Abstracts

The degree of neuronal vulnerability following seizures is genetically determined, however the exact identity of the genes and their role in contributing to differences in cell survival is not known. We used differential display of hippocampal mRNAs to compare seizure-induced gene expression related to differences in neuronal damage found between a sensitive and resistant inbred mouse strain. We identified changes in MCL1 (Myeloid cell leukemia-1) expression, a Bcl2-related anti-apoptotic protein originally isolated from differentiating human myeloid leukemia cells. Unlike Bcl2, expression has not been previously reported in CNS neurons, but may contribute to inherited differences in threshold for seizure-induced brain damage.
C3H/HeJ (250 mg/kg) and C57BL/6J (350 mg/kg) mice, the animals were sacrificed 12 and 24 hours after a 6 hour episode of pilocarpine-induced status epilepticus (SE). TUNEL labeling and cresyl violet staining were used to verify apoptosis and neuronal cell loss. Mcl1 was identified using the differential display technique, 12 hours after SE, These results were confirmed by subsequent in situ hybridization and RPA. RTPCR for Mcl1 was used in order to identify which isoform of Mcl1 was being expressed in the brain. Mcl1 immunohistochemistry and western blotting were used for subcellular localization and quantification of the protein in mouse and human tissues Klenow fragment labeling and caspase 3 immunohistochemistry were performed in Mcl1 deficient mice 24 hours after pilocarpine-induced seizures in order to verify apoptosis and the type of DNA damage.
We isolated Mcl1 using a direct screen for candidate modifier genes of neuronal vulnerability by differential display of mRNAs upregulated following prolonged seizures in two mouse strains with contrasting levels of hippocampal cell death. Mcl1 is widely expressed in neurons, and mRNA transcription is rapidly induced in both strains. In resistant C57BL/6 mice, Mcl1 protein levels remain persistently elevated. In contrast, Mcl1 levels in C3H/HeJ hippocampus fall rapidly after seizures, coinciding with extensive neuronal apoptosis. DNA damage and caspase-mediated cell death were strikingly increased in [italic]Mcl1[/italic] deficient mice when compared to +/+ littermates after similar seizures.
Our data identify [italic]Mcl1[/italic] as a neuronal immediate early gene responsive to excitotoxic insult in the brain, and link relative levels of Mcl1 expression to inherited differences in neuronal thresholds for apoptosis. This strategy suggests a method of identifying molecular candidates for modifier gene loci related to seizure-induced neuronal vulnerability without pursuing genetic mapping studies.
[Supported by: NS29709 (JLN), FAPESP, American Epilepsy Society and Milken Family Foundation Fellowship (MM)]