Conditional deletion of ROCK2 induces anxiety-like behaviors and alters dendritic spine density and morphology on CA1 pyramidal neurons

Audrey J Weber,Jeremy H Herskowitz,Cameron D Freeman,Julia P Andrade,Kelsey M Greathouse,Courtney K Walker,Jung Vin Seo,Rosaria J Rae,Ashley B Adamson

doi:10.1186/s13041-021-00878-4

Abstract

Rho-associated kinase isoform 2 (ROCK2) is an attractive drug target for several neurologic disorders. A critical barrier to ROCK2-based research and therapeutics is the lack of a mouse model that enables investigation of ROCK2 with spatial and temporal control of gene expression. To overcome this, we generated ROCK2fl/fl mice. Mice expressing Cre recombinase in forebrain excitatory neurons (CaMKII-Cre) were crossed with ROCK2fl/fl mice (Cre/ROCK2fl/fl), and the contribution of ROCK2 in behavior as well as dendritic spine morphology in the hippocampus, medial prefrontal cortex (mPFC), and basolateral amygdala (BLA) was examined. Cre/ROCK2fl/fl mice spent reduced time in the open arms of the elevated plus maze and increased time in the dark of the light–dark box test compared to littermate controls. These results indicated that Cre/ROCK2fl/fl mice exhibited anxiety-like behaviors. To examine dendritic spine morphology, individual pyramidal neurons in CA1 hippocampus, mPFC, and the BLA were targeted for iontophoretic microinjection of fluorescent dye, followed by high-resolution confocal microscopy and neuronal 3D reconstructions for morphometry analysis. In dorsal CA1, Cre/ROCK2fl/fl mice displayed significantly increased thin spine density on basal dendrites and reduced mean spine head volume across all spine types on apical dendrites. In ventral CA1, Cre/ROCK2fl/fl mice exhibited significantly increased spine length on apical dendrites. Spine density and morphology were comparable in the mPFC and BLA between both genotypes. These findings suggest that neuronal ROCK2 mediates spine density and morphology in a compartmentalized manner among CA1 pyramidal cells, and that in the absence of ROCK2 these mechanisms may contribute to anxiety-like behaviors.

Highlights

Rho-associated coiled-coil containing kinase isoform 2 (ROCK2) belongs to the AGC serine/threonine kinase family and was originally identified as an interacting partner of GTP-bound RhoA
ROCK2 deficiency induces anxiety‐like behaviors ROCK2fl/fl mice were generated from C57BL/6NRock2tm1a(KOMP)Wtsi ES cells that were purchased from the NIH Knock-Out Mouse Project (KOMP) and obtained from the KOMP Repository
To eliminate ROCK2 from forebrain excitatory neurons, ROCK2fl/fl mice were crossed with B6.CgTg(Camk2a-cre)T29-1Stl/J mice (CaMKII-Cre) to yield experimental animals that are positive for Cre recombinase (Cre/ROCK2fl/fl) or ROCK2fl/fl littermate controls that are negative for Cre recombinase (Fig. 1a) [35]

Summary

Introduction

Rho-associated coiled-coil containing kinase isoform 2 (ROCK2) belongs to the AGC serine/threonine kinase family and was originally identified as an interacting partner of GTP-bound RhoA. ROCK1 and ROCK2 are established mediators of actin–myosin cytoskeleton contractility [1,2,3,4,5]. While both kinase isoforms share similar expression patterns, ROCK1 transcript levels are most abundant in thymus and blood, and ROCK2 is most prominent in brain [6]. Special emphasis is placed on ROCK2 as a target for neurologic conditions, including Alzheimer’s disease, Frontotemporal Dementia, Parkinson’s disease, Amyotrophic Lateral Sclerosis, glaucoma, and stroke [13,14,15,16,17,18,19,20,21,22]

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Conclusion