Apoptosome inactivation rescues proneural and neural cells from neurodegeneration

Abstract

Deficiency of the apoptosome component Apaf1 leads to accumulation of supernumerary brain cells in mouse embryos. We observed that neural precursor cells (NPCs) in Apaf1 À/À embryos escape programmed cell death, proliferate and retain their potential to differentiate. To evaluate the circumstances of Apaf1 À/À NPC survival and investigate their fate under neurodegenerative conditions, we established cell lines of embryonic origin (ETNA). We found that Apaf1 À/À NPCs resist common apoptotic stimuli and neurodegenerative inducers such as amyloid-b peptide (typical of Alzheimer's disease) and mutant G93A superoxide dismutase 1 (typical of familial amyotrophic lateral sclerosis). Similar results were obtained in Apaf1 À/À primary cells. When death is prevented by Apaf1 deficiency, cytochrome c is released from mitochondria and rapidly degraded by the proteasome, but mitochondria remain intact. Under these conditions, neither activation by cleavage of initiator caspases nor release of alternative apoptotic inducers from mitochondria takes place. In addition, NPCs can still differentiate, as revealed by neurite outgrowth and expression of differentiation markers. Our findings imply that the mitochondrion/apoptosome pathway is the main route of proneural and neural cells to death and that its inhibition prevents them from dismantling in neurodegenerative conditions. Indeed, the ETNA cell model is ideally suited for exploring the potential of novel cell therapies for the treatment of human neurodegenerations.

Faf1 is expressed during neurodevelopment and is involved in Apaf1-dependent caspase-3 activation in proneural cells

“…To gain an understanding of the role of Faf1 in developing brain, we took advantage of the embryonic neural ETNA cells that were established from the brain primordia isolated from e14 wild-type embryos [15]. We transfected ETNA cells with the murine Faf1 cDNA cloned in the mammalian expressing vector pcDNA3.1/myc-his and we selected some clones (referred as clones 22, 44 and 54), which express levels of Faf1-myc 50 % (clones 22, 44) and 100 % (clone 54) higher than control ETNA cells ( Fig.…”

“…ETNA cells were obtained as described in [15]. They were routinely grown in DMEM + 10 % FCS, at 338C (permissive temperature for large T antigen expression) in an atmosphere of 5 % CO 2 in air, and used between passages 2 and 7.…”

“…Caspase-3 activity was assayed by incubating equal amounts (30 mg) of clear cell lysates with 20 mM Ac-DEVD-AMC (Calbiochem, San Diego, CA) in lysis buffer for 30 min at 378C. The amount of 7-amido-4-methylcoumarin (AMC; Calbiochem) release was quantified using a fluorescence plate reader, as described in [15].…”

“…In this work, we analyzed the specific involvement of Faf1 in neurodevelopment by means of the heterozygous Faf1 gene trap (gt) mouse strain (Faf1 +/ gt ) and by a proneural cell line generated in our laboratory (Embryonic telencephalic naïve Apaf1 cells, ETNA). This cell line derives from the brain primordia isolated from wild-type embryos at embryonic stage (e) 14 (referred as control ETNA cells) or from e14 Apaf1 -/-embryos that lack the Apaf1 gene and therefore are impaired in apoptosome formation (referred as ETNA -/-cells) [15]. Despite the fact that Faf1 is widely expressed in many adult and embryonic tissues and in tumor cell lines [2,3], we found that it is predominantly expressed in specific areas of mouse brain at various embryonic stages.…”

Bcl2a1 serves as a switch in death of motor neurons in amyotrophic lateral sclerosis

“…7 In addition, survival of the G93A-SOD1 ALS mice is prolonged by treatment with the pan-caspase inhibitor ZVAD-fmk and by overexpression of Bcl2, and we have demonstrated that overexpression of G93A-SOD1 requires the expression of Apaf1 to induce cell death. 8,9 Intriguingly, evidence of a direct link between SOD1 and an apoptotic pathway has been provided by the demonstration that both wild-type (WT) and mutant SOD1 can bind Bcl2. 10 In this study, we have analysed the expression of genes in the Bcl2-family in transgenic mice expressing G93A-SOD1 and focused on Bcl2a1, the mouse homologue of human Bfl-1 gene.…”