PINK1 and Parkin target Miro for phosphorylation and degradation to arrest mitochondrial motility.

Cells keep their energy balance and avoid oxidative stress by regulating mitochondrial movement, distribution, and clearance. We report here that two Parkinson's disease proteins, the Ser/Thr kinase PINK1 and ubiquitin ligase Parkin, participate in this regulation by arresting mitochondrial movement. PINK1 phosphorylates Miro, a component of the primary motor/adaptor complex that anchors kinesin to the mitochondrial surface. The phosphorylation of Miro activates proteasomal degradation of Miro in a Parkin-dependent manner. Removal of Miro from the mitochondrion also detaches kinesin from its surface. By preventing mitochondrial movement, the PINK1/Parkin pathway may quarantine damaged mitochondria prior to their clearance. PINK1 has been shown to act upstream of Parkin, but the mechanism corresponding to this relationship has not been known. We propose that PINK1 phosphorylation of substrates triggers the subsequent action of Parkin and the proteasome.

Correction: Mutations in LRRK2 linked to Parkinson disease sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia.

[This corrects the article DOI: 10.1371/journal.pbio.3001480.].

Mutations in LRRK2 linked to Parkinson disease sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal dominant Parkinson disease (PD), while polymorphic LRRK2 variants are associated with sporadic PD. PD-linked mutations increase LRRK2 kinase activity and induce neurotoxicity in vitro and in vivo. The small GTPase Rab8a is a LRRK2 kinase substrate and is involved in receptor-mediated recycling and endocytic trafficking of transferrin, but the effect of PD-linked LRRK2 mutations on the function of Rab8a is poorly understood. Here, we show that gain-of-function mutations in LRRK2 induce sequestration of endogenous Rab8a to lysosomes in overexpression cell models, while pharmacological inhibition of LRRK2 kinase activity reverses this phenotype. Furthermore, we show that LRRK2 mutations drive association of endocytosed transferrin with Rab8a-positive lysosomes. LRRK2 has been nominated as an integral part of cellular responses downstream of proinflammatory signals and is activated in microglia in postmortem PD tissue. Here, we show that iPSC-derived microglia from patients carrying the most common LRRK2 mutation, G2019S, mistraffic transferrin to lysosomes proximal to the nucleus in proinflammatory conditions. Furthermore, G2019S knock-in mice show a significant increase in iron deposition in microglia following intrastriatal LPS injection compared to wild-type mice, accompanied by striatal accumulation of ferritin. Our data support a role of LRRK2 in modulating iron uptake and storage in response to proinflammatory stimuli in microglia.

Regulation of a distinct activated RIPK1 intermediate bridging complex I and complex II in TNFα-mediated apoptosis.

Stimulation of cells with TNFα can promote distinct cell death pathways, including RIPK1-independent apoptosis, necroptosis, and RIPK1-dependent apoptosis (RDA)-the latter of which we still know little about. Here we show that RDA involves the rapid formation of a distinct detergent-insoluble, highly ubiquitinated, and activated RIPK1 pool, termed "iuRIPK1." iuRIPK1 forms after RIPK1 activation in TNF-receptor-associated complex I, and before cytosolic complex II formation and caspase activation. To identify regulators of iuRIPK1 formation and RIPK1 activation in RDA, we conducted a targeted siRNA screen of 1,288 genes. We found that NEK1, whose loss-of-function mutations have been identified in 3% of ALS patients, binds to activated RIPK1 and restricts RDA by negatively regulating formation of iuRIPK1, while LRRK2, a kinase implicated in Parkinson's disease, promotes RIPK1 activation and association with complex I in RDA. Further, the E3 ligases APC11 and c-Cbl promote RDA, and c-Cbl is recruited to complex I in RDA, where it promotes prodeath K63-ubiquitination of RIPK1 to lead to iuRIPK1 formation. Finally, we show that two different modes of necroptosis induction by TNFα exist which are differentially regulated by iuRIPK1 formation. Overall, this work reveals a distinct mechanism of RIPK1 activation that mediates the signaling mechanism of RDA as well as a type of necroptosis.

Lysosome and Inflammatory Defects in GBA1-Mutant Astrocytes Are Normalized by LRRK2 Inhibition.

BACKGROUND: Autosomal recessive mutations in the glucocerebrosidase gene, Beta-glucocerebrosidase 1 (GBA1), cause the lysosomal storage disorder Gaucher's disease. Heterozygous carriers of most GBA1 mutations have dramatically increased Parkinson's disease (PD) risk, but the mechanisms and cells affected remain unknown. Glucocerebrosidase expression is relatively enriched in astrocytes, yet the impact of its mutation in these cells has not yet been addressed. OBJECTIVES: Emerging data supporting non-cell-autonomous mechanisms driving PD pathogenesis inspired the first characterization of GBA1-mutant astrocytes. In addition, we asked whether LRRK2, likewise linked to PD and enriched in astrocytes, intersected with GBA1 phenotypes. METHODS: Using heterozygous and homozygous GBA1 D409V knockin mouse astrocytes, we conducted rigorous biochemical and image-based analyses of lysosomal function and morphology. We also examined basal and evoked cytokine response at the transcriptional and secretory levels. RESULTS: The D409V knockin astrocytes manifested broad deficits in lysosomal morphology and function, as expected. This, however, is the first study to show dramatic defects in basal and TLR4-dependent cytokine production. Albeit to different extents, both the lysosomal dysfunction and inflammatory responses were normalized by inhibition of LRRK2 kinase activity, suggesting functional intracellular crosstalk between glucocerebrosidase and LRRK2 activities in astrocytes. CONCLUSIONS: These data demonstrate novel pathologic effects of a GBA1 mutation on inflammatory responses in astrocytes, indicating the likelihood of broader immunologic changes in GBA-PD patients. Our findings support the involvement of non-cell-autonomous mechanisms contributing to the pathogenesis of GBA1-linked PD and identify new opportunities to correct these changes with pharmacological intervention. © 2020 International Parkinson and Movement Disorder Society.

Miro phosphorylation sites regulate Parkin recruitment and mitochondrial motility.

The PTEN-induced putative kinase 1 (PINK1)/Parkin pathway can tag damaged mitochondria and trigger their degradation by mitophagy. Before the onset of mitophagy, the pathway blocks mitochondrial motility by causing Miro degradation. PINK1 activates Parkin by phosphorylating both Parkin and ubiquitin. PINK1, however, has other mitochondrial substrates, including Miro (also called RhoT1 and -2), although the significance of those substrates is less clear. We show that mimicking PINK1 phosphorylation of Miro on S156 promoted the interaction of Parkin with Miro, stimulated Miro ubiquitination and degradation, recruited Parkin to the mitochondria, and via Parkin arrested axonal transport of mitochondria. Although Miro S156E promoted Parkin recruitment it was insufficient to trigger mitophagy in the absence of broader PINK1 action. In contrast, mimicking phosphorylation of Miro on T298/T299 inhibited PINK1-induced Miro ubiquitination, Parkin recruitment, and Parkin-dependent mitochondrial arrest. The effects of the T298E/T299E phosphomimetic were dominant over S156E substitution. We propose that the status of Miro phosphorylation influences the decision to undergo Parkin-dependent mitochondrial arrest, which, in the context of PINK1 action on other substrates, can restrict mitochondrial dynamics before mitophagy.

Familial knockin mutation of LRRK2 causes lysosomal dysfunction and accumulation of endogenous insoluble α-synuclein in neurons.

Missense mutations in the multi-domain kinase LRRK2 cause late onset familial Parkinson's disease. They most commonly with classic proteinopathy in the form of Lewy bodies and Lewy neurites comprised of insoluble α-synuclein, but in rare cases can also manifest tauopathy. The normal function of LRRK2 has remained elusive, as have the cellular consequences of its mutation. Data from LRRK2 null model organisms and LRRK2-inhibitor treated animals support a physiological role for LRRK2 in regulating lysosome function. Since idiopathic and LRRK2-linked PD are associated with the intraneuronal accumulation of protein aggregates, a series of critical questions emerge. First, how do pathogenic mutations that increase LRRK2 kinase activity affect lysosome biology in neurons? Second, are mutation-induced changes in lysosome function sufficient to alter the metabolism of α-synuclein? Lastly, are changes caused by pathogenic mutation sensitive to reversal with LRRK2 kinase inhibitors? Here, we report that mutation of LRRK2 induces modest but significant changes in lysosomal morphology and acidification, and decreased basal autophagic flux when compared to WT neurons. These changes were associated with an accumulation of detergent-insoluble α-synuclein and increased neuronal release of α-synuclein and were reversed by pharmacologic inhibition of LRRK2 kinase activity. These data demonstrate a critical and disease-relevant influence of native neuronal LRRK2 kinase activity on lysosome function and α-synuclein homeostasis. Furthermore, they also suggest that lysosome dysfunction, altered neuronal α-synuclein metabolism, and the insidious accumulation of aggregated protein over decades may contribute to pathogenesis in this late-onset form of familial PD.

Membrane recruitment of endogenous LRRK2 precedes its potent regulation of autophagy.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial and idiopathic Parkinson's disease. However, the mechanisms for activating its physiological function are not known, hindering identification of the biological role of endogenous LRRK2. The recent discovery that LRRK2 is highly expressed in cells of the innate immune system and genetic association is a risk factor for autoimmune disorders implies an important role for LRRK2 in pathology outside of the central nervous system. Thus, an examination of endogenous LRRK2 in immune cells could provide insight into the protein's function. Here, we establish that stimulation of specific Toll-like receptors results in a complex biochemical activation of endogenous LRRK2, with early phosphorylation of LRRK2 preceding its dimerization and membrane translocation. Membrane-associated LRRK2 co-localized to autophagosome membranes following either TLR4 stimulation or mTOR inhibition with rapamycin. Silencing of endogenous LRRK2 expression resulted in deficits in the induction of autophagy and clearance of a well-described macroautophagy substrate, demonstrating the critical role of endogenous LRRK2 in regulating autophagy. Inhibition of LRRK2 kinase activity also reduced autophagic degradation and suggested the importance of the kinase domain in the regulation of autophagy. Our results demonstrate a well-orchestrated series of biochemical events involved in the activation of LRRK2 important to its physiological function. With similarities observed across multiple cell types and stimuli, these findings are likely relevant in all cell types that natively express endogenous LRRK2, and provide insights into LRRK2 function and its role in human disease.

Pathologic and therapeutic implications for the cell biology of parkin.

Mutations in the E3 ligase parkin are the most common cause of autosomal recessive Parkinson's disease (PD), but it is believed that parkin dysfunction may also contribute to idiopathic PD. Since its discovery, parkin has been implicated in supporting multiple neuroprotective pathways, many revolving around the maintenance of mitochondrial health quality control and governance of cell survival. Recent advances across the structure, biochemistry, and cell biology of parkin have provided great insights into the etiology of parkin-linked and idiopathic PD and may ultimately generate novel therapeutic strategies to slow or halt disease progression. This review describes the various pathways in which parkin acts and the mechanisms by which parkin may be targeted for therapeutic intervention. This article is part of a Special Issue entitled 'Neuronal Protein'.

The ubiquitin E3 ligase parkin regulates the proapoptotic function of Bax.

Autosomal recessive loss-of-function mutations within the PARK2 gene functionally inactivate the E3 ubiquitin ligase parkin, resulting in neurodegeneration of catecholaminergic neurons and a familial form of Parkinson disease. Current evidence suggests both a mitochondrial function for parkin and a neuroprotective role, which may in fact be interrelated. The antiapoptotic effects of parkin have been widely reported, and may involve fundamental changes in the threshold for apoptotic cytochrome c release, but the substrate(s) involved in parkin dependent protection had not been identified. Here, we demonstrate the parkin-dependent ubiquitination of endogenous Bax comparing primary cultured neurons from WT and parkin KO mice and using multiple parkin-overexpressing cell culture systems. The direct ubiquitination of purified Bax was also observed in vitro following incubation with recombinant parkin. We found that parkin prevented basal and apoptotic stress-induced translocation of Bax to the mitochondria. Moreover, an engineered ubiquitination-resistant form of Bax retained its apoptotic function, but Bax KO cells complemented with lysine-mutant Bax did not manifest the antiapoptotic effects of parkin that were observed in cells expressing WT Bax. These data suggest that Bax is the primary substrate responsible for the antiapoptotic effects of parkin, and provide mechanistic insight into at least a subset of the mitochondrial effects of parkin.

Genetic deletion of the GATA1-regulated protein α-synuclein reduces oxidative stress and nitric oxide synthase levels in mature erythrocytes.

α-Synuclein is highly expressed in neural tissue and during erythropoiesis, where the key erythroid regulator GATA1 has been found to modulate its expression. While specific α-synuclein (SNCA) mutations are known to cause autosomal dominant familial Parkinson's disease, its wild-type function remains under debate. To investigate the role of α-synuclein in murine hematopoiesis and erythropoiesis, we utilized Snca knock-out mice and analyzed erythroid compartments for maturation defects, in vivo erythrocyte survival, and erythrocyte-based reactive oxygen species (ROS) and nitric oxide synthase (NOS) levels. Our findings show that while bone marrow and spleen erythropoiesis and peripheral blood erythrocyte survival in Snca(-/-) mice was comparable to controls, the levels of ROS and of NOS-2 were significantly decreased in mature erythrocytes in these animals. These results indicate a role for α-synuclein in regulating oxidative stress in erythrocytes in vivo and could open new avenues for the investigation of its function in non-neural tissue.

The LRRK2 kinase substrates RAB8a and RAB10 contribute complementary but distinct disease-relevant phenotypes in human neurons.

Mutations in the LRRK2 gene cause familial Parkinson's disease presenting with pleomorphic neuropathology that can involve α-synuclein or tau accumulation. LRRK2 mutations are thought to converge upon a pathogenic increase in LRRK2 kinase activity. A subset of small RAB GTPases has been identified as LRRK2 substrates, with LRRK2-dependent phosphorylation resulting in RAB inactivation. We used CRISPR-Cas9 genome editing to generate a novel series of isogenic iPSC lines deficient in the two most well-validated LRRK2 substrates, RAB8a and RAB10, from deeply phenotyped healthy control lines. Thorough characterization of NGN2-induced neurons revealed opposing effects of RAB8a and RAB10 deficiency on lysosomal pH and Golgi organization, with isolated effects of RAB8a and RAB10 ablation on α-synuclein and tau, respectively. Our data demonstrate largely antagonistic effects of genetic RAB8a or RAB10 inactivation, which provide discrete insight into the pathologic features of their biochemical inactivation by pathogenic LRRK2 mutation in human disease.

The mitochondrial disease associated protein Ndufaf2 is dispensable for Complex-1 assembly but critical for the regulation of oxidative stress.

Deficiency in human mitochondrial Complex-1 has been linked to a wide variety of neurological disorders. Homozygous deletion of the Complex-1 associated protein, Ndufaf2, leads to a severe juvenile onset encephalopathy involving degeneration of the substantia nigra and other sub-cortical regions resulting in adolescent lethality. To understand the precise role of Ndufaf2 in Complex-1 function and its links to neurologic disease, we studied the effects on Complex-1 assembly and function, as well as pathological consequences at the cellular level, in multiple in vitro models of Ndufaf2 deficiency. Using both Ndufaf2-deficient human neuroblastoma cells and primary fibroblasts cultured from Ndufaf2 knock-out mice we found that Ndufaf2-deficiency selectively reduces Complex-1 activity. While Ndufaf2 is traditionally referred to as an assembly factor of Complex-1, surprisingly, however, Ndufaf2-deficient cells were able to assemble a fully mature Complex-1 enzyme, albeit with reduced kinetics. Importantly, no evidence of intermediate or incomplete assembly was observed. Ndufaf2 deficiency resulted in significant increases in oxidative stress and mitochondrial DNA deletion, consistent with contemporary hypotheses regarding the pathophysiology of inherited mutations in Complex-1 disorders. These data suggest that Ndufaf2, unlike other Complex-1 assembly factors, may be more accurately described as a chaperone involved in proper folding during Complex-1 assembly, since it is dispensable for Complex-1 maturation but not its proper function.

Polymerization of recombinant tau core fragments in vitro and seeding studies in cultured cells.

The relative polymerization of specific tau protein cores that define Alzheimer's disease, Pick's disease and corticobasal degeneration were investigated using amyloid fluorometry and electron microscopy. In addition, the relative prion-like activities of polymers comprised of these respective tau protein segments were investigated in a cell-based assay. It is demonstrated that the seeding activities of specific tau core fibrils are affected by the presence of pathogenic tau missense mutations and the microtubule binding domain composition of tau. The unique impact of tau phosphorylation on seeding propensity was also investigated by altering stretches of phospho-mimetic and phospho-null residues in the presence of Alzheimer's disease tau core fibrils. These results have important mechanistic implications for mutation and isoform-specific driven pathogenesis.

The LRRK2 kinase substrates Rab8a and Rab10 contribute complementary but distinct disease-relevant phenotypes in human neurons.

Mutations in the LRRK2 gene cause familial Parkinson's disease presenting with pleomorphic neuropathology that can involve α-synuclein or tau accumulation. LRRK2 mutations are thought to converge toward a pathogenic increase in LRRK2 kinase activity. A subset of small Rab GTPases have been identified as LRRK2 substrates, with LRRK2-dependent phosphorylation resulting in Rab inactivation. We used CRISPR/Cas9 genome editing to generate a novel series of isogenic iPSC lines deficient in the two most well validated LRRK2 substrates, Rab8a and Rab10, from two independent, deeply phenotyped healthy control lines. Thorough characterization of NGN2-induced neurons revealed divergent effects of Rab8a and Rab10 deficiency on lysosomal pH, LAMP1 association with Golgi, α-synuclein insolubility and tau phosphorylation, while parallel effects on lysosomal numbers and Golgi clustering were observed. Our data demonstrate largely antagonistic effects of genetic Rab8a or Rab10 inactivation which provide discrete insight into the pathologic features of their biochemical inactivation by pathogenic LRRK2 mutation.

Cellular and subcellular localization of Rab10 and phospho-T73 Rab10 in the mouse and human brain.

Autosomal dominant pathogenic mutations in Leucine-rich repeat kinase 2 (LRRK2) cause Parkinson's disease (PD). The most common mutation, G2019S-LRRK2, increases the kinase activity of LRRK2 causing hyper-phosphorylation of its substrates. One of these substrates, Rab10, is phosphorylated at a conserved Thr73 residue (pRab10), and is one of the most abundant LRRK2 Rab GTPases expressed in various tissues. The involvement of Rab10 in neurodegenerative disease, including both PD and Alzheimer's disease makes pinpointing the cellular and subcellular localization of Rab10 and pRab10 in the brain an important step in understanding its functional role, and how post-translational modifications could impact function. To establish the specificity of antibodies to the phosphorylated form of Rab10 (pRab10), Rab10 specific antisense oligonucleotides were intraventricularly injected into the brains of mice. Further, Rab10 knock out induced neurons, differentiated from human induced pluripotent stem cells were used to test the pRab10 antibody specificity. To amplify the weak immunofluorescence signal of pRab10, tyramide signal amplification was utilized. Rab10 and pRab10 were expressed in the cortex, striatum and the substantia nigra pars compacta. Immunofluorescence for pRab10 was increased in G2019S-LRRK2 knockin mice. Neurons, astrocytes, microglia and oligodendrocytes all showed Rab10 and pRab10 expression. While Rab10 colocalized with endoplasmic reticulum, lysosome and trans-Golgi network markers, pRab10 did not localize to these organelles. However, pRab10, did overlap with markers of the presynaptic terminal in both mouse and human cortex, including α-synuclein. Results from this study suggest Rab10 and pRab10 are expressed in all brain areas and cell types tested in this study, but pRab10 is enriched at the presynaptic terminal. As Rab10 is a LRRK2 kinase substrate, increased kinase activity of G2019S-LRRK2 in PD may affect Rab10 mediated membrane trafficking at the presynaptic terminal in neurons in disease.

Parkin coregulates glutathione metabolism in adult mammalian brain.

We recently discovered that the expression of PRKN, a young-onset Parkinson disease-linked gene, confers redox homeostasis. To further examine the protective effects of parkin in an oxidative stress model, we first combined the loss of prkn with Sod2 haploinsufficiency in mice. Although adult prkn-/-//Sod2± animals did not develop dopamine cell loss in the S. nigra, they had more reactive oxidative species and a higher concentration of carbonylated proteins in the brain; bi-genic mice also showed a trend for more nitrotyrosinated proteins. Because these redox changes were seen in the cytosol rather than mitochondria, we next explored the thiol network in the context of PRKN expression. We detected a parkin deficiency-associated increase in the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) in murine brain, PRKN-linked human cortex and several cell models. This shift resulted from enhanced recycling of GSSG back to GSH via upregulated glutathione reductase activity; it also correlated with altered activities of redox-sensitive enzymes in mitochondria isolated from mouse brain (e.g., aconitase-2; creatine kinase). Intriguingly, human parkin itself showed glutathione-recycling activity in vitro and in cells: For each GSSG dipeptide encountered, parkin regenerated one GSH molecule and was S-glutathionylated by the other (GSSG + P-SH [Formula: see text] GSH + P-S-SG), including at cysteines 59, 95 and 377. Moreover, parkin's S-glutathionylation was reversible by glutaredoxin activity. In summary, we found that PRKN gene expression contributes to the network of available thiols in the cell, including by parkin's participation in glutathione recycling, which involves a reversible, posttranslational modification at select cysteines. Further, parkin's impact on redox homeostasis in the cytosol can affect enzyme activities elsewhere, such as in mitochondria. We posit that antioxidant functions of parkin may explain many of its previously described, protective effects in vertebrates and invertebrates that are unrelated to E3 ligase activity.

Dopamine covalently modifies and functionally inactivates parkin.

Inherited mutations in PARK2, the gene encoding parkin, cause selective degeneration of catecholaminergic neurons in the substantia nigra and locus coeruleus of the brainstem, resulting in early-onset parkinsonism. But the role of parkin in common, sporadic forms of Parkinson disease remains unclear. Here we report that the neurotransmitter dopamine covalently modifies parkin in living dopaminergic cells, a process that increases parkin insolubility and inactivates its E3 ubiquitin ligase function. In the brains of individuals with sporadic Parkinson disease, we observed decreases in parkin solubility consistent with its functional inactivation. Using a new biochemical method, we detected catechol-modified parkin in the substantia nigra but not other regions of normal human brain. These findings show a vulnerability of parkin to modification by dopamine, the principal transmitter lost in Parkinson disease, suggesting a mechanism for the progressive loss of parkin function in dopaminergic neurons during aging and sporadic Parkinson disease.

Aph-1 associates directly with full-length and C-terminal fragments of gamma-secretase substrates.

Gamma-secretase is a ubiquitous, multiprotein enzyme composed of presenilin, nicastrin, Aph-1, and Pen-2. It mediates the intramembrane proteolysis of many type 1 proteins, plays an essential role in numerous signaling pathways, and helps drive the pathogenesis of Alzheimer disease by excising the hydrophobic, aggregation-prone amyloid beta-peptide from the beta-amyloid precursor protein. A central unresolved question is how its many substrates bind and enter the gamma-secretase complex. Here, we provide evidence that both the beta-amyloid precursor protein holoprotein and its C-terminal fragments, the immediate substrates of gamma-secretase, can associate with Aph-1 at overexpressed as well as endogenous protein levels. This association was observed using bi-directional co-immunoprecipitation in multiple systems and detergent conditions, and an beta-amyloid precursor protein-Aph-1 complex was specifically isolated following in situ cross-linking in living cells. In addition, another endogenous canonical gamma-substrate, Jagged, showed association of both its full-length and C-terminal fragment forms with Aph-1. We were also able to demonstrate that this interaction with substrates was conserved across the multiple isoforms of Aph-1 (beta, alphaS, and alphaL), as they were all able to bind beta-amyloid precursor protein with similar affinity. Finally, two highly conserved intramembrane histidines (His-171 and His-197) within Aph-1, which were recently shown to be important for gamma-secretase activity, are required for efficient binding of substrates. Taken together, our data suggest a dominant role for Aph-1 in interacting with gamma-secretase substrates prior to their processing by the proteolytic complex.

Parkin selectively alters the intrinsic threshold for mitochondrial cytochrome c release.

Autosomal-recessive mutations in the Parkin gene are the second most common cause of familial Parkinson's disease (PD). Parkin deficiency leads to the premature demise of the catecholaminergic neurons of the ventral midbrain in familial PD. Thus, a better understanding of parkin function may elucidate molecular aspects of their selective vulnerability in idiopathic PD. Numerous lines of evidence suggest a mitochondrial function for parkin and a protective effect of ectopic parkin expression. Since mitochondria play a critical role in cell survival/cell death through regulated cytochrome c release and control of apoptosis, we sought direct evidence of parkin function in this pathway. Mitochondria were isolated from cells expressing either excess levels of human parkin or shRNA directed against endogenous parkin and then treated with peptides corresponding to the active Bcl-2 homology 3 (BH3) domains of pro-apoptotic proteins and the threshold for cytochrome c release was analyzed. Data obtained from both rodent and human neuroblastoma cell lines showed that the expression levels of parkin were inversely correlated with cytochrome c release. Parkin was found associated with isolated mitochondria, but its binding per se was not sufficient to inhibit cytochrome c release. In addition, pathogenic parkin mutants failed to influence cytochrome c release. Furthermore, PINK1 expression had no effect on cytochrome c release, suggesting a divergent function for this autosomal recessive PD-linked gene. In summary, these data demonstrate a specific autonomous effect of parkin on mitochondrial mechanisms governing cytochrome c release and apoptosis, which may be relevant to the selective vulnerability of certain neuronal populations in PD.