18 Aug 2006

The sticky mouse gets its name from the unkempt appearance of its fur, but the animal has more than a cosmetic flaw. Starting at 6 weeks old, sticky mice develop mild tremors, which progress to ataxia because of Purkinje cell neurodegeneration.

In tracking down the sticky mutation, Susan Ackerman and colleagues at the Jackson Lab in Bar Harbor, Maine, along with Paul Schimmel and coworkers at the Scripps Research Institute, La Jolla, California, made an unexpected discovery. The sticky mouse’s problems result from a defect in general protein synthesis, which leads to the mistaken substitution of serine for alanine in proteins. They show that this misreading of the genetic code, even at low levels, causes the accumulation of misfolded proteins, which triggers cell stress and leads to neurodegeneration.

The work, which was published online August 13 in Nature, reveals a novel mechanism for the pathological generation of misfolded proteins. In addition, the sticky mouse phenotype testifies to the exquisite sensitivity of neurons, and Purkinje cells in particular, to the buildup of protein garbage. While most of the cells in the mouse appear to tolerate small infidelities in protein synthesis, neurons are hit early and hard by the production of misfolded proteins.

Lead author Jeong Woong Lee and coworkers traced the sticky mouse’s troubles to a single nucleotide change in the alanine aminoacyl tRNA synthetase (AlaRS) gene. AlaRS is the matchmaker for the amino acid alanine and its corresponding transfer RNA (tRNAAla), producing a properly charged acyl-tRNA for polypeptide synthesis. AlaRS can also couple serine to tRNAAla, an error that occurs about one time in 500 reactions. To minimize the chance that serine will be mistakenly incorporated into protein instead of alanine, the synthetase also contains a separate editing function, which removes the mischarged serine, reducing the error rate to less than one in 1,000.

The sticky (sti) mutation, a single amino acid substitution in the editing domain of AlaRS, impairs this proofreading function. The researchers showed that recombinant mutant enzyme displayed an editing defect in the test tube, as its ability to hydrolyze serine from tRNAAla was reduced by 40-50 percent in the case of the human enzyme, with a smaller change in the mouse enzyme. The ability to load alanine or serine was unchanged, and the mutant did not remove alanine from the tRNA. The result was an accumulation of ser-charged tRNAAla in vitro reactions using either human or mouse mutant enzymes.

Even a low level of mischarged tRNAs in cells might theoretically lead to an accumulation of misfolded proteins, and experiments with sti mutant cells indicated that that is exactly what happens. Sti/sti embryonic fibroblasts showed elevated levels of polyubiquitinated proteins, as well as the stress-inducible cytosolic chaperone HSP72. In addition, the sti mutant cells were selectively sensitive to high concentrations of serine in the media. The toxicity of elevated serine, but not other amino acids, is consistent with the editing defect demonstrated in vitro.

But what of the in vivo consequences of AlaRS infidelity? In sticky mice, Purkinje cell loss begins early, becoming noticeable at 3 weeks, and extensive by 6 weeks. By 4 weeks of age, apoptotic neurons appear, and by 1 year, all the Purkinje cells are gone. Examination of Purkinje cells from 3-week-old mice by electron microscopy revealed cells under stress, with structures that looked like autophagosomes and protein inclusions. Intense punctuate ubiquitin staining showed up in cytosol, in axons and dendrites, and in the nucleolus, suggesting the presence of misfolded proteins throughout the cells. Increased cytosolic chaperone proteins were evident, including HSP72, HSC70, and HSP40. Cells from young mice also contained markers of endoplasmic reticulum stress and the unfolded protein response, including a transient upregulation of the chaperone BiP at 2 weeks of age, and a sustained increase of the stress-activated transcription factor CHOP, a situation that may be a prelude to apoptosis.

Despite its devastating effects on Purkinje cells and the resulting ataxia, the AlaRS mutation did not prevent the mice from reproducing, and caused no other visible problems besides persistent bad hair. The focal degeneration of Purkinje cells provides yet another example of the sensitivity of neurons to the accumulation of misfolded proteins. As Herve Roy and Michael Ibba of Ohio State University (Columbus) put it in their commentary accompanying the paper, the sticky mouse “shows just how fine the line is between a tolerable degree of error and a catastrophic loss of accuracy in protein synthesis. The loss in editing activity caused by the mutation AlaRS has no discernible effect on the efficiency of protein synthesis in non-neuronal cells, but has a detrimental effect on Purkinje cells.”

While the reasons for this differential sensitivity are not clear, common wisdom holds that postmitotic neurons are susceptible to misfolded protein accumulation because they do not divide. Purkinje cells in particular are a common target of protein misfolding diseases—many human repeat expansion diseases are associated with ataxia and Purkinje cell loss. In their discussion, Ackerman and colleagues speculate that in the case of the AlaRS mutants, perhaps other cells express additional translational editing functions that Purkinje cells lack. Alternatively, it is possible that Purkinje neurons are intrinsically worse at clearing folded proteins, or are more sensitive to the toxic effects of those proteins.

Could mutations in AlaRS, or other tRNA synthetases cause human neurodegenerative disease? The sticky mouse results suggest the answer is yes, the authors say. “Because editing domains of tRNA synthetases are functionally independent of those for the essential aminoacylation function, mild defects in editing could be transmitted from generation to generation without disruption of protein synthesis,” they write, “raising the possibility that some heritable diseases are connected to mild mutations in tRNA synthetase editing functions that, in turn, generate misfolded proteins.”—Pat McCaffrey

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