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Summary The protein α-synuclein accumulates in the brain of patients with sporadic Parkinson’s disease (PD), and increased gene dosage causes a severe, dominantly inherited form of PD, but we know little about the effects of synuclein that precede degeneration. α-Synuclein localizes to the nerve terminal, but the knockout has little if any effect on synaptic transmission. In contrast, we now find that the modest over-expression of α-synuclein, in the range predicted for gene multiplication and in the absence of overt toxicity, markedly inhibits neurotransmitter release. The mechanism, elucidated by direct imaging of the synaptic vesicle cycle, involves a specific reduction in size of the synaptic vesicle recycling pool. Ultrastructural analysis demonstrates reduced synaptic vesicle density at the active zone, and imaging further reveals a defect in the reclustering of synaptic vesicles after endocytosis. Increased levels of α-synuclein thus produce a specific, physiological defect in synaptic vesicle recycling that precedes detectable neuropathology.

The protein ␣-synuclein has a central role in Parkinson disease, but the mechanism by which it contributes to neural degeneration remains unknown. We now show that the expression of ␣-synuclein in mammalian cells, including neurons in vitro and in vivo, causes the fragmentation of mitochondria. The effect is specific for synuclein, with more fragmentation by ␣-than ␤-or ␥-isoforms, and it is not accompanied by changes in the morphology of other organelles or in mitochondrial membrane potential. However, mitochondrial fragmentation is eventually followed by a decline in respiration and neuronal death. The fragmentation does not require the mitochondrial fission protein Drp1 and involves a direct interaction of synuclein with mitochondrial membranes. In vitro, synuclein fragments artificial membranes containing the mitochondrial lipid cardiolipin, and this effect is specific for the small oligomeric forms of synuclein. ␣-Synuclein thus exerts a primary and direct effect on the morphology of an organelle long implicated in the pathogenesis of Parkinson disease.Many observations have implicated mitochondria in the pathogenesis of PD.2 Mitochondria from the substantia nigra of affected patients show a selective reduction in the activity of respiratory chain complex I (1). Somatic mutations also accumulate with age and PD in the mitochondrial DNA of substantia nigra neurons (2). In addition, the neurotoxins MPTP and rotenone, which produce models of PD, both act by disrupting mitochondrial function. Genetic evidence further supports a primary role for mitochondria in the pathogenesis of PD. Mutations in parkin and the mitochondrial kinase PINK1 both cause autosomal recessive PD (3), and these genes appear required for the normal clearance of defective mitochondria by autophagy (4). However, the molecular mechanisms responsible for mitochondrial dysfunction in the much more common sporadic forms of PD have remained unclear. Several observations suggest a central role for the protein ␣-synuclein in the pathogenesis of sporadic PD. Point mutations in synuclein produce a rare autosomal dominant form of PD (5-7), indicating a causative role for the protein. ␣-Synuclein also accumulates in the Lewy bodies and dystrophic neurites of essentially all patients with idiopathic PD (8), implicating the protein in sporadic as well as familial forms of the disease. Furthermore, duplication and particularly triplication of the SNCA (␣-synuclein) gene cause a severe, highly penetrant form of PD (9, 10), indicating a dose-dependent pathogenic role for the wild type protein when overexpressed and suggesting that the accumulation of synuclein in sporadic PD is the primary pathogenic event. However, the mechanism by which ␣-synuclein causes PD remains poorly understood. Expressed in yeast and Drosophila, human ␣-synuclein produces severe toxicity (11-14), but these model organisms lack endogenous synuclein, and the overexpression of wild type synuclein in mammalian systems causes remarkably little if any consistent toxicity (15-18).Althou...

␣-Synuclein contributes to the pathogenesis of Parkinson's disease (PD), but its precise role in the disorder and its normal function remain poorly understood. Consistent with a presumed role in neurotransmitter release and its prominent deposition in the dystrophic neurites of PD, ␣-synuclein localizes almost exclusively to the nerve terminal. In brain extracts, however, ␣-synuclein behaves as a soluble, monomeric protein. Using a binding assay to characterize the association of ␣-synuclein with cell membranes, we find that ␣-synuclein binds saturably and with high affinity to characteristic intracellular structures that double label for components of lipid rafts. Biochemical analysis demonstrates the interaction of ␣-synuclein with detergent-resistant membranes and reveals a shift in electrophoretic mobility of the raft-associated protein. In addition, the A30P mutation associated with PD disrupts the interaction of ␣-synuclein with lipid rafts. Furthermore, we find that both the A30P mutation and raft disruption redistribute ␣-synuclein away from synapses, indicating an important role for raft association in the normal function of ␣-synuclein and its role in the pathogenesis of PD.

Summary Ubiquitin-mediated targeting of intracellular bacteria to the autophagy pathway is a key innate defense mechanism against invading microbes, including the important human pathogen Mycobacterium tuberculosis. However, the ubiquitin ligases responsible for catalyzing ubiquitin chains that surround intracellular bacteria are poorly understood. PARKIN is a ubiquitin ligase with a well-established role in mitophagy, and mutations in the PARKIN gene (Park2) lead to increased susceptibility to Parkinson’s disease. Surprisingly, genetic polymorphisms in the Park2 regulatory region are also associated with increased susceptibility to intracellular bacterial pathogens in humans, including Mycobacterium leprae and Salmonella typhi, but the function of PARKIN in immunity remains unexplored. Here we show that PARKIN plays a role in ubiquitin-mediated autophagy of M. tuberculosis. Both PARKIN-deficient mice and flies are sensitive to various intracellular bacterial infections, suggesting PARKIN plays a conserved role in metazoan innate defense. Moreover, our work reveals an unexpected functional link between mitophagy and infectious disease.

It has been only 15 years since studies began on the molecular mechanisms underlying mitochondrial fission and fusion using simple model organisms such as Drosophila, yeast, and C. elegans. Beyond the primary functions of mitochondrial fission and fusion in controlling organelle shape, size and number, it became clear that these dynamic processes are also critical for regulating cell death, mitophagy and organelle distribution. Now, studies suggest that prominent changes occur in mitochondrial dynamics in a broad array of neurodegenerative disease, and there is substantial evidence suggesting a key role in disease pathogenesis, as neurons are among the most energy-consuming cell types and have a highly developed cell shape. Here, we review the recent findings on mitochondrial dynamics in neurodegeneration.

TRAP1 (TNF receptor-associated protein), a member of the HSP90 chaperone family, is found predominantly in mitochondria. TRAP1 is broadly considered to be an anticancer molecular target. However, current inhibitors cannot distinguish between HSP90 and TRAP1, making their utility as probes of TRAP1-specific function questionable. Some cancers express less TRAP1 than do their normal tissue counterparts, suggesting that TRAP1 function in mitochondria of normal and transformed cells is more complex than previously appreciated. We have used TRAP1-null cells and transient TRAP1 silencing/overexpression to show that TRAP1 regulates a metabolic switch between oxidative phosphorylation and aerobic glycolysis in immortalized mouse fibroblasts and in human tumor cells. TRAP1-deficiency promotes an increase in mitochondrial respiration and fatty acid oxidation, and in cellular accumulation of tricarboxylic acid cycle intermediates, ATP and reactive oxygen species. At the same time, glucose metabolism is suppressed. TRAP1-deficient cells also display strikingly enhanced invasiveness. TRAP1 interaction with and regulation of mitochondrial c-Src provide a mechanistic basis for these phenotypes. Taken together with the observation that TRAP1 expression is inversely correlated with tumor grade in several cancers, these data suggest that, in some settings, this mitochondrial molecular chaperone may act as a tumor suppressor.M olecular chaperones help to maintain cellular homeostasis.The heat-shock protein 90 (HSP90) family of molecular chaperones is highly conserved from bacteria to mammals. HSP90 itself is an essential molecular chaperone found in the cytoplasm and nucleus of all eukaryotic cells (1, 2). In multicellular eukaryotes, the HSP90 family includes the mitochondrial chaperone TRAP1 (TNF receptor-associated protein), which shares 50% sequence similarity with HSP90. Although TRAP1 binds and hydrolyzes ATP in an analogous manner to HSP90 (3), its cellular function is less well understood. Thus, although many HSP90-dependent proteins ("clients") and interacting cochaperones have been described (www.picard.ch/downloads/Hsp90interactors.pdf), the validated list of TRAP1-dependent clients is quite small and TRAP1-interacting cochaperones, if they exist, have yet to be identified (4).Several studies have suggested that TRAP1 plays a cytoprotective role by buffering reactive oxygen species (ROS)-mediated oxidative stress (5, 6), and others have reported that TRAP1 overexpression attenuates ROS production (7). The antioxidant properties of TRAP1, together with its reported ability to regulate opening of the mitochondrial permeability transition pore (8, 9), may contribute to its antiapoptotic activity (4). For these reasons, TRAP1 has been proposed as an anticancer molecular target, and first-generation inhibitors have shown some anticancer activity in preclinical models (10). However, these inhibitors do not distinguish between HSP90 and TRAP1 (11), and TRAP1 expression in cancer is variable but HSP90 comprises as much as 5% of...

Summary Mitochondria have long been implicated in the pathogenesis of Parkinson’s disease (PD). Mutations in the mitochondrial kinase PINK1 that reduce kinase activity are associated with mitochondrial defects and result in an autosomal recessive form of early onset PD. Therapeutic approaches for enhancing the activity of PINK1 have not been considered since no allosteric regulatory sites for PINK1 are known. Here we show that an alternative strategy, a neo-substrate approach involving the ATP analog kinetin triphosphate (KTP), can be used to increase the activity of both PD related mutant PINK1G309D and PINK1wt. Moreover, we show that application of the KTP precursor kinetin to cells results in biologically significant increases in PINK1 activity, manifest as higher levels of Parkin recruitment to depolarized mitochondria, reduced mitochondrial motility in axons, and lower levels of apoptosis. Discovery of neo-substrates for kinases could provide a heretofore-unappreciated modality for regulating kinase activity.