The persistence of drug-resistant epilepsy highlights the need for new therapies that target novel mechanisms while maintaining synaptic strength and minimizing side effects. Small molecules of natural origin with anti-epileptogenic potential can influence altered synaptic deficits and reducing seizure threshold, offering a strategy to restore cellular energy homeostasis and slow the progression of epilepsy and associated comorbidities. We used a combination of approaches including behavioural assays, immunostaining and electrophysiological recordings to study the synaptic contributions to reduced seizure-threshold in both Drosophila and mouse models of epilepsy. In addition, we used affinity-purification with synthesized beads specific for our small molecule to identify its cellular partners. We found that administration of Urolithin-A (UA), a small molecule of natural origin, mitigated behavioural and synaptic dysfunctions under chemically and genetically induced epileptic conditions in Drosophila. Additionally, chronic treatment with UA attenuated the altered single-cell and network synaptic transmission in PTZ-induced chronic kindling of mice. However, UA exerted no seizure-suppressive effects in in vitro epileptic conditions, suggesting the absence of acute anti-epileptic effects. In addition, we identified the direct interactor of UA as the complex of tubulin dimer and VDAC1. Molecular docking analyses also predicted that urolithin A interacts with VDAC1. Consistent with the neuroprotective role of VDAC1 downregulation reported in previous studies, neuron-specific knockdown of VDAC1 in Drosophila phenocopied the effects of UA. Collectively, our data show that UA exerts disease-modifying anti-epileptic effects in both Drosophila and mouse models with VDAC1 identified as a critical functional mediator; however, direct functional modulation of VDAC1 and associated partners by UA will require further experimental validation. These findings provide the blueprint for future drug discovery targeting VDAC1-dependent pathways.

Tauopathies represent a major class of neurodegenerative disorders associated with intracellular aggregates of the microtubule-associated protein Tau. To identify molecular modulators of Tau toxicity, we used a genetic screen to identify protein chaperones whose RNAi-mediated knockdown could modulate hTauV337M-induced eye-ommatidial degeneration in Drosophila. This screen identified the Prefoldins Pfdn5 and Pfdn6 as strong modifiers of hTauV337M cytotoxicity. Consistent with the known function of Pfdn as a cotranslational chaperone for tubulin, Pfdn5 mutants showed substantially reduced levels of tubulin monomer. However, additional microtubule-related functions were indicated by the robust unexpected association of Pfdn5 with axonal microtubules in vivo, as well as binding with stabilized microtubules in biochemical assays. Loss of Pfdn5 resulted in neuromuscular junctions (NMJ) defects similar to those previously described in hTau-expressing flies: namely, increased supernumerary boutons and fewer microtubule loops within mature presynaptic boutons. Significantly, synaptic phenotypes caused by hTauV337M overexpression were also strongly enhanced in a Pfdn5 mutant background. Consistent with a role in modulating Tau toxicity, not only did loss of Pfdn5 result in increased accumulations of Tau aggregates in hTauV337M-expressing neurons, but also neuronal overexpression of Prefoldin strikingly ameliorated age-dependent neurodegeneration and memory deficits induced by pathological hTau. Together, these and other observations described herein: (a) provide new insight into Prefoldin-microtubule interactions; (b) point to essential post-translational roles for Pfdn5 in controlling Tau toxicity in vivo; and (c) demonstrate that Pfdn5 overexpression is sufficient to restrict Tau-induced neurodegeneration.

Clathrin-mediated endocytosis is essential for neural development and function. Recent studies have linked de novo mutations in the clathrin heavy-chain gene to a range of neurodevelopmental disorders. In this study, we have modeled two pathogenic mutations, L1047P and W1108R, in Drosophila melanogaster and examined their effects on vesicle dynamics, ligand uptake, neuronal development, and memory formation. Our data show that expression of these mutant forms of clathrin heavy chain results in reduced survival and defective learning when expressed ubiquitously or exclusively in neurons. These mutations also disrupted vesicle dynamics and reduced ligand uptake under conditions of heat stress. While no obvious defects in neuronal morphology and function were observed at the larval neuromuscular junction, we noticed significant disruptions in the expression of markers associated with synapse maturation. Overall, our study establishes a model that can provide insights into the cellular basis of clathrin heavy-chain-related neurodevelopmental disorders.

Calcium release from intracellular stores influences synaptic response timing and magnitude. Despite the critical role of inositol trisphosphate (IP3)- and ryanodine receptor (RyR)-dependent calcium release in regulating synaptic strength, the upstream signaling mechanisms that govern IP3 receptor or RyR activity remain elusive. Here, we provide evidence that the ArfGAP-containing protein Asap modulates NMJ morphogenesis and synaptic calcium homeostasis by activating IP3-mediated calcium release from the endoplasmic reticulum (ER) via the phospholipase C-beta (PLCβ) signaling pathway. Using CRISPR/Cas9-engineered Asap mutants and genetically encoded calcium sensors, we demonstrate that loss of Asap leads to elevated resting synaptic calcium, resulting in increased evoked amplitude, elevated spontaneous miniature frequency, and reduced synaptic failures under low extracellular calcium conditions. Additional pharmacological and genetic manipulations of calcium regulatory pathways further support the role of increased resting intracellular calcium in driving enhanced neurotransmission in Asap-deficient synapses. Consistent with the role of Asap's ArfGAP domain in NMJ morphogenesis and intracellular calcium regulation, expressing a GDP-locked form of Arf6 (Arf6DN) or knocking down Arf6 in Asap mutants not only rescues Asap-associated synaptic defects but also normalizes synaptic calcium levels. Furthermore, epistatic analysis revealed that attenuation of IP3-signaling components in animals constitutively expressing Arf6CA normalized the NMJ morphological defects and synaptic functions. Together, these findings provide novel insights into the role of Asap-Arf6-PLCβ signaling in IP3-regulated calcium dynamics, sustaining both structural and functional synaptic plasticity.

Clathrin-mediated endocytosis is essential for neural development and function. Recent studies have linked de novo mutations in the clathrin heavy chain (Chc) gene to a range of neurodevelopmental disorders. In this study we have modelled two pathogenic mutations: L1047P and W1108R in Drosophila melanogaster and examined their effects on vesicle dynamics, ligand uptake, neuronal development and memory formation. Our data shows that expression of these mutant forms of Chc result in reduced survival and defective learning when expressed ubiquitously or exclusively in neurons. Our analysis also reveals that these mutations have the ability to disrupt vesicle dynamics and reduce ligand uptake in cells. Although we do not see a defect in neuronal morphology and function at the larval neuro-muscular junction, we see an increase in the number of Dlg-negative boutons, and a significant reduction in Spectrin expression, indicative of disruptions in the process of synapse maturation. Overall, this study provides mechanistic insights into the cellular and molecular basis of Chc-related neurodevelopmental disorders. ### Competing Interest Statement The authors have declared no competing interest. Department of Biotechnology, https://ror.org/03tjsyq23, BT/PR43190/MED/97/573/2021 Department of Science and Technology, https://ror.org/0101xrq71, CRG/2021/00599

Endocytosis regulates the retrieval of synaptic membranes and the trafficking of growth signaling receptors. While Drosophila endocytic mutants show synaptic overgrowth at the neuromuscular junctions (NMJs), the signaling pathways by which endocytosis restricts synapse growth remain poorly understood. Here, we demonstrate that sigma2-adaptin, one of the obligate subunits of the AP2 complex, facilitates the degradation and trafficking of E3-ubiquitin ligase Highwire (Hiw)/PHR1 and inhibits the c-Jun N-terminal kinase (JNK) signaling. This function of sigma2-adaptin is independent of its Bone Morphogenetic Protein (BMP) signaling regulation. Loss of sigma2-adaptin leads to Hiw accumulation and mislocalization in the neuronal cell body, leading to elevated MAP3K Wallenda levels. Stabilizing Hiw by expressing Rae1 or genetically blocking the JNK signaling suppresses the synaptic overgrowth defects observed in sigma2-adaptin mutants. Remarkably, blocking BMP and JNK signaling pathways suppressed the synaptic overgrowth observed in the sigma2-adaptin mutant to the wild-type levels. Finally, we show that loss of Rab11 but not Rab5 or Rab7 leads to accumulation/mislocalization of Hiw in the neuronal cell body akin to sigma2-adaptin mutants. We propose a model in which endocytosis regulates Rab11-mediated Hiw trafficking and attenuates JNK signaling in a pathway parallel to the BMP signaling to restrict synaptic growth. ### Competing Interest Statement The authors have declared no competing interest.

Intracellular membrane tubules play a crucial role in diverse cellular processes, and their regulation is facilitated by Bin-Amphiphysin-Rvs (BAR) domain-containing proteins. This study investigates the roles of Drosophila ICA69 (dICA69) (an N-BAR protein) and Drosophila CIP4 (dCIP4) (an F-BAR protein), focusing on their impact on in vivo membrane tubule organization. In contrast to the prevailing models of BAR-domain protein function, we observed colocalization of endogenous dICA69 with dCIP4-induced tubules, indicating their potential recruitment for tubule formation and maintenance. Moreover, actin-regulatory proteins such as Wasp, SCAR, and Arp2/3 were recruited at the site of CIP4-induced tubule formation. An earlier study indicated that F-BAR proteins spontaneously segregate from the N-BAR domain proteins during membrane tubule formation. In contrast, our observation supports a model in which different BAR-domain family members can associate with the same tubule and cooperate to fine-tune the tubule width, possibly by recruiting actin modulators during the generation of tubules. Our data suggests that cooperative activities of distinct BAR-domain family proteins may determine the length and width of the membrane tubule in vivo.

Membrane protrusions are fundamental to cellular functions like migration, adhesion, and communication and depend upon dynamic reorganization of the cytoskeleton. GAP‐dependent GTP hydrolysis of Arf proteins regulates actin‐dependent membrane remodeling. Here, we show that dAsap regulates membrane protrusions in S2R+ cells by a mechanism that critically relies on its ArfGAP domain and relocalization of actin regulators, SCAR, and Ena. While our data reinforce the preference of dAsap for Arf1 GTP hydrolysis in vitro , we demonstrate that induction of membrane protrusions in S2R+ cells depends on Arf6 inactivation. This study furthers our understanding of how dAsap‐dependent GTP hydrolysis maintains a balance between active and inactive states of Arf6 to regulate cell shape.

The appropriate growth of the neurons, accurate organization of their synapses, and successful neurotransmission are indispensable for sensorimotor activities. These processes are highly dynamic and tightly regulated. Extensive genetic, molecular, physiological, and behavioural studies have identified many molecular players and investigated their roles in various neuromuscular processes. In this paper, we show that Beadex (Bx), the Drosophila LIM only (LMO) protein, is required for motor activities and neuromuscular growth of Drosophila . Bx 7 , a null allele, adult flies are flightless, with reduced walking and jumping activities. The larvae of Bx 7 , and the RNAi-mediated neuronal-specific knockdown of Bx show drastically reduced crawling behaviour, a diminished synaptic span of the neuromuscular junctions and an increased spontaneous neuronal firing with altered motor patterns in the central pattern generators (CPGs). Microarray studies identified multiple targets of Beadex that are involved in different cellular and molecular pathways, including those associated with the cytoskeleton and mitochondria, that could be responsible for the observed neuromuscular defects. With genetic interaction studies, we further show that Highwire ( Hiw ), a negative regulator of synaptic growth at the NMJs, negatively regulates Bx , as the latter’s deficiency was able to rescue the phenotype of the Hiw null mutant, Hiw DN . Thus, our data indicates that Beadex functions downstream of Hiw to regulate the larval synaptic growth and physiology.

Membrane protrusions are fundamental to cellular functions like migration, adhesion, and communication and depend upon the dynamic reorganization of the cytoskeleton. The GAP-dependent GTP hydrolysis of Arf proteins regulates actin-dependent membrane remodeling. Here, we show that the dAsap regulates membrane protrusions in S2R+ cells by a mechanism that critically relies on its ArfGAP domain and re-localization of actin regulators, SCAR, and Ena. While our data reinforce the preference of dAsap for Arf1 GTP hydrolysis in vitro , we demonstrate that induction of membrane protrusions in S2R+ cells depends on Arf6 inactivation. This study furthers our understanding of how dAsap-dependent GTP hydrolysis maintains a balance between active and inactive states of dArf6 to regulate cell shape.### Competing Interest StatementThe authors have declared no competing interest.

We demonstrate the real-time sensing of high frequency vibrations with sub-micron amplitudes using the Hong-Ou-Mandel interferometer of narrow dip width resulted from the broadband parametric down-converted photons generated in non-linear crystals of short interaction length.

Synaptic morphogenesis involves an interplay of multiple signaling pathways and requires membrane remodeling and cytoskeleton dynamics. We identified the BAR-domain protein dAsap (Arf GAP, SH3, Ankyrin repeat, and PH domain) as one of the regulators of synaptic morphogenesis. Loss of dAsap results in decreased bouton numbers, increased inter-bouton diameter, and disrupted microtubule organization at the nerve terminals. Electrophysiological analysis of the mutants revealed a gain in neurotransmission compared to control neuromuscular junctions (NMJs). dAsap mutant NMJs have increased evoked amplitude, increased spontaneous miniature frequency, and significantly fewer synaptic failures in low calcium. Consistent with these observations, dAsap mutants have increased active zone number. Additional pharmacological and genetic manipulations that are known to impair calcium release from stores suppress the dAsap phenotypes. Finally, we show that expressing a GDP-locked form of Arf6 in dAsap mutants restored the NMJ morphological defects, disrupted cytoskeleton, and aberrant neurotransmission. Thus, we propose a model in which dAsap regulates NMJ morphogenesis and synaptic calcium homeostasis through Arf6-dependent neuronal signaling.

AbstractIntracellular membrane tubules play a crucial role in diverse cellular processes, and their regulation is facilitated by Bin-Amphiphysin-Rvs (BAR) domain-containing proteins. This study investigates the roles of dICA69N-BARand dCIP4F-BARin vivo, focusing on their impact onin vivotubule organization. Through cell culture and immunofluorescence staining, we observed co-localization of endogenous dICA69 with dCIP4-induced membrane tubules, indicating their potential recruitment for tubule formation and maintenance. Additionally, dCIP4-positive tubules exhibit enrichment of actin regulatory proteins such as Wasp, SCAR, Arp2, Arp3, and Syndapin. Overexpressing dICA69N-BARin S2R+ cells reveals distinct punctate patterns in the perinuclear region. An earlier study indicated that F-BAR proteins spontaneously segregate from the N-BAR domain-containing proteins during membrane tubule formation. In contrast, our observation supports a model in which different BAR-domain family members can associate with the same tubule and cooperate to fine-tune the tubule width. Moreover, our analysis highlights how dCIP4F-BARfacilitates the redistribution of dICA69N-BARpunctae, leading to altered patterns within the cells. These cooperative activities of dICA69N-BARand dCIP4F-BARare vital for the precise organization of intracellular tubules. Understanding the underlying mechanisms governing this cooperation provides valuable insights into cellular dynamics and the organization of membrane tubules. The implications extend to various physiological and pathological conditions related to intracellular membrane dynamics.

Intracellular membrane tubules play a crucial role in diverse cellular processes, and their regulation is facilitated by Bin-Amphiphysin-Rvs (BAR) domain-containing proteins. This study investigates the roles of dICA69N-BAR and dCIP4F-BAR in vivo , focusing on their impact on in vivo tubule organization. Through cell culture and immunofluorescence staining, we observed co-localization of endogenous dICA69 with dCIP4-induced membrane tubules, indicating their potential recruitment for tubule formation and maintenance. Additionally, dCIP4-positive tubules exhibit enrichment of actin regulatory proteins such as Wasp, SCAR, Arp2, Arp3, and Syndapin. Overexpressing dICA69N-BAR in S2R+ cells reveals distinct punctate patterns in the perinuclear region. An earlier study indicated that F-BAR proteins spontaneously segregate from the N-BAR domain-containing proteins during membrane tubule formation. In contrast, our observation supports a model in which different BAR-domain family members can associate with the same tubule and cooperate to fine-tune the tubule width. Moreover, our analysis highlights how dCIP4F-BAR facilitates the redistribution of dICA69N-BAR punctae, leading to altered patterns within the cells. These cooperative activities of dICA69N-BAR and dCIP4F-BAR are vital for the precise organization of intracellular tubules. Understanding the underlying mechanisms governing this cooperation provides valuable insights into cellular dynamics and the organization of membrane tubules. The implications extend to various physiological and pathological conditions related to intracellular membrane dynamics.### Competing Interest StatementThe authors have declared no competing interest.

Several proteins contain signaling domains that can regulate the cell membrane dynamics as well as the underlying cytoskeleton. Among these, Bin-Amphiphysin-Rvs (BAR) domain-containing proteins, with their membrane deforming properties, have emerged as the key players in regulating neuronal morphology and inducing neuronal signaling that can modulate synaptic architecture. While the biochemical and structural basis of membrane deformation by the BAR-domain proteins has been extensively studied, the in vivo contexts in which these proteins function remain to be elucidated. Despite the discovery of BAR-domain proteins over 25 years ago, most of the studies have primarily focused on understanding the structural and biochemical properties and cell biological processes regulated by these proteins. Understanding the functional requirements of these proteins at the level of multicellular organisms and the way these proteins regulate biological processes remains a topic of intensive study. In this review, we discuss the functional roles of BAR-domain proteins in the context of membrane dynamics and cellular signaling. We highlight recent developments describing the functional role of these proteins in neuronal morphogenesis, synaptic function, and disease.

Compromised endocytosis in neurons leads to synapse overgrowth and altered organization of synaptic proteins. However, the molecular players and the signaling pathways which regulate the process remain poorly understood. Here, we show that σ2-adaptin, one of the subunits of the AP2-complex, genetically interacts with Mad, Medea and Dad (components of BMP signaling) to control neuromuscular junction (NMJ) growth in Drosophila Ultrastructural analysis of σ2-adaptin mutants show an accumulation of large vesicles and membranous structures akin to endosomes at the synapse. We found that mutations in σ2-adaptin lead to an accumulation of Tkv receptors at the presynaptic membrane. Interestingly, the level of small GTPase Rab11 was significantly reduced in the σ2-adaptin mutant synapses. However, expression of Rab11 does not restore the synaptic defects of σ2-adaptin mutations. We propose a model in which AP2 regulates Tkv internalization and endosomal recycling to control synaptic growth.

Compromised endocytosis in neurons leads to synapse overgrowth and altered organization of synaptic proteins. However, the molecular players and the signaling pathways which regulate the process remains poorly understood. Here we show that σ2-adaptin, one of the subunits of the AP2-complex, genetically interacts with BMP type I receptor, Thickveins (Tkv), and Daughter against decapentaplegic (Dad), two of the components of BMP signaling. We found that mutations in σ2-adaptin lead to an accumulation of Tkv receptors at the NMJ and results in a significant reduction in Tkv-positive early endosomes in the presynaptic terminals. Interestingly, the level of small GTPase Rab11 was significantly reduced in the σ2-adaptin mutant synapses. Consistent with the role of σ2-adaptin and Rab11 in the regulation of the same signaling pathway, a mutation in Rab11 or overexpression of a GDP-locked form of Rab11 (Rab11S25N) phenocopies the morphological and signaling defects of the σ2-adaptin mutants. Finally, we demonstrate that σ2-adaptin mutants show an accumulation of large vesicles and massive membranous structures, akin to endosomes at the synapse. Thus, we propose a model in which AP2 regulates Tkv internalization and recycling through a process that requires Rab11 activity to control the synaptic growth.