult Drosophila.

The Sap47 gene of Drosophila melanogaster encodes a highly abundant 47 kDa synaptic vesicle-associated protein. Sap47 null mutants show defects in synaptic plasticity and larval olfactory associative learning but the molecular function of Sap47 at the synapse is unknown. We demonstrate that Sap47 modulates the phosphorylation of another highly abundant conserved presynaptic protein, synapsin. Site-specific phosphorylation of Drosophila synapsin has repeatedly been shown to be important for behavioural plasticity but it was not known where these phospho-synapsin isoforms are localized in the brain. Here, we report the distribution of serine-6-phosphorylated synapsin in the adult brain and show that it is highly enriched in rings of synapses in the ellipsoid body and in large synapses near the lateral triangle. The effects of knockout of Sap47 or synapsin on olfactory associative learning/memory support the hypothesis that both proteins operate in the same molecular pathway. We therefore asked if this might also be true for other aspects of their function. We show that knockout of Sap47 but not synapsin reduces lifespan, whereas knockout of Sap47 and synapsin, either individually or together, affects climbing proficiency, as well as plasticity in circadian rhythms and sleep. Furthermore, electrophysiological assessment of synaptic properties at the larval neuromuscular junction (NMJ) reveals increased spontaneous synaptic vesicle fusion and reduced paired pulse facilitation in Sap47 and synapsin single and double mutants. Our results imply that Sap47 and synapsin cooperate non-uniformly in the control of synaptic properties in different behaviourally relevant neuronal networks of the fruitfly.

RNP-Granule Assembly via Ataxin-2 Disordered Domains Is Required for Long-Term Memory and Neurodegeneration.

Human Ataxin-2 is implicated in the cause and progression of amyotrophic lateral sclerosis (ALS) and type 2 spinocerebellar ataxia (SCA-2). In Drosophila, a conserved atx2 gene is essential for animal survival as well as for normal RNP-granule assembly, translational control, and long-term habituation. Like its human homolog, Drosophila Ataxin-2 (Atx2) contains polyQ repeats and additional intrinsically disordered regions (IDRs). We demonstrate that Atx2 IDRs, which are capable of mediating liquid-liquid phase transitions in vitro, are essential for efficient formation of neuronal mRNP assemblies in vivo. Remarkably, ΔIDR mutants that lack neuronal RNP granules show normal animal development, survival, and fertility. However, they show defects in long-term memory formation/consolidation as well as in C9ORF72 dipeptide repeat or FUS-induced neurodegeneration. Together, our findings demonstrate (1) that higher-order mRNP assemblies contribute to long-term neuronal plasticity and memory, and (2) that a targeted reduction in RNP-granule formation efficiency can alleviate specific forms of neurodegeneration.

The Long 3'UTR mRNA of CaMKII Is Essential for Translation-Dependent Plasticity of Spontaneous Release in Drosophila melanogaster.

A null mutation of the Drosophila calcium/calmodulin-dependent protein kinase II gene (CaMKII) was generated using homologous recombination. Null animals survive to larval and pupal stages due to a large maternal contribution of CaMKII mRNA, which consists of a short 3'-untranslated region (UTR) form lacking regulatory elements that guide local translation. The selective loss of the long 3'UTR mRNA in CaMKII-null larvae allows us to test its role in plasticity. Development and evoked function of the larval neuromuscular junction are surprisingly normal, but the resting rate of miniature excitatory junctional potentials (mEJPs) is significantly lower in CaMKII mutants. Mutants also lack the ability to increase mEJP rate in response to spaced depolarization, a type of activity-dependent plasticity shown to require both transcription and translation. Consistent with this, overexpression of miR-289 in wild-type animals blocks plasticity of spontaneous release. In addition to the defects in regulation of mEJP rate, CaMKII protein is largely lost from synapses in the mutant. All phenotypes are non-sex-specific and rescued by a fosmid containing the entire wild-type CaMKII locus, but only viability and CaMKII localization are rescued by genomic fosmids lacking the long 3'UTR. This suggests that synaptic CaMKII accumulates by two distinct mechanisms: local synthesis requiring the long 3'UTR form of CaMKII mRNA and a process that requires zygotic transcription of CaMKII mRNA. The origin of synaptic CaMKII also dictates its functionality. Locally translated CaMKII has a privileged role in regulation of spontaneous release, which cannot be fulfilled by synaptic CaMKII from the other pool.SIGNIFICANCE STATEMENT As a regulator of synaptic development and plasticity, CaMKII has important roles in both normal and pathological function of the nervous system. CaMKII shows high conservation between Drosophila and humans, underscoring the usefulness of Drosophila in modeling its function. Drosophila CaMKII-null mutants remain viable throughout development, enabling morphological and electrophysiological characterization. Although the structure of the synapse is normal, maternally contributed CaMKII does not localize to synapses. Zygotic production of CaMKII mRNA with a long 3'-untranslated region is necessary for modulating spontaneous neurotransmission in an activity-dependent manner, but not for viability. These data argue that regulation of CaMKII localization and levels by local transcriptional processes is conserved. This is the first demonstration of distinct functions for Drosophila CaMKII mRNA variants.

Inhibitory engrams in perception and memory.

Nervous systems use excitatory cell assemblies to encode and represent sensory percepts. Similarly, synaptically connected cell assemblies or "engrams" are thought to represent memories of past experience. Multiple lines of recent evidence indicate that brain systems create and use inhibitory replicas of excitatory representations for important cognitive functions. Such matched "inhibitory engrams" can form through homeostatic potentiation of inhibition onto postsynaptic cells that show increased levels of excitation. Inhibitory engrams can reduce behavioral responses to familiar stimuli, thereby resulting in behavioral habituation. In addition, by preventing inappropriate activation of excitatory memory engrams, inhibitory engrams can make memories quiescent, stored in a latent form that is available for context-relevant activation. In neural networks with balanced excitatory and inhibitory engrams, the release of innate responses and recall of associative memories can occur through focused disinhibition. Understanding mechanisms that regulate the formation and expression of inhibitory engrams in vivo may help not only to explain key features of cognition but also to provide insight into transdiagnostic traits associated with psychiatric conditions such as autism, schizophrenia, and posttraumatic stress disorder.

Long-term memory consolidation: The role of RNA-binding proteins with prion-like domains.

Long-term and short-term memories differ primarily in the duration of their retention. At a molecular level, long-term memory (LTM) is distinguished from short-term memory (STM) by its requirement for new gene expression. In addition to transcription (nuclear gene expression) the translation of stored mRNAs is necessary for LTM formation. The mechanisms and functions for temporal and spatial regulation of mRNAs required for LTM is a major contemporary problem, of interest from molecular, cell biological, neurobiological and clinical perspectives. This review discusses primary evidence in support for translational regulatory events involved in LTM and a model in which different phases of translation underlie distinct phases of consolidation of memories. However, it focuses largely on mechanisms of memory persistence and the role of prion-like domains in this defining aspect of long-term memory. We consider primary evidence for the concept that Cytoplasmic Polyadenylation Element Binding (CPEB) protein enables the persistence of formed memories by transforming in prion-like manner from a soluble monomeric state to a self-perpetuating and persistent polymeric translationally active state required for maintaining persistent synaptic plasticity. We further discuss prion-like domains prevalent on several other RNA-binding proteins involved in neuronal translational control underlying LTM. Growing evidence indicates that such RNA regulatory proteins are components of mRNP (RiboNucleoProtein) granules. In these proteins, prion-like domains, being intrinsically disordered, could mediate weak transient interactions that allow the assembly of RNP granules, a source of silenced mRNAs whose translation is necessary for LTM. We consider the structural bases for RNA granules formation as well as functions of disordered domains and discuss how these complicate the interpretation of existing experimental data relevant to general mechanisms by which prion-domain containing RBPs function in synapse specific plasticity underlying LTM.

Repression of Pumilio Protein Expression by Rbfox1 Promotes Germ Cell Differentiation.

RNA-binding Fox (Rbfox) proteins have well-established roles in regulating alternative splicing, but specific Rbfox isoforms lack nuclear localization signals and accumulate in the cytoplasm. The potential splicing-independent functions of these proteins remain unknown. Here we demonstrate that cytoplasmic Drosophila Rbfox1 regulates germ cell development and represses the translation of mRNAs containing (U)GCAUG elements within their 3'UTRs. During germline cyst differentiation, Rbfox1 targets pumilio mRNA for destabilization and translational silencing, thereby promoting germ cell development. Mis-expression of pumilio results in the formation of germline tumors, which contain cysts that break down and dedifferentiate back to single, mitotically active cells. Together, these results reveal that cytoplasmic Rbfox family members regulate the translation of specific target mRNAs. In the Drosophila ovary, this activity provides a genetic barrier that prevents germ cells from reverting back to an earlier developmental state. The finding that Rbfox proteins regulate mRNA translation has implications for Rbfox-related diseases.

σ2-Adaptin Facilitates Basal Synaptic Transmission and Is Required for Regenerating Endo-Exo Cycling Pool Under High-Frequency Nerve Stimulation in Drosophila.

The functional requirement of adapter protein 2 (AP2) complex in synaptic membrane retrieval by clathrin-mediated endocytosis is not fully understood. Here we isolated and functionally characterized a mutation that dramatically altered synaptic development. Based on the aberrant neuromuscular junction (NMJ) synapse, we named this mutation angur (a Hindi word meaning "grapes"). Loss-of-function alleles of angur show more than twofold overgrowth in bouton numbers and a dramatic decrease in bouton size. We mapped the angur mutation to σ2-adaptin, the smallest subunit of the AP2 complex. Reducing the neuronal level of any of the subunits of the AP2 complex or disrupting AP2 complex assembly in neurons phenocopied the σ2-adaptin mutation. Genetic perturbation of σ2-adaptin in neurons leads to a reversible temperature-sensitive paralysis at 38°. Electrophysiological analysis of the mutants revealed reduced evoked junction potentials and quantal content. Interestingly, high-frequency nerve stimulation caused prolonged synaptic fatigue at the NMJs. The synaptic levels of subunits of the AP2 complex and clathrin, but not other endocytic proteins, were reduced in the mutants. Moreover, bone morphogenetic protein (BMP)/transforming growth factor ß (TGFß) signaling was altered in these mutants and was restored by normalizing σ2-adaptin in neurons. Thus, our data suggest that (1) while σ2-adaptin facilitates synaptic vesicle (SV) recycling for basal synaptic transmission, its activity is also required for regenerating SVs during high-frequency nerve stimulation, and (2) σ2-adaptin regulates NMJ morphology by attenuating TGFß signaling.