Semi-quantitative distribution of excitatory amino acid (glutamate) transporters 1-3 (EAAT1-3) and the cystine-glutamate exchanger (xCT) in the adult murine spinal cord.

Proper glutamatergic neurotransmission requires a balance between glutamate release and removal. The removal is mainly catalyzed by the glutamate transporters EAAT1-3, while the glutamate-cystine exchanger (system xc- with specific subunit xCT) represents one of the release mechanisms. Previous studies of the spinal cord have focused on the cellular distribution of EAAT1-3 with special reference to the dorsal horn, but have not provided quantitative data and have not systematically compared multiple segments. Here we have studied the distribution of EAAT1-3 and xCT in sections of multiple spinal cord segments using knockout tissue as negative controls. EAAT2 and EAAT3 were evenly expressed in all gray matter areas at all segmental levels, albeit with slightly higher levels in laminae 1-4 (dorsal horn). Somewhat higher levels of EAAT2 were also seen in lamina 9 (ventral horn), while EAAT3 was also detected in the lateral spinal nucleus. EAAT1 was concentrated in laminae 1-3, lamina 10, the intermediolateral nucleus and the sacral parasympathetic nucleus, while xCT was concentrated in laminae 1-3, lamina 10 and the leptomeninges. The levels of these four transporters were low in white matter, which represents 42% of the spinal cord volume. Quantitative immunoblotting revealed that the average level of EAAT1 in the whole spinal cord was 0.6 ± 0.1% of that in the cerebellum, while the levels of EAAT2, EAAT3 and xCT were, respectively, 41.6 ± 12%, 39.8 ± 7.6%, and 30.8 ± 4.3% of the levels in the hippocampus (mean values ± SEM). Conclusions: Because the hippocampal tissue content of EAAT2 protein is two orders of magnitude higher than the content of the EAAT3, it follows that most of the gray matter in the spinal cord depends almost exclusively on EAAT2 for glutamate removal, while the lamina involved in the processing of autonomic and nociceptive information rely on a complex system of transporters.

Selective deletion of glutamine synthetase in the mouse cerebral cortex induces glial dysfunction and vascular impairment that precede epilepsy and neurodegeneration.

Glutamate-ammonia ligase (glutamine synthetase; Glul) is enriched in astrocytes and serves as the primary enzyme for ammonia detoxification and glutamate inactivation in the brain. Loss of astroglial Glul is reported in hippocampi of epileptic patients, but the mechanism by which Glul deficiency might cause disease remains elusive. Here we created a novel mouse model by selectively deleting Glul in the hippocampus and neocortex. The Glul deficient mice were born without any apparent malformations and behaved unremarkably until postnatal week three. There were reductions in tissue levels of aspartate, glutamate, glutamine and GABA and in mRNA encoding glutamate receptor subunits GRIA1 and GRIN2A as well as in the glutamate transporter proteins EAAT1 and EAAT2. Adult Glul-deficient mice developed progressive neurodegeneration and spontaneous seizures which increased in frequency with age. Importantly, progressive astrogliosis occurred before neurodegeneration and was first noted in astrocytes along cerebral blood vessels. The responses to CO2-provocation were attenuated at four weeks of age and dilated microvessels were observed histologically in sclerotic areas of cKO. Thus, the abnormal glutamate metabolism observed in this model appeared to cause epilepsy by first inducing gliopathy and disrupting the neurovascular coupling.

Genetic association of the cytochrome c oxidase-related genes with Alzheimer's disease in Han Chinese.

Alzheimer's disease (AD) is the most common cause of dementia. Mitochondrial dysfunction has been widely reported in AD due to its important role in cellular metabolism and energy production. Complex IV (cytochrome c oxidase, COX) of mitochondrial electron transport chain, is particularly vulnerable in AD. Defects of COX in AD have been well documented, but there is little evidence to support the genetic association of the COX-related genes with AD. In this study, we investigated the genetic association between 17 nuclear-encoded COX-related genes and AD in 1572 Han Chinese. The whole exons of these genes were also screened in 107 unrelated AD patients with a high probability of hereditarily transmitted AD. Variants in COX6B1, NDUFA4, SURF1, and COX10 were identified to be associated with AD. An integrative analysis with data of eQTL, expression and pathology revealed that most of the COX-related genes were significantly downregulated in AD patients and mouse models, and the AD-associated variants in COX6B1, SURF1, and COX10 were linked to altered mRNA levels in brain tissues. Furthermore, mRNA levels of Ndufa4, Cox5a, Cox10, Cox6b2, Cox7a2, and Lrpprc were significantly correlated with Aß plaque burden in hippocampus of AD mice. Convergent functional genomics analysis revealed strong supportive evidence for the roles of COX6B1, COX10, NDUFA4, and SURF1 in AD. As the result of our comprehensive analysis of the COX-related genes at the genetic, expression, and pathology levels, we have been able to provide a systematic view for understanding the relationships of the COX-related genes in the pathology of AD.

The cystine-glutamate exchanger (xCT, Slc7a11) is expressed in significant concentrations in a subpopulation of astrocytes in the mouse brain.

The cystine-glutamate exchanger (xCT) promotes glutathione synthesis by catalyzing cystine uptake and glutamate release. The released glutamate may modulate normal neural signaling and contribute to excitotoxicity in pathological situations. Uncertainty, however, remains as neither the expression levels nor the distribution of xCT have been unambiguously determined. In fact, xCT has been reported in astrocytes, neurons, oligodendrocytes and microglia, but most of the information derives from cell cultures. Here, we show by immunohistochemistry and by Western blotting that xCT is widely expressed in the central nervous system of both sexes. The labeling specificity was validated using tissue from xCT knockout mice as controls. Astrocytes were selectively labeled, but showed greatly varying labeling intensities. This astroglial heterogeneity resulted in an astrocyte domain-like labeling pattern. Strong xCT labeling was also found in the leptomeninges, along some blood vessels, in selected circumventricular organs and in a subpopulation of tanycytes residing the lateral walls of the ventral third ventricle. Neurons, oligodendrocytes and resting microglia, as well as reactive microglia induced by glutamine synthetase deficiency, were unlabeled. The concentration of xCT protein in hippocampus was compared with that of the EAAT3 glutamate transporter by immunoblotting using a chimeric xCT-EAAT3 protein to normalize xCT and EAAT3 labeling intensities. The immunoblots suggested an xCT/EAAT3 ratio close to one (0.75 ± 0.07; average ± SEM; n = 4) in adult C57BL6 mice. CONCLUSIONS: xCT is present in select blood/brain/CSF interface areas and in an astrocyte subpopulation, in sufficient quantities to support the notion that system xc- provides physiologically relevant transport activity.

Expression of Glutamate Transporters in Mouse Liver, Kidney, and Intestine.

Glutamate transport activities have been identified not only in the brain, but also in the liver, kidney, and intestine. Although glutamate transporter distributions in the central nervous system are fairly well known, there are still uncertainties with respect to the distribution of these transporters in peripheral organs. Quantitative information is mostly lacking, and few of the studies have included genetically modified animals as specificity controls. The present study provides validated qualitative and semi-quantitative data on the excitatory amino acid transporter (EAAT)1-3 subtypes in the mouse liver, kidney, and intestine. In agreement with the current view, we found high EAAT3 protein levels in the brush borders of both the distal small intestine and the renal proximal tubules. Neither EAAT1 nor EAAT2 was detected at significant levels in murine kidney or intestine. In contrast, the liver only expressed EAAT2 (but 2 C-terminal splice variants). EAAT2 was detected in the plasma membranes of perivenous hepatocytes. These cells also expressed glutamine synthetase. Conditional deletion of hepatic EAAT2 did neither lead to overt neurological disturbances nor development of fatty liver.

The OPA1 Gene Mutations Are Frequent in Han Chinese Patients with Suspected Optic Neuropathy.

While many patients with hereditary optic neuropathies are caused by mitochondrial DNA (mtDNA) mutations of Leber's hereditary optic neuropathy (LHON), a significant proportion of them does not have mtDNA mutation and is caused by mutations in genes of the nuclear genome. In this study, we investigated whether the OPA1 gene, which is a pathogenic gene for autosomal dominant optic atrophy (ADOA), is frequently mutated in these patients. We sequenced all 29 exons of the OPA1 gene in 105 Han Chinese patients with suspected LHON. mtDNA copy number was quantified in blood samples from patients with and without OPA1 mutation and compared to healthy controls. In silico program-affiliated prediction, evolutionary conservation analysis, and in vitro cellular assays were performed to show the potential pathogenicity of the mutations. We identified nine OPA1 mutations in eight patients; six of them are located in exons and three are located in splicing sites. Mutation c.1172T > G has not been reported before. When we combined our data with 193 reported Han Chinese patients with optic neuropathy and compared to the available data of 4327 East Asians by the Exome Aggregation Consortium (ExAC), we found a significant enrichment of potentially pathogenic OPA1 mutations in Chinese patients. Cellular assays for OPA1 mutants c.869G > A and c.2708_2711del showed abnormalities in OPA1 isoforms, mitochondrial morphology, and cellular reactive oxygen species (ROS) level. Our results indicated that screening OPA1 mutation is needed for clinical diagnosis of patients with suspected optic neuropathy.

Mitochondrial DNA Haplogroup A Decreases the Risk of Drug Addiction but Conversely Increases the Risk of HIV-1 Infection in Chinese Addicts.

Drug addiction is one of the most serious social problems in the world today and addicts are always at a high risk of acquiring HIV infection. Mitochondrial impairment has been reported in both drug addicts and in HIV patients undergoing treatment. In this study, we aimed to investigate whether mitochondrial DNA (mtDNA) haplogroup could affect the risk of drug addiction and HIV-1 infection in Chinese. We analyzed mtDNA sequence variations of 577 Chinese intravenous drug addicts (289 with HIV-1 infection and 288 without) and compared with 2 control populations (n = 362 and n = 850). We quantified the viral load in HIV-1-infected patients with and without haplogroup A status and investigated the potential effect of haplogroup A defining variants m.4824A > G and m.8794C > T on the cellular reactive oxygen species (ROS) levels by using an allotopic expression assay. mtDNA haplogroup A had a protective effect against drug addiction but appeared to confer an increased risk of HIV infection in addicts. HIV-1-infected addicts with haplogroup A had a trend for a higher viral load, although the mean viral load was similar between carriers of haplogroup A and those with other haplogroup. Hela cells overexpressing allele m.8794 T showed significantly decreased ROS levels as compared to cells with the allele m.8794C (P = 0.03). Our results suggested that mtDNA haplogroup A might protect against drug addiction but increase the risk of HIV-1 infection. The contradictory role of haplogroup A might be caused by an alteration in mitochondrial function due to a particular mtDNA ancestral variant.

Validating GWAS-Identified Risk Loci for Alzheimer's Disease in Han Chinese Populations.

In recent years, genome-wide association studies (GWASs) have identified many novel susceptible genes/loci for Alzheimer's disease (AD). However, most of these studies were conducted in European and populations of European origin, and limited studies have been performed in Han Chinese. In this study, we genotyped 14 single-nucleotide polymorphisms (SNPs) in eight GWAS-reported AD risk genes in 1509 individuals comprising two independent Han Chinese case-control cohorts. Four SNPs (rs11234495, rs592297, rs676733, and rs3851179) in the PICALM gene were significantly associated with late-onset (LO)-AD in populations from Southwest China, whereas SNPs rs744373 (BIN1), rs9331942 (CLU), and rs670139 (MS4A4E) were linked to LO-AD in populations from East China. In the combined Han Chinese population, positive associations were observed between PICALM, CLU, MS4A4E genes, and LO-AD. The association between rs3851179 (PICALM), rs744373 (BIN1), and AD was further confirmed by meta-analysis of Asian populations. Our study verified the association between PICALM, BIN1, CLU, and MS4A4E variants and AD susceptibility in Han Chinese populations. We also discerned some regional differences concerning AD susceptibility SNPs.

Analysis of the complete mitochondrial genome and characterization of diverse NUMTs of Macaca leonina.

As a non-human primate, the pig-tailed macaque has received wide attention because it can be infected by HIV-1. In this study, we determined the complete mtDNA sequence of the northern pig-tailed macaque (Macaca leonina). Unexpectedly, during the amplification of the mtDNA control region (D-loop region) we observed several D-loop-like sequences, which were NUMTs (nuclear mitochondrial sequences) and a total of 14 D-loop-like NUMT haplotypes were later identified in five individuals. The neighbor-joining tree and estimated divergence time based on these D-loop-like NUMT sequences of M. leonina provide some insights into the understanding of the evolutionary history of NUMTs. D-loop-like haplotypes G and H, which also exist in the nuclear genome of mulatta, appear to have been translocated into the nuclear genome before the divergence of M. mulatta and M. leonina. The other D-loop-like NUMT haplotypes were translocated into the nuclear genome of M. leonina after the divergence of the two species. Later sequence conversion was predicted to occur among these 14 D-loop-like NUMT haplotypes. The overall structure of the mtDNA of M. leonina was found to be similar to that seen in other mammalian mitochondrial genomes. Phylogenetic analysis based on the maximum likelihood method shows M. leonina clustered with Macaca silenus among the analyzed mammalian species.

Mitochondrial DNA haplogroup B5 confers genetic susceptibility to Alzheimer's disease in Han Chinese.

Mitochondrial dysfunction has been widely reported in psychiatric and neurodegenerative diseases. We aimed to investigate the association between matrilineal structures of Han Chinese populations and Alzheimer's disease (AD) by a 2-stage case-control study: A total of 341 AD patients and 435 normal individuals from Southwest China were analyzed for mitochondrial DNA sequence variations and were classified into respective haplogroups. A total of 371 AD patients and 470 normal individuals from East China, as validation samples, were genotyped for the variants defining the risk haplogroup. Haplogroup B5 had a significantly higher frequency in AD patients (7.33%) than in control subjects (3.68%) from Southwest China, and we found a similar pattern of higher frequency of B5 in patients in the case-control sample from East China. In the combined population, association of haplogroup B5 with AD risk was strengthened (p = 0.02; odds ratio = 1.74; 95% confidence interval = 1.10-2.76). In lymphoblastoid cell lines belonging to haplogroup B5a, we observed significantly increased reactive oxygen species and decreased mitochondrial mass. Hela cells with stable expression of the MT-ATP6 gene with B5-defining variant m.8584G>A also showed a significantly decreased mitochondrial function. Taken together, our results indicated that haplogroup B5 conferred genetic susceptibility to AD in Han Chinese, and this effect was most likely mediated by ancient variant m.8584G>A. The predisposing effect of B5 to AD is consistent with the ancestral-susceptibility model of complex diseases.

No association of the LRRK2 genetic variants with Alzheimer's disease in Han Chinese individuals.

The leucine-rich repeat kinase-2 (LRRK2) gene has been regarded as 1 of the most common genetic causes of Parkinson's disease (PD). We hypothesized that LRRK2-susceptible allele(s) for PD might pose a risk for Alzheimer's disease (AD). In this study, we screened 12 LRRK2 gene variants in 2 independent cohorts from southwestern China (341 AD patients and 435 normal individuals) and eastern China (297 AD patients and 384 normal individuals), to discern the potential association between this gene and AD. No variant was identified to be associated with AD in either case-control sample. As both of the cohorts were of Han Chinese origin, we combined the LRRK2 variant data for the 2 sample sets together (a total of 638 AD patients and 819 normal individuals) and still found no association between the LRRK2 gene and AD, suggesting that LRRK2 gene variants may not affect the development of AD in Han Chinese individuals.

Mutation p.G83R in the transthyretin gene is associated with hereditary vitreous amyloidosis in Han Chinese families.

PURPOSE: Hereditary vitreous amyloidosis (HVA) is a genetic ophthalmological disorder. The purpose of this study was to investigate whether a mutation in the transthyretin (TTR) gene is associated with HVA in Han Chinese families. METHODS: We performed clinical evaluation of three Han Chinese families with HVA and sequenced the entire exon of the TTR gene in probands and normal individuals from the families. The identified mutation was further genotyped in 196 unrelated healthy controls. Evolutionary conservation analysis and structural prediction were used to infer the potential pathogenicity of the mutation. RESULTS: Clinical penetrance of HVA varied in the three families (11/30 in Family A, 8/83 in Family B, and 7/47 in Family C). A comprehensive medical examination of the patients showed no signs of abnormality except ophthalmologic symptoms, in which floccular turbidity and high echo in both vitreous bodies were observed in all probands. Further histochemical examination of the vitrectomy specimen with Congo red staining identified amyloid deposits. A heterozygous mutation c.307G>C (p.G83R) in exon 3 of the TTR gene was identified in all patients, but not in some unaffected family members. Screening of 196 unrelated normal controls revealed no presence of this mutation. This mutation changed the highly conserved glycine to arginine in the 83(rd) position and altered the tertiary structure of the TTR protein. CONCLUSIONS: Mutation p.G83R in the TTR protein is associated with HVA in Chinese families. The seemingly specific distribution of this mutation in Han Chinese may be used for clinical diagnosis.

Polycyclic aromatic hydrocarbons in surface sediments of the Jialu River.

The Jialu River, an important branch of the Huaihe River in China, was seriously polluted because of rapid economic growth and urbanization. In order to evaluate the potential for serious environmental consequences as a result of anthropogenic contamination, the distribution of polycyclic aromatic hydrocarbons (PAHs) has been investigated in surface sediment samples collected in connection with field surveys of 19 sites along the Jialu River. The total concentration of the 16 USEPA priority PAHs ranged from 466.0 to 2605.6 ng/g dry weight with a mean concentration of 1363.2 ng/g. Sediment samples with the highest PAH concentrations were from the upper reaches of the river, where Zhengzhou City is located; the PAH levels in the middle and lower reaches were relatively low. According to the observed molecular indices, PAHs originated largely from the high-temperature pyrolytic process. According to the numerical effect-based sediment quality guidelines (SQGs) of the United States, the levels of PAHs in the Jialu River should not exert adverse biological effects. The total benzo[a]pyrene toxicity equivalent (TEQ) values calculated for samples varied from 50.4 to 312.8 ng/g dry weight with an average of 167.4 ng/g. The relationships between PAHs and environmental factors, including chemical properties of sediments, water quality, aquatic organisms, hydrological conditions, and anthropogenic activities, are also discussed. PAHs exerted a potential negative impact on the benthos. Settlement percentage, population density and industrial GDP per capita had a significant influence on the distribution of PAHs.