Apolipoprotein E is an important protein of the cholesterol transport system which is synthesized by a variety of tissues. Following secretion, apoE is incorporated in a variety of lipoprotein particles (chylomicron remnants, VLDL, IDL and a subfraction of HDL) and directs their catabolism by cell receptors. Common genetic mutations have been identified in apoE which give rise to three homozygous (E4/4, E3/3 and E2/2) and three heterozygous (E4/3 E3/2 and E4/2) phenotypes. The apo E4 containing phenotypes occur with increased frequency in patients with late onset of Alzheimer's Disease (AD). It is our hypothesis based on existing experimental data and models, that the abnormal interactions of apoE with secreted (Abeta) or intracellular proteins in the brain (tau, cytoskeletal protein) depend on alterations int he three-dimensional arrangement and stabilizing interactions of the amphipathic aminoterminal apoE helices and their interaction with the carboxy terminal domain which is predicted to contain similar helical bundles. Mutations which can disrupt the conformation of apoE may affect its physiochemical and functional properties in vitro and may lead to AD in vivo. To test this hypothesis we propose the following specific aims; 1) to mutagenize the E3 gene and generate by transfection and selection will lines which express the variant E forms. Six categories of mutations have been carefully selected to disrupt the electrostatic or hydrophobic interactions which stabilize helices 2,3 and 4 of apoE as well as the carboxy terminal domain of apoE which is predicted to contain similar helical bundles. 2) To grow the normal and variant apoE forms on a large scale (Bioreactor roller bottles) and purify the mutant proteins from the culture media for physicochemical and functional analyses and toxicity tests on neuronal cell cultures. The conformation of the mutant apoE forms as well as of the Abeta and apoE interactions will be monitored by physicochemical methods. Selected constructs will be introduced by electroporation or transfection in neuronal cell cultures in order to assess the effect of apoE on neuronal cell growth, extension and branching as well as on other morphological and cytoskeletal changes. 3) To study the thermodynamic stability of the variant forms of apoE as well as the interactions of apoE with Abeta and tau in order to establish the molecular details which are important for the maintenance of the secondary and tertiary conformation required for these interactions. 4) To generate animal models of Alzheimer's disease a) by expressing the apoE4 gene and the APP751 (Swedish mutation Met 1 yields Leu, Lys-2 yields Asn) in the apoE deficient mouse strain under the control of astrocyte specific and neuronal specific promoters. For comparison a mouse line will be generated expressing apoE3 and normal APP751. b) by studying the brain changes occurring in mouse lines expressing apoE and APP. Understanding the structural elements of apoE which determine its conformation is essential to understand athe normal and aberrant function of apoE and its role in AD.
