Neuronal identity defines α-synuclein and tau toxicity.

Pathogenic α-synuclein and tau are critical drivers of neurodegeneration, and their mutations cause neuronal loss in patients. Whether the underlying preferential neuronal vulnerability is a cell-type-intrinsic property or a consequence of increased expression levels remains elusive. Here, we explore cell-type-specific α-synuclein and tau expression in human brain datasets and use deep phenotyping as well as brain-wide single-cell RNA sequencing of >200 live neuron types in fruit flies to determine which cellular environments react most to α-synuclein or tau toxicity. We detect phenotypic and transcriptomic evidence of differential neuronal vulnerability independent of α-synuclein or tau expression levels. Comparing vulnerable with resilient neurons in Drosophila enabled us to predict numerous human neuron subtypes with increased intrinsic susceptibility to pathogenic α-synuclein or tau. By uncovering synapse- and Ca2+ homeostasis-related genes as tau toxicity modifiers, our work paves the way to leverage neuronal identity to uncover modifiers of neurodegeneration-associated toxic proteins.

Parkinsonism mutations in DNAJC6 cause lipid defects and neurodegeneration that are rescued by Synj1.

Recent evidence links dysfunctional lipid metabolism to the pathogenesis of Parkinson's disease, but the mechanisms are not resolved. Here, we generated a new Drosophila knock-in model of DNAJC6/Auxilin and find that the pathogenic mutation causes synaptic dysfunction, neurological defects and neurodegeneration, as well as specific lipid metabolism alterations. In these mutants, membrane lipids containing long-chain polyunsaturated fatty acids, including phosphatidylinositol lipid species that are key for synaptic vesicle recycling and organelle function, are reduced. Overexpression of another protein mutated in Parkinson's disease, Synaptojanin-1, known to bind and metabolize specific phosphoinositides, rescues the DNAJC6/Auxilin lipid alterations, the neuronal function defects and neurodegeneration. Our work reveals a functional relation between two proteins mutated in Parkinsonism and implicates deregulated phosphoinositide metabolism in the maintenance of neuronal integrity and neuronal survival.

EndophilinA-dependent coupling between activity-induced calcium influx and synaptic autophagy is disrupted by a Parkinson-risk mutation.

Neuronal activity causes use-dependent decline in protein function. However, it is unclear how this is coupled to local quality control mechanisms. We show in Drosophila that the endocytic protein Endophilin-A (EndoA) connects activity-induced calcium influx to synaptic autophagy and neuronal survival in a Parkinson disease-relevant fashion. Mutations in the disordered loop, including a Parkinson disease-risk mutation, render EndoA insensitive to neuronal stimulation and affect protein dynamics: when EndoA is more flexible, its mobility in membrane nanodomains increases, making it available for autophagosome formation. Conversely, when EndoA is more rigid, its mobility reduces, blocking stimulation-induced autophagy. Balanced stimulation-induced autophagy is required for dopagminergic neuron survival, and a variant in the human ENDOA1 disordered loop conferring risk to Parkinson disease also blocks nanodomain protein mobility and autophagy both in vivo and in human-induced dopaminergic neurons. Thus, we reveal a mechanism that neurons use to connect neuronal activity to local autophagy and that is critical for neuronal survival.

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.

Torsin and NEP1R1-CTDNEP1 phosphatase affect interphase nuclear pore complex insertion by lipid-dependent and lipid-independent mechanisms.

The interphase nuclear envelope (NE) is extensively remodeled during nuclear pore complex (NPC) insertion. How this remodeling occurs and why it requires Torsin ATPases, which also regulate lipid metabolism, remains poorly understood. Here, we show that Drosophila Torsin (dTorsin) affects lipid metabolism via the NEP1R1-CTDNEP1 phosphatase and the Lipin phosphatidic acid (PA) phosphatase. This includes that Torsins remove NEP1R1-CTDNEP1 from the NE in fly and mouse cells, leading to subsequent Lipin exclusion from the nucleus. NEP1R1-CTDNEP1 downregulation also restores nuclear pore membrane fusion in post-mitotic dTorsinKO fat body cells. However, dTorsin-associated nuclear pore defects do not correlate with lipidomic abnormalities and are not resolved by silencing of Lipin. Further testing confirmed that membrane fusion continues in cells with hyperactivated Lipin. It also led to the surprising finding that excessive PA metabolism inhibits recruitment of the inner ring complex Nup35 subunit, resulting in elongated channel-like structures in place of mature nuclear pores. We conclude that the NEP1R1-CTDNEP1 phosphatase affects interphase NPC biogenesis by lipid-dependent and lipid-independent mechanisms, explaining some of the pleiotropic effects of Torsins.

Lowering Synaptogyrin-3 expression rescues Tau-induced memory defects and synaptic loss in the presence of microglial activation.

Tau is a major driver of neurodegeneration and is implicated in over 20 diseases. Tauopathies are characterized by synaptic loss and neuroinflammation, but it is unclear if these pathological events are causally linked. Tau binds to Synaptogyrin-3 on synaptic vesicles. Here, we interfered with this function to determine the role of pathogenic Tau at pre-synaptic terminals. We show that heterozygous knockout of synaptogyrin-3 is benign in mice but strongly rescues mutant Tau-induced defects in long-term synaptic plasticity and working memory. It also significantly rescues the pre- and post-synaptic loss caused by mutant Tau. However, Tau-induced neuroinflammation remains clearly upregulated when we remove the expression of one allele of synaptogyrin-3. Hence neuroinflammation is not sufficient to cause synaptic loss, and these processes are separately induced in response to mutant Tau. In addition, the pre-synaptic defects caused by mutant Tau are enough to drive defects in cognitive tasks.

Excess Lipin enzyme activity contributes to TOR1A recessive disease and DYT-TOR1A dystonia.

TOR1A/TorsinA mutations cause two incurable diseases: a recessive congenital syndrome that can be lethal, and a dominantly-inherited childhood-onset dystonia (DYT-TOR1A). TorsinA has been linked to phosphatidic acid lipid metabolism in Drosophila melanogaster. Here we evaluate the role of phosphatidic acid phosphatase (PAP) enzymes in TOR1A diseases using induced pluripotent stem cell-derived neurons from patients, and mouse models of recessive Tor1a disease. We find that Lipin PAP enzyme activity is abnormally elevated in human DYT-TOR1A dystonia patient cells and in the brains of four different Tor1a mouse models. Its severity also correlated with the dosage of Tor1a/TOR1A mutation. We assessed the role of excess Lipin activity in the neurological dysfunction of Tor1a disease mouse models by interbreeding these with Lpin1 knock-out mice. Genetic reduction of Lpin1 improved the survival of recessive Tor1a disease-model mice, alongside suppressing neurodegeneration, motor dysfunction, and nuclear membrane pathology. These data establish that TOR1A disease mutations cause abnormal phosphatidic acid metabolism, and suggest that approaches that suppress Lipin PAP enzyme activity could be therapeutically useful for TOR1A diseases.

TBC1D24-TLDc-related epilepsy exercise-induced dystonia: rescue by antioxidants in a disease model.

Genetic mutations in TBC1D24 have been associated with multiple phenotypes, with epilepsy being the main clinical manifestation. The TBC1D24 protein consists of the unique association of a Tre2/Bub2/Cdc16 (TBC) domain and a TBC/lysin motif domain/catalytic (TLDc) domain. More than 50 missense and loss-of-function mutations have been described and are spread over the entire protein. Through whole genome/exome sequencing we identified compound heterozygous mutations, R360H and G501R, within the TLDc domain, in an index family with a Rolandic epilepsy exercise-induced dystonia phenotype (http://omim.org/entry/608105). A 20-year long clinical follow-up revealed that epilepsy was self-limited in all three affected patients, but exercise-induced dystonia persisted into adulthood in two. Furthermore, we identified three additional sporadic paediatric patients with a remarkably similar phenotype, two of whom had compound heterozygous mutations consisting of an in-frame deletion I81_K84 and an A500V mutation, and the third carried T182M and G511R missense mutations, overall revealing that all six patients harbour a missense mutation in the subdomain of TLDc between residues 500 and 511. We solved the crystal structure of the conserved Drosophila TLDc domain. This allowed us to predict destabilizing effects of the G501R and G511R mutations and, to a lesser degree, of R360H and potentially A500V. Next, we characterized the functional consequences of a strong and a weak TLDc mutation (TBC1D24G501R and TBC1D24R360H) using Drosophila, where TBC1D24/Skywalker regulates synaptic vesicle trafficking. In a Drosophila model neuronally expressing human TBC1D24, we demonstrated that the TBC1D24G501R TLDc mutation causes activity-induced locomotion and synaptic vesicle trafficking defects, while TBC1D24R360H is benign. The neuronal phenotypes of the TBC1D24G501R mutation are consistent with exacerbated oxidative stress sensitivity, which is rescued by treating TBC1D24G501R mutant animals with antioxidants N-acetylcysteine amide or α-tocopherol as indicated by restored synaptic vesicle trafficking levels and sustained behavioural activity. Our data thus show that mutations in the TLDc domain of TBC1D24 cause Rolandic-type focal motor epilepsy and exercise-induced dystonia. The humanized TBC1D24G501R fly model exhibits sustained activity and vesicle transport defects. We propose that the TBC1D24/Sky TLDc domain is a reactive oxygen species sensor mediating synaptic vesicle trafficking rates that, when dysfunctional, causes a movement disorder in patients and flies. The TLDc and TBC domain mutations' response to antioxidant treatment we observed in the animal model suggests a potential for combining antioxidant-based therapeutic approaches to TBC1D24-associated disorders with previously described lipid-altering strategies for TBC domain mutations.

Hsp90 Mediates Membrane Deformation and Exosome Release.

Hsp90 is an essential chaperone that guards proteome integrity and amounts to 2% of cellular protein. We now find that Hsp90 also has the ability to directly interact with and deform membranes via an evolutionarily conserved amphipathic helix. Using a new cell-free system and in vivo measurements, we show this amphipathic helix allows exosome release by promoting the fusion of multivesicular bodies (MVBs) with the plasma membrane. We dissect the relationship between Hsp90 conformation and membrane-deforming function and show that mutations and drugs that stabilize the open Hsp90 dimer expose the helix and allow MVB fusion, while these effects are blocked by the closed state. Hence, we structurally separated the Hsp90 membrane-deforming function from its well-characterized chaperone activity, and we show that this previously unrecognized function is required for exosome release.

Tau association with synaptic vesicles causes presynaptic dysfunction.

Tau is implicated in more than 20 neurodegenerative diseases, including Alzheimer's disease. Under pathological conditions, Tau dissociates from axonal microtubules and missorts to pre- and postsynaptic terminals. Patients suffer from early synaptic dysfunction prior to Tau aggregate formation, but the underlying mechanism is unclear. Here we show that pathogenic Tau binds to synaptic vesicles via its N-terminal domain and interferes with presynaptic functions, including synaptic vesicle mobility and release rate, lowering neurotransmission in fly and rat neurons. Pathological Tau mutants lacking the vesicle binding domain still localize to the presynaptic compartment but do not impair synaptic function in fly neurons. Moreover, an exogenously applied membrane-permeable peptide that competes for Tau-vesicle binding suppresses Tau-induced synaptic toxicity in rat neurons. Our work uncovers a presynaptic role of Tau that may be part of the early pathology in various Tauopathies and could be exploited therapeutically.

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.

Cardiolipin promotes electron transport between ubiquinone and complex I to rescue PINK1 deficiency.

PINK1 is mutated in Parkinson's disease (PD), and mutations cause mitochondrial defects that include inefficient electron transport between complex I and ubiquinone. Neurodegeneration is also connected to changes in lipid homeostasis, but how these are related to PINK1-induced mitochondrial dysfunction is unknown. Based on an unbiased genetic screen, we found that partial genetic and pharmacological inhibition of fatty acid synthase (FASN) suppresses toxicity induced by PINK1 deficiency in flies, mouse cells, patient-derived fibroblasts, and induced pluripotent stem cell-derived dopaminergic neurons. Lower FASN activity in PINK1 mutants decreases palmitate levels and increases the levels of cardiolipin (CL), a mitochondrial inner membrane-specific lipid. Direct supplementation of CL to isolated mitochondria not only rescues the PINK1-induced complex I defects but also rescues the inefficient electron transfer between complex I and ubiquinone in specific mutants. Our data indicate that genetic or pharmacologic inhibition of FASN to increase CL levels bypasses the enzymatic defects at complex I in a PD model.

Skywalker-TBC1D24 has a lipid-binding pocket mutated in epilepsy and required for synaptic function.

Mutations in TBC1D24 cause severe epilepsy and DOORS syndrome, but the molecular mechanisms underlying these pathologies are unresolved. We solved the crystal structure of the TBC domain of the Drosophila ortholog Skywalker, revealing an unanticipated cationic pocket conserved among TBC1D24 homologs. Cocrystallization and biochemistry showed that this pocket binds phosphoinositides phosphorylated at the 4 and 5 positions. The most prevalent patient mutations affect the phosphoinositide-binding pocket and inhibit lipid binding. Using in vivo photobleaching of Skywalker-GFP mutants, including pathogenic mutants, we showed that membrane binding via this pocket restricts Skywalker diffusion in presynaptic terminals. Additionally, the pathogenic mutations cause severe neurological defects in flies, including impaired synaptic-vesicle trafficking and seizures, and these defects are reversed by genetically increasing synaptic PI(4,5)P2 concentrations through synaptojanin mutations. Hence, we discovered that a TBC domain affected by clinical mutations directly binds phosphoinositides through a cationic pocket and that phosphoinositide binding is critical for presynaptic function.

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.

Torsins Are Essential Regulators of Cellular Lipid Metabolism.

Torsins are developmentally essential AAA+ proteins, and mutation of human torsinA causes the neurological disease DYT1 dystonia. They localize in the ER membranes, but their cellular function remains unclear. We now show that dTorsin is required in Drosophila adipose tissue, where it suppresses triglyceride levels, promotes cell growth, and elevates membrane lipid content. We also see that human torsinA at the inner nuclear membrane is associated with membrane expansion and elevated cellular lipid content. Furthermore, the key lipid metabolizing enzyme, lipin, is mislocalized in dTorsin-KO cells, and dTorsin increases levels of the lipin substrate, phosphatidate, and reduces the product, diacylglycerol. Finally, genetic suppression of dLipin rescues dTorsin-KO defects, including adipose cell size, animal growth, and survival. These findings identify that torsins are essential regulators of cellular lipid metabolism and implicate disturbed lipid biology in childhood-onset DYT1 dystonia.

Mitochondrial uncouplers inhibit clathrin-mediated endocytosis largely through cytoplasmic acidification.

ATP production requires the establishment of an electrochemical proton gradient across the inner mitochondrial membrane. Mitochondrial uncouplers dissipate this proton gradient and disrupt numerous cellular processes, including vesicular trafficking, mainly through energy depletion. Here we show that Endosidin9 (ES9), a novel mitochondrial uncoupler, is a potent inhibitor of clathrin-mediated endocytosis (CME) in different systems and that ES9 induces inhibition of CME not because of its effect on cellular ATP, but rather due to its protonophore activity that leads to cytoplasm acidification. We show that the known tyrosine kinase inhibitor tyrphostinA23, which is routinely used to block CME, displays similar properties, thus questioning its use as a specific inhibitor of cargo recognition by the AP-2 adaptor complex via tyrosine motif-based endocytosis signals. Furthermore, we show that cytoplasm acidification dramatically affects the dynamics and recruitment of clathrin and associated adaptors, and leads to reduction of phosphatidylinositol 4,5-biphosphate from the plasma membrane.

Shawn, the Drosophila Homolog of SLC25A39/40, Is a Mitochondrial Carrier That Promotes Neuronal Survival.

Mitochondria play an important role in the regulation of neurotransmission, and mitochondrial impairment is a key event in neurodegeneration. Cells rely on mitochondrial carrier proteins of the SLC25 family to shuttle ions, cofactors, and metabolites necessary for enzymatic reactions. Mutations in these carriers often result in rare but severe pathologies in the brain, and some of the genes, including SLC25A39 and SLC25A40, reside in susceptibility loci of severe forms of epilepsy. However, the role of most of these carriers has not been investigated in neurons in vivo. We identified shawn, the Drosophila homolog of SLC25A39 and SLC25A40, in a genetic screen to identify genes involved in neuronal function. Shawn localizes to mitochondria, and missense mutations result in an accumulation of reactive oxygen species, mitochondrial dysfunction, and neurodegeneration. Shawn regulates metal homeostasis, and we found in shawn mutants increased levels of manganese, calcium, and mitochondrial free iron. Mitochondrial mutants often cannot maintain synaptic transmission under demanding conditions, but shawn mutants do, and they also do not display endocytic defects. In contrast, shawn mutants harbor a significant increase in neurotransmitter release. Our work provides the first functional annotation of these essential mitochondrial carriers in the nervous system, and the results suggest that metal imbalances and mitochondrial dysfunction may contribute to defects in synaptic transmission and neuronal survival. SIGNIFICANCE STATEMENT: We describe for the first time the role of the mitochondrial carrier Shawn/SLC25A39/SLC25A40 in the nervous system. In humans, these genes reside in susceptibility loci for epilepsy, and, in flies, we observe neuronal defects related to mitochondrial dysfunction and metal homeostasis defects. Interestingly, shawn mutants also harbor increased neurotransmitter release and neurodegeneration. Our data suggest a connection between maintaining a correct metal balance and mitochondrial function to regulate neuronal survival and neurotransmitter release.

Hsc70-4 Deforms Membranes to Promote Synaptic Protein Turnover by Endosomal Microautophagy.

Synapses are often far from their cell bodies and must largely independently cope with dysfunctional proteins resulting from synaptic activity and stress. To identify membrane-associated machines that can engulf synaptic targets destined for degradation, we performed a large-scale in vitro liposome-based screen followed by functional studies. We identified a presynaptically enriched chaperone Hsc70-4 that bends membranes based on its ability to oligomerize. This activity promotes endosomal microautophagy and the turnover of specific synaptic proteins. Loss of microautophagy slows down neurotransmission while gain of microautophagy increases neurotransmission. Interestingly, Sgt, a cochaperone of Hsc70-4, is able to switch the activity of Hsc70-4 from synaptic endosomal microautophagy toward chaperone activity. Hence, Hsc70-4 controls rejuvenation of the synaptic protein pool in a dual way: either by refolding proteins together with Sgt, or by targeting them for degradation by facilitating endosomal microautophagy based on its membrane deforming activity.

Reduced synaptic vesicle protein degradation at lysosomes curbs TBC1D24/sky-induced neurodegeneration.

Synaptic demise and accumulation of dysfunctional proteins are thought of as common features in neurodegeneration. However, the mechanisms by which synaptic proteins turn over remain elusive. In this paper, we study Drosophila melanogaster lacking active TBC1D24/Skywalker (Sky), a protein that in humans causes severe neurodegeneration, epilepsy, and DOOR (deafness, onychdystrophy, osteodystrophy, and mental retardation) syndrome, and identify endosome-to-lysosome trafficking as a mechanism for degradation of synaptic vesicle-associated proteins. In fly sky mutants, synaptic vesicles traveled excessively to endosomes. Using chimeric fluorescent timers, we show that synaptic vesicle-associated proteins were younger on average, suggesting that older proteins are more efficiently degraded. Using a genetic screen, we find that reducing endosomal-to-lysosomal trafficking, controlled by the homotypic fusion and vacuole protein sorting (HOPS) complex, rescued the neurotransmission and neurodegeneration defects in sky mutants. Consistently, synaptic vesicle proteins were older in HOPS complex mutants, and these mutants also showed reduced neurotransmission. Our findings define a mechanism in which synaptic transmission is facilitated by efficient protein turnover at lysosomes and identify a potential strategy to suppress defects arising from TBC1D24 mutations in humans.

HDAC6 is a Bruchpilot deacetylase that facilitates neurotransmitter release.

Presynaptic densities are specialized structures involved in synaptic vesicle tethering and neurotransmission; however, the mechanisms regulating their function remain understudied. In Drosophila, Bruchpilot is a major constituent of the presynaptic density that tethers vesicles. Here, we show that HDAC6 is necessary and sufficient for deacetylation of Bruchpilot. HDAC6 expression is also controlled by TDP-43, an RNA-binding protein deregulated in amyotrophic lateral sclerosis (ALS). Animals expressing TDP-43 harboring pathogenic mutations show increased HDAC6 expression, decreased Bruchpilot acetylation, larger vesicle-tethering sites, and increased neurotransmission, defects similar to those seen upon expression of HDAC6 and opposite to hdac6 null mutants. Consequently, reduced levels of HDAC6 or increased levels of ELP3, a Bruchpilot acetyltransferase, rescue the presynaptic density defects in TDP-43-expressing flies as well as the decreased adult locomotion. Our work identifies HDAC6 as a Bruchpilot deacetylase and indicates that regulating acetylation of a presynaptic release-site protein is critical for maintaining normal neurotransmission.