University of Utah
The mission of our laboratory is the development of therapeutics for inherited diseases of the nervous system with an emphasis on spinocerebellar ataxias, amyotrophic lateral sclerosis (ALS), and Parkinsonīs disease.
Our History of SCA2
Our work on SCA2 represents an effort funded more than two decades by the NINDS. It began when Dr. Pulst and a colleague Dr. Sid Starkman visited a large SCA2 family in Syracuse NY in the early 1990’s. Later with other groups the SCA2 gene was mapped to Chr. 12, and Drs. Pulst and Starkman demonstrated anticipation in the original family suggesting that the causative gene mutation was likely a repeat expansion (Pulst et al., 1993). The gene discovery was published shortly thereafter (Pulst et al., 1996). The discovery demonstrated that ATXN2 had an expanded CAG repeat encoding a polyglutamine, and that ATXN2 is a highly evolutionarily conserved gene. At the time, the only hint on the function of ATXN2 was that it had RNA binding domains. Subsequent work to characterize ATXN2 followed two directions of investigation, discovery of ATXN2 interacting proteins, and the production and characterization of SCA2 mouse models. Chronologically there was much overlap in these efforts.
ATXN2 interacting proteins
Initial efforts to discover ATXN2 interacting proteins was by employing use of the yeast two-hybrid (Y2H) system, a method adopted by Dr. Scoles in the lab between ’95-‘96 to identify interactors to the NF2 protein (Scoles et al., 1998; Scoles et al., 2000; Scoles et al., 2006). By using Y2H we discovered the RNA binding protein ATXN2 binding protein 1 (A2BP1 / RBFOX1) as an ATXN2 interactor further supporting ATXN2 having a role in regulation of RNAs (Shibata et al., 2000). Later work done collaboratively with Ilya Bezprozvanny demonstrated that ATXN2 interacts with the inotisol triphosphate receptor protein (ITPR1) linking ATXN2 to a role in regulating calcium internal storage (Liu et al., 2009). Our studies on ATXN2 regulation of calcium movement is ongoing in collaboration with Tom Otis and Meera Pratap (UCLA) with focus primarily on hyperexcitability of SCA2 neurons (Meera et al., 2016).
SCA2 mouse models
We have two primary SCA2 mouse models in the lab, including ATXN2-Q127 and BAC-Q72. The ATXN2-Q127 mouse has the human ATXN2 cDNA driven by a promoter for specific expression in Purkinje cells (Pcp2) while the BAC-Q72 mouse has the human ATXN2 gene with all its introns and exons driven by 16kb of human upstream sequence. Both of these models have very similar age-dependent SCA2 phenotypes, including progressively reduced rotarod performance, progressive reduction of expression of specific Purkinje cell genes that we initially discovered by transcriptome analyses, and progressive reduction of Purkinje cell intrinsic firing frequency (Hanson et al., 2013; Dansithong et al., 2015).
SCA2 antisense oligonucleotide therapy
Toward developing a therapy for SCA2 we developed antisense oligonucleotides (ASOs) targeting ATXN2, and tested in a proof of concept study their efficacy for restoring the SCA2 phenotypes of our ATXN2-Q127 and BAC-Q72 mouse models. Our approach employs an ASO targeting the mutant and normal copy of the ATXN2 gene mRNA which we believe would be well-tolerated because mice null for the Atxn2 gene have no neurodegeneration (Kiehl et al., 2006). We treated SCA2 mice at approximately the time of phenotype onset at 8 weeks of age by intracerebroventricular (ICV) injection, and evaluated mice on the rotarod at specific timepoints thereafter out to 21 weeks of age. We observed that the ASO treated mice had a significantly better rotarod performance than control mice. At the study endpoints we evaluated the cerebellar proteins finding for ASO treated animals that the expressions of 6 key proteins were restored to levels as in wild-type. We also observed that the intrinsic Purkinje cell firing frequencies were restored by ASO treatment. The effect of the ATXN2 ASO for improving SCA2 phenotypes was seen in both SCA2 mouse models (Scoles et al., 2017). This proof of concept study supports our ongoing efforts to refine our ASO approach for targeting ATXN2 as a therapy for SCA2.
ALS in SCA2
Some SCA2 patients look like ALS patients but with added ataxia. This prompted Aaron Gitler to investigate CAG repeat structures in ATXN2 in ALS patients, revealing that some ALS patients have intermediate CAG length expansions (Elden et al., 2010). Thereafter we published a meta-analysis that showed that ALS risk is significantly associated with ATXN2 repeat length, where intermediate repeat lengths of 27-28 were associated with low risk, but for 32-33 repeats ALS risk reached a maximum (Neuenschwander et al., 2014). We then partnered with Dr. Gitler to show that survival of TDP43 ALS mice was improved when normal Atxn2 gene expression was reduced by crossing the TDP43 mouse with our Atxn2 knockout mouse. The study also showed that the same result could be achieved using an ASO targeting expression of the mouse Atxn2 gene, and that the underlying pathology was reduction of TDP43 positive stress granules with reduced Atxn2 expression (Becker et al., 2017).
Stress granules and targeting ATXN2 in other neurodegenerative diseases
We have found that stress granule production is stimulated when ATXN2 is polyglutamine expanded. By studying SCA2 mouse cerebellar transcriptomes we have also discovered SCA2-related mRNAs that localize to SCA stress granules where their fates are determined. One example includes the sequestration of the PCP2 mRNA to stress granules in SCA2 patient derived fibroblasts where the message decays, accounting for the reduced Pcp2 expression that we observed in SCA2 mouse cerebella.
Quantitative high throughput compound screening (qHTS)
Past funding to Dr. Scoles and Dr. Pulst included screening for compounds lowering ATXN2 expression toward developing small molecule therapeutics for SCA2. This work was performed as a cooperative agreement with National Institutes of Health (NCATS). We with Dr. Duong Huynh presently have another qHTS effort with NCATS to identify small molecule therapeutics lowering alpha synuclein expression for Parkinson’s disease, funded by the Michael J. Fox Foundation. The alpha synuclein study follows up on another MJFF funded study for creation of the cell line screening assay (Dansithong et al., 2015). Studies are ongoing.
NINDS, R37-NS033123 & R01-NS097903
Michael J Fox Foundation