RBFOX1 is regulated by the adenosine 2a receptor and is ubiquitinated in tau tangles in Alzheimer’s disease

Abstract

We recently discovered that common variants in RBFOX1 correlated with β-amyloid load in preclinical Alzheimer's disease (AD) as determined by β-amyloid PET imaging. We found RBFOX1, which is usually cytoplasmic and nuclear in neurons, is mislocalized in human AD brain to tau tangles and neuropil threads in dystrophic neurites. Little is known about the molecular consequences of this mislocalization, but the link RBFOX1 provides between β-amyloid accumulation and tau pathology is intriguing, so we conducted a series of experiments to determine the molecular regulation of RBFOX1 in axons.We optimized and thoroughly validated a proximity ligation assay (PLA) for use in post-mortem human tissue to enable us to interrogate post-translational protein modifications in situ in diseased brain. Deeply characterized brain tissue was obtained from patients with Alzheimer's disease and elderly, neurological controls through the Vanderbilt Brain and Biospecimen Bank and PLA was used to evaluate the presence and distribution of ubiquitinated RBFOX1. Experiments were also performed in neuronal and non-neuronal cell models to characterize the regulation of RBFOX1 ubiquitination by the adenosine 2a receptor (A2AR).Ubiquitination of RBFOX1 in AD brain is markedly increased (over three-fold, p < 0.01, compared to control by two-tailed t-test), particularly in axons, tangles and neuropil threads. The A2AR is also enriched in axons in AD, and colocalizes with tau pathology. In cell cultures, A2AR agonist CGS21680 (100 nM) treatment resulted in marked increase in RBFOX1 ubiquitination compared to controls (p < 0.001, n=4 experimental replicates). Finally, we present evidence that RBFOX1 is involved in promoting autophagy, which may be impaired by ubiquitination.RBFOX1 is ubiquitinated in tangles and neuropil threads and this, in part, appears to be regulated by A2AR signaling. RBFOX1 may have a role in axonal proteostasis, thus clarifying its molecular regulation may help us understand this emerging genetic AD risk factor and potentially identify new pharmacological targets. Optimization of the PLA for use in aged human brain tissue is a broadly applicable technical achievement which expands the range of molecular relationships which can be sensitively and precisely quantitated in situ in human neuropathological material.NIA 1K76AG060001, R01-AG059716, P20-AG068082, NSF 1706155.