Metabolomics and cognition in African American adults in midlife: the atherosclerosis risk in communities study.

Clinical studies have shown alterations in metabolic profiles when patients with mild cognitive impairment and Alzheimer's disease dementia were compared to cognitively normal subjects. Associations between 204 serum metabolites measured at baseline (1987-1989) and cognitive change were investigated in 1035 middle-aged community-dwelling African American participants in the biracial Atherosclerosis Risk in Communities (ARIC) Study. Cognition was evaluated using the Delayed Word Recall Test (DWRT; verbal memory), the Digit Symbol Substitution Test (DSST; processing speed) and the Word Fluency Test (WFT; verbal fluency) at visits 2 (1990-1992) and 4 (1996-1998). In addition, Cox regression was used to analyze the metabolites as predictors of incident hospitalized dementia between baseline and 2011. There were 141 cases among 1534 participants over a median 17.1-year follow-up period. After adjustment for established risk factors, one standard deviation increase in N-acetyl-1-methylhistidine was significantly associated with greater 6-year change in DWRT scores (ß=-0.66 words; P=3.65 × 10-4). Two metabolites (one unnamed and a long-chain omega-6 polyunsaturated fatty acid found in vegetable oils (docosapentaenoate (DPA, 22:5 n-6)) were significantly associated with less decline on the DSST (DPA: ß=1.25 digit-symbol pairs, P=9.47 × 10-5). Two unnamed compounds and three sex steroid hormones were associated with an increased risk of dementia (all P<3.9 × 10-4). The association of 4-androstene-3beta, 17beta-diol disulfate 1 with dementia was replicated in European Americans. These results demonstrate that screening the metabolome in midlife can detect biologically plausible biomarkers that may improve risk stratification for cognitive impairment at older ages.

GWAS for executive function and processing speed suggests involvement of the CADM2 gene.

To identify common variants contributing to normal variation in two specific domains of cognitive functioning, we conducted a genome-wide association study (GWAS) of executive functioning and information processing speed in non-demented older adults from the CHARGE (Cohorts for Heart and Aging Research in Genomic Epidemiology) consortium. Neuropsychological testing was available for 5429-32,070 subjects of European ancestry aged 45 years or older, free of dementia and clinical stroke at the time of cognitive testing from 20 cohorts in the discovery phase. We analyzed performance on the Trail Making Test parts A and B, the Letter Digit Substitution Test (LDST), the Digit Symbol Substitution Task (DSST), semantic and phonemic fluency tests, and the Stroop Color and Word Test. Replication was sought in 1311-21860 subjects from 20 independent cohorts. A significant association was observed in the discovery cohorts for the single-nucleotide polymorphism (SNP) rs17518584 (discovery P-value=3.12 × 10(-8)) and in the joint discovery and replication meta-analysis (P-value=3.28 × 10(-9) after adjustment for age, gender and education) in an intron of the gene cell adhesion molecule 2 (CADM2) for performance on the LDST/DSST. Rs17518584 is located about 170 kb upstream of the transcription start site of the major transcript for the CADM2 gene, but is within an intron of a variant transcript that includes an alternative first exon. The variant is associated with expression of CADM2 in the cingulate cortex (P-value=4 × 10(-4)). The protein encoded by CADM2 is involved in glutamate signaling (P-value=7.22 × 10(-15)), gamma-aminobutyric acid (GABA) transport (P-value=1.36 × 10(-11)) and neuron cell-cell adhesion (P-value=1.48 × 10(-13)). Our findings suggest that genetic variation in the CADM2 gene is associated with individual differences in information processing speed.

Genetic contributions to variation in general cognitive function: a meta-analysis of genome-wide association studies in the CHARGE consortium (N=53949).

General cognitive function is substantially heritable across the human life course from adolescence to old age. We investigated the genetic contribution to variation in this important, health- and well-being-related trait in middle-aged and older adults. We conducted a meta-analysis of genome-wide association studies of 31 cohorts (N=53,949) in which the participants had undertaken multiple, diverse cognitive tests. A general cognitive function phenotype was tested for, and created in each cohort by principal component analysis. We report 13 genome-wide significant single-nucleotide polymorphism (SNP) associations in three genomic regions, 6q16.1, 14q12 and 19q13.32 (best SNP and closest gene, respectively: rs10457441, P=3.93 × 10(-9), MIR2113; rs17522122, P=2.55 × 10(-8), AKAP6; rs10119, P=5.67 × 10(-9), APOE/TOMM40). We report one gene-based significant association with the HMGN1 gene located on chromosome 21 (P=1 × 10(-6)). These genes have previously been associated with neuropsychiatric phenotypes. Meta-analysis results are consistent with a polygenic model of inheritance. To estimate SNP-based heritability, the genome-wide complex trait analysis procedure was applied to two large cohorts, the Atherosclerosis Risk in Communities Study (N=6617) and the Health and Retirement Study (N=5976). The proportion of phenotypic variation accounted for by all genotyped common SNPs was 29% (s.e.=5%) and 28% (s.e.=7%), respectively. Using polygenic prediction analysis, ~1.2% of the variance in general cognitive function was predicted in the Generation Scotland cohort (N=5487; P=1.5 × 10(-17)). In hypothesis-driven tests, there was significant association between general cognitive function and four genes previously associated with Alzheimer's disease: TOMM40, APOE, ABCG1 and MEF2C.

Mitochondrial toxin 3-nitropropionic acid induces cardiac and neurotoxicity differentially in mice.

We investigated the effects of 3-nitropropionic acid (3NPA), a previously characterized neurotoxin, in four strains of mice to better understand the molecular basis of variable host responses to this agent. Unexpectedly, we found significant cardiac toxicity that always accompanied the neurotoxicity in all strains of mice in acute and subacute/chronic toxicity testing. Caudate putamen infarction never occurred without cardiac toxicity. All mouse strains tested are sensitive to 3NPA although the C57BL/6 and BALB/c mice require more exposure than 129SVEMS and FVB/n mice. Cardiac toxicity alone was found in 50% of symptomatic mice tested and morphologically, the cardiac toxicity is characterized by diffuse swelling of cardiomyocytes and multifocal coagulative contraction band necrosis. In subacute to chronic exposure, atrial thrombosis, cardiac mineralization, cell loss, and fibrosis are combined with cardiomyocyte swelling and necrosis. Ultrastructurally, mitochondrial swelling occurs initially, followed by disruption of myofilaments. Biochemically, isolated heart mitochondria from the highly sensitive 129SVEMS mice have a significant reduction of succinate dehydrogenase activity, succinate oxygen consumption rates, and heart adenosine triphosphate after 3NPA treatment. The severity of morphological changes parallels the biochemical alterations caused by 3NPA, consistent with cardiac toxicity being a consequence of the effects of 3NPA on succinate dehydrogenase. These experiments show, for the first time, that 3NPA has important cardiotoxic effects as well as neurotoxic effects, and that cardiac toxicity possibly resulting from inhibition of the succinate dehydrogenase in heart mitochondria, contributes to the cause of death in 3NPA poisoning in acute and subacute/chronic studies in mice.

Involvement of DMT1 in uptake of Cd in MDCK cells: role of protein kinase C.

The involvement of iron (Fe) transporters in the uptake of cadmium (Cd) was examined in Madin-Darby kidney cells (MDCK). The uptake of Cd displayed properties that are associated with the Fe transporter divalent metal transporter 1 (DMT1). For example, the uptake of Cd and Fe was reduced by altering the cell membrane potential. The uptake of Cd was blocked by Fe, and the uptake of Fe was blocked by Cd. Also, the uptake of Cd and Fe was higher in MDCK cells bathed in a buffer at low pH. Increased uptake of Fe and Cd was observed in the HEK-293 cell line overexpressing DMT1. Overnight treatment of MDCK cells with the protein kinase C activator phorbol 12,13-dibutyrate (PDBu) resulted in increased uptake of Cd and Fe and an increase in DMT1 mRNA. An increase in newly transcribed DMT1 mRNA was not observed, suggesting that PDBu does not increase DMT1 mRNA by activating transcription. Rather, the increase was most likely due to greater stability of DMT1 mRNA, because the rate of degradation of DMT1 mRNA was slower in MDCK cells treated with PDBu. Our results suggest that Fe and Cd are transported in MDCK cells by a transporter with biochemical properties similar to those of DMT1.

Associations of lead exposure and dose measures with erythrocyte protein kinase C activity in 212 current Korean lead workers.

Lead can replace calcium in enzyme assays that measure protein kinase C activity and lead activates protein kinase C in human erythrocytes after exposure to lead in vitro. To examine the relevance of these observations to lead exposure in humans, we studied the associations of lead found in blood or tibia with activation of protein kinase C in erythrocytes isolated from workers in the lead industry. We examined erythrocytes among 212 lead workers, with a mean (+/-SD) age of 39.1 (10.0) years and exposure duration of 8.1 (6.5) years and measured protein kinase C activation by an in vitro back-phosphorylation assay. After adjustment for potential confounding factors (age and sex), tibia lead and exposure duration were significantly associated with erythrocyte protein kinase C activation (both p values < 0.05). No associations were observed between protein kinase C activation and blood-lead or zinc-protoporphyrin levels. These findings suggest that human exposure to lead results in activation of erythrocyte protein kinase C, which may be directly relevant to the neurotoxicity of lead.

The SNRPN promoter is not required for genomic imprinting of the Prader-Willi/Angelman domain in mice.

In mice and humans, the locus encoding the gene for small nuclear ribonucleoprotein N (SNRPN/Snrpn), as well as other loci in the region are subject to genomic imprinting. The SNRPN promoter is embedded in a maternally methylated CpG island, is expressed only from the paternal chromosome and lies within an imprinting center that is required for switching to and/or maintenance of the paternal epigenotype. We show here that a 0.9-kb deletion of exon 1 of mouse Snrpn did not disrupt imprinting or elicit any obvious phenotype, although it did allow the detection of previously unknown upstream exons. In contrast, a larger, overlapping 4.8-kb deletion caused a partial or mosaic imprinting defect and perinatal lethality when paternally inherited.

Relation of alleles of the sodium-potassium adenosine triphosphatase alpha 2 gene with blood pressure and lead exposure.

Lead is associated with elevated blood pressure, although the mechanism of action is unknown. Genetic differences in sodium-potassium adenosine triphosphatase (Na(+)-K(+)ATPase) could explain some of the variation in the strength of the blood pressure-blood lead relation that has been observed in previous studies. In 1996-1997, the authors studied the association of blood pressure, hypertension prevalence, and polymorphisms in the gene for the alpha 2 subunit of Na(+)-K(+)ATPase (ATP1A2) among 220 former organolead manufacturing workers from New Jersey. Subjects were genotyped for a restriction fragment length polymorphism (RFLP) on the ATP1A2 gene. The association between blood lead and blood pressure was stronger among persons who were homozygous for the variant allele. Genotype was also associated with hypertension (adjusted odds ratio = 7.7; 95% confidence interval: 1.9, 31.4). Finally, the variant allele was 1.8 times more common among African Americans than among Caucasians. The RFLP may indicate susceptibility to the effect of lead on blood pressure. Moreover, the alpha 2 gene (or a closely linked gene) may contribute to the pathophysiology of hypertension. However, because the number of subjects (especially African Americans) with the susceptible genotype in this study was small, these observations should be considered preliminary.

Maitotoxin stimulates Cd influx in Madin-Darby kidney cells by activating Ca-permeable cation channels.

This study examined the role of calcium channels for the uptake of cadmium (Cd) into Madin-Darby canine kidney (MDCK) cells. Maitotoxin, an activator of different types of calcium channels, increased accumulation of 109Cd and 45Ca in MDCK cells. We found that maitotoxin increased accumulation by stimulating 109Cd influx because it did not affect efflux. An inhibitor of store-operated Ca channels, SKF96365, partially blocked 45Ca influx but did not affect 109Cd influx. Ni and Mn, and loperamide and proadifen (SKF 525a), inhibited 45Ca and 109Cd influx in cells stimulated with maitotoxin, but La and nifedipine did not. Overnight treatment with phorbol 12, 13-ibutyrate (PDBu) to activate protein kinase C resulted in a decrease in the concentration of maitotoxin needed to stimulate 45Ca and 109Cd influx. The effect of PDBu was blocked by treating cells with the protein kinase C inhibitor GF109203X. Additionally, the effect of PDBu was lost in cells treated with an inhibitor of RNA synthesis actinomycin D. These results suggest that a Ca permeable cation channel different from voltage-dependent and store-operated Ca channels mediates the uptake of Cd in MDCK cells. The expression of this channel is regulated by protein kinase C.

Regulation and developmental expression of the divalent metal-ion transporter in the rat brain.

Divalent metal ion transporter 1 (DMT1) is a recently identified metal-ion transporter that appears to mediate the absorption of iron in the intestine. DMT1 mRNA is also present in discrete areas of the brain. In this study, we examined the expression of DMT1 mRNA in developing rat brain. DMT1 mRNA was found by in situ hybridization in the striatum, cortex, hippocampus and cerebellum. During development, DMT1 mRNA was found in Purkinje and granule cells in the cerebellum at post-natal day (PND) 14 and PND 30. DMT1 mRNA was also expressed in the external granular layer of the cerebellum at PND 14. No change in the level of DMT1 mRNA was observed by Northern analysis in the cerebellum at different ages between PND 1 and 21. DMT1 was found by Northern analysis in cultures of rat astrocytes. Activation of protein kinase C increased the expression of DMT1 in kidney epithelial cells but not astrocytes from newborn rats. Because DMT1 is expressed in a wide variety of types of cells, we suggest that it plays an important role in metal homeostasis in the brain.

The role of anion exchange in the uptake of Pb by human erythrocytes and Madin-Darby canine kidney cells.

Anion exchange (AE) plays a critical role in regulating intracellular pH in erythrocytes and epithelial cells and has been suggested to facilitate the transport of lead (Pb) across the erythrocyte cell membrane. In this study we examined the role of AE in the uptake of Pb by human erythrocytes and by Madin-Darby canine kidney (MDCK) cells, the kidney epithelial cell line. Functional AE in MDCK cells was evidenced by: increased uptake of SO(4)(2-) at pH 6.0 over pH 7.0, and inhibition of SO(4)(2-) uptake by the AE inhibitor 4, 4'-diisothiocyanostilbene-2, 2'- disulfonic acid (DIDS) as well as by non-halide anions. Accumulation of Pb into MDCK cells was time and temperature dependent. DIDS inhibited uptake of Pb into human erythrocytes but not MDCK cells. In conclusion, uptake of Pb into erythrocytes but not kidney epithelial cells occurs through AE.

Immediate early gene expression in PC12 cells exposed to lead: requirement for protein kinase C.

We previously demonstrated induction of c-fos mRNA in PC12 cells exposed to lead that was dependent on new transcription. In the current work, we examined two signal transduction mechanisms that are activated by lead and have been shown to mediate induction of c-fos mRNA. One mechanism involves protein kinase C, and the other requires calmodulin-dependent protein kinase II. Significant increases in the levels of c-fos, c-jun, and egr-1 but not NGFIB mRNA were observed in PC12 cells exposed to lead or phorbol 12-myristate 13-acetate. In contrast, PC12 cells depolarized with 56 mM K+ displayed an increase in c-fos, egr-1, and NGFIB but not c-jun mRNA. Similar to other activators of protein kinase C, lead increased AP-1 and Egr-1 DNA binding activity. Additionally, lead increased luciferase activity in cerebellar granule cells transfected with an AP-1 luciferase reporter construct. Lead did not increase c-fos mRNA in PC12 cells that were depleted of protein kinase C by a 24-h treatment with phorbol 12,13-dibutyrate or incubated with the protein kinase C inhibitor H-7. In contrast, an inhibitor of calmodulin-dependent protein kinase, KN-62, and an inhibitor of calmodulin, W-7, did not block the induction of c-fos mRNA by lead. An increase in serum-response element DNA-binding activity was observed in nuclear extracts from PC12 cells exposed to lead. It is interesting that lead activated protein kinase C isoforms delta and epsilon, but not isoforms alpha and beta. In conclusion, lead appears to induce the expression of immediate early genes by a mechanism that requires protein kinase C.

Increased AP-1 DNA binding activity in PC12 cells treated with lead.

The possibility that the mechanism of lead neurotoxicity may be at the level of transcription was investigated in PC12 cells. In electrophoretic mobility gel shift assays Pb2+ was found to increase activator protein-1 complex (AP-1) DNA binding activity in PC12 cells; the increase was time- and concentration-dependent. Exposure to Pb2+ also resulted in an increase in AP-1-driven transcription in cerebellar granule cells transfected with a luciferase gene reporter construct. The increase in AP-1 DNA binding activity by Pb2+ required protein synthesis. The increase was mediated by protein kinase C because depletion of protein kinase C and an inhibitor of protein kinase C prevented the increase in AP-1 DNA binding activity by Pb2+. Fra-2 and JunD were found in supershift assays to be the major components of the AP-1 that was increased by Pb2+. In summary, our studies indicate that Pb2+ increases AP-1 DNA binding activity in PC12 cells by a pathway that requires protein kinase C and new protein synthesis.

Paternal deletion from Snrpn to Ube3a in the mouse causes hypotonia, growth retardation and partial lethality and provides evidence for a gene contributing to Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is caused by paternal deficiency of human chromosome 15q11-q13. There is conflicting evidence from human translocations regarding the direct involvement of SNRPN in the pathogenesis of PWS and it is not known if the phenotypic features result from the loss of expression of a single imprinted gene or multiple genes. In an attempt to dissect genotype/phenotype correlations for the homologous region of mouse chromosome 7C, we prepared three mutant genotypes: (i) mice with a deletion of Snrpn exon 2, which removes a portion of a small, upstream open reading frame (ORF); (ii) mice with double targeting for Snrpn exon 2 and Ube3a; (iii) mice deleted from Snrpn to Ube3a, removing coding exons for both loci and intervening genes. Mice deleted for Snrpn exon 2 have no obvious phenotypic abnormalities and switching of the genomic imprint for the region is conserved. Mice carrying the Snrpn - Ube3a deletion on the paternal chromosome showed severe growth retardation, hypotonia and approximately 80% lethality before weaning. The surviving mice were fertile and were not obese up to 14 months of age. The deletion was transmitted for multiple generations and continued to cause partial lethality when inherited paternally, but not when inherited maternally. The normal imprinted expression and methylation patterns of necdin, a gene outside the deletion region, indicate that the deletion is not an imprinting mutation. The data suggest the presence of a paternally expressed structural gene between Snrpn and Ipw whose deficiency causes lethality, although other possibilities exist, including position effects on expression of imprinted genes or that simultaneous deficiency of both ORFs of Snrpn causes lethality.

Molecular mechanisms of lead neurotoxicity.

Epidemiological studies have shown a strong relationship between the level of lead in blood and bone as assessed by performance on IQ tests and other psychometric tests. Approximately 1 out of 10 children in the United States have blood lead levels above 10 microg/dl, which has been established as the level of concern. Studies on experimental animals exposed to lead after birth have shown learning deficits at similar blood lead levels. Since learning requires the remodeling of synapses in the brain, lead may specifically affect synaptic transmission. Although the molecular targets for lead are unknown, a vast amount of evidence accumulated over many years has shown that lead disrupts processes that are regulated by calcium. Our laboratory has been studying the effect of lead on protein kinase C, a family of isozymes some of which require calcium for activity. We and others have shown that picomolar concentrations of lead can replace micromolar concentrations of calcium in a protein kinase C enzyme assay. Furthermore, lead activates protein kinase C in intact cells and induces the expression of new genes by a mechanism dependent on protein kinase C. We propose that the learning deficits caused by lead are due to events regulated by protein kinase C that most likely occur at the synapse.

Imprinting in Angelman and Prader-Willi syndromes.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are caused by deficiencies of gene expression from paternal or maternal chromosome 15q11-q13, respectively. Many advances have occurred during the past year. The gene for necdin was mapped in the PWS candidate region and found to be paternally expressed in mouse and human. The bisulfite method for analysis of methylation was established for genomic sequencing and diagnostics, and the methylation of Snrpn was studied in detail in the mouse. A region near the Snrpn promoter was shown to function as a silencer in Drosophila. Point mutations were found in the gene for E6-AP ubiquitin-protein ligase (UBE3A) identifying it as the AS gene, and tissue-specific imprinting (maternal expression) was shown in the human brain and in hippocampal neurons and Purkinje cells in the mouse.

Induction of c-fos mRNA by lead in PC 12 cells.

Addition of lead acetate to PC 12 pheochromocytoma cells elicits induction of c-fos, an immediate early response gene. Induction of c-fos was concentration- and time-dependent: the lowest concentration of lead acetate tested that induced c-fos was 10 microM; induction was observed after a 30 min incubation and remained high after 90 min. Treatment with lead acetate and cycloheximide superinduced c-fos mRNA. Actinomycin D, an inhibitor of mRNA transcription, decreased the level of c-fos mRNA induced by lead acetate by almost 80%. Cadmium chloride and zinc chloride did not induce c-fos mRNA. Since the c-fos gene encodes a transcription factor, Pb2+ has the potential to deregulate the expression of other genes.

The E6-Ap ubiquitin-protein ligase (UBE3A) gene is localized within a narrowed Angelman syndrome critical region.

Angelman syndrome (AS) and Prader-Willi syndrome (PWS) are distinct clinical phenotypes resulting from maternal and paternal deficiencies, respectively, in human chromosome 15qll-q13. Although several imprinted, paternally expressed transcripts have been identified within the PWS candidate region, no maternally expressed gene has yet been identified within the AS candidate region. We have developed an integrated physical map spanning the PWS and AS candidate regions and localized two breakpoints, including a cryptic t(14;15) translocation associated with AS and a non-AS 15q deletion, which substantially narrow the AS candidate region to approximately 250 kb. Mapping data indicate that the entire transcriptional unit of the E6-AP ubiquitin-protein ligase (UBE3A) gene lies within the AS region. The UBE3A locus expresses a transcript of approximately 5 kb at low to moderate levels in all tissues tested. The mouse homolog of UBE3A was cloned and sequenced revealing a high degree of conservation at nucleotide and protein levels. Northern and RT-PCR analysis of Ube3a expression in mouse tissues from animals with segmental, paternal uniparental disomy failed to detect substantially reduced or absent expression compared to control animals, failing to provide any evidence for maternal-specific expression from this locus. Recent identification of de novo truncating mutations in UBE3A taken with these observations indicates that mutations in UBE3A can lead to AS and suggests that this locus may encode both imprinted and biallelically expressed products.