Abbreviations used : AdV, adenoviral vector system ; AdV-LacZ, adenoviral construct encoding LacZ ; DEVD-amc, N-acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin ; HIAP, human inhibitor of apoptosis protein ; IAP, inhibitor of apoptosis protein ; MK-801, dizocilpine ; MOI, multiplicity of infection ; NAIP, neuronal apoptosis inhibitory protein ; nt, nucleotides ; PBS, phosphate-buffered saline ; RIAP, rat IAP ; SDS, sodium dodecyl sulfate ; SMA, spinal muscular atrophy ; TNF, tumor necrosis factor ; TUNEL, terminal transferase-mediated dUTP-biotin DNA nick end-labeling ; XIAP, X chromosome-linked IAP ; zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone.
Adenovirus-Mediated Gene Transfer of Inhibitors of Apoptosis Proteins Delays Apoptosis in Cerebellar Granule Neurons
Article first published online: 18 JAN 2002
Journal of Neurochemistry
Volume 72, Issue 1, pages 292–301, January 1999
How to Cite
Simons, M., Beinroth, S., Gleichmann, M., Liston, P., Korneluk, R. G., MacKenzie, A. E., Bähr, M., Klockgether, T., Robertson, G. S., Weller, M. and Schulz, J. B. (1999), Adenovirus-Mediated Gene Transfer of Inhibitors of Apoptosis Proteins Delays Apoptosis in Cerebellar Granule Neurons. Journal of Neurochemistry, 72: 292–301. doi: 10.1046/j.1471-4159.1999.0720292.x
- Issue published online: 18 JAN 2002
- Article first published online: 18 JAN 2002
- Cerebellar granule neurons;
- Inhibitors of apoptosis;
Abstract : The inhibitor of apoptosis (IAP) family of anti-apoptotic genes, originally discovered in baculovirus, exists in animals ranging from insects to humans. Here, we investigated the ability of IAPs to suppress cell death in both a neuronal model of apoptosis and excitotoxicity. Cerebellar granule neurons undergo apoptosis when switched from 25 to 5 mM potassium, and excitotoxic cell death in response to glutamate. We examined the endogenous expression of four members of the IAP family, X chromosome-linked IAP (XIAP), rat IAP1 (RIAP1), RIAP2, and neuronal apoptosis inhibitory protein (NAIP), by semiquantitative reverse PCR and immunoblot analysis in cultured cerebellar granule neurons. Cerebellar granule neurons express significant levels of RIAP2 mRNA and protein, but expression of RIAP1, NAIP, and XIAP was not detected. RIAP2 mRNA content and protein levels did not change when cells were switched from 25 to 5 mM potassium. To determine whether ectopic expression of IAP influenced neuronal survival after potassium withdrawal or glutamate exposure, we used recombinant adenoviral vectors to target XIAP, human IAP1 (HIAP1), HIAP2, and NAIP into cerebellar granule neurons. We demonstrate that forced expression of IAPs efficiently blocked potassium withdrawal-induced N-acetly-Asp-Glu-Val-Asp-specific caspase activity and reduced DNA fragmentation. However, neurons were only protected from apoptosis up to 24 h after potassium withdrawal, not at later time points suggesting that IAPS delay but do not block apoptosis in cerebellar granule neurons. In contrast, treatment with 100 μM or 1 mM glutamate did not induce caspase activity and adenoviral-mediated expression of IAPs had no influence on subsequent excitotoxic cell death.