Ataxia research

Stem cells play a key role not only in development and growth of organisms but also in building new tissues and replacing others after injury. Thus they have great potential in regenerative medicine for the development of new therapies in a variety of different disease types. We employed fibroblasts from patients with A-T to reprogram into bona fide A-T induced pluripotent stem cells (A-T iPSCs). We showed that A-T iPSCs recapitulate key features of the A-T cellular phenotype, including radiation-induced cell cycle defects and increased sensitivity to radiation. We are at present in the process of differentiating these iPSCs into cerebellar neurons in collaboration with Prof. Ernst Wolvetang, AIBN.
 

Before the A-T stem cells can be used to correct the defect in patients, it is necessary to restore normal ATM activity. This can now be achieved using gene editing to correct the defective gene. One such approach is the CRISPR-Cas9 assisted homologous recombination mediated gene repair. CRISPR-Cas9 operates through targeted introduction of dsDNA breaks that allow strand invasion of a repair template. Since ATM is centrally involved in dsDNA break repair, homologous recombination in A-T stem cells may prove to be particularly challenging. These resulting, corrected stem cells will need to be checked for their efficacy and safety especially with respect to genomic sequence in animal models to confirm their therapeutic use in patients. These corrected stem cells from patients represent an important resource for use as an isogenic cell model for differentiation into neuronal cells of interest to screen for therapeutic agents.

Animal models are valuable to study in vivo aspects of A-T which are difficult or impossible to investigate in cell culture. Over the years many labs including ours created various animal models of A-T. ATM knockout (Atm-/-) mouse displayed some of A-T phenotypic characteristics: high propensity to develop lymphoid tumours, immunological abnormalities and infertility. We showed that Atm-/- murine Purkinje neurons have a reduced number of dendrites when cultured in vitro, however the Atm-/- mice did not recapitulate characteristic ataxia and neurodegeneration seen in human A-T patients. Therefore it is difficult to use Atm-/- mouse models to study functions of defective ATM protein in the nervous system. We have recently created a novel rat model characterized by some of the neurodegenerative phenotypes seen in patients with A-T. We are currently investigating mechanisms of neurodegeneration in this model.

The MRN complex (Mre11/Rad50/Nbs1) rapidly recognises and localizes to DNA DSBs where it acts to recruit and assist in ATM activation. ATM, in the company of several other DNA damage response proteins, in turn phosphorylates all three members of the MRN complex to initiate downstream signalling. We have recently shown that Rad50 plays a novel role in entry to and exit from mitosis, independent of its role in the MRN complex. Investigations are underway to unravel the specific mechanism involved.

To date, only patient-derived lymphoblastoid cells, fibroblasts, and SETX knockdown cells have mostly been used to investigate AOA2, all of which are not the most suitable model to study the neurological phenotype. Recent disruption of the Setx gene in mice did not lead to neurobehavioral defects or neurodegeneration, making it difficult to study the etiology of AOA2. Thus, we have recently developed the first AOA2 neuronal model by reprogramming AOA2 patient fibroblasts using iPSC technology to study the role of senataxin in gene regulation and neurodegeneration in this syndrome.

We generated the first AOA2 mouse model and demonstrated that senataxin is essential for spermatogenesis, functioning at two stages in meiosis- during crossing-over in homologous recombination and in meiotic sex chromosome inactivation (MSCI). We uncovered a critical role for senataxin in transcriptional silencing and chromatin remodeling during meiosis, providing greater insight into its critical role in gene regulation to protect against neurodegeneration.

A serum tube has been developed for analyte determination to overcome problems with conventional serum tubes such as lack of clotting with anticoagulated patients and cellular contamination. A snake prothrombin activator, OsPA, has been added to these tubes to enhance clotting. More recently, a recombinant form of the prothrombin activator has been developed to replace native prothrombin activator to enhance blood clotting and analyte determination.