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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 StatementThe 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.

Objective Neuroimaging and other biomarker assays suggest that the pathological processes of Alzheimer’s disease (AD) initiate years prior to clinical dementia onset. However some 30%–50% of older individuals that harbor AD pathology do not become symptomatic in their lifetime. It is hypothesized that such individuals exhibit cognitive resilience that protects against AD dementia. We hypothesized that in cases with AD pathology structural changes in dendritic spines would distinguish individuals that had or did not have clinical dementia.

Alzheimer's disease (AD) is the leading cause of dementia and mitigating amyloid-β (Aβ) levels may serve as a rational therapeutic avenue to slow AD progression. Pharmacologic inhibition of the Rho-associated protein kinases (ROCK1 and ROCK2) is proposed to curb Aβ levels, and mechanisms that underlie ROCK2's effects on Aβ production are defined. How ROCK1 affects Aβ generation remains a critical barrier. Here, we report that ROCK1 protein levels were elevated in mild cognitive impairment due to AD (MCI) and AD brains compared to controls. Aβ42 oligomers marginally increased ROCK1 and ROCK2 protein levels in neurons but strongly induced phosphorylation of Lim kinase 1 (LIMK1), suggesting that Aβ42 activates ROCKs. RNAi depletion of ROCK1 or ROCK2 suppressed endogenous Aβ40 production in neurons, and Aβ40 levels were reduced in brains of ROCK1 heterozygous knock-out mice compared to wild-type littermate controls. ROCK1 knockdown decreased amyloid precursor protein (APP), and treatment with bafilomycin accumulated APP levels in neurons depleted of ROCK1. These observations suggest that reduction of ROCK1 diminishes Aβ levels by enhancing APP protein degradation. Collectively, these findings support the hypothesis that both ROCK1 and ROCK2 are therapeutic targets to combat Aβ production in AD. Mitigating amyloid-β (Aβ) levels is a rational strategy for Alzheimer's disease (AD) treatment, however, therapeutic targets with clinically available drugs are lacking. We hypothesize that Aβ accumulation in mild cognitive impairment because of AD (MCI) and AD activates the RhoA/ROCK pathway which in turn fuels production of Aβ. Escalation of this cycle over the course of many years may contribute to the buildup of amyloid pathology in MCI and/or AD.

Progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) are neurodegenerative four-repeat tauopathies with no cure. Mitigating pathogenic tau levels is a rational strategy for tauopathy treatment, but therapeutic targets with clinically available drugs are lacking. Here, we report that protein levels of the Rho-associated protein kinases (ROCK1 and ROCK2), p70 S6 kinase (S6K), and mammalian target of rapamycin (mTOR) were increased in PSP and CBD brains. RNAi depletion of ROCK1 or ROCK2 reduced tau mRNA and protein level in human neuroblastoma cells. However, additional phenotypes were observed under ROCK2 knockdown, including decreased S6K and phosphorylated mTOR levels. Pharmacologic inhibition of Rho kinases in neurons diminished detergent-soluble and -insoluble tau through a combination of autophagy enhancement and tau mRNA reduction. Fasudil, a clinically approved ROCK inhibitor, suppressed rough eye phenotype and mitigated pathogenic tau levels by inducing autophagic pathways in aDrosophilamodel of tauopathy. Collectively, these findings highlight the Rho kinases as rational therapeutic targets to combat tau accumulation in PSP and CBD.SIGNIFICANCE STATEMENTStudies of progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) suggest that mitigating pathogenic tau levels is a rational strategy for tauopathy treatment. In this report, the Rho-associated protein kinases (ROCK1 and ROCK2) are identified as novel drug targets for PSP and CBD. We show that elevated insoluble tau levels are associated with increased ROCK1 and ROCK2 in PSP and CBD brains, whereas experiments in cellular and animal models identify pharmacologic inhibition of ROCKs as a mechanism-based approach to reduce tau levels. Our study correlates bona fide changes in PSP and CBD brains with cellular models, identifies drug targets, and tests the therapeuticin vivo.

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. The ROCKs heavily influence neuronal architecture and structural plasticity, and ROCKs are putative drug targets for various brain disorders. While the pan-ROCK inhibitor Fasudil has been clinically approved to treat hypertension, heart failure, glaucoma, spinal cord injury, and stroke, a barrier to progress on this therapeutic avenue is the lack of experimental comparisons between pharmacologic and genetic manipulation of ROCKs. Our study begins to address this question using parallel approaches to study behavior in mice that were treated with Fasudil or were heterozygous for ROCK1 or ROCK2. Adult mice treated with Fasudil for thirty days displayed reduced time spent in the open arms of the elevated plus maze, whereas activity in the open field was more analogous to mock-treated animals. Both male and female adult ROCK1(+/−) and ROCK2(+/−) mice exhibited reduced time spent in open arms of the elevated plus maze compared to littermate controls. However, ROCK1 or ROCK2 heterozygosity did not alter performance in the open field or Y-maze. These results indicate that chronic treatment with Fasudil induces anxiety-like behaviors that are likely the consequence of ROCK1 and/or ROCK2 inhibition. Our findings may have implications for several ongoing clinical trials using Fasudil or other ROCK-based therapeutics.

Communication among neurons is mediated through synaptic connections between axons and dendrites, and most excitatory synapses occur on actin-rich protrusions along dendrites called dendritic spines. Dendritic spines are structurally dynamic, and synapse strength is closely correlated with spine morphology. Abnormalities in the size, shape, and number of dendritic spines are prevalent in neurologic diseases, including autism spectrum disorders, schizophrenia, and Alzheimer disease. However, therapeutic targets that influence spine morphology are lacking. Rho-associated coiled-coil containing protein kinases (ROCK) 1 and ROCK2 are potent regulators of the actin cytoskeleton and highly promising drug targets for central nervous system disorders. In this report, we addressed how pharmacologic inhibition of ROCK1 and ROCK2 affects dendritic spine morphology. Hippocampal neurons were transfected with plasmids expressing fluorescently labeled Lifeact, a small actin binding peptide, and then incubated with or without Y-27632, an established pan-ROCK small molecule inhibitor. Using an automated 3D spine morphometry analysis method, we showed that inhibition of ROCK1 and ROCK2 significantly increased the mean protrusion density and significantly reduced the mean protrusion width. A trending increase in mean protrusion length was observed following Y-27632 treatment, and novel effects were observed among spine classes. Exposure to Y-27632 significantly increased the number of filopodia and thin spines, while the numbers of stubby and mushroom spines were similar to mock-treated samples. These findings support the hypothesis that pharmacologic inhibition of ROCK1 and ROCK2 may convey therapeutic benefit for neurologic disorders that feature dendritic spine loss or aberrant structural plasticity.