Environmental enrichment alters protein expression as well as the proteomic response to cocaine in rat nucleus accumbens

Abstract

Prior research demonstrated that environmental enrichment creates individual differences in behavior leading to a protective addiction phenotype in rats. Understanding the mechanisms underlying this phenotype will guide selection of targets for much-needed novel pharmacotherapeutics. The current study investigates differences in proteome expression in the nucleus accumbens of enriched and isolated rats and the proteomic response to cocaine self-administration using a liquid chromatography mass spectrometry (LCMS) technique to quantify 1917 proteins. Results of complementary Ingenuity Pathways Analyses (IPA) and gene set enrichment analyses (GSEA), both performed using protein quantitative data, demonstrate that cocaine increases vesicular transporters for dopamine and glutamate as well as increasing proteins in the RhoA pathway. Further, cocaine regulates proteins related to ERK, CREB and AKT signaling. Environmental enrichment altered expression of a large number of proteins implicated in a diverse number of neuronal functions (e.g., energy production, mRNA splicing, and ubiquitination), molecular cascades (e.g., protein kinases), psychiatric disorders (e.g., mood disorders), and neurodegenerative diseases (e.g., Huntington's and Alzheimer's diseases). Upregulation of energy metabolism components in EC rats was verified using RNA sequencing. Most of the biological functions and pathways listed above were also identified in the Cocaine X Enrichment interaction analysis, providing clear evidence that enriched and isolated rats respond quite differently to cocaine exposure. The overall impression of the current results is that enriched saline-administering rats have a unique proteomic complement compared to enriched cocaine-administering rats as well as saline and cocaine-taking isolated rats. These results identify possible mechanisms of the protective phenotype and provide fertile soil for developing novel pharmacotherapeutics. Proteomics data are available via ProteomeXchange with identifier PXD000990.

Keywords: cocaine; differential rearing; drug abuse; drug addiction; label-free quantification; proteomics.

Figure 1

IPA and GSEA findings for cocaine main effect. A complete list of results can be found in Supplementary Materials, as can a complete list of all proteins in each pathway. (A) A selection of significant biological functions and diseases for cocaine main effect as determined by IPA. The y axis represents the −log(p-value) for each function/disease. (B) A selection of significant canonical pathways for cocaine main effect, as determined by IPA. Orange line represents a ratio of regulated proteins to all proteins in the pathway. (C) Combined glutamate and dopamine signaling pathways. Proteins whose expression levels were significantly increased are highlighted in red. SLC17A: vesicular glutamate transporter. GRIK: glutamate receptor (kainate). (D) GSEA plot demonstrating enrichment of proteins associated with the Protein Interaction Database's ras homolog family member A (RhoA) pathway. Protein regulation is ranked from those with highest correlation of expression to saline rats to the left to those with the highest correlation of expression in cocaine rats to the right. Vertical black tick marks represent relative rank of proteins in the RhoA pathway. (E) Significantly regulated proteins associated with cocaine-related behaviors. Proteins significantly increased are highlighted in red, while those that are significantly decreased are highlighted in green. (F) Network of regulated proteins related to kinase cascades. Solid lines represent direct interactions and dashed lines represent indirect. JNK: cJun N-terminal kinase. ERK: extracellular signal-regulated kinase. PKC: Protein kinase C. MAPK: mitogen-activated protein kinase. PI3K: phosphoinositide-3-kinase. AKT: v-AKT murine thymoma viral oncogene. (G) Network of regulated proteins related to ubiquitin C (UBC), hepatocyte nuclear factor 4alpha (HNF4A) and estradiol. Proteins significantly increased are highlighted in red, while those significantly decreased are highlighted in green. As a whole, this figure highlights previously-known effects of cocaine (dopamine signaling, RhoA signaling, glutamate signaling) and known kinase pathways (ERK, JNK, AKT, etc.) as well as novel findings (HNF4A, ubiquitination, etc.).

Figure 2

IPA and GSEA findings for environmental enrichment main effect. A complete list of results can be found in Supplementary Materials, as can a complete list of all proteins in each pathway. (A) A selection of significant biological functions and diseases for environmental enrichment main effect, as determined by IPA. The y axis represents the −log(p) value for each function/disease. (B) A selection of significant canonical pathways for environmental enrichment main effect, as determined by IPA. Orange line represents a ratio of regulated proteins to all proteins in the pathway. (C) Significantly regulated proteins associated with mood disorders as a subset of psychological disorders in Panel A. Proteins with a measured increase in expression level are highlighted in red, while those with a measured decrease in expression level are highlighted in green. (D) Protein ubiquitination canonical pathway. Proteins highlighted in red showed a measured increase in expression level. Symbols with purple outline are members of the heat-shock family of proteins (HSPs). DUB: de-ubiquitinating enzyme. (E) GSEA analysis of the KEGG Spliceosome gene set, showing a decreased expression of proteins relating to splicing in EC rats. Vertical black tick marks denote proteins in the gene set among ranked expression list of proteins. Heat map displays relative expression level of proteins in the set. Red denotes higher expression and blue denotes lower. (F) GSEA analysis using Ribonucleoprotein complex GO gene set, showing a decreased expression of proteins relating to splicing in EC rats. (G) GSEA analysis using GO Substrate-specific transporter activity gene set, showing increased expression of proteins related to transporter activity in EC rats. (H) GSEA analysis using GO Protein kinase activity gene set, showing upregulation in EC rats. This figure highlights the breadth of regulation of different novel pathways and protein sets regulated by enrichment.

Figure 3

IPA and GSEA findings illustrating the regulation of energy metabolism proteins by environmental enrichment. A complete list of results can be found in Supplementary Materials, as can a complete list of all proteins in each pathway. (A) Canonical pathways involved in energy metabolism from IPA. Proteins whose expression was significantly increased in EC rats are highlighted in red. TCA: tricarboxylic acid. Complexes I–V: mitochondrial respiratory chain complexes. (B) Proteins involved in ATP synthesis and degradation, a subset from the IPA biological functions analysis of Energy production (Figure 2A). Proteins whose expression is increased are shown in red, while those with decreased expression are shown in green. (C–E) Proteins from Upstream Analysis in IPA whose expression supports upregulation of (C) PPARgamma coactivator protein 1alpha (PPARGC1A) and estrogen-related receptor alpha (ESRRA) activity, (D) insulin receptor (INSR), and (E) huntingtin (HTT). (F) GSEA analysis using Reactome TCA Cycle and Electron Transport gene set. (G) GSEA analysis using proteins upregulated by ESRRA from Stein et al. (2008). This figure focuses specifically on the depth of regulation of energy metabolism proteins by environmental enrichment.

Figure 4

IPA and GSEA findings for the Enrichment X Cocaine interaction. A complete list of results can be found in Supplementary Materials, as can a complete list of all proteins in each pathway. (A) A selection of significant biological functions and diseases, as determined by IPA. The y axis represents the −log(p) value for each function/disease. (B) A selection of significant canonical pathways for Enrichment X Cocaine interaction, as determined by IPA. Orange line represents a ratio of regulated proteins to all proteins in the pathway. (C) RhoA signaling canonical pathway from IPA (from B). Proteins with a measured positive effect size are highlighted in red, while those with a measured negative effect size are highlighted in green. (D) Protein interaction network highlighting ERK, MAPK kinase 1/2 (MAP2K1/2) and cAMP response element binding protein (CREB) signaling. (E) Protein interaction network highlighting PI3K, MAPK, ERK and nuclear factor kappa B (NFkB) as hubs. (F) Protein interaction network demonstrating changes in expression of ubiquitin targets. The interaction term is important because it specifically identifies targets and pathways differentially regulated by cocaine in EC and IC rats.

Figure 5

RNA-seq upregulation of energy metabolism gene expression. Transcripts whose expression was significantly increased in EC rats (main effect) are highlighted in red. Green denotes a decrease in expression. N = 7–8. This figure is an RNA validation of enrichment-induced upregulation of energy metabolism proteins (compare to Figure 3A). A complete list of all proteins in this pathway can be found in Supplementary Materials. Right: heat map of relative expression intensity of energy metabolism transcripts. Red denotes higher expression and blue denotes lower.