Distinct and Complementary Functions of Rho kinase isoforms ROCK1 and ROCK2 in Prefrontal Cortex Structural Plasticity

Abstract Twenty-nine protein kinase inhibitors have been used to treat human diseases. Out of these, two are Rho-associated protein kinase (ROCK) 1 and 2 inhibitors. ROCKs are attractive drug targets for a range of neurologic disorders; however a critical barrier to ROCK-based therapeutics is ambiguity over whether there are isoform-specific roles for ROCKs in neuronal structural plasticity. Here, we used a genetics approach to address this long-standing question. Both male and female adult ROCK1+/− and ROCK2+/− mice exhibited anxiety-like behaviors compared to littermate controls. Individual pyramidal neurons in the medial prefrontal cortex (mPFC) were targeted for iontophoretic microinjection of fluorescent dye, followed by high-resolution confocal microscopy and neuronal 3D reconstructions for morphometry analysis. Increased apical and basolateral dendritic length and intersections were observed in ROCK1+/− but not ROCK2+/− mice. Although dendritic spine densities were comparable among genotypes, apical spine extent was decreased in ROCK1+/− but increased in ROCK2+/− mice. Spine head and neck diameter were reduced similarly in ROCK1+/− and ROCK2+/− mice; however certain spine morphologic subclasses were more affected than others in a genotype-dependent manner. Biochemical analyses of ROCK substrates revealed that phosphorylation of LIM kinase was reduced in synaptic fractions from ROCK1+/− or ROCK2+/− mice, correlating to overlapping spine morphology phenotypes. Collectively, these observations implicate ROCK1 as a novel regulatory factor of neuronal dendritic structure and detail distinct and complementary roles of ROCKs in mPFC dendritic spine structural plasticity. This study provides a fundamental basis for current and future development of isoform-selective ROCK inhibitors to treat neurologic disorders. Significance Statement The Rho-associated protein kinases (ROCK) 1 and 2 heavily influence neuronal architecture and synaptic plasticity. ROCKs are exciting drug targets and pan-ROCK inhibitors are clinically approved to treat hypertension, heart failure, glaucoma, spinal cord injury, and stroke. However development of isoform-specific ROCK inhibitors is hampered due to ambiguity over ROCK1- or ROCK2-specific functions in the brain. Our study begins to address this critical barrier and demonstrates that ROCK1 can mediate the dendritic arbor of neurons while both ROCK1 and ROCK2 heavily influence dendritic spine morphology. This study highlights distinct and complementary roles for ROCK1 and ROCK1 in prefrontal cortex structural plasticity and provides a fundamental basis for future development of isoform-selective ROCK inhibitors to treat neurologic disorders.