RNA Deep Sequencing Analysis Reveals That Nicotine Restores Impaired Gene Expression by Viral Proteins in the Brains of HIV-1 Transgenic Rats

Persons infected with HIV-1 often develop neurologic disorders despite receiving highly active anti-retroviral therapy. Although the underlying mechanism is largely undetermined, our previous RNA-seq-based study showed that the expression of many genes was altered in the central nervous system (CNS) of HIV-1 transgenic (HIV-1Tg) rats. Because nicotine, a natural agonist of nicotinic acetylcholine receptors, exhibits a neuroprotective effect, we presently tested the hypothesis that nicotine restores the expression of altered genes in the CNS of HIV-1Tg rats. Adult male HIV-1Tg and F344 control strain rats were injected with either nicotine (0.25 mg/kg) or saline subcutaneously twice a day for 17 days. Gene expression in the prefrontal cortex (PFC), dorsal hippocampus (HIP), and dorsal striatum (STR) was evaluated using the RNA deep sequencing technique. We found that about 20% of the altered genes in the HIV-1Tg rat were affected by nicotine in each brain region, with the expression of most restored. Analysis of the restored genes showed distinct pathways corrected by nicotine in different brain regions of HIV-1Tg rats. Specifically, the two most significantly restored pathways were Wnt/β-catenin signaling and ephrin B signaling in the PFC, cAMP-responsive element-binding protein (CREB) signaling and glutathione metabolism pathway in the HIP, and tricarboxylic acid (TCA) cycle and calcium signaling in the STR. Together, our findings indicate that cholinergic modulators such as nicotine have beneficial effects on HIV-1-induced neurologic deficits.     

Proteomic profiling of proteins associated with methamphetamine-induced neurotoxicity in different regions of rat brain

It is well documented that methamphetamine (MA) can cause obvious damage to the brain, but the exact mechanism is still unknown. In the present study, proteomic methods of two-dimensional gel electrophoresis in combination with mass spectrometry analysis were used to identify global protein profiles associated with MA-induced neurotoxicity. For the first time, 30 protein spots have been found differentially expressed in different regions of rat brain, including 14 in striatum, 12 in hippocampus and 4 in frontal cortex. The proteins identified by tandem mass spectrometry were Cu, Zn superoxide dismutase, dimethylarginine dimethylaminohydrolase 1, alpha synuclein, ubiquitin-conjugating enzyme E2N, stathmin 1, calcineurin B, cystatin B, subunit of mitochondrial H-ATP synthase, ATP synthase D chain, mitochondrial, NADH dehydrogenase(ubiquinone) Fe-S protein 8, glia maturation factor, beta, Ash-m, neurocalcin delta, myotrophin, profiling IIa, D-dopachrome tautomerase, and brain lipid binding protein. The known functions of these proteins were related to the pathogenesis of MA-induced neurotoxicity, including oxidative stress, degeneration/apoptosis, mitochontrial/energy metabolism and others. Of these proteins, alpha-synuclein was up-regulated, and ATP synthase D chain, mitochondrial was down-regulated in all brain regions. Two proteins, Cu, Zn superoxide dismutase, subunit of mitochondrial H-ATPsynthase were down-regulated and Ubiquitin-conjugating enzyme E2N, NADH dehydrogenase (ubiquinone) Fe-S protein 8 were up-regulated simultaneously in striatum and hippocaltum. The expression of dimethylarginine dimethylaminohydrolase 1 (DDAH 1) increased both in striatum and frontal cortex. The parallel expression patterns of these proteins suggest that the pathogenesis of MA neurotoxicity in different brain regions may share some same pathways.     

Alterations in ionotropic glutamate receptor subunits during binge cocaine self-administration and withdrawal in rats

Chronic cocaine use in humans and animal models is known to lead to pronounced alterations in glutamatergic function in brain regions associated with reinforcement. Previous studies have examined ionotropic glutamate receptor (iGluR) subunit protein level changes following acute and chronic experimenter-administered cocaine or after withdrawal periods from experimenter-administered cocaine. To evaluate whether alterations in expression of iGluRs are associated with cocaine reinforcement, protein levels were assessed after binge (8 h/day, 15 days; 24-h access, days 16-21) cocaine self-administration and following 2 weeks of abstinence from this binge. Western blotting was used to compare levels of iGluR protein expression (NR1-3B, GluR1-7, KA2) in the ventral tegmental area (VTA), substantia nigra (SN), nucleus accumbens (NAc), striatum and prefrontal cortex (PFC) of rats. iGluR subunits were altered in a time-dependent manner in all brain regions studied; however, selective alterations in certain iGluR subtypes appeared to be associated with binge cocaine self-administration and withdrawal in a region-specific manner. In the SN and VTA, alterations in iGluR protein levels compared with controls occurred only following binge access, whereas in the striatum and PFC, iGluR alterations occurred with binge access and following withdrawal. In the NAc, GluR2/3 levels were increased following withdrawal compared with binge access, and were the only changes observed in this region. Because subunit composition determines the functional properties of iGluRs, the observed changes may indicate alterations in the excitability of dopamine transmission underlying long-term biochemical and behavioral effects of cocaine.     

Proteomic analysis of rat prefrontal cortex in three phases of morphine-induced conditioned place preference

Morphological alterations of synapse are found after morphine administration, suggesting that regulation of synaptic plasticity may be one of the mechanisms of neuroadaptation in addiction. However, the molecular basis underlying the abnormal synapse morphological and physiological changes in the morphine-induced dependence, withdraw, and relapse is not well understood. As prefrontal cortex (PFC) is one of the most important brain regions, which provides executive control over drug use and is severely impaired in many addicts, systematic analysis of the biochemical and molecular alteration of synaptic fraction of PFC in morphine-induced neuroadaptation is necessary. In this study, differential protein expression profiling of synaptic fraction of rat PFC based on morphine-induced conditioned place preference (CPP) model was performed with two-dimensional gel electrophoresis (2-DE). Our results showed that a total of 80 proteins were differentially expressed by 2-DE analysis during three phases of CPP assay. Of them, 58 were further identified by mass spectrometry. These proteins were classified into multiple categories, such as energy metabolism, signal transduction, synaptic transmission, cytoskeletal proteins, chaperones, and local synaptic protein synthetic machinery according to their biological functions. Our study provides a global view of synaptic-related molecular networking in PFC under morphine-induced dependence, withdraw, and relapse, indicative of a concerted biological process in neuroadaptation under chronic morphine exposure.     

Difference in the Pattern of Soluble Proteins from Rat Brain Regions

The electrophoretic pattern of soluble proteins from seven rat brain regions (amygdala, cerebellum, corpus striatum, cortex, hypothalamus, medulla, and midbrain) was examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Although the number of protein bands (36) was identical in all brain regions studied, there were differences in their relative densities, the greatest variation occurring in the low-molecular-weight region of the electrophoretogram. The bulk of the soluble proteins had molecular weights between 23,000 and 90,000 daltons. The medulla and amygdala showed the greatest range of protein band concentration. A large number of protein bands in the midbrain and corpus striatum showed a greater concentration of protein compared to the same bands in the other regions. A protein band that migrated with the same characteristic as albumin was found. It was consistently high in all regions, the midbrains showing a 1.5-fold greater concentration compared to other regions. Linear regression analysis of wet weight of regional brain tissue against protein concentration yielded a regression coefficient (r2) of 0.77. Midbrain and corpus striatum showed a relatively higher protein concentration: weight ratio than other regions.     

Quantitative Proteome Analysis of Brain Subregions and Spinal Cord from Experimental Autoimmune Encephalomyelitis Mice by TMT‐Based Mass Spectrometry

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS); its cause is unknown. To understand the pathogenesis of MS, researchers often use the experimental autoimmune encephalomyelitis (EAE) mouse model. Here, the aim is to build a proteome map of the biological changes that occur during MS at the major onset sites—the brain and the spinal cord. Quantitative proteome profiling is performed in five specific brain regions and the spinal cord of EAE and healthy mice with high‐resolution mass spectrometry based on tandem mass tags. On average, 7400 proteins per region are quantified, with the most differentially expressed proteins in the spinal cord (1691), hippocampus (104), frontal cortex (83), cerebellum (63), brainstem (50), and caudate nucleus (41). Moreover, region‐specific and commonly expressed proteins in each region are identified and bioinformatics analysis is performed. Pathway analysis reveals that protein clusters resemble their functions in disease pathogenesis (i.e., by inducing inflammatory responses, immune activation, and cell–cell adhesion). In conclusion, the study provides an understanding of the pathogenesis of MS in the EAE animal model. It is expected that the comprehensive proteome map of the brain and spinal cord can be used to identify biomarkers for the pathogenesis of MS.     

Cross‐species analysis of nicotine‐induced proteomic alterations in pancreatic cells

Toxic compounds in tobacco, such as nicotine, may adversely affect pancreatic function. We aim to determine nicotine‐induced protein alterations in pancreatic cells, thereby revealing links between nicotine exposure and pancreatic disease. We compared the proteomic alterations induced by nicotine treatment in cultured pancreatic cells (mouse, rat, and human stellate cells and human duct cells) using MS‐based techniques, specifically SDS‐PAGE (gel) coupled with LC‐MS/MS and spectral counting. We identified thousands of proteins in pancreatic cells, hundreds of which were identified exclusively or in higher abundance in either nicotine‐treated or untreated cells. Interspecies comparisons of stellate cell proteins revealed several differentially abundant proteins (in nicotine treated versus untreated cells) common among the three species. Proteins appearing in all nicotine‐treated stellate cells include amyloid beta (A4), procollagen type VI alpha 1, integral membrane protein 2B, and toll‐interacting protein. Proteins that were differentially expressed upon nicotine treatment across cell lines were enriched in certain pathways, including nicotinic acetylcholine receptor, cytokine, and integrin signaling. At this analytical depth, we conclude that similar pathways are affected by nicotine, but alterations at the protein level among stellate cells of different species vary. Further interrogation of such pathways will lead to insights into the potential effect of nicotine on pancreatic cells at the biomolecular level and the extension of this concept to the effect of nicotine on pancreatic disease.     

Locally Administered Low Nicotine-Induced Neurotransmitter Changes in Areas of Cognitive Function

The present study examined the effect of a low-dose of nicotine; below that one expects to be achieved from a single cigarette, on brain regional heterogeneity and sensitivity of catecholaminergic responses. 1 μM nicotine was infused into six brain areas via a microdialysis probe: the dorsal and ventral hippocampus, the medial temporal and prefrontal cortex, the basolateral amygdala, and the ventral tegmental area (VTA). The nicotine concentration in the brain tissue near the probe site was approximately 0.1 μM. Nicotine-induced increases and decreases could be noted in dopamine (DA), norepinephrine (NE), and serotonin (5HT) levels. In particular, DA and 5HT decreased in both hippocampal areas, while NE increased in the dorsal and decreased in the ventral hippocampus. In the cortical areas, DA and NE increased and 5HT was not significantly altered. In the amygdala all three neurotransmitters increased and in the VTA, all three decreased. Many of the nicotine-induced changes in neurotransmitter concentrations were reversed in the presence of atropine. Where nicotine induced decreases in DA and 5HT in the VTA, increases were observed in the presence of atropine. A similar reversal was seen with NE in the VTA and ventral hippocampus. In contrast, the increases in DA observed in the cortex and amygdala and the increases in NE observed in the cortex, amygdala and dorsal hippocampus were inhibited by the presence of atropine. 5HT was also significantly decreased in the amygdala and both cortical areas in the presence of atropine, where nicotine alone had no significant effect. We conclude, that at low doses, nicotine significantly alters the release of DA, NE, and 5HT – in some areas increasing, in others decreasing endogenous neurotransmitter levels. This data, in conjunction with previous experiments, indicates that the effects of nicotine are regionally heterogeneous and arise from both direct and indirect actions on various receptors and neurotransmitter systems and nicotine’s effects at low doses differ from that at higher doses. The changes in effects in the presence of atropine suggest that muscarinic acetylcholine receptors play a major role in nicotine’s actions on neurotransmitter systems.     

Stimulated D(1) dopamine receptors couple to multiple Galpha proteins in different brain regions

Previous studies have revealed that activation of rat striatal D(1) dopamine receptors stimulates both adenylyl cyclase and phospholipase C via G(s) and G(q), respectively. The differential distribution of these systems in brain supports the existence of distinct receptor systems. The present communication extends the study by examining other brain regions: hippocampus, amygdala, and frontal cortex. In membrane preparations of these brain regions, selective stimulation of D(1) dopamine receptors increases the hydrolysis of phosphatidylinositol/phosphatidylinositol 4,5-biphosphate. In these brain regions, D(1) dopamine receptors couple differentially to multiple Galpha protein subunits. Antisera against Galpha(q) blocks dopamine-stimulated PIP(2) hydrolysis in hippocampal and in striatal membranes. The binding of [(35)S]GTPgammaS or [alpha-(32)P]GTP to Galpha(i) was enhanced in all brain regions. Dopamine also increased the binding of [(35)S]GTPgammaS or [alpha-(32)P]GTP to Galpha(q) in these brain regions: hippocampus = amygdala > frontal cortex. However, dopamine-stimulated binding of [(35)S]GTPgammaS to Galphas only in the frontal cortex and striatum. This differential coupling profile in the brain regions was not related to a differential regional distribution of the Galpha proteins. Dopamine induced increases in GTPgammaS binding to Galpha(s) and Galpha(q) was blocked by the D(1) antagonist SCH23390 but not by D(2) receptor antagonist l-sulpiride, suggesting that D(1) dopamine receptors couple to both Galpha(s) and Galpha(q) proteins. Co-immunoprecipitation of Galpha proteins with receptor-binding sites indicate that in the frontal cortex, D(1) dopamine-binding sites are associated with both Galpha(s) and Galpha(q) and, in hippocampus or amygdala, D(1) dopamine receptors couple solely to Galpha(q). The results indicate that in addition to the D(1)/G(s)/adenylyl cyclase system, brain D(1)-like dopamine receptor sites activate phospholipase C through Galpha(q) protein.     

Acute stress responsive RGS proteins in the mouse brain

Regulator of G-protein signaling (RGS) proteins play an important role in G-protein coupled receptor (GPCR) signaling and the activity of some GPCRs is modulated via RGS protein levels during stress response. The aim of this study was to investigate changes in RGS protein mRNA expressions in the mouse brain after 2h restraint stress. The mRNA level of 19 RGS proteins was analyzed using real-time PCR in six brain regions, which included the prefrontal cortex, amygdala, hippocampus, hypothalamus, striatum, and pituitary gland, from control and stressed mouse. We found that the level of mRNA of each RGS varied according to brain region and that two to eight RGS proteins exhibited changes in mRNA levels in each brain region by restraint stress. It was also revealed that RGS4 protein amount was consistent with mRNA level, indicating RGS4 protein may have regulatory roles in the acute stress response.     

Gel-based proteomics of unilateral irradiated striatum after gamma knife surgery

Gamma knife surgery (GKS) is used for the treatment of various brain disorders. The biological effects of focal gamma ray irradiation on targeted or surrounding areas in the brain are not well-known. In the present study, we evaluated protein expression changes in the unilateral irradiated (60 Gy) striatum in rat. Striata of irradiated and control brains were dissected 16 h post-irradiation for analysis by large-format two-dimensional gel electrophoresis (2-DGE). In parallel, we also examined the un-targeted contralateral striatum over the control for potential changes in proteins patterns that may have occurred due to the effects of irradiation to the unilateral striatum. A total of 17 reproducible and differentially expressed silver nitrate-stained protein spots in the irradiated striatum was detected on 2-D gel. Their subsequent analysis by tandem mass spectrometry (nESI-LC-MS/MS) resulted in the identification of 13 nonredundant proteins. Interestingly, out of these 13 changed proteins, 2 proteins were also detected in the contralateral striatum. Some of the significantly changed proteins identified were creatine kinase, protein disulfide isomerase A3 precursor (PDA3), and peroxiredoxin 2 (Prx2). Western analysis with anti-PDA3 and anti-Prx2 antibodies revealed 4 and 2 cross-reacting protein spots on 2-D gel blots. Interestingly, after GKS, in the irradiated and un-irradiated striata, these spots showed a shift toward the acidic side, suggesting post-translational modifications. Taken together, these results indicate that unilateral irradiation during GKS triggers molecular changes in the bilateral striata.     

Dopamine and Serotonin Metabolism in Brain Sites Ipsi- and Contralateral to Direction of Conditioned Turning in Rats

Concentrations of dopamine, serotonin, and some of their metabolites were analyzed by means of HPLC in brain samples obtained from rats operantly conditioned to turn in circles to obtain water reinforcement. In experiment 1 using Wistar rats, no differences in the levels of transmitters or metabolites were detected between brain samples (frontal cortex, ventral striatum, dorsal striatum, septum, amygdala, substantia nigra) from the hemispheres located ipsi- and contralateral to the direction of turning. A higher dopamine metabolism (indicated by higher metabolite/transmitter ratios) in ventral striatum, dorsal striatum, and amygdala was found after 15 min than after 5 min of turning in both hemispheres. A higher dopamine metabolism was found in water-deprived rats compared to nondeprived rats independently of whether or not deprived rats were trained to turn for water reinforcement. In two additional experiments, no differences in dopamine metabolism were found between the ipsi- and contralateral striatum of Wistar rats after 25 min and Sprague-Dawley rats after 10 min of operantly conditioned turning. The present results confirm that dopamine metabolism can change with different behavioral or physiological states; they do not support the hypothesis that conditioned turning is correlated with asymmetrical changes in the metabolism of dopamine or serotonin in the brain.     

Proteomic changes in rat serum, polymorphonuclear and mononuclear leukocytes after chronic nicotine administration

In order to gain information about the effect triggered at the molecular level by nicotine, its neuroimmunomodulatory properties and its impact on the pathogenesis of inflammatory diseases, peripheral blood serum and leukocytes of rat submitted to passive nicotine administration were subjected to proteomic investigation. Serum, polymorphonuclear (PMN) and mononuclear (MN) leukocytes from chronically treated animals and from control animals were analysed by a two-dimensional (2-D) gel electrophoresis/mass spectrometry approach to detect differentially expressed proteins. The nicotine regimen selected is known to have a stimulatory effect on locomotor activity and to produce a sensitisation of the mesolimbic dopamine system mechanism involved in addiction development. After 2-D gel analysis and matching, 36 spots in serum, seven in PMN and five in MN were found to display a statistical difference in their expression and were subjected to matrix-assisted laser desorption/ionization-time of flight-mass spectrometry peptide fingerprinting for protein identification. Fifteen different proteins were identified. The results indicate an overall impact of nicotine on proteins involved in a variety of cellular and metabolic pathways, including acute phase response (suggesting the effect on inflammatory cascades and more in general on the immune system), oxidative stress metabolism and assembly and regulation of cytoskeleton. In particular, the observed changes imply a general reduction in the inflammatory response with a concomitant increased unbalance of the oxidative stress metabolism in the periphery and point to a number of potential noninvasive markers for the central nervous system (CNS) and non-CNS mediated activities of nicotine.     

应用蛋白质组学方法初步筛选与鼻咽癌放射敏感相关的蛋白

目的:通过筛选放射敏感性不同的鼻咽癌细胞中差异表达蛋白,以发现与鼻咽癌放射敏感相关的蛋白。方法:放射处理并结合流式细胞术检测及比较5-8F和6-10B细胞的放射敏感性。提取细胞总蛋白,进行双向凝胶电泳、MALDI-TOF肽质指纹图分析、质谱数据的蛋白质库搜寻鉴定。应用Western Blot检测细胞中蛋白质表达。应用免疫组织化学方法检测鼻咽癌组织中相关蛋白的表达。结果:双向凝胶电泳后对胶上的部分分辨较好的差异蛋白质点进行肽质谱指纹图分析和鉴定,在两种细胞中差异表达最为显著的蛋白质有9个。Western Blot证实CK19和P73在5-8F和6-10B表达与蛋白质组结果一致。P73在鼻咽癌放射敏感组和不敏感组中的表达阳性率分别为90%、57.5%,存在显著性差异。结论:放射敏感性不同的鼻咽癌细胞中存在一些差异表达蛋白,这些蛋白可能与鼻咽癌放射敏感性有关,其中P73可能成为放射敏感性预测的侯选标志物。     

Development of a novel proteomic approach for mitochondrial proteomics from cardiac tissue from patients with atrial fibrillation

Atrial fibrillation (AF) is the most common cardiac arrhythmia affecting approximately 2.2 million Americans. Because several studies have suggested that changes in mitochondrial function and morphology may contribute to AF, we developed a novel proteomic workflow focused on the identification of differentially expressed mitochondrial proteins in AF patients. Right human atrial tissue was collected from 20 patients, 10 with and 10 without AF, and the tissue was subjected to hydrostatic pressure cycling-based lysis followed by label-free mass spectrometric (MS) analysis of mitochondrial enriched isolates. Approximately 5% of the 700 proteins identified by MS analysis were differentially expressed between the AF and non-AF samples. We chose four differentially abundant proteins for further verification using reverse phase protein microarray analysis based on their known importance in energy production and regulatory association with atrial ion channels: four and a half LIM, destrin, heat shock protein 2, and chaperonin-containing TCP1. These initial study results provide evidence that a workflow to identify AF-related proteins that combines a powerful upfront tissue cell lysis with high resolution MS for discovery and protein array technology for verification may be an effective strategy for discovering candidate markers in highly fibrous tissue samples.