Distinct and complementary functions of rho kinase isoforms ROCK1 and ROCK2 in prefrontal cortex structural plasticity

Kelsey M. Greathouse, Benjamin D. Boros, Josue F. Deslauriers, Benjamin W. Henderson, Kendall A. Curtis, Erik G. Gentry, Jeremy H. Herskowitz

Rho-associated protein kinases (ROCK) 1 and 2 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 by analyzing both male and female adult ROCK1+/- and ROCK2+/- mice 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 basal dendritic length and intersections were observed in ROCK1+/- but not ROCK2+/- mice. Although dendritic spine densities were comparable among genotypes, apical spine length 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 in synaptic fractions revealed that phosphorylation of LIM kinase and cofilin were reduced in ROCK1+/- and ROCK2+/- mice, while phosphorylation of myosin light chain was decreased exclusively in ROCK1+/- mice. 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 structure.