MAPK Pathway Components: Modulators of Ataxin1 Toxicity in SCA1
Background: Targeting Disease-Driving Proteins
As neurodegenerative disorders become more common in an aging population, understanding the molecular pathways that drive them matters more than ever. The mammalian brain is extremely complex, which makes designing better targeted treatments difficult. One of the biggest obstacles in neurodegenerative disease research is the lack of viable biomarkers for older patients. Disorders like Alzheimer’s, Parkinson’s, and the polyglutamine diseases share a common pathogenic feature: the accumulation of misfolded proteins, caused either by mutations that resist degradation or by over-expression of the normal (wild type) protein.
In the May 2013 issue of Nature, Dr. Zoghbi and colleagues at Baylor College of Medicine devised a strategy for finding therapeutic entry points that influence the levels of disease-driving proteins. They applied this approach to spinocerebellar ataxia type 1 (SCA1), a disease caused by expansion of the polyglutamine tract in ataxin 1 (ATXN1), using modulation of the ATXN1 pathway as a proof-of-principle. They chose this model for three reasons:
- Neurodegeneration in SCA1 tracks with the level of mutant ATXN1 protein.
- Over-expressing wild type ATXN1 also causes neurodegeneration.
- SCA1’s pathogenic mechanisms are already well characterized.
A Genetic Screen for ATXN1 Modifiers
To find regulators of ATXN1 levels, the authors built a human medulloblastoma-derived cell line carrying the transgene glutamine-expanded ATXN1 fused to red fluorescent protein (mRFP-ATXN1(82Q)). To separate modifiers that regulate ATXN1 protein levels from those that just affect transgene transcription, they added an internal ribosomal entry site followed by yellow fluorescent protein downstream of ATXN1 (mRFP-ATXN1(82Q)-IRES-YFP). The screen focused entirely on kinases and kinase-like genes, since ATXN1 phosphorylation is known to be critical for its toxicity and kinases can be targeted with drugs. The team tested 1,908 small interfering RNAs (siRNAs) targeting 638 genes, using ATXN1 levels as the readout. From there, they selected 50 siRNAs (corresponding to 45 genes) that reduced the ratio of RFP to YFP by 2 standard deviations from the mean.
A parallel genetic screen used the Drosophila SCA1 model expressing ATXN1(82Q), which can be identified by an external eye phenotype. Here, the team screened 704 alleles (337 kinase-encoding, including shRNA and loss-of-function mutations) for those that modified ATXN1 levels. Based on morphological and histological assessments, they found 51 alleles (49 genes) that suppressed ATXN1 toxicity in vivo. Human cell-based screens also identified 10 human modifier genes that reduced ATXN1 and its toxicity, corresponding to 8 Drosophila modifiers.
The MAPK Pathway Emerges
Network analysis showed that the MAPK cascade was the most enriched pathway in both Drosophila and human screens — 6 of the 10 human genes identified belonged to the canonical MAPK pathway (ERK1, ERK2, MED2, MEK3, MEK6, and MSK1).
Since ATXN1(82Q) is known to impair motor performance, the team ran a motor performance test in Drosophila to check the MAPK pathway’s effect on the central nervous system. Decreasing the MEK, ERK1/2, and MSK1 Drosophila homologues by siRNA increased motor performance and lifespan. Decreasing upstream MAPK pathway homologues suppressed ATXN1(82Q) eye defects and improved both motor performance and lifespan. Conversely, constitutively active RAS made ATXN1 eye degeneration worse. In human cells, decreasing HRAS and FNTA lowered ATXN1 protein levels, and decreasing RAS homologues reduced ATXN1 in vivo.
Validating MSK1 as a Regulator
Earlier work from Dr. Zoghbi’s group had shown that ATXN1 levels were sensitive to S776 phosphorylation. Based on that, the team determined that MSK1 — among the MAPK kinases identified here — could phosphorylate the consensus sequence associated with S776. To confirm this, they ran an in vitro kinase assay with purified MSK1 and ATXN1, and found robust ATXN1-S776 phosphorylation in both mutant and wild-type protein forms. Cerebellar fractionation assays in wild-type mice then showed that MSK1 was enriched, with increased activity, in S776-phosphorylated fractions. Conversely, immunodepletion of MSK1 from mouse cerebellar extracts reduced S776 phosphorylation.
Next, the team tested whether the MAPK pathway could serve as a pharmacological target for SCA1. Human cells expressing ATXN1(82Q) were treated with PD184352 (a MEK1/2 inhibitor), GW5704 (a RAF1 inhibitor), and Ro31-8220 (an MSK1 inhibitor). Pharmacological inhibition of the MAPK pathway decreased ATXN1(82Q) levels, and adding MAPK inhibitors to cerebellar slices also decreased ATXN1 levels.
Finally, to test the genetic interaction between ATXN1 and MSK1, the team bred ATXN1(154Q) knock-in mice (Atxn154Q/+) with Msk1+/- Msk2+/- mice. Atxn154Q/+ mice develop a motor phenotype by 9 weeks of age that can be measured with a rotarod test. Breeding Atxn154Q/+ Msk1+/- Msk2+/- mice produced better rotarod performance. Since ATXN1 alterations cause Purkinje cell degeneration, the team also tested whether removing one copy of MSK1 could rescue Purkinje cell loss in a second ATXN1(82Q) mouse model, B05/+. A single-copy deletion of Msk1 partially suppressed the Purkinje cell loss phenotype, and deleting one copy each of MSK1 and MSK2 (B05/+Msk1+/- Msk2+/-) reduced ATXN1 levels even further.
What This Means for SCA1 and Beyond
Dr. Zoghbi’s group has developed a proof-of-principle strategy that opens new paths for identifying modifiers of neurodegenerative disease. They combined cross-species genetic screens to identify novel modifiers of ATXN1, and validated their findings in human, mouse, and Drosophila models. The study focused on an early event in disease pathogenesis that could potentially delay onset and progression for this class of disorders. The RAS-MAPK-MSK1 pathway identified here — acting through phosphorylation of S776-ATXN1 — offers a new pharmacological target for SCA1, and opens the door to combination therapies. Most neurodegenerative disease research has focused on treating advanced symptoms. It would be worth exploring what therapeutic benefit targeting the RAS-MAPK-MSK1 pathway offers at a more advanced stage of disease, and whether it could partially reverse motor defects.
References:
References
Park, J., et al., RAS-MAPK-MSK1 pathway modulates ataxin 1 protein levels and toxicity in SCA1. Nature.
Emamian, E.S. et al. Serine776 of ataxin-1 is critical for polyglutamine-induced disease in SCA1 transgenic mice. Neuron 38, 375–387 (2003).
Jorgensen, N. D. et al. Phosphorylation of ATXN1 at Ser776 in the cerebellum. J. Neurochem. 110, 675–686 (2009).
