Phosphoproteomic analysis of the striatum from pleiotrophin knockout and midkine knockout mice treated with cocaine reveals regulation of oxidative stress-related proteins potentially underlying cocaine-induced neurotoxicity and neurodegeneration

Abstract

The neurotrophic factors pleiotrophin (PTN) and midkine (MK) are highly upregulated in different brain areas relevant to drug addiction after administrations of different drugs of abuse, including psychostimulants. We have previously demonstrated that PTN and MK modulate amphetamine-induced neurotoxicity and that PTN prevents cocaine-induced cytotoxicity in NG108-15 and PC12 cells. In an effort to dissect the different mechanisms of action triggered by PTN and MK to exert their protective roles against psychostimulant neurotoxicity, we have now used a proteomic approach to study protein phosphorylation, in which we combined phosphoprotein enrichment, by immobilized metal affinity chromatography (IMAC), with two-dimensional gel electrophoresis and mass spectrometry, in order to identify the phosphoproteins regulated in the striatum of PTN knockout, MK knockout and wild type mice treated with a single dose of cocaine (15mg/kg, i.p.). We identified 7 differentially expressed phosphoproteins: 5'(3')-deoxyribonucleotidase, endoplasmic reticulum resident protein 60 (ERP60), peroxiredoxin-6 (PRDX6), glutamate dehydrogenase 1 (GLUD1), aconitase and two subunits of hemoglobin. Most of these proteins are related to neurodegeneration processes and oxidative stress and their variations specially affect the PTN knockout mice, suggesting a protective role of endogenous PTN against cocaine-induced neural alterations. Further studies are needed to validate these proteins as possible targets against neural alterations induced by cocaine.

Keywords: 2D-PAGE; 5′(3′)-deoxyribonucleotidase; Cocaine-induced neurotoxicity; ERK; ERP29; ERP60; GLUD1; HBB1; HBB2; IMAC; MALDI-TOF; MC; MK; MK−/− cocaine; MK−/− saline; MS; Midkine; NT5C; Oxidative stress; PC; PD; PRDX6; PS; PTN; PTN−/− cocaine; PTN−/− saline; Parkinson's disease; Phosphoproteomics; Pleiotrophin; RPTP; WC; WS; WT; WT cocaine; WT saline; endoplasmic reticulum resident protein 29; endoplasmic reticulum resident protein 60; extracellular signalregulated kinase; glutamate dehydrogenase 1; hemoglobin subunit beta-1; hemoglobin subunit beta-2; immobilized metal affinity chromatography; matrix-assisted laser desorption/ionization time-of-flight; midkine; peroxiredoxin-6; pleiotrophin; receptor protein tyrosine phosphatase; two-dimensional polyacrylamide gel electrophoresis; wild type.