Roberto Hodara

Peroxynitrite reactions and formation in mitochondria

Mitochondria constitute a primary locus for the intracellular formation and reactions of peroxynitrite, and these interactions are recognized to contribute to the biological and pathological effects of both nitric oxide ((*)NO) and peroxynitrite. Extra- or intramitochondrially formed peroxynitrite can diffuse through mitochondrial compartments and undergo fast direct and free radical-dependent target molecule reactions. These processes result in oxidation, nitration, and nitrosation of critical components in the matrix, inner and outer membrane, and intermembrane space. Mitochondrial scavenging and repair systems for peroxynitrite-dependent oxidative modifications operate but they can be overwhelmed under enhanced cellular (*)NO formation as well as under conditions that lead to augmented superoxide formation by the electron transport chain. Peroxynitrite can lead to alterations in mitochondrial energy and calcium homeostasis and promote the opening of the permeability transition pore. The effects of peroxynitrite in mitochondrial physiology can be largely rationalized based on the reactivities of peroxynitrite and peroxynitrite-derived carbonate, nitrogen dioxide, and hydroxyl radicals with critical protein amino acids and transition metal centers of key mitochondrial proteins. In this review we analyze (i) the existing evidence for the intramitochondrial formation and reactions of peroxynitrite, (ii) the key reactions and fate of peroxynitrite in mitochondria, and (iii) their impact in mitochondrial physiology and signaling of cell death.

Dopamine-modified α-synuclein blocks chaperone-mediated autophagy

Altered degradation of alpha-synuclein (alpha-syn) has been implicated in the pathogenesis of Parkinson disease (PD). We have shown that alpha-syn can be degraded via chaperone-mediated autophagy (CMA), a selective lysosomal mechanism for degradation of cytosolic proteins. Pathogenic mutants of alpha-syn block lysosomal translocation, impairing their own degradation along with that of other CMA substrates. While pathogenic alpha-syn mutations are rare, alpha-syn undergoes posttranslational modifications, which may underlie its accumulation in cytosolic aggregates in most forms of PD. Using mouse ventral medial neuron cultures, SH-SY5Y cells in culture, and isolated mouse lysosomes, we have found that most of these posttranslational modifications of alpha-syn impair degradation of this protein by CMA but do not affect degradation of other substrates. Dopamine-modified alpha-syn, however, is not only poorly degraded by CMA but also blocks degradation of other substrates by this pathway. As blockage of CMA increases cellular vulnerability to stressors, we propose that dopamine-induced autophagic inhibition could explain the selective degeneration of PD dopaminergic neurons.

Nitric oxide and peroxynitrite interactions with mitochondria.

Abstract Nitric oxide (NO) and peroxynitrite (ONOO) avidly interact with mitochondrial components, leading to a range of biological responses spanning from the modulation of mitochondrial respiration, mitochondrial dysfunction to the signaling of apoptotic cell death. Physiological levels of NO primarily interact with cytochrome c oxidase, leading to a competitive and reversible inhibition of mitochondrial oxygen uptake. In turn, this leads to alterations in electrochemical gradients, which affect calcium uptake and may regulate processes such as mitochondrial transition pore (MTP) opening and the release of proapoptotic proteins. Large or persistent levels of NO in mitochondria promote mitochondrial oxidant formation. Peroxynitrite formed either extra or intramitochondrially leads to oxidative damage, most notably at complexes I and II of the electron transport chain, ATPase, aconitase and Mnsuperoxide dismutase. Mitochondrial scavenging systems for peroxynitrite and peroxynitritederived radicals such as carbonate (CO3.) and nitrogen dioxide radicals (NO2) include cytochrome c oxidase, glutathione and ubiquinol and serve to partially attenuate the reactions of these oxidants with critical mitochondrial targets. Detection of nitrated mitochondrial proteins in vivo supports the concept that mitochondria constitute central loci of the toxic effects of excess reactive nitrogen species. In this review we will provide an overview of the biochemical mechanisms by which NO and ONOO regulate or alter mitochondrial functions.

Reversible Inhibition of α-Synuclein Fibrillization by Dopaminochrome-mediated Conformational Alterations

Previous studies demonstrated that α-synuclein (α-syn) fibrillization is inhibited by dopamine, and studies to understand the molecular basis of this process were conducted (Conway, K. A., Rochet, J. C., Bieganski, R. M., and Lansbury, P. T., Jr. (2001) Science 294, 1346–1349). Dopamine inhibition of α-syn fibrillization generated exclusively spherical oligomers that depended on dopamine autoxidation but not α-syn oxidation, because mutagenesis of Met, His, and Tyr residues in α-syn did not abrogate this inhibition. However, truncation of α-syn at residue 125 restored the ability of α-syn to fibrillize in the presence of dopamine. Mutagenesis and competition studies with specific synthetic peptides identified α-syn residues 125–129 (i.e. YEMPS) as an important region in the dopamine-induced inhibition of α-syn fibrillization. Significantly, the dopamine oxidation product dopaminochrome was identified as a specific inhibitor of α-syn fibrillization. Dopaminochrome promotes the formation of spherical oligomers by inducing conformational changes, as these oligomers regained the ability to fibrillize by simple denaturation/renaturation. Taken together, these data indicate that dopamine inhibits α-syn fibrillization by inducing structural changes in α-syn that can occur through the interaction of dopaminochrome with the 125YEMPS129 motif of α-syn. These results suggest that the dopamine autoxidation can prevent α-syn fibrillization in dopaminergic neurons through a novel mechanism. Thus, decreased dopamine levels in substantia nigra neurons might promote α-syn aggregation in Parkinsons disease.

Distinct cleavage patterns of normal and pathologic forms of α‐synuclein by calpain I in vitro

Parkinsons disease (PD) is characterized by fibrillary neuronal inclusions called Lewy bodies (LBs) consisting largely of alpha‐synuclein (α‐syn), the protein mutated in some patients with familial PD. The mechanisms of α‐syn fibrillization and LB formation are unknown, but may involve aberrant degradation or turnover. We examined the ability of calpain I to cleave α‐syn in vitro. Calpain I cleaved wild‐type α‐syn predominantly after amino acid 57 and within the non‐amyloid component (NAC) region. In contrast, calpain I cleaved fibrillized α‐syn primarily in the region of amino acid 120 to generate fragments like those that increase susceptibility to dopamine toxicity and oxidative stress. Further, while calpain I cleaved wild‐type α‐syn after amino acid 57, this did not occur in mutant A53T α‐syn. This paucity of proteolysis could increase the stability of A53T α‐syn, suggesting that calpain I might protect cells from forming LBs by specific cleavages of soluble wild‐type α‐syn. However, once α‐syn has polymerized into fibrils, calpain I may contribute to toxicity of these forms of α‐syn by cleaving at aberrant sites within the C‐terminal region. Elucidating the role of calpain I in the proteolytic processing of α‐syn in normal and diseased brains may clarify mechanisms of neurodegenerative α‐synucleinopathies.

Oxidative Stress and Protein Deposition Diseases

Despite divergent opinions on whether protein inclusions represent a protective or harmful mechanism in the progression of diseases such as Alzheimer’s disease, Parkinson’s disease, and prion diseases, oxidative and nitrative modifications may play a critical role in development of the diseases. Oxidative and nitrative modifications may occur early during the disease initiation, or later, compromising cellular functions once the inclusions have become sufficiently large at later stages of disease progression. These assertions are supported by exciting published but preliminary evidence, which requires extensive validation by experiments employing simple in vitro systems, cellular and animal models. Experiments in progress should inform us of the significance of oxidative and nitrative chemistries in the pathological mechanisms of diseases characterized by protein inclusions.