Inhibition of CD33-sialic acid binding in Late-Onset Alzheimer's Disease

PROJECT SUMMARY Alzheimer’s disease (AD) is a debilitating neurodegenerative disease characterized by progressive cognitive decline and pre-symptomatic accumulation of amyloid-beta (Aβ) and tau pathology with older age. AD pathogenesis is poorly understood, however genetic studies have clearly indicated a causal role for microglia, the innate immune cells of the CNS. Indeed, GWAS established the innate immune gene CD33 as a risk factor for Late-Onset AD (LOAD) in 2011. The CD33 LOAD associated rs3865444CC risk genotype is associated with diminished internalization of amyloid-β1-42 peptide, accumulation of neuritic amyloid pathology and fibrillar amyloid on in vivo imaging, and increased numbers of human microglia with small, thick processes, and a rounded morphology compared to the rs3865444AA protective genotype. Individuals with the LOAD associated rs3865444CC risk genotype have increased expression of full-length CD33, the isoform containing the sialic acid binding domain, compared to those with the rs3865444AA protective genotype, suggesting that the sialic- acid binding domain may be critical to the genetic association to AD. Based on the known inhibitory function of CD33, my hypothesis is that sialic acid mediated CD33 activation leads to microglial suppression, resulting in the inability to resolve inflammatory processes and mitigate pathogenic amyloid plaques, which may heighten susceptibility to neuronal loss and contribute to AD progression. To test this hypothesis, our lab developed an alternative approach for inhibiting CD33 function, by using peptides designed to bind to sialic acid to act as “decoy” receptors for sialic acid to reduce it’s binding to CD33 and inhibit CD33 signaling. I hypothesize that competitive inhibition of CD33 with sialic acid will reduce CD33 activation, restore microglial responsiveness, and increase Aβ uptake. The overarching objective of this proposal is to characterize the binding properties of these peptides and their inhibitory effects on CD33 activation and signaling (Aim 1) and then examine the functional responses of these peptides on immune pathways in vitro (Aim 2). In Aim 1, I will determine the potency and selectivity of peptides 1 and 2 in inhibiting CD33-sialic acid binding in a solid-phase binding assay I have optimized (Aim1a), determine the critical residues for efficacy through an alanine screen (Aim1b), and investigate the effects of the peptides on CD33 activation and signaling (Aim1c). In Aim 2, I will look at how human microglia functionally respond to treatment with peptides 1 and 2 in the context of AD using primary microglia-like cells from healthy donor and AD patients expressing the CD33 risk and protective genotypes by measuring gene expression changes in markers of microglial activation and amyloid uptake (Aim2a) and expression changes in key LOAD-associated immune-related proteins (Aim 2b).These experiments will provide insights to not only the mechanism of action of these peptides, but also to further clarify the role of CD33 and Siglec receptors in microglial activation, immune signaling pathways, and the pathogenesis of Alzheimer’s disease.