Regulatory non-coding RNAs (ncRNAs), including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), and RNA binding proteins (RBPs) represent important modulators of cell fate identity. In particular, ncRNAs and RBPs are remarkably abundant in the nervous system where they exert important regulatory functions.
The present proposal stems from a 2014 ERC Starting Grant project (acronym: NeuRNA) aimed at studying ncRNAs involved in human early neural development. Here we propose to extend this aim by systematically identifying ncRNAs that play a role during neural development AND neurodegeneration, using the motoneuron disease Amyotrophic Lateral Sclerosis (ALS) as a paradigm. In the last few years, indeed, it has been proposed that dysregulation of RNA metabolism could be a trigger to motoneuron degeneration. We will therefore explore the possibility that neural ncRNAs, which we name NeuRNAs (for Neural untranslated RNA), might be under the control of RBPs mutated in ALS, such as FUS. Our lab has recently established a collection of human induced Pluripotent Stem Cells (iPSCs) with ALS mutations in the FUS gene. We have also set up proper protocols for differentiating iPSCs into different subtypes of motor and cortical neurons. Together with our expertise in the non-coding RNA field, these tools put us in the ideal position to accomplish the proposed aims.
In the first aim we will profile NeuRNAs from differentiating iPSCs, identifying specific neural signatures, and provide a functional and mechanistic characterization of relevant candidates. In the second aim we will assess whether NeuRNAs are under the control of ALS-linked RBPs and whether ALS mutations affect their activity. Our work will investigate about the contribution of the non protein-coding portion of our genome to fundamental processes such as neural development and neurodegeneration.
Our proposal is innovative and original, because it relies on appropriate cellular model systems, consisting of a collection of human iPSCs with mutations in disease-relevant RBPs, for the study of neurogenesis and neurodegeneration in human. This will allow the discovery and molecular characterization of the function and mechanisms of action of regulatory NeuRNAs with a role in the development of the nervous system and possibly involved in pathways leading to neurodegeneration by interplaying with disease-linked RBPs.
A crucial biological question drives our research program: pluripotent stem cells are the only cell type that can generate all tissues and organs, however our understanding of their biology remains fragmentary. We can now derive iPSCs by reprogramming of adult somatic cells, maintain these cells in vitro thanks to their unlimited self-renew potential, and differentiate them in a variety of specialized cell types. The therapeutic potential of human iPSCs is remarkable and they also represent useful tools to answer basic biological questions, as they can be considered as an in vitro model of human embryonic development. It is clearly imperative to study the molecular regulators of iPSC differentiation to fully exploit their potential. A better understanding of the molecular pathways underlying their differentiation along the neural lineage is crucial to obtain ¿high quality¿ neuronal cell types with proper functional properties. Our research will shed light on the role played by regulatory RNAs during neurogenesis. The regulatory network underlying neural differentiation is made of transcription factors, chromatin modifiers, microRNAs and signal transducers. We will add a missing piece in this puzzle by identifying and characterizing new NeuRNAs that act in concert with other factors to dictate neuronal cell fates. For all these reasons, our proposal has the potential to enhance our control of human iPSCs differentiation for biomedical applications and shed light on the molecular determinants that orchestrate human neural development.
We have recently shown that the activity of miR-375, a neural miRNA, is impaired in iPSC-derived motoneurons carrying an ALS-linked mutation in the RBP FUS (De Santis et al., 2017). In turn, dysregulation of miR-375 causes an aberrant increase of the levels of another RBP, ELAVL4. We consider the axis FUS/miR-375/ELAVL4 as a paradigm illustrating how ALS mutations in RBPs might have profound effects by perturbing the activity of ncRNAs and other RBPs. Notably, FUS mutations are associated to particularly severe and juvenile ALS cases. Despite increasing evidence points to the gain of toxic functions of the mutant protein as the major pathological mechanism, at present the pathways leading to neurodegeneration downstream of FUS mutations remain largely unknown. A better comprehension of the aberrant activities acquired by mutant FUS in the specific cell type primarily affected by the pathology, i.e. the human motoneuron, is mandatory to design proper therapeutic strategies. In particular, it is important to define the repertoire of RNA molecules, both coding and non-coding, bound by mutant FUS and its effects on the expression of these genes. Our model system, consisting in human iPSC-derived motor neurons carrying ALS FUS mutations (Lenzi et al., 2015; De Santis et al., 2017), allows to fill this gap. Defining at the global level the genes altered by mutant FUS will provide important pieces of information on the pathological mechanisms of FUS ALS and possible therapeutic targets. In the long term, based on the results obtained with the present project, our iPSC-derived human motor neurons might be used as a system to screen in vitro for drugs that restore the normal levels and activity of coding genes and NeuRNAs. Importantly, upon accomplishment of the present project we will produce publicly available datasets that could be mined by members of the ALS community for their own pathway of interest. Finally, the outcomes of this project, focused on FUS, could be broader and relevant for other RNA binding proteins involved in ALS.