DESCRIPTION (provided by applicant): Alzheimer's disease (AD) in a neurodegenerative disorder with amyloid-beta (Aβ) aggregates as a core pathology. Accumulation of Aβ in the brain is believed to be an important force in AD pathogenesis since every mutation linked to familial AD influences some aspect of Aβ metabolism. Despite the known role of Aβ in AD pathogenesis the downstream events that contribute to neurotoxicity and cognitive decline still remain fundamental and poorly understood. Recently our group discovered that numerous serine/arginine (SR) splicing proteins including U1-70K, DDX46, and Luc7L aggregate in AD, but not other non-AD neurodegenerative diseases. Unspliced RNA species were also found significantly elevated in AD brain further supporting the role of SR-protein loss-of-function in AD U1-70K and other SR-protein aggregates are also observed in familial cases of AD caused by mutations in amyloid precursor protein (APP), which strongly links Aβ deposition with SR protein aggregation. Given the critical role of SR-protein in regulation of RNA splicing, and the large scale RNA splicing deficits in AD brain, better understanding SR protein dysfunction may reveal new signaling mechanisms linked to Aβ and AD pathogenesis. To this end, we provide compelling preliminary data showing that arginine/serine (RS) phosphorylation is dramatically increased in AD brain extracts, which agrees with previous studies indicating that serine/arginine protein kinase-2 (SRPK2) is hyperactive in AD. SRPK2 specifically phosphorylates intrinsically disordered RS domains like those found in U1-70K and other SR-proteins. Moreover, we provide evidence that RS domains are necessary for U1-70K self-association, granule assembly and aggregation in AD brain. These data support my hypothesis that SRPK2 influences U1-70K protein interactions and assembly via phosphorylation of RS domains. Rigorously testing this hypothesis will serve as a paradigm to study how RS phosphorylation contributes to SR-protein aggregation observed in AD. While RS phosphorylation regulates many aspects of SR-protein biology the exact location and function of RS phosphorylation sites is largely unknown due to the technological challenges of sequencing arginine-rich domains via conventional `bottom-up' proteomic approaches. To overcome this limitation I developed a novel `middle-down' proteomic approach by coupling limited trypsin digestion with electron transfer dissociation (ETD). This new method has provided the unprecedented ability to sequence RS phosphorylation sites from cell lines and human tissue. I will exploit this approach to map and determine the role of RS phosphorylation on U1-70K assembly and quantify levels of RS phosphorylation in AD brain linked to Aβ deposition. Based on our preliminary studies our central hypothesis will be tested through two complementary aims that seek to: i) assess the physiological function of phosphorylation within the U1-70K RS domain and ii) identify and quantify levels of RS phosphorylation in AD brain. Completing these goals may lead to new therapeutic approaches that modify SR protein aggregation and global splicing defects observed in AD.

PUBLIC HEALTH RELEVANCE: Amyloid-β peptide (Aβ), the core component of senile plaques, has a central role in Alzheimer's disease (AD) pathogenesis. Recent evidence indicates that the aggregation of a structurally unique set of RNA splicing factors, which cause profound defects in neuronal maintenance, is linked to Aβ. Our goal is to understand the downstream signaling mechanisms linking Aβ to the aggregation of this select group of RNA splicing factors.