Endophilin-A/SH3GL2 calcium switch for synaptic autophagy induction is impaired by a Parkinson's risk variant.

At the synapse, proteins are reused several times during neuronal activity, causing a decline in protein function over time. Although emerging evidence supports a role of autophagy in synaptic function, the precise molecular mechanisms linking neuronal activity, autophagy and synaptic dysfunction are vastly unknown. We show how extracellular calcium influx in the pre-synaptic terminal constitutes the initial stimulus for autophagosome formation in response to neuronal activity. This mechanism likely acts to rapidly support synaptic homeostasis and protein quality control when intense neuronal activity challenges the synaptic proteome. We identified a residue in the flexible region of EndoA (Endophilin A) that dictates calcium-dependent EndoA mobility from the plasma membrane to the cytosol, where this protein interacts with autophagic membranes to promote autophagosome formation. We discovered that a novel Parkinson's disease-risk mutation in SH3GL2 (SH3 domain containing GRB2 like 2, endophilin A1) disrupts the calcium sensing of SH3GL2, leading to an immobile protein that cannot respond to calcium influx and therefore disrupting autophagy induction at synapses. Our work shows how neuronal activity is connected with autophagy to maintain synaptic homeostasis and survival.

Targeting Macroautophagy as a Therapeutic Opportunity to Treat Parkinson's Disease.

Macroautophagy, an evolutionary conserved catabolic process in the eukaryotic cell, regulates cellular homeostasis and plays a decisive role in self-engulfing proteins, protein aggregates, dysfunctional or damaged organelles, and invading pathogens. Growing evidence from in vivo and in vitro models shows that autophagy dysfunction plays decisive role in the pathogenesis of various neurodegenerative diseases, including Parkinson's disease (PD). PD is an incurable and second most common neurodegenerative disease characterised by neurological and motor dysfunction accompanied of non-motor symptoms that can also reduce the life quality of patients. Despite the investment in research, the aetiology of the disease is still unknown and the therapies available are aimed mostly at ameliorating motor symptoms. Hence, therapeutics regulating the autophagy pathway might play an important role controlling the disease progression, reducing neuronal loss and even ameliorating non-motor symptoms. In this review, we highlight potential therapeutic opportunities involved in different targeting options like an initiation of autophagy, Leucine-rich repeat kinase 2 (LRRK2) inhibition, mitophagy, lysosomes, lipid metabolism, immune system, gene expression, biomarkers, and also non-pharmacological interventions. Thus, strategies to identify therapeutics targeting the pathways modulating autophagy might hold a future for therapy development against PD.

Endophilin-B regulates autophagy during synapse development and neurodegeneration.

Synapses are critical for neuronal communication and brain function. To maintain neuronal homeostasis, synapses rely on autophagy. Autophagic alterations cause neurodegeneration and synaptic dysfunction is a feature in neurodegenerative diseases. In Parkinson's disease (PD), where the loss of synapses precedes dopaminergic neuron loss, various PD-causative proteins are involved in the regulation of autophagy. So far only a few factors regulating autophagy at the synapse have been identified and the molecular mechanisms underlying autophagy at the synapse is only partially understood. Here, we describe Endophilin-B (EndoB) as a novel player in the regulation of synaptic autophagy in health and disease. We demonstrate that EndoB is required for autophagosome biogenesis at the synapse, whereas the loss of EndoB blocks the autophagy induction promoted by the PD mutation LRRK2G2019S. We show that EndoB is required to prevent neuronal loss. Moreover, loss of EndoB in the Drosophila visual system leads to an increase in synaptic contacts between photoreceptor terminals and their post-synaptic synapses. These data confirm the role of autophagy in synaptic contact formation and neuronal survival.

Macros to Quantify Exosome Release and Autophagy at the Neuromuscular Junction of Drosophila Melanogaster.

Automatic quantification of image parameters is a powerful and necessary tool to explore and analyze crucial cell biological processes. This article describes two ImageJ/Fiji automated macros to approach the analysis of synaptic autophagy and exosome release from 2D confocal images. Emerging studies point out that exosome biogenesis and autophagy share molecular and organelle components. Indeed, the crosstalk between these two processes may be relevant for brain physiology, neuronal development, and the onset/progression of neurodegenerative disorders. In this context, we describe here the macros "Autophagoquant" and "Exoquant" to assess the quantification of autophagosomes and exosomes at the neuronal presynapse of the Neuromuscular Junction (NMJ) in Drosophila melanogaster using confocal microscopy images. The Drosophila NMJ is a valuable model for the study of synapse biology, autophagy, and exosome release. By use of Autophagoquant and Exoquant, researchers can have an unbiased, standardized, and rapid tool to analyze autophagy and exosomal release in Drosophila NMJ. Code available at: https://github.com/IreneSaMi/Exoquant-Autophagoquant.

The role of lipids in autophagy and its implication in neurodegeneration.

Neurodegenerative diseases are, at present, major socio-economic burdens without effective treatments and their increasing prevalence means that these diseases will be a challenge for future generations. Neurodegenerative diseases may differ in etiology and pathology but are often caused by the accumulation of dysfunctional and aggregation-prone proteins. Autophagy, a conserved cellular mechanism, deals with cellular stress and waste product build-up and has been shown to reduce the accumulation of dysfunctional proteins in animal models of neurodegenerative diseases. Historically, progress in understanding the precise function of lipids has traditionally been far behind other biological molecules (like proteins) but emerging works demonstrate the importance of lipids in the autophagy pathway and how the disturbance of lipid metabolism is connected to neurodegeneration. Here we review how altered autophagy and the disturbance of lipid metabolism, particularly of phosphoinositols and sphingolipids, feature in neurodegenerative diseases and address work from the field that suggests that these potentially offer an opportunity of therapeutic intervention.

Parkinson's disease: convergence on synaptic homeostasis.

Parkinson's disease, the second most common neurodegenerative disorder, affects millions of people globally. There is no cure, and its prevalence will double by 2030. In recent years, numerous causative genes and risk factors for Parkinson's disease have been identified and more than half appear to function at the synapse. Subtle synaptic defects are thought to precede blunt neuronal death, but the mechanisms that are dysfunctional at synapses are only now being unraveled. Here, we review recent work and propose a model where different Parkinson proteins interact in a cell compartment-specific manner at the synapse where these proteins regulate endocytosis and autophagy. While this field is only recently emerging, the work suggests that the loss of synaptic homeostasis may contribute to neurodegeneration and is a key player in Parkinson's disease.

The SAC1 domain in synaptojanin is required for autophagosome maturation at presynaptic terminals.

Presynaptic terminals are metabolically active and accrue damage through continuous vesicle cycling. How synapses locally regulate protein homeostasis is poorly understood. We show that the presynaptic lipid phosphatase synaptojanin is required for macroautophagy, and this role is inhibited by the Parkinson's disease mutation R258Q. Synaptojanin drives synaptic endocytosis by dephosphorylating PI(4,5)P2, but this function appears normal in SynaptojaninRQ knock-in flies. Instead, R258Q affects the synaptojanin SAC1 domain that dephosphorylates PI(3)P and PI(3,5)P2, two lipids found in autophagosomal membranes. Using advanced imaging, we show that SynaptojaninRQ mutants accumulate the PI(3)P/PI(3,5)P2-binding protein Atg18a on nascent synaptic autophagosomes, blocking autophagosome maturation at fly synapses and in neurites of human patient induced pluripotent stem cell-derived neurons. Additionally, we observe neurodegeneration, including dopaminergic neuron loss, in SynaptojaninRQ flies. Thus, synaptojanin is essential for macroautophagy within presynaptic terminals, coupling protein turnover with synaptic vesicle cycling and linking presynaptic-specific autophagy defects to Parkinson's disease.

EndoA/Endophilin-A creates docking stations for autophagic proteins at synapses.

Synapses are very specialized compartments with high metabolic demand to maintain neurotransmission, an essential step for basic brain function. Neurons are post-mitotic and synapses need to stay functional over time-sometimes over decades. Given that synapses are often at a long distance from the cell body, they must use local mechanisms to regulate protein quality control. We show that macroautophagy/autophagy is one of these local processes and found that it is under strict control of the synapse-enriched protein EndoA/Endophilin-A, previously only implicated in endocytosis. Metabolic and neuronal stimulation induce synaptic autophagy and phosphorylation of EndoA by the Parkinson disease kinase Lrrk/LRRK2 is essential to promote the process. EndoA induces membrane curvature in vitro, and, mechanistically, phosphorylated EndoA creates curved membrane-protein docking sites that are capable of recruiting Atg3. Our work reveals a synapse-enriched branch of autophagy under the control of EndoA that may be deregulated in Parkinson disease.

Endophilin-A Deficiency Induces the Foxo3a-Fbxo32 Network in the Brain and Causes Dysregulation of Autophagy and the Ubiquitin-Proteasome System.

Endophilin-A, a well-characterized endocytic adaptor essential for synaptic vesicle recycling, has recently been linked to neurodegeneration. We report here that endophilin-A deficiency results in impaired movement, age-dependent ataxia, and neurodegeneration in mice. Transcriptional analysis of endophilin-A mutant mice, complemented by proteomics, highlighted ataxia- and protein-homeostasis-related genes and revealed upregulation of the E3-ubiquitin ligase FBXO32/atrogin-1 and its transcription factor FOXO3A. FBXO32 overexpression triggers apoptosis in cultured cells and neurons but, remarkably, coexpression of endophilin-A rescues it. FBXO32 interacts with all three endophilin-A proteins. Similarly to endophilin-A, FBXO32 tubulates membranes and localizes on clathrin-coated structures. Additionally, FBXO32 and endophilin-A are necessary for autophagosome formation, and both colocalize transiently with autophagosomes. Our results point to a role for endophilin-A proteins in autophagy and protein degradation, processes that are impaired in their absence, potentially contributing to neurodegeneration and ataxia.

A LRRK2-Dependent EndophilinA Phosphoswitch Is Critical for Macroautophagy at Presynaptic Terminals.

Synapses are often far from the soma and independently cope with proteopathic stress induced by intense neuronal activity. However, how presynaptic compartments turn over proteins is poorly understood. We show that the synapse-enriched protein EndophilinA, thus far studied for its role in endocytosis, induces macroautophagy at presynaptic terminals. We find that EndophilinA executes this unexpected function at least partly independent of its role in synaptic vesicle endocytosis. EndophilinA-induced macroautophagy is activated when the kinase LRRK2 phosphorylates the EndophilinA-BAR domain and is blocked in animals where EndophilinA cannot be phosphorylated. EndophilinA-phosphorylation promotes the formation of highly curved membranes, and reconstitution experiments show these curved membranes serve as docking stations for autophagic factors, including Atg3. Functionally, deregulation of the EndophilinA phosphorylation state accelerates activity-induced neurodegeneration. Given that EndophilinA is connected to at least three Parkinson's disease genes (LRRK2, Parkin and Synaptojanin), dysfunction of EndophilinA-dependent synaptic macroautophagy may be common in this disorder.

DLin-7 is required in postsynaptic lamina neurons to prevent light-induced photoreceptor degeneration in Drosophila.

Inherited retinal degeneration in humans is caused by mutations in a wide spectrum of genes that regulate photoreceptor development and homeostasis. Many of these genes are structurally and functionally conserved in Drosophila, making the fly eye an ideal system in which to study the cellular and molecular basis of blindness. DLin-7, the ortholog of vertebrate MALS/Veli, is a core component of the evolutionarily conserved Crumbs complex. Mutations in any core member of the Crb complex lead to retinal degeneration in Drosophila. Strikingly, mutations in the human ortholog, CRB1, result in retinitis pigmentosa 12 (RP12) and Leber congenital amaurosis, two severe retinal dystrophies. Unlike Crumbs, DLin-7 is expressed not only in photoreceptor cells but also in postsynaptic lamina neurons. Here, we show that DLin-7 is required in postsynaptic neurons, but not in photoreceptors such as Crumbs, to prevent light-dependent retinal degeneration. At the photoreceptor synapse, DLin-7 acts as part of a conserved DLin-7/CASK/DlgS97 complex required to control the number of capitate projections and active zones, important specializations in the photoreceptor synapse that are essential for proper neurotransmission. These results are the first to demonstrate that a postsynaptically acting protein prevents light-dependent photoreceptor degeneration and describe a novel, Crumbs-independent mechanism for photoreceptor degeneration.

Uptake of the necrotic serpin in Drosophila melanogaster via the lipophorin receptor-1.

The humoral response to fungal and Gram-positive infections is regulated by the serpin-family inhibitor, Necrotic. Following immune-challenge, a proteolytic cascade is activated which signals through the Toll receptor. Toll activation results in a range of antibiotic peptides being synthesised in the fat-body and exported to the haemolymph. As with mammalian serpins, Necrotic turnover in Drosophila is rapid. This serpin is synthesised in the fat-body, but its site of degradation has been unclear. By "freezing" endocytosis with a temperature sensitive Dynamin mutation, we demonstrate that Necrotic is removed from the haemolymph in two groups of giant cells: the garland and pericardial athrocytes. Necrotic uptake responds rapidly to infection, being visibly increased after 30 mins and peaking at 6-8 hours. Co-localisation of anti-Nec with anti-AP50, Rab5, and Rab7 antibodies establishes that the serpin is processed through multi-vesicular bodies and delivered to the lysosome, where it co-localises with the ubiquitin-binding protein, HRS. Nec does not co-localise with Rab11, indicating that the serpin is not re-exported from athrocytes. Instead, mutations which block late endosome/lysosome fusion (dor, hk, and car) cause accumulation of Necrotic-positive endosomes, even in the absence of infection. Knockdown of the 6 Drosophila orthologues of the mammalian LDL receptor family with dsRNA identifies LpR1 as an enhancer of the immune response. Uptake of Necrotic from the haemolymph is blocked by a chromosomal deletion of LpR1. In conclusion, we identify the cells and the receptor molecule responsible for the uptake and degradation of the Necrotic serpin in Drosophila melanogaster. The scavenging of serpin/proteinase complexes may be a critical step in the regulation of proteolytic cascades.