As a leading neurological disorder, acute cerebral ischemia accounts for approximately 80% of all human strokes and has a major impact on public health. Understanding the pathophysiology is essential to develop therapeutic avenues to minimize brain damage. Thus, the project goal is to determine the novel role of astrocytes in a mouse model of ischemia-induced neuronal death and brain damage. Our central hypothesis is that astrocytes induce neuronal excitotoxic responses through enhanced Ca2+-dependent glutamate release (gliotransmission) and consequently contribute to ischemia-induced neuronal death and brain damage. A variety of state-of-the-art technologies including 2-P microscopy, electrophysiology, viral transduction and transgenic mice will be used to test this hypothesis. We have three SPECIFIC hypotheses: 1) Focal ischemia induces enhanced Ca2+ excitability in astrocytes in the ischemic core as well as in the penumbra and mediates glutamate release from these glial cells. Using 2-P in vivo Ca2+ imaging we will study the spatial and temporal dynamics of astrocytic Ca2+ signaling in the ischemic region and characterize the properties of Ca2+ oscillations. Using pharmacological interventions as well as astrocyte-specific molecular genetic approaches including viral transduction and transgenic mice, we will identify the molecular basis and the properties of astrocytic Ca2+ excitability that follows photothrombosis. 2) Astrocytes stimulate N-methyl-D-aspartate receptors (NMDARs)-mediated neuronal excitation during the period of their Ca2+ hyperexcitability following ischemia. Using 2-P microscopy and electrophysiology, we will determine the effects of gliotransmission on neuronal excitation following ischemia. Specifically, we will determine whether astrocytes stimulate the NR2B- containing NMDAR (NR2B-NMDAR)-mediated neuronal excitation after ischemia. 3) Astrocytes exacerbate ischemia-induced delayed neuronal death and brain damage through Ca2+-dependent gliotransmission. Using immunohistochemistry and a neuronal death assay, we will determine the role of gliotransmission in mediating neuronal death and brain damage. Furthermore we will test whether NR2B-NMDARs are involved in gliotransmission-mediated neuronal death. Although there are many studies suggesting the potential role of astrocytes in brain damage following ischemic injury, the lack of knowledge of biological properties of this type of glial cell together with the virtual absence of in vivo astrocyte-specific manipulations has hampered our progress in understanding their role in pathogenesis. By examining the novel hypothesis that alterations in Ca2+ signaling within and among astrocytes induce delayed neuronal death through gliotransmission, our study will provide entirely new insights into the physiological and pathological role of astrocytes in regulating neuronal excitability and excitotoxicity. Results from this project will advance the field of glial biology and provide therapeutic avenues and targets that could potentially ameliorate neuronal death and brain damage following ischemia.

Public Health Relevance

Cerebral ischemia is a leading neurological disorder and has a major impact on public health. Understanding the cellular and molecular mechanism by which ischemia induces brain damage is essential for providing potential therapeutic avenues to minimize the damage. The proposed project will determine the novel role of a non-neuronal cell of the brain called astrocyte in regulating neuronal death and brain damage following ischemia. Results from this project will derive entirely new insights into the causes of ischemia-induced neurodegeneration.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Cellular and Molecular Biology of Glia Study Section (CMBG)
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Bosetti, Francesca
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University of Missouri-Columbia
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Choudhury, Gourav Roy; Ding, Shinghua (2016) Reactive astrocytes and therapeutic potential in focal ischemic stroke. Neurobiol Dis 85:234-44
Wang, Xiaowan; Li, Hailong; Ding, Shinghua (2016) Pre-B-cell colony-enhancing factor protects against apoptotic neuronal death and mitochondrial damage in ischemia. Sci Rep 6:32416
Li, Hailong; Xie, Yicheng; Zhang, Nannan et al. (2015) Disruption of IP₃R2-mediated Ca²⁺ signaling pathway in astrocytes ameliorates neuronal death and brain damage while reducing behavioral deficits after focal ischemic stroke. Cell Calcium 58:565-76
Li, Hailong; Roy Choudhury, Gourav; Zhang, Nannan et al. (2015) Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice. J Vis Exp :e53161
Ding, Shinghua (2014) Ca(2+) signaling in astrocytes and its role in ischemic stroke. Adv Neurobiol 11:189-211
Wang, Xiaowan; Li, Hailong; Ding, Shinghua (2014) The effects of NAD+ on apoptotic neuronal death and mitochondrial biogenesis and function after glutamate excitotoxicity. Int J Mol Sci 15:20449-68
Li, Hailong; Wang, Xiaowan; Zhang, Nannan et al. (2014) Imaging of mitochondrial Ca2+ dynamics in astrocytes using cell-specific mitochondria-targeted GCaMP5G/6s: mitochondrial Ca2+ uptake and cytosolic Ca2+ availability via the endoplasmic reticulum store. Cell Calcium 56:457-66
Li, Hailong; Zhang, Nannan; Lin, Hsin-Yun et al. (2014) Histological, cellular and behavioral assessments of stroke outcomes after photothrombosis-induced ischemia in adult mice. BMC Neurosci 15:58
Ding, Shinghua (2013) In vivo astrocytic Ca(2+) signaling in health and brain disorders. Future Neurol 8:529-554
Li, Hailong; Zhang, Nannan; Sun, Grace et al. (2013) Inhibition of the group I mGluRs reduces acute brain damage and improves long-term histological outcomes after photothrombosis-induced ischaemia. ASN Neuro 5:195-207

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