Xiuzhen Fan

Psychological stress, cocaine and natural reward each induce endoplasmic reticulum stress genes in rat brain.

Our prior research has shown that the transcription of endoplasmic reticulum (ER) stress transcription factors activating transcription factor 3 (ATF3) and ATF4 are induced by amphetamine and restraint stress in rat striatum. However, presently the full extent of ER stress responses to psychological stress or cocaine, and which of the three ER stress pathways is activated is unknown. The current study examines transcriptional responses of key ER stress target genes subsequent to psychological stress or cocaine. Rats were subjected to acute or repeated restraint stress or cocaine treatment and mRNA was isolated from dorsal striatum, medial prefrontal cortex and nucleus accumbens brain tissue. ER stress gene mRNA expression was measured using quantitative polymerase chain reaction (PCR) and RNA sequencing. Restraint stress and cocaine-induced transcription of the classic ER stress-induced genes (BIP, CHOP, ATF3 and GADD34) and of two other ER stress components x-box binding protein 1 (XBP1) and ATF6. In addition, rats living in an enriched environment (large group cage with novel toys changed daily) exhibited rapid induction of GADD34 and ATF3 after 30 min of exploring novel toys, suggesting these genes are also involved in normal non-pathological signaling. However, environmental enrichment, a paradigm that produces protective addiction and depression phenotypes in rats, attenuated the rapid induction of ATF3 and GADD34 after restraint stress. These experiments provide a sensitive measure of ER stress and, more importantly, these results offer good evidence of the activation of ER stress mechanisms from psychological stress, cocaine and natural reward. Thus, ER stress genes may be targets for novel therapeutic targets for depression and addiction.

Protein adducts of malondialdehyde and 4-hydroxynonenal contribute to trichloroethene-mediated autoimmunity via activating Th17 cells: dose- and time-response studies in female MRL+/+ mice.

Trichloroethene (TCE), a common occupational and environmental toxicant, is known to induce autoimmunity. Previous studies in our laboratory showed increased oxidative stress in TCE-mediated autoimmunity. To further establish the role of oxidative stress and to investigate the mechanisms of TCE-mediated autoimmunity, dose- and time-response studies were conducted in MRL+/+ mice by treating them with TCE via drinking water at doses of 0.5, 1.0 or 2.0mg/ml for 12, 24 or 36 weeks. TCE exposure led to dose-related increases in malondialdehyde (MDA)-/hydroxynonenal (HNE)-protein adducts and their corresponding antibodies in the sera and decreases in GSH and GSH/GSSG ratio in the kidneys at 24 and 36 weeks, with greater changes at 36 weeks. The increases in these protein adducts and decreases in GSH/GSSG ratio were associated with significant elevation in serum anti-nuclear- and anti-ssDNA-antibodies, suggesting an association between TCE-induced oxidative stress and autoimmune response. Interestingly, splenocytes from mice treated with TCE for 24 weeks secreted significantly higher levels of IL-17 and IL-21 than did splenocytes from controls after stimulation with MDA-mouse serum albumin (MSA) or HNE-MSA adducts. The increased release of these cytokines showed a dose-related response and was more pronounced in mice treated with TCE for 36 weeks. These studies provide evidence that MDA- and or HNE-protein adducts contribute to TCE-mediated autoimmunity, which may be via activation of Th17 cells.

Overexpression of DeltaFosB in nucleus accumbens mimics the protective addiction phenotype, but not the protective depression phenotype of environmental enrichment

Environmental enrichment produces protective addiction and depression phenotypes in rats. ΔFosB is a transcription factor that regulates reward in the brain and is induced by psychological stress as well as drugs of abuse. However, the role played by ΔFosB in the protective phenotypes of environmental enrichment has not been well studied. Here, we demonstrate that ΔFosB is differentially regulated in rats reared in an isolated condition (IC) compared to those in an enriched condition (EC) in response to restraint stress or cocaine. Chronic stress or chronic cocaine treatment each elevates ΔFosB protein levels in the nucleus accumbens (NAc) of IC rats, but not of EC rats due to an already elevated basal accumulation of ΔFosB seen under EC conditions. Viral-mediated overexpression of ΔFosB in the NAc shell of pair-housed rats (i.e., independent of environmental enrichment/isolation) increases operant responding for sucrose when motivated by hunger, but decreases responding in satiated animals. Moreover, ΔFosB overexpression decreases cocaine self-administration, enhances extinction of cocaine seeking, and decreases cocaine-induced reinstatement of intravenous cocaine self-administration; all behavioral findings consistent with the enrichment phenotype. In contrast, however, ΔFosB overexpression did not alter responses of pair-housed rats in several tests of anxiety- and depression-related behavior. Thus, ΔFosB in the NAc the shell mimics the protective addiction phenotype, but not the protective depression phenotype of environmental enrichment.

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

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., Huntingtons and Alzheimers 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.

Dynamic proteomics of nucleus accumbens in response to acute psychological stress in environmentally enriched and isolated rats.

Our prior research has shown that environmental enrichment (i.e. rats reared in an environment with novel objects, social contact with conspecifics) produces a protective antidepressant-like phenotype in rats and decreases neurobiological effects of acute psychological stress. Although CREB activity has been identified as a major player, the downstream molecular mechanisms remain largely unexplored. Thus, the current study investigates proteomic differences in the accumbens of rats raised in an enriched condition (EC) versus those raised in an isolated control condition (IC) under basal conditions and after 30 min of acute restraint stress. Results showed that under basal conditions, EC rats generally expressed less mitochondria-related proteins, particularly those involved in TCA cycle and electron transport compared to IC rats. After 30 min of acute stress, EC rats displayed increased expression of energy metabolism enzymes (among others) while IC rats exhibited decreased expression of similar proteins. Further, network and pathway analyses also identified links to AKT signaling proteins, 14-3-3 family proteins, heat-shock proteins, and ubiquitin-interacting proteins. The protein ENO1 showed marked differential expression and regulation; EC rats expressed higher levels under basal conditions that increased subsequent to stress, while the basal IC expression was lower and decreased further still after stress. The results of this study define differential protein expression in a protective rat model for major depression and additionally identify a dynamic and coordinated differential response to acute stress between the two groups. These results provide new avenues for exploration of the molecular determinants of depression and the response to acute stress.

Differential Phosphoproteome Regulation of Nucleus Accumbens in Environmentally Enriched and Isolated Rats in Response to Acute Stress

Increasing evidence shows that stress contributes to the pathogenesis of major depressive disorder which is a severe neuropsychiatric disorder and influences over 10% of the worlds population. Our previous studies revealed that rats reared in an enriched environment display less depression-related behavior compared to rats raised in an isolated environment, which implies that environmental enrichment produces an antidepressant-like behavioral phenotype. However, the molecular mechanisms are not fully understood. Protein phosphorylation rapidly changes signaling pathway function and alters the function of proteins associated with the stress-induced depressive disorder. Thus, in this study, a phosphoproteomic approach was used to uncover differential phosphoprotein regulation in rat nucleus accumbens between isolated (IC) and enriched environmental conditions (EC) under basal conditions, and in response to acute stress. We found 23 phosphoproteins were regulated in EC vs. IC rats under basal conditions; 10 phosphoproteins regulated by stress in IC rats; and 15 regulated by stress in EC rats. Among all significantly regulated phosphoproteins, 11 of them were represented in at least two conditions. The regulated phosphoproteins represent signaling pathway proteins (including ERK2), enzymes, transcriptional regulators, protein translation regulators, transporters, chaperones and cytoskeletal proteins. These findings provide a global view for further understanding the contribution of protein phosphorylation in depression pathogenesis and antidepressant action.

Aniline-induced nitrosative stress in rat spleen: Proteomic identification of nitrated proteins

Aniline exposure is associated with toxicity to the spleen which is characterized by splenomegaly, hyperplasia, fibrosis, and a variety of sarcomas on chronic exposure in rats. However, mechanisms by which aniline elicits splenotoxic responses are not well understood. Earlier we have shown that aniline exposure leads to increased nitration of proteins in the spleen. However, nitrated proteins remain to be characterized. Therefore, in the current study using proteomic approaches, we focused on characterizing the nitrated proteins in the spleen of aniline-exposed rats. Aniline exposure led to increased tyrosine nitration of proteins, as determined by 2D Western blotting with anti-3-nitrotyrosine specific antibody, compared to the controls. The analyzed nitrated proteins were found in the molecular weight range of 27.7 to 123.6kDa. A total of 37 nitrated proteins were identified in aniline-treated and control spleens. Among them, 25 were found only in aniline-treated rats, 11 were present in both aniline-treated and control rats, while one was found in controls only. The nitrated proteins identified mainly represent skeletal proteins, chaperones, ferric iron transporter, enzymes, nucleic acids binding protein, and signaling and protein synthesis pathways. Furthermore, aniline exposure led to significantly increased iNOS mRNA and protein expression in the spleen, suggesting its role in increased reactive nitrogen species formation and contribution to increased nitrated proteins. The identified nitrated proteins provide a global map to further investigate alterations in their structural and functional properties, which will lead to a better understanding of the role of protein nitration in aniline-mediated splenic toxicity.

5-HT2C Receptor Knockdown in the Amygdala Inhibits Neuropathic-Pain-Related Plasticity and Behaviors

Neuroplasticity in the amygdala drives pain-related behaviors. The central nucleus (CeA) serves major amygdala output functions and can generate emotional-affective behaviors and modulate nocifensive responses. The CeA receives excitatory and inhibitory inputs from the basolateral nucleus (BLA) and serotonin receptor subtype 5-HT2CR in the BLA, but not CeA, has been implicated anxiogenic behaviors and anxiety disorders. Here, we tested the hypothesis that 5-HT2CR in the BLA plays a critical role in CeA plasticity and neuropathic pain behaviors in the rat spinal nerve ligation (SNL) model. Local 5-HT2CR knockdown in the BLA with stereotaxic injection of 5-HT2CR shRNA AAV vector decreased vocalizations and anxiety- and depression-like behaviors and increased sensory thresholds of SNL rats, but had no effect in sham controls. Extracellular single-unit recordings of CeA neurons in anesthetized rats showed that 5-HT2CR knockdown blocked the increase in neuronal activity (increased responsiveness, irregular spike firing, and increased burst activity) in SNL rats. At the synaptic level, 5-HT2CR knockdown blocked the increase in excitatory transmission from BLA to CeA recorded in brain slices from SNL rats using whole-cell patch-clamp conditions. Inhibitory transmission was decreased by 5-HT2CR knockdown in control and SNL conditions to a similar degree. The findings can be explained by immunohistochemical data showing increased expression of 5-HT2CR in non-GABAergic BLA cells in SNL rats. The results suggest that increased 5-HT2CR in the BLA contributes to neuropathic-pain-related amygdala plasticity by driving synaptic excitation of CeA neurons. As a rescue strategy, 5-HT2CR knockdown in the BLA inhibits neuropathic-pain-related behaviors. SIGNIFICANCE STATEMENT Neuroplasticity in the amygdala has emerged as an important pain mechanism. This study identifies a novel target and rescue strategy to control abnormally enhanced amygdala activity in an animal model of neuropathic pain. Specifically, an integrative approach of gene transfer, systems and brain slice electrophysiology, behavior, and immunohistochemistry was used to advance the novel concept that serotonin receptor subtype 5-HT2C contributes critically to the imbalance between excitatory and inhibitory drive of amygdala output neurons. Local viral vector-mediated 5-HT2CR knockdown in the amygdala normalizes the imbalance, decreases neuronal activity, and inhibits neuropathic-pain-related behaviors. The study provides valuable insight into serotonin receptor (dys)function in a limbic brain area.

Transcriptomics of Environmental Enrichment Reveals a Role for Retinoic Acid Signaling in Addiction

There exists much variability in susceptibility/resilience to addiction in humans. The environmental enrichment paradigm is a rat model of resilience to addiction-like behavior, and understanding the molecular mechanisms underlying this protective phenotype may lead to novel targets for pharmacotherapeutics to treat cocaine addiction. We investigated the differential regulation of transcript levels using RNA sequencing of the rat nucleus accumbens after environmental enrichment/isolation and cocaine/saline self-administration. Ingenuity Pathways Analysis and Gene Set Enrichment Analysis of 14,309 transcripts demonstrated that many biofunctions and pathways were differentially regulated. New functional pathways were also identified for cocaine modulation (e.g., Rho GTPase signaling) and environmental enrichment (e.g., signaling of EIF2, mTOR, ephrin). However, one novel pathway stood out above the others, the retinoic acid (RA) signaling pathway. The RA signaling pathway was identified as one likely mediator of the protective enrichment addiction phenotype, an interesting result given that nine RA signaling-related genes are expressed selectively and at high levels in the nucleus accumbens shell (NAcSh). Subsequent knockdown of Cyp26b1 (an RA degradation enzyme) in the NAcSh of rats confirmed this role by increasing cocaine self-administration as well as cocaine seeking. These results provide a comprehensive account of enrichment effects on the transcriptome and identify RA signaling as a contributing factor for cocaine addiction.

Convergent transcriptomics and proteomics of environmental enrichment and cocaine identifies novel therapeutic strategies for addiction.

Transcriptomic and proteomic approaches have separately proven effective at identifying novel mechanisms affecting addiction-related behavior; however, it is difficult to prioritize the many promising leads from each approach. A convergent secondary analysis of proteomic and transcriptomic results can glean additional information to help prioritize promising leads. The current study is a secondary analysis of the convergence of recently published separate transcriptomic and proteomic analyses of nucleus accumbens (NAc) tissue from rats subjected to environmental enrichment vs. isolation and cocaine self-administration vs. saline. Multiple bioinformatics approaches (e.g. Gene Ontology (GO) analysis, Ingenuity Pathway Analysis (IPA), and Gene Set Enrichment Analysis (GSEA)) were used to interrogate these rich data sets. Although there was little correspondence between mRNA vs. protein at the individual target level, good correspondence was found at the level of gene/protein sets, particularly for the environmental enrichment manipulation. These data identify gene sets where there is a positive relationship between changes in mRNA and protein (e.g. glycolysis, ATP synthesis, translation elongation factor activity, etc.) and gene sets where there is an inverse relationship (e.g. ribosomes, Rho GTPase signaling, protein ubiquitination, etc.). Overall environmental enrichment produced better correspondence than cocaine self-administration. The individual targets contributing to mRNA and protein effects were largely not overlapping. As a whole, these results confirm that robust transcriptomic and proteomic data sets can provide similar results at the gene/protein set level even when there is little correspondence at the individual target level and little overlap in the targets contributing to the effects.