Project 2: New Gene Expression in Cell Culture Models of Ischemia In Project 2, primary neurons cell culture models will be used to investigate the neurocidal or neuroprotective effects of gene products whose expression is altered by cerebral ischemia, as identified in Project 1. The long-term objective is to identify endogenous mechanisms of neuronal death and neuroprotection that can be exploited in the treatment of stroke. These hypothesis are: (A) some of the same gene products whose expression is altered by cerebral ischemia in vivo are affected similarly in cell culture models; (B) these models can be used to determine which gene products influence whether ischemic neurons survive or die; (C) the ability of altered gene expression to modify neuronal injury in cell culture can predict effects of altered expression in vivo.
The specific aims are to: (1) determine which candidate neurocidal and neuroprotective gene products show altered expression in cell culture models of neuronal ischemia and cell death; (2) investigate which gene products exhibiting increased expression have neurocidal or neuroprotective functional effects, using antisense oligodeoxynucleotides (ODNs) to block their expression; and (3) investigate which gene products exhibiting decreased expression have functional effects, using viral vectors to enhance their expression. Northern and Western analysis will be used to detect expression of candidate genes and gene products, and Alamar blue fluorescence and lactate dehydrogenase will be used to quantify neurotoxicity. A spectrum of cell culture models-hypoxia with glucose deprivation in cortical neuron cultures, excitatory amino acid exposure in cortical neuron cultures, and K+/- withdrawal-induced apoptosis in cerebellar granule cell cultures-will be employed. Phosphorothioate antisense ODNs will be used to inhibit translation of, and single- and double-mutant replication-defective HSV vectors will be used to overexpress, candidate gene products. The most promising of these will be tested in vivo, using antisense and gene transfer techniques, in Project 3.
| Simon, Roger P (2016) Epigenetic modulation of gene expression governs the brain's response to injury. Neurosci Lett 625:16-9 |
| Simon, Roger P; Meller, Robert; Zhou, An et al. (2012) Can genes modify stroke outcome and by what mechanisms? Stroke 43:286-91 |
| Stevens, Susan L; Leung, Philberta Y; Vartanian, Keri B et al. (2011) Multiple preconditioning paradigms converge on interferon regulatory factor-dependent signaling to promote tolerance to ischemic brain injury. J Neurosci 31:8456-63 |
| Stapels, Martha; Piper, Chelsea; Yang, Tao et al. (2010) Polycomb group proteins as epigenetic mediators of neuroprotection in ischemic tolerance. Sci Signal 3:ra15 |
| Marsh, B J; Williams-Karnesky, R L; Stenzel-Poore, M P (2009) Toll-like receptor signaling in endogenous neuroprotection and stroke. Neuroscience 158:1007-20 |
| West, G Alexander; Golshani, Kiarash J; Doyle, Kristian P et al. (2009) A new model of cortical stroke in the rhesus macaque. J Cereb Blood Flow Metab 29:1175-86 |
| Stevens, Susan L; Ciesielski, Thomas M P; Marsh, Brenda J et al. (2008) Toll-like receptor 9: a new target of ischemic preconditioning in the brain. J Cereb Blood Flow Metab 28:1040-7 |
| Doyle, Kristian P; Yang, Tao; Lessov, Nikola S et al. (2008) Nasal administration of osteopontin peptide mimetics confers neuroprotection in stroke. J Cereb Blood Flow Metab 28:1235-48 |
| Khan, Adil A; Mao, Xiao Ou; Banwait, Surita et al. (2008) Regulation of hypoxic neuronal death signaling by neuroglobin. FASEB J 22:1737-47 |
| Pignataro, Giuseppe; Maysami, Samaneh; Studer, Francesca E et al. (2008) Downregulation of hippocampal adenosine kinase after focal ischemia as potential endogenous neuroprotective mechanism. J Cereb Blood Flow Metab 28:17-23 |
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