Project Summary / Abstract Signaling by cAMP-dependent protein kinase (PKA) is essential for learning and memory and cAMP-elevating phosphodiesterase (PDE) inhibitors are in clinical development for Alzheimer’s disease (AD) and AD-related dementias (ADRD). RIβ is a brain-specific regulatory subunit of PKA and several RIβ mutations, among them. R335W and L50R, have recently been shown to cause neurological symptoms, including intellectual disability in children and dementia in adults. We recently discovered that RIα, the other RI regulatory subunit of PKA, undergoes liquid-liquid phase separation (LLPS) as a function of cAMP signaling to dynamically buffer cAMP, which is critical for the specificity of the signaling pathway. In our preliminary studies, we showed that cAMP- bound RIβ also undergoes LLPS and the L50R and R335W mutations significantly alter the properties of RIβ condensates. Importantly, PKA is also involved in regulating tau condensates, as phosphorylation of tau by a suite of protein kinases including PKA has been shown to trigger tau LLPS. In addition, specific PKA signaling is critical for CREB mediated transcription and protein degradation, dysregulation of which contribute to AD/ADRD. However, the functional impact of RIβ condensates in AD/ADRD and the interplay between tau and RIβ condensates have not been explored. We assembled a multi-PI team with complementary expertise from two institutions to investigate the molecular and cellular mechanisms governing PKA/RIβ phase-separation and protection from tauopathy and cognitive decline during aging and in disease. To this end, we generated mice carrying epitope-tagged wild-type and pathogenic R335W and L50R alleles of Prkar1b, the gene encoding RIβ. We hypothesize that defective RIβ LLPS and dysregulated interplays between RIβ and tau condensates are critically linked to AD/ADRD. In aim 1, we will use multiple cell types, including cortical cultures from Prkar1b-mutant mice to study the interplay between tau and RIβ condensates using biophysical and imaging approaches. Aim 2 seeks to answer the complementary question how the two phase-separating proteins interact functionally in vivo to influence tau pathology and memory loss in a mouse model of frontotemporal dementia (hTau P301L). Our proposed study will not only develop novel tools and mouse models to study the impact of defective RIβ LLPS and interplays between RIβ and tau condensates, but also establish a collaboration framework for further mechanistic studies of dysregulated biomolecular condensates in AD/ADRD. A better understanding of underlying mechanisms is paramount to identify new therapeutic targets and to improve upon the efficacy of therapeutics under development, such as PDE inhibitors.