REST, also known as NRSF, is a gene silencing factor that is widely expressed in pluripotent stem cells and neural progenitors, where it actively represses a large array of coding and non-coding genes important to synaptic plasticity and structural remodeling including microRNAs. The interplay between REST and microRNAs is a new, emerging topic1-4. Emerging evidence indicates that microRNAs play a key role in regulation of K+ channel expression. Our findings that neuronal insults trigger induction of REST in mature hippocampal neurons and REST- dependent silencing/attenuation of miR-132 and Kv7.1 expression, target genes essential for neuronal function, implicate REST in the pathogenesis of ischemic stroke11. Our finding that overexpression of miR-132 in hippocampal neurons affords robust protection documents a causal relation between loss of miR-132 and neuronal death and underscores the clinical relevance of our work11. The overall objective is to examine a potential role for two validated REST targets miR132 and KCNQ1/Kv7.1, which we recently discovered in neurons, in the neurodegeneration and cognitive impairment associated with global ischemia. The central hypothesis driving the research is that global ischemia upregulates REST in adult neurons and that REST promotes silencing of a subset of transcriptionally-responsive and gene networks that maintain the balance between neuronal death and survival. We seek to test this hypothesis in the following Specific Aims: 1) Examine a role for miR-132, a candidate REST target, in ischemia-induced neuronal death. Experiments will 1) verify that miR-132 exhibits REST-dependent epigenetic remodeling and altered gene expression in response to ischemic insults; 2) Determine whether REST is causally related to ischemia-induced alterations in miR-132 expression and/or function in insulted CA1 neurons by overexpression or inhibition of REST, including a floxed REST KO mouse; 3) Determine whether miR-132 is causally related to ischemia-induced neuronal death; 4) Identify novel miR-132 targets that promote neuronal death by miR pull-down assay; 5) Examine the ability of ischemic preconditioning to prevent ischemia-induced activation of REST, suppression of miR-132 and rescue of neuronal death and cognitive deficits 2) Determine whether the Kv7/KCNQ family of voltage-gated K+ channels, novel REST target genes, are causally related to ischemia-induced neuronal death. Experiments will: 1) Document the presence of functional KCNQ1 channels, a candidate REST target, in under control and ischemic conditions; 2) Validate the impact of ischemia on altered REST binding and epigenetic modifications at Kv7.1-3 promoters by ChIP; 3) Validate alterations in gene expression of Kv7.1-3 by qPCR, protein by Westerns and function by electrophysiology; 4) Examine the examine ability of genetic manipulation of REST to mimic or prevent ischemia-induced alterations in Kv7.1-3 expression and/or function by genetic manipulation, including a REST cKO mouse; 5) Examine the ability of genetic manipulation of Kv7.1 to influence neuronal death and cognitive deficits; and 6) Examine the ability of novel microRNAs, such as 133 and 375, expressed in neurons and putative targets of REST, to regulate KCNQ1 expression and function in insulted neurons.
Global ischemia arising as a consequence of cardiac arrest affects ~200,000 Americans per year and in many cases results in neurological deficits. To date, treatment of the neurodegeneration and cognitive deficits associated with global ischemia is an unmet need. These translational studies will accelerate the development of novel therapeutic strategies to ameliorate this serious cause of human morbidity and mortality. Findings from these studies have broad implications not only for ischemic stroke, but also for other neurodegenerative disorders including spinal cord injury, ALS, Huntington's disease, Parkinson's disease and Alzheimer's disease.
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