P4–430: Antisense oligonucleotide‐mediated inhibition of miR‐33 in cultured neurons, astrocytes and microglia: Effects on ABCA1 expression, APOE lipidation, cellular cholesterol and beta‐amyloid neurotoxicity

Increasing the expression of ATP-binding cassette transporter A1 (ABCA1) reduces plaque deposits and improves memory performance in mouse models of Alzheimer's disease (AD). This has predominantly been attributed to ABCA1-mediated lipidation of apolipoprotein E (apoE) in the brain and facilitating amyloid clearance. Despite the beneficial effects of LXR agonists - therapeutic compounds that increase ABCA1 expression- in preclinical studies, their utility for the treatment of AD is limited by serious side effects underscoring the need for discovering alternative therapeutic approaches. MicroRNAs (miRNAs) are endogenous small, noncoding RNA molecules that bind to target mRNAs leading to translational repression. MicroRNA-33 (miR-33) regulates the expression of key genes involved in cellular cholesterol metabolism including ABCA1 and has emerged as a promising target in experimental models of atherosclerosis and diabetes. The objective of this study is to evaluate the effects of increasing ABCA1 in brain cells by inhibiting miR-33 and study the cellular and molecular mechanisms relevant to AD pathogenesis. Antisense oligonucleotides (ASOs), primary neurons, astrocytes and microglia, western immunoblotting, cellular cholesterol, cholesterol efflux, phagocytosis and cytokine release assays, neuronal viability assays, intracerebroventricular infusion, APP/PS1 mice and mouse neuropathology. Treatment of cultured neurons, astrocytes and microglia with an ASO targeting miR-33 increases ABCA1 expression in a concentration dependant manner. This is accompanied by increased cholesterol and ApoE efflux by astrocytes into the cultured medium. Interestingly, increasing ABCA1 expression did not change the total cellular cholesterol content in neurons and astrocytes, neither did it protect neurons against Aβ toxicity. Moreover, i.c.v. infusion of miR-33 ASO also increased hippocampal and striatal ABCA1 expression in wild type mice. We demonstrate that ASO mediated inhibition of miR-33 can successfully be employed towards increasing ABCA1 expression in cultured brain cells and in vivo. Our data suggest that increasing ABCA1 expression enhances cholesterol and ApoE efflux, which may indirectly promote amyloid clearance. It remains to be determined whether miR-33 inhibition affects APP processing or has any beneficial effects on the development and progression of AD like neuropathology in relevant animal models. We are confident that these findings will have significant impact on the development of novel therapies for AD.

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