Project description:Neurodegenerative brain disorders become more common in the aged. Most of these disorders are associated with or caused by selective death of certain neuronal subpopulations. The mechanisms underlying the differential vulnerability of certain neuronal populations are still largely unexplored and few neuroprotective treatments are available to date. Elucidation of these mechanisms may lead to a greater understanding of the pathogenesis and treatment of neurodegenerative diseases. Moreover, preconditioning by a short seizure confers neuroprotection following a subsequent prolonged seizure. Our goal is to identify pathways that confer vulnerability and resistance to neurotoxic conditions by comparing the basal and preconditioned gene expression profiles of three differentially vulnerable hippocampal neuron populations. Hippocampal CA1 and CA3 pyramidal neurons are highly susceptible to seizures and ischemia, whereas dentate gyrus granule cells are relatively resistant. A brief preconditioning seizure confers protection to the pyramidal cells. We will first determine gene expression profiles of untreated rat CA1 and CA3 pyramidal cells, and dentate granule cells, using laser capture microscopy to obtain region-specific neuronal mRNA. We will then determine the effect of a brief preconditioning seizure, which is neuroprotective in CA1 and CA3, on these expression profiles. We hypothesize that common molecular mechanisms exist in neurons that determine their susceptibility to seizure-induced injury. Intrinsic differences in gene expression exist between hippocampal glutamatergic CA1 and CA3 pyramidal neurons, on the one hand, and dentate granule cells on the other, which contribute to the greater susceptibility of pyramidal neurons to degeneration in experimental stroke and epilepsy. We specifically hypothesize that differences in basal energy metabolism genes may confer differential susceptibility to neurodegeneration produced by seizures and ischemia. Anesthetized animals will be sacrificed by decapitation, and frozen 10 micron sections will be lightly stained with cresyl violet to identify cell layers in the hippocampus. Approximately 1000 neurons from each of the three cell layers will be isolated by LCM. Poly-A RNA will be amplified using a modified Eberwine protocol. The quality of our aRNA will be evaluated by quantitative RT-PCR of GluR6 and KA2 mRNA levels before we send the samples to the Center for labeling and hybridization to Affymetrix rat 230A arrays. We will provide a one-round amplification cDNA product to the center for labeling and hybridization. This protocol is identical to a previously approved study by Jim Greene in our laboratory.

Project description:Neurodegenerative brain disorders become more common in the aged. Most of these disorders are associated with or caused by selective death of certain neuronal subpopulations. The mechanisms underlying the differential vulnerability of certain neuronal populations are still largely unexplored and few neuroprotective treatments are available to date. Elucidation of these mechanisms may lead to a greater understanding of the pathogenesis and treatment of neurodegenerative diseases. Moreover, preconditioning by a short seizure confers neuroprotection following a subsequent prolonged seizure. Our goal is to identify pathways that confer vulnerability and resistance to neurotoxic conditions by comparing the basal and preconditioned gene expression profiles of three differentially vulnerable hippocampal neuron populations. Hippocampal CA1 and CA3 pyramidal neurons are highly susceptible to seizures and ischemia, whereas dentate gyrus granule cells are relatively resistant. A brief preconditioning seizure confers protection to the pyramidal cells. We will first determine gene expression profiles of untreated rat CA1 and CA3 pyramidal cells, and dentate granule cells, using laser capture microscopy to obtain region-specific neuronal mRNA. We will then determine the effect of a brief preconditioning seizure, which is neuroprotective in CA1 and CA3, on these expression profiles. We hypothesize that common molecular mechanisms exist in neurons that determine their susceptibility to seizure-induced injury. Intrinsic differences in gene expression exist between hippocampal glutamatergic CA1 and CA3 pyramidal neurons, on the one hand, and dentate granule cells on the other, which contribute to the greater susceptibility of pyramidal neurons to degeneration in experimental stroke and epilepsy. We specifically hypothesize that differences in basal energy metabolism genes may confer differential susceptibility to neurodegeneration produced by seizures and ischemia. Anesthetized animals will be sacrificed by decapitation, and frozen 10 micron sections will be lightly stained with cresyl violet to identify cell layers in the hippocampus. Approximately 1000 neurons from each of the three cell layers will be isolated by LCM. Poly-A RNA will be amplified using a modified Eberwine protocol. The quality of our aRNA will be evaluated by quantitative RT-PCR of GluR6 and KA2 mRNA levels before we send the samples to the Center for labeling and hybridization to Affymetrix rat 230A arrays. We will provide a one-round amplification cDNA product to the center for labeling and hybridization. This protocol is identical to a previously approved study by Jim Greene in our laboratory. Keywords: other

Project description:Injury of the CA1 subregion induced by a single injection of kainic acid (1M-CM-^WKA) is attenuated when juvenile animals (P20) have a history of two sustained neonatal seizures on P6 and P9. To identify gene candidates involved in the spatially protective effects produced by early life conditioning seizures, we profiled and compared the transcriptomes of CA1 subregions from control, 1M-CM-^WKA, and 3M-CM-^WKA treated animals. More genes were regulated following 3M-CM-^WKA (9.6%) than after 1M-CM-^WKA (7.1%). Following 1M-CM-^WKA, genes supporting oxidative stress, growth, development, inflammation, and neurotransmission were upregulated (e.g., Cacng1, Nadsyn1, Kcng1, Aven, S100a4, GFAP, Vim, Hrsp12, Grik1). After 3M-CM-^WKA, protective genes were differentially over-expressed (e.g., Cat, Gpx7, GAD1, Hspa12A, Foxn1, adenosine A1 receptor, Ca2+ adaptor and homeostatic proteins, Cacnb4, Atp2b2, anti-apoptotic Bcl-2 gene members, intracellular trafficking protein, Grasp, suppressor of cytokine signaling (Socs3)). Distinct anti-inflammatory interleukins not observed in adult tissues (e.g., IL6 transducer, IL23 and IL33 or their receptors (ILF2)) were also over-expressed. Several transcripts were validated by real-time polymerase chain reaction (QPCR) and immunohistochemistry. QPCR showed that casp 6 was increased after 1M-CM-^WKA but reduced after 3M-CM-^WKA; pro-inflammatory gene cox1 was either upregulated or unchanged after 1M-CM-^WKA but reduced by ~70% after 3M-CM-^WKA. Enhanced GFAP immunostaining following 1M-CM-^WKA was selectively attenuated in the CA1 subregion after 3M-CM-^WKA. The observed differential transcriptional responses may contribute to early life seizure-induced pre-conditioning and neuroprotection by reducing glutamate receptor-mediated Ca2+ permeability of the hippocampus and redirecting inflammatory and apoptotic pathways which could lead to new genetic therapies for epilepsy. The transcriptomes of the hippocampal CA1 region of Sprague Dawley 23-day-old male rats after 1 or 3 seizures induced by kainic acid injection were compared to the corresponding controls (injected with PBS) using Duke 27k oligonucleotide arrays.

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