Mechanistic Insight into the Pathology of Polyalanine Expansion Disorders Revealed by a Mouse Model for X Linked Hypopituitarism

Abstract

Polyalanine expansions in transcription factors have been associated with eight distinct congenital human diseases. It is thought that in each case the polyalanine expansion causes misfolding of the protein that abrogates protein function. Misfolded proteins form aggregates when expressed in vitro; however, it is less clear whether aggregation is of relevance to these diseases in vivo. To investigate this issue, we used targeted mutagenesis of embryonic stem (ES) cells to generate mice with a polyalanine expansion mutation in Sox3 ( Sox3-26ala) that is associated with X-linked Hypopituitarism (XH) in humans. By investigating both ES cells and chimeric mice, we show that endogenous polyalanine expanded SOX3 does not form protein aggregates in vivo but rather is present at dramatically reduced levels within the nucleus of mutant cells. Importantly, the residual mutant protein of chimeric embryos is able to rescue a block in gastrulation but is not sufficient for normal development of the hypothalamus, a region that is functionally compromised in Sox3 null embryos and individuals with XH. Together, these data provide the first definitive example of a disease-relevant PA mutant protein that is both nuclear and functional, thereby manifesting as a partial loss-of-function allele.

Author Summary

Alanine is one of the 20 amino acid building blocks from which proteins are generated. Nearly 500 human proteins contain stretches of consecutive alanine residues ranging from 4 to 20 amino acids in length. Whilst the function of these polyalanine (PA) tracts remains unknown, they are interesting because DNA changes (mutations) that increase their length above a threshold are responsible for nine different human disorders. In vitro studies indicate that expanded PA proteins misfold and aggregate, suggesting that there may be a common “gain-of-function” mechanism that underpins this group of disorders. However, these data are difficult to reconcile with genetic studies, which indicate that most PA mutations cause protein loss-of-function. Therefore, to investigate the pathological mechanism of PA disorders we generated a mouse model of X-linked Hypopituitarism (XH), a disease caused by PA expansion in the SOX3 protein. Strikingly, we found that the mouse version of the disease-causing protein was almost completely cleared from cells and that aggregates do not form in vivo. These data explain why this type of mutation causes protein loss-of-function and reveals why nature limits the length of PA stretches.