M. Maral Mouradian

Degradation of alpha-synuclein by proteasome.

Mutations in α-synuclein are known to be associated with Parkinsons disease (PD). The coexistence of this neuronal protein with ubiquitin and proteasome subunits in Lewy bodies in sporadic disease suggests that alterations of α-synuclein catabolism may contribute to the pathogenesis of PD. The degradation pathway of α-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 α-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 α-synuclein was 50% longer than that of the wild-type protein (p < 0.01). The degradation of both recombinant proteins and endogenous α-synuclein in these cells was blocked by the selective proteasome inhibitor β-lactone (40 μm), indicating that both wild-type and A53T mutant α-synuclein are degraded by the ubiquitin-proteasome pathway. The slower degradation of mutant α-synuclein provides a kinetic basis for its intracellular accumulation, thus favoring its aggregation.

Repression of α-synuclein expression and toxicity by microRNA-7

α-Synuclein is a key protein in Parkinsons disease (PD) because it accumulates as fibrillar aggregates in pathologic hallmark features in affected brain regions, most notably in nigral dopaminergic neurons. Intraneuronal levels of this protein appear critical in mediating its toxicity, because multiplication of its gene locus leads to autosomal dominant PD, and transgenic animal models overexpressing human α-synuclein manifest impaired function or decreased survival of dopaminergic neurons. Here, we show that microRNA-7 (miR-7), which is expressed mainly in neurons, represses α-synuclein protein levels through the 3′-untranslated region (UTR) of α-synuclein mRNA. Importantly, miR-7-induced down-regulation of α-synuclein protects cells against oxidative stress. Further, in the MPTP-induced neurotoxin model of PD in cultured cells and in mice, miR-7 expression decreases, possibly contributing to increased α-synuclein expression. These findings provide a mechanism by which α-synuclein levels are regulated in neurons, have implications for the pathogenesis of PD, and suggest miR-7 as a therapeutic target for PD and other α-synucleinopathies.

Motor fluctuations in Parkinson's disease: central pathophysiological mechanisms, Part II.

The duration of the antiparkinsonian action of levodopa was studied in 48 patients with various response patterns to the oral administration of the dopamine precursor. Deterioration in motor scores after abrupt cessation of a steady‐state intravenous levodopa infusion occurred at two successive rates: an initial rapid phase followed by a terminal slower phase. Efficacy half‐time Decemberreased and initial efficacy Decemberay slope increased with progression of levodopa response groups from never treated to stable responders, and then to fluctuating responders of the wearing‐off type and finally of the on‐off type. Efficacy half‐time exceeded plasma levodopa half‐life in the 2 nonfluctuating groups, approximated it in those patients with wearing‐off responses, and was significantly shorter in patients with fluctuations of the on‐off type. The half‐times for the Decemberline in antiparkinsonian efficacy and dyskinesia severity differed significantly, suggesting different pharmacological mechanisms. Motor fluctuation severity correlated best with initial efficacy Decemberay slope, and both were best predicted by parkinsonian symptom severity. The dyskinesia Decemberay rate correlated most closely with levodopa dose. These results support the view that progressive dopamine neuron degeneration reduces the brains ability to buffer shifts in levodopa availability attending its periodic oral administration; the clinical result is wearing‐off phenomenon. The on‐off phenomenon as well as dyskinesia apparently reflects additional secondary changes related to levodopa therapy and occurring postsynaptically.

The Role of Oxidative Stress in Parkinson’s Disease

Oxidative stress plays an important role in the degeneration of dopaminergic neurons in Parkinsons disease (PD). Disruptions in the physiologic maintenance of the redox potential in neurons interfere with several biological processes, ultimately leading to cell death. Evidence has been developed for oxidative and nitrative damage to key cellular components in the PD substantia nigra. A number of sources and mechanisms for the generation of reactive oxygen species (ROS) are recognized including the metabolism of dopamine itself, mitochondrial dysfunction, iron, neuroinflammatory cells, calcium, and aging. PD causing gene products including DJ-1, PINK1, parkin, alpha-synuclein and LRRK2 also impact in complex ways mitochondrial function leading to exacerbation of ROS generation and susceptibility to oxidative stress. Additionally, cellular homeostatic processes including the ubiquitin-proteasome system and mitophagy are impacted by oxidative stress. It is apparent that the interplay between these various mechanisms contributes to neurodegeneration in PD as a feed forward scenario where primary insults lead to oxidative stress, which damages key cellular pathogenetic proteins that in turn cause more ROS production. Animal models of PD have yielded some insights into the molecular pathways of neuronal degeneration and highlighted previously unknown mechanisms by which oxidative stress contributes to PD. However, therapeutic attempts to target the general state of oxidative stress in clinical trials have failed to demonstrate an impact on disease progression. Recent knowledge gained about the specific mechanisms related to PD gene products that modulate ROS production and the response of neurons to stress may provide targeted new approaches towards neuroprotection.

Adenosine A2A receptor antagonist treatment of Parkinson’s disease

Background: Observations in animal models suggest that A2A antagonists confer benefit by modulating dopaminergic effects on the striatal dysfunction associated with motor disability. This double-blind, placebo-controlled, proof-of-principle study evaluated the pathogenic contribution and therapeutic potential of adenosine A2A receptor–mediated mechanisms in Parkinson disease (PD) and levodopa-induced motor complications. Methods: Fifteen patients with moderate to advanced PD consented to participate. All were randomized to either the selective A2A antagonist KW-6002 or matching placebo capsules in a 6-week dose-rising design (40 and 80 mg/day). Motor function was rated on the Unified PD Rating Scale. Results: KW-6002 alone or in combination with a steady-state IV infusion of each patient’s optimal levodopa dose had no effect on parkinsonian severity. At a low dose of levodopa, however, KW-6002 (80 mg) potentiated the antiparkinsonian response by 36% (p < 0.02), but with 45% less dyskinesia compared with that induced by optimal dose levodopa alone (p < 0.05). All cardinal parkinsonian signs improved, especially resting tremor. In addition, KW-6002 prolonged the efficacy half-time of levodopa by an average of 47 minutes (76%; p < 0.05). No medically important drug toxicity occurred. Conclusions: The results support the hypothesis that A2A receptor mechanisms contribute to symptom production in PD and that drugs able to selectively block these receptors may help palliate symptoms in levodopa-treated patients with this disorder.

Recent advances in the genetics and pathogenesis of Parkinson disease

The identification of three genes and several additional loci associated with inherited forms of levodopa-responsive PD has confirmed that this is not a single disorder. Yet, analyses of the structure and function of these gene products point to the critical role of protein aggregation in dopaminergic neurons of the substantia nigra as the common mechanism leading to neurodegeneration in all known forms of this disease. The three specific genes identified to date—α-synuclein, Parkin, and ubiquitin C terminal hydrolase L1—are either closely involved in the proper functioning of the ubiquitin-proteasome pathway or are degraded by this protein-clearing machinery of cells. Knowledge gained from genetically transmitted PD also has clear implications for nonfamilial forms of the disease. Lewy bodies, even in sporadic PD, contain these three gene products, particularly abundant amounts of fibrillar α-synuclein. Increased aggregation of α-synuclein by oxidative stress, as well as oxidant-induced proteasomal dysfunction, link genetic and potential environmental factors in the onset and progression of the disease. The biochemical and molecular cascades elucidated from genetic studies in PD can provide novel targets for curative therapies.

Tissue transglutaminase-induced aggregation of α-synuclein: Implications for Lewy body formation in Parkinson's disease and dementia with Lewy bodies

Proteinaceous aggregates containing α-synuclein represent a feature of neurodegenerative disorders such as Parkinsons disease, dementia with Lewy bodies, and multiple system atrophy. Despite extensive research, the mechanisms underlying α-synuclein aggregation remain elusive. Previously, tissue transglutaminase (tTGase) was found to contribute to the generation of aggregates by cross-linking pathogenic substrate proteins in Huntingtons and Alzheimers diseases. In this article, the role of tTGase in the formation of α-synuclein aggregates was investigated. Purified tTGase catalyzed α-synuclein cross-linking, leading to the formation of high molecular weight aggregates in vitro, and overexpression of tTGase resulted in the formation of detergent-insoluble α-synuclein aggregates in cellular models. Immunocytochemical studies demonstrated the presence of α-synuclein-positive cytoplasmic inclusions in 8% of tTGase-expressing cells. The formation of these aggregates was significantly augmented by the calcium ionophore A23187 and prevented by the inhibitor cystamine. Immunohistochemical studies on postmortem brain tissue confirmed the presence of transglutaminase-catalyzed ɛ(γ-glutamyl)lysine cross-links in the halo of Lewy bodies in Parkinsons disease and dementia with Lewy bodies, colocalizing with α-synuclein. These findings, taken together, suggest that tTGase activity leads to α-synuclein aggregation to form Lewy bodies and perhaps contributes to neurodegeneration.

Human α-Synuclein over-expression increases intracellular reactive oxygen species levels and susceptibility to dopamine

alpha-Synuclein is a major component of Lewy bodies found in the brains of patients with Parkinsons disease (PD). Two point mutations in alpha-synuclein (A53T and A30P) are identified in few families with dominantly inherited PD. Yet the mechanism by which this protein is involved in nigral cell death remains poorly understood. Mounting evidence suggests the importance of oxidative stress in the pathogenesis of PD. Here we investigated the effects of wild-type and two mutant forms of alpha-synuclein on intracellular reactive oxygen species (ROS) levels using clonal SH-SY5Y cells engineered to over-express these proteins. All three cell lines, and particularly mutant alpha-synuclein-expressing cells, had increased ROS levels relative to control LacZ-engineered cells. In addition, cell viability was significantly curtailed following the exposure of all three alpha-synuclein-engineered cells to dopamine, but more so with mutant alpha-synuclein. These results suggest that over-expression of alpha-synuclein, and especially its mutant forms, exaggerates the vulnerability of neurons to dopamine-induced cell death through excess intracellular ROS generation. Thus, these findings provide a link between mutations or over-expression of alpha-synuclein and apoptosis of dopaminergic neurons by lowering the threshold of these cells to oxidative damage.

Enhanced vulnerability to oxidative stress by α-synuclein mutations and C-terminal truncation

α-Synuclein is a key component of Lewy bodies found in the brains of patients with Parkinson’s disease and two point mutations in this protein, Ala53Thr and Ala30Pro, are associated with rare familial forms of the disease. Several lines of evidence suggest the involvement of oxidative stress in the pathogenesis of nigral neuronal death in Parkinson’s disease. In the present work we studied the effects of changes in the α-synuclein sequence on the susceptibility of cells to reactive oxygen species. Human dopaminergic neuroblastoma SH-SY5Y cells were stably transduced with various isoforms of α-synuclein and their survival following exposure to hydrogen peroxide or to the dopaminergic neurotoxin MPP+ was assessed. Cells expressing the two point mutant isoforms of α-synuclein were significantly more vulnerable to oxidative stress, with the Ala53Thr engineered cells faring the worst. In addition, cells expressing C-terminally truncated α-synuclein, particularly the 1–120 residue protein, were more susceptible than control β-galactosidase engineered cells. The present experiments indicate that point mutations and C-terminal truncation of α-synuclein exaggerate the susceptibility of dopaminergic cells to oxidative damage. Thus, these observations provide a pathogenetic link between α-synuclein aberrations and a putative cell death mechanism in Parkinson’s disease.

Mitochondrial localization of DJ-1 leads to enhanced neuroprotection

Mutations in DJ‐1 (PARK7) cause recessively inherited Parkinsons disease. DJ‐1 is a multifunctional protein with antioxidant and transcription modulatory activity. Its localization in cytoplasm, mitochondria, and nucleus is recognized, but the relevance of this subcellular compartmentalization to its cytoprotective activity is not fully understood. Here we report that under basal conditions DJ‐1 is present mostly in the cytoplasm and to a lesser extent in mitochondria and nucleus of dopaminergic neuroblastoma SK‐N‐BE(2)C cells. Upon oxidant challenge, more DJ‐1 translocates to mitochondria within 3 hr and subsequently to the nucleus by 12 hr. The predominant DJ‐1 species in both mitochondria and nucleus is a dimer believed to be the functional form. Mutating cysteine 106, 53, or 46 had no impact on the translocation of DJ‐1 to mitochondria. To study the relative neuroprotective activity of DJ‐1 in mitochondria and nucleus, DJ‐1 cDNA constructs fused to the appropriate localization signal were transfected into cells. Compared with 30% protection against oxidant‐induced cell death in wild‐type DJ‐1‐transfected cells, mitochondrial targeting of DJ‐1 provided a significantly stronger (55%) cytoprotection based on lactate dehydrogenase release. Nuclear targeting of DJ‐1 preserved cells equally as well as the wild‐type protein. These observations suggest that the time frame for the translocation of DJ‐1 from the cytoplasm to mitochondria and to the nucleus following oxidative stress is quite different and that dimerized DJ‐1 in mitochondria is functional as an antioxidant not related to cysteine modification. These findings further highlight the multifaceted functions of DJ‐1 as a cytoprotector in different cellular compartments.