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. Methods We compared dendritic spines within layers 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 brightfield microscopy images which enabled accurate three-dimensional digital reconstruction of dendritic structure for morphologic analyses. Results Spine density was similar among control and CAD cases but 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.

Subtle alterations in dendritic spine morphology can induce marked effects on connectivity patterns of neuronal circuits and subsequent cognitive behavior. Past studies of rodent and non-human primate aging revealed reductions in spine density with concomitant alterations in spine morphology among pyramidal neurons in the prefrontal cortex. In this report, we visualized and digitally reconstructed the three-dimensional morphology of dendritic spines from the dorsolateral prefrontal cortex in cognitively normal individuals aged 40–94 years. Linear models defined relationships between spines and age, Mini–Mental State Examination (MMSE), APOE ε4 allele status, and Alzheimer’s disease (AD) pathology. Similar to findings in other mammals, spine density correlated negatively with human aging. Reduced spine head diameter associated with higher MMSE scores. Individuals harboring an APOE ε4 allele displayed greater numbers of dendritic filopodia and structural alterations in thin spines. The presence of AD pathology correlated with increased spine length, reduced thin spine head diameter, and increased filopodia density. Our study reveals how spine morphology in the prefrontal cortex changes in human aging and highlights key structural alterations in selective spine populations that may promote cognitively normal function despite harboring the APOE ε4 allele or AD pathology.

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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.

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.

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