Qiping Chen

Induction of α-Synuclein Aggregation by Intracellular Nitrative Insult

Brain lesions containing filamentous and aggregated α-synuclein are hallmarks of neurodegenerative synucleinopathies. Oxidative stress has been implicated in the formation of these lesions. Using HEK 293 cells stably transfected with wild-type and mutant α-synuclein, we demonstrated that intracellular generation of nitrating agents results in the formation of α-synuclein aggregates. Cells were exposed simultaneously to nitric oxide- and superoxide-generating compounds, and the intracellular formation of peroxynitrite was demonstrated by monitoring the oxidation of dihydrorhodamine 123 and the nitration of α-synuclein. Light microscopy using antibodies against α-synuclein and electron microscopy revealed the presence of perinuclear aggregates under conditions in which peroxynitrite was generated but not when cells were exposed to nitric oxide- or superoxide-generating compounds separately. α-Synuclein aggregates were observed in 20–30% of cells expressing wild-type or A53T mutant α-synuclein and in 5% of cells expressing A30P mutant α-synuclein. No evidence of synuclein aggregation was observed in untransfected cells or cells expressing β-synuclein. In contrast, selective inhibition of the proteasome resulted in the formation of aggregates detected with antibodies to ubiquitin in the majority of the untransfected cells and cells expressing α-synuclein. However, α-synuclein did not colocalize with these aggregates, indicating that inhibition of the proteasome does not promote α-synuclein aggregation. In addition, proteasome inhibition did not alter the steady-state levels of α-synuclein, but addition of the lysosomotropic agent ammonium chloride significantly increased the amount of α-synuclein, indicating that lysosomes are involved in degradation of α-synuclein. Our data indicate that nitrative and oxidative insult may initiate pathogenesis of α-synuclein aggregates.

Basal and Stimulated Protein S-Nitrosylation in Multiple Cell Types and Tissues

There is substantial evidence that proteinS-nitrosylation provides a significant route through which nitric oxide (NO)-derived bioactivity is conveyed. However, most examples of S-nitrosylation have been characterized on the basis of analysis in vitro, and relatively little progress has been made in assessing the participant forms of nitric-oxide synthase (NOS) or the dynamics of protein S-nitrosylationin situ. Here we utilize antibodies specific for the nitrosothiol (SNO) moiety to provide an immunohistochemical demonstration that protein S-nitrosylation is coupled to the activity of each of the major forms of NOS. In cultured endothelial cells, SNO-protein immunoreactivity increases in response to Ca2+-stimulated endothelial NOS (eNOS) activity, and in aortic rings, endothelium-derived and eNOS-mediated relaxation (EDRF) is coupled to increased protein S-nitrosylation in both endothelial and associated smooth muscle cells. In cultured macrophages, SNO-protein levels increase upon cytokine induction of induced NOS (iNOS), and in PC12 cells, increased proteinS-nitrosylation is linked to nerve growth factor induction of neuronal NOS (nNOS). In addition, we describe developmental and pathophysiological increases in SNO-protein immunoreactivity within human lung. These results, which demonstrate Ca2+, neurohumoral, growth factor, cytokine, and developmental regulation of protein S-nitrosylation that is coupled to NOS expression and activity, provide unique evidence for the proposition that this ubiquitous NO-derived post-translational protein modification serves as a major effector of NO-related bioactivity.

Widespread Nitration of Pathological Inclusions in Neurodegenerative Synucleinopathies

Reactive nitrogen species may play a mechanistic role in neurodegenerative diseases by posttranslationally altering normal brain proteins. In support of this hypothesis, we demonstrate that an anti-3-nitrotyrosine polyclonal antibody stains all of the major hallmark lesions of synucleinopathies including Lewy bodies, Lewy neurites and neuraxonal spheroids in dementia with Lewy bodies, the Lewy body variant of Alzheimers disease, and neurodegeneration with brain iron accumulation type 1, as well as glial and neuronal cytoplasmic inclusions in multiple system atrophy. This antibody predominantly recognized nitrated alpha-synuclein when compared to other in vitro nitrated constituents of these pathological lesions, such as neurofilament subunits and microtubules. Collectively, these findings imply that alpha-synuclein is nitrated in pathological lesions. The widespread presence of nitrated alpha-synuclein in diverse intracellular inclusions suggests that oxidation/nitration is involved in the onset and/or progression of neurodegenerative diseases.

Oxidative post-translational modifications of α-synuclein in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease

Structural and functional alterations of α‐synuclein is a presumed culprit in the demise of dopaminergic neurons in Parkinsons disease (PD). α‐Synuclein mutations are found in familial but not in sporadic PD, raising the hypothesis that effects similar to those of familial PD‐linked α‐synuclein mutations may be achieved by oxidative post‐translational modifications. Here, we show that wild‐type α‐synuclein is a selective target for nitration following peroxynitrite exposure of stably transfected HEK293 cells. Nitration of α‐synuclein also occurs in the mouse striatum and ventral midbrain following administration of the parkinsonian neurotoxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP). Conversely, β‐synuclein and synaptophysin were not nitrated in MPTP‐intoxicated mice. Our data demonstrate that α‐synuclein is a target for tyrosine nitration, which, by disrupting its biophysical properties, may be relevant to the putative role of α‐synuclein in the neurodegeneration associated with MPTP toxicity and with PD.

Dynamic regulation of metabolism and respiration by endogenously produced nitric oxide protects against oxidative stress.

One of the many biological functions of nitric oxide is the ability to protect cells from oxidative stress. To investigate the potential contribution of low steady state levels of nitric oxide generated by endothelial nitric oxide synthase (eNOS) and the mechanisms of protection against H2O2, spontaneously transformed human ECV304 cells, which normally do not express eNOS, were stably transfected with a green fluorescent-tagged eNOS cDNA. The eNOS-transfected cells were found to be resistant to injury and delayed death following a 2-h exposure to H2O2 (50–150 μM). Inhibition of nitric oxide synthesis abolished the protective effect against H2O2 exposure. The ability of nitric oxide to protect cells depended on the presence of respiring mitochondria as ECV304+eNOS cells with diminished mitochondria respiration (ρ−) are injured to the same extent as nontransfected ECV304 cells and recovery of mitochondrial respiration restores the ability of nitric oxide to protect against H2O2-induced death. Nitric oxide also found to have a profound effect in cell metabolism, because ECV304+eNOS cells had lower steady state levels of ATP and higher utilization of glucose via the glycolytic pathway than ECV304 cells. However, the protective effect of nitric oxide against H2O2 exposure is not reproduced in ECV304 cells after treatment with azide and oligomycin suggesting that the dynamic regulation of respiration by nitric oxide represent a critical and unrecognized primary line of defense against oxidative stress.

Reactive Nitrogen Species and Proteins: Biological Significance and Clinical Relevance

Nitric oxide and reactive nitrogen species such as nitrogen dioxide, dinitrogen trioxide and peroxynitrite react selectively with different proteins causing covalent structural modifications that alter protein function (1–3). The predominant post-translational modification mediated by nitric oxide is the S-nitrosylation of cysteine residues whereas reactive nitrogen intermediates primarily oxidize cysteine and nitrate tyrosine residues. Glyceraldehyde-3-phosphate dehydrogenase, ryanodine receptor, p21ras, hemoglobin and caspase 3 are modified by Snitrosylation of cysteine residues in vivo (4–10). The S-nitrosylation of the cysteine residues provides a selective and reversible covalent modification that regulates protein function and explains the ability of nitric oxide to regulate simultaneously different cellular pathways.