Apoptosis is an evolutionarily conserved form of programmed cell death that is essential for morphogenesis, tissue homeostasis and elimination of cells that are potentially dangerous to the rest of the organism. As a consequence, alterations in apoptotic pathways contribute to the pathology of a number of human diseases. Insufficient apoptosis is necessary for tumourigenesis and favours viral infection or autoimmunity, while increased apoptosis is evident in neurodegenerative diseases, infertility and AIDS.
Mitochondria play an essential role in the regulation of apoptosis. The release of cytochrome c from mitochondria, that is controlled by the proteins of the Bcl-2 family, triggers the activation of caspases which are responsible for cell death.
The signalling pathways that control the survival/death fate of cells and that converge onto mitochondria remain poorly understood. Identifying these mechanisms remains of crucial importance. Indeed, in most cases, permeabilization of the outer mitochondrial membrane is the point of no return and cell death can only be prevented by acting upstream or at the level of mitochondria per se.
Since the early 1990s, accumulating evidence indicates that the ubiquitin-proteasome system plays an important role in the regulation of apoptosis by controlling the level or the function of key regulatory proteins. Notably, in several neuronal types, short-term treatment with proteasome inhibitors has been shown to prevent apoptosis upstream of the mitochondrial control point. This suggests that essential anti-apoptotic proteins have to be eliminated by the proteasome for the cell death process to be initiated. However, only a few of these proteins have been identified so far and the specific E3 ubiquitin-ligases responsible for their degradation are mostly unknown.
Our main objective is to identify novel molecular mechanisms regulating apoptosis upstream of the mitochondrial control point. For this purpose, we have focused on E3 ubiquitin-ligases of the TRIM family.
We have identified the E3 ubiquitin-ligase TRIM17 in primary cultures of mouse cerebellar granule neurons that constitute one of the best characterized in vitro models of neuronal apoptosis (Fig.1). We have found that TRIM17 is both necessary and sufficient for inducing neuronal apoptosis. Moreover, TRIM17 acts upstream of mitochondria and its pro-apoptotic effect depends on the RING domain that confers its E3 ubiquitin-ligase activity (Lassot et al. 2010).
In order to elucidate the mechanisms of action of TRIM17, we undertook to identify its partners and substrates by both a hypothesis-driven approach and a Yeast two-hybrid screen. We have shown that, in neurons, TRIM17 is an E3 ubiquitin-ligase for Mcl-1. This anti-apoptotic protein of the Bcl-2 family is essential for the survival of multiple cell lineages and contributes to the tumourigenesis and drug resistance of many human cancers. Our results indicate that the proteasomal degradation of Mcl-1 induced by TRIM17 is necessary for the triggering of neuronal apoptosis (Magiera et al. 2013). In parallel, we have found that TRIM17 inhibits the transcription factors NFATc3 and NFATc4 by binding them and by preventing their nuclear translocation. Moreover, NFATc3 favours neuronal apoptosis by inducing TRIM17 expression, with the resulting feed-back loop providing a fine regulation of neuronal apoptosis (Mojsa et al. 2015).
In addition, a series of studies on other TRIM17 partners has revealed an original mode of action: TRIM17 can modulate the level of certain protein substrates by inhibiting other E3 ubiquitin-ligases of the TRIM family. For example, we have shown that TRIM17 inhibits the ubiquitination and degradation of the anti-apoptotic protein BCL2A1 mediated by TRIM28, by preventing the binding of the E3 ubiquitin-ligase TRIM28 to its substrate BCL2A1. These mechanisms seem to have a direct effect on the survival of cells that depend on BCL2A1, because we could restore the sensitivity towards apoptosis in a chemoresistant melanoma cell line by overexpressing TRIM28 or by silencing TRIM17 (Lionnard et al. 2018). We have found a similar mechanism for the transcription factor ZSCAN21 : TRIM17 inhibits the degradation of ZSCAN21 by inhibiting the E3 ubiquitin-ligase TRIM41. This is of particular interest as ZSCAN21 stimulates the expression of α-synuclein, a protein that plays an essential role in Parkinson disease, when it accumulates, by inducing the death of dopaminergic neurons of the substantia nigra. Indeed, we have identified mutations in the genes of TRIM41 and ZSCAN21 in patients with familial forms of Parkinson’s disease. These genetic variations lead to the stabilization of ZSCAN21, suggesting that a dysregulation of the mechanisms that we have identified could be involved in the pathogenesis of Parkinson’s disease, by increasing the expression of α-synuclein (Lassot et al. 2018, Fig. 2).
Our current work is focusing on transcriptional regulation and neurotoxic mechanisms of α-synuclein. Our first obejective is to determine the role of the TRIM17/TRIM41/ZSCAN21 pathway in the pathogenesis of Parkinson’s disease using different models of the disease. In parallel, we are trying to elucidate the molecular mechanisms through which TRIM17 inhibits TRIM41 and thereby increases α-synuclein expression. In the longer term, our results could lead to novel therapeutic strategies that could normalize α-synuclein level and therefore protect dopaminergic neurons from apoptosis. In addition, we are developing a novel project aimed at better understanding the molecular mechanisms through which α-synuclein induces apoptosis in dopaminergic neurons, notably by acting at the mitochondria.