RNA binding proteins (RBPs) play multiple roles in RNA metabolism and their mutation, delocalization and/or altered expression have been proposed to cause familial and sporadic ALS. Elucidating the underlying molecular mechanisms is of crucial importance to design effective therapeutic approaches. Recently, it has been identified a new RBP involved in FUS-ALS: HuD/ELAVL4 (De Santis et al., 2017; De Santis et al., 2019). New preliminary data show that altered HuD levels are associated with competition between mutant FUS and FMRP for HuD 3'UTR binding. This evidence could induce increased levels of NRN1 and GAP43 and aberrant axon regeneration phenotype (Garone et al. in preparation). This pilot project aims at increasing the scientific confidence in the role of the molecular circuitry involving HuD, FMRP, NRN1 and GAP43 in the context of FUS-ALS.
I expect to clarify the contribution of HuD and its targets, including (but not limited to) NRN1 and GAP43, to early dysfunctions possibly leading to ALS. In particular, I would like to define the contribution of dysregulated HuD to the alteration of RNA metabolism in FUS mutant motoneurons. Moreover, I wish to clarify if the competition between mutant FUS and FMRP represents a pathogenic mechanism downstream of FUS mislocalization in the cytoplasm. Finally, mutant mouse ESC lines generated in this project will be next used to produce mouse models of both FUS and sporadic ALS. So, these evidences could be used for designing new therapeutic strategies and/or as biomarkers in future pre-clinical studies.
The reason why mutated genes encoding RNA-binding proteins cause ALS is not clear. RBPs share structural: RNA recognition motifs (RRMs), a low complexity domain (LCD), and a nuclear localization signal (NLS). In particular, LCD permit promiscuous binding not only to RNA but also to other proteins. The ALS-related mutations increase this binding bent, leading to self-assembly of the proteins and the formation of aggregates (Kim et al., 2013), that are toxic and induce self-propagating of these structures that spread disease within and between cells like prion proteins. These proteases also share many functions that affect RNA homeostasis such as transcription, mRNA splicing and polyadenylation, miRNA biogenesis, mRNA stability and transport, regulation of translation and miRNA processing. So, altered activity of an individual RBP results in defective RNA metabolism in the MN.
Interestingly, several works identified a crosstalk between FUS and other RBPs (Nakaya et al., 2013; Zhou et al., 2013; Masuda et al., 2015; Blokhuis et al., 2016; De Santis et al., 2017; De Santis et al., 2019), suggesting that exists a RBPs network in the nervous system, where ubiquitous and neural-specific RBPs cooperate in regulation of RNA homeostasis.
Here I propose to address this topic by using the FUS-HuD-FMRP axis as a paradigm. Despite this project is focused on ALS-FUS models, the discovery obtained here could be relevant for other mutant genes involved in familial and sporadic ALS.
Furthermore, preliminary data suggest that there is an aberrant axonal growth phenotype caused upstream by the mutation in the FUS gene and consequently downstream by the altered expression of dal NEURITIN and GAP-43. These two targets could be used as biomarkers for designing new therapeutic strategies. Finally, mESC lines generated in the framework of this Pilot Grant project will be used to generate mouse models that reproduce overexpression of HuD, NRN1, and GAP43 in MNs, independently from FUS mutations. Since HuD is altered also in sporadic ALS devoid of FUS mutations (De Santis et al., 2019), these mice might be used as sporadic ALS models for the future pre-clinical studies.