[en] Stem cells have the ability to either self-renew, thereby maintaining their stem cell status, or to undergo differentiation. The balance between stem cell maintenance and differentiation is tightly regulated by a complex interplay of various signaling pathways, cell fate determinants and non-coding RNAs, including microRNAs (miRNAs). One important cell fate determinant during embryonic and adult neurogenesis is the TRIM-NHL family member TRIM32. In neural stem cells (NSCs), TRIM32 inhibits proliferation and induces neuronal differentiation by two mechanisms. On the one hand, TRIM32 ubiquitinates the transcription factor c-Myc, thereby targeting it for proteasomal degradation and inducing cell-cycle exit. On the other hand, TRIM32 associates with the RNA-induced silencing complex (RISC) and increases the activity of certain miRNAs such as Let-7a. However, the exact mechanism of miRNA regulation by TRIM32 during neuronal differentiation remains elusive. Here, we used a mass spectrometry approach to identify novel protein-protein interaction networks centred on TRIM32 during neuronal differentiation. By applying bioinformatic tools, we identified an enrichment of proteins involved in neurogenesis and RNA-related processes among the TRIM32-associated proteins. One of the candidates discovered in this screen was the RNA helicase DDX6, which has been implicated in miRNA regulation. Here, we demonstrate, that DDX6 colocalises with TRIM32 in NSCs and neurons and that it increases the activity of Let-7a. Furthermore, we provide evidence that DDX6 is necessary and sufficient for neuronal differentiation and that it functions in cooperation with TRIM32.
Similar to TRIM32, the Parkinson’s disease (PD)-associated leucine-rich repeat kinase 2 (LRRK2) protein plays crucial roles in adult neurogenesis and miRNA regulation. LRRK2 is expressed in the stem cell niches of the embryonic and adult mouse brain. Moreover, neurogenesis is impaired in mice carrying the PD-associated LRRK2(G2019S) mutation, suggesting that LRRK2 plays an important role in neurogenesis. However, the precise role of LRRK2 in neuronal differentiation and neurogenesis is poorly understood. Previously, LRRK2 has been shown to associate with RISC, thereby repressing the activity of miRNAs such as Let-7a, and this repression was further enhanced by the G2019S mutation. We addressed the role of LRRK2 in miRNA-mediated neuronal differentiation by using NSCs derived from mice carrying the PD-associated LRRK2(R1441G) mutation as well as from LRRK2 knock-out mice. Here, we demonstrate that LRRK2(R1441G) NSCs display increased levels of oxidative stress and are more susceptible to cell death upon neuronal differentiation. Consequently, neuronal differentiation is greatly impaired, while NSC self-renewal is unaffected in these cells. In contrast, LRRK2 deficiency in NSCs produced the opposite effect on oxidative stress, cell survival and neuronal differentiation. Interestingly, the expression levels of the miRNAs Let-7a and miR-9 were downregulated in LRRK2(R1441G) NSCs, possibly accounting for the observed neuronal differentiation defect in these cells. These data indicate that LRRK2 regulates miRNAs to modulate neuronal differentiation with opposing effects compared to TRIM32. Thus, LRRK2 may antagonize the function of TRIM32 in miRNA-mediated neuronal differentiation.
Besides its role in the brain, TRIM32 also plays an important role in skeletal muscle, as it is implicated in the aetiology of Limb girdle muscular dystrophy type 2H (LGMD2H). LGMD2H is an inherited autosomal recessive disease of skeletal muscle caused by a mutation in the TRIM32 gene. Despite several studies addressing the function of TRIM32 in skeletal muscle, the contribution of TRIM32 to the pathogenesis of LGMD2H is poorly understood. Typically the regeneration process of adult skeletal muscle during growth or following injury is controlled by a tissue specific stem cell population termed satellite cells. Given that TRIM32 regulates the fate of mammalian NSCs by controlling their differentiation, we hypothesized that TRIM32 could also be essential for the regulation of satellite cells. Here we demonstrate for the first time that TRIM32 is expressed in the skeletal muscle stem cell lineage of adult mice and that it is strongly induced during muscle differentiation in vitro and during muscular regeneration in vivo. We show that TRIM32 is necessary and sufficient for myoblast differentiation in muscle progenitor cells, a function that is dependent on the ubiquitination and degradation of its binding partner c-Myc. Finally, we demonstrate that loss of TRIM32 in mice strongly impairs skeletal muscle regeneration in vivo similar to LGMD2H patients. Our studies indicate that loss of TRIM32 results in dysfunctional muscle stem cells, which could contribute to the development of LGMD2H.
Overall, this work provides important insights into the mechanisms, by which TRIM32 regulates the balance between stem cell maintenance and differentiation in the adult brain and skeletal muscle.