Mevalonate Pathway Regulation of Astrocyte ApoE

Abstract The ApoE 4 isoform is the strongest genetic link to Alzheimer’s disease for unclear reasons. ApoE is a lipid carrying protein. Both the content of the ApoE particles and their release from astrocytes varies by ApoE isoform. We have found that the mevalonate pathway can regulate both the release of ApoE from astrocytes and the lipid content of those ApoE particles. Depending on the part of the pathway that we inhibit we can separate these two functions. We hypothesize that targeting individual components of the mevalonate pathway can be used to manipulate the secretion and composition of ApoE in order to reduce Aβ synthesis and improve neuron survival. Further, we will explore if reducing the lipid content of the ApoE particles, without impacting secretion of the particles, can help shift the behavior of ApoE4 particles towards the behavior of lower risk ApoE genotypes. Through the proposed experiments we will test if: Aim 1. Secretion of ApoE is regulated via the mevalonate pathway in quiescent and activated astrocytes in an isoform specific manner. There are 3 well-characterized isoforms of ApoE in humans; 2, 3 and 4, which carry varying risks for developing AD. The quantity and composition of lipids carried by ApoE is influenced by ApoE genotype and astrocyte activation state. We will use mice expressing humanized ApoE 2, 3 or 4 or an ApoE knockout control to grow primary cultures of cortical astrocytes. Astrocytes will be grown in serum-free media to mimic the quiescent state or treated with TNF, IL-1α and C1q to mimic the activated state. Utilizing chemical inhibitors, shRNAs and conditional knockouts, we will determine the contributions of the cholesterol and prenylation arms of the mevalonate pathway to regulation of ApoE secretion by astrocytes. Aim 2. Inhibition of the mevalonate pathway can improve the abnormal lipid droplet accumulation and lipid secretion profile in ApoE4 astrocytes. Using the tools from Aim 1, we will manipulate the mevalonate pathway in astrocytes from ApoE knockout and ApoE 2, 3 or 4 knock-in mice. Intracellular ApoE and lipid droplets will be assessed by confocal microscopy. The lipid composition of astrocyte conditioned media from quiescent and activated astrocytes with and without inhibitors to cholesterol synthesis, prenylation or both will be measured. Changes in cholesterol-lipid carrier interactions will be determined using a cholesterol probe. Aim 3. Selective inhibition of the mevalonate pathway in astrocytes will reduce amyloid-beta cleavage and neuron death. We predict that inhibition of cholesterol, while preserving prenylation, will reduce AD pathology, with the greatest effect in ApoE4 astrocytes. Conditioned media from astrocytes treated with inhibitors of cholesterol synthesis, prenylation or both will be applied to primary neurons. We will then measure clustering of APP with lipid rafts by super resolution imaging, Aβ secretion by ELISA, activation of the lipoapoptotic pathway by western and cell death by ethidium incorporation.