Bax siRNA promotes survival of cultured and allografted granule cell precursors through blockade of caspase-3 cleavage

Authors:
Zhokhov SS, Desfeux A, Aubert N, Falluel-Morel A, Fournier A, Laudenbach V, Vaudry H, Gonzalez BJ
In:
Source: Cell Death Differ
Publication Date: (2008)
Issue: 15(6): 1042-53
Research Area:
Neurobiology
Cells used in publication:
Granule cell (CGC), rat
Species: rat
Tissue Origin: brain
Platform:
Nucleofector® I/II/2b
Experiment
Precursors of granule cells (4x10^6) were transfected with 15 µg of two different siRNA against Bax (siBax-1, siBax-2). For GFP expressing of cells they were transfected with 0.4 µg pmaxFP-Green-N vector. Knocked-down or GFP-labelled cells were injected into cerebella of 8-day-old rats for immunohistochemical detection.
Abstract
Transplantation of neuronal precursor cells (NPCs) into the central nervous system could represent a powerful therapeutical tool against neurodegenerative diseases. Unfortunately, numerous NPCs die shortly after transplantation, predominantly due to caspase-dependent apoptosis. Using a culture of cerebellar neuronal precursors, we have previously demonstrated protective effect of the neuropeptide PACAP, which suppresses ceramide-induced apoptosis by blockade of the mitochondrial apoptotic pathway. The main objective of this study was to determine whether Bax repression can promote survival of NPCs allotransplanted into a host animal. In vivo and ex vivo experiments revealed that C2-ceramide increases Bax expression, while PACAP reverses this effect. In vitro tests using cerebellar NPCs demonstrated that the Bax-specific small interfering RNA (siRNA) could reduce their death and caspase-3 cleavage within the first 24 h. BrdU-labelled NPCs were subjected to transfection procedure with or without siRNA introduction before using for in vivo transplantation. Twenty-four hours after, the allografted NPCs containing siRNA showed significantly reduced level of caspase-3 cleavage, and the volume of their implants was almost twofold higher than in the case of empty-transfected precursors. These data evidence an important role of Bax in life/death decision of grafted NPCs and suggest that RNA interference strategy may be applicable for maintaining NPCs survival within the critical first hours after their transplantation.