1.Regulation of neuronal apoptosis by the E3 ubiquitin-ligases of the TRIM family
Apoptosis is an evolutionarily conserved form of programmed cell death. It plays an essential role in morphogenesis, tissue homeostasis and the elimination of cells that are potentially dangerous to the organism. As a consequence, alterations in apoptotic pathways contribute to many of human diseases. In the nervous system, apoptosis plays a crucial role during development. It determines the final shape and size of the brain, and is necessary for the formation of adequate neuronal networks by eliminating neurons that have not established adequate synaptic connections. In addition, many studies indicate that neurons that are lost during neurodegenerative diseases or after cerebral ischemia die through apoptosis. The ubiquitin-proteasome system plays an important role in the regulation of apoptosis by controlling the level or function of many regulatory proteins. The E3 ubiquitin-ligases confer a high level of specificity to this system by recognizing target proteins and mediating their ubiquitination. This post-translational modification can have various consequences on protein function, localization, interactions with partners or degradation by the proteasome. TRIM proteins represent one of the largest classes of E3 ubiquitin-ligases with a RING domain.
We identified TRIM17 as one of the most upregulated genes during early apoptosis in primary cultures of mouse cerebellar granule neurons (Desagher et al. 2005). At that time, its cellular function was unknown. Since then, we have shown that TRIM17 is both necessary and sufficient for apoptosis in various types of neurons and that its pro-apoptotic activity depends on its RING domain that confers it its E3 ubiquitin-ligase activity (Lassot et al. 2010). We have also shown that TRIM17 participates in the ubiquitination and degradation of Mcl-1 in neurons (Magiera et al. 2013). This anti-apoptotic Bcl-2 family protein plays a critical role in the survival of a myriad of cell types and contributes to tumorigenesis and chemoresistance in a large number of cancers (Mojsa et al. 2014). In parallel, we have shown that TRIM17 inhibits the activity of the transcription factors NFATc3 and NFATc4 by preventing their nuclear translocation. In addition, NFATc3 promotes neuronal apoptosis by activating TRIM17 expression, thereby forming a negative feedback loop (Mojsa et al. 2015). In addition, a series of studies on other TRIM17 partners led us to discribe a novel mode of action: TRIM17 can modulate the level of certain proteins by inhibiting other E3 ubiquitin-ligases of the TRIM family (Figure 1). For example, we showed that TRIM17 inhibits TRIM28-mediated ubiquitination and degradation of the anti-apoptotic protein BCL2A1, thereby promoting the survival of BCL2A1-dependent cells, including chemotherapy-resistant melanoma cells (Lionnard et al. 2019). We identified a similar mechanism for the transcription factor ZSCAN21: TRIM17 inhibits ZSCAN21 degradation by inhibiting the E3 ubiquitin-ligase TRIM41 (Lassot et al. 2018, Figures 1,3). More recently, we identified TRIM39 as a novel SUMO-dependent E3 ubiquitin-ligase (STUbL) that induces the ubiquitination and degradation of NFATc3 in neurons and is inhibited by TRIM17 (Basu-Shrivastava et al. 2020; Figure 2). Taken as a whole, our work has thus made an important contribution to the understanding of the cellular functions of TRIM17 (Basu-Shrivastava et al. 2021).
2.
Regulation of α-synuclein expression in Parkinson’s disease
α-Synuclein is an abundant neuronal protein that can form pathological proteinaceous inclusions in a group of neurodegenerative diseases called synucleinopathies. Parkinson's disease is the main synucleinopathy and the second most common neurodegenerative disease after Alzheimer's disease. Numerous studies suggest that α-synuclein plays a direct role in the pathogenesis of Parkinson's disease by inducing neuronal dysfunction and the death of the most vulnerable neurons: the dopaminergic neurons of the substantia nigra, whose loss is responsible for the motor symptoms of the disease. Indeed, mutations in the SNCA gene, which encodes α-synuclein, cause familial forms of Parkinson's disease and variations in the SNCA locus are among the highest genetic risk factors for sporadic Parkinson’s disease. Furthermore, accumulating data indicate that even a limited increase in the level of α-synuclein can cause both inherited and sporadic forms of the disease. Notably, duplications and triplications of the wild-type SNCA gene have been identified in families in which the severity of the disease is directly correlated to the amount of α-synuclein expressed. In addition, polymorphisms that result in increased SNCA gene transcription are associated with a high risk of developing the disease. Reducing α-synuclein expression therefore appears to be a promising therapeutic strategy and some studies have shown beneficial effects in animals. However, the deep reduction of α-synuclein expression by RNA interference has also shown toxic effects. A subtle regulation of α-synuclein expression therefore appears to be required for the function and survival of dopaminergic neurons in the substantia nigra. This highlights the need for elucidating the mechanisms and factors that regulate SNCA gene transcription. However, these have been poorly studied and our knowledge on this subject is very limited.
The main goal of our current project is to better understand the mechanisms underlying the transcriptional regulation of α-synuclein in Parkinson's disease.
In a previous study (Lassot et al. 2018), we showed that the transcription factor ZSCAN21 increases α-synuclein expression and that TRIM41 is an E3 ubiquitin-ligase of ZSCAN21. Our results also indicate that TRIM17 inhibits the ubiquitination and degradation of ZSCAN21 mediated by TRIM41, thereby promoting α-synuclein expression (Figure 3). In addition, we identified rare variants in the TRIM41 and ZSCAN21 genes, in patients with familial forms of Parkinson's disease. These genetic variations result in the stabilization of the ZSCAN21 protein and thus should induce α-synuclein accumulation (Figure 3). These results therefore suggest that a dysregulation of the TRIM17/TRIM41/ZSCAN21 pathway may be involved in the pathogenesis of Parkinson's disease. Our first objective is now to test this hypothesis in different cellular or animal models. This project is supported by the TRIM-NET ITN (a H2020 Marie Sklodowska-Curie action from the European Commission).
In particular, we use the human neuronal precursors LUHMES that can be homogeneously differentiated into post-mitotic dopaminergic neurons (Figure 4). Nearly 100% of the differentiated LUHMES cells express the dopaminergic markers tyrosine hydroxylase and dopamine transporter, which makes them sensitive to the MPP+ neurotoxin or other stimuli associated with Parkinson's disease (Fig. 4A). We differentiate LUHMES cells in monolayers (Fig. 4A,C) or spheroids (Fig. 4B), and we use CRISPR-Cas9-based techniques to edit their genome (Fig. 4C). We also use primary cultures of neurons, as well as in vivo mouse models through collaborations.
In the longer term, our goal is to identify and characterize other transcription factors involved in the increased expression of α-synuclein during Parkinson's disease, as well as the signaling pathways leading to their activation. By contributing to elucidate the molecular mechanisms regulating the expression of α-synuclein, our work could lead to the development of new therapeutic strategies aiming at normalizing the level of this protein in order to protect the dopaminergic neurons of the substantia nigra and to slow down or even stop the progression of Parkinson's disease.
