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An induced pluripotent stem cell-based neuronal model of Spinocerebellar ataxia

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 project iPSCAtaxia aimed to develop state-of-the-art human cell models for the study of diseases affecting the cerebellum region of the brain. In particular, the project focused on the spinocerebellar ataxias (SCAs), a group of devastating, inherited neurodegenerative diseases for which there is currently no known cure. Research into the SCAs, and neurological disease research in general, has typically been hindered by two main challenges. The first is a lack of lack of accessible, disease-relevant cells for study, particularly given the difficulties associated with obtaining patient brain tissue. We sought to overcome this obstacle by harnessing groundbreaking induced pluripotent stem cell (iPSC) technology to establish a novel human cell model of cerebellar disease. iPSCs are stem cells derived from adult cells, which have been reprogrammed to an embryonic-like state through the introduction of genetically modified viruses. They are thus capable of differentiation into any cell type of the body following treatment with specific combinations of growth factors, offering the unique opportunity to study human neurodegeneration in the laboratory. The first research objective was thus to differentiate iPSCs into the brain cell types most affected by cerebellar disease, for use both in understanding the complex disease mechanisms underlying the SCAs, and as future drug discovery tools.

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.
Over the course of the project, our research group has generated and characterised multiple iPSC lines from patients with two genetic subtypes of SCA (SCA14 and SCA41), in collaboration with colleagues at the Sir William Dunn School of Pathology (University of Oxford) and University of California, Los Angeles. Using these lines, we have successfully developed a novel and highly reproducible protocol for differentiating iPSCs into cerebellar neurons, with a particular focus on the generation of Purkinje cells, the neurons most affected in SCA. During the course of differentiation, these cells robustly expressed early markers of hindbrain (the developmental lineage from which Purkinje cells are derived), and went on to show morphology and protein expression characteristic of this specialised neuronal subtype.
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 our knowledge, we are the first in Europe to generate iPSC-derived cerebellar neurons, and the first to establish a disease-relevant human model for the study of common pathways underlying SCA. These results were recognised by the Society for Research on the Cerebellum and Ataxias, who awarded us the Masao Ito Prize at their International Symposium in 2017.

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.
hiPSC-derived Purkinje cells from SCA41 patient (G: Calbindin, R: Human Nuclear Antigen, B: Nuclei)