RNA-binding protein Staufen1 conspires with Atxn2 in stress granules to cause neurodegeneration by dysregulating RNA metabolism

By Jamshed Arslan Pharm.D.

Spinocerebellar ataxia type 2 (SCA2) is a movement disorder characterized by neurodegeneration. The cause of this autosomal dominant disease is a mutation in the RNA processing gene Atxn2, which leads to polyglutamine (polyQ) expansion in ATXN2 protein. The association between mutant polyQ proteins, neurodegeneration, and dysfunctional autophagy has been extensively studied in amyotrophic lateral sclerosis (ALS), autism, and Huntington’s disease, but not in SCA2. It is noteworthy that ATXN2 is widely expressed in our nervous system, partly as a constituent of stress granules (SGs), which are cytosolic aggregations of proteins and RNAs. Investigating the interaction between ATXN2 with RNA-binding proteins that could modulate SG dynamics and RNA metabolism could be useful in SCA2 therapy. One such protein Staufen1 (STAU1) caught the attention of the University of Utah researchers. They found increased STAU1 levels in SCA2 patients and animal models, and reported that STAU1 interacts with mutated ATXN2 to cause aberrations in SCA2-related RNA metabolism.

Staufen1 physically interacts with ATXN2 in SCA2

The team first reported the co-localization of STAU1 and ATXN2 in SGs. Double immunostaining revealed that STAU1 and ATXN2 co-localized to SG-like aggregates in cerebellar Purkinje cells from SCA2 patients and mouse model. To test the physical interaction between STAU1 and ATXN2, immunoprecipitation of HEK-293 cell extracts (with/without RNAse A treatment) expressing Flag-tagged ATXN2 was performed with Flag antibody beads. Western blotting revealed that STAU1 interacted with ATXN2 in an RNA-dependent manner. Similarly, an increased STAU1 level was found in the cerebellar extracts of ATXN2-mutant mouse model of SCA2.

After demonstrating the STAU1-ATXN2 interaction in SCA2, the researchers moved on to investigate the role of this interaction in dysregulated RNA metabolism seen in SCA2.

Immunocytochemistry/Immunofluorescence: Aldolase C Antibody (4A9) [NBP2-25145] - A. Immunofluorescence of rat cerebellum section stained with mouse mAb to aldolase C, NBP2-25145, dilution 1:1,000, in green, and co-stained with rabbit pAb to GFAP, dilution 1:5,000 in red. In both A and B blue is Hoechst staining of nuclear DNA. aldolase C antibody selectively labels the perikarya and dendrites of Purkinje cells, while GFAP antibody stains processes of Bergman glia and astrocytic cells. B. Immunofluorescence of E20 rat cortical culture stained with NBP2-25145, dilution 1:1,000 in green, and co-stained with chicken pAb to MAP2, dilution 1:10,000 in red. The aldolase C antibody labels cytosolic protein expressed in glial cells, while MAP2 antibody stains dendrites and perikarya of mature neurons.

Increased Staufen1 causes aberrant RNA processing in SCA2

The researchers found a progressive reduction in Pcp2 and Calb1 proteins in SCA2 mice. They showed that mutant ATXN2 significantly reduces Pcp2 mRNA in SCA2 fibroblasts. Interestingly, overexpressing STAU1 in HEK293 cells without altering endogenous ATXN2 levels had similar effects: reduced Pcp2 protein and decreased Calb1 and Pcp2 mRNA abundance. Using fluorescent in situ hybridization (FISH) with PCP2-Cy3 probe, the team showed that Pcp2 mRNA co-localizes with STAU1 aggregates, indicating a direct STAU1-Pcp2 interaction in SGs. Moreover, SCA2 fibroblasts also showed increased levels of LC3-II and p62, indicating that impaired autophagy is also responsible for Stau1/Atxn2-induced pathology.

These data show that STAU1 plays a central role in SCA2 pathogenesis. The next step was to see if decreasing STAU1 could reduce problems associated with SCA2.

Reducing Staufen1 ameliorates SCA2 pathology

Researchers downregulated STAU1 by introducing siRNA in SCA2 cells that expressed mutant ATXN2. In contrast to normal cells, STAU1 reduction restored Pcp2 transcription levels in SCA2 cells. To explore the utility of this approach, the team generated four genotypes by crossing mice expressing mutant ATXN2 with mice deficient in one Stau1 allele (Stau1 haploinsufficiency). As expected, Stau1 reduction led to improved motor behavior in SCA2 mice. These mice have a characteristically low mRNA and protein levels for Pcp2, Pcp4, Calb1, Rgs8, Fam107b, and Homer3, along with SG-like aggregates positive for Stau1 and ATXN2. It was encouraging to see that Stau1 haploinsufficiency restored the levels of these proteins to normalcy and reduced those aggregates to almost none.

All in all, Stau1 reduction can potentially restore the motor, morphological and proteomic features of SCA2 pathology.

Significance

This study sheds light on the SCA2 biology and presents STAU1 as a novel therapeutic target for SCA2. Discovering an interactor (Stau1) of a mutant polyQ protein (ATXN2) in this paper has implications for other neurodegenerative diseases associated with elevated Stau1, mutant polyQ proteins, SGs and/or dysfunctional autophagy, such as ALS.

Explore Stress Granule Pathway

Jamshed Arslan, Pharm D.
University of Alabama at Birmingham, School of Medicine
Dr. Arslan studies cell signaling in mitochondrial defects in C. elegans
and transgenic mice.

References

Kobayashi, Tamae, et al. "PKCα Binds G3BP2 and Regulates Stress Granule Formation following Cellular Stress." PLoS ONE, vol. 7, no. 4, 2012, n.pag. https://doi.org/10.1371/journal.pone.0035820

Paul, Sharan, et al. "Staufen1 Links RNA Stress Granules and Autophagy in a Model of Neurodegeneration." Nature Communications, vol. 9, 2018, n. pag. https://doi.org/10.1038/s41467-018-06041-3

Xie, Xuan, et al. "Deubiquitylases USP5 and USP13 are Recruited to and Regulate Heat-Induced Stress Granules through their Deubiquitylating Activities." Journal of Cell Science, vol. 131, no. 8, 2018, n.pag. https://doi.org/10.1242/jcs.210856