alpha-Synuclein aggregates interfere with Parkin solubility and distribution: role in the pathogenesis of Parkinson disease

Parkinson disease (PD) belongs to a heterogeneous group of neurodegenerative disorders with movement alterations, cognitive impairment, and alpha-synuclein accumulation in cortical and subcortical regions. Jointly, these disorders are denominated Lewy body disease. Mutations in the parkin gene are the most common cause of familial parkinsonism, and a growing number of studies have shown that stress factors associated with sporadic PD promote parkin accumulation in the insoluble fraction. alpha-Synuclein and parkin accumulation and mutations in these genes have been associated with familial PD. To investigate whether alpha-synuclein accumulation might be involved in the pathogenesis of these disorders by interfering with parkin solubility, synuclein-transfected neuronal cells were transduced with lentiviral vectors expressing parkin. Challenging neurons with proteasome inhibitors or amyloid-beta resulted in accumulation of insoluble parkin and, to a lesser extent, alpha-tubulin. Similarly to neurons in the brains of patients with Lewy body disease, in co-transduced cells alpha-synuclein and parkin colocalized and co-immunoprecipitated. These effects resulted in decreased parkin and alpha-tubulin ubiquitination, accumulation of insoluble parkin, and cytoskeletal alterations with reduced neurite outgrowth. Taken together, accumulation of alpha-synuclein might contribute to the pathogenesis of PD and other Lewy body diseases by promoting alterations in parkin and tubulin solubility, which in turn might compromise neural function by damaging the neuronal cytoskeleton. These studies provide a new perspective on the potential nature of pathogenic alpha-synuclein and parkin interactions in Parkinson disease.     

Parkin cleaves intracellular alpha-synuclein inclusions via the activation of calpain

Mutations in the alpha-synuclein and parkin genes cause heritable forms of Parkinson's disease. In the present study, we examined the possible functional relationship between the parkin and alpha-synuclein genes in a conditionally immortalized embryonic hippocampal cell (H19-7) line. Whereas transient transfection of alpha-synuclein into neuronal H19-7 cells caused the formation of its intracytoplasmic inclusions and a significant cell death, the combined overexpression of parkin restored the alpha-synuclein-induced decrease in cell viability to control levels. In addition, the overexpression of parkin was found to generate selective cleavage of alpha-synuclein. Furthermore, the cytoprotective effect of parkin on alpha-synuclein-induced cell death was not inhibited in the presence of a proteasome inhibitor. Interestingly, the overexpression of parkin induced the activation of an intracellular cysteine protease, calpain, but not caspase, and the cytoprotective effect of parkin on alpha-synuclein cytotoxicity was significantly inhibited by the presence of calpain-specific inhibitors. In conclusion, our results suggest that parkin accelerates the degradation of alpha-synuclein via the activation of the nonproteasomal protease, calpain, leading to the prevention of alpha-synuclein-induced cell death in embryonic hippocampal progenitor cells.     

Mutant alpha-synuclein-induced degeneration is reduced by parkin in a fly model of Parkinson's disease.

Parkinson's disease (PD) patients show a characteristic loss of motor control caused by the degeneration of dopaminergic neurons. Mutations in the genes that encode alpha-synuclein and parkin have been linked to inherited forms of this disease. The parkin protein functions as a ubiquitin ligase that targets proteins for degradation. Expression of isoforms of human alpha-synuclein in the Drosophila melanogaster nervous system forms the basis of an excellent genetic model that recapitulates phenotypic and behavioural features of PD. Using this model, we analysed the effect of parkin co-expression on the climbing ability of aging flies, their life span, and their retinal degeneration. We have determined that co-expression of parkin can suppress phenotypes caused by expression of mutant alpha-synuclein. In the developing eye, parkin reduces retinal degeneration. When co-expressed in the dopaminergic neurons, the ability to climb is extended over time. If conserved in humans, we suggest that upregulation of parkin may prove a method of suppression for PD induced by mutant forms of alpha-synuclein.     

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.     

Degradation of alpha-synuclein by proteasome

Mutations in alpha-synuclein are known to be associated with Parkinson's disease (PD). The coexistence of this neuronal protein with ubiquitin and proteasome subunits in Lewy bodies in sporadic disease suggests that alterations of alpha-synuclein catabolism may contribute to the pathogenesis of PD. The degradation pathway of alpha-synuclein has not been identified nor has the kinetics of this process been described. We investigated the degradation kinetics of both wild-type and A53T mutant 6XHis-tagged alpha-synuclein in transiently transfected SH-SY5Y cells. Degradation of both isoforms followed first-order kinetics over 24 h as monitored by the pulse-chase method. However, the t((1)/(2)) of mutant alpha-synuclein was 50% longer than that of the wild-type protein (p < 0.01). The degradation of both recombinant proteins and endogenous alpha-synuclein in these cells was blocked by the selective proteasome inhibitor beta-lactone (40 microM), indicating that both wild-type and A53T mutant alpha-synuclein are degraded by the ubiquitin-proteasome pathway. The slower degradation of mutant alpha-synuclein provides a kinetic basis for its intracellular accumulation, thus favoring its aggregation.     

Parkin ubiquitinates the alpha-synuclein-interacting protein, synphilin-1: implications for Lewy-body formation in Parkinson disease

Parkinson disease is a common neurodegenerative disorder characterized by the loss of dopaminergic neurons and the presence of intracytoplasmic-ubiquitinated inclusions (Lewy bodies). Mutations in alpha-synuclein (A53T, A30P) and parkin cause familial Parkinson disease. Both these proteins are found in Lewy bodies. The absence of Lewy bodies in patients with parkin mutations suggests that parkin might be required for the formation of Lewy bodies. Here we show that parkin interacts with and ubiquitinates the alpha-synuclein-interacting protein, synphilin-1. Co-expression of alpha-synuclein, synphilin-1 and parkin result in the formation of Lewy-body-like ubiquitin-positive cytosolic inclusions. We further show that familial-linked mutations in parkin disrupt the ubiquitination of synphilin-1 and the formation of the ubiquitin-positive inclusions. These results provide a molecular basis for the ubiquitination of Lewy-body-associated proteins and link parkin and alpha-synuclein in a common pathogenic mechanism through their interaction with synphilin-1.     

Parkin suppresses wild-type alpha-synuclein-induced toxicity in SHSY-5Y cells

Current hypotheses concerning the mechanism of neuronal cell death in Parkinson's disease (PD) and related synucleopathies propose a functional interaction between parkin and alpha-synuclein (alphaS). Recently parkin was shown to suppress mutant alphaS-induced toxicity in primary neurons, providing a basis for an association between these proteins and neuronal loss [Neuron 36 (2000) 1007-1019]. We have asked if a similar association could be made between wild-type (wt) alphaS and parkin. We examined inducible over-expression of alphaS in SHSY-5Y cells through adenoviral infection under conditions which produce cellular toxicity through a reduction in ATP levels. We demonstrate that parkin suppresses toxicity induced by mutant (A53T) and wt alphaS. Parkin over-expression was also associated with the appearance of higher molecular weight alphaS-immunoreactive bands by Western blot analysis. These data, consistent with a protective role for parkin, extend previous findings to include a functional association between parkin and the wt form of alphaS.     

Parkin facilitates the elimination of expanded polyglutamine proteins and leads to preservation of proteasome function

Parkin, the most commonly mutated gene in familial Parkinson's disease, encodes an E3 ubiquitin ligase. A number of candidate substrates have been identified for parkin ubiquitin ligase action including CDCrel-1, o-glycosylated alpha-synuclein, Pael-R, and synphilin-1. We now show that parkin promotes the ubiquitination and degradation of an expanded polyglutamine protein. Overexpression of parkin reduces aggregation and cytotoxicity of an expanded polyglutamine ataxin-3 fragment. Using a cellular proteasome indicator system based on a destabilized form of green fluorescent protein, we demonstrate that parkin reduces proteasome impairment and caspase-12 activation induced by an expanded polyglutamine protein. Parkin forms a complex with the expanded polyglutamine protein, heat shock protein 70 (Hsp70) and the proteasome, which may be important for the elimination of the expanded polyglutamine protein. Hsp70 enhances parkin binding and ubiquitination of expanded polyglutamine protein in vitro suggesting that Hsp70 may help to recruit misfolded proteins as substrates for parkin E3 ubiquitin ligase activity. We speculate that parkin may function to relieve endoplasmic reticulum stress by preserving proteasome activity in the presence of misfolded proteins. Loss of parkin function and the resulting proteasomal impairment may contribute to the accumulation of toxic aberrant proteins in neurodegenerative diseases including Parkinson's disease.     

Alpha-Synuclein is degraded by both autophagy and the proteasome

Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra and the formation of aggregates (Lewy bodies) in neurons. alpha-Synuclein is the major protein in Lewy bodies and rare mutations in alpha-synuclein cause early-onset PD. Consequently, alpha-synuclein is implicated in the pathogenesis of PD. Here, we have investigated the degradation pathways of alpha-synuclein, using a stable inducible PC12 cell model, where the expression of exogenous human wild-type, A30P, or A53T alpha-synuclein can be switched on and off. We have used a panel of inhibitors/stimulators of autophagy and proteasome function and followed alpha-synuclein degradation in these cells. We found that not only is alpha-synuclein degraded by the proteasome, but it is also degraded by autophagy. A role for autophagy was further supported by the presence of alpha-synuclein in organelles with the ultrastructural features of autophagic vesicles. Since rapamycin, a stimulator of autophagy, increased clearance of alpha-synuclein, it merits consideration as a potential therapeutic for Parkinsons disease, as it is designed for chronic use in humans.     

The effects of oxidative stress on parkin and other E3 ligases

Autosomal recessive mutations within the Parkin gene are associated with degeneration of the substantia nigra and locus coeruleus and an inherited form of Parkinson's disease (PD). As loss-of-function mutations in parkin are responsible for a familial variant of PD, conditions that affect wild-type parkin are likely to be associated with increased risk of idiopathic disease. Previous studies uncovered a unique vulnerability of the parkin protein to dopamine (DA)-induced aggregation and inactivation. In this study, we compared several proteins that share structural elements or ubiquitinating activity with parkin. We report that oxidative stress in several cell lines and primary neurons induces the aggregation of parkin into high molecular weight species, at least a portion of which are self-associated homo-multimers. While parkin was preferentially affected by excess DA, each of the E3 proteins tested were made more insoluble by oxidative stress, and they varied in degree of susceptibility (e.g. parkin > HHARI ≅ CHIP > c-Cbl > E6AP). These conditions of oxidative stress were also associated with decreased parkin E3 ligase activity. Similar to recently conducted studies on α-synuclein processing, both macroautophagy and the proteasome participate in parkin degradation, with the proteasome playing the predominant role for normal parkin turnover and macroautophagy being more important in the degradation of aggregated parkin. These data further highlight the selective vulnerability of parkin to DA-induced modifications, demonstrating for the first time the ability of both endogenous and ectopically expressed parkin to transition into an insoluble state in part through self-association and oligomer formation.     

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.     

Methionine sulfoxide reductase A protects dopaminergic cells from Parkinson's disease-related insults

Parkinson's disease (PD) is a neurologic disorder characterized by dopaminergic cell death in the substantia nigra. PD pathogenesis involves mitochondrial dysfunction, proteasome impairment, and alpha-synuclein aggregation, insults that may be especially toxic to oxidatively stressed cells including dopaminergic neurons. The enzyme methionine sulfoxide reductase A (MsrA) plays a critical role in the antioxidant response by repairing methionine-oxidized proteins and by participating in cycles of methionine oxidation and reduction that have the net effect of consuming reactive oxygen species. Here, we show that MsrA suppresses dopaminergic cell death and protein aggregation induced by the complex I inhibitor rotenone or mutant alpha-synuclein, but not by the proteasome inhibitor MG132. By comparing the effects of MsrA and the small-molecule antioxidants N-acetylcysteine and vitamin E, we provide evidence that MsrA protects against PD-related stresses primarily via methionine sulfoxide repair rather than by scavenging reactive oxygen species. We also demonstrate that MsrA efficiently reduces oxidized methionine residues in recombinant alpha-synuclein. These findings suggest that enhancing MsrA function may be a reasonable therapeutic strategy in PD.     

Pael-R transgenic mice crossed with parkin deficient mice displayed progressive and selective catecholaminergic neuronal loss

Parkin, a ubiquitin ligase, is responsible for autosomal recessive juvenile parkinsonism (AR-JP). We identified parkin-associated endothelin receptor-like receptor (Pael-R) as a substrate of parkin, whose accumulation is thought to induce unfolded protein response (UPR) -mediated cell death, leading to dopaminergic neurodegeneration. To create an animal model of AR-JP, we generated parkin knockout/Pael-R transgenic (parkin-ko/Pael-R-tg) mice. parkin-ko/Pael-R-tg mice exhibited early and progressive loss of dopaminergic as well as noradrenergic neurons without formation of inclusion bodies, recapitulating the pathological features of AR-JP. Evidence of activation of UPR and up-regulation of dopamine and its metabolites were observed throughout the lifetime. Moreover, complex I activity of mitochondria isolated from parkin-ko/Pael-R-tg mice was significantly reduced later in life. These findings suggest that persistent induction of unfolded protein stress underlies chronic progressive catecholaminergic neuronal death, and that dysfunction of mitochondrial complex I and oxidative stress might be involved in the progression of Parkinson's disease. parkin-ko/Pael-R-tg mice represents an AR-JP mouse model displaying chronic and selective loss of catecholaminergic neurons.     

Rotenone selectively kills serotonergic neurons through a microtubule-dependent mechanism

As a major co-morbidity of Parkinson's disease (PD), depression is associated with the loss of serotonergic neurons. Our recent study has shown that midbrain dopaminergic neurons are particularly vulnerable to microtubule-depolymerizing agents including rotenone, an environmental toxin linked to PD. Here we show that rotenone also selectively killed serotonergic neurons in midbrain neuronal cultures. Its selective toxicity was significantly decreased by the microtubule-stabilizing drug taxol and mimicked by microtubule-depolymerizing agents such as colchicine and nocodazole. Microtubule depolymerization induced by rotenone or colchicine caused vesicle accumulation in the soma and killed serotonergic neurons through a mechanism dependent on serotonin metabolism in the cytosol. Blocking serotonin synthesis or degradation, as well as application of antioxidants, significantly reduced the selective toxicity of rotenone or colchicine. Inhibition of vesicular sequestration of serotonin exerted a selective toxicity on serotonergic neurons that was mitigated by blocking serotonin metabolism. Over-expression of parkin, a protein-ubiquitin E3 ligase that strongly binds to microtubules, greatly attenuated the selective toxicity of rotenone or colchicine. The protective effects of parkin were abrogated by its PD-linked mutations. Together, our results suggest that rotenone and parkin affect the survival of serotonergic neurons by impacting on microtubules in opposing manners.     

alpha-synuclein is required for the fibrillar nature of ubiquitinated inclusions induced by proteasomal inhibition in primary neurons

Proteasomal dysfunction may underlie certain neuro-degenerative conditions such as Parkinson disease. We have shown that pharmacological inhibition of the proteasome in cultured neuronal cells leads to apoptotic death and formation of cytoplasmic ubiquitinated inclusions. These inclusions stain for alpha-synuclein and assume a fibrillar structure, as assessed by thioflavine S staining, and therefore resemble Lewy bodies. alpha-Synuclein is thought to be a central component of Lewy bodies. Whether alpha-synuclein is required for inclusion formation or apoptotic death has not been formally assessed. The present study examines whether alpha-synuclein deficiency in neurons alters their sensitivity to proteasomal inhibition-induced apoptosis or inclusion formation. Cortical neurons derived from alpha-synuclein-null mice showed a similar sensitivity to death induced by the proteasomal inhibitor lactacystin compared with neurons derived from wild-type mice. Furthermore, the absence of alpha-synuclein did not influence the percentage of lactacystin-treated neurons harboring cytoplasmic ubiquitinated inclusions or alter the solubility of such inclusions. In contrast, however, ubiquitinated inclusions in alpha-synuclein-deficient neurons lacked amyloid-like fibrillization, as determined by thioflavine S staining. This indicates that although alpha-synuclein deficiency does not affect the formation of ubiquitinated inclusions, it does significantly alter their structure. The lack of effect on survival in alpha-synuclein knock-out cultures further suggests that the fibrillar nature of the inclusions does not contribute to neuronal degeneration in this model.     

Parkin: a multipurpose neuroprotective agent?

An autosomal recessive juvenile-onset form of Parkinson's disease (AR-JP) is caused by loss-of-function mutations of the parkin gene, which encodes a ubiquitin-protein ligase. Three recent reports demonstrate that parkin can protect neurons from diverse cellular insults, including alpha-synuclein toxicity, proteasomal dysfunction, Pael-R accumulation, and kainate-induced excitotoxicity. These findings suggest a central role for parkin in maintaining dopaminergic neuronal integrity and strengthen the link between AR-JP and the more common sporadic form of Parkinson's disease.     

Parkin-deficient mice exhibit nigrostriatal deficits but not loss of dopaminergic neurons

Loss-of-function mutations in parkin are the major cause of early-onset familial Parkinson's disease. To investigate the pathogenic mechanism by which loss of parkin function causes Parkinson's disease, we generated a mouse model bearing a germline disruption in parkin. Parkin-/- mice are viable and exhibit grossly normal brain morphology. Quantitative in vivo microdialysis revealed an increase in extracellular dopamine concentration in the striatum of parkin-/- mice. Intracellular recordings of medium-sized striatal spiny neurons showed that greater currents are required to induce synaptic responses, suggesting a reduction in synaptic excitability in the absence of parkin. Furthermore, parkin-/- mice exhibit deficits in behavioral paradigms sensitive to dysfunction of the nigrostriatal pathway. The number of dopaminergic neurons in the substantia nigra of parkin-/- mice, however, is normal up to the age of 24 months, in contrast to the substantial loss of nigral neurons characteristic of Parkinson's disease. Steady-state levels of CDCrel-1, synphilin-1, and alpha-synuclein, which were identified previously as substrates of the E3 ubiquitin ligase activity of parkin, are unaltered in parkin-/- brains. Together these findings provide the first evidence for a novel role of parkin in dopamine regulation and nigrostriatal function, and a non-essential role of parkin in the survival of nigral neurons in mice.     

Proteasomal inhibition causes the formation of protein aggregates containing a wide range of proteins,including nitrated proteins

Mutations in Cu,Zn-superoxide dismutase (SOD-1) are associated with some familial cases of amyotrophic lateral sclerosis (ALS), but it is not known how they result in cell death. We examined effects of overexpression of wild-type SOD-1 or the G37R or G85R mutations on the accumulation of ubiquitinated and nitrated proteins, and on loss of cell viability induced by the proteasome inhibitor, lactacystin. Wild-type SOD-1 had no effect on proteasomal activity, but the mutants decreased it somewhat. Treatment with lactacystin (1 micro m) caused only limited cell viability loss, even though it induced a marked inhibition of proteasomal activities. However, viability loss due to apoptosis was substantial in response to lactacystin when cells were overexpressing a mutant SOD-1. The frequency of cells showing immunoreactivity against ubiquitinated- or nitrated-proteins was enhanced when wild-type and mutant SOD-1 s were overexpressed. Ubiquitinated or nitrated alpha-tubulin, SOD-1, alpha-synuclein and 68K neurofilaments were observed in the aggregates. Similar aggregates were observed in cells overexpressing mutant parkin (Del3-5, T240R and Q311'X). The nitric oxide synthase inhibitor, l-NAME, decreased viability loss and aggregation, suggesting that nitration of proteins may play an important role in aggregation and in the cell death accompanying it.