1Reseach Programs Unit, Molecular Neurology, University of Helsinki, Helsinki, Finland; 2Clinical Neurosciences, Neurology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland; 3Neuromuscular Research Center, University of Tampere and Tampere University Hospital, Tampere, Finland
Inherited axon degeneration diseases such as axonal Charcot-Marie-Tooth disease (CMT2) and hereditary spastic paraplegia (HSP) are clinically and genetically highly heterogeneous diseases. Tens of different disease genes have been identified as causative for these disorders when mutated. The genes function in multiple intracellular pathways that are required for the maintenance of axon shapes and functions. Studies of detailed molecular mechanisms have often been complicated by the lack of human neuronal models with endogenous expression levels of the mutant proteins. We have characterized the genetic landscape of CMT2 and HSP in Finland, and identified two founder mutations, as well as tens of other mutations in different genes. To investigate the molecular mechanisms of the most interesting disease genes and mutations, we have reprogrammed patient-specific skin fibroblasts into induced pluripotent stem cells, and differentiated those into spinal motor neuron cultures. We currently use the droplet based 10x Genomics Chromium system to capture single motor neurons from mixed cultures to profile the patient-specific transcriptomics of these cells. We aim to identify mutation-specific altered neuronal pathways to elucidate disease mechanisms, which could aid in finding targets for therapeutic approaches. As a proof of principle, we profiled single motor neurons from a patient with a NEFL mutation predicted to affect the mRNA level of this strictly neuron-specific intermediate filament subunit, neurofilament light. The profile of the patient’s neurons revealed that less than five percent of NEFL mRNA was retained in comparison to control neurons. This particular setting allows a rare opportunity to investigate the transcriptomic alterations of human neurons lacking NEFL, which is largely considered to be essential for axonal architecture.
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