1994 — 1996 |
Strittmatter, Warren J |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Apolipoprotein E/Tau Interactions in Alzheimers Disease
Apolipoprotein E (apoE) has been recently implicated in the pathogenesis of Alzheimer's disease (A.D.) One of the apoE alleles, epsilon4, behaves as an autosomal co-dominant trait in the majority of late-onset familial and sporadic A.D. The apoE4 gene dose is a major risk factor susceptibility gene for A.D. with homozygosity for this allele virtually sufficient to cause disease by age 80. ApoE is found in the defining pathologic structures in the A.D. brain, neurofibrillary tangle-bearing neurons, the neuritic plaque, and congophilic angiopathy. The amount of Abeta peptide amyloid deposited in the senile plaque is increased in A.D. patients homozygous for the apoE4 allele. In vitro, apoE avidly binds Abeta peptide and binding properties are isoform-specific. The presence of apoE extracellularly in the senile plaque and angiopathy and intracellularly in neurons bearing neurofibrillary tangles implicates apoE in the biology of these lesions. The number of neurofibrillary tangles in A.D. correlates better with the degree of dementia than does the number of senile plaques. Therefore the mechanism of formation of these structures appears important in the biochemical mechanisms of this disease. Neurofibrillary tangles are pathologic filamentous structures formed from tau protein, which normally binds and stabilizes microtubules. The assembly of tau into paired helical filaments is preceded by extensive phosphorylation, and avid binding of the tandem-repeat binding domains of tau. We propose to examine the hypothesis that the isoform-specific interaction of apoE with tau alters the normal interaction of tau with the microfilament, alters the kinetics of tau phosphorylation, and alters the ability of tau to assemble the pathologic paired helical filament. We have demonstrated in vitro that apoE3, but not apoE4, binds tau to form a complex which is not dissociated by boiling in SDS. The demonstrated inability of apoE4 to bind tau may therefore result in increased tau phosphorylation, and the subsequent enhanced assembly of the pathologic paired helical filament. Pilot experiments on cultured neurons demonstrate that the addition of exogenous purified and delipidated apoE3 increases neurite extension, which is dependent on microtubules. We will determine whether exogenous apoE3 binds tau in the cultured neuron, as previously demonstrated in vitro, and will determine if apoE4 affects neurite outgrowth. In vitro experiments will determine whether the isoform-specific apoE interaction with tau alters tau binding to the microtubule, alters tau phosphorylation, or alters the formation of the paired helical filament. Alterations of tau metabolism or function by isoform-specific interaction with apoE may explain the mechanism of pathogenesis of apoE4 associated with late-onset Alzheimer's disease.
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1997 — 1999 |
Strittmatter, Warren J |
P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
Isoform Specific Interactions of Apoe in Vitro
Apolipoprotein E (apoE) has been recently implicated in the pathogenesis of Alzheimer's disease (A.D.) One of the apoE alleles, epsilon4, behaves as an autosomal co-dominant trait in the majority of late-onset and sporadic A.D.. The apoE4 gene dose is a major risk-factor susceptibility gene for A.D. with homozygosity for this allele virtually sufficient to cause disease by age 80, and with 50% of homozygous patients developing disease by age 68. In contrast, the epsilon2 and epsilon3 alleles decrease the probability of disease, and increase the age of onset, with the protective effect of epsilon2 greater than epsilon3. Thus the inherited apoE allele determines in part, the risk of developing Alzheimer disease, and determines the rate of disease progression. Interactions of apoE protein with other molecules is therefore critical int he disease process, with isoform-specific interactions of apoE determining the probability,a nd rate, of disease expression. The three common protein isoforms of apoE; E2, E3, E4, differ from each other by one amino acid, which determines their profoundly differing interactions with other proteins. In vitro, apoE4 binds betaA peptide (the primary constituent of the neuritic plaque) faster, and with a different pH dependence, than does aplE3. This isoform-specific difference observed in vitro correlates with the greater betaA peptide amyloid burden deposited in situ in homozygous epsilon4 A.D. patients, compared with homozygous epsilon3 A.D. patients. Paired-helical filaments of the neurofibrillary tangle are composed of tau protein. ApoE3 avidly binds tau in vitro, forming a complex not dissociated by boiling in SDS. In contrast, apoE4 does not form such a complex. Isoform-specific interactions of apoE with tau cold alter tau function or metabolism. ApoE is found in populations of neurons, some of which contain neurofibrillary tangles. In vitro interactions of the apoE isoforms have been studied only semi- quantitatively under harshly denaturing conditions. The technique of surface plasmon resonance will be employed to characterize interactions of the apoE isoforms with other molecules quantitatively and under physiologic conditions. This technique permits the characterization of protein interactions including affinity measurements to 10-6M sec-1. No derivitization of the proteins is required, and the sensitivity of detection is picomolar. Quantitative characterization of isoform-specific interactions of apoE with other molecules under physiologic conditions is essential to identify molecular targets by which apoE is relevant to A.D.
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