Alpha-synuclein suppresses mitochondrial protease ClpP to trigger mitochondrial oxidative damage and neurotoxicity.
ABSTRACT: Both ?-Synuclein (?Syn) accumulation and mitochondrial dysfunction have been implicated in the pathology of Parkinson's disease (PD). Although studies suggest that ?Syn and its missense mutant, A53T, preferentially accumulate in the mitochondria, the mechanisms by which ?Syn and mitochondrial proteins regulate each other to trigger mitochondrial and neuronal toxicity are poorly understood. ATP-dependent Clp protease (ClpP), a mitochondrial matrix protease, plays an important role in regulating mitochondrial protein turnover and bioenergetics activity. Here, we show that the protein level of ClpP is selectively decreased in ?Syn-expressing cell culture and neurons derived from iPS cells of PD patient carrying ?Syn A53T mutant, and in dopaminergic (DA) neurons of ?Syn A53T mice and PD patient postmortem brains. Deficiency in ClpP induces an overload of mitochondrial misfolded/unfolded proteins, suppresses mitochondrial respiratory activity, increases mitochondrial oxidative damage and causes cell death. Overexpression of ClpP reduces ?Syn-induced mitochondrial oxidative stress through enhancing the level of Superoxide Dismutase-2 (SOD2), and suppresses the accumulation of ?Syn S129 phosphorylation and promotes neuronal morphology in neurons derived from PD patient iPS cells carrying ?Syn A53T mutant. Moreover, we find that ?Syn WT and A53T mutant interact with ClpP and suppress its peptidase activity. The binding of ?Syn to ClpP further promotes a distribution of ClpP from soluble to insoluble cellular fraction in vitro and in vivo, leading to reduced solubility of ClpP. Compensating for the loss of ClpP in the substantia nigra of ?Syn A53T mice by viral expression of ClpP suppresses mitochondrial oxidative damage, and reduces ?Syn pathology and behavioral deficits of mice. Our findings provide novel insights into the mechanism underlying ?Syn-induced neuronal pathology, and they suggest that ClpP might be a useful therapeutic target for PD and other synucleinopathies.
Project description:Mutations in alpha-synuclein (alpha-Syn) cause Parkinson's disease (PD) in a small number of pedigrees with familial PD. Moreover, alpha-Syn accumulates as a major component of Lewy bodies and Lewy neurites, intraneuronal inclusions that are neuropathological hallmarks of PD. To better understand the pathogenic relationship between alterations in the biology of alpha-Syn and PD-associated neurodegeneration, we generated multiple lines of transgenic mice expressing high levels of either wild-type or familial PD-linked Ala-30 --> Pro (A30P) or Ala-53 --> Thr (A53T) human alpha-Syns. The mice expressing the A53T human alpha-Syn, but not wild-type or the A30P variants, develop adult-onset neurodegenerative disease with a progressive motoric dysfunction leading to death. Pathologically, affected mice exhibit neuronal abnormalities (in perikarya and neurites) including pathological accumulations of alpha-Syn and ubiquitin. Consistent with abnormal neuronal accumulation of alpha-Syn, brain regions with pathology exhibit increases in detergent-insoluble alpha-Syn and alpha-Syn aggregates. Our results demonstrate that the A53T mutant alpha-Syn causes significantly greater in vivo neurotoxicity as compared with other alpha-Syn variants. Further, alpha-Syn-dependent neurodegeneration is associated with abnormal accumulation of detergent-insoluble alpha-Syn.
Project description:Parkinson's disease (PD) is characterized by accumulation of alpha-synuclein (alpha-syn) and degeneration of neuronal populations in cortical and subcortical regions. Mitochondrial dysfunction has been considered a potential unifying factor in the pathogenesis of the disease. Mutations in genes linked to familial forms of PD, including SNCA encoding alpha-syn and Pten-induced putative kinase 1 (PINK1), have been shown to disrupt mitochondrial activity. We investigated the mechanisms through which mutant Pink1 might disrupt mitochondrial function in neuronal cells with alpha-syn accumulation. For this purpose, a neuronal cell model of PD was infected with virally-delivered Pink1, and was analyzed for cell survival, mitochondrial activity and calcium flux. Mitochondrial morphology was analyzed by confocal and electron microscopy. These studies showed that mutant (W437X) but not wildtype Pink1 exacerbated the alterations in mitochondrial function promoted by mutant (A53T) alpha-syn. This effect was associated with increased intracellular calcium levels. Co-expression of both mutant Pink1 and alpha-syn led to alterations in mitochondrial structure and neurite outgrowth that were partially ameliorated by treatment with cyclosporine A, and completely restored by treatment with the mitochondrial calcium influx blocker Ruthenium Red, but not with other cellular calcium flux blockers. Our data suggest a role for mitochondrial calcium influx in the mechanisms of mitochondrial and neuronal dysfunction in PD. Moreover, these studies support an important function for Pink1 in regulating mitochondrial activity under stress conditions.
Project description:Parkinson's disease (PD) is the second most common neurodegenerative disease. A key pathological feature of PD is Lewy bodies, of which the major protein component is ?-synuclein (?-syn). Human genetic studies have shown that mutations (A53T, A30P, E46K) and multiplication of the ?-syn gene are linked to familial PD. Mice overexpressing the human A53T mutant ?-syn gene develop severe movement disorders. However, the molecular mechanisms of ?-syn toxicity are not well understood. Recently, mitochondrial dysfunction has been linked with multiple neurodegenerative diseases including Parkinson's disease. Here we investigated whether mitochondrial motility, dynamics and respiratory function are affected in primary neurons from a mouse model expressing the human A53T mutation. We found that mitochondrial motility was selectively inhibited in A53T neurons while transport of other organelles was not affected. In addition, A53T expressing neurons showed impairment in mitochondrial membrane potential and mitochondrial respiratory function. Furthermore, we found that rapamycin, an autophagy inducer, rescued the decreased mitochondrial mobility. Taken together, these data demonstrate that A53T ?-syn impairs mitochondrial function and dynamics and the deficit of mitochondrial transport is reversible, providing further understanding of the disease pathogenesis and a potential therapeutic strategy for PD.
Project description:Transgenic (Tg) mouse models of Parkinson's disease (PD) generated to date have primarily been designed to overexpress human alpha-synuclein (alpha-syn) to recapitulate PD-like motor impairments as well as PD-like nigrostriatal degeneration and alpha-syn pathology. However, cognitive impairments and cortical alpha-syn pathology are also common in PD patients. To model these features of PD, we created forebrain-specific conditional Tg mice that overexpress human wild type (WT) or A53T mutant alpha-syn. Here we show that both WT and A53T mutant alpha-syn lead to massive degeneration of postmitotic neurons in the hippocampal dentate gyrus (DG) during postnatal development, with hippocampal synapse loss as evidenced by reduced levels of pre- and postsynaptic markers. However, when mutant and WT alpha-syn expression was repressed until the Tg mice were mature postnatally and then induced for several months, no hippocampal neuron loss was observed. These data imply that developing neurons are more vulnerable to degenerate than mature neurons as a consequence of forebrain WT and mutant alpha-syn overexpression.
Project description:Parkinson's disease (PD) is an age-dependent neurodegenerative disease that can be caused by genetic mutations in ?-synuclein (?-syn) or duplication of wild-type ?-syn; PD is characterized by the deposition of ?-syn aggregates, indicating a gain of toxicity from accumulation of ?-syn. Although the major neuropathologic feature of PD is the degeneration of dopaminergic (DA) neurons in the substantia nigra, non-motor symptoms including anxiety, cognitive defect and sleep disorder precede the onset of motor impairment, and many clinical symptoms of PD are not caused by degeneration of DA neurons. Non-human primate models of PD are important for revealing the early pathology in PD and identifying effective treatments. We established transgenic PD rhesus monkeys that express mutant ?-syn (A53T). Six transgenic A53T monkeys were produced via lentiviral vector expressing A53T in fertilized monkey eggs and subsequent embryo transfer to surrogates. Transgenic A53T is expressed in the monkey brain and causes age-dependent non-motor symptoms, including cognitive defects and anxiety phenotype, without detectable sleeping disorders. The transgenic ?-syn monkeys demonstrate the specific early symptoms caused by mutant ?-syn and provide insight into treatment of early PD.
Project description:Parkinson's disease (PD) is a movement disorder caused by neurodegeneration in neocortex, substantia nigra and brainstem, and synucleinopathy. Some inherited PD is caused by mutations in ?-synuclein (?Syn), and inherited and idiopathic PD is associated with mitochondrial perturbations. However, the mechanisms of pathogenesis are unresolved. We characterized a human ?Syn transgenic mouse model and tested the hypothesis that the mitochondrial permeability transition pore (mPTP) is involved in the disease mechanisms. C57BL/6 mice expressing human A53T-mutant ?Syn driven by a thymic antigen-1 promoter develop a severe, age-related, fatal movement disorder involving ataxia, rigidity, and postural instability. These mice develop synucleinopathy and neocortical, substantia nigra, and cerebello-rubro-thalamic degeneration involving mitochondriopathy and apoptotic and non-apoptotic neurodegeneration. Interneurons undergo apoptotic degeneration in young mice. Mutant ?Syn associated with dysmorphic neuronal mitochondria and bound voltage-dependent anion channels. Genetic ablation of cyclophilin D, an mPTP modulator, delayed disease onset, and extended lifespans of mutant ?Syn mice. Thus, mutant ?Syn transgenic mice on a C57BL/6 background develop PD-like phenotypes, and the mPTP is involved in their disease mechanisms.
Project description:Background: Mutations in glucocerebrosidase (GBA), a lysosomal enzyme are the most common genetic risk factor for developing Parkinson's disease (PD). We studied how reduced GCase activity affects ?-synuclein (?-syn) and its mutants (A30P and A53T) aggregation, neurodegeneration, sleep and locomotor behavior in a fly model of PD. Methods: We developed drosophila with GBA gene knockdown (RNAi) (with reduced GCase activity) that simultaneously expresses either wildtype (WT) or mutants such as A30P or A53T ?-syn. Western blot and confocal microscopy were performed to study the ?-syn aggregation and neurodegeneration in these flies. We also studied the sleep and locomotor activity of those flies using Drosophila activity monitor (DAM) system. Results: Western blot analysis showed that GBA RNAi A53T ?-syn flies (30 days old) had an increased level of Triton insoluble synuclein (that corresponds to ?-syn aggregates) compared to corresponding A53T flies without GBA RNAi (control), while mRNA expression of ?-syn remained unchanged. Confocal imaging of whole brain staining of 30 days old drosophila showed a statistically significant decrease in neuron numbers in PPL1 cluster in flies expressing ?-syn WT, A30P and A53T in the presence GBA RNAi compared to corresponding control. Staining with conformation specific antibody for ?-syn aggregates showed an increased number of neurons staining for ?-syn aggregates in A53T fly brain with GBA RNAi compared to control A53T flies, thus confirming our protein analysis finding that under decreased GBA enzyme activity, mutant A53T aggregates more than the control A53T without GBA silencing. Sleep analysis revealed decreased total activity in GBA silenced flies expressing mutant A53T compared to both A53T control flies and GBA RNAi flies without synuclein expression. Conclusion: In A53T flies with reduced GCase activity, there is increased ?-syn aggregation and dopamine (DA) neuronal loss. This study demonstrates that reduced GCase activity both in the context of heterozygous GBA1 mutation associated with PD and in old age, contribute to increased aggregation of mutant ?-syn A53T and exacerbates the phenotype in a fly model of PD.
Project description:Lipid peroxidation is a key to a portfolio of neurodegenerative diseases and plays a central role in ?-synuclein (?-syn) toxicity, mitochondrial dysfunction and neuronal death, all key processes in the pathogenesis of Parkinson's disease (PD). Polyunsaturated fatty acids (PUFAs) are important constituents of the synaptic and mitochondrial membranes and are often the first molecular targets attacked by reactive oxygen species (ROS). The rate-limiting step of the chain reaction of ROS-initiated PUFAs autoxidation involves hydrogen abstraction at bis-allylic sites, which can be slowed down if hydrogens are replaced with deuteriums. In this study, we show that targeted overexpression of human A53T ?-syn using an AAV vector unilaterally in the rat substantia nigra reproduces some of pathological features seen in PD patients. Chronic dietary supplementation with deuterated PUFAs (D-PUFAs), specifically 0.8% D-linoleic and 0.3% H-linolenic, produced significant disease-modifying beneficial effects against ?-syn-induced motor deficits, synaptic pathology, oxidative damage, mitochondrial dysfunction, disrupted trafficking along axons, inflammation and DA neuronal loss. These findings support the clinical evaluation of D-PUFAs as a neuroprotective therapy for PD.
Project description:Apolipoprotein E (APOE) ?4 genotype is associated with increased risk of dementia in Parkinson's disease (PD), but the mechanism is not clear, because patients often have a mixture of ?-synuclein (?Syn), amyloid-? (A?), and tau pathologies. APOE ?4 exacerbates brain A? pathology, as well as tau pathology, but it is not clear whether APOE genotype independently regulates ?Syn pathology. In this study, we generated A53T ?Syn transgenic mice (A53T) on Apoe knockout (A53T/EKO) or human APOE knockin backgrounds (A53T/E2, E3, and E4). At 12 months of age, A53T/E4 mice accumulated higher amounts of brainstem detergent-insoluble phosphorylated ?Syn compared to A53T/EKO and A53T/E3; detergent-insoluble ?Syn in A53T/E2 mice was undetectable. By immunohistochemistry, A53T/E4 mice displayed a higher burden of phosphorylated ?Syn and reactive gliosis compared to A53T/E2 mice. A53T/E2 mice exhibited increased survival and improved motor performance compared to other APOE genotypes. In a complementary model of ?Syn spreading, striatal injection of ?Syn preformed fibrils induced greater accumulation of ?Syn pathology in the substantia nigra of A53T/E4 mice compared to A53T/E2 and A53T/EKO mice. In two separate cohorts of human patients with PD, APOE ?4/?4 individuals showed the fastest rate of cognitive decline over time. Our results demonstrate that APOE genotype directly regulates ?Syn pathology independent of its established effects on A? and tau, corroborate the finding that APOE ?4 exacerbates pathology, and suggest that APOE ?2 may protect against ?Syn aggregation and neurodegeneration in synucleinopathies.
Project description:Dopaminergic neurodegeneration during Parkinson disease (PD) involves several pathways including proteasome inhibition, alpha-synuclein (alpha-syn) aggregation, mitochondrial dysfunction, and glutathione (GSH) depletion. We have utilized a systems biology approach and built a dynamic model to understand and link the various events related to PD pathophysiology. We have corroborated the modeling data by examining the effects of alpha-syn expression in the absence and presence of proteasome inhibition on GSH metabolism in dopaminergic neuronal cultures. We report here that the expression of the mutant A53T form of alpha-syn is neurotoxic and causes GSH depletion in cells after proteasome inhibition, compared to wild-type alpha-syn-expressing cells and vector control. Modeling data predicted that GSH depletion in these cells was due to ATP loss associated with mitochondrial dysfunction. ATP depletion elicited by combined A53T expression and proteasome inhibition results in decreased de novo synthesis of GSH via the rate-limiting enzyme gamma-glutamyl cysteine ligase. Based on these data and other recent reports, we propose a novel dynamic model to explain how the presence of mutated alpha-syn protein or proteasome inhibition may individually impact on mitochondrial function and in combination result in alterations in GSH metabolism via enhanced mitochondrial dysfunction.