Two related papers in the April 20 Nature offer support for antisense oligonucleotide (ASO) therapy in two different neurodegenerative diseases. Curiously, both strategies target the same protein. Scientists in the lab of Stefan Pulst, University of Utah, Salt Lake City, reported that reducing the ataxin-2 protein with ASOs prevents motor impairment and cerebellar problems in mice that model spinocerebellar ataxia type 2 (SCA2). Reducing ataxin-2 maintained normal gene expression and firing patterns in cells of the cerebellum. Scientists led by Aaron Gitler at Stanford University School of Medicine, California, also used ASOs against ataxin-2, but to treat amyotrophic lateral sclerosis (ALS). They claim that ataxin-2 helps recruit TDP-43 into stress granules, where it accumulates. Knocking down ataxin-2 reduced pathology and motor problems in mouse models of motorneuron disease. Together, the papers suggest that targeting one protein could prevent pathology and motor impairments in two separate diseases.

“These two papers herald a new wave of valuable preclinical research,” wrote Ke Zhang and Jeffrey Rothstein, Johns Hopkins University, Baltimore, in an accompanying News and Views, adding, “It is hoped that these successes can be translated to patients.” Philip Van Damme, KU Leuven, Belgium, agreed. “The translational value of the role of ataxin-2 in ALS and SCA2 is very much strengthened by these two papers.” Van Damme was not involved in either study.

Stress reduction

Knocking down ataxin-2 (right) kept stress granules smaller, immature, and reduced the number containing TDP-43 (white arrows) than seen in control human epithelial cells (left). [Becker et al., Nature.]

ASOs are approximately 20 base-pair, artificial DNA- or RNA-like sequences that bind complementary mRNA and mark it for destruction. Their purpose is to lower specific protein products. Both of the present studies used ASOs to diminish levels of ataxin-2, a protein involved in mRNA transport, processing, and translation, as well as stress granule assembly. Ataxin-2 harboring an expanded CAG repeat region leads to an enlarged polyglutamine domain at the protein’s N-terminus, which leads to SCA2. Patients with this disease develop degeneration in their cerebellum, as well as attendant symptoms such as uncoordinated movement. Could quashing ataxin-2 levels alleviate their pathology and symptoms?

To find out, Pulst’s group, including first author Daniel Scoles, collaborated with Ionis Pharmaceuticals in Carlsbad, California, to design ASO7, which targets exon 11 of ataxin-2 mRNA. Well tolerated in mice, ASO7 lowered ataxin-2 protein levels in the cerebellum without activating microglia or astrocytes.

The researchers tested this ASO in ATXN2-Q127, a mouse model of SCA2 that expresses a human ataxin-2 transgene with 127 CAG repeats in Purkinje cells in the cerebellum. At around 8 weeks of age, the mice develop motor difficulties, the firing of Purkinje cells wanes, and Purkinje cells degenerate. Scoles and colleagues injected either ASO7 or saline into the cerebral ventricles of these mice at eight weeks of age. Then at and five, nine, and 13 weeks afterward, they tested how long the mice were able to walk on an accelerating rotating cylinder before sliding off. Compared to untreated transgenic mice, the treated ones stayed aloft longer. A week after the last rotarod assessment, the researchers sacrificed the mice and homogenized their cerebella, using qPCR to find that mRNA levels of the transgenic human ataxin-2 had dropped 75 percent in mice treated with ASO7 compared to untreated controls. The group saw similar results when they tested the ASO in a second mouse model, the BAC-Q72 mouse, which has 72 glutamines in ataxin-2.

How did the ASO work? Pulst’s group previously reported gene expression changes over the course of disease in multiple SCA2 mouse models (Dansithong et al., 2015). They reasoned that ASO7 might prevent those disease-related changes. The scientists monitored transcription of the top six mRNAs—Rgs8, Pcp2, Pcp4, Cep76, Homer3, and Fam107b—all of which decline with disease in the Purkinje cells of these models. The genes are involved in calcium release and homeostasis, synapse maintenance, and centriole function. ASO7 restored expression of all six proteins, adding to evidence that mutated ataxin-2 disrupts protein translation.

There was a change in neural function, too. Whereas the baseline firing rate of Purkinje cells slows as disease progresses in ATXN2-Q127 mice, treatment with ASO7 returned firing to normal. This effect lasted at least 14 weeks after a single injection.

In the second paper, first author Lindsay Becker and colleagues wanted to diminish ataxin-2 in ALS models that accumulate TDP-43 in cells. Previous work from this group suggested that having less ataxin-2 alleviates TDP-43 toxicity in yeast and flies (Elden et al., 2010). Becker wondered if knocking down this protein would similarly bolster mice against excess TDP-43.

First, she took a genetic approach. She started with a mouse model that overexpresses human TDP-43 in neurons and develops aggregates of ubiquitinated and phosphorylated forms of the protein. Mice that carry two copies of the human transgene develop severe motor problems, tremor, hunched posture, and paralysis by three weeks of age, and die shortly thereafter. They also lose almost a third of neurons in layer V of the cortex and lower motor neurons. The researchers crossed these transgenic mice with ataxin-2 knockouts, which have no apparent phenotype except mild obesity late in life. The crosses survived on average 80 percent longer than the TDP-43 mice, with some animals living more than 55 weeks. Motor impairments, hunched posture, and tremor did develop, but slowly and to a much milder degree. In addition, lacking ataxin-2 largely preserved layer V neurons, but only trended toward rescuing lower motor neurons. Immunohistochemistry in mouse spinal cords revealed 45 to 75 percent fewer inclusions positive for phosphorylated TDP-43 compared to the TDP-43 mice.

Ataxin-2 knockout did all of this without changing the levels of TDP-43 protein or its mRNA. How was that possible? Knowing that ataxin-2 binds RNA and regulates the assembly of stress granules, the authors guessed that it might help concentrate TDP-43 in stress granules. To test this idea, they reduced ataxin-2 in cultured human cells with small interfering RNA, and peered at them through a fluorescent microscope. Stress granules formed more slowly; they stayed smaller and more numerous, suggesting they didn’t fuse and mature as they normally would. These stress granules also carried less insoluble TDP-43 (see image above). Becker said that since TDP-43 appears to enter mature stress granules, it is possible that delaying or preventing that process may help keep TDP-43 at bay.

To test ataxin-2 reduction in a therapeutically relevant paradigm, the researchers also turned to ASOs by Ionis. The type they used—called Atxn2 ASO—targets the mouse version of the protein. They injected Atxn2 ASO into the cerebral ventricles of either wild-type or TDP-43 mice when they were one day old. Three weeks later, the researchers measured a 77 percent reduction in ataxin-2 mRNA in the brain along with a reduced gait impairment score. Treated mice lived 35 percent longer than untreated littermates, some up to 120 days.

Together, the data suggest lowering ataxin-2 in TDP-43 mice keeps TDP-43 out of stress granules, lowers its aggregation, and prevents neurodegeneration and motor impairments.

“This is the latest and most convincing demonstration that reducing ataxin-2 can improve disease outcomes in this model of ALS,” said Sami Barmada, University of Michigan Medical School, Ann Arbor. He was puzzled that some knockouts mice lived an exceptionally long time, while others lived only modestly longer than TDP-43 mice. He agreed with the authors that there must be some effect of genetic background they have yet to understand. He also pointed out that the lack of rescue of lower motor neurons did not match the large clinical benefit seen in the mice.

The strategy could be relevant for most ALS patients, as 97 percent have TDP-43 pathology, the authors wrote.

Though many therapeutics developed in mice fail in humans, there is reason to hope when it comes to ASOs. The FDA recently approved the ASO therapy nusinersen for a childhood form of neurodegeneration called spinal muscular atrophy (see Nov 2016 news). Scientists are now testing an ASO against mutant SOD1, which appeared to be well tolerated in a Phase 1 trial with ALS patients (see May 2013 news). “Taken together, the data shows ASOs have quite a bit of therapeutic potential for the treatment of these neurodegenerative diseases,” said Barmada. “That is very exciting for patients and clinicians.”

“There is no reason that the current studies should not provide a comparable foundation for ASO therapy in SCA2 and in TDP-43-mediated neurodegenerative diseases,” wrote Zhang and Rothstein. They cautioned, however, that ongoing neurodegeneration or microglial or astroglial activation could undermine the impact of ASOs in SCA2 patients, saying treatment may have to be early in the disease process. They suggested testing the ASO for ALS in other mouse models, such as those with a C9orf72 mutation, or in cells derived from human patients.

Van Damme said that ASO therapy protected against TDP-43 overexpression, but it is unclear whether it would work when TDP-43 is expressed at normal levels. He also noted that scientists need to determine whether reducing ataxin-2 is safe in people. “That’s a major hurdle.” Even so, he said translation of these ASOs to people is feasible.

“The future challenges are on the dose finding and bio-distribution of these anti-sense oligonucleotides in patients,” said Sheng-Han Kuo, Columbia University Medical Center, New York. “I think that we are very close to human clinical trials.”

A separate line of research independently reinforced the idea that given the tight packing of proteins in a cell, reducing the concentration of one protein can strongly affect the behavior of others. In the April 10 Proceedings of the National Academy of Sciences, scientists led by Prajwal Ciryam, Columbia University Medical Center, New York, and Justin Yerbury, University of Wollongong, Australia, show an ALS protein network that incudes ataxin-2 and TPD-43—both RNA binding proteins (see image below). All the proteins pictured are found in ALS inclusions in motor neurons.

Supersaturated Network

TDP-43 (TARDBP) and ataxin-2 (both lower left corner) fit into a network of ALS-related proteins found in inclusions. Their high concentrations in motor neurons make them prone to co-aggregation. [Ciryam et al., PNAS 2016]

The researchers calculated the concentration of each of these proteins and compared it to their respective threshold for aggregating. They found that the vast majority were metastable because their levels exceeded those at which they stay soluble. This property makes them more likely to aggregate when proteostatic mechanisms weaken with age or disease. The authors hypothesize that this explains why this particular subset of proteins—which otherwise are not directly related—is found jumbled together in inclusions of motor neurons in patients with ALS.—Gwyneth Dickey Zakaib



No Available Comments

Make a Comment

To make a comment you must login or register.


News Citations

  1. Positive Trials of Spinal Muscular Atrophy Bode Well for Antisense Approach
  2. Paper Alert: Antisense Oligonucleotide Therapy Safe for ALS?

Paper Citations

  1. . Ataxin-2 regulates RGS8 translation in a new BAC-SCA2 transgenic mouse model. PLoS Genet. 2015 Apr;11(4):e1005182. Epub 2015 Apr 22 PubMed.
  2. . Ataxin-2 intermediate-length polyglutamine expansions are associated with increased risk for ALS. Nature. 2010 Aug 26;466(7310):1069-75. PubMed.

Further Reading


  1. . ISS-N1 makes the First FDA-approved Drug for Spinal Muscular Atrophy. Transl Neurosci. 2017 Jan;8:1-6. Epub 2017 Jan 26 PubMed.
  2. . Antisense Oligonucleotide-Based Therapy for Neuromuscular Disease. Molecules. 2017 Apr 5;22(4) PubMed.

Primary Papers

  1. . Antisense oligonucleotide therapy for spinocerebellar ataxia type 2. Nature. 2017 Apr 20;544(7650):362-366. Epub 2017 Apr 12 PubMed.
  2. . Therapeutic reduction of ataxin-2 extends lifespan and reduces pathology in TDP-43 mice. Nature. 2017 Apr 20;544(7650):367-371. Epub 2017 Apr 12 PubMed.
  3. . Spinal motor neuron protein supersaturation patterns are associated with inclusion body formation in ALS. Proc Natl Acad Sci U S A. 2017 May 16;114(20):E3935-E3943. Epub 2017 Apr 10 PubMed.