Serine 421 regulates mutant huntingtin toxicity and clearance in mice
Ian H. Kratter, … , Eliezer Masliah, Steven Finkbeiner
Ian H. Kratter, … , Eliezer Masliah, Steven Finkbeiner
Published September 1, 2016; First published August 15, 2016
Citation Information: J Clin Invest. 2016;126(9):3585-3597. https://doi.org/10.1172/JCI80339.
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Research Article Neuroscience

Serine 421 regulates mutant huntingtin toxicity and clearance in mice

Abstract

Huntington’s disease (HD) is a progressive, adult-onset neurodegenerative disease caused by a polyglutamine (polyQ) expansion in the N-terminal region of the protein huntingtin (HTT). There are no cures or disease-modifying therapies for HD. HTT has a highly conserved Akt phosphorylation site at serine 421, and prior work in HD models found that phosphorylation at S421 (S421-P) diminishes the toxicity of mutant HTT (mHTT) fragments in neuronal cultures. However, whether S421-P affects the toxicity of mHTT in vivo remains unknown. In this work, we used murine models to investigate the role of S421-P in HTT-induced neurodegeneration. Specifically, we mutated the human mHTT gene within a BAC to express either an aspartic acid or an alanine at position 421, mimicking tonic phosphorylation (mHTT-S421D mice) or preventing phosphorylation (mHTT-S421A mice), respectively. Mimicking HTT phosphorylation strongly ameliorated mHTT-induced behavioral dysfunction and striatal neurodegeneration, whereas neuronal dysfunction persisted when S421 phosphorylation was blocked. We found that S421 phosphorylation mitigates neurodegeneration by increasing proteasome-dependent turnover of mHTT and reducing the presence of a toxic mHTT conformer. These data indicate that S421 is a potent modifier of mHTT toxicity and offer in vivo validation for S421 as a therapeutic target in HD.

Authors

Ian H. Kratter, Hengameh Zahed, Alice Lau, Andrey S. Tsvetkov, Aaron C. Daub, Kurt F. Weiberth, Xiaofeng Gu, Frédéric Saudou, Sandrine Humbert, X. William Yang, Alex Osmand, Joan S. Steffan, Eliezer Masliah, Steven Finkbeiner

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Figure 1

Generation and characterization of mHTT transgenic mice with phosphomimetic (S421D) and phosphoresistant (S421A) mutations at S421.

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Generation and characterization of mHTT transgenic mice with phosphomime...
(A) Schematic of the recombineered BAC cassette, in which S421D or S421A mutations within the original BACHD construct were generated. (B) Representative Western blot demonstrating expression levels of mHTT from cortical lysates of 2-month-old BACHD, mHTT-S421D, and mHTT-S421A mice. The blot was probed with anti-expanded polyQ mAb 4H7H7 and anti–γ-tubulin as a loading control. (C) Quantification of expression levels of mHTT from cortical lysates of 2-month-old BACHD, mHTT-S421D, and mHTT-S421A mice. Values are based on the mean of 3 independent 4H7H7 blots, each with lysates from at least 3 mice per line compared across different blots by normalization to BACHD samples. Each value was first normalized for input using the anti–γ-tubulin control. F = 36.71, S421A vs. BACHD/S421D, P < 0.0001. (D) Quantification of the levels of cortical mHTT transcript in BACHD, mHTT-S421D, and mHTT-S421A mice by qRT-PCR. The results are from 4 independent samples per transgenic line, each run in quadruplicate. Values are normalized to BACHD mice. F = 51.8, P < 0.0001; S421A vs. BACHD/S421D, P < 0.0001. **P < 0.01; ***P < 0.001; ****P < 0.0001; all by 1-way ANOVA.