Regulating ataxin-1 expression as a therapeutic avenue for SCA1

Written by Dr. Hannah Shorrock   Edited by Dr. Hayley McLoughlin

Nitschke and colleagues identify a microRNA that regulates ataxin-1 levels and rescues motor deficits in a mouse model of SCA1

What if you could use systems already in place in the cell to regulate levels of toxic proteins in disease? This is the approach that Nitschke and colleagues took to identify the cellular pathways that regulate ataxin-1 levels. Through this strategy, the group found a microRNA, a small single-stranded RNA, called miR760, that regulates levels of ataxin-1 by directly binding to its mRNA and inhibiting expression. By increasing levels of miR760 in a mouse model of SCA1, ataxin-1 protein levels decreased and motor function improved. This approach has the potential to identify possible therapies for SCA1. It may also help identify disease-causing mutations in ataxia patients with unknown genetic causes.

Spinocerebellar Ataxia type 1 (SCA1) is an autosomal dominant disease characterized by a loss of coordination and balance. SCA1 is caused by a CAG repeat expansion in the ATXN1 gene. This results in the ataxin-1 protein containing an expanded polyglutamine tract. With the expanded polyglutamine tract, ataxin-1 is toxic to cells in the brain and leads to dysfunction and death of neurons in the cerebellum and brainstem.

As with all protein-coding genes, surrounding the protein coding region of ATXN1 gene are the 5’ (before the coding sequence) and 3’ (after the coding sequence) untranslated regions (UTRs). These regions are not translated into the final ataxin-1 protein product but are important for the regulation of this process. Important regulation factors called enhancers and repressors of translation located in 5’ and 3’ UTRs. ATXN1 has a long 5’ UTR. Genes that require fine regulation, such as growth factors, are often found to have long 5’ UTRs: the longer a 5’ UTR, the more opportunity for regulation of gene expression. The group, therefore, tested the hypothesis that the 5’ UTR is involved in regulating the expression of ataxin-1.

In their initial studies, Nitschke and colleagues identified that the ATXN1 5’UTR is capable of reducing both protein and RNA levels when placed in front of (5’ to) a reporter coding sequence. One common mechanism through which this regulation of gene expression could be occurring is the binding of microRNAs, or miRNAs, to the ATXN1 5’UTR. miRNAs are short single-stranded RNAs that form base pairs with a specific sequence to which the miRNA has a complementary sequence; this leads to regulation of expression of the mRNA to which the miRNA is bound.

3d illustration of single-strand ribonucleic acid
Artist drawing of single-stranded RNA. Photo used under license by nobeastsofierce/

Using an online microRNA target prediction database called miRDB, the group identified two microRNAs that could be responsible for these changes in gene expression through binding to the ATXN1 5’ UTR. By increasing the expression of one of these microRNAs, called miR760, ataxin-1 protein levels were reduced in cell culture. Conversely, using a miR760 inhibitor so that the miRNA could not perform its normal functions led to increased levels of ataxin-1. Together this shows that miR760 negatively regulates ataxin-1 expression.

Next, the group sought to understand how miR760 regulates the expression of ataxin-1. The group predicted that miR760 directly binds to a specific sequence in the 5’ UTR of ATXN1 RNA. By deleting three bases of this sequence, the regulation effect of miR760 on ataxin-1 levels was lost. This shows that direct binding of miR760 to this specific sequence is required for the microRNA to regulate ataxin-1 expression.

To understand how the direct binding of miR760 regulates ataxin-1 levels, the group looked at levels of ATXN1 mRNA. They studied the relationship between ATXN1 mRNA and the translation machinery. They found that miR760 regulates ataxin-1 expression by causing degradation of ATXN1 mRNA and by reducing the efficiency of ataxin-1 translation. Together, this indicates that the direct interaction of miR760 with the ATXN1 5’UTR leads to decreased regulation of ataxin-1 expression. This decrease is caused by both mRNA degradation and translational inhibition.

In SCA1, ataxin-1 contains an expanded polyglutamine tract which makes the protein toxic to cells. Because miR760 can reduce levels of this toxic protein, the group studied the therapeutic potential of miR760 in a mouse model of SCA1. They used viral gene therapy to increase levels of miR760 in the cerebellum of SCA1 mice. By doing this, levels of the toxic ataxin-1 protein reduced by approximately 25%. This led to an improvement in the motor function of the treated mice five weeks after treatment. Together this shows that increasing levels of miR760 in the cerebellum can rescue motor function in SCA1 and represents a potential therapeutic approach.

Each person has two different copies, or alleles, of the ATXN1 gene. Both copires contain the 5’UTR that has the miR760 binding site. In SCA1 patients, one of these alleles has the CAG repeat expansion. This allele produces the toxic ataxin-1 protein that contains an expanded polyglutamine tract; the other allele produces normal functional ataxin-1 protein that is not toxic to cells.

Because miR760 binds to the 5’UTR of ATXN1 mRNA, it indiscriminately reduces the levels of both the toxic ataxin-1 protein and the normal ataxin-1 protein: miR760 is, therefore, non-allele specific.

To understand if the reduction of normal ataxin-1 protein levels is problematic, the group performed viral gene therapy of miR760 in wild-type mice that do not have repeat expansions. The group found no difference in the motor function of miR760 treated and control injected wild-type mice. This suggests that miR760 reduction of normal ataxin-1 levels does not cause motor defects, at least in the short term. While miR760 represents a promising therapeutic approach, further studies are needed to understand the broader molecular effects. Researchers will also need to examine the long-term safety and efficacy of miR760 treatment.

Tight regulation of ataxin-1 expression is incredibly important for maintaining brain health. Even in the absence of the polyglutamine expansion, small increases in ataxin-1 levels can cause motor coordination defects and ataxia in mice. On the other hand, reduced levels of ataxin-1 have been implicated as a risk factor for Alzheimer’s disease. This shows that the levels of ataxin-1 need to be tightly regulated within an optimal range as alterations in its levels may increase disease susceptibility.

Given the requirement for tight regulation of ataxin-1 levels, understanding how its levels are regulated in the cell will be essential for developing therapies for SCA1. Identifying the different regions and sequences involved in regulating levels of ataxin-1 may also help to identify disease-causing mutations in ataxia patients with unknown genetic causes. For example, mutations in the binding site for miR760 that prevent miR760 from binding to ataxin-1 could result in increased levels of ataxin-1 and cause ataxia. Understanding regulatory regions and mechanisms of gene regulation is incredibly important for both therapy development and investigating potential disease-causing mutations.

Key Terms

microRNA: MicroRNAs (miRNAs) are small single-stranded noncoding RNAs that regulate gene expression.

Untranslated region: Regions of the messenger RNA either side of the coding sequence that are not translated into protein. The untranslated region (UTR) before the start of the coding sequence is called the 5’ UTR and the UTR after the coding sequence is called the 3’ UTR. The 5’ UTR and the 3’ UTR play important roles in translation and regulation of gene expression.

Conflict of Interest Statement

The author and editor declare no conflict of interest. L. Nitschke, A. Tewari, S. Coffin, and E. Xhako are authors on the scientific paper who also volunteer for SCAsource. They did not contribute to the writing of this piece.

Citation of Article Reviewed

Nitschke, L. et al. miR760 regulates ATXN1 levels via interaction with its 5′ untranslated region. Genes Dev 34, 1147-1160, doi:10.1101/gad.339317.120 (2020).