Both serum cholesterol levels and the cholesterol carrying protein ApoE have been implicated in the development of Alzheimer?s disease. Despite intensive research, there is not a consensus for why hypercholesterolemia and ApoE are risk factors. In the brain, the majority of cholesterol and ApoE are made by astrocytes. We will use a recently created cholesterol probe as a novel approach to investigate how ApoE isoform impacts trafficking of cholesterol out of the astrocyte and into the neuron. This probe contains a photoactivatable group which creates covalent bonds to adjacent proteins. In addition, it has a click-chemistry group which allows probe-protein complex purification for mass spectrometry analysis. We will add the cholesterol probe to astrocyte lines expressing each of the human ApoE isoforms. With this approach we will answer the questions: 1. What are the cholesterol-protein interactions within astrocyte-secreted ApoE particles and how are these interactions impacted by ApoE isoform? 2. What are the cholesterol-protein interactions in the neuron which can be linked to Alzheimer?s disease associated genes, and are these interactions in the neuron impacted by ApoE isoform? Using this approach, we believe we will identify how some known Alzheimer?s disease risk alleles are connected to cholesterol/ApoE, and identify proteins not previously implicated in Alzheimer?s disease that are modified by cholesterol and may be contributing to the development of the disease.
Alzheimer?s disease is a devastating disease of the brain that has been linked to cholesterol, but why cholesterol is important in Alzheimer?s disease remains unclear. This project will use a cholesterol probe to track how cholesterol leaves cells in the brain, called astrocytes, and enters neurons. By better understanding this process, and how it goes wrong in Alzheimer?s disease, it could provide new paths for developing therapies to treat the disease.