Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and incurable neurodegenerative disease, characterized by the progressive death of motor neurons (MNs) for which the early cellular and molecular events remain poorly understood. As a consequence, no effective treatments have been developed for this devastating disease where most patients die within 3-5 years (with 30% dying in just 12 months of diagnosis). We recently uncovered cytoplasmic accumulation of aberrant intron retaining transcripts (termed ‘aCIRT’ hereafter) as the earliest detectable molecular phenotype in ALS and revealed universal nuclear-to-cytoplasmic mislocalisation of the SFPQ protein in human stem cell cultures, transgenic mouse models and human post-mortem tissues from sporadic cases, thus encompassing the full spectrum of ALS. This suggests that aCIRT and the nuclear loss of SFPQ are unifying molecular hallmarks. However the regulatory role of CIRT on cell behaviour, as well as the impact of aCIRT on protein mislocalization and aggregation remains unknown. Alternative splicing and the presence of CIRTs in astrocytes (ACs) and microglia is understudied, yet ACs are now established as playing pivotal roles in ALS pathogenesis.
The overall aim of this project is to systematically study how aberrant mRNA metabolism in the cytoplasm shapes MN and AC cellular phenotype including protein localisation early in ALS pathogenesis. In order to do this we will 1) characterise the nuclear versus cytoplasmic mRNA metabolism of developing MNs and ACs with a focus on 3’ UTR and intronic sequences, 2) develop and adapt computational methods to high content microscopy data of developing MN and AC to extract hundreds of nuclear, cytoplasmic, neurite-related single-cell measurements i.e. shape and morphometric descriptors, fluorescent intensity data and signal distribution across various channels, 3) model the cellular trajectories of developing control and ALS-mutant MNs and ACs from high-content single-cell profiling data to discover the earliest and universally identifiable cellular processes occurring in ALS MNs/ACs, 4) develop statistical models that integrate RNA-sequencing with cell imaging to test and model the association between aberrant mRNA metabolism in the cytoplasm and aberrant cell phenotype including ALS-related RBP mislocalization in early ALS development, and 5) validate comprehensively using orthogonal approaches including multiple mutant iPSC lines, transgenic mouse models and human post mortem tissues, so ensuring high-confidence findings of translational value.
In the four years time-framework of this project, we will build a transcriptomic-phenotypic map of early ALS development enabling the identification of key molecular and cellular processes that best discriminate the ALS from control trajectories. Thus we will not only propose a novel combination of cellular and molecular hallmarks of early ALS development but we will also shed light into the initiating molecular and cellular events of ALS through this novel multi-modal and integrated approach. Capturing the earliest cellular changes underlying the disease could in turn point to new pathways for targeted therapies. In addition, although this proposal focuses on ALS, results might be transferable to other neurological disorders.