Common anti-apoptotic roles of parkin and alpha-synuclein in human dopaminergic cells

Parkin, a product of the gene responsible for autosomal recessive juvenile parkinsonism (AR-JP), is an important player in the pathogenic process of Parkinson's disease (PD). Despite numerous studies including search for the substrate of parkin as an E3 ubiquitin-protein ligase, the mechanism by which loss-of-function of parkin induces selective dopaminergic neuronal death remains unclear. Related to this issue, here we show that antisense knockdown of parkin causes apoptotic cell death of human dopaminergic SH-SY5Y cells associated with caspase activation and accompanied by accumulation of oxidative dopamine (DA) metabolites due to auto-oxidation of DOPA and DA. Forced expression of alpha-synuclein (alpha-SN), another familial PD gene product, prevented accumulation of oxidative DOPA/DA metabolites and cell death caused by parkin loss. Our findings indicate that both parkin and alpha-SN share a common pathway in DA metabolism whose abnormality leads to accumulation of oxidative DA metabolites and subsequent cell death.     

Progress in the pathogenesis and genetics of Parkinson's disease

Recent progresses in the pathogenesis of sporadic Parkinson's disease (PD) and genetics of familial PD are reviewed. There are common molecular events between sporadic and familial PD, particularly between sporadic PD and PARK1-linked PD due to alpha-synuclein (SNCA) mutations. In sporadic form, interaction of genetic predisposition and environmental factors is probably a primary event inducing mitochondrial dysfunction and oxidative damage resulting in oligomer and aggregate formations of alpha-synuclein. In PARK1-linked PD, mutant alpha-synuclein proteins initiate the disease process as they have increased tendency for self-aggregation. As highly phosphorylated aggregated proteins are deposited in nigral neurons in PD, dysfunctions of proteolytic systems, i.e. the ubiquitin-proteasome system and autophagy-lysosomal pathway, seem to be contributing to the final neurodegenerative process. Studies on the molecular mechanisms of nigral neuronal death in familial forms of PD will contribute further on the understanding of the pathogenesis of sporadic PD.     

14-3-3eta is a novel regulator of parkin ubiquitin ligase

Mutation of the parkin gene, which encodes an E3 ubiquitin-protein ligase, is the major cause of autosomal recessive juvenile parkinsonism (ARJP). Although various substrates for parkin have been identified, the mechanisms that regulate the ubiquitin ligase activity of parkin are poorly understood. Here we report that 14-3-3eta, a chaperone-like protein present abundantly in neurons, could bind to parkin and negatively regulate its ubiquitin ligase activity. Furthermore, 14-3-3eta could bind to the linker region of parkin but not parkin with ARJP-causing R42P, K161N, and T240R mutations. Intriguingly, alpha-synuclein (alpha-SN), another familial Parkinson's disease (PD) gene product, abrogated the 14-3-3eta-induced suppression of parkin activity. alpha-SN could bind tightly to 14-3-3eta and consequently sequester it from the parkin-14-3-3eta complex. PD-causing A30P and A53T mutants of alpha-SN could not bind 14-3-3eta, and failed to activate parkin. Our findings indicate that 14-3-3eta is a regulator that functionally links parkin and alpha-SN. The alpha-SN-positive and 14-3-3eta-negative control of parkin activity sheds new light on the pathophysiological roles of parkin.     

Oxidative modifications and down-regulation of ubiquitin carboxyl-terminal hydrolase L1 associated with idiopathic Parkinson's and Alzheimer's diseases

Alzheimer's disease (AD) and Parkinson's disease (PD) are the two most common neurodegenerative diseases that occur either in relatively rare, familial forms or in common, sporadic forms. The genetic defects underlying several monogenic familial forms of AD and PD have recently been identified, however, the causes of other AD and PD cases, particularly sporadic cases, remain unclear. To gain insights into the pathogenic mechanisms involved in AD and PD, we used a proteomic approach to identify proteins with altered expression levels and/or oxidative modifications in idiopathic AD and PD brains. Here, we report that the protein level of ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), a neuronal de-ubiquitinating enzyme whose mutation has been linked to an early-onset familial PD, is down-regulated in idiopathic PD as well as AD brains. By using a combination of two-dimensional gel electrophoresis and mass spectrometry, we have identified three human brain UCH-L1 isoforms, a full-length form and two amino-terminally truncated forms. Our proteomic analyses reveal that the full-length UCH-L1 is a major target of oxidative damage in AD and PD brains, which is extensively modified by carbonyl formation, methionine oxidation, and cysteine oxidation. Furthermore, immunohistochemical studies show that prominent UCH-L1 immunostaining is associated with neurofibrillary tangles and that the level of soluble UCH-L1 protein is inversely proportional to the number of tangles in AD brains. Together, these results provide evidence supporting a direct link between oxidative damage to the neuronal ubiquitination/de-ubiquitination machinery and the pathogenesis of sporadic AD and PD.     

Pathogenesis of familial Parkinson's disease: new insights based on monogenic forms of Parkinson's disease

Parkinson's disease (PD) is one of the most common movement disorders caused by the loss of dopaminergic neuronal cells. The molecular mechanisms underlying neuronal degeneration in PD remain unknown; however, it is now clear that genetic factors contribute to the pathogenesis of this disease. Approximately, 5% of patients with clinical features of PD have clear familial etiology, which show a classical recessive or dominant Mendelian mode of inheritance. Over the decade, more than 15 loci and 11 causative genes have been identified so far and many studies shed light on their implication in not only monogenic but also sporadic form of PD. Recent studies revealed that PD-associated genes play important roles in cellular functions, such as mitochondrial functions, ubiquitin-proteasomal system, autophagy-lysosomal pathway and membrane trafficking. Furthermore, the proteins encoded by PD-associated genes can interact with each other and such gene products may share a common pathway that leads to nigral degeneration. However, their precise roles in the disease and their normal functions remain poorly understood. In this study, we review recent progress in knowledge about the genes associated with familial PD.     

Mutant Pink1 induces mitochondrial dysfunction in a neuronal cell model of Parkinson's disease by disturbing calcium flux

Parkinson's disease (PD) is characterized by accumulation of α-synuclein (α-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 α-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 α-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) α-syn. This effect was associated with increased intracellular calcium levels. Co-expression of both mutant Pink1 and α-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.     

Mitochondrial translocation of alpha-synuclein is promoted by intracellular acidification

Mitochondrial dysfunction plays a central role in the selective vulnerability of dopaminergic neurons in Parkinson's disease (PD) and is influenced by both environmental and genetic factors. Expression of the PD protein α-synuclein or its familial mutants often sensitizes neurons to oxidative stress and to damage by mitochondrial toxins. This effect is thought to be indirect, since little evidence physically linking α-synuclein to mitochondria has been reported. Here, we show that the distribution of α-synuclein within neuronal and non-neuronal cells is dependent on intracellular pH. Cytosolic acidification induces translocation of α-synuclein from the cytosol onto the surface of mitochondria. Translocation occurs rapidly under artificially-induced low pH conditions and as a result of pH changes during oxidative or metabolic stress. Binding is likely facilitated by low pH-induced exposure of the mitochondria-specific lipid cardiolipin. These results imply a direct role for α-synuclein in mitochondrial physiology, especially under pathological conditions, and in principle, link α-synuclein to other PD genes in regulating mitochondrial homeostasis.     

Neuroinflammation and oxidative stress: Co-conspirators in the pathology of Parkinson’s disease

Parkinson’s disease (PD) is a complex disease, with genetics and environment contributing to the disease onset. Recent studies of causative PD genes have confirmed the involvement of cellular mechanisms engaged in mitochondrial and UPS dysfunction, oxidative stress and apoptosis in the progressive degeneration of the dopaminergic neurons in PD. In addition, clinical, epidemiological and experimental evidence has implicated neuroinflammation in the disease progression. This review will discuss neuroinflammation in PD, with particular focus on the genetic and toxin-based models of the disease. These studies have confirmed elevated oxidative stress and the pro-inflammatory response occurs early in the disease and these processes contribute to and/or exacerbate the nigro-striatal degeneration. In addition, the experimental models discussed here have also provided strong evidence that these pathways are an important link between the familial and sporadic causes of PD. The potential application of anti-inflammatory interventions in limiting the dopaminergic neuronal cell death in these models is discussed with evidence suggesting that the further investigation of their use as part of multi-targeted clinical trials is warranted.     

Embryonic stem cell-derived neuron models of Parkinson's disease exhibit delayed neuronal death

Establishment of a Parkinson's disease (PD) neuron model was attempted with mouse embryonic stem (ES) cells. ES cell lines over-expressing mouse nuclear receptor-related 1 (Nurr1), together with human wild-type and alanine 30 --> proline (A30P) and alanine 53 --> threonine (A53T) mutant alpha-synuclein were established and subjected to differentiation into dopaminergic neurons. The ES cell-derived dopaminergic neurons expressing wild-type or mutant alpha-synuclein exhibited the fundamental characteristics consistent with dopaminergic neurons in the substantia nigra. The ES cell-derived PD model neurons exhibited increased susceptibility to oxidative stress, proteasome inhibition, and mitochondrial inhibition. Cell viability of PD model neurons and the control neurons was similar until 28 days after differentiation. Nonetheless, after that time, PD model neurons gradually began to undergo neuronal death over the course of 1 month, showing cytoplasmic aggregate formation and an increase of insoluble alpha-synuclein protein. Such delayed neuronal death was observed in a mutant alpha-synuclein protein level-dependent manner, which was slightly inhibited by a c-jun N-terminal kinase inhibitor and a caspase inhibitor. Such cell death was not observed when the same ES cell lines were differentiated into oligodendrocytes. The ES cell-derived PD model neurons are considered as prospective candidates for a new prototype modelling PD that would allow better investigation of the underlying neurodegenerative pathophysiology.     

Parkinson's disease-associated DJ-1 mutations impair mitochondrial dynamics and cause mitochondrial dysfunction

Mitochondrial dysfunction represents a critical event during the pathogenesis of Parkinson's disease (PD) and expanding evidences demonstrate that an altered balance in mitochondrial fission/fusion is likely an important mechanism leading to mitochondrial and neuronal dysfunction/degeneration. In this study, we investigated whether DJ-1 is involved in the regulation of mitochondrial dynamics and function in neuronal cells. Confocal and electron microscopic analysis demonstrated that M17 human neuroblastoma cells over-expressing wild-type DJ-1 (WT DJ-1 cells) displayed elongated mitochondria while M17 cells over-expressing PD-associated DJ-1 mutants (R98Q, D149A and L166P) (mutant DJ-1 cells) showed significant increase of fragmented mitochondria. Similar mitochondrial fragmentation was also noted in primary hippocampal neurons over-expressing PD-associated mutant forms of DJ-1. Functional analysis revealed that over-expression of PD-associated DJ-1 mutants resulted in mitochondria dysfunction and increased neuronal vulnerability to oxidative stress (H(2) O(2)) or neurotoxin. Further immunoblot studies demonstrated that levels of dynamin-like protein (DLP1), also known as Drp1, a regulator of mitochondrial fission, was significantly decreased in WT DJ-1 cells but increased in mutant DJ-1 cells. Importantly, DLP1 knockdown in these mutant DJ-1 cells rescued the abnormal mitochondria morphology and all associated mitochondria/neuronal dysfunction. Taken together, these studies suggest that DJ-1 is involved in the regulation of mitochondrial dynamics through modulation of DLP1 expression and PD-associated DJ-1 mutations may cause PD by impairing mitochondrial dynamics and function.     

Reprint of: Revisiting oxidative stress and mitochondrial dysfunction in the pathogenesis of Parkinson disease—resemblance to the effect of amphetamine drugs of abuse

Parkinson disease (PD) is a chronic and progressive neurological disease associated with a loss of dopaminergic neurons. In most cases the disease is sporadic but genetically inherited cases also exist. One of the major pathological features of PD is the presence of aggregates that localize in neuronal cytoplasm as Lewy bodies, mainly composed of α-synuclein (α-syn) and ubiquitin. The selective degeneration of dopaminergic neurons suggests that dopamine itself may contribute to the neurodegenerative process in PD. Furthermore, mitochondrial dysfunction and oxidative stress constitute key pathogenic events of this disorder. Thus, in this review we give an actual perspective to classical pathways involving these two mechanisms of neurodegeneration, including the role of dopamine in sporadic and familial PD, as well as in the case of abuse of amphetamine-type drugs. Mutations in genes related to familial PD causing autosomal dominant or recessive forms may also have crucial effects on mitochondrial morphology, function, and oxidative stress. Environmental factors, such as MPTP and rotenone, have been reported to induce selective degeneration of the nigrostriatal pathways leading to α-syn-positive inclusions, possibly by inhibiting mitochondrial complex I of the respiratory chain and subsequently increasing oxidative stress. Recently, increased risk for PD was found in amphetamine users. Amphetamine drugs have effects similar to those of other environmental factors for PD, because long-term exposure to these drugs leads to dopamine depletion. Moreover, amphetamine neurotoxicity involves α-syn aggregation, mitochondrial dysfunction, and oxidative stress. Therefore, dopamine and related oxidative stress, as well as mitochondrial dysfunction, seem to be common links between PD and amphetamine neurotoxicity.     

Systematic Comparison of the Effects of Alpha-synuclein Mutations on Its Oligomerization and Aggregation

Aggregation of alpha-synuclein (ASYN) in Lewy bodies and Lewy neurites is the typical pathological hallmark of Parkinson''s disease (PD) and other synucleinopathies. Furthermore, mutations in the gene encoding for ASYN are associated with familial and sporadic forms of PD, suggesting this protein plays a central role in the disease. However, the precise contribution of ASYN to neuronal dysfunction and death is unclear. There is intense debate about the nature of the toxic species of ASYN and little is known about the molecular determinants of oligomerization and aggregation of ASYN in the cell. In order to clarify the effects of different mutations on the propensity of ASYN to oligomerize and aggregate, we assembled a panel of 19 ASYN variants and compared their behaviour. We found that familial mutants linked to PD (A30P, E46K, H50Q, G51D and A53T) exhibited identical propensities to oligomerize in living cells, but had distinct abilities to form inclusions. While the A30P mutant reduced the percentage of cells with inclusions, the E46K mutant had the opposite effect. Interestingly, artificial proline mutants designed to interfere with the helical structure of the N-terminal domain, showed increased propensity to form oligomeric species rather than inclusions. Moreover, lysine substitution mutants increased oligomerization and altered the pattern of aggregation. Altogether, our data shed light into the molecular effects of ASYN mutations in a cellular context, and established a common ground for the study of genetic and pharmacological modulators of the aggregation process, opening new perspectives for therapeutic intervention in PD and other synucleinopathies.     

Distinct cleavage patterns of normal and pathologic forms of alpha-synuclein by calpain I in vitro

Parkinson's disease (PD) is characterized by fibrillary neuronal inclusions called Lewy bodies (LBs) consisting largely of alpha-synuclein (alpha-syn), the protein mutated in some patients with familial PD. The mechanisms of alpha-syn fibrillization and LB formation are unknown, but may involve aberrant degradation or turnover. We examined the ability of calpain I to cleave alpha-syn in vitro. Calpain I cleaved wild-type alpha-syn predominantly after amino acid 57 and within the non-amyloid component (NAC) region. In contrast, calpain I cleaved fibrillized alpha-syn primarily in the region of amino acid 120 to generate fragments like those that increase susceptibility to dopamine toxicity and oxidative stress. Further, while calpain I cleaved wild-type alpha-syn after amino acid 57, this did not occur in mutant A53T alpha-syn. This paucity of proteolysis could increase the stability of A53T alpha-syn, suggesting that calpain I might protect cells from forming LBs by specific cleavages of soluble wild-type alpha-syn. However, once alpha-syn has polymerized into fibrils, calpain I may contribute to toxicity of these forms of alpha-syn by cleaving at aberrant sites within the C-terminal region. Elucidating the role of calpain I in the proteolytic processing of alpha-syn in normal and diseased brains may clarify mechanisms of neurodegenerative alpha-synucleinopathies.     

LRRK2 Affects Vesicle Trafficking,Neurotransmitter Extracellular Level and Membrane Receptor Localization

The leucine-rich repeat kinase 2 (LRRK2) gene was found to play a role in the pathogenesis of both familial and sporadic Parkinson’s disease (PD). LRRK2 encodes a large multi-domain protein that is expressed in different tissues. To date, the physiological and pathological functions of LRRK2 are not clearly defined. In this study we have explored the role of LRRK2 in controlling vesicle trafficking in different cellular or animal models and using various readouts. In neuronal cells, the presence of LRRK2^G2019S pathological mutant determines increased extracellular dopamine levels either under basal conditions or upon nicotine stimulation. Moreover, mutant LRRK2 affects the levels of dopamine receptor D1 on the membrane surface in neuronal cells or animal models. Ultrastructural analysis of PC12-derived cells expressing mutant LRRK2^G2019S shows an altered intracellular vesicle distribution. Taken together, our results point to the key role of LRRK2 to control vesicle trafficking in neuronal cells.     

Dopaminergic neuronal loss, reduced neurite complexity and autophagic abnormalities in transgenic mice expressing G2019S mutant LRRK2

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant familial Parkinson's disease (PD) and also contribute to idiopathic PD. LRRK2 mutations represent the most common cause of PD with clinical and neurochemical features that are largely indistinguishable from idiopathic disease. Currently, transgenic mice expressing wild-type or disease-causing mutants of LRRK2 have failed to produce overt neurodegeneration, although abnormalities in nigrostriatal dopaminergic neurotransmission have been observed. Here, we describe the development and characterization of transgenic mice expressing human LRRK2 bearing the familial PD mutations, R1441C and G2019S. Our study demonstrates that expression of G2019S mutant LRRK2 induces the degeneration of nigrostriatal pathway dopaminergic neurons in an age-dependent manner. In addition, we observe autophagic and mitochondrial abnormalities in the brains of aged G2019S LRRK2 mice and markedly reduced neurite complexity of cultured dopaminergic neurons. These new LRRK2 transgenic mice will provide important tools for understanding the mechanism(s) through which familial mutations precipitate neuronal degeneration and PD.     

The antioxidant Trolox helps recovery from the familial Parkinson's disease-specific mitochondrial deficits caused by PINK1- and DJ-1-deficiency in dopaminergic neuronal cells

The nature of mitochondrial dysfunction in dopaminergic neurons in familial Parkinson's disease (PD) is unknown. We characterized the pathophenotypes of dopaminergic neuronal cells that were deficient in PINK1 or DJ-1, genes with mutations linked to familial PD. Both PINK1- and DJ-1-deficient dopaminergic neurons had the increased production of ROS, severe mitochondrial structural damages and complex I deficits. A striking decrease in complex IV activity was also prominent by the PINK1-deficiency. The complex I deficits were relatively PD-specific and were significantly improved by an antioxidant Trolox. These data suggest that mitochondrial deficits are severe in dopaminergic neurons in familial PD and antioxidant-mediated functional recovery is feasible.     

Effect of single amino acid substitution on oxidative modifications of the Parkinson's disease-related protein, DJ-1

Mutations in the gene encoding DJ-1 have been identified in patients with familial Parkinson's disease (PD) and are thought to inactivate a neuroprotective function. Oxidation of the sulfhydryl group to a sulfinic acid on cysteine residue C106 of DJ-1 yields the "2O " form, a variant of the protein with enhanced neuroprotective function. We hypothesized that some familial mutations disrupt DJ-1 activity by interfering with conversion of the protein to the 2O form. To address this hypothesis, we developed a novel quantitative mass spectrometry approach to measure relative changes in oxidation at specific sites in mutant DJ-1 as compared with the wild-type protein. Treatment of recombinant wild-type DJ-1 with a 10-fold molar excess of H(2)O(2) resulted in a robust oxidation of C106 to the sulfinic acid, whereas this modification was not detected in a sample of the familial PD mutant M26I exposed to identical conditions. Methionine oxidized isoforms of wild-type DJ-1 were depleted, presumably as a result of misfolding and aggregation, under conditions that normally promote conversion of the protein to the 2O form. These data suggest that the M26I familial substitution and methionine oxidation characteristic of sporadic PD may disrupt DJ-1 function by disfavoring a site-specific modification required for optimal neuroprotective activity. Our findings indicate that a single amino acid substitution can markedly alter a protein's ability to undergo oxidative modification, and they imply that stimulating the conversion of DJ-1 to the 2O form may be therapeutically beneficial in familial or sporadic PD.     

Superoxide dismutase 1 encoding mutations linked to ALS adopts a spectrum of misfolded states

Background

Oxidative stress is a key pathophysiological mechanism contributing to degenerative processes in many neurodegenerative diseases and therefore, unraveling molecular mechanisms underlying various stages of oxidative neuronal damage is critical to better understanding the diseases and developing new treatment modalities. We previously showed that protein kinase C delta (PKCδ) proteolytic activation during the late stages of oxidative stress is a key proapoptotic signaling mechanism that contributes to oxidative damage in Parkinson's disease (PD) models. The time course studies revealed that PKCδ activation precedes apoptotic cell death and that cells resisted early insults of oxidative damage, suggesting that some intrinsic compensatory response protects neurons from early oxidative insult. Therefore, the purpose of the present study was to characterize protective signaling pathways in dopaminergic neurons during early stages of oxidative stress.

Results

Herein, we identify that protein kinase D1 (PKD1) functions as a key anti-apoptotic kinase to protect neuronal cells against early stages of oxidative stress. Exposure of dopaminergic neuronal cells to H₂O₂ or 6-OHDA induced PKD1 activation loop (PKD1S744/748) phosphorylation long before induction of neuronal cell death. Blockade of PKCδ cleavage, PKCδ knockdown or overexpression of a cleavage-resistant PKCδ mutant effectively attenuated PKD1 activation, indicating that PKCδ proteolytic activation regulates PKD1 phosphorylation. Furthermore, the PKCδ catalytic fragment, but not the regulatory fragment, increased PKD1 activation, confirming PKCδ activity modulates PKD1 activation. We also identified that phosphorylation of S916 at the C-terminal is a preceding event required for PKD1 activation loop phosphorylation. Importantly, negative modulation of PKD1 by the RNAi knockdown or overexpression of PKD1^S916A phospho-defective mutants augmented oxidative stress-induced apoptosis, while positive modulation of PKD1 by the overexpression of full length PKD1 or constitutively active PKD1 plasmids attenuated oxidative stress-induced apoptosis, suggesting an anti-apoptotic role for PKD1 during oxidative neuronal injury.

Conclusion

Collectively, our results demonstrate that PKCδ-dependent activation of PKD1 represents a novel intrinsic protective response in counteracting early stage oxidative damage in neuronal cells. Our results suggest that positive modulation of the PKD1-mediated compensatory protective mechanism against oxidative damage in dopaminergic neurons may provide novel neuroprotective strategies for treatment of PD.