Microtubule dysfunction precedes transport impairment and mitochondria damage in MPP+ -induced neurodegeneration.

Dysfunction of the microtubule (MT) system is an emerging theme in the pathogenesis of Parkinson's disease. This study was designed to investigate the putative role of MT dysfunction in dopaminergic neuron death induced by the neurotoxin 1-methyl-4-phenylpiridinium (MPP(+)). In nerve growth factor-differentiated PC12 cells, we have analyzed post-translational modifications of tubulin known to be associated with differently dynamic MTs and show that MPP(+) causes a selective loss of dynamic MTs and a concomitant enrichment of stable MTs. Through a direct live cell imaging approach, we show a significant reduction of MT dynamics following exposure to MPP(+) and a reorientation of MTs. Furthermore, these alterations precede the impairment of intracellular transport as revealed by changes in mitochondria movements along neurites and their accumulation into varicosities. We have also analyzed activation of caspase 3 and mitochondrial injury, well-known alterations induced by MPP(+), and found that they are noticeable only when MT dysfunction is already established. These data provide the first evidence that axonal transport impairment and mitochondrial damage might be a consequence of MT dysfunction in MPP(+) -induced neurodegeneration, lending support to the concept that alterations of MT organization and dynamics could play a pivotal role in neuronal death in Parkinson's disease.

The nitration of tau protein in neurone-like PC12 cells.

Tyrosine nitration of proteins is emerging as a post-translational modification playing a role in physiological conditions. Looking for the molecular events triggered by nitric oxide in nerve growth factor-induced neuronal differentiation, we now find that nitration occurs on the microtubule-associated protein tau. In differentiated PC12 cells, we have identified as tau a nitrated protein that co-immunoprecipitates with alpha-tubulin and indicated that the modified protein is associated with the cytoskeleton but it is confined to a restricted cell region. This paper supplies the first evidence that nitration of tau occurs in a physiological process and suggests that it could play a role in neuronal differentiation.

Characterization of nitroproteome in neuron-like PC12 cells differentiated with nerve growth factor: identification of two nitration sites in alpha-tubulin.

Nitric oxide (NO) is a precursor of reactive nitrating species, peroxynitrite and nitrogen dioxide, which modify proteins to generate oxidized species such as 3-nitrotyrosine that has been used as a hallmark of peroxynitrite-mediated oxidative stress on proteins. In the last few years however, a growing body of evidence indicates that NO also regulates a myriad of physiologic responses by modifying tyrosine residues. Looking for the molecular event triggered by NO in nerve growth factor (NGF)-induced neuronal differentiation, we recently reported that in differentiating PC12 cells, the cytoskeleton becomes the main cellular fraction containing nitrotyrosinated proteins, and alpha-tubulin is the major target. In the present work, we focus on the investigation of the sites of tyrosine nitration in alpha-tubulin purified by two-dimensional gel electrophoresis following anti-alpha-tubulin immunoprecipitation of protein extract from NGF-treated PC12 cells. Using Western blotting and matrix-assisted laser desorption/ionization-time of flight analysis, we show for the first time, both in vivo and in vitro, that nitration can occur on alpha-tubulin at sites other than the C-terminus and we positively identify Tyr 161 and Tyr 357 as two specific amino acids endogenously nitrated.

Protein tyrosine nitration is triggered by nerve growth factor during neuronal differentiation of PC12 cells.

Nitric oxide (NO) is a signaling molecule implicated in a spectrum of cellular processes including neuronal differentiation. The signaling pathway triggered by NO in physiological processes involves the activation of soluble guanylate cyclase and S-nitrosylation of proteins, and, as recently proposed, nitration of tyrosine residues in proteins. However, little is known about the mechanisms involved and the target proteins for endogenous NO during the progression of neuronal differentiation. To address this question, we investigated the presence, localization, and subcellular distribution of nitrated proteins during neurotrophin-induced differentiation of PC12 cells. We find that some proteins show basal levels of tyrosine nitration in PC12 cells grown in the absence of nerve growth factor (NGF) and that nitration levels increase significantly after 2 days of incubation with this neurotrophin. Nitrated proteins accumulate over a period of several days in the presence of NGF. We demonstrate that this nitration is coupled to activation of nitric oxide synthase. The subcellular distribution of nitrated proteins changes during PC12 cell differentiation, displaying a shift from the cytosolic to the cytoskeletal fraction and we identified alpha-tubulin as the major target of nitration in PC12 cells by N-terminal sequence and MALDI-TOF analyses. We conclude that tyrosine nitration of proteins could be a novel molecular mechanism involved in the signaling pathway by which NO modulates NGF-induced differentiation in PC12 cells.