ncRNAs and PPARy-Induced Sympathetic Neuroprotection in an NHP Model of Cardiac Neurodegeneration

University Of Wisconsin-Madison, Madison WI

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Abstract

PROJECT SUMMARY Cardiac sympathetic innervation is critical to regulating heart activity in response to changes in posture or exercise that require adjustments in blood pressure or heart rate. Neurodegeneration of the cardiac sympathetic system is a common feature of diabetes, idiopathic REM sleep disorder, and Parkinson's disease (PD) that affects patients' well being. For example, cardiac sympathetic loss is found in 80-90% of PD patients, is associated with increased risk of injury due to falls and is worsened by dopamine replacement therapy. Our research group developed a nonhuman primate (NHP) model of cardiac sympathetic denervation induced by systemic administration of the catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA). Using cardiac positron emission tomography (PET) in this model we demonstrated that the peroxisome proliferator-activated receptor gamma (PPAR?) agonist pioglitazone induces neuroprotection in association with decreased inflammation and oxidative stress. NFkappaB (NF?B) is a transcription factor considered the `master switch' of pro-inflammatory effector molecules that contribute to neurodegeneration. PPAR? activation has been shown to inhibit NF?B by inducing transcription of non-protein coding RNAs (ncRNAs) that target NF?B. We hypothesize that PPAR? activation during peripheral sympathetic neurodegeneration in NHPs induces differential expression of ncRNA transcripts that regulate NF?B in leukocytes, resulting in decreased pro- inflammatory cytokines and reactive oxygen species, leading to preservation of cardiac sympathetic innervation. Our specific aims are: 1) To assess expression levels of NF?B and downstream pro-inflammatory effector molecules (IL1?, TNF?, IL8, IL6, iNOS, and NADPH oxidase) in total peripheral blood by mRNA microarray and in the left ventricle by immunohistochemistry in 6-OHDA-treated NHPs treated with either pioglitazone or placebo and, 2) To identify in peripheral blood of 6-OHDA-treated NHPs differentially expressed overtime miRNA and lncRNA transcripts that target the NF?B pathway associated with PPAR? activation. Immunohistochemical analysis will include quantification of myocardial nerves positive for tyrosine hydroxylase, PGP9.5 and NF?B pathway markers. RNA will be extracted from total peripheral blood and analyzed by mRNA, microRNA (miRNA) and long non-coding RNA (lncRNA) microarrays. Downstream bioinformatics analysis of miRNA data will focus on identification of transcripts targeting NF?B. Analysis of differentially expressed lncRNAs will include sequence comparison to exons of genes known to be part of the NF?B pathway and analysis of lncRNA upstream promoter sequences to identify potential regulators. Immunohistochemistry and molecular data will be correlated with in vivo PET imaging to provide an integrated vision of mechanisms of sympathetic cardiac neurodegeneration and neuroprotection. Elucidation of these mechanisms will be critical to the development of novel neuroprotective strategies and associated biomarkers of target validation and therapeutic efficacy.