We have previously identified and functionally characterized pCharme, a muscle-specific, evolutionary conserved lncRNA acting in the nucleus as an epigenetic regulator. In mouse, pCharme ablation severely impairs myogenic differentiation at early onset, triggering muscle hyperplasia and cardiac remodeling. Mechanistically, pCharme supervises, through direct interaction, the chromatin localization of MATR3, a multifunctional DNA/RNA-binding protein whose mutations were identified in patients affected by myopathies or motor neuron diseases. In particular, mutations that specifically compromise the MATR3 binding to RNA result in the formation of non-physiological, MATR3-containing condensates, which are also hallmarks of neurodegeneration. In line with this, mice expressing a cytoplasmic version of pCharme show mislocalised MATR3 distribution and a significant impairement of neuromotor abilities. This phenotype suggests unpredicted roles for pCharme in maintaining both muscles and neurons and evokes an intriguing "dying-back" phenomenon by which the impairment of muscles would produce retrograde signals contributing to motor neuropathies.
On these premises, we sought to investigate the involvement of the human pCharme into neuromuscular circuitries and neurodegeneration. To this aim, we plan to apply an experimental design where the synergy between CRISPR-Cas9 gene editing and induced pluripotent stem cells technology will allow the study of muscle-nerve connections through the generation of pCharme-KO neuromuscular organoids (NMOs), in which the spinal cord and the skeletal muscle counterparts develop in parallel and functionally interact. Our study is expected to provide an innovative platform to study the human neuromuscular networks and trace the developmental contribution of new classes of genes to neurodegenerative disorders, particularly those in which the formation of a functional neuromuscular junction (NMJ) is early impaired.
Muscle homeostasis is critically required for the maintenance and repair of adult tissues. Any deviation from the dynamic equilibrium that keeps muscle fibers in a constant state of complexity may evolve into different types of diseases. Over the past few years, several groundbreaking studies have focused fresh attention on this field by exploring the impact of novel regulators in muscle pathophysiology. Intriguingly, our research revealed the essential contribution of non-coding RNA (ncRNA)-mediated circuitries to the regulation of myogenic gene expression. The chemical features of RNA as a single-stranded polymer become fully exploited with lncRNAs, whose i) nuclear/cytoplasmic dual localization, ii) extraordinary cell-type and timely regulated expression, iii) ability to base-pair nucleic acids and to simultaneously interact with proteins, foster their biological relevance (Ballarino et al, JCI 2016). For instance, we found that the functional ablation of the muscle-specific lncRNA pCharme causes skeletal and cardiac muscle hyperplasia and premature death of mice around one year of age. Besides the progressive myopathy, mutant animals also experience motor deficits ascribable to neurodegeneration. As the functional interplay between muscle and nerve is crucial for both counterparts to survive and adequately function (Lepore et al, Cells 2019), these preliminary results are consistent with altered muscle-nerve transmission and imply pCharme as a candidate gene implicated in neuromuscular and neurodegenerative disorders.
While we put substantial efforts to elucidate the mechanisms of pCharme regulation (Ballarino et al, EMBO J 2018, Desideri et al, Cell Rep. 2020), our studies mostly focused on skeletal and cardiac muscles, while the role of the lncRNA in developing NMJs, the chemical bridge where motor neurons and muscle communicate, remains virtually unknown. The study of such diseases affecting a process in which more than a single tissue participates, represents a great challenge in practice. In our case, the generation of all the NMJ components with a precise positional identity is mandatory for studying the involvement of pCharme in neuromuscular circuitries in vivo. While the use of our mouse models represents a concrete opportunity for analysing the influence of the murine pCharme to NMJs (ongoing collaboration with Czech Centre of Phenogenomics and Laboratory of Transgenic Models of Diseases, Institute of Molecular Genetics of the Czech Academy of Sciences), the same is not easily addressable in humans. The development of induced pluripotent stem cells (iPSCs) (Takahashi et al, 2007) indeed provided unprecedented opportunities to decipher pathophysiological mechanisms and to model human diseases in which a single, specific tissue is affected (Rowe and Daley, Nat Rev Genet. 2019). However, cellular interactions in the tridimensional space are also crucial to define tissue architectures. This fact makes the 3D organoids among the most promising biological systems for tissue and disease modeling and, in the present work programme, crucial means for understanding pCharme function.
Moving beyond the plan described here, we expect to be innovative at both conceptual and methodological levels. The synergistic match among the longstanding skills of our teams in non-coding RNAs, stem cells, neuro and muscle biology will ensure a productive, multi-disciplinary approach for the successful realization of this plan. The effectiveness of this collaborative structure was recently proved in previous collaborations as well as in the successful completion of the preliminary data presented in this application.
Since we are extremely motivated in addressing our biological questions, we have already applied a strict set-up of the main protocols and methodologies, to maximize the chances of success and minimize project failure. Our CRISPR-Cas9 pipelines have been already standardized and showed fully feasibility (Ballarino et al, EMBO J 2018; Desideri et al, Cell Reports 2020). Another challenging step will certainly be the generation of hiPSC-derived neuromuscular organoids. Nonetheless, the direct involvement in the project of Prof. Rosa, who has already set-up the protocols, confers robustness to the proposed experimental plan. Overall, the optimization of these tools represents a valuable starting point that greatly increases the probability of success of the experimental path.
My long-term goal is to augment our understanding of the RNA biology of lncRNAs, with direct implications for interpreting variation in their sequences in the context of clinical genetics, as well as for their use in diagnosis. As neuromuscular pathologies are genetically heterogeneous, most disease-causing mutations being still unidentified, this will be particularly relevant for those cases lacking any identified causing mutation.