Philipp Lachenmann: DELPHI Rationale


Schoeller Junkmann Prize 2007

Sarah F. Funderburk and Liubov Shatkina

Research Center Karlsruhe, Institute for Toxicology and Genetics

N-terminal mutations of androgen receptor alter aggregate formation and polyglutamine toxicity in SMBA models

Kennedy's disease or X-linked spinal and bulbar muscular atrophy (SBMA) is a genetically inherited neurodegenerative disorder that affects males during mid-adult life. Those afflicted with SBMA experience muscle weakness and wasting of the extremities (legs/arms), face, and throat, as well as hormonal abnormalities. The disorder results from a trinucleotide repeat expansion in the androgen receptor protein (AR) of the sequence CAG, which codes for the amino acid glutamine. Normally the AR contains 9 to 38 copies of the CAG sequence while a pathogenic AR can have anywhere from 40 to 88 copies. Eight other neurodegenerative diseases, including Huntington's, are also caused by CAG repeat expansions in various proteins. Together with SBMA, they are commonly referred to as the polyglutamine (polyQ) disorders.

A hallmark of the polyQ disorders is misfolding and aggregation of the mutant protein. Much of the focus of the toxicity of the polyQ regions is on aggregation, a dynamic and complex process. An aggregated protein can range from a soluble oligomeric structure to a very dense insoluble structure similar to the amyloid fibrils found in Alzheimer's and Parkinson's. It is believed that the oligomeric intermediates may actually be the toxic components of SBMA and the other polyQ disorders.

Each of the polyQ disorders is unique in regards to the types of neurons affected, and therefore each presents a distinct set of symptoms. This ?uniqueness? is most likely brought about by what is referred to as "protein context". For example, the environment of the polyQ region in the huntingtin protein is different from that of the AR.

In previous studies, we identified two putative phosphorylation sites of the AR that when mutated appeared to cause conformational change in the AR. We mutated these sites in the context of a normal AR with 22 glutamines (ARQ22) and a pathogenic AR containing 77 glutamines (ARQ77). Their effects on aggregation and toxicity were then examined in cell culture and in a Drosophila model of SBMA. The Drosophila model has the advantage of correlating aggregation with toxicity as well as replicating movement impairments as seen in human cases.

With mutation of the two sites in the AR, we were indeed able to cause a conformational change in the protein. Surprisingly, the effects of this conformational change differed depending on the length of the polyQ stretch. Mutations in the ARQ22 resulted in a marked increase in aggregation as well as decreased survival rates and altered locomotion behavior in Drosophila. These results were similar but not as severe as the ARQ77/SBMA model. In quite the opposite manner, mutations in the ARQ77 caused a decrease in aggregation and a lessened toxic effect in Drosophila. We have therefore identified two distinct amino acid sites that profoundly modulate polyQ toxicity in the AR. These results can be further utilized to understand the conformational changes in the AR that lead to aggregation as well as the types of aggregates that lead to toxicity.



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