The role of age-associated alpha-synuclein aggregation in a conditional transgenic mouse model of Parkinson's disease: Implications for Lewy body formation.

Parkinson's disease (PD) is a common neurodegenerative disorder that is affecting an increasing number of older adults. Although PD is mostly sporadic, genetic mutations have been found in cohorts of families with a history of familial PD (FPD). The first such mutation linked to FPD causes a point mutation (A53T) in α-synuclein (α-syn), a major component of Lewy bodies, which are a classical pathological hallmark of PD. These findings suggest that α-syn is an important contributor to the development of PD. In our previous study, we developed an adenoviral mouse model of PD and showed that the expression of wild-type (WT) α-syn or a mutant form with an increased propensity to aggregate, designated as WT-CL1 α-syn, could be used to study how α-syn aggregation contributes to PD. In this study, we established a transgenic mouse model that conditionally expresses WT or WT-CL1 α-syn in dopaminergic (DA) neurons and found that the expression of either WT or WT-CL1 α-syn was associated with an age-dependent degeneration of DA neurons and movement dysfunction. Using this model, we were able to monitor the process of α-syn aggregate formation and found a correlation between age and the number and sizes of α-syn aggregates formed. These results provide a potential mechanism by which age-dependent α-syn aggregation may lead to the formation of Lewy bodies in PD pathogenesis.

Novel enhancement mechanism of tyrosine hydroxylase enzymatic activity by nitric oxide through S-nitrosylation.

Tyrosine hydroxylase (TH) is a rate-limiting step enzyme in the synthesis of catecholamines. Catecholamines function both as hormone and neurotransmitters in the peripheral and central nervous systems, therefore TH's expression and enzymatic activity is tightly regulated by various mechanisms. Several post-translational modifications have been shown to regulate TH's enzymatic activity such as phosphorylation, nitration and S-glutathionylation. While phosphorylation at N-terminal of TH can activate its enzymatic activity, nitration and S-glutathionylation can inactivate TH. In this study, we found that TH can also be S-nitrosylated by nitric oxide (NO). S-nitrosylation is a reversible modification of cysteine (cys) residue in protein and is known to be an emerging signaling mechanism mediated by NO. We found that TH can be S-nitrosylated at cys 279 and TH S-nitrosylation enhances its enzymatic activity both in vitro and in vivo. These results provide a novel mechanism of how NO can modulate TH's enzymatic activity through S-nitrosylation.

Studying nitrosative stress in Parkinson's disease.

Parkinson's disease (PD) is marked by a selective degeneration of dopaminergic neurons in the brain stem and it is the second most common neurodegenerative disorder. The pathogenic mechanism of PD is not completely known but it is believed that oxidative stress involving the imbalance of nitric oxide (NO) signaling is involved. Recent studies have suggested that NO, through the modification of protein's cysteine residues can contribute to the pathogenesis of PD. This NO modification, designated as S-nitrosylation, is emerging as an important signaling mechanism that regulates increasing number of cellular processes such as vesicle trafficking, receptor mediated signal transduction, gene transcription, and cell death. In our studies, we found that increased nitrosative stress promotes the S-nitrosylation of neuroprotective proteins and compromises their function which contributes to the development of PD. One of the obstacles in studying S-nitrosylation signaling is how to detect this modification in biological samples. Here, two simple and commonly used methods in detecting S-nitrosylated proteins are introduced for the study of this NO signaling mechanism.

S-nitrosylation of XIAP at Cys 213 of BIR2 domain impairs XIAP's anti-caspase 3 activity and anti-apoptotic function.

X-linked inhibitor of apoptosis (XIAP) is a protein that possesses anti-apoptotic function and dysregulation of it has been linked to a number human disease such as cancers and neurodegenerative disorders. In our previous study, we have found that nitric oxide (NO) can modulate the anti-apoptotic function of XIAP and found that this can contribute to the pathogenesis of Parkinson's disease. Specifically, we found that modification of baculoviral IAP repeat 2 of XIAP by S-nitrosylation can compromise XIAP's anti-caspase 3 and anti-apoptotic function. In this study, we report that cysteine (Cys) 90, Cys 213 and Cys 327 can be specifically S-nitrosylated by NO. We found that mutations of Cys 90 and Cys 327 affect the normal structure of XIAP. More importantly, we found that S-nitrosylation of XIAP Cys 213 impairs the anti-caspase 3 and anti-apoptotic function of XIAP that we observed in our previous study.

The role of alpha-synuclein oligomerization and aggregation in cellular and animal models of Parkinson's disease.

α-Synuclein (α-syn) is a synaptic protein in which four mutations (A53T, A30P, E46K and gene triplication) have been found to cause an autosomal dominant form of Parkinson's disease (PD). It is also the major component of intraneuronal protein aggregates, designated as Lewy bodies (LBs), a prominent pathological hallmark of PD. How α-syn contributes to LB formation and PD is still not well-understood. It has been proposed that aggregation of α-syn contributes to the formation of LBs, which then leads to neurodegeneration in PD. However, studies have also suggested that aggregates formation is a protective mechanism against more toxic α-syn oligomers. In this study, we have generated α-syn mutants that have increased propensity to form aggregates by attaching a CL1 peptide to the C-terminal of α-syn. Data from our cellular study suggest an inverse correlation between cell viability and the amount of α-syn aggregates formed in the cells. In addition, our animal model of PD indicates that attachment of CL1 to α-syn enhanced its toxicity to dopaminergic neurons in an age-dependent manner and induced the formation of Lewy body-like α-syn aggregates in the substantia nigra. These results provide new insights into how α-syn-induced toxicity is related to its aggregation.

Alpha-synuclein impairs normal dynamics of mitochondria in cell and animal models of Parkinson's disease.

Alpha-synuclein (α-syn) is a synaptic protein that mutations have been linked to Parkinson's disease (PD), a common neurodegenerative disorder that is caused by the degeneration of the dopaminergic neurons in the substantia nigra pars compacta (SNc). How α-syn can contribute to neurodegeneration in PD is not conclusive but it is agreed that mutations or excessive accumulation of α-syn can lead to the formation of α-syn oligomers or aggregates that interfere with normal cellular function and contribute to the degeneration of dopaminergic neurons. In this study, we found that α-syn can impair the normal dynamics of mitochondria and this effect is particular prominent in A53T α-syn mutant. In mice expressing A53T α-syn, age-dependent changes in both mitochondrial morphology and proteins that regulate mitochondrial fission and fusion were observed. In the cellular model of PD, we found that α-syn reduces the movement of mitochondria in both SH-SY5Y neuroblastoma and hippocampal neurons. Taken together, our study provides a new mechanism of how α-syn can contribute to PD through the impairment of normal dynamics of mitochondria.

Modulation of pro-survival proteins by S-nitrosylation: implications for neurodegeneration.

Nitric oxide (NO) is a gaseous signaling molecule in the biological system. It mediates its function through the direct modification of various cellular targets, such as through S-nitrosylation. The process of S-nitrosylation involves the attachment of NO to the cysteine residues of proteins. Interestingly, an increasing number of cellular pathways are found to be regulated by S-nitrosylation, and it has been proposed that this redox signaling pathway is comparable to phosphorylation in cells. However, imbalance of NO metabolism has also been linked to a number of human diseases. For instance, NO is known to contribute to neurodegeneration by causing protein nitration, lipid peroxidation and DNA damage. Moreover, recent studies show that NO can also contribute to the process of neurodegeneration through the impairment of pro-survival proteins by S-nitrosylation. Thus, further understanding of how NO, through S-nitrosylation, can compromise neuronal survival will provide potential therapeutic targets for neurodegenerative diseases.

The role of ubiquitin linkages on alpha-synuclein induced-toxicity in a Drosophila model of Parkinson's disease.

Parkinson's disease (PD) is a common movement disorder marked by the loss of dopaminergic (DA) neurons in the brain stem and the presence of intraneuronal inclusions designated as Lewy bodies (LB). The cause of neurodegeneration in PD is not clear, but it has been suggested that protein misfolding and aggregation contribute significantly to the development of the disease. Misfolded and aggregated proteins are cleared by ubiquitin proteasomal system (UPS) and autophagy lysosomal pathway (ALP). Recent studies suggested that different types of ubiquitin linkages can modulate these two pathways in the process of protein degradation. In this study, we found that co-expression of ubiquitin can rescue neurons from alpha-syn-induced neurotoxicity in a Drosophila model of PD. This neuroprotection is dependent on the formation of lysine 48 polyubiquitin linkage which is known to target protein degradation via the proteasome. Consistent with our results that we observed in vivo, we found that ubiquitin co-expression in the cell can facilitate cellular protein degradation by the proteasome in a lysine 48 polyubiquitin-dependent manner. Taken together, these results suggest that facilitation of proteasomal protein degradation can be a potential therapeutic approach for PD.

S-nitrosylation of XIAP compromises neuronal survival in Parkinson's disease.

Inhibitors of apoptosis (IAPs) are a family of highly-conserved proteins that regulate cell survival through binding to caspases, the final executioners of apoptosis. X-linked IAP (XIAP) is the most widely expressed IAP and plays an important function in regulating cell survival. XIAP contains 3 baculoviral IAP repeats (BIRs) followed by a RING finger domain at the C terminal. The BIR domains of XIAP possess anticaspase activities, whereas the RING finger domain enables XIAP to function as an E3 ubiquitin ligase in the ubiquitin and proteasomal system. Our previous study showed that parkin, a protein that is important for the survival of dopaminergic neurons in Parkinson's disease (PD), is S-nitrosylated both in vitro and in vivo in PD patients. S-nitrosylation of parkin compromises its ubiquitin E3 ligase activity and its protective function, which suggests that nitrosative stress is an important factor in regulating neuronal survival during the pathogenesis of PD. In this study we show that XIAP is S-nitrosylated in vitro and in vivo in an animal model of PD and in PD patients. Nitric oxide modifies mainly cysteine residues within the BIR domains. In contrast to parkin, S-nitrosylation of XIAP does not affect its E3 ligase activity, but instead directly compromises its anticaspase-3 and antiapoptotic function. Our results confirm that nitrosative stress contributes to PD pathogenesis through the impairment of prosurvival proteins such as parkin and XIAP through different mechanisms, indicating that abnormal S-nitrosylation plays an important role in the process of neurodegeneration.

Comparison of intensive and low-dose atorvastatin therapy in the reduction of carotid intimal-medial thickness in patients with coronary heart disease.

BACKGROUND: Intensive statin therapy has been shown to improve prognosis in patients with coronary heart disease (CHD). It is unknown whether such benefit is mediated through the reduction of atherosclerotic plaque burden. AIM: To examine the efficacy of high-dose atorvastatin in the reduction of carotid intimal-medial thickness (IMT) and inflammatory markers in patients with CHD. DESIGN: Randomised trial. SETTING: Single centre. PATIENTS: 112 patients with angiographic evidence of CHD. INTERVENTIONS: A high dose (80 mg daily) or low dose (10 mg daily) of atorvastatin was given for 26 weeks. MAIN OUTCOME MEASURES: Carotid IMT, C-reactive protein (CRP) and proinflammatory cytokine levels were assessed before and after therapy. RESULTS: The carotid IMT was reduced significantly in the high-dose group (left: mean (SD), 1.24 (0.48) vs 1.15 (0.35) mm, p = 0.02; right: 1.12 (0.41) vs 1.01 (0.26) mm, p = 0.01), but was unchanged in the low-dose group (left: 1.25 (0.55) vs 1.20 (0.51) mm, p = NS; right: 1.18 (0.54) vs 1.15 (0.41) mm, p = NS). The CRP levels were reduced only in the high-dose group (from 3.92 (6.59) to 1.35 (1.83) mg/l, p = 0.01), but not in the low-dose group (from 2.25 (1.84) to 3.36 (6.15) mg/l, p = NS). A modest correlation was observed between the changes in carotid IMT and CRP (r = 0.21, p = 0.03). CONCLUSIONS: In patients with CHD, intensive atorvastatin therapy results in regression of carotid atherosclerotic disease, which is associated with reduction in CRP levels. On the other hand, a low-dose regimen only prevents progression of the disease.

Say NO to neurodegeneration: role of S-nitrosylation in neurodegenerative disorders.

Nitric oxide (NO) is an important signaling molecule that controls a wide range of biological processes. One of the signaling mechanisms of NO is through the S-nitrosylation of cysteine residues on proteins. S-nitrosylation is now regarded as an important redox signaling mechanism in the regulation of different cellular and physiological functions. However, deregulation of S-nitrosylation has also been linked to various human diseases such as neurodegenerative disorders. Nitrosative stress has long been considered as a major mediator in the development of neurodegeneration, but the molecular mechanism of how NO can contribute to neurodegeneration is not completely clear. Early studies suggested that nitration of proteins, which can induce protein aggregation might contribute to the neurodegenerative process. However, several recent studies suggest that S-nitrosylation of proteins that are important for neuronal survival contributes substantially in the development of various neurodegenerative disorders. Thus, in-depth understanding of the mechanism of neurodegeneration in relation to S-nitrosylation will be critical for the development of therapeutic treatment against these neurodegenerative diseases.

Accumulation of the authentic parkin substrate aminoacyl-tRNA synthetase cofactor, p38/JTV-1, leads to catecholaminergic cell death.

Autosomal-recessive juvenile parkinsonism (AR-JP) is caused by loss-of-function mutations of the parkin gene. Parkin, a RING-type E3 ubiquitin ligase, is responsible for the ubiquitination and degradation of substrate proteins that are important in the survival of dopamine neurons in Parkinson's disease (PD). Accordingly, the abnormal accumulation of neurotoxic parkin substrates attributable to loss of parkin function may be the cause of neurodegeneration in parkin-related parkinsonism. We evaluated the known parkin substrates identified to date in parkin null mice to determine whether the absence of parkin results in accumulation of these substrates. Here we show that only the aminoacyl-tRNA synthetase cofactor p38 is upregulated in the ventral midbrain/hindbrain of both young and old parkin null mice. Consistent with upregulation in parkin knock-out mice, brains of AR-JP and idiopathic PD and diffuse Lewy body disease also exhibit increased level of p38. In addition, p38 interacts with parkin and parkin ubiquitinates and targets p38 for degradation. Furthermore, overexpression of p38 induces cell death that increases with tumor necrosis factor-alpha treatment and parkin blocks the pro-cell death effect of p38, whereas the R42P, familial-linked mutant of parkin, fails to rescue cell death. We further show that adenovirus-mediated overexpression of p38 in the substantia nigra in mice leads to loss of dopaminergic neurons. Together, our study represents a major advance in our understanding of parkin function, because it clearly identifies p38 as an important authentic pathophysiologic substrate of parkin. Moreover, these results have important implications for understanding the molecular mechanisms of neurodegeneration in PD.

Parkin and Hsp70 sacked by BAG5.

Loss-of-function mutations in the parkin gene, which encodes an E3 ubiquitin ligase, are the major cause of early-onset Parkinson's disease (PD). In this issue of Neuron, Kalia et al. show that the bcl-2-associated athanogene 5 (BAG5) enhances dopamine neuron death in an in vivo model of PD through inhibiting the E3 ligase activity of parkin and the chaperone activity of Hsp70.

S-nitrosylation of parkin regulates ubiquitination and compromises parkin's protective function.

Parkin is an E3 ubiquitin ligase involved in the ubiquitination of proteins that are important in the survival of dopamine neurons in Parkinson's disease (PD). We show that parkin is S-nitrosylated in vitro, as well as in vivo in a mouse model of PD and in brains of patients with PD and diffuse Lewy body disease. Moreover, S-nitrosylation inhibits parkin's ubiquitin E3 ligase activity and its protective function. The inhibition of parkin's ubiquitin E3 ligase activity by S-nitrosylation could contribute to the degenerative process in these disorders by impairing the ubiquitination of parkin substrates.

Survey of smokeless tobacco use in basic trainees and armor basic course officers.

In this study an all-male population of 256 U.S. Army basic trainees and 98 armor officer basic course students were surveyed concerning extent of use, knowledge of, and factors contributing to the use of smokeless tobacco (ST). The survey was administered at a dental clinic at Fort Knox, Kentucky. The data were analyzed using the SPSS computer software program. Results showed that more than 17% of this population were either occasional or regular users of ST. Nearly 24% had tried ST and more than 9% were former users. Exactly one-half had not tried ST. A typical user was white, had started ST use at age 14, had used it for 4 years, and was most likely to use snuff or dip. Higher educational level achieved was associated with increased likelihood of ST use. Nearly 22% of college graduates used ST, whereas only 15.3% of high school graduates or those with some college were users. Knowledge about the potential health effects of ST was generally high or moderately high, except for its ability to cause hypertension. This was true for both high school and college graduates, although college graduates were somewhat more informed. Few in this population had a personal attitude of approval toward ST use, and very few perceived that either their parents or their superiors in the military approved of ST use. Perceived attitude of approval was highest from friends and peers. The principle reasons for starting ST use were use by friends and curiosity about taste and effects. Influence from public figures, such as athletes, and from advertising was minimal. Enjoyment of flavor and taste was found to be the main reason for continued ST use. Health professionals were identified by the majority of respondents as the single most important information source on ST. Relatively few (27%), however, stated that their dentist or hygienist had ever discussed ST with them.