Cocaine-induced changes of synaptic transmission in the striatum are modulated by adenosine A2A receptors and involve the tyrosine phosphatase STEP.

The striatum is a brain area implicated in the pharmacological action of drugs of abuse. Adenosine A2A receptors (A2ARs) are highly expressed in the striatum and mediate, at least in part, cocaine-induced psychomotor effects in vivo. Here we studied the synaptic mechanisms implicated in the pharmacological action of cocaine in the striatum and investigated the influence of A2ARs. We found that synaptic transmission was depressed in corticostriatal slices after perfusion with cocaine (10 µM). This effect was reduced by the A2AR antagonist ZM241385 and almost abolished in striatal A2AR-knockout mice (mice lacking A2ARs in striatal neurons, stA2ARKO). The effect of cocaine on synaptic transmission was also prevented by the protein tyrosine phosphatases (PTPs) inhibitor sodium orthovanadate (Na3VO4). In synaptosomes prepared from striatal slices, we found that the activity of striatal-enriched protein tyrosine phosphatase (STEP) was upregulated by cocaine, prevented by ZM241385, and absent in synaptosomes from stA2ARKO. The role played by STEP in cocaine modulation of synaptic transmission was investigated in whole-cell voltage clamp recordings from medium spiny neurons of the striatum. We found that TAT-STEP, a peptide that renders STEP enzymatically inactive, prevented cocaine-induced reduction in AMPA- and NMDA-mediated excitatory post-synaptic currents, whereas the control peptide, TAT-myc, had no effect. These results demonstrate that striatal A2ARs modulate cocaine-induced synaptic depression in the striatum and highlight the potential role of PTPs and specifically STEP in the effects of cocaine.

Phosphorylation and nitration of tyrosine residues affect functional properties of Synaptophysin and Dynamin I, two proteins involved in exo-endocytosis of synaptic vesicles.

Phosphorylation and nitration of protein tyrosine residues are thought to play a role in signaling pathways at the nerve terminal and to affect functional properties of proteins involved in the synaptic vesicle (SV) exo-endocytotic cycle. We previously demonstrated that the tyrosine residues in the C-terminal domain of the SV protein Synaptophysin (SYP) are targets of peroxynitrite (PN). Here, we have characterized the association between SYP and c-src tyrosine kinase demonstrating that phosphorylation of Tyr(273) in the C-terminal domain of SYP is crucial in mediating SYP binding to and activation of c-src. SYP forms a complex with Dynamin I (DynI), a GTPase required for SV endocytosis, which may be regulated by tyrosine phosphorylation of SYP. We here report that, in rat brain synaptosomes treated with PN, the formation of SYP/DynI complex was impaired. Noteworthy, we found that DynI was also modified by PN. DynI tyrosine phosphorylation was down-regulated in a dose-dependent manner, while DynI tyrosine nitration increased. Using mass spectrometry analysis, we identified Tyr(354) as one nitration site in DynI. In addition, we tested DynI self-assembly and GTPase activity, which are enhanced by c-src-dependent tyrosine phosphorylation of DynI, and found that both were inhibited by PN. Our results suggest that the site-specific tyrosine residue modifications may modulate the association properties of SV proteins and serve as a regulator of DynI function via control of self-assembly, thus influencing the physiology of the exo-endocytotic cycle.

Peroxynitrite induces tyrosine residue modifications in synaptophysin C-terminal domain, affecting its interaction with src.

Peroxynitrite is a potent oxidant that contributes to tissue damage in neurodegenerative disorders. We have previously reported that treatment of rat brain synaptosomes with peroxynitrite induced post-translational modifications in pre- and post-synaptic proteins and stimulated soluble N-ethylmaleimide sensitive fusion proteins attachment receptor complex formation and endogenous glutamate release. In this study we show that, following peroxynitrite treatment, the synaptic vesicle protein synaptophysin (SYP) can be both phosphorylated and nitrated in a dose-dependent manner. We found that tyrosine-phosphorylated, but not tyrosine-nitrated, SYP bound to the src tyrosine kinase and enhanced its catalytic activity. These effects were mediated by direct and specific binding of the SYP cytoplasmic C-terminal tail with the src homology 2 domain. Using mass spectrometry analysis, we mapped the SYP C-terminal tail tyrosine residues modified by peroxynitrite and found one nitration site at Tyr250 and two phosphorylation sites at Tyr263 and Tyr273. We suggest that peroxynitrite-mediated modifications of SYP may be relevant in modulating src signalling of synaptic terminal in pathophysiological conditions.

Peroxynitrite-dependent upregulation of SRC kinases in red blood cells: strategies to study the activation mechanisms.

Several studies have demonstrated that treatment of cells with oxidants, and in particular with peroxynitrite, may cause the upregulation of tyrosine phosphorylation signaling. In erythrocytes, peroxynitrite induces tyrosine phosphorylation of the major intrinsic membrane protein, band 3. A closer look at the enzymes involved revealed that the effect of peroxynitrite was due to the inhibition of phosphotyrosine phosphatases and/or to the activation of src kinases. The activity of src kinases is modulated not only by phosphatases and other kinases but also through redox modification of cysteine residues: Peroxynitrite can, thus, affect src kinase activity by means of direct and indirect mechanisms. In this chapter, we describe the different pathways leading to src kinase activation and the experimental procedures that can be performed to reveal the activation mechanism. The aim is to provide a more general strategy adaptable to different cell types and different oxidants.