Dendritic spines provide cognitive resilience against Alzheimer's disease.

OBJECTIVE: Neuroimaging and other biomarker assays suggest that the pathological processes of Alzheimer's disease (AD) begin years prior to clinical dementia onset. However, some 30 to 50% of older individuals who 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 who had or did not have clinical dementia. METHODS: We compared dendritic spines within layer II and III pyramidal neuron dendrites in Brodmann area 46 dorsolateral prefrontal cortex using the Golgi-Cox technique in 12 age-matched pathology-free controls, 8 controls with AD pathology (CAD), and 21 AD cases. We used highly optimized methods to trace impregnated dendrites from bright-field microscopy images that enabled accurate 3-dimensional digital reconstruction of dendritic structure for morphologic analyses. RESULTS: Spine density was similar among control and CAD cases but was reduced significantly in AD. Thin and mushroom spines were reduced significantly in AD compared to CAD brains, whereas stubby spine density was decreased significantly in CAD and AD compared to controls. Increased spine extent distinguished CAD cases from controls and AD. Linear regression analysis of all cases indicated that spine density was not associated with neuritic plaque score but did display negative correlation with Braak staging. INTERPRETATION: These observations provide cellular evidence to support the hypothesis that dendritic spine plasticity is a mechanism of cognitive resilience that protects older individuals with AD pathology from developing dementia. Ann Neurol 2017;82:602-614.

Rho Kinase Inhibition as a Therapeutic for Progressive Supranuclear Palsy and Corticobasal Degeneration.

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 a Drosophila model of tauopathy. Collectively, these findings highlight the Rho kinases as rational therapeutic targets to combat tau accumulation in PSP and CBD. SIGNIFICANCE STATEMENT: Studies 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 therapeutic in vivo.

Rho-associated protein kinase 1 (ROCK1) is increased in Alzheimer's disease and ROCK1 depletion reduces amyloid-ß levels in brain.

Alzheimer's disease (AD) is the leading cause of dementia and mitigating amyloid-ß (Aß) 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 Aß levels, and mechanisms that underlie ROCK2's effects on Aß production are defined. How ROCK1 affects Aß 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. Aß42 oligomers marginally increased ROCK1 and ROCK2 protein levels in neurons but strongly induced phosphorylation of Lim kinase 1 (LIMK1), suggesting that Aß42 activates ROCKs. RNAi depletion of ROCK1 or ROCK2 suppressed endogenous Aß40 production in neurons, and Aß40 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 Aß levels by enhancing APP protein degradation. Collectively, these findings support the hypothesis that both ROCK1 and ROCK2 are therapeutic targets to combat Aß production in AD. Mitigating amyloid-ß (Aß) levels is a rational strategy for Alzheimer's disease (AD) treatment, however, therapeutic targets with clinically available drugs are lacking. We hypothesize that Aß accumulation in mild cognitive impairment because of AD (MCI) and AD activates the RhoA/ROCK pathway which in turn fuels production of Aß. Escalation of this cycle over the course of many years may contribute to the buildup of amyloid pathology in MCI and/or AD.

Disparities in spatial access to neurological care in Appalachia: a cross-sectional health services analysis.

Background: Appalachia is rural and socioeconomically deprived with a heavy burden of neurological disorders and poor access to healthcare providers. Rates of neurological disorders are increasing over time without equal increases in providers, indicating that Appalachian disparities are likely to worsen. Spatial access to neurological care has not been robustly explored for U.S. areas, so we aimed to examine disparities in the vulnerable Appalachian region. Methods: Using 2022 CMS Care Compare physician data, we conducted a cross-sectional health services analysis, where we computed spatial accessibility of neurologists for all census tracts in the 13 states with Appalachian counties. We stratified access ratios by state, area deprivation, and rural-urban commuting area (RUCA) codes then utilized Welch two-sample t-tests to compare Appalachian tracts with non-Appalachian tracts. Using stratified results, we identified Appalachian areas where interventions would have the largest impact. Findings: Appalachian tracts (n = 6169) had neurologist spatial access ratios between 25% and 35% lower than non-Appalachian tracts (n = 18,441; p < 0.001). When stratified by rurality and deprivation, three-step floating catchment area spatial access ratios for Appalachian tracts remained significantly lower in the most urban (RUCA = 1 [p < 0.0001) and most rural tracts (RUCA = 9 [p = 0.0093]; RUCA = 10 [p = 0.0227]). We identified 937 Appalachian census tracts where interventions can be targeted. Interpretation: After stratifying by rural status and deprivation, significant disparities in spatial access to neurologists remained for Appalachian areas, indicating both poorer access in Appalachia and that neurologist accessibility cannot be determined solely by remoteness and socioeconomic status. These findings and our identified disparity areas have broad implications for policymaking and intervention targeting in Appalachia. Funding: R.B.B. was supported by NIH Award Number T32CA094186. M.P.M. was supported by NIH-NCATS Award Number KL2TR002547.

Fasudil or genetic depletion of ROCK1 or ROCK2 induces anxiety-like behaviors.

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.

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

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.

ROCK1 and ROCK2 inhibition alters dendritic spine morphology in hippocampal neurons.

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.