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Inhibition of mitochondrial protein import by mutant huntingtin

Nature Neuroscience volume 17, pages 822–831 (2014)Cite this article

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Abstract

Mitochondrial dysfunction is associated with neuronal loss in Huntington's disease (HD), a neurodegenerative disease caused by an abnormal polyglutamine expansion in huntingtin (Htt). However, the mechanisms linking mutant Htt and mitochondrial dysfunction in HD remain unknown. We identify an interaction between mutant Htt and the TIM23 mitochondrial protein import complex. Remarkably, recombinant mutant Htt directly inhibited mitochondrial protein import in vitro. Furthermore, mitochondria from brain synaptosomes of presymptomatic HD model mice and from mutant Htt-expressing primary neurons exhibited a protein import defect, suggesting that deficient protein import is an early event in HD. The mutant Htt–induced mitochondrial import defect and subsequent neuronal death were attenuated by overexpression of TIM23 complex subunits, demonstrating that deficient mitochondrial protein import causes mutant Htt-induced neuronal death. Collectively, these findings provide evidence for a direct link between mutant Htt, mitochondrial dysfunction and neuronal pathology, with implications for mitochondrial protein import–based therapies in HD.

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Figure 1: Mutant Htt interacts with the TIM23 complex.
Figure 2: Mutant Htt directly inhibits mitochondrial protein import.
Figure 3: Mitochondria isolated from mutant Htt-expressing striatal cells and mouse brain exhibit decreased protein import.
Figure 4: Mutant Htt-expressing primary neurons show impaired mitochondrial protein import.
Figure 5: Global and TIM23-driven mitochondrial protein import is necessary for survival of primary neurons.
Figure 6: Augmentation of mitochondrial protein import rescues neurons from mutant Htt-induced death.

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Acknowledgements

We are grateful to J.T. Greenamyre, B. Kristal, I.G. Stavrovskaya and members of the Friedlander laboratory for discussion and technical support. We thank M. Lesort for assistance generating recombinant GST-Httex1. This work was supported by US National Institutes of Health grants R01 NS039324 and NS077748 (to R.M.F.) and K01 AG033724 (to H.Y.), Huntington's Disease Society of America (Coalition for the Cure) (R.M.F.), the Brain & Behavior Research Foundation (NARSAD Young Investigator Award) (H.Y.) and the DSF Charitable Foundation (R.M.F.).

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Authors and Affiliations

  1. Department of Neurological Surgery, Neuroapoptosis Laboratory, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

    Hiroko Yano, Sergei V Baranov, Oxana V Baranova, Jinho Kim, Svitlana Yablonska, Diane L Carlisle, Robert J Ferrante & Robert M Friedlander

  2. Department of Neurological Surgery, Washington University School of Medicine, St. Louis, Missouri, USA

    Hiroko Yano, Yanchun Pan & Albert H Kim

  3. Department of Neurology, Washington University School of Medicine, St. Louis, Missouri, USA

    Hiroko Yano & Albert H Kim

  4. Department of Genetics, Washington University School of Medicine, St. Louis, Missouri, USA

    Hiroko Yano

  5. Department of Developmental Biology, Washington University School of Medicine, St. Louis, Missouri, USA

    Albert H Kim

  6. University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA

    Robert M Friedlander

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Contributions

H.Y. designed and performed experiments, analyzed data and wrote the manuscript. S.V.B. contributed to brain mitochondrial preparation, functional and imaging analysis of mitochondria, and manuscript writing. O.V.B. performed preparation of GST fusion proteins and primary neuron cultures and assisted in experiments designed by S.V.B. and H.Y. J.K. and R.J.F. performed immunofluorescence experiments with human HD samples and cell lines. Y.P. performed viral transduction of primary neurons and assisted with neuron viability assays, preparation of GST fusion proteins and coimmunoprecipitation experiments. S.Y. performed Tim23 knockdown experiments for live imaging. D.L.C. contributed to the design of experiments. A.H.K. contributed to neuronal death experiments, experimental design and manuscript writing. R.M.F. contributed to the development of the project, supervised the analysis of all experiments and contributed to manuscript writing.

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Correspondence to Robert M Friedlander.

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Integrated supplementary information

Supplementary Figure 1 Endogenous huntingtin is not required for mitochondrial protein import in embryonic stem (ES) cells.

Mitochondria prepared from wild-type (Htt+/+) and Htt knockout (Htt–/–) ES cells were subjected to in vitro pOTC import assay. A representative gel image is shown (top). Immunoblot analysis confirmed the absence of Htt protein in Htt–/– ES cells (bottom left). Htt+/+ and Htt–/– mitochondria demonstrated similar pOTC import activity (bottom right, n = 3). Mean ± s.e.m.

Supplementary Figure 2 N-terminal mutant huntingtin fragments impair mitochondrial protein import.

Mitochondria were prepared from rat striatal ST14A cells transiently transfected with the plasmid encoding the N-terminal 170-amino acid fragments of wild-type (N170wt) or mutant (N170mut) Htt and subjected to protein import assay. Expression of the N-terminal mutant Htt fragments decreased pOTC import into mitochondria (unpaired t-test (two-tailed); *P = 0.0355, t = 5.187, d.f. = 1.99, n = 2 independent experiments). Mean ± s.e.m. Representative gel image is shown (bottom). D: a mitochondrial uncoupler, 2,4-dinitrophenol was added to mitochondria before starting the import reaction.

Supplementary Figure 3 Mutant huntingtin decreases the rate of mitochondria-targeted GFP accumulation in cells.

(a) Time-lapse imaging shows an increase in mitochondria-targeted GFP (mtGFP) fluorescence intensity over time in ST-HdhQ7/Q7(Q7) cells expressing mtGFP using the BacMam system (Life Technologies) (green). Polarized active mitochondria were identified by mitochondria selective TMRM dye (red). Bar = 10 μm. A representative result of 3 independent experiments is shown. See also Supplementary Video 1. (b) mtGFP fluorescence intensity in Q7 cells in experiments (a) was recorded and quantified using MetaMorph™ (Molecular Devices) and ImageJ (Wayne Rasband, NIH (rsb.info.nih.gov) software and was plotted against time. tlag: the time between transfection and the detection of GFP fluorescence. Slope = rate of mtGFP accumulation. (c) The rate of increase in mtGFP fluorescence intensity in mutant ST-HdhQ111/Q111 (Q111) cells were significantly lower compared to that of WT Q7 cells (unpaired t-test (two-tailed), P = 0.02, d.f. = 24, t = 2.539, n = 49 cells per group). Vertical bars represent the range of values.

Supplementary Figure 4 Respiratory function is not altered in forebrain mitochondria isolated from presymptomatic R6/2 mice.

(a) Synaptosomal and nonsynaptosomal mitochondria were isolated from 3–4 week-old 150CAG R6/2 and control WT mice. Respiratory function was measured using the complex I substrate, glutamate-malate (top), or complex II substrate, succinate (bottom). The respiratory control ratios (RCR) were calculated as the ratios of state 3 to state 2 respiration rates (RCR3/2) or the ratios of state 3 to state 4 respiration rates (RCR3/4). Both synaptosomal and nonsynaptosomal mitochondria from 150CAG R6/2 forebrain show no significant difference in respiratory function compared to that of WT controls (n = 3 and n = 4 independent experiments for synaptosomal and nonsynaptosomal mitochondria, respectively). (b) Synaptosomal and nonsynaptosomal mitochondria isolated from 5–6 week-old 195CAG R6/2 and control WT mice were subjected to respiratory function analysis with the complex I substrate as in (a). Synaptosomal and nonsynaptosomal mitochondria from 195CAG R6/2 forebrain show no significant difference in respiratory function compared to that of WT controls (n = 3 independent experiments for both synaptosomal and nonsynaptosomal mitochondria). Data (a,b) are presented as mean + s.e.m.

Supplementary Figure 5 Mutant huntingtin expression and oxidative stress attenuate mitochondrial protein import in primary cortical neurons.

(a) HD mouse primary neurons expressing mutant Httex1 show impaired mitochondrial protein import. Primary cortical neurons were prepared from R6/2 and WT littermate embryos at E15.5. Neurons prepared from each embryo were plated into separate dishes. Mitochondria isolated from each individual culture at 7–8 days culture in vitro (DIV 7–8) were subjected to pOTC import assay (30 min at 25°C). mOTC was quantified, and the data were scaled to WT import (set equal to 1). R6/2 neuron mitochondria showed a significant reduction in pOTC import compared to that of WT neurons (unpaired t-test (two-tailed); *P = 0.0079, t = 2.805, d.f. = 37.21, total 18 WT and 24 R6/2 neuron cultures prepared from distinct embryos in 6 independent experiments). The vertical bars represent the range of values. (b,c) Dose-response histogram for the effect of hydrogen peroxide (H2O2) on mitochondrial protein import and cell death in WT primary neurons. Mitochondria were isolated from primary cortical neurons (DIV 7–8) treated with the indicated concentrations of H2O2 for 2 h, and subjected to pOTC import assay (b). Primary cortical neurons were incubated with the indicated concentrations of H2O2 for 1 day, and neuronal death was analyzed by lactate dehydrogenase (LDH) assay (c). Data (b,c) are presented as mean + s.e.m.

Supplementary Figure 6 The effect of polyglutamine proteins on mitochondrial protein import and cell viability in primary neurons.

(a) Mouse primary cortical neurons were transduced with lentiviruses expressing Htt exon1 or the androgen receptor (AR N-terminal 127-amino acid fragment) with normal or pathological length of polyQ repeats, Htt25Q, Htt72Q, AR22Q, and AR65Q, at DIV 5. Mitochondria were isolated at DIV 10 and subjected to pOTC import assays (30 min at 25 °C). mOTC was quantified, and the data were scaled to pOTC import in Htt25Q neuron mitochondria (set equal to 1). Primary neurons expressing Htt72Q showed diminished mitochondrial protein import compared to that of Htt25Q. In contrast, neurons expressing AR65Q did not exhibit a decrease in pOTC import compared to control neurons expressing AR22Q. One-way ANOVA followed by Bonferroni post hoc test; *P < 0.0001, F3,30 = 14.76, n = 8 (Htt25Q, Htt72Q, and AR22Q), n = 10 (AR65Q) mitochondria samples from four independent experiments. The expression of the Htt and AR proteins was confirmed by immunoblotting (data not shown). (b) Primary cortical neurons infected with lentiviruses at DIV 5 as in a were subjected to MTS assays at DIV 14. Neurons expressing Htt72Q and AR65Q showed decreased mitochondrial metabolic activity compared to those expressing Htt25Q and AR22Q, respectively (one-way ANOVA followed by Bonferroni test; *P < 0.0001 compared to their WT counterparts, F3,79 = 35.95, n = 20 (Htt-25Q), n = 21 (Htt72Q, AR22Q, and AR65Q) neuron cultures per group from three independent experiments).

Supplementary Figure 7 Tom40 knockdown reduces Tom40 protein levels and mitochondrial protein import.

(a) Lysates from 293T cells transfected with three distinct Tom40 RNAi plasmids (U6-Tom40.1, U6-Tom40.2, and U6-Tom40.3) or control U6 plasmid were immunoblotted with indicated antibodies. The U6-Tom40.1 plasmid had little to no effect on Tom40 levels and was not used for further analysis. (b) Primary cortical neurons cotransfected with Tom40-GFP and RFP expression plasmids together with the Tom40 RNAi (U6-Tom40.2 or U6-Tom40.3) or control U6 plasmid were fixed and subjected to fluorescence microscopy. Bar = 50 μm. Tom40 RNAi reduced Tom40 protein levels in primary neurons. (c) 293T cells transfected with the indicated Tom40 RNAi plasmids or control U6 plasmid were subjected to pOTC import assay. mOTC was quantified, and data were scaled to mOTC in control U6 mitochondria at the maximum reaction time (set equal to 100).

Supplementary Figure 8 A model for the mutant huntingtin-induced mitochondrial protein import defect.

Mutant Htt binds the TIM23 complex and prevents the import of nuclear-encoded proteins into neuronal mitochondria early in HD pathogenesis, causing mitochondrial dysfunction and subsequent neuronal dysfunction and death in HD. Inhibition of mitochondrial protein import in R6/2 mice is tissue- and age-dependent.

Supplementary Figure 9 Full-length images of blots and gels presented in the main figures.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9 and Supplementary Table 1 (PDF 19544 kb)

Supplementary Methods Checklist (PDF 496 kb)

Time-lapse imaging of ST-HdhQ7/Q7 cells transfected with mitochondria-targeted GFP.

A representative video for Supplementary Fig. 3a experiments is shown. Transfected cells were loaded with 200 nM of TMRM to visualize mitochondria and placed into an on-stage incubator for imaging using IX-81-DCU fluorescence microscope for a period of 24 h. (MOV 1158 kb)

Tim23 knockdown triggers loss of mitochondrial membrane potential and subsequent cell death in primary cortical neurons.

Mouse primary cortical neurons (DIV 5) were transfected with the Tim23 RNAi plasmid (pBSU6-Tim23/CMV-GFP) or control scrambled pBSU6/CMV-GFP plasmid. Three days after transfection, neurons loaded with the red fluorescent TMRM dye, which accumulates in polarized mitochondria, and exposed to the far-red nuclear dye, RedDot2, were subjected to time-lapse multichannel fluorescent imaging using confocal microscopy. Representative videos of control and Tim23 knockdown (Tim23 KD) neurons with and without the GFP channel are shown side-by-side. Red and blue indicate polarized mitochondria and dead cell nuclei, respectively. (MOV 2081 kb)

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Yano, H., Baranov, S., Baranova, O. et al. Inhibition of mitochondrial protein import by mutant huntingtin. Nat Neurosci 17, 822–831 (2014). https://doi.org/10.1038/nn.3721

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