Rho Kinase Pathway Alterations in the Brain and Leukocytes in Huntington's Disease.

K Lakshmi Narayanan,Vanita Chopra,Keith Malarick,Steven Hersch,H Diana Rosas

doi:10.1007/s12035-015-9147-9

Abstract

Huntington’s disease (HD) is a fatal neurodegenerative disease caused by an expanded polyglutamine tract in the huntingtin gene. Therapeutic approaches targeting mutant huntingtin (mtHtt) or its downstream toxic consequences are under development, including Rho kinase pathway inhibition. We investigated the messenger RNA (mRNA) expression of Rho kinase pathway genes, including RhoA (Ras homolog family member A), ROCK1 (Rho-associated kinase1), PRK2 (protein kinase C-related protein kinase 2), Profilin1, cofilin1, MYPT1 (myosin phosphatase target subunit 1), and LIMK1 (LIM domain kinase 1) in HD human blood leukocytes, postmortem brain, and in R6/2 HD mouse brain tissue using qPCR. RhoA, ROCK1, PRK2, Profilin1, cofilin1, and MYPT1 were significantly increased in HD blood compared to controls. In frontal cortex of HD postmortem brain tissue, the expression of RhoA, ROCK1, PRK2, Profilin1, and MYPT1 were also significantly increased. In the brain from 4-week-old R6/2 mice, the expression of Rock1, Prk2, Cofilin1, and MYPT1 was significantly increased while RhoA, Rock1, Profilin1, Cofilin1, and Mypt1 were increased and Limk1 mRNA decreased in 13-week-old R6/2 mice. Western blot analysis using human postmortem tissues for ROCK1 and Profilin1 demonstrated significantly increased protein levels, which correlated with the mRNA increases. Collectively, we have shown the panel of Rho kinase pathway genes to be highly altered in human HD blood, postmortem brain tissue, and in R6/2 mice. These studies confirm that HD upregulates the Rho kinase pathway and identifies mRNAs that could serve as peripheral markers in HD patients and translational markers in HD mouse models.

Highlights

Huntington’s disease (HD) is a fatal, autosomal dominant disorder caused by a pathologic expansion of CAG repeats in the IT15 gene, which encodes the ubiquitously expressed huntingtin protein [1]
To determine whether Rho kinase pathway genes are altered in HD, we extracted RNA from age-matched symptomatic HD and healthy controls blood samples. Complementary DNA (cDNA) were synthesized and we carried out qPCR for the panel of genes in the Rho kinase pathway
To assess whether the Rho kinase pathway alterations in the blood are consistent with changes in HD brain, we extracted RNA from the frontal cortex of postmortem HD brain tissue and controls. cDNAwas synthesized and we carried out qPCR for the panel of genes from the Rho kinase pathway

Summary

Introduction

Huntington’s disease (HD) is a fatal, autosomal dominant disorder caused by a pathologic expansion of CAG repeats in the IT15 gene, which encodes the ubiquitously expressed huntingtin protein [1]. While there are not yet any disease modifying treatments that have been demonstrated to delay or slow HD in patients, a number of potential targets have been identified and validated preclinically in HD models. One such promising target is inhibition of the Rho kinase pathway, which reduces mtHtt aggregation, enhances huntingtin clearance, and improves motor function in the R6/2 transgenic model of HD [3, 4], perhaps by modulating p21-activated kinase (Pak1), a downstream target protein of the Rho family that enhances mtHtt aggregation in cell culture and colocalizes with mtHtt in postmortem HD brain [5]. To assess the potential of Rho kinase genes as potential peripheral markers for HD in human or as pharmacodynamics markers to enable developing treatments targeting the Rho kinase pathway, we used quantitative real-time PCR of RhoA, ROCK1, PRK2, Profilin, cofilin, MYPT1, and LIMK1 to examine the pathway in more detail in leukocytes from HD patients as well as in human postmortem and R6/2 transgenic mouse brain tissue

Methods

Results

Conclusion