Cholesterol Metabolites as Regulators of Nicastrin

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Abstract Brain cholesterol is disrupted in aging and Alzheimer’s disease. We have found that cholesterol can directly interact with the protein Nicastrin to alter gamma-secretase complex function. Understanding this interaction, and the mechanisms by which it regulates complex function, could facilitate efforts to develop therapies to prevent Alzheimer’s disease. We hypothesize that cholesterol and its metabolites differentially interact with the Nicastrin subunit of gamma-secretase to change accessory protein recruitment and alter selectivity and function of the complex. Through the proposed experiments we will: Aim 1. Identify the cholesterol binding site on Nicastrin. Nicastrin has a large extracellular domain which has been proposed to contain a substrate recognition domain, a juxtamembrane domain which interacts with Presenilin and a single transmembrane domain. Using the cholesterol probe coupled to mass spectrometry, we will identify the cholesterol-Nicastrin adducts. Proposed sites of interaction will be mutated on human Nicastrin and then wildtype and mutated Nicastrin will be immunoprecipitated and probe interaction confirmed. Aim 2. Determine the composition of accessory proteins recruited to Nicastrin in response to cholesterol and cholesterol metabolites. We hypothesize that the form of cholesterol bound to Nicastrin will change the recruitment of accessory proteins to the complex, which will alter function. We will transduce Nicastrin knockout primary neuron cultures with human Nicastrin. Cells will then be treated with cholesterol metabolites and the complexes immunoprecipitated. Isolated proteins will be measured by mass spectrometry. Using dSTORM super-resolution imaging we will determine if cholesterol metabolites differentially influence accessory protein/gamma-secretase co-localization. Aim 3. Analyze how processing of substrates of the gamma-secretase complex is altered by different cholesterol metabolites. Our preliminary data suggest that cholesterol metabolites are able to act as endogenous gamma-secretase modulators. These metabolites could alter maturation of the complex, substrate selection and/or processivity of the complex. We will test cholesterol metabolites for their ability to influence complex maturation, differential cleavage of the substrates APP and Notch and size of APP products generated, using western blot, reporter assays, ELISA and mass spectrometry. These studies will provide insights into how cholesterol acts as an endogenous modulator of gamma-secretase through a novel interaction with the Nicastrin subunit and provide the basis for future interventions to modulate specific cholesterol metabolites for the prevention of Alzheimer’s disease.