Determining the Roles of the Intrinsic versus the Extrinsic Pathway in Regulating Neuronal Programmed Cell Death In Vivo

Programmed cell death (PCD) is a highly evolved mechanism of cellular suicide that is aberrantly activated following neural injury. Two fundamental PCD signaling pathways termed the extrinsic (caspase-8-mediated) and intrinsic (caspase-9-mediated) pathways, have been described. While each pathway is initiated by distinct cellular stimuli, both pathways culminate in the activation of downstream executioner caspases. Previous efforts to isolate the in vivo contribution of each pathway have been hindered by the embryonic lethality of casp8 and casp9 null mice. In the present study, I overcame this obstacle to directly assess the contribution of each pathway following two well-characterized forms of acute neural injury; excitotoxic destruction of CA1 pyramidal neurons, and the loss of motor neurons following facial nerve transection. To determine the role of caspase-8, I constructed several lines of mice in which caspase-8 was conditionally ablated within the relevant neuronal populations. The results obtained from these animals definitively demonstrate that caspase-8 is not required by either motor neurons or CA1 pyramidal neurons to undergo PCD following injury. Therefore, these findings have provided the first direct experimental evidence to counter the widely held dogma of caspase-8 as the central effector of death receptor-mediated signaling within neurons. With respect to the intrinsic pathway, several lines of evidence suggest that the apoptosome predominantly regulates the death of motor neurons. I tested this hypothesis by performing facial axotomies in mice containing a point mutation introduced (“knocked in”) into the genomic locus of cytochrome c which abolishes its ability to activate the intrinsic pathway. Homozygous cytochrome c knock-in mice displayed a significant enhancement in motor neuron survival in comparison to control littermates following injury. However, the level of motor neuron protection differed from that previously reported in mice either overexpressing anti-apoptotic or lacking pro-apoptotic members of the Bcl-2 family. Therefore, the results of this study directly demonstrate the influence of the apoptosome on injury-induced neuronal PCD isolated from upstream Bcl-2 family-mediated effects. In addition, my results have provided the first evidence that activation of the apoptosome is required for the release of apoptosis inducing factor (AIF) from the mitochondria of injured motor neurons in vivo.Ph