Abstract
Phosphorylation has been shown to have a significant impact on expanded huntingtin-mediated cellular toxicity. Several phosphorylation sites have been identified on the huntingtin (Htt) protein. To find new potential therapeutic targets for Huntington's Disease (HD), we used mass spectrometry to identify novel phosphorylation sites on N-terminal Htt, expressed in HEK293 cells. Using site-directed mutagenesis we introduced alterations of phosphorylation sites in a N586 Htt construct containing 82 polyglutamine repeats. The effects of these alterations on expanded Htt toxicity were evaluated in primary neurons using a nuclear condensation assay and a direct time-lapse imaging of neuronal death. As a result of these studies, we identified several novel phosphorylation sites, validated several known sites, and discovered one phospho-null alteration, S116A, that had a protective effect against expanded polyglutamine-mediated cellular toxicity. The results suggest that S116 is a potential therapeutic target, and indicate that our screening method is useful for identifying candidate phosphorylation sites.
Highlights
Huntington’s disease (HD) is a fatal progressive neurodegenerative disorder involving movement, cognitive and emotional symptoms, with no current neuroprotective therapy [1,2,3,4,5,6,7,8,9,10]
HD is caused by a CAG triplet repeat expansion in the Huntingtin gene on chromosome 4 coding for a polyglutamine repeat expansion in the Huntingtin protein (Htt) [15]
In order to identify novel phosphorylation sites in a region of Htt using mass spectrometry, we chose a fragment of 511 amino acids for study
Summary
Huntington’s disease (HD) is a fatal progressive neurodegenerative disorder involving movement, cognitive and emotional symptoms, with no current neuroprotective therapy [1,2,3,4,5,6,7,8,9,10]. HD is caused by a CAG triplet repeat expansion in the Huntingtin gene on chromosome 4 coding for a polyglutamine repeat expansion in the Huntingtin protein (Htt) [15]. There is a correlation between repeat length and the severity and age of onset of the disease. Longer repeats cause earlier onset and more widespread neurodegeneration. The pathogenesis of HD is still incompletely understood, but is believed to arise predominantly via a genetic gain of toxic function due to the CAG repeat expansion [9,16,17]. The expanded protein can be cleaved into N-terminal fragments, which in most experimental systems, are more toxic that full-length Htt [18,19,20,21,22]. Transgenic mouse models expressing the caspase 6 fragment or other shorter fragments generally have more striking and robust phenotypes than transgenic mouse models expressing full-length Htt [20,24,25,26,27]
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