O2‐12‐04: Rho kinase II (ROCK2) inhibition reduces tau levels via autophagy in neurons

Abstract

Background: A predominant pathology of sporadic and familial Frontotemporal dementia (FTD) is neuronal inclusions, comprised of hyperphosphorylated, aggregated microtubule-associated protein tau. Aggregated tau can be toxic to neurons, reducing synaptic transmission, promoting synapse loss, and driving cell death. It is proposed that activating intracellular protein degradation pathways may be a rational therapeutic avenue to reduce tau aggregation in Tauopathies. Activity of Rho-associated coiled-coil containing protein kinases (ROCK) 1 and ROCK2 is increased in sporadic FTD and Alzheimer's disease (AD) cases, and drug inhibition of ROCKs can induce protein degradation pathways in mammalian cells. Methods: Models: primary neuronal cultures, human neuroblastoma cells. Methodologies and Reagents: small molecule kinase inhibitors, lentivirus delivery of shRNA, site-directed mutagenesis, biochemical analysis of postmortem human brain tissue from late-onset AD, age-matched control cases, and FTD-tau, including FTDP-17 (P301L), Pick's disease, corticobasal degeneration (CBD), progressive supranuclear palsy (PSP). Results: In neurons, treatment with SR3677, a selective drug inhibitor of ROCK2, dramatically reduced endogenous tau protein level in a dose-dependent manner. In contrast, Fasudil, a pan-ROCK inhibitor, had more modest effects. Exposure to SR3677, but not Fasudil, decreased p62, phospho-mTOR, and p70 S6 Kinase levels in neurons, suggesting that ROCK2 inhibition promotes autophagymediated degradation of tau. ROCK2 depletion by shRNA mimicked effects of SR3677 on tau levels as well as autophagy induction in human neuroblastoma cells. ROCK1 depletion reduced tau levels but had no observable effect on autophagy. ROCK2-shRNA rescue experiments revealed that ROCK2 mutants with amino acid substitutions in the ATP-binding site could not restore tau levels, indicating that ROCK2 kinase activity is required for effects on tau. Finally, immunoblot analysis of postmortem human prefrontal cortex tissue samples from FTD patients, including FTDP-17 (P301L), Pick's disease, CBD, and PSP, revealed changes in autophagy markers as well as Rho kinases compared to age-matched controls. Conclusions: Our findings highlight ROCK2 as a potentially new and exciting therapeutic target to reduce tau protein level in Tauopathies.