Abstract

Huntington's disease (HD) is caused in large part by a polyglutamine expansion within the huntingtin (Htt) protein. Post-translational modifications (PTMs) control and regulate many protein functions and cellular pathways, and PTMs of mutant Htt are likely important modulators of HD pathogenesis. Alterations of selected numbers of PTMs of Htt fragments have been shown to modulate Htt cellular localization and toxicity. In this study, we systematically introduced site-directed alterations in individual phosphorylation and acetylation sites in full-length Htt constructs. The effects of each of these PTM alteration constructs were tested on cell toxicity using our nuclear condensation assay and on mitochondrial viability by measuring mitochondrial potential and size. Using these functional assays in primary neurons, we identified several PTMs whose alteration can block neuronal toxicity and prevent potential loss and swelling of the mitochondria caused by mutant Htt. These PTMs included previously described sites such as serine 116 and newly found sites such as serine 2652 throughout the protein. We found that these functionally relevant sites are clustered in protease-sensitive domains throughout full-length Htt. These findings advance our understanding of the Htt PTM code and its role in HD pathogenesis. Because PTMs are catalyzed by enzymes, the toxicity-modulating Htt PTMs identified here may be promising therapeutic targets for managing HD.

Highlights

  • Huntington’s disease (HD) is caused in large part by a polyglutamine expansion within the huntingtin (Htt) protein

  • Using these functional assays in primary neurons, we identified several Posttranslational modifications (PTMs) whose alteration can block neuronal toxicity and prevent potential loss and swelling of the mitochondria caused by mutant Htt

  • These PTMs included previously described sites such as serine 116 and newly found sites such as serine 2652 throughout the protein. We found that these functionally relevant sites are clustered in protease-sensitive domains throughout full-length Htt. These findings advance our understanding of the Htt PTM code and its role in HD pathogenesis

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Summary

PTM modulation of huntingtin toxicity

Palmitoylation of Htt by HIP14 at cysteine 214 mediates membrane attachment [15], and a reduced palmitoylation of mutant Htt can be responsible for Htt mislocalization and aggregation. In a previous study of Htt PTMs, we identified several phosphorylation sites within the first 586-amino acid (aa) (N586) fragment of Htt [17]. Other alterations previously shown to reduce Htt toxicity include serines 421, 536, 1181, and 1201 [17]. We identified close to 40 PTMs, including 17 novel sites, throughout full-length Htt. In the current study, we characterized the functional effects of PTM site modifications on mutant Htt. We introduced in full-length Htt alterations preventing PTMs of the amino acids that we have found modified in human or mouse brain, to determine whether abrogation of single PTM sites could reduce mutant Htt–induced toxicity and mitochondrial dysfunction. We were able to identify several PTM sites that can modulate expanded Htt toxicity and its effects on mitochondria.

Results
Alterations of PTM modulate Htt toxicity
Identification source
Mitochondrial abnormalities caused by expanded Htt are modulated by PTMs
Discussion
Plasmid generation and mutagenesis
Primary neuron preparation
Transfection of primary neurons
Nuclear condensation cell death assay
Mitochondrial function assays
Full Text
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