Motor neuron disease (MND) is an incurable neurodegenerative disease that culminates in death following rapid loss of motor neurons and muscle function. The hallmark pathology of MND is the cytoplasmic mislocalisation of a predominantly nuclear protein – TDP-43. In healthy individuals, TDP-43 regulates RNA processing in the nucleus, a key component of which is the suppression of cryptic exon inclusion in mRNA. In MND, the cytoplasmic shift of TDP-43 leads to loss of nuclear function and therefore, dysregulated RNA processing. Recently, retention of a cryptic exon in Stathmin-2 (STMN2) was detected in MND patient spinal cord tissue and iNeuron cell culture. This cryptic exon results in nonsense-mediated decay of STMN2 mRNA and, consequently, reduced STMN2 protein levels. This was a seminal discovery, which provided new insight into MND pathomechanisms. More excitingly, STMN2 has also emerged as an enticing therapeutic target as restoration of normal STMN2 in human motor neurons derived from pluripotent stem cells rescued axonal regeneration post-injury.
We seek to establish a platform to investigate the role of cryptic exons in MND, and other TDP-43 proteinopathies, using a zebrafish model system. Zebrafish are suited to high-throughput analyses and therapeutic testing due to high scalability (lay large numbers of eggs) and a developed motor system by 2 days of age. This allows efficient analysis compared to other vertebrate models and allow in vivo analyses unlike human neuronal cell models currently in use.
We have established transgenic zebrafish models that express fluorescently-tagged human TDP-43 (wild-type, MND-causative mutation p.G294V or nuclear depleted dNLS) in motor neurons. The G294V and dNLS models display MND-relevant pathologies such as reduced motor function as early as 6 days of age, compared to the wild-type model. In vivo study of cryptic exons in MND have only recently commenced and, to date, a motor neuron specific zebrafish transcriptome and its changes in MND has yet to be established. To address this, we have established a workflow to isolate motor neurons from the TDP-43 zebrafish models using flow cytometry. Ongoing work will optimize downstream RNA extraction and perform RNAseq on our TDP-43 zebrafish models to allow the identification of novel and known cryptic exons. This not only further characterises our preclinical models of MND but also identifies new targets for investigations in human neuronal lines. Targets validated in human neurons can then be investigated for their therapeutic potential in the TDP-43 models of which the field is sorely lacking.