Hui Zhu

A Comparative Proteomics Analysis of Rat Mitochondria from the Cerebral Cortex and Hippocampus in Response to Antipsychotic Medications

An increasing number of experiments have found anomalies in mitochondria in the brains of psychotics, which suggests that mitochondrial dysfunction or abnormal cerebral energy metabolism might play an important role in the pathophysiology of schizophrenia (SCZ). We adopted a proteomic approach to identify the differential effects on the cerebral cortex and hippocampus mitochondrial protein expression of Sprague-Dawley (SD) rats by comparing exposure to typical and atypical antipsychotic medications. Differential mitochondrial protein expressions were assessed using two-dimensional (2D) gel electrophoresis for three groups with Chlorpromazine (CPZ), Clozapine (CLZ), quetiapine (QTP) and a control group. A total of 14 proteins, of which 6 belong to the respiratory electron transport chain (ETC) of oxidative phosphorylation (OXPHOS), showed significant changes in quantity including NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 10 (Ndufa10), NADH dehydrogenase (ubiquinone) flavoprotein 2 (Ndufv2), NADH dehydrogenase (ubiquinone) Fe-S protein 3 (Ndufs3), F1-ATPase beta subunit (Atp5b), ATPase, H+ transporting, lysosomal, beta 56/58 kDa, isoform 2 (Atp6v1b2) and ATPase, H+ transporting, V1 subunit A, isoform 1 (Atp6v1a1). The differential proteins subjected to 2D were assessed for levels of mRNA using quantitative real time PCR (Q-RT-PCR), and we also made partial use of Western blotting for assessing differential expression. The results of our study may help to explain variations in SD rats as well as in human response to antipsychotic drugs. In addition, they should improve our understanding of both the curative effects and side effects of antipsychotics and encourage new directions in SCZ research.

Abnormal changes of plasma acute phase proteins in schizophrenia and the relation between schizophrenia and haptoglobin (Hp) gene.

Summary.In this study we focused on detecting schizophrenia related changes of plasma proteins using proteomic technology and examining the relation between schizophrenia and haptoglobin (Hp) genotype. We investigated plasma proteins from schizophrenic subjects (n = 42) and healthy controls (n = 46) by two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry. To further reveal the genetic relationship between acute phase proteins (APPs) and schizophrenia disease, we tested Hp α1/Hp α2 (Hp 1/2) polymorphism and two single nucleotide polymorphisms (SNPs) of Hp, rs2070937 and rs5473, for associations with schizophrenia in the Chinese Han population. With the relatively high number of samples for 2-DE work, we found that four proteins in the family of positive APPs were all up-regulated in patients. In genetic association study, we found significant associations existing between schizophrenia and Hp polymorphisms, Hp 1/2 and rs2070937 variants. Schizophrenia is accompanied by both an altered expression of Hp protein and a different genotype distribution of Hp gene, demonstrating that Hp is associated with schizophrenia. The results from proteomic and genomic aspects both indicate that acute phase reaction is likely to be an aetiological agent in the pathophysiology of schizophrenia, but not just an accompanying symptom. The positive APPs are schizophrenic related proteins, with the highly concordant results on four positive APPs.

Decreased levels of apolipoprotein A-I in plasma of schizophrenic patients

SummaryThis study aims to identify the effects of antipsychotics on plasma proteins, and on the proteins associated with schizophrenia. We applied proteomics technology to screen protein aberrations in Sprague-Dawley rats treated with antipsychotics and schizophrenic patients undergoing medication. ApoA-I was found significantly increased in the chlorpromazine-treated rats and decreased in the patients with treatment-resistant schizophrenia, which suggest that decreased levels of apoA-I might be associated with the pathology of schizophrenia and that chlorpromazine increases apoA-I levels as part of its therapeutic action.

Hippocampus protein profiling reveals aberration of malate dehydrogenase in chlorpromazine/clozapine treated rats

Chlorpromazine and clozapine are antipsychotic agents used to relieve symptoms of early-diagnosed schizophrenia. In this study, we used proteomics technology to screen the protein aberration in Sprague-Dawley rats treated with these two antipsychotic agents. Our goal was to identify the effects of antipsychotics on hippocampal proteins in rats. Protein expression profiles were compared in each experimental group using two-dimensional gel electrophoresis and identified using matrix-assisted laser desorption/ionisation time of flight mass spectrometry. Malate dehydrogenase, peroxiredoxin 3, vacuolar ATP synthase subunit beta and mitogen-activated protein kinase kinase 1 were found to have altered expression levels in the groups treated with antipsychotics compared with the matched controls. These findings should contribute to identifying new targets for disease preventing pharmacological agents and be beneficial for the development of more effective agents.

NMDA Receptor Hypofunction Induces Dysfunctions of Energy Metabolism And Semaphorin Signaling in Rats: A Synaptic Proteome Study

There is considerable evidence to suggest that aberrations of synapse connectivity contribute to the pathophysiology of schizophrenia and that N-methyl-D-aspartate (NMDA) receptor-mediated glutamate transmission is especially important. Administration of MK-801 ([+]-5-methyl-10, 11-dihydro-5H-dibenzo-[a, d]-cycloheptene-5, 10-iminehydrogenmaleate) induces hypofunction of NMDA receptors in rats, which are widely used as a model for schizophrenia. We investigated synaptosomal proteome expression profiling of the cerebral cortex of MK-801-treated Sprague-Dawley rats using the 2-dimensional difference gel electrophoresis method, and 49 differentially expression proteins were successfully identified using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight/Time-of-Flight mass spectrometry. We carried out a literature search for further confirmation of subsynaptic locations and to explore the relevance to the diseases of differentially expressed proteins. Ingenuity Pathways Analysis (IPA) was used to further examine the underlying relationship between the changed proteins. The network encompassing cell morphology, cell-to-cell signaling and interaction, nervous system development and function was found to be significantly altered in the MK-801-treated rats. Energy metabolism and semaphorin signaling in neurons are the most significant IPA canonical pathways to be affected by MK-801 treatment. Using western blots, we confirmed the differential expression of Camk2a, Crmp2, Crmp5, Dnm1, and Ndufs3 in both synaptosome proteins and total proteins in the cerebral cortex of the rats. Our study identified the change and/or response of the central nervous transmission system under the stress of NMDA hypofunction, underlining the importance of the synaptic function in schizophrenia.

Metabolomic Analysis Reveals Metabolic Disturbance in the Cortex and Hippocampus of Subchronic MK-801 Treated Rats

Background Although a number of proteins and genes relevant to schizophrenia have been identified in recent years, few are known about the exact metabolic pathway involved in this disease. Our previous proteomic study has revealed the energy metabolism abnormality in subchronic MK-801 treated rat, a well-established animal model for schizophrenia. This prompted us to further investigate metabolite levels in the same rat model to better delineate the metabolism dysfunctions and provide insights into the pathology of schizophrenia. Methods Metabolomics, a high-throughput investigatory strategy developed in recent years, can offer comprehensive metabolite-level insights that complement protein and genetic findings. In this study, we employed a nondestructive metabolomic approach (1H-MAS-NMR) to investigate the metabolic traits in cortex and hippocampus of MK-801 treated rats. Multivariate statistics and ingenuity pathways analyses (IPA) were applied in data processing. The result was further integrated with our previous proteomic findings by IPA analysis to obtain a systematic view on our observations. Results Clear distinctions between the MK-801 treated group and the control group in both cortex and hippocampus were found by OPLS-DA models (with R2Xu200a=u200a0.441, Q2Yu200a=u200a0.413 and R2Xu200a=u200a0.698, Q2Yu200a=u200a0.677, respectively). The change of a series of metabolites accounted for the separation, such as glutamate, glutamine, citrate and succinate. Most of these metabolites fell in a pathway characterized by down-regulated glutamate synthesis and disturbed Krebs cycle. IPA analysis further confirmed the involvement of energy metabolism abnormality induced by MK-801 treatment. Conclusions Our metabolomics findings reveal systematic changes in pathways of glutamate metabolism and Krebs cycle in the MK-801 treated rats’ cortex and hippocampus, which confirmed and improved our previous proteomic observation and served as a valuable reference to the etiology research of schizophrenia.

Positive association between ALDH1A2 and schizophrenia in the Chinese population

Vitamin A (retinol), in the biologically active form of retinoic acid (RA), has been proposed as involved in the pathogenesis of schizophrenia. We hypothesized that genetic basis of genes encoding RA metabolism enzymes, which control the cellular RA level, might be associated with this disease. This cascade genetic association model, using markers in genes of synthesis and degradation enzymes within the retinoid cascade, would better fit the biological character of the retinoid hypothesis than the single gene strategy. In the present study we chose to investigate 7 genes involved in the synthesis, degradation and transportation of RA, ALDH1A1, ALDH1A2, ALDH1A3, CYP26A1, CYP26B1, CYP26C1 and Transthyretin (TTR), for their roles in the development of schizophrenia. We genotyped 18 single nucleotide polymorphisms (SNPs) in the regulatory and coding regions of these 7 genes using LDR technology in the 617 Chinese Han subjects. Case-control analyses were performed to detect association of these 7 genes with schizophrenia. Association analyses using both allelic and genotypic single-locus tests revealed no significant association between the risk for each of investigated gene and schizophrenia. However, analyses of multiple-locus haplotypes indicated that the overall frequency of rs4646642-rs4646580 of ALDH1A2 gene showed significant difference between patients and control subjects (p=0.0055). We also employed multifactor dimensionality reduction method to detect multilocus effects. In summary, in this work we show multiple candidate genes involved in retinoid cascade in schizophrenics. In addition, our results suggest a positive association between ALDH1A2 and schizophrenics in the Chinese population and support the retinoid hypothesis of schizophrenia.

Differential expression profiling of the synaptosome proteome in a rat model of antipsychotic resistance

This study used a comparative proteomics approach to identify the effects of the antipsychotic drugs, Chlorpromazine (CPZ), Clozapine (CLZ), and Quetiapine (QTP) on the synaptosomal protein of the cerebral cortex of Sprague-Dawley (SD) rats. The multivariate statistical test partial least squares-discriminant analysis (PLS-DA) was applied to build the models for screening out the variable important plot (VIP). The PLS-DA models were able to distinguish each drug treatment group and the control group; more importantly, the univariate differentially expressed protein spots were capable of being verified by the VIP of the models. The interrelationships among the identified proteins were analyzed using Pearsons correlation analysis and pathway analysis. Through the synaptosome proteome experiments, we established that the energy production of the mitochondrial function and Glycolysis/Gluconeogenesis were involved in the response to antipsychotic medications. Furthermore, the G protein-coupled signal transduction system was also inhibited by antipsychotic medications. The result of our study should contribute to the understanding of the effects of antipsychotic drugs on synaptic function.

Proteome alterations of cortex and hippocampus tissues in mice subjected to vitamin A depletion.

Vitamin A regulates the development and maintenance of the central nervous system. Studies of vitamin A depletion (VAD) and mutations of retinoid receptors in rodents have revealed a dysfunction of motor and cognitive abilities. However, the molecular mechanisms underlying these behavioral changes are not well understood. In this study, VAD mice were examined and abnormal motor behavior related to psychosis symptoms was found. With the use of two-dimensional gel electrophoresis (2-DE), two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mass spectrometric (MS) technologies, 44 and 23 altered protein spots were identified in the cortex and hippocampus, respectively, in VAD mice. By Western blot, the up-regulation of mitogen-activated protein kinase 1 (MAPK1) and proteasome subunit beta type 2 (PSMB2) in the cortex and that of dihydropyrimidinase-related protein 2 (DPYSL2) and PSMB2 in the hippocampus were observed in VAD mice. Bioinformatic analysis using DAVID revealed that altered proteins induced by VAD showed significant enrichment of (i) glycolysis, cytoskeleton, mitochondrion and glutamate metabolism in the cortex; and (ii) actin binding, dopamine receptor signaling and transmission of nerve impulse in the hippocampus. The up-regulations of DPYSL2, MAPK1 and PSMB2 may indicate the activated neuronal defensive mechanism in VAD brain regions, which may underlie the VAD-related psychosis behavior.

Proteome alteration of U251 human astrocytoma cell after inhibiting retinoic acid synthesis

Retinoic acid (Ra) is crucial for the patterning and neuronal differentiation in the central nervous system (CNS). Ra deficiency in animals disrupts the motor activities and memory abilities. The molecular mechanisms underlying these behavior abnormalities remain largely unknown. In the current study, we treated the astrocytoma cells with citral, an inhibitor of Ra synthesis. We analyzed the differences in the protein concentrations between the treated and untreated astrocytoma cells by two-dimensional gel electrophoresis (2-DE), Imagemaster software, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In total, 39 of 46 altered protein spots with significant mascot scores were identified representing 36 proteins, that were involved in significantly altered glutamate metabolism, lipid metabolism, mitochrondrial function, and oxidative stress response by Ingenuity Pathway Analysis (IPA). Altered 3-phosphoglycerate dehydrogenase (PHGDH) was also observed in western blot. These data provide some clues for explaining the behavioral changes caused by Ra deficiency, and support the hypothesis that Ra signaling is associated with some symptoms of neurodegenerative disorders and schizophrenia.