dAsap regulates cellular protrusions via an Arf6-dependent actin regulatory pathway in S2R+ cells.

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.

Madm/NRBP1 mediates synaptic maintenance and neurodegeneration-induced presynaptic homeostatic potentiation.

The precise regulation of synaptic connectivity and function is essential to maintain neuronal circuits. Here, we show that the Drosophila pseudo-kinase Madm/NRBP1 (Mlf-1-adapter-molecule/nuclear-receptor-binding protein 1) is required presynaptically to maintain synaptic stability and to coordinate synaptic growth and function. Presynaptic Madm mediates these functions by controlling cap-dependent translation via the target of rapamycin (TOR) effector 4E-BP/Thor (eukaryotic initiation factor 4E binding protein/Thor). Strikingly, at degenerating neuromuscular synapses, postsynaptic Madm induces a compensatory, transsynaptic signal that utilizes the presynaptic homeostatic potentiation (PHP) machinery to offset synaptic release deficits and to delay synaptic degeneration. Madm is not required for canonical PHP but induces a neurodegeneration-specific form of PHP and acts via the regulation of the cap-dependent translation regulators 4E-BP/Thor and S6-kinase. Consistently, postsynaptic induction of canonical PHP or TOR activation can compensate for postsynaptic Madm to alleviate functional and structural synaptic defects. Our results provide insights into the molecular mechanisms underlying neurodegeneration-induced PHP with potential neurotherapeutic applications.

Fragile X premutation rCGG repeats impair synaptic growth and synaptic transmission at Drosophila larval neuromuscular junction.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disease that develops in some premutation (PM) carriers of the FMR1 gene with alleles bearing 55-200 CGG repeats. The discovery of a broad spectrum of clinical and cell-developmental abnormalities among PM carriers with or without FXTAS and in model systems suggests that neurodegeneration seen in FXTAS could be the inevitable end-result of pathophysiological processes set during early development. Hence, it is imperative to trace early PM-induced pathological abnormalities. Previous studies have shown that transgenic Drosophila carrying PM-length CGG repeats are sufficient to cause neurodegeneration. Here, we used the same transgenic model to understand the effect of CGG repeats on the structure and function of the developing nervous system. We show that presynaptic expression of CGG repeats restricts synaptic growth, reduces the number of synaptic boutons, leads to aberrant presynaptic varicosities, and impairs synaptic transmission at the larval neuromuscular junctions. The postsynaptic analysis shows that both glutamate receptors and subsynaptic reticulum proteins were normal. However, a high percentage of boutons show a reduced density of Bruchpilot protein, a key component of presynaptic active zones required for vesicle release. The electrophysiological analysis shows a significant reduction in quantal content, a measure of total synaptic vesicles released per excitation potential. Together, these findings suggest that synapse perturbation caused by riboCGG (rCGG) repeats mediates presynaptically during larval neuromuscular junction development. We also suggest that the stress-activated c-Jun N-terminal kinase protein Basket and CIDE-N protein Drep-2 positively mediate Bruchpilot active zone defects caused by rCGG repeats.

Drosophila Mon1 and Rab7 interact to regulate glutamate receptor levels at the neuromuscular junction.

Regulation of post-synaptic receptors plays an important role in determining synaptic strength and plasticity. The Drosophila larval neuromuscular junction (nmj) has been used extensively as a model to understand some of these processes. In this context, we are interested in the role of Drosophila Monensin sensitivity protein 1 (DMon1) in regulating glutamate receptor (GluRIIA) levels at the nmj. DMon1 is an evolutionarily conserved protein which, in complex with calcium caffeine zinc sensitivity1 (CCZ1), regulates the conversion of early endosomes to late endosomes through recruitment of Rab7. C-terminal deletion mutants of Dmon1 (Dmon1Δ181) exhibit lethality. The escapers have a short life span and exhibit severe motor defects. At the nmj, these mutants show defects in synaptic morphology and a strong increase in GluRIIA levels. The mechanism by which Dmon1 regulates GluRIIA is unclear. In this study, we have characterized an EMS mutant referred to as pog1 and demonstrate it to be an allele of Dmon1. Further, we have examined the role of rab7 in regulating GluRIIA. We show that similar to Dmon1, knock-down of rab7 using RNAi in neurons, but not muscles, leads to an increase in GluRIIA. Loss of one copy each of Dmon1 and rab7 leads to a synergistic increase in receptor expression. Further, overexpression of an activated Rab7 can rescue the GluRIIA phenotype observed in Dmon1 Δ181 mutants. Together, these results highlight a neuronal role for Rab7 in GluRIIA regulation and underscore the importance of the endo-lysosomal pathway in this process.

Selective Imaging of Lipids in Adipocytes by Using an Imidazolyl Derivative of Perylene Bisimide.

A small molecule, perylene bisimide imidazolyl derivative (PBI-ID), has been identified and developed as a specific marker for labelling multifunctional fat bodies in various organisms, including Drosophila and mammalian adipocytes. Interestingly, PBI-ID neither labels the plasma membranes nor cell nuclei by trapping into it. A remarkable feature of unbound PBI-ID is diminished fluorescence, which reduces the background emission noise, while contrasting the bound state effectively.

Stable Positioning of Unc13 Restricts Synaptic Vesicle Fusion to Defined Release Sites to Promote Synchronous Neurotransmission.

Neural information processing depends on precisely timed, Ca2+-activated synaptic vesicle exocytosis from release sites within active zones (AZs), but molecular details are unknown. Here, we identify that the (M)Unc13-family member Unc13A generates release sites and show the physiological relevance of their restrictive AZ targeting. Super-resolution and intravital imaging of Drosophila neuromuscular junctions revealed that (unlike the other release factors Unc18 and Syntaxin-1A) Unc13A was stably and precisely positioned at AZs. Local Unc13A levels predicted single AZ activity. Different Unc13A portions selectively affected release site number, position, and functionality. An N-terminal fragment stably localized to AZs, displaced endogenous Unc13A, and reduced the number of release sites, while a C-terminal fragment generated excessive sites at atypical locations, resulting in reduced and delayed evoked transmission that displayed excessive facilitation. Thus, release site generation by the Unc13A C terminus and their specific AZ localization via the N terminus ensure efficient transmission and prevent ectopic, temporally imprecise release.

Regulation of neuromuscular junction organization by Rab2 and its effector ICA69 in Drosophila.

The mechanisms underlying synaptic differentiation, which involves neuronal membrane and cytoskeletal remodeling, are not completely understood. We performed a targeted RNAi-mediated screen of Drosophila BAR-domain proteins and identified islet cell autoantigen 69 kDa (ICA69) as one of the key regulators of morphological differentiation of the larval neuromuscular junction (NMJ). We show that Drosophila ICA69 colocalizes with α-Spectrin at the NMJ. The conserved N-BAR domain of ICA69 deforms liposomes in vitro Full-length ICA69 and the ICAC but not the N-BAR domain of ICA69 induce filopodia in cultured cells. Consistent with its cytoskeleton regulatory role, ICA69 mutants show reduced α-Spectrin immunoreactivity at the larval NMJ. Manipulating levels of ICA69 or its interactor PICK1 alters the synaptic level of ionotropic glutamate receptors (iGluRs). Moreover, reducing PICK1 or Rab2 levels phenocopies ICA69 mutation. Interestingly, Rab2 regulates not only synaptic iGluR but also ICA69 levels. Thus, our data suggest that: (1) ICA69 regulates NMJ organization through a pathway that involves PICK1 and Rab2, and (2) Rab2 functions genetically upstream of ICA69 and regulates NMJ organization and targeting/retention of iGluRs by regulating ICA69 levels.

Altered translational repression of an RNA-binding protein, Elav by AOA2-causative Senataxin mutation.

Mutations in Senataxin (SETX) gene causes two types of neurological disorders, Amyotrophic Lateral Sclerosis (ALS4) and Ataxia with Oculomotor Apraxia type 2 (AOA2). Recent studies in cultured cells suggest that SETX plays a crucial role at the interface of transcription and the DNA damage response. Whether SETX can alter translational of specific RNA is not known. In this study, we report that expressing AOA2-causative truncated form of human SETX in Drosophila neurons alters the development of neuromuscular junction (NMJ) synapses. Interestingly, we found that expressing this truncated form of SETX in Drosophila muscles resulted in an alteration of translational repression of an RNA-binding protein, Embryonic Lethal Abnormal Vision (Elav). Elav is transcribed in all tissues but remains translationally repressed except in neurons. Thus, our data suggest that an altered repression profile of RNA by SETX mutants could be one of the mechanisms underlying ALS4 or AOA2 pathogenesis.

Human Senataxin Modulates Structural Plasticity of the Neuromuscular Junction in Drosophila through a Neuronally Conserved TGFß Signalling Pathway.

BACKGROUND: Mutations in the human Senataxin (hSETX) gene have been shown to cause two forms of neurodegenerative disorders - a dominant form called amyotrophic lateral sclerosis type 4 (ALS4) and a recessive form called ataxia with oculomotor apraxia type 2 (AOA2). SETX is a putative DNA/RNA helicase involved in RNA metabolism. Although several dominant mutations linked with ALS4 have been identified in SETX, their contribution towards ALS4 pathophysiology is still elusive. METHOD: In order to model ALS4 in Drosophila and to elucidate the morphological, physiological and signalling consequences, we overexpressed the wild-type and pathological forms of hSETX in Drosophila. RESULTS AND CONCLUSIONS: The pan-neuronal expression of wild-type or mutant forms of hSETX induced morphological plasticity at neuromuscular junction (NMJ) synapses. Surprisingly, we found that while the NMJ synapses were increased in number, the neuronal function was normal. Analysis of signalling pathways revealed that hSETX modulates the Highwire (Hiw; a conserved neuronal E3 ubiquitin ligase)-dependent bone morphogenetic protein/TGFß pathway. Thus, our study could pave the way for a better understanding of ALS4 progression by SETX through the regulation of neuronal E3 ubiquitin pathways.

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.