Researchers in Gitler’s laboratory previously discovered that carrying between 27 and 33 glutamine codons in the ataxin-2 gene is a risk factor for ALS (see ARF related news story on Elden et al., 2010). Most people have 22 or 23 repeats. Curiously, having more than 34 repeats causes a higher likelihood not of ALS, but of another disease, spinocerebellar ataxia.

Ataxin-2 protein associates with TDP-43 in stress granules. These are transient protein accumulations that cells create in times of trouble. Stress granules have been repeatedly linked to ALS (see ARF Webinar; Liu-Yesucevitz et al., 2010). Pathogenic TDP-43 in cytoplasmic stress granules tends to be both fragmented and phosphorylated. Hart and Gitler had a hunch that the extra repeats might enhance the ataxin-2-TDP-43 interaction, effectively imprisoning TDP-43 in stress granules and promoting disease.

Hart tested the hypothesis in three cell types: the HEK293T human embryonic kidney line, lymphoblasts from people with ALS, and differentiated BE(2)-M17 human neuroblastoma cells. The lymphoblasts included samples from people with ALS who had either normal or intermediate-length expansions, as well as one person with spinocerebellar ataxia due to 40 repeats. In the case of the cell lines, Hart transfected them with ataxin-2 toting 22, 31, or 39 repeats.

Simply adding ataxin-2, of any repeat size, did not affect TDP-43. To stress the cells and to force TDP-43 out of its normal nuclear location into cytoplasmic stress granules, Hart heated the cultures to 42 degrees Celsius for one hour. Then he immunoblotted with an antibody specific for phosphorylated TDP-43. Heat shock drove up the amount of phosphorylated, insoluble protein, which was present as a fragment of approximately 30 kilodaltons. The intermediate-length repeats approximately doubled the concentration of this pathogenic TDP-43, compared to ataxin-2 with longer or shorter repeat sequences.

Since only fragmented TDP-43 was phosphorylated, Hart suspected that the cleavage would precede, and might even promote, the phosphate acquisition. Other researchers have shown that caspase-3 cleaves TDP-43 (see ARF related news story on Zhang et al., 2007), and indeed caspase activation is a normal function of short-repeat ataxin-2 (Wiedemeyer et al., 2003). This led Hart to examine his immunoblots with antibodies specific to activated caspases.

The cells with intermediate-length ataxin repeats turned on caspase-3; cells expressing longer or shorter versions did not. It appears that the medium-length repeats somehow lower the cell’s threshold for stress, making it prone to activate caspase and place TDP-43 in stress granules. Treating the cells with a caspase inhibitor diminished accumulation of phosphorylated TDP-43 fragments—suggesting that caspase activation occurs upstream of the TDP-43 aggregation.

Finally, Hart examined spinal cord sections from eight people who died of ALS. Specifically in the samples from four people with intermediate ataxin expansions, he observed cytoplasmic inclusions of activated caspase-3 in the motor neurons. “We think this is a new feature of ALS pathology that is specific to cases of ALS that harbor ataxin-2 expansions,” Gitler concluded.

The paper supports the current hypothesis that accumulated TDP-43 fragments cause disease, and offers a pathological pathway specific to people with the ataxin-2 risk factor, said Randal Tibbetts of the University of Wisconsin, Madison, who was not involved in the paper. In people without the expansions, other genetic or environmental factors, such as TDP-43 mutations, could lead to the same “common endpoint” of TDP-43 pathology, he suggested.

Subduing the caspase might treat ALS, Gitler suggested. Unfortunately, caspase inhibitors have fared poorly in ALS trials (Gordon et al., 2007). Perhaps the therapy would be effective for the subpopulation of people with ataxin expansions, Gitler speculated. Alternatively, Tibbetts suggested, scientists might try to alter the pathway between ataxin-2 repeats and caspase activation, but researchers still have to work out that chain of events.

How can just a few excess amino acids make such a striking difference in ataxin’s activity? The simplest explanation is that they alter the protein’s conformation, suggested Leonard Petrucelli of the Mayo Clinic in Jacksonville, Florida, who was not involved in the study. The expansion might cause a form of toxic gain of function, allowing ataxin to interact differently with normal partners or even recruit new binding partners. For his part, Gitler suspects that the intermediate repeats enhance some normal function of ataxin, such as its caspase-activating ability, while the long repeats that cause spinocerebellar ataxia create a new, poisonous property.—Amber Dance.

Reference:
Hart MP, Gitler AD. ALS-associated ataxin-2 polyQ expansions enhance stress-induced caspase-3 activation and increase TDP-43 pathological modifications. J Neurosci. 2012 Jul 4;32(27):9133-42. Abstract