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.

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.

Development of a monoclonal antibody against a tumor-associated antigen.

A monoclonal antibody-producing hybrid cell line was obtained by fusing mouse myeloma cells with spleen cells from a mouse immunized with C6 glioma cells. This antibody binds to a specific cell-surface antigen that is present on C6 rat glioma cells, transformed astrocytes and oligodendrocytes, and a human glioma cell line but is absent on a normal glial cell line, fibroblasts, and primary cultures of astrocytes and oligodendrocytes. The antigen also appears on tumor tissue of transformed oligodendrocytes but not on normal brain tissue.

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.

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.

Neoplastic transformation of newborn rat oligodendrocytes in culture.

We have developed a model to study the neoplastic transformation of rat oligodendrocytes in culture. This procedure utilizes a technique previously developed by McCarthy and de Vellis which allows the preparation of 99% pure astrocyte and oligodendrocyte populations from 1- to 2-day-old rat cerebral cortices. Pregnant rats on the 19th day of gestation were given injections with either ethyl nitrosourea (10 micrograms/g body weight) in phosphate-buffered saline or phosphate-buffered saline, and oligodendrocyte cultures were prepared. Oligodendrocytes appear to be unstable in culture since transformation was observed with cells derived from either pups from pregnant rats either treated with nitrosourea or phosphate-buffered saline. Transformation required 78 to 108 days and 3 to 9 passages, at which time a marked increase in cellular proliferation was observed. The possibility that the transformed cells were derived from a nonoligodendroglial cell was excluded by the following evidence. Light and scanning electron micrographs of the transformed cells revealed cytological features essentially similar to those of primary oligodendroglial cultures. Furthermore, 2 biochemical oligodendroglial markers, the induction of lactate dehydrogenase by N6,O6-dibutyryl cyclic adenosine 3':5'-monophosphate and the presence of 2',3'-cyclic nucleotide 3' phosphohydrolase, were also retained. Conversely, another oligodendroglial marker, the hydrocortisone induction of glycerol phosphate dehydrogenase, was not found in any of the cell lines. These transformed cells grew as tumors when injected intracranially into 21-day-old rats. Histologically, these tumors did not appear as classical oligodendrogliomas, but their oligodendroglial origin was confirmed since the tumor tissue contained 2':3'-cyclic nucleotide 3'-phosphohydrolase activity, and the cells which grew from tumor explant cultures morphologically appeared similar to the parent cell line. The transformed cells were also characterized for in vitro properties which correlate with the expression of tumorigenicity. The transformed cells exhibited anchorage-independent growth and were agglutinated by concanavalin A treatment. Changes in fibrinolytic activity were not an exclusive property of transformed glial cells. This model should now allow us to study various mechanisms involved in the neoplastic transformation of oligodendrocytes.

Analysis of ras oncogenes in malignant melanoma and precursor lesions: correlation of point mutations with differentiation phenotype.

This study examined noncultured and cultured melanomas and related precursor specimens for (i) mutated ras genes using polymerase chain reaction (PCR) methodology, (ii) correlation of mutated ras genes with differentiation related phenotypic characteristics, (iii) expression of ras-encoded p21 proteins in tissues by immunoperoxidase analysis, (iv) quantitative expression of mutated and wild-type ras encoded p21 proteins by flow cytometry, and (v) correlation between p21 expression, the occurrence of ras mutations, and cell cycle kinetics. The results of these studies are (1) 24% of cultured malignant melanomas have activated ras genes, with N-ras being activated ten times as frequently as Harvey (Ha)-ras. Each example of an activated ras gene showed a mutation at the 61st codon of the protein, with the exception of one melanoma which showed a mutation at codon 13 of the N-ras gene; (2) all the melanomas displaying an activated ras gene had a similar cell surface phenotype and appear to come from a similar phase of differentiation; (3) 5-6% of noncultured primary and metastatic melanomas have mutated ras genes; (4) no ras gene mutations were found in any precursor lesion, specifically normal nevi and dysplastic nevi; (5) immunoperoxidase analysis of paraffin-embedded specimens indicated no quantitative or qualitative alterations in p21 expression that correlate with tumor progression; (6) there were no observable differences in p21 expression between melanoma cells growing exponentially or in plateau phase, or between melanoma cells with or without ras mutations; nor were any cell kinetic differences found between cells with and without mutated ras genes. These studies suggest that the role of ras mutations may be limited to an indirect involvement in the transformation of a subset of melanomas.

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.

Second stage tumor promoters: differences in biological potency and phorbol ester receptor affinity in C6 cells.

We have shown that the second stage tumor promoters mezerein (MEZ) and phorbol 12-retinoate 13-acetate (PRA) inhibit the gluccocorticoid-induced increase in glycerol phosphate dehydrogenase (GPDH) activity in C6 rat glioma cells with ED 50-values of 3.9 and 2.9 nM, respectively. Phorbol 12-myristate 13-acetate (PMA) was 10-fold less potent. MEZ was likewise more potent than PMA for inhibition of cAMP formation in response to isoproterenol. Binding competition studies using [3H]phorbol 12,13-dibutyrate ([3H]PDBu) yielded apparent Ki-values for MEZ and PRA of 50-70 nM. The large difference between the biological potencies of MEZ and PRA and their affinity for the major phorbol ester receptor suggest they may be acting through a more complicated mechanism in these cells.

Mechanisms of lead neurotoxicity.

During the past several years, there has been a renewed interest in the mechanisms by which lead poisoning disrupts brain function. In part, this is related to clinical observations that imply an absence of threshold for toxicity in the immature brain. Many of the neurotoxic effects of lead appear related to the ability of lead to mimic or in some cases inhibit the action of calcium as a regulator of cell function. At a neuronal level, exposure to lead alters the release of neurotransmitter from presynaptic nerve endings. Spontaneous release is enhanced and evoked release is inhibited. The former may be due to activation of protein kinases in the nerve endings and the latter to blockade of voltage-dependent calcium channels. This disruption of neuronal activity may, in turn, alter the developmental processes of synapse formation and result in a less efficient brain with cognitive deficits. Brain homeostatic mechanisms are disrupted by exposure to higher levels of lead. The final pathway appears to be a breakdown in the blood-brain barrier. Again, the ability of lead to mimic or mobilize calcium and activate protein kinases may alter the behavior of endothelial cells in immature brain and disrupt the barrier. In addition to a direct toxic effect upon the endothelial cells, lead may alter indirectly the microvasculature by damaging the astrocytes that provide signals for the maintenance of blood-brain barrier integrity.

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.

Neoplastic transformation of newborn rat astrocytes in culture.

A subpopulation of rat glial cells, derived from the astroglial population of newborn cerebral cortex cell cultures, spontaneously transformed in culture. Unlike the pretransformed cells, the transformed cells formed pile-up colonies, exhibited anchorage-independent growth, and were tumorigenic in young rats. Both the pretransformed and transformed cells exhibited differentiated properties characteristic of glial cells. For example, the pretransformed cells possessed hydrocortisone-inducible glutamine synthetase (GS), a property restricted to astrocytes in the central nervous system. As was anticipated, these cells did not exhibit either of two oligodendroglial characteristics, hydrocortisone-inducible glycerol phosphate dehydrogenase (GPDH) or the induction of lactate dehydrogenase (LDH) by N6,O6-dibutyryl cyclic adenosine 3':5'monophosphate (Bt2 cAMP). Unexpectedly, the transformed cells expressed the induction of glycerol phosphate dehydrogenase and lactate dehydrogenase but lost the glutamine synthetase induction. Both the pretransformed and transformed cells were examined ultrastructurally. Neither cell type exhibited glial filaments (9-10 nm), a structure typical of astrocytes. Rather, the pretransformed cells were characterized by distinct longitudinal filaments near the cell surface and the absence of microtubules. On the other hand, the only cytoskeletal element visible in transformed cells were microtubules. Our work demonstrates that, like other rodent cell types, rat glial cells can spontaneously transform in culture. It also shows that the expression of differentiated properties are sensitive to the transformation process.

Chemotaxis of rat brain astrocytes to platelet derived growth factor.

Using a purified population of rat brain astrocytes prepared from neonatal cortex, we investigated the chemotaxis of astroglia to several well characterized growth factors. Chemotactic activity for astrocytes was found for the platelet derived growth factor with a half maximal response occurring at 0.5 ng/ml as compared with a value of 2-3 ng/ml obtained for NIH/3T3 fibroblasts in control experiments. Other growth factors including epidermal growth factor, fibroblast growth factor and insulin were inactive as chemoattractants. Affinity purified fibronectin was also found to stimulate the migration of astroglia, with half maximal doses of approximately 1 microgram/ml relative to maximal responses to platelet derived growth factor.

Dexamethasone reduces vascular density and plasminogen activator activity in 9L rat brain tumors.

Angiogenesis, a process dependent upon perivascular proteolysis, is required for solid tumor growth and is inhibited by certain steroids including glucocorticoids. We examined the relationship between tumor growth and vessel density in experimental rat brain 9L glial tumors following chronic treatment with the glucocorticoid dexamethasone. Tumor growth was inhibited by intraperitoneal administration of 3 mg/kg/day dexamethasone. Maximal cross-sectional areas of post-implantation day 9 tumors were 4.6 +/- 1.0 mm2 in dexamethasone-treated animals and 17.0 +/- 3.4 mm2 in controls (P < 0.01). Microvessel density assessed by laminin immunohistochemistry was 59% lower in dexamethasone-treated tumors (P < 0.01). Plasminogen activator (PA) activity, a proteolytic enzyme related to endothelial migration and vessel growth, was 4.2 +/- 0.9 IU/micrograms protein in dexamethasone-treated tumors and 9.0 +/- 1.0 IU/micrograms protein in control tumors (P < 0.01). Exposure of cultured 9L and central nervous system microvessel endothelial cells to dexamethasone concentrations comparable to those achieved in vivo had no effect on cell growth, but reduced the PA activity of culture supernatant fractions by 78% and 99%, respectively. These findings suggest that inhibition of proteolytic steps involved in vessel growth may underlie, in part, the mechanism by which glucocorticoids decrease brain tumor growth.

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.

Transforming growth factor-beta-like activity in tumors of the central nervous system.

Transforming growth factor type beta (TGF-beta) is a ubiquitous peptide with wide-ranging regulatory functions. This paper reports the initial isolation of TGF-beta activity from human glial and mesenchymally derived tumors and a human glial tumor cell line. While its physiological function at the molecular level is not yet defined, it is believed that this peptide plays a central role in the control of growth and transformation, with the exact role it plays being a function of the entire set of growth factors present in a given cell.

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.

Effect of ganglioside GM1 on arachidonic acid release in bovine aortic endothelial cells.

A role for the ganglioside GM1 in arachidonic acid release in bovine aortic endothelial cells (BAEC) was investigated. [3H]Arachidonic acid-labeled BAEC were preincubated with GM1 and incubated with one of four different stimulators. GM1 inhibited arachidonic acid release when stimulated with maitotoxin or melittin but not with ionomycin or thapsigargin. A 10 microM GM1 concentration achieved a 50% and 100% inhibition of the maitotoxin and melittin responses, respectively. The selective inhibition displayed by GM1 on the maitotoxin and melittin responses was not due to its ability to bind calcium since all four drugs, maitotoxin, melittin, ionomycin, and thapsigargin, required extracellular calcium. The effect of GM1 was not specific to arachidonic acid release. GM1 at 50 microM inhibited phosphatidylinositol polyphosphate (PIP) hydrolysis mediated by melittin, but did not affect hydrolysis mediated by ionomycin. Moreover, the activity of GM1 was not restricted to phospholipid metabolism since it also inhibited calcium influx that was stimulated by maitotoxin or melittin but not by ionomycin. We conclude that GM1 is not a specific inhibitor of phospholipases in bovine aortic endothelial cells, but rather its activity is dependent on the type of stimulant used to activate the cell.