One of the most feared disabilities in survivors of cardiac arrest is neurological impairment. Even brief global cerebral ischemia causes delayed loss of CA1 hippocampal neurons while sparing nearby hippocampal dentate gyrus (DG) neurons. Lack of consideration of the role of astrocytes is thought to be a factor in the failure o many potential stroke therapies aimed only at neuronal survival (Nedergaard and Dirnagl, 2005). In the prior funding period we demonstrated selective dysfunction of hippocampal CA1 astrocytes at early reperfusion times, long before CA1 neuron death. We published the first demonstration that potent protection of CA1 neurons can be achieved by targeting protective proteins selectively to hippocampal astrocytes. Although delayed CA1 neuronal death after transient ischemia is apoptotic, the regulation of the critically important BCL2 family following cerebral ischemia is not well understood. BCL2 family proteins are central regulators of the life/death decision in cells (Adams et al, 2007), and via their regulation of mitochondrial membrane integrity and function control apoptosis signaling. MicroRNAs (miRNAs) are a novel, abundant class of ~22-nucleotide RNAs that control gene expression post- transcriptionally. Although hundreds of miRNAs have been cloned, little is known about their real targets and functions. Numerous miRNAs are expressed in a cell-specific manner and the miR-29 family has been suggested to be astrocyte related (Smirnova et al, 2005). In this proposal we will investigate the role of miR-29 in brain ischemia and neuroprotection, evaluate manipulation of miR-29 as a novel target for neuroprotection, and assess potential targets of miR-29 in the BCL2 family. Using computational miRNA target prediction algorithms we found that miR-29 could potentially target messenger RNAs of several BCL2 family members, both pro- and anti-apoptotic.
Aims 1 and 2 focus on the role of miR-29 in ischemic brain injury. We will first investigate how altering miR-29 expression changes ischemia-induced cell death in vivo and in vitro.
Aim 2 will determine changes in miR-29 family member levels in response to ischemia, including cell type and hippocampal region specific changes with forebrain ischemia. We will then address the mechanisms of the miR-29 effects on cells focusing on mitochondrial function and oxidative stress. The role of miR-29 in selective CA1 astrocyte dysfunction will be tested. In addition to focusing on the role of miR-29 in outcome from injury, we need to investigate downstream targets.
In Aim 3 we focus on 5 likely miR-29 targets, all members of the BCL2 family, focusing especially on pro-apoptotic PUMA. These are likely candidates for effects of miR-29 on mitochondrial function and oxidative stress. Overall this proposal will test the hypothesis that specific microRNAs are a novel and effective target for protection from ischemic brain injury, as well as advancing our understanding of the contributions of astrocyte regulation and impairment to neurological outcome following cardiac arrest and resuscitation.

Public Health Relevance

Cardiac arrest and resuscitation affects about 450,000 patients/year and currently has few treatment options to reduce the devastating neurological complications that often occur. This research is focused on developing novel ways to rapidly change gene expression to protect the brain and improve neurological outcome. This new treatment can be used to target individual cell types that are affected, and might be able to translate rapidly to clinical applications.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-MDCN-E (03))
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Bosetti, Francesca
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Stanford University
Schools of Medicine
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Bell, Josh D; Cho, Jang-Eun; Giffard, Rona G (2017) MicroRNA Changes in Preconditioning-Induced Neuroprotection. Transl Stroke Res 8:585-596
Stary, Creed M; Xu, Lijun; Li, Le et al. (2017) Inhibition of miR-181a protects female mice from transient focal cerebral ischemia by targeting astrocyte estrogen receptor-?. Mol Cell Neurosci 82:118-125
Stary, Creed M; Sun, Xiaoyun; Ouyang, YiBing et al. (2016) miR-29a differentially regulates cell survival in astrocytes from cornu ammonis 1 and dentate gyrus by targeting VDAC1. Mitochondrion 30:248-54
Xu, Li-Jun; Ouyang, Yi-Bing; Xiong, Xiaoxing et al. (2015) Post-stroke treatment with miR-181 antagomir reduces injury and improves long-term behavioral recovery in mice after focal cerebral ischemia. Exp Neurol 264:1-7
Ouyang, Yi-Bing; Stary, Creed M; White, Robin E et al. (2015) The use of microRNAs to modulate redox and immune response to stroke. Antioxid Redox Signal 22:187-202
Stary, Creed M; Sun, Xiaoyun; Giffard, Rona G (2015) Astrocytes Protect against Isoflurane Neurotoxicity by Buffering pro-brain-derived Neurotrophic Factor. Anesthesiology 123:810-9
Stary, Creed M; Giffard, Rona G (2015) Advances in astrocyte-targeted approaches for stroke therapy: an emerging role for mitochondria and microRNAS. Neurochem Res 40:301-7
Ouyang, Yi-Bing; Xu, Lijun; Liu, Siwei et al. (2014) Role of Astrocytes in Delayed Neuronal Death: GLT-1 and its Novel Regulation by MicroRNAs. Adv Neurobiol 11:171-88
Ouyang, Yi-Bing; Giffard, Rona G (2014) MicroRNAs affect BCL-2 family proteins in the setting of cerebral ischemia. Neurochem Int 77:2-8
Ouyang, Yi-Bing; Xu, Lijun; Yue, Sibiao et al. (2014) Neuroprotection by astrocytes in brain ischemia: importance of microRNAs. Neurosci Lett 565:53-8

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