Youming Lu, M.D., Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): Ischemic stroke is the third leading cause of death in developed countries. A critical feature of the disease is a highly selective pattern of neuronal loss; certain identifiable subsets of neurons, particularly CA1 pyramidal neurons in the hippocampus, are severely damaged while others remain intact. A step in this selective neuronal injury involves Ca2+ entry through Ca2+-permeable AMPA receptor channels. AMPA receptors are a major subtype of glutamate receptors (GluRs) that are assembled from GluR1-4 subunits. Ca2+ permeability of the channels is dominated by GluR2 RNA editing at the Q/R site; edited GluR2(R) subunits form Ca2+-impermeable channels, whereas unedited GluR2(Q) channels allow Ca2+ entry. In most CA1 neurons, AMPA receptor channels contain GluR2(R), and thus are impermeable to Ca2+ flow. Recently, we have identified that transient forebrain ischemia selectively disrupts GluR2 Q/R site editing and hence induces injurious Ca2+ entry through AMPA receptor channels into vulnerable CA1 neurons. We have also shown that impaired GluR2 Q/R site editing is closely correlated with reduced expression of ADAR2 (short for adenosine deaminase acting on RNA) gene, a nuclear enzyme responsible for GluR2 Q/R site editing. We thus hypothesize that reduced expression of ADAR2 gene is responsible for the impaired GluR2 Q/R site editing. To address this hypothesis directly, we will determine if restoration of ADAR2 gene expression rescues GluR2 Q/R site editing and in turn blocks Ca2+ permeability of AMPA receptor channels, leading to the survival of vulnerable neurons in the post-ischemic rats. Overall, this project will have two specific aims: Specific Aim 1: To determine whether restoration of ADAR2 gene expression blocks Ca2+ entry through AMPA receptor channels and rescues vulnerable neurons in the post-ischemic rats. Specific Aim 2: To determine if generation of stable ADAR2 gene silencing induces degeneration of ischemia-insensitive neurons, and if degeneration of ADAR2-deficient neurons results from RNA editing deficits of one or more glutamate receptor subunits. Together, this project will identify that ADAR2-dependent GluR2 Q/R site editing determines vulnerability of neurons to ischemia. Thus, this work will define a promising target for stoke therapy. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): N-methyl-D-aspartate (NMDA) receptors constitute the major subtype of glutamate receptors and normally participate in rapid excitatory synaptic transmission throughout the CNS. To date, a variety of NMDA receptor subunit proteins (NR1, NR2A-D) have been cloned. Native NMDA receptors appear to be heteroligomeric complexes consisting of an essential NR1 subunit and one or more regulatory NR2 subunits (NR2A-D) and possibly the more recently identified NR3 subunit. Activation of NR2A and NR2B receptor channels are permeable to Na+ and K+ and also to Ca2+, which triggers multiple intracellular catabolic processes, leading to the irreversible death of neuronal cells. Recently, we have used reverse phase nano-LC-MS/MS mass spectrometry to analyze protein components in the NR2B receptor complex from forebrain of mice that had been subjected to sham or focal cerebral ischemia. We have shown that cerebral ischemia recruits death-associated protein kinase (DAPK1) into the NR2B receptor complex. DAPK1 is one member of Ca2+/calmodulin (CaM)-dependent serine/threonine kinase family and functions as a critical mediator of cell death. Whole-cell patch clamp recordings have demonstrated that activation of DAPK1 increases the NR1/NR2B receptor-mediated currents. Subsequently, we have generated genetically modified mice (cdDAPK1), in which catalytic domain of DAPK1 is selectively deleted. We have found that neurons in the forebrain of the cdDAPK1 mutant mice are resistant to ischemic insults. Thus, we hypothesize that DAPK1 physically and functionally interacts with NR2B receptors and this interaction contributes to neuronal injury in ischemic stroke. Proposed studies will address this question. To date, all clinical stroke trials targeting glutamate receptors (AMPA or NMDA) have failed, possibly because receptor antagonists block the physiological actions of glutamate as well. This proposal describes, for the first time, a molecular approach to selectively block the pathological effects of NR2B receptors by targeting DAPK1. Thus, this approach should not affect the physiological actions of glutamate receptors in the brain, thereby defining a promising target for stroke therapy. PUBLIC HEALTH RELEVANCE: Stroke is a third leading cause of death in the United States. A critical feature of the disease is selective degeneration of neurons in the brain by activation of glutamate receptor channels. To date, all clinical stroke trials targeting glutamate receptors have, however, failed, because receptor antagonists block the physiological actions of glutamate as well. This proposal describes, for the first time, a molecular approach to selectively block the pathological effects of glutamate receptors by targeting DAPK1 enzyme. Thus, this approach should not affect the physiological actions of glutamate receptors in the brain, thereby defining a promising target for stroke therapy. [unreadable] [unreadable] [unreadable]