Donald E. Schmechel

Mitochondrial Polymorphisms Significantly Reduce the Risk of Parkinson Disease

Mitochondrial (mt) impairment, particularly within complex I of the electron transport system, has been implicated in the pathogenesis of Parkinson disease (PD). More than half of mitochondrially encoded polypeptides form part of the reduced nicotinamide adenine dinucleotide dehydrogenase (NADH) complex I enzyme. To test the hypothesis that mtDNA variation contributes to PD expression, we genotyped 10 single-nucleotide polymorphisms (SNPs) that define the European mtDNA haplogroups in 609 white patients with PD and 340 unaffected white control subjects. Overall, individuals classified as haplogroup J (odds ratio [OR] 0.55; 95% confidence interval [CI] 0.34-0.91; P=.02) or K (OR 0.52; 95% CI 0.30-0.90; P=.02) demonstrated a significant decrease in risk of PD versus individuals carrying the most common haplogroup, H. Furthermore, a specific SNP that defines these two haplogroups, 10398G, is strongly associated with this protective effect (OR 0.53; 95% CI 0.39-0.73; P=.0001). SNP 10398G causes a nonconservative amino acid change from threonine to alanine within the NADH dehydrogenase 3 (ND3) of complex I. After stratification by sex, this decrease in risk appeared stronger in women than in men (OR 0.43; 95% CI 0.27-0.71; P=.0009). In addition, SNP 9055A of ATP6 demonstrated a protective effect for women (OR 0.45; 95% CI 0.22-0.93; P=.03). Our results suggest that ND3 is an important factor in PD susceptibility among white individuals and could help explain the role of complex I in PD expression.

Apolipoprotein E ∈4 allele distributions in late-onset Alzheimer's disease and in other amyloid-forming diseases

The frequency of the allele for apolipoprotein E type 4 (epsilon 4) is increased in late-onset familial and sporadic Alzheimers disease (AD). We have examined epsilon 4 frequencies in four distinct, normal, elderly control groups and, most importantly, in patients with amyloid-forming diseases whose epsilon 4 distributions were not previously known (Creutzfeldt-Jakob disease, familial amyloidotic polyneuropathy, Downs syndrome). There were no differences between any of these controls and published control series, cementing the relevance of epsilon 4 for late-onset AD. The increase in late-onset AD was confirmed in two new series.

Production of a specific antiserum to rat brain glutamic acid decar☐ylase by injection of an antigen-antibody complex

Abstract Production of an antiserum specific to rat brain l -glutamic acid decar☐ylase is described, featuring the injection of an antigen-antibody complex. Partial purification of glutamate decar☐ylase was first achieved through differential centrifugation, ammonium sulfate fractionation, chromatography on Sephadex G-150, preparative isoelectric focusing in sucrose gradient and polyacrylamide gel electrophoresis for the production of a preliminary polyvalent, so called ‘trapping’ glutamate decar☐ylase antiserum in sheep. In rat brain homogenate supernatant this antiserum maximally inhibited glutamate decar☐ylase activity by 80% and totally precipitated enzyme activity on centrifugation. This antiserum, however, was polyvalent, as in crossed immunoelectrophoresis it detected four antigens in rat brain homogenate supernatant and three antigens in a partially purified preparation of glutamate decar☐ylase. One of these three precipitin lines could be radioactively labelled by 2-[ 3 H]-γ-acetylenic γ-aminobutyrate, an irreversible inhibitor of glutamate decar☐ylase. Injection of this antigen-antibody-precipitin line into a new non-immunized sheep yielded a new antiserum, which slightly inhibited but maximally precipitated 85% of the glutamate decar☐ylase activity in rat brain homogenate supernatant. In crossed immunoelectrophoresis the latter antiserum detected one antigen in the partially purified preparation of glutamate decar☐ylase. In crossed immunoelectrophoresis with intermediate gel the antiserum altered the mobility of a single antigen in brain homogenate supernatant. Thus, according to precipitating characteristics, the second antiserum appears to be specific to glutamate decar☐ylase.

Hypothesis: Microtubule Instability and Paired Helical Filament Formation in the Alzheimer Disease Brain Are Related to Apolipoprotein E Genotype

A genetic classification of Alzheimer disease(s) (AD) is presented. We describe a potential metabolic process in individuals who inherit apolipoprotein E-epsilon 4 (APOE4, gene; apoE4, protein) alleles, leading to increased risk and earlier age of onset of late-onset Alzheimer disease. Apolipoprotein E-epsilon 3 (apoE3) binds to tau protein, possibly slowing the initial rate of tau phosphorylation and self-assembly into paired helical filaments (PHFs); apoE4 does not bind tau. Tau promotes microtubule assembly and stabilizes microtubules; hyperphosphorylated tau does not bind, thereby destabilizing microtubules. Hyperphosphorylated tau may self-assemble into PHFs. Over time a bias toward destabilization of microtubules and the formation of neurofibrillary tangles may occur in individuals who inherit APOE4 alleles, leading to a shorter functional neuronal life span. This hypothesis focuses attention on two important aspects of AD research design: (1) Although the inheritance of APOE4 is associated with increased risk and decreased age of onset, apoE4 does not directly cause the disease. Our data point to the absence of an important function of apoE3 or apoE2 in individuals who do not inherit these alleles as the genetically relevant metabolic factor. This has important implications for design of experiments directed toward understanding the relevant neuronal metabolism. (2) Should this hypothesis be proven and confirmed, targets for pharmaceutical therapy designed to mimic the metabolic function of apoE3 or apoE2 become a realistic preventive strategy.

Immunocytochemical localization of glutamate decar☐ylase in rat cerebellum with a new antiserum

Abstract An antiserum prepared in sheep against rat brain glutamic acid decar☐ylase, the biosynthetic enzyme for γ-aminobutyric acid, has been employed to localize, at the light and electronmicroscopic levels, neurons containing glutamate decar☐ylase in the cerebellum of adult rats using the unlabeled antibody enzyme method of S ternberger (1979). With high dilutions of the primary antiserum (1:4000 and higher) glutamate decar☐ylase-immunoreactivity was localized in synaptic terminals of basket, stellate and Golgi cells and Purkinje cell axon recurrent collaterals in the cerebellar cortex, as well as in axosomatic and axodendritic synaptic terminals in the deep cerebellar nuclei, all of which contain pleomorphic synaptic vesicles. The reaction product was present in the axoplasm and at the cytoplasmic aspect of the plasmamembrane, the membrane of synaptic vesicles and axoplasmic reticulum, the outer mitochondrial membrane and also the microtubules. Lower dilutions of antiserum (1:1500) revealed light, specific immunoreactivity in somata of Purkinje, basket, stellate and Golgi-cells of the cerebellar cortex. We obtained specimens in which the vast majority of the members of these cells were stained. Staining of stellate cells was the least intense of all positive neurons in the cerebellar cortex. Very light labeling of some small cell bodies of the deep cerebellar nuclei was also observed. Granule cells, parallel fibers, mossy fibers and climbing fibers in the cerebellar cortex, the large neurons of the cerebellar nuclei, and glial cells were negative. When the antiserum was absorbed against acetone-dried rat liver powder, the same staining pattern was obtained, however, dilutions of 1:500 were necessary to obtain equally strong staining of neuronal cell bodies. These data indicate the specificity of the new anti-serum to rat brain glutamate decar☐ylase. They confirm and complement earlier immunocytochemical work with an antiserum to mouse brain glutamate decar☐ylase. The new antiserum not only shows that nearly all inhibitory axons in the cerebellum are glutamate decar☐ylase positive but also detects immunoreactivity in neuronal cell bodies in the cerebellar cortex without administration of blockers of axoplasmic transport.

SNPing Away at Complex Diseases: Analysis of Single-Nucleotide Polymorphisms around APOE in Alzheimer Disease

There has been great interest in the prospects of using single-nucleotide polymorphisms (SNPs) in the search for complex disease genes, and several initiatives devoted to the identification and mapping of SNPs throughout the human genome are currently underway. However, actual data investigating the use of SNPs for identification of complex disease genes are scarce. To begin to look at issues surrounding the use of SNPs in complex disease studies, we have initiated a collaborative SNP mapping study around APOE, the well-established susceptibility gene for late-onset Alzheimer disease (AD). Sixty SNPs in a 1.5-Mb region surrounding APOE were genotyped in samples of unrelated cases of AD, in controls, and in families with AD. Standard tests were conducted to look for association of SNP alleles with AD, in cases and controls. We also used family-based association analyses, including recently developed methods to look for haplotype association. Evidence of association (P</=.05) was identified for 7 of 13 SNPs, including the APOE-4 polymorphism, spanning 40 kb on either side of APOE. As expected, very strong evidence for association with AD was seen for the APOE-4 polymorphism, as well as for two other SNPs that lie <16 kb from APOE. Haplotype analysis using family data increased significance over that seen in single-locus tests for some of the markers, and, for these data, improved localization of the gene. Our results demonstrate that associations can be detected at SNPs near a complex disease gene. We found that a high density of markers will be necessary in order to have a good chance of including SNPs with detectable levels of allelic association with the disease mutation, and statistical analysis based on haplotypes can provide additional information with respect to tests of significance and fine localization of complex disease genes.

Neurons switch from non-neuronal enolase to neuron-specific enolase during differentiation

The enolase (EC 4.2.1.11) isoenzymes, neuron-specific enolase (NSE, gamma gamma) and non-neuronal enolase (NNE, alpha alpha), are markers for neurons and glia, respectively, in adult mammalian brain. In developing fetal and early postnatal brain, levels of non-neuronal enolase (NNE) are high. Neuron-specific enolase (NSE) appears only after neurogenesis begins in a given region and only slowly attains adult levels. Immunocytochemistry in developing rat and rhesus monkey brain reveals that proliferative zones that give rise to neurons are NNE(+). Thus, nerve cells must undergo a switch from NNE to NSE. In addition, study of neurons in cerebellum and neocortex reveals that they are NNE(+) during migration and only become NSE(+) in their final location, presumably after making full synaptic connections. Such migrating cells may contain hybrid enolase (alpha gamma) and some (e.g. cerebellar stellate/basket cells) may not completely switch over to NSE even in the adult. Neuron-specific enolase is not only a specific molecular marker for mature nerve cells, but is closely correlated to the differentiated state.

Age at onset in two common neurodegenerative diseases is genetically controlled.

To identify genes influencing age at onset (AAO) in two common neurodegenerative diseases, a genomic screen was performed for AAO in families with Alzheimer disease (AD; n=449) and Parkinson disease (PD; n=174). Heritabilities between 40%--60% were found in both the AD and PD data sets. For PD, significant evidence for linkage to AAO was found on chromosome 1p (LOD = 3.41). For AD, the AAO effect of APOE (LOD = 3.28) was confirmed. In addition, evidence for AAO linkage on chromosomes 6 and 10 was identified independently in both the AD and PD data sets. Subsequent unified analyses of these regions identified a single peak on chromosome 10q between D10S1239 and D10S1237, with a maximum LOD score of 2.62. These data suggest that a common gene affects AAO in these two common complex neurodegenerative diseases.

Alzheimer's disease is associated with reduced expression of energy metabolism genes in posterior cingulate neurons

Alzheimers disease (AD) is associated with regional reductions in fluorodeoxyglucose positron emission tomography (FDG PET) measurements of the cerebral metabolic rate for glucose, which may begin long before the onset of histopathological or clinical features, especially in carriers of a common AD susceptibility gene. Molecular evaluation of cells from metabolically affected brain regions could provide new information about the pathogenesis of AD and new targets at which to aim disease-slowing and prevention therapies. Data from a genome-wide transcriptomic study were used to compare the expression of 80 metabolically relevant nuclear genes from laser-capture microdissected non-tangle-bearing neurons from autopsy brains of AD cases and normal controls in posterior cingulate cortex, which is metabolically affected in the earliest stages; other brain regions metabolically affected in PET studies of AD or normal aging; and visual cortex, which is relatively spared. Compared with controls, AD cases had significantly lower expression of 70% of the nuclear genes encoding subunits of the mitochondrial electron transport chain in posterior cingulate cortex, 65% of those in the middle temporal gyrus, 61% of those in hippocampal CA1, 23% of those in entorhinal cortex, 16% of those in visual cortex, and 5% of those in the superior frontal gyrus. Western blots confirmed underexpression of those complex I–V subunits assessed at the protein level. Cerebral metabolic rate for glucose abnormalities in FDG PET studies of AD may be associated with reduced neuronal expression of nuclear genes encoding subunits of the mitochondrial electron transport chain.

Specificity, sensitivity, and predictive value of apolipoprotein-E genotyping for sporadic Alzheimer's disease

BACKGROUND We aimed to determine the specificity, sensitivity, and predictive value of apolipoprotein E (APOE) genotyping in 67 consecutive patients with clinical diagnoses of sporadic Alzheimers disease (AD) who underwent necropsy. METHODS We studied patients who attended the Duke Memory Disorders Clinic and were diagnosed as having probable AD. These patients were followed up until they died. APOE genotyping was done during life in most cases, but in some brain tissue obtained at necropsy was used. Members of known AD families were excluded. FINDINGS After neuropathological examination 57 (85%) of 67 of our patients were confirmed as having AD including all 43 who had at least one APOE-epsilon 4 allele. None of the patients found not to have AD carried an epsilon 4 allele. In this series, the specificity of the epsilon 4 allele was 100%, the sensitivity 75%, the positive predictive value 100%, and the negative predictive value 42%. In this necropsy-confirmed series, the epsilon 4/epsilon 4 genotype predicted AD with 100% accuracy. The epsilon 3/epsilon 4 and epsilon 2/epsilon 4 genotypes were also unexpectedly highly specific for AD. INTERPRETATION Data from hundreds of necropsy-confirmed non-AD patients in other longitudinal necropsy series will allow the predictive value of APOE genotypes to be assessed with useful confidence limits.