Eunsung Junn

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.

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.

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.

Apoptotic signaling in dopamine-induced cell death: the role of oxidative stress, p38 mitogen-activated protein kinase, cytochrome c and caspases

Oxidative stress generated by dopamine (DA) oxidation could be one of the factors underlying the selective vulnerability of nigral dopaminergic neurons in Parkinsons diseases. Here we show that DA induces apoptosis in SH‐SY5Y neuroblastoma cells demonstrated by activation of caspase‐9 and caspase‐3, cleavage of poly(ADP‐ribose) polymerase as well as nuclear condensation. We also show that p38 mitogen‐activated protein kinase is activated within 10 min of DA treatment, which precedes the onset of apoptosis because the potent p38 kinase inhibitor SB203580 protects against DA‐induced cell death as well as against caspase‐9 and caspase‐3 activation. In addition, the antioxidant N‐acetyl‐l‐cysteine (NAC) effectively blocks DA‐induced p38 kinase activation, caspase‐9 and caspase‐3 cleavage and subsequent apoptosis, indicating that DA triggers apoptosis via a signaling pathway that is initiated by the generation of reactive oxygen species (ROS). Dopamine exerts its toxicity principally intracellularly as the DA uptake inhibitor, nomifensine significantly reduces DA‐induced cell death as well as activation of p38 kinase and caspase‐3. Furthermore, DA induces mitochondrial cytochrome c release, which is dependent on p38 kinase activation and precedes the cleavage of caspases. These observations indicate that DA induces apoptosis primarily by generating ROS, p38 kinase activation, cytochrome c release followed by caspase‐9 and caspase‐3 activation.

α-Synuclein Activates Microglia by Inducing the Expressions of Matrix Metalloproteinases and the Subsequent Activation of Protease-Activated Receptor-1

The mutation or overexpression of α-synuclein protein plays a pivotal role in the pathogenesis of Parkinson’s disease. In our preliminary experiments, we found that α-synuclein induced the expression of matrix metalloproteinases (MMPs) (MMP-1, -3, -8, and -9) in rat primary cultured microglia. Thus, the current study was undertaken to determine the roles of MMPs in α-synuclein–induced microglial activation. The inhibition of MMP-3, -8, or -9 significantly reduced NO and reactive oxygen species levels and suppressed the expression of TNF-α and IL-1β. Notably, MMP-8 inhibitor suppressed TNF-α production more efficaciously than MMP-3 or MMP-9 inhibitors. Inhibition of MMP-3 or -9 also suppressed the activities of MAPK, NF-κB, and AP-1. Previously, protease-activated receptor-1 (PAR-1) has been associated with the actions of MMPs, and thus, we further investigated the role of PAR-1 in α-synuclein–induced inflammatory reactions. A PAR-1–specific inhibitor and a PAR-1 antagonist significantly suppressed cytokine levels, and NO and reactive oxygen species production in α-synuclein–treated microglia. Subsequent PAR-1 cleavage assay revealed that MMP-3, -8, and -9, but not α-synuclein, cleaved the synthetic peptide containing conventional PAR-1 cleavage sites. These results suggest that MMPs secreted by α-synuclein–stimulated microglia activate PAR-1 and amplify microglial inflammatory signals in an autocrine or paracrine manner. Furthermore, our findings suggest that modulation of the activities of MMPs and/or PAR-1 may provide a new therapeutic strategy for Parkinson’s disease.

MicroRNAs in neurodegenerative diseases and their therapeutic potential

MicroRNAs (miRNAs) are abundant, endogenous, short, noncoding RNAs that act as important post-transcriptional regulators of gene expression by base-pairing with their target mRNA. During the last decade, substantial knowledge has accumulated regarding the biogenesis of miRNAs, their molecular mechanisms and functional roles in a variety of cellular contexts. Altered expression of certain miRNA molecules in the brains of patients with neurodegenerative diseases such as Alzheimer and Parkinson suggests that miRNAs could have a crucial regulatory role in these disorders. Polymorphisms in miRNA target sites may also constitute an important determinant of disease risk. Additionally, emerging evidence points to specific miRNAs targeting and regulating the expression of particular proteins that are key to disease pathogenesis. Considering that the amount of these proteins in susceptible neuronal populations appears to be critical to neurodegeneration, miRNA-mediated regulation represents a new target of significant therapeutic prospects. In this review, the implications of miRNAs in several neurodegenerative disorders and their potential as therapeutic interventions are discussed.

DJ-1 induces thioredoxin 1 expression through the Nrf2 pathway

DJ-1, which is linked to recessively inherited Parkinsons disease when mutated, is a multi-functional protein with anti-oxidant and transcription regulatory activities. However, the mechanism(s) through which DJ-1 and the genes it regulates provide neuroprotection is not fully understood. Here, we show that wild-type DJ-1 induces the expression of thioredoxin 1 (Trx1), a protein disulfide oxidoreductase, whereas pathogenic mutant isoforms L166P and M26I cannot. Conversely, DJ-1 knockdown in SH-SY5Y cells and DJ-1 knockout in mice result in significant decrease in Trx1 protein and mRNA expression levels. The importance of Trx1 in the cytoprotective function of DJ-1 is confirmed using a pharmacological inhibitor of Trx reductase, 1-chloro-2,4-dinitrobenzene, and Trx1 siRNA. Both approaches result in partial loss of DJ-1-mediated protection. Additionally, knockdown of Trx1 significantly abrogates DJ-1-dependent, hydrogen peroxide-induced activation of the pro-survival factor AKT. Promoter analysis of the human Trx1 gene identified an antioxidant response element (ARE) that is required for DJ-1-dependent induction of Trx1 expression. The transcription factor Nuclear factor erythroid-2 related factor 2 (Nrf2), which is a critical inducer of ARE-mediated expression, is regulated by DJ-1. Overexpression of DJ-1 results in increased Nrf2 protein levels, promotes its translocation into the nucleus and enhances its recruitment onto the ARE site in the Trx1 promoter. Further, Nrf2 knockdown abolishes DJ-1-mediated Trx1 induction and cytoprotection against hydrogen peroxide, indicating the critical role of Nrf2 in carrying out the protective functions of DJ-1 against oxidative stress. These findings provide a new mechanism to support the antioxidant function of DJ-1 by increasing Trx1 expression via Nrf2-mediated transcriptional induction.

Three-amino acid Extension Loop Homeodomain Proteins Meis2 and TGIF Differentially Regulate Transcription

Three-amino acid extension loop (TALE) homeobox proteins are highly conserved transcription regulators. We report that two members of this family, Meis2 and TGIF, which frequently have overlapping consensus binding sites on complementary DNA strands in opposite orientations, can function competitively. For example, in theD1A gene, which encodes the predominant dopamine receptor in the striatum, Meis2 and TGIF bind to the activator sequence ACT (−1174 to −1154) and regulate transcription differentially in a cell type-specific manner. Among the five cloned splice variants of Meis2, isoforms Meis2a–d activate theD1A promoter in most cell types tested, whereas TGIF competes with Meis2 binding to DNA and represses Meis2-induced transcription activation. Consequently, Meis2 cannot activate theD1A promoter in a cell that has abundant TGIF expression. The Meis2 message is highly co-localized with the D1A message in adult striatal neurons, whereas TGIF is barely detectable in the adult brain. Our observations provide in vitro and in vivo evidence that Meis2 and TGIF differentially regulate their target genes. Thus, the delicate ratio between Meis2 and TGIF expression in a given cell type determines the cell-specific expression of theD1A gene. We also found that splice variant Meis2e, which has a truncated homeodomain, cannot bind to theD1A ACT sequence or activate transcription. However, Meis2e is an effective dominant negative regulator by blocking Meis2d-induced transcription activation. Thus, truncated homeoproteins with no DNA binding domains can have important regulatory functions.

DJ-1 protects against oxidative damage by regulating the thioredoxin/ASK1 complex.

DJ-1 is a multifunctional protein linked to recessively inherited Parkinsons disease (PD) due to loss of function mutations. Among its activities is anti-oxidant property leading to cytoprotection under oxidative stress conditions. A key effector of oxidant-induced cell death is the MAP3 kinase apoptosis signal-regulating kinase 1 (ASK1) which is bound to and inhibited by thioredoxin 1 (Trx1) under basal conditions. Upon oxidative stimuli, however, ASK1 dissociates from this physiological inhibitor and is activated. In the present study, we investigated the role of DJ-1 in regulating Trx1/ASK1 interaction. Over-expression of DJ-1 suppressed ASK1 activation in response to H(2)O(2) in a time-dependent manner. Wild-type DJ-1, but not the PD-associated L166P mutant, prevented the dissociation of ASK1 from Trx1 in response to H(2)O(2). Among cysteine mutants of DJ-1, C46S, C53S, and C106S, only C106S failed to inhibit this dissociation implying that cysteine 106 is essential for Trx1/ASK1 regulation. Furthermore, compared to wild-type mice, DJ-1 null mouse brain homogenates and embryonic fibroblasts were more susceptible to oxidant-induced dissociation of ASK1 from Trx1, activation of the downstream kinase c-Jun N-terminal kinase, and to cell death. These findings point to yet another mechanism through which DJ-1 has anti-oxidant and cytoprotective properties by regulating the Trx1/ASK1 complex and controlling the availability of ASK1 to effect apoptosis.