Periodic Reporting for period 1 - iPSCAtaxia (An induced pluripotent stem cell-based neuronal model of Spinocerebellar ataxia)
Reporting period: 2016-07-01 to 2018-06-30
The second key challenge facing this research is the vast clinical and genetic heterogeneity of the conditions under study. To date, more than 40 causative genes have been identified for the SCAs, making the task of unravelling disease pathology and developing effective, targeted therapies particularly difficult. To help make sense of this complexity, iPSCAtaxia focused on a single candidate, the mGluR1-TRPC3 signalling pathway, defects in which have been linked to SCA1, 3, 5, 14, and 41, as well as the Moonwalker mouse model of ataxia. Since recent transcriptomic analysis of human brain tissue has also identified TRPC3 as part of a significant SCA-enriched co-expression module, we reasoned that TRPC3-mediated signalling represented a strong candidate for a common ataxia pathway, which could serve as a novel therapeutic target. The second research objective was therefore to elucidate the precise molecular mechanisms underlying such common SCA-causing disease pathways, using our disease-relevant human iPSC-based model.
The final research objective involved the adaptation of the model as a tool for therapeutic screening and drug development. Although SCAs and other neurodegenerative diseases are traditionally thought of as late-onset conditions, we and others have demonstrated developmental abnormalities in several ataxic mouse models, predominantly affecting the Purkinje cells of the cerebellum. Our iPSC-derived models, which are capable of recapitulating early developmental events in vitro, will therefore be vital in unravelling the neurodevelopmental aspects of these diseases in humans, in order to develop early-intervention therapies.
Microscopy analysis of Purkinje cells generated from SCA14 patients revealed the presence of a disease-associated phenotype characterised by reduced survival, and abnormal cellular development and morphology. The identification of a morphological phenotype in SCA41 patient cells proved less straightforward. However, our preliminary results indicate that disease-associated aberrations closely resembling those previously observed in the Moonwalker mouse (a model of TRPC3-mediated cerebellar ataxia) may manifest as early as the iPSC stage. This is the first evidence of a disease phenotype in SCA41 human cells, suggesting that defective TRPC3 signalling early in development may underlie the cerebellar dysfunction associated with disease. Since iPSCs are more easily manipulated in culture than cerebellar neurons, these results offer the additional possibility of high throughput screening to identify potential therapeutic strategies. Using a similar approach, our research group has subsequently also identified a disease phenotype in SCA14 iPSCs, which closely resembles human brain pathology.
Building on the results obtained from the cerebellar differentiation protocol mentioned above, we have also developed a pipeline for the generation of three-dimensional cerebellar organoids from iPSCs. Preliminary data is promising, suggesting that these cells are capable of long-term survival and differentiation, making them an ideal next-generation disease model. Work is currently underway to characterise the composition and maturity of these organoids using a combination of immunocytochemistry and single-cell sequencing.
To enable its broader use for the study of other neurodegenerative conditions, our differentiation method was published in 2018 in the journal The Cerebellum, where it has attracted considerable international attention, with research groups from numerous different countries expressing their interest in collaborating or reproducing our results. This paper was also recently selected as ""Paper of the Year 2018"" by the editorial board of the Journal. In addition, our interdisciplinary research has drawn together several clinical and basic science departments within the University of Oxford – a collaboration which resulted in the identification of a novel genetic subtype of SCA (SCA44, caused by mutations in mGluR1) in 2017.
The models generated over the course of iPSCAtaxia have provided us with the unique opportunity to study human cerebellar neurons and cerebellar development in the laboratory, leading to a better understanding of disease mechanisms in cerebellar ataxia. Given their great potential as drug screening tools, these cells are now poised to help in the discovery of novel treatments for these devastating neurodegenerative conditions.