Patrik Verstreken

Synaptic Mitochondria Are Critical for Mobilization of Reserve Pool Vesicles at Drosophila Neuromuscular Junctions

In a forward screen for genes affecting neurotransmission in Drosophila, we identified mutations in dynamin-related protein (drp1). DRP1 is required for proper cellular distribution of mitochondria, and in mutant neurons, mitochondria are largely absent from synapses, thus providing a genetic tool to assess the role of mitochondria at synapses. Although resting Ca2+ is elevated at drp1 NMJs, basal synaptic properties are barely affected. However, during intense stimulation, mutants fail to maintain normal neurotransmission. Surprisingly, FM1-43 labeling indicates normal exo- and endocytosis, but a specific inability to mobilize reserve pool vesicles, which is partially rescued by exogenous ATP. Using a variety of drugs, we provide evidence that reserve pool recruitment depends on mitochondrial ATP production downstream of PKA signaling and that mitochondrial ATP limits myosin-propelled mobilization of reserve pool vesicles. Our data suggest a specific role for mitochondria in regulating synaptic strength.

Drosophila parkin mutants have decreased mass and cell size and increased sensitivity to oxygen radical stress

Mutations in the gene parkin in humans (PARK2) are responsible for a large number of familial cases of autosomal-recessive Parkinson disease. We have isolated a Drosophila homolog of human PARK2 and characterized its expression and null phenotype. parkin null flies have 30% lower mass than wild-type controls which is in part accounted for by a reduced cell size and number. In addition, these flies are infertile, show significantly reduced longevity, and are unable to jump or fly. Rearing mutants on paraquat, which generates toxic free radicals in vivo, causes a further reduction in longevity. Furthermore, loss of parkin results in progressive degeneration of most indirect flight muscle (IFM) groups soon after eclosion, accompanied by apoptosis. However, parkin mutants have normal neuromuscular junction recordings during the third larval instar stage, suggesting that larval musculature is intact and that parkin is required only in pupal and adult muscle. parkin flies do not show an age-dependent dopaminergic neuron loss in the brain, even after aging adults for 3 weeks. Nevertheless, degeneration of IFMs demonstrates the importance of parkin in maintaining specific cell groups, perhaps those with a high-energy demand and the concomitant production of high levels of free radicals. parkin mutants will be a valuable model for future analysis of the mechanisms of cell and tissue degeneration.

Synaptojanin Is Recruited by Endophilin to Promote Synaptic Vesicle Uncoating

We describe the isolation and characterization of Drosophila synaptojanin (synj) mutants. synj encodes a phosphatidylinositol phosphatase involved in clathrin-mediated endocytosis. We show that Synj is specifically localized to presynaptic terminals and is associated with synaptic vesicles. The electrophysiological and ultrastructural defects observed in synj mutants are strikingly similar to those found in endophilin mutants, and Synj and Endo colocalize and interact biochemically. Moreover, synj; endo double mutant synaptic terminals exhibit properties that are very similar to terminals of each single mutant, and overexpression of Endophilin can partially rescue the functional defects in partial loss-of-function synj mutants. Interestingly, Synj is mislocalized and destabilized at synapses devoid of Endophilin, suggesting that Endophilin recruits and stabilizes Synj on newly formed vesicles to promote vesicle uncoating. Our data also provide further evidence that kiss-and-run is able to maintain neurotransmitter release when synapses are not extensively challenged.

Endophilin Mutations Block Clathrin-Mediated Endocytosis but Not Neurotransmitter Release

We have identified mutations in Drosophila endophilin to study its function in vivo. Endophilin is required presynaptically at the neuromuscular junction, and absence of Endophilin dramatically impairs endocytosis in vivo. Mutant larvae that lack Endophilin fail to take up FM1-43 dye in synaptic boutons, indicating an inability to retrieve synaptic membrane. This defect is accompanied by an expansion of the presynaptic membrane, and a depletion of vesicles from the bouton lumen. Interestingly, mutant larvae are still able to sustain release at 15%-20% of the normal rate during high-frequency stimulation. We propose that kiss-and-run maintains neurotransmission at active zones of the larval NMJ in endophilin animals.

Parkinson's disease mutations in PINK1 result in decreased Complex I activity and deficient synaptic function

Mutations of the mitochondrial PTEN (phosphatase and tensin homologue)‐induced kinase1 (PINK1) are important causes of recessive Parkinson disease (PD). Studies on loss of function and overexpression implicate PINK1 in apoptosis, abnormal mitochondrial morphology, impaired dopamine release and motor deficits. However, the fundamental mechanism underlying these various phenotypes remains to be clarified. Using fruit fly and mouse models we show that PINK1 deficiency or clinical mutations impact on the function of Complex I of the mitochondrial respiratory chain, resulting in mitochondrial depolarization and increased sensitivity to apoptotic stress in mammalian cells and tissues. In Drosophila neurons, PINK1 deficiency affects synaptic function, as the reserve pool of synaptic vesicles is not mobilized during rapid stimulation. The fundamental importance of PINK1 for energy maintenance under increased demand is further corroborated as this deficit can be rescued by adding ATP to the synapse. The clinical relevance of our observations is demonstrated by the fact that human wild type PINK1, but not PINK1 containing clinical mutations, can rescue Complex 1 deficiency. Our work suggests that Complex I deficiency underlies, at least partially, the pathogenesis of this hereditary form of PD. As Complex I dysfunction is also implicated in sporadic PD, a convergence of genetic and environmental causes of PD on a similar mitochondrial molecular mechanism appears to emerge.

The v-ATPase V0 Subunit a1 Is Required for a Late Step in Synaptic Vesicle Exocytosis in Drosophila

The V(0) complex forms the proteolipid pore of an ATPase that acidifies vesicles. In addition, an independent function in membrane fusion has been proposed largely based on yeast vacuolar fusion experiments. We have isolated mutations in the largest V(0) component vha100-1 in flies in an unbiased genetic screen for synaptic malfunction. The protein is only required in neurons, colocalizes with markers for synaptic vesicles as well as active zones, and interacts with t-SNAREs. Loss of vha100-1 leads to vesicle accumulation in synaptic terminals, suggesting a deficit in release. The amplitude of spontaneous release events and release with hypertonic stimulation indicate normal levels of neurotransmitter loading, yet mutant embryos display severe defects in evoked synaptic transmission and FM1-43 uptake. Our data suggest that Vha100-1 functions downstream of SNAREs in synaptic vesicle fusion.

Shar-pei mediates cell proliferation arrest during imaginal disc growth in Drosophila

During animal development, organ size is determined primarily by the amount of cell proliferation, which must be tightly regulated to ensure the generation of properly proportioned organs. However, little is known about the molecular pathways that direct cells to stop proliferating when an organ has attained its proper size. We have identified mutations in a novel gene, shar-pei, that is required for proper termination of cell proliferation during Drosophila imaginal disc development. Clones of shar-pei mutant cells in imaginal discs produce enlarged tissues containing more cells of normal size. We show that this phenotype is the result of both increased cell proliferation and reduced apoptosis. Hence, shar-pei restricts cell proliferation and promotes apoptosis. By contrast, shar-pei is not required for cell differentiation and pattern formation of adult tissue. Shar-pei is also not required for cell cycle exit during terminal differentiation, indicating that the mechanisms directing cell proliferation arrest during organ growth are distinct from those directing cell cycle exit during terminal differentiation. shar-pei encodes a WW-domain-containing protein that has homologs in worms, mice and humans, suggesting that mechanisms of organ growth control are evolutionarily conserved.

Drosophila fragile X protein, DFXR, regulates neuronal morphology and function in the brain.

Mental retardation is a pervasive societal problem, 25 times more common than blindness for example. Fragile X syndrome, the most common form of inherited mental retardation, is caused by mutations in the FMR1 gene. Fragile X patients display neurite morphology defects in the brain, suggesting that this may be the basis of their mental retardation. Drosophila contains a single homolog of FMR1, dfxr (also called dfmr1). We analyzed the role of dfxr in neurite development in three distinct neuronal classes. We find that DFXR is required for normal neurite extension, guidance, and branching. dfxr mutants also display strong eclosion failure and circadian rhythm defects. Interestingly, distinct neuronal cell types show different phenotypes, suggesting that dfxr differentially regulates diverse targets in the brain.

Dap160/Intersectin Acts as a Stabilizing Scaffold Required for Synaptic Development and Vesicle Endocytosis

We describe the isolation of mutations in dynamin-associated protein 160 kDa (dap160), the Drosophila homolog of intersectin, a putative adaptor for proteins involved in endocytosis, cytoskeletal regulation, and signaling. We show that partial loss-of-function mutants display temperature-sensitive (ts) paralysis, whereas null mutants show ts defects in endocytosis. Loss-of-function mutants exhibit bouton overgrowth at larval neuromuscular junctions (NMJs), but evoked neurotransmission is normal. Mutant NMJs show a mild endocytic defect at 22 degrees C, which is strongly enhanced at 34 degrees C. The levels of dynamin, synaptojanin and endophilin are severely reduced in dap160 mutant NMJs, suggesting that Dap160 serves to stabilize an endocytic macromolecular complex. Electron microscopy reveals fewer vesicles, aberrant large vesicles, and an accumulation of endocytic intermediates at active and periactive zones in mutant terminals. Our data suggest that Dap160, like dynamin, is involved in synaptic vesicle retrieval at active and periactive zones.

Variants of the elongator protein 3 (ELP3) gene are associated with motor neuron degeneration

Amyotrophic lateral sclerosis (ALS) is a spontaneous, relentlessly progressive motor neuron disease, usually resulting in death from respiratory failure within 3 years. Variation in the genes SOD1 and TARDBP accounts for a small percentage of cases, and other genes have shown association in both candidate gene and genome-wide studies, but the genetic causes remain largely unknown. We have performed two independent parallel studies, both implicating the RNA polymerase II component, ELP3, in axonal biology and neuronal degeneration. In the first, an association study of 1884 microsatellite markers, allelic variants of ELP3 were associated with ALS in three human populations comprising 1483 people (P = 1.96 × 10−9). In the second, an independent mutagenesis screen in Drosophila for genes important in neuronal communication and survival identified two different loss of function mutations, both in ELP3 (R475K and R456K). Furthermore, knock down of ELP3 protein levels using antisense morpholinos in zebrafish embryos resulted in dose-dependent motor axonal abnormalities [Pearson correlation: −0.49, P = 1.83 × 10−12 (start codon morpholino) and −0.46, P = 4.05 × 10−9 (splice-site morpholino), and in humans, risk-associated ELP3 genotypes correlated with reduced brain ELP3 expression (P = 0.01). These findings add to the growing body of evidence implicating the RNA processing pathway in neurodegeneration and suggest a critical role for ELP3 in neuron biology and of ELP3 variants in ALS.