Linghan Gao

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.

Two FSHR variants, haplotypes and meta-analysis in Chinese women with premature ovarian failure and polycystic ovary syndrome

In this study, two polymorphisms of follicle stimulating hormone receptor (FSHR) gene were analysed in the case-control sample using 40 premature ovarian failure (POF) patients, 60 polycystic ovary syndrome (PCOS) patients and 92 healthy controls. All subjects were unrelated Han Chinese from Shanghai. No difference was observed on the allelic or genotypic distribution of FSHR gene polymorphisms between the groups. However, the two-marker haplotypes covering components Thr307Ala (rs6165) G and Asn680Ser (rs6166) A were observed to be significantly associated with PCOS (p=0.007, corrected p=0.042). Meanwhile, a meta-analysis including our study (altogether six POF and eight PCOS studies) showed significant association between rs6166 marker and PCOS (p<0.05). The results suggest that FSH receptor might play a role in genetic susceptibility to PCOS. However, confirmatory studies in independent samples are needed.

Association between the PDE4D gene and ischaemic stroke in the Chinese Han population.

Recent findings suggests that PDE4D (gene encoding phosphodiesterase 4D) is a stroke-related gene in the Icelandic population, but it is still very controversial as to whether it is a susceptible gene for stroke in other populations. In the present study, we attempted to explore the role of the gene in the pathogenesis of stroke in the Chinese Han population of eastern China. A total of 649 ischaemic stroke patients and 761 unrelated control individuals with no history of stroke or transient ischaemic attack were examined in a case-control study. Four SNPs (single nucleotide polymorphisms) rs152312 (C/T), SNP56 (A/T), SNP83 (C/T) and SNP87 (C/T) with a minor allele frequency over 5% were genotyped and the corresponding haplotypes were constructed. In an analysis of the combined cardiogenic and carotid stroke group, both the allele (P=0.0060) and genotype (P=0.0160) frequencies between cases and controls at SNP83 showed significant differences. However, no difference in haplotype frequencies was observed between cases and controls at rs152312 and SNP56. In the analysis of the small-artery-occlusive stroke group, no difference in allele or genotype frequencies was observed at any marker between cases and controls; the global haplotype frequency in rs152312 and SNP56 had a significant difference between cases and controls (P=0.0162); the frequency of haplotype C-A was higher in cases than in controls (P=0.0122). In conclusion, our present findings show that polymorphisms in the PDE4D gene are associated with an increased risk of ischaemic stroke in the Chinese Han population. The present study adds further support to the role of PDE4D in stroke.

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 R2X = 0.441, Q2Y = 0.413 and R2X = 0.698, Q2Y = 0.677, 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.

No association between EGR gene family polymorphisms and schizophrenia in the Chinese population

Early growth response (EGR) genes are thought to have a role in the pathogenesis of schizophrenia because of their conserved DNA binding domain and biologically activity in neuronal plasticity. This zinc-finger motif could influence gene post-translational modification and expression. The multigenetic association model, using markers in genes of similar or antagonistic biological effects within a signal pathway or gene family, might be more appropriate to this aspect of the schizophrenia hypothesis than the single gene strategy. In this study we investigated the role of EGR1, EGR2, EGR3 and EGR4 within the EGR family. Taqman technology was used to examine 12 single nucleotide polymorphisms (SNPs) covering these four genes in 2044 Chinese Han subjects. Case-control analyses were performed to detect association of these 4 genes with schizophrenia and multifactor dimensionality reduction (MDR) analysis was employed to examine their potential gene-gene interaction in schizophrenia. Neither allelic nor genotypic single-locus tests revealed any significant association between EGR1-4 and the risk of schizophrenia nor was any such association found with regard to interaction within EGR1-4 (p(min)=0.623, CV Consistency=10/10). We concluded that although multiple candidate genes are involved in schizophrenogenic development, the EGR family may not play a major role in schizophrenia susceptibility in the Chinese Han population.

Indian hedgehog mutations causing brachydactyly type A1 impair Hedgehog signal transduction at multiple levels.

Brachydactyly type A1 (BDA1), the first recorded Mendelian autosomal dominant disorder in humans, is characterized by a shortening or absence of the middle phalanges. Heterozygous missense mutations in the Indian Hedgehog (IHH) gene have been identified as a cause of BDA1; however, the biochemical consequences of these mutations are unclear. In this paper, we analyzed three BDA1 mutations (E95K, D100E, and E131K) in the N-terminal fragment of Indian Hedgehog (IhhN). Structural analysis showed that the E95K mutation changes a negatively charged area to a positively charged area in a calcium-binding groove, and that the D100E mutation changes the local tertiary structure. Furthermore, we showed that the E95K and D100E mutations led to a temperature-sensitive and calcium-dependent instability of IhhN, which might contribute to an enhanced intracellular degradation of the mutant proteins via the lysosome. Notably, all three mutations affected Hh binding to the receptor Patched1 (PTC1), reducing its capacity to induce cellular differentiation. We propose that these are common features of the mutations that cause BDA1, affecting the Hh tertiary structure, intracellular fate, binding to the receptor/partners, and binding to extracellular components. The combination of these features alters signaling capacity and range, but the impact is likely to be variable and mutation-dependent. The potential variation in the signaling range is characterized by an enhanced interaction with heparan sulfate for IHH with the E95K mutation, but not the E131K mutation. Taken together, our results suggest that these IHH mutations affect Hh signaling at multiple levels, causing abnormal bone development and abnormal digit formation.

Analysis of ischemic neuronal injury in Cav2.1 channel α1 subunit mutant mice.

One of the main instigators leading to cell death and brain damage following ischemia is Ca(2+) dysregulation. Neuronal membrane depolarization results in the activation of voltage-gated Ca(2+) (CaV) channels and intracellular Ca(2+) influx. We investigated the physiological role of the CaV2.1 (P/Q-type) channel in ischemic neuronal injury using CaV2.1 channel α1 subunit mutant mice, rolling Nagoya and leaner mice. The in vivo ischemia model with a complete occlusion of the middle cerebral artery showed that the infarct area at 24h was significantly smaller in rolling Nagoya (27.1±3.5% of total brain volume) and leaner (20.1±3.5%) mice compared to wild-type (42.9±4.5%) mice. In an in vitro Ca(2+) imaging study, oxygen-glucose deprivation using a hippocampal slice induced a significantly slower rate of increase in intracellular Ca(2+) concentration ([Ca(2+)]i) in rolling Nagoya (0.083±0.007/min) and leaner (0.062±0.006/min) mice compared to wild-type (0.105±0.008/min) mice. These results demonstrate that the mutant CaV2.1 channel in rolling Nagoya and leaner mice plays a different protective role in a ([Ca(2+)]i)-dependent manner in ischemic models and indicate that CaV2.1 channel blockers may be used preventively against ischemic injury.

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.

Identification of Duplication Downstream of BMP2 in a Chinese Family with Brachydactyly Type A2 (BDA2)

Brachydactyly type A2 (BDA2, MIM 112600) is characterized by the deviation and shortening of the middle phalange of the index finger and the second toe. Using genome-wide linkage analysis in a Chinese BDA2 family, we mapped the maximum candidate interval of BDA2 to a ∼1.5 Mb region between D20S194 and D20S115 within chromosome 20p12.3 and found that the pairwise logarithm of the odds score was highest for marker D20S156 (Zmax = 6.09 at θ = 0). Based on functional and positional perspectives, the bone morphogenetic protein 2 (BMP2) gene was identified as the causal gene for BDA2 in this region, even though no point mutation was detected in BMP2. Through further investigation, we identified a 4,671 bp (Chr20: 6,809,218–6,813,888) genomic duplication downstream of BMP2. This duplication was located within the linked region, co-segregated with the BDA2 phenotype in this family, and was not found in the unaffected family members and the unrelated control individuals. Compared with the previously reported duplications, the duplication in this family has a different breakpoint flanked by the microhomologous sequence GATCA and a slightly different length. Some other microhomologous nucleotides were also found in the duplicated region. In summary, our findings support the conclusions that BMP2 is the causing gene for BDA2, that the genomic location corresponding to the duplication region is prone to structural changes associated with malformation of the digits, and that this tendency is probably caused by the abundance of microhomologous sequences in the region.

No association between the KCNH1, KCNJ10 and KCNN3 genes and schizophrenia in the Han Chinese population.

Schizophrenia is a common severe mental illness affecting 0.3-2.0% of the worlds population. The potassium channels are thought to have a role in modulating electrical excitability in neurons, regulating calcium signaling in oligodendrocytes and regulating action potential duration in presynaptic terminals and GABA release. Previous studies have reported that some potassium channel genes might be candidate genes for susceptibility to schizophrenia. In the present study, we chose three potassium channel genes, KCNH1, KCNJ10, KCNN3 to investigate the role of potassium channels in schizophrenia by genotyping 23 SNPs (9 in KCNH1, 5 in KCNJ10 and 9 in KCNN3) in a Han Chinese sample consisting of 893 schizophrenia patients and 611 healthy controls. No significant difference in allelic or genotypic frequency was revealed between schizophrenia patients and healthy individuals. Nor was a significant difference in haplotypic distribution detected. MDR analysis revealed no gene-gene interaction within the three potassium channel genes. Our study suggests that the 23 SNPs within the three potassium genes we examined do not play a major role in schizophrenia in the Han Chinese population.