Programmed cell death, or apoptosis, plays an essential role during the development of the nervous system. It not only determines the size and the shape of the brain but also enables the establishment of correct synaptic connections. Accumulating data also suggest that the loss of specific sets of neurons, that occurs after stroke or during neurodegenerative diseases, involve the same mechanisms as developmental neuronal death. We use cultures of mouse neurons to elucidate these mechanisms and identify new molecular signalling pathways controlling the survival/death fate of neurons.
Neurons use the same apoptotic machinery as other cells but signalling pathways that lead to neuronal death are more specific. Much progress has been made in characterizing these signalling pathways on one hand and the cell death machinery on the other hand. Nonetheless, little is known regarding the interactions between upstream signalling pathways and the activation of the downstream apoptotic machinery. However, these mechanisms are potentially the most specific therapeutic targets for successful treatment of neurodegenerative diseases. Increasing data suggest that the ubiquitin-proteasome system may constitute one of these links. This system is a pivotal mechanism through which cells identify, target and degrade cellular proteins, particularly short-lived regulatory proteins and damaged or misfolded proteins. Under the sequential action of E1, E2 and E3 enzymes, ubiquitin is covalently conjugated to target proteins. Proteins decorated with polyubiquitin chains are usually degraded by the proteasome, a large multicatalytic complex. Since the early 1990s, accumulating evidence indicates that the ubiquitin-proteasome system is involved in the control of apoptosis by regulating the protein level of key regulatory molecules. Notably, in several neuronal types, short-term treatment with proteasome inhibitors has been shown to prevent apoptosis. This suggests that the “road-blockers” of the apoptotic pathways have to be eliminated by the UPS for the death process to be initiated in neurons. The main goal of our project is to identify new regulatory mechanisms linking the apoptotic machinery to signalling pathways triggered by neuronal survival/death factors, in connection to the ubiquitin-proteasome system. The rationale is that targeting the very selective interaction between E3 enzymes and their substrates might be used to specifically block the degradation of anti-apoptotic proteins without interfering with the elimination of toxic proteins by the proteasome. We use mainly primary cultures of mouse cerebellar granule neurons (CGN) that constitute one of the best characterized in vitro models of neuronal apoptosis. These neurons can survive and differentiate in culture in the presence of serum and depolarizing levels of extracellular KCl. Lowering KCl level in the absence of serum triggers an apoptotic process that can be blocked by proteasome inhibitors. The processes underlying apoptosis in neurodegenerative diseases are likely to result from the reactivation of mechanisms involved in developmental neuronal death mimicked by this model. In this system, we have identified a novel E3 enzyme that plays a crucial role in the triggering of apoptosis. Our results indicate that this enzyme is induced during neuronal apoptosis in several models of neuronal apoptosis both in vitro and in vivo. Moreover we have shown that expression of this protein is necessary and sufficient for inducing neuronal apoptosis and that its pro-apoptotic effect depends on its E3 activity. Our objective is now to elucidate the mechanisms of regulation of this enzyme, to characterize its substrates and to determine its role in different in vivo models of both physiological and pathological apoptosis. In parallel, we are seeking to identify the anti-apoptotic proteins whose degradation is prerequisite to the initiation of apoptosis. Our work should ultimately contribute to the elucidation of new molecular pathways controlling the survival/death fate of neurons and to the identification of novel therapeutic targets for the treatment of neurodegenerative diseases.