Papers in tomorrow’s Cell and last week's Neuron reveal that protein phosphorylation plays a key role in spinocerebellar ataxia type 1 (SCA-1), an ultimately fatal neurodegenerative disease marked by progressive loss of muscle control. The finding may lead to a new approach for therapeutics, the authors suggest.
SCA-1 is an inherited disease caused by mutations in the ataxin-1 gene. These mutations lead to unusually long polyglutamine tracts in the ataxin protein, which aggregates and forms inclusions in the nucleus. The latter are widely thought to be the direct cause of neuronal damage, but now, collaboration between Huda Zoghbi's lab at Baylor College of Medicine in Houston, Texas, and Harry Orr's at the University of Minnesota in Minneapolis, has uncovered that this process depends on phosphorylation.
In Neuron, first author Effat Emamian and colleagues show that modification of one amino acid, serine 776, is crucial for ataxin-1 aggregation, inclusion formation, and disease progression. In ataxin containing an 82-glutamine tract, the authors replaced serine 776 with alanine, which is impervious to phosphorylation, and introduced the modified protein into cultured cells and the mouse genome.
Less than 0.1 percent of cells harboring the alanine-776-ataxin contained nuclear inclusions compared with cells expressing the serine-776-ataxin, but it was in the transgenic mice where results were dramatic. The authors found that mice expressing serine-776-ataxin had nuclear inclusions of this protein in Purkinje cells by five weeks of age, whereas five-week-old mice expressing alanine-776-ataxin did not. Progression of SCA-1 is slow by nature, and the authors traced the accumulation of inclusions as the mice aged. By 30 weeks, 100 percent of examined Purkinje cells of serine-776-ataxin mice had inclusions, compared with 16 percent in alanine-776-ataxin mice. The latter showed no signs of neurodegeneration, whereas serine-776-ataxin mice had the weak gait normally associated with SCA-1 mouse models. The alanine-776-ataxin mice’s behavior was indistinguishable from that of normal littermates, and balance testing on an accelerating rotarod produced no statistical performance differences between the alanine-776-ataxin and wild-type mice. In contrast, S776-ataxin mice performed poorly on the rotarod, lasting only one quarter as long as the other mice.
Hung-Kai Chen and colleagues extend these observations by asking what the role of the phosphorylated serine at position 776 could be. To identify proteins that may interact with this amino acid, the authors used ataxin antibodies to isolate it and associated molecules. They found two proteins, with molecular masses of 28 kDa and 30 kDa, which bind tightly to S776-ataxin, but not to A776-ataxin. The authors used mass spectroscopic analysis to determine that these are both isoforms of the regulatory factor 14-3-3.
Chen and colleagues found that 14-3-3 aggravates the disease process. In cultured cells expressing both serine-776-ataxin with an 82 polyglutamine tract and 14-3-3, inclusions were larger and more numerous than in cells without this regulatory factor. 14-3-3 had no effect on cells expressing alanine-776-ataxin, which were mostly devoid of inclusions. Chen et al. go on to demonstrate that in fruit fly models, this interaction results in deformation of the ommatidia and retina of the compound eye, a common yardstick for measuring neurodegeneration in flies. The authors found that expressing 14-3-3 alone in the eye caused no detectable effects, but it exacerbated the degeneration caused by serine-776-ataxin.
All these experiments indicated that phosphorylated ataxin and 14-3-3 interact to hasten the disease process. But part of the picture was still missing-what phosphorylates ataxin? To answer this question, Chen and colleagues turned to bioinformatics, putting the ataxin sequence through a motif search program (Scansite http://scansite.mit.edu). The results of the search suggested that serine 776 and its surrounding amino acids make up a sequence recognized and phosphorylated by the kinase Akt. The authors tested the veracity of this prediction in vitro by mixing ataxin and 14-3-3 in the absence or presence of Akt. Only in the latter case did ataxin and 14-3-3 interact. In addition, as Akt is activated by the common phosphatidylinositol-3-kinase pathway, Chen determined that activation of this pathway worsens ataxin-mediated neurodegeneration in fruit flies.
Overall, these experiments introduce some new players in the polyglutamine expansion story and emphasize how important it is to study the nonglutamine parts of proteins such as ataxin and huntingtin. The surprising new insight, according to the authors, is "that although the N-terminal polyglutamine tract is critical for pathogenesis, it is not sufficient, even when the protein is in the nucleus."
The experiments also raise the tantalizing possibility of controlling polyglutamine diseases independently of the glutamine tract. "The identification of factors modulating SCA-1 pathology may lead to therapeutic interventions such as interfering with ataxin-1/14-3-3 interaction using small peptides, or reducing PI3K/Akt signaling by specific kinase inhibitors," conclude the authors.—Tom Fagan
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