Shinghua Ding - US grants

DESCRIPTION (provided by applicant): 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.

? DESCRIPTION (provided by applicant): Focal ischemic stroke is a leading neural disorder with limited choices for clinical treatment, thus identifying new molecular pathways that can reduce neuronal death and improve stroke outcomes remains an intensive research area. The project goal is to elucidate the role and mechanisms by which Pre-B-Cell Colony- Enhancing Factor (PBEF) exerts brain protection following focal ischemic stroke. PBEF is a rate limiting enzyme in the salvage pathway of nicotinamide adenosine dinucleotide (NAD+) biosynthesis in mammals and is mainly expressed in neurons in the brain under normal conditions. We hypothesize that PBEF plays a critical role in ameliorating neuronal death and brain damage and promoting behavioral recovery following focal ischemic stroke. Using a combination of state-of-the art technologies including PBEF conditional knockout (Pbef-/- cKO) mice, viral transduction, photothrombosis (PT) ischemia models, in vivo two-photon (2-P) microscopy, electrophysiology, mitochondrial assay, we propose three SPECIFIC aims to test our hypothesis. Aim 1: To test the hypothesis that PBEF ameliorates acute neuronal death and brain damage after ischemia by facilitating NAD+ synthesis. We will generate inducible and neuron-specific Pbef-/- cKO mice (i.e., Thy1-Pbef-/- cKO mice) and viral overexpression of WT and mutant enzymatic activity-deficient mutant PBEF for loss- and gain-of functional studies. We will image dendrite beading, glutamate release and Ca2+ overloading in live mice with in vivo 2-P microscopy, and conduct infarct volume measurement and, neuronal death assay to determine the effect of PBEF on neuronal injury and death in the acute phase of ischemia. We will determine whether neuronal PBEF is involved in PARP-1-mediated neuronal death (i.e., parthanatos) after ischemia. Aim 2: To test the hypothesis that mitochondrial NAD+ salvage pathway plays a predominant role in mediating neuronal protection after ischemia We will determine the effect of neuronal PBEF on subcompartment NAD+ pools and mitochondrial function under normal and ischemic conditions. Using subcompartment-targeting molecular expression of PBEF in cytoplasm, nuclei, and mitochondria, we will determine whether the mitochondrial NAD+ salvage pathway plays a critical role in neuroprotection after ischemia. Aim 3: To test the hypothesis that PBEF improves long-term stroke outcomes through promoting neuronal (synaptic) plasticity. We will evaluate the effect of neuronal PBEF on long-term stroke outcomes using multiple approaches. We will conduct infarct volume measurements, tissue loss, apoptotic neuronal death, behavior tests, and neurological evaluations on Thy1-Pbef-/- cKO and WT mice over a long- term period ranging from one day to a few weeks following ischemia. We will determine the effect of neuronal PBEF on synaptic plasticity in the peri-infarct region using long-term repeated in vivo 2-P imaging and electrophysiology. Results from our project will provide new insights into potential therapeutic targets for translational research on stroke treatment.

Project Summary The adult brain has a remarkable capacity to recover from focal ischemic stroke (FIS). Astrocytes are the most numerous and diverse glial cells in CNS and intimately interact with neurons to support and regulate their functions. After FIS, astrocytes in the PIR exhibit dynamic changes in morphology, proliferation and gene expression especially in the peri-infarct region (PIR). These astrocytes are called reactive astrocytes (RAs). However, whether and how reactive astrocytes (RAs) affect brain recovery after FIS in the context of astrocyte?neuron interactions largely remain unexplored. In our preliminary study, we found GDNF, a potent neurotrophic factor, is dramatically upregulated in the ischemic hemisphere and RAs after photothrombosis (PT)-induced FIS. Furthermore, we found that deletion of astrocytic GDNF reduces adult neurogenesis in normal brain, and increases brain infarction and attenuates cell proliferation in the PIR after PT. Based on these strong preliminary results, we hypothesize that RAs-derived GDNF plays an important role in neural regeneration and functional brain recovery after FIS. The prohect goal is to determine whether and how RAs- derived GDNF stimulates synaptic regeneration and remodeling of surviving neurons in the PIR and improves long-term stroke outcomes after FIS. To achieve this goal, we have developed interdisciplinary technologies including self-complementary adeno-associated virus (scAAV) vectors and Glast-CreERT2:GDNFf/f mice to specifically overexpress or delete GDNF in RAs during post FIS time, in vivo two photon (2-P) long-term microscopy, electrophysiology, immunocytochemistry, Western blot (WB) analysis, brain damage and neuronal death assays and behavioral tests. We propose three specific aims. In Aim 1, we will test the hypothesis that RAs-derived GDNF can enhance synaptogenesis to stimulate neural regeneration in the PIR after FIS. We will determine the effects of RAs-derived GDNF on the expression of neuronal proteins involving synaptic function and plasticity in the PIR; using TRAP (translating ribosome affinity purification) method we will further identify neuronal transcript changes at translational status in the PIR. In Aim 2, we will test the hypothesis that RAs- derived GDNF can promote structural and functional synaptic remodeling of surviving neurons in the PIR after FIS. Using in vivo long-term 2-P imaging we will determine the effect of RAs-derived GDNF on spine turnover (i.e., spine formation and elimination), glutamate release and Ca2+ signaling in the same dendrites of surviving neurons in the PIR. We will conduct patch-clamp recording on surviving neurons in the PIR to determine the effect of RAs-derived GDNF on functional synaptic plasticity. In Aim 3, we will test the hypothesis that astrocytic GDNF can improve long-term stroke outcomes. We will evaluate the effect of RAs-derived GDNF on long-term histological and behavioral outcomes. Our project will provide novel molecular, cellular and functional insights into the brain recovery processes after FIS in the context of glia-neuron interactions, reveal potential strategies for stroke therapy, and thus has both scientific and translational significances.