Structural and functional insights into naturally occurring apolipoprotein E variants with protective effects against Alzheimer's disease

Apolipoprotein E (apoE), a major protein for lipid transport in circulation and the brain, has three common isoforms, apoE2, apoE3 and apoE4. APOE4 is the strongest genetic risk factor for late-onset Alzheimer's disease (AD). Recently identified rare apoE variants, the apoE3(R136S)-Christchurch, apoE3(V236E)-Jacksonville and apoE4(R251G), appear to exert protective functions against AD and reduce the disease risk, but the molecular basis behind these effects is unknown. ApoE is a structurally dynamic protein, undergoing significant rearrangements that are important for its biological function. To examine the structural basis behind the properties of the protective apoE variants, we analyzed their structural and thermodynamic integrity both in APOE3 and APOE4 allelic backgrounds compared to their wild-type counterparts. Circular dichroism spectroscopy showed that only the V236E variation significantly alters the secondary structure of apoE3 and apoE4 in lipid-free form. This variant was also less prone to oligomerization. Chemical denaturation analysis indicated changes in the unfolding profile of V236E and R251G apoE variants in lipid-free form. Thermal unfolding analysis revealed small thermodynamic alterations in each variant compared to their wild-type apoE counterparts in lipid-free form, but a thermodynamic stabilization in lipoprotein-associated form. Additionally, following lipidation, all protective apoE variants were found to enhance the viability of SK-N-SH neuroblastoma cells and reduce the production of TNFα from BV2 microglia cells. Overall, these findings suggest that the specific amino acid substitutions found in AD-protective apoE variants can induce changes in the molecule's stability and conformation that may underlie common functional consequences, which are independent of the apoE background.