Abstracts

RATIONALE: At the end of this activity, participants should be able to discuss which genes are induced by injury in astrocytes.
Temporal lobe epilepsy (TLE) is characterized by cell loss, circuit rearrangements, reactive gliosis and expression alterations of multiple genes. Reactive gliosis is manifested by increased number of astrocytes with aberrant morphology. The increased number and altered intrinsic astrocyte properties demonstrated in human epilepsy suggest a role of these cells in epileptogenesis. We assessed gene expression alterations in a scratch-injury reactive glia model using microchip technology.
METHODS: Reactive glia were created by scratching purified mouse cortical astrocyte cultures with a dissection pin comb, and harvested 2 hours, 2 and 7 days post injury (PI). Total RNA was isolated, cDNA synthesized, cRNA labeled and hybridized to Murine Genome U74Av2 arrays (Affymetrix).
RESULTS: High-density oligonucleotide arrays containing 12,473 gene probe sets were used to monitor injury-associated gene expression alterations. After 2 h PI (n=4) 1.6% of gene probe sets exhibited altered expression levels, while 2 and 7 days PI (n=3 each), 0.8% and 0.1% gene probe sets exhibited altered expression compared to control (n=5). In 2 h PI astrocytes a 2 - 21 fold increase in expression was detected for transcription-related mRNAs including Nurr77, fosB, fra-1, LRG-21, Kruppel-like factor, Krox-20, Krox-24, GIF, NFIL/E4BP4, Nurr1, junB, Hox2.4, bHLH, c/EBP, PEBP2, ATF4, pip92, mTGIF. Also induced were inflammation (griPGHS, cytokine A2), apoptosis (TDAG51, MyD116, caspase 9), injury (PAI-1), stress response (mafF, HSP70), signal transduction (SOCS1, stathmin-like protein RB3, Ldlr, serum inducible kinase, Ras-like GTP-binding protein), and growth factor genes (EGF-like growth factor, TGF- , NGF). Downregulated genes included caspase 6 and pyruvate dehydrogenase kinase. In 2 days PI cultures a 2 - 6 fold increase in expression was detected for cell cycle (cyclin B1, Cdc25, Ki-67, Plk, Bub1, Mad2, Cenp-a, CHO1/MKLP1), and vesicular transport genes (rabkinesin-6). Decreased expression was detected for complement C1q B chain, colony stimulating factor 1 receptor, G-protein coupled receptor. In 7 days PI cultures altered genes included 4.6 fold increased metalloelastase and decreased heme oxygenase.
CONCLUSIONS: This reactive glia culture model demonstrated a fast (within 2 hours) complex genomic response to the injury. The identity of the genes exhibiting altered expression revealed that injury alters many aspects of cell physiology, including transcriptional regulation, signal transduction, stress responses, apoptosis, inflammation and the cell cycle. At 2h post injury, 25% of genes with altered expression were transcription factors and immediate early genes. These alterations were absent in astrocyte cultures 2 and 7 days post injury, and were replaced by changes in cell cycle genes. This pattern of gene induction in reactive glia may facilitate TLE-associated reactive gliosis and affect epileptogenesis.
[Supported by: DAC: NS 32403, NS 38572. PGH: NS43142, NS37585]