Selective drug inhibition of rho kinase II (ROCK2) reduces beta-amyloid production in the brain

Oral Sessions: O1-11: Therapeutics: Preclinical Studies—APP, Beta-Amyloid and APOE worse than apoE4-TR mice. Ab12-28P treatment of APP SW/PS1 dE9/ apoE4-TR mice normalized OR behavior and improved RAM performance to the level of apoE4-TR mice. Levels of soluble and insoluble Ab x-40 and Ab x-42 measured by ELISA in the whole brain extract and the burden of Thioflavin-S positive plaques were significantly higher in APP SW/PS1 dE9/apoE4-TR than in APP SW/PS1 dE9/apoE2-TR mice. Ab12-28P treatment was associated with significant reduction in soluble and insoluble Ab x-40 and Ab x-42 levels and lower Thioflavin-S plaque burden in the neocortex and in the hippocampus in both APP SW/PS1 dE9/apoE2-TR and APP SW/PS1 dE9/apoE4-TR lines. Conclusions: Our results indicate that future therapies targeting the apoE/Ab interaction could produce favorable outcome in both APOE2 and APOE4 carriers. O1-11-06

SELECTIVE DRUG INHIBITION OF RHO KINASE II (ROCK2) REDUCES BETA-AMYLOID PRODUCTION IN THE BRAIN

Jeremy Herskowitz1, Yangbo Feng2, Lenora Higginbotham1, Nicholas Seyfried1, Allan Levey3, James Lah1, 1Emory University, Atlanta, Georgia, United States; 2The Scripps Research Institute, Jupiter, Florida, United States; 3Emory University School of Medicine, Atlanta, Georgia, United States. Contact e-mail: [email protected] Background: There is strong evidence to support the hypothesis that overproduction and accumulation of amyloid-b (Ab) is a driving factor in Alzheimer’s disease (AD) progression. Ab can reduce synaptic transmission and promote synapse loss, and therefore targeting proteins that simultaneously influence synaptic plasticity and Ab production may lead to the de-

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velopment of therapeutics. The Rho GTPases regulate neuronal morphology and synaptic plasticity through their principle downstream effectors, Rhoassociated coiled-coil containing protein kinases (ROCK) 1 and ROCK2. ROCK substrates are elevated in sporadic AD brains and drug inhibition of ROCKs can reduce Ab levels. Methods: Models: human neuroblastoma cells, primary neuronal cultures, 5XFAD mice Methodologies and Reagents: lentivirus delivery of shRNA, small molecule kinase inhibitors, stereotaxic injection, phosphoproteomics, in vitro kinase assay, site-directed mutagenesis, biochemical analysis of postmortem human brain tissue from asymptomatic AD, mild cognitive impairment (MCI), sporadic AD, and age-matched control cases. Results: We report that shRNA-mediated depletion of ROCK1 increased production of endogenous Ab in human neuroblastoma cells, whereas ROCK2 depletion reduced Ab levels and promoted degradation of the amyloid precursor protein (APP). Similarly in neurons, treatment with SR3677, a selective drug inhibitor of ROCK2, reduced full-length APP and Ab levels in a dose-dependent manner, and stereotaxis injection of SR3677 decreased brain levels of soluble Ab in 5XFAD mice. In vitro experiments revealed that ROCK2 can directly phosphorylate the cytoplasmic tail of APP at threonine 654 (T654) and characterization of APP mutants suggest that T654 is critical for the generation of Ab in neurons. Finally, immunoblot analysis of asymptomatic AD, mild cognitive impairment (MCI), and sporadic AD brains revealed that ROCK1 and ROCK2 protein levels are increased in disease cases compared to age-matched controls. Conclusions: Our findings highlight ROCK2 as a potentially new and exciting therapeutic target to combat the production of Ab in AD progression.