Serine 421 regulates mutant huntingtin toxicity and clearance in mice - PubMed (original) (raw)

. 2016 Sep 1;126(9):3585-97.

doi: 10.1172/JCI80339. Epub 2016 Aug 15.

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

Serine 421 regulates mutant huntingtin toxicity and clearance in mice

Ian H Kratter et al. J Clin Invest. 2016.

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.

PubMed Disclaimer

Figures

Figure 1

Figure 1. Generation and characterization of mHTT transgenic mice with phosphomimetic (S421D) and phosphoresistant (S421A) mutations at S421.

(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 m_HTT_ 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.

Figure 2

Figure 2. Essential functions of HTT in development can be rescued by expression of transgenic mHTT with phosphomimetic or phosphoresistant mutations at S421.

Western blots of cortical lysates probed with anti-HTT (mAb 2166), anti-expanded polyQ (4H7H7), and anti–γ-tubulin confirm that mice rescued by phosphomimetic (A) or phosphoresistant (B) m_HTT_ transgene express only mHTT and lack endogenous murine HTT. Some residual signal after membrane stripping can be appreciated in the 4H7H7 blots.

Figure 3

Figure 3. mHTT-S421D mice, but not mHTT-S421A mice, have less severe motor and psychiatric-like behavioral deficits than BACHD mice.

(A) Analysis of motor performance on the accelerating rotarod of a cohort of BACHD (n = 7), mHTT-S421D (n = 17), and WT (n = 31) mice at 3, 6, and 12 months of age. (B) Analysis of hind-limb gaits of BACHD, mHTT-S421D, and WT mice at 12 months of age with the CatWalk XT (Noldus). (C and D) Comparison of total activity (C) and rearing activity (D) in BACHD, mHTT-S421D, and WT mice in the open field for 10 minutes at 12 months of age. (E) Comparison of anxiety-like behavior in the light-dark box over 10 minutes in BACHD, mHTT-S421D, and WT mice at 12 months of age. (F) Analysis of motor performance on the accelerating rotarod of a cohort of mHTT-S421A (n = 13) and WT (n = 19) mice at 6 and 12 months of age. In A and F, 2-way repeated-measures ANOVA statistical analyses were used, with 1-way ANOVA used for B–E. Bonferroni post hoc tests were used for all pairwise comparison. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 4

Figure 4. Phosphomimetic mutation at S421 rescues the neurodegeneration caused by expression of mHTT at 12 months of age without accompanying loss of inclusion body formation.

(A) Measurement of striatal volume by unbiased stereology in BACHD (n = 6), mHTT-S421D (n = 7), and WT (n = 6) mice. (B) Measurement of striatal NeuN counts with correction for volume changes in the same cohort. (C) Immunohistochemistry with polyclonal sheep antibody S830 reveals prominent inclusion bodies in BACHD and mHTT-S421D striatum and cingulate cortex but not in WT littermate controls. Example smaller (black) and larger (white) inclusion bodies are indicated by arrowheads. The scale bar represents 220 μm. In A and B, 1-way ANOVA statistical analysis was used with Bonferroni post hoc tests for all pairwise comparisons. *P < 0.05.

Figure 5

Figure 5. Phosphomimetic mutation at S421 decreases steady-state levels of mHTT in the striatum.

(A and B) Immunohistochemistry of striatal sections of BACHD (n = 6), mHTT-S421D (n = 7), and WT (n = 5) mice with mAb 4H7H7 (A) and subsequent quantification of signal intensity (B). Scale bar: 20 μm. (C) Representative Western blot of BACHD and mHTT-S421D striatal lysates with 4H7H7 and anti–γ-tubulin as a loading control. (D) Quantification of expression levels of mHTT in striatal lysates of BACHD and mHTT-S421D mice. Values are based on the mean of 3 independent 4H7H7 blots of 4 mice per transgenic line, compared across different blots by normalization to BACHD samples. Each value was first normalized for input using the anti–γ-tubulin controls. (E) Quantification of the levels of striatal m_HTT_ transcript in BACHD and mHTT-S421D mice by qRT-PCR. The results are from 4 independent samples per transgenic line, each run in quadruplicate. Values are normalized to BACHD. Means were tested with an unpaired t test in D and E. ****P < 0.0001.

Figure 6

Figure 6. Phosphomimetic mutation at S421 increases the turnover of an N-terminal fragment of HTT in striatal cells via the proteasome.

(A) Representative Western blot of St14A cell lysates generated 48 hours after transfection with HTT-N480-17Q (probed with mAb 5492) and myc-actin. (B) Quantification of the effect of S421 mutation on HTT-N480 levels in St14A cell lysate. (C) Quantification of the fold change in levels of HTT-N480 with and without S421 mutation after treatment with the specific proteasome inhibitor epoxomicin or lysosome inhibitors ammonium chloride and leupeptin. Values in B and C are based on the mean of 3 independent experiments after normalization for transfection efficiency and loading with the myc-actin control. (D) Representative Western blot after coimmunoprecipitation of HTT-N480-17Q with anti-HA rabbit polyclonal or IgG control from St14A cell lysates after transfection of HTT-N480-17Q, myc-actin, and HA-ubiquitin and treatment with epoxomicin and PR619 (an inhibitor of deubiquitinases) to enhance accumulation of ubiquitin. One-twentieth whole-cell lysates (WCL) were run as controls on the same gel. (E) Quantification of the effect of S421 mutation on HTT-N480 coimmunoprecipitation by HA-ubiquitin in St14A cell lysate. Values are expressed as a normalized ratio of immunoprecipitated to whole-cell control levels per construct. Final data represent 2 independent experiments each run in triplicate. Statistical analysis in each case was by 1-way ANOVA with Bonferroni post hoc tests for all pairwise comparisons. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

References

    1. Walker FO. Huntington’s disease. Lancet. 2007;369(9557):218–228. doi: 10.1016/S0140-6736(07)60111-1. -DOI -PubMed
    1. Finkbeiner S. Huntington’s disease. Cold Spring Harb Perspect Biol. 2011;3(6): -PMC -PubMed
    1. Vonsattel JP, DiFiglia M. Huntington disease. J Neuropathol Exp Neurol. 1998;57(5):369–384. doi: 10.1097/00005072-199805000-00001. -DOI -PubMed
    1. A novel gene containing a trinucleotide repeat that is expanded unstable on Huntington’s disease chromosomes The Huntington’s Disease Collaborative Research Group. Cell. 1993;72(6):971–983. doi: 10.1016/0092-8674(93)90585-E. -DOI -PubMed
    1. Borrell-Pagès M, Zala D, Humbert S, Saudou F. Huntington’s disease: from huntingtin function and dysfunction to therapeutic strategies. Cell Mol Life Sci. 2006;63(22):2642–2660. doi: 10.1007/s00018-006-6242-0. -DOI -PMC -PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources