Molecular mechanisms regulating PDE11A4 age-related liquid-liquid phase separation and its reversal by selective PDE11A4-targeted small molecules in cells and brain

Age-related increases in hippocampal PDE11A expression occur in humans and rodents, causing age-related cognitive decline of social memories. Interestingly, this age-related increase triggers PDE11A4 liquid-liquid phase separation (LLPS), causing the enzyme to condensate in neurons forming filamentous structures termed "ghost axons". Here we sought to identify molecular mechanisms regulating PDE11A4 LLPS and its reversal by therapeutic agents. PDE11A4 LLPS was increased by broad kinase inhibition and overactivation of the trans-Golgi network (via TGN38 or RhoB) but suppressed by PDE11A4-pS163, PDE11A4-pS239 or a D355A mutation affecting allosteric cGMP-GAF-A interactions. PDE11Ai inhibitors across three scaffolds rapidly reversed PDE11A LLPS independently of their catalytic effects, pS163 or pS239, but rather--at least in part--by reducing PDE11 homodimerization at the GAF-B domain. PDE11Ais blocked exacerbated LLPS caused by age-related PDE11A4 phosphorylation, kinase disruption, and altered proteostasis in hippocampal HT22 neuronal cells. Further, in hippocampus and amygdala of old mice, oral SMQ-03-20 reversed age-related increases in PDE11A4 condensation in ghost axons and protein expression of immunoglobulin light chains (a marker of increased blood-brain permeability), with direct correlation between the two measures. These findings suggest PDE11A4 LLPS is a regulated and druggable mechanism contributing to age-related hippocampal pathology.\