Changes in Protein Glycosylation Due to Translation of Mannosidases Encoded by Circular RNAs

Changes of glycosylation in Alzheimer’s disease due to circular RNA translation The formation of amyloid plaques and neurofibrillary tangles, formed by amyloid precursor protein (APP) and microtubule associated protein tau (MAPT) are hallmarks of Alzheimer’s disease. The glycosylation of APP and possibly tau, as well as of other proteins are changed in AD. Mannosidases are key enzymes in the formation of N-glycans, as they remove mannose residues from high mannose- precursors allowing the formation of complex, mature N-glycans. Two key human alpha mannosidases, MAN2A1 and MAN1A2 encode highly expressed circular RNAs (circRNAs), ranking #24 and #2, respectively. The expression of circMAN2A1 correlates with AD progression in parietal and entorhinal cortex, as well as hippocampus. Using a circRNA-specific antiserum, we found that like other circRNAs, endogenous circMAN2A1 is translated into a protein after undergoing Adenosine to Inosine RNA editing, catalyzed by ADAR1-p150, an ADAR1 isoform that is induced by inflammation-dependent alpha interferons, which are known to be upregulated in AD. The sequence of circMAN2A1-protein overlaps with the active center of the linear MAN2A1 protein, and transfection experiments indicate that circMAN2A1 influences glycosylation. We hypothesize that due to an increase in its RNA editing, caused by interferon alpha/ ADAR1-p150, circMAN2A1 is increasingly translated in AD, resulting in a change in N-glycosylation. This hypothesis will be tested in two specific aims: #1: Determine the changes in circRNA and protein expression of circMAN2A1 in AD brain, where we use immunohistochemistry, Western blot and RNAseq to measure changes in circMAN2A1 in human brain regions affected by AD, determine the intracellular localization of endogenous circMAN2A1 and test its suitability as a biomarker. #2: Aim#2: Determine the influence of circMAN2A1 expression on protein glycosylation, where we use mass-spectrometry to determine the glycan changes in HEK293 cells and iPS-cell derived human neurons after circMAN2A1 expression and determine the changes of N-glycans on AD-relevant proteins. These studies are significant, as they could unveil the molecular cause for the change of glycosylation in AD, which could identify therapeutic targets, the studies are innovative, as they are the first functional characterizations of proteins encoded by circular RNAs. The work is feasible by combining a team consisting of RNA biologist who discovered circRNA translation (Stamm), an expert in mass-spectrometry of glycans (Gentry) and a neuropathologist (Nelson).