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. 2012 Jan 15;21(2):406-20.
doi: 10.1093/hmg/ddr475. Epub 2011 Oct 13.

Mutant huntingtin's interaction with mitochondrial protein Drp1 impairs mitochondrial biogenesis and causes defective axonal transport and synaptic degeneration in Huntington's disease

Ulziibat P Shirendeb  1 , Marcus J Calkins, Maria Manczak, Vishwanath Anekonda, Brett Dufour, Jodi L McBride, Peizhong Mao, P Hemachandra Reddy
Affiliations

Mutant huntingtin's interaction with mitochondrial protein Drp1 impairs mitochondrial biogenesis and causes defective axonal transport and synaptic degeneration in Huntington's disease

Ulziibat P Shirendeb et al. Hum Mol Genet. .

Abstract

The purpose of this study was to investigate the link between mutant huntingtin (Htt) and neuronal damage in relation to mitochondria in Huntington's disease (HD). In an earlier study, we determined the relationship between mutant Htt and mitochondrial dynamics/synaptic viability in HD patients. We found mitochondrial loss, abnormal mitochondrial dynamics and mutant Htt association with mitochondria in HD patients. In the current study, we sought to expand on our previous findings and further elucidate the relationship between mutant Htt and mitochondrial and synaptic deficiencies. We hypothesized that mutant Htt, in association with mitochondria, alters mitochondrial dynamics, leading to mitochondrial fragmentation and defective axonal transport of mitochondria in HD neurons. In this study, using postmortem HD brains and primary neurons from transgenic BACHD mice, we identified mutant Htt interaction with the mitochondrial protein Drp1 and factors that cause abnormal mitochondrial dynamics, including GTPase Drp1 enzymatic activity. Further, using primary neurons from BACHD mice, for the first time, we studied axonal transport of mitochondria and synaptic degeneration. We also investigated the effect of mutant Htt aggregates and oligomers in synaptic and mitochondrial deficiencies in postmortem HD brains and primary neurons from BACHD mice. We found that mutant Htt interacts with Drp1, elevates GTPase Drp1 enzymatic activity, increases abnormal mitochondrial dynamics and results in defective anterograde mitochondrial movement and synaptic deficiencies. These observations support our hypothesis and provide data that can be utilized to develop therapeutic targets that are capable of inhibiting mutant Htt interaction with Drp1, decreasing mitochondrial fragmentation, enhancing axonal transport of mitochondria and protecting synapses from toxic insults caused by mutant Htt.

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Figures

Figure 1.
Figure 1.
Identification of Htt in BACHD mice. Mutant Htt proteins were identified in cortical tissues from 2-month-old BACHD mice and control mice using two different Htt antibodies: MAB2166 (which recognizes both WT and mutant Htt) and 1C2 (which identifies expanded polyQ proteins). As shown, both full-length WT and mutant Htt proteins were found in the 2-month-old BACHD mice (lanes 2 and 3), and only full-length WT Htt was observed in the WT mice (lane 1). Western blot analysis using the 1C2 antibody (in the middle panel) showed that the BACHD mice cortex expressed four distinct bands: <350 kDa full-length mutant Htt, and 230, 115 and 82 kDa cleaved products of mutant Htt. A 115 kDa protein was identified in the WT mice. Bottom panel shows β-actin for equal loading.
Figure 2.
Figure 2.
IP analysis in HD patients using the Drp1 antibody. (A) IP and immunoblotting analyses of Drp1 in cortical tissues from control subjects (lanes 1 and 2) and HD patients (lanes 3 and 4). (B) Immunoblotting analysis of Drp1 in control subjects (lanes 5 and 6) and HD patients (7 and 8). The Drp1 antibody recognizes the 82 kDa protein.
Figure 3.
Figure 3.
Co-IP analysis of Drp1 and mutant Htt antibodies in HD patients and control subjects. (A) IP with the Drp1antibody and immunoblotting with the mutant Htt-specific 1C2 antibody in control subjects (lanes 1 and 2) and HD patients (3 and 4). (B) Immunoblotting with the 1C2 antibody using protein lysates from control subjects (lanes 5 and 6) and HD patients (lanes 7 and 8). Mutant Htt-specific 1C2 antibody immunoreacted with two proteins: one with 82 kDa and the other with 40 kDa in IP elutes from HD patients.
Figure 4.
Figure 4.
IP analysis in BACHD mice using the Drp1 antibody. (A) IP and immunoblotting analyses of Drp1 in cortical tissues from WT mice (lane 1) and BACHD mice (lane 2). (B) Immunoblotting analysis of Drp1 in WT mice (lane 3) and BACHD mice (lane 4).
Figure 5.
Figure 5.
Co-IP analysis of Drp1 and mutant Htt antibodies in WT mice and BACHD mice. (A) IP with the Drp1 antibody and immunoblotting with the mutant Htt-specific 1C2 antibody in WT mice (lane 1) and BACHD mice (lane 2). (B) Immunoblotting with the 1C2 antibody using protein lysates from WT mice (lane 1) and BACHD mice (lane 2). The mutant Htt-specific 1C2 antibody immunoreacted with two proteins: one with 82 kDa and the other with 40 kDa in IP elutes from the BACHD mice.
Figure 6.
Figure 6.
Time-course, double-labeling analysis of Drp1 and mutant Htt in cortical primary neurons from BACHD mice. Drp1 (green) and mutant Htt (1C2 in red) accumulate in the cell neurites and soma over time. Drp1 co-localization increased with mutant Htt in DIV 14.
Figure 7.
Figure 7.
Increased GTPase Drp1 enzymatic activity in HD patients and BACHD mice. (A) Significantly increased GTPase Drp1 enzymatic activity in the cortex of HD patients relative to control subjects. (B) Significantly increased Drp1 activity in the striatum and cortex of 2-month-old BACHD mice relative to non-transgenic littermates.
Figure 8.
Figure 8.
Primary neurons from BACHD mice show reduced motile mitochondria and also anterograde axonal transport of mitochondria. (A) Percent of motile mitochondria in neuronal cultures of BACHD and WT mice. (B) Mitochondrial speed in BACHD and WT neurons. (C) Kymographs show anterograde movement of mitochondria. *P < 0.02; **P < 0.0005.
Figure 9.
Figure 9.
Mitochondria are more fragmented in primary cortical neurons from BACHD mice. Upper panel shows DsRed-mito (A) and GFP (B) transfected (C, merged) cortical neurons from WT mice, and lower panel shows DsRed-mito (D) and GFP (E) transfected (F, merged) cortical neurons from BACHD mice.
Figure 10.
Figure 10.
Mitochondrial fragmentation is prevented in neurons transfected with the Drp1 dominant negative mutation K38A. (A) Hippocampal primary neuron showing mitochondrial fragmentation. (B) Primary neurons transfected with WT Drp1 showing increased fragmentation. (C) Elongated mitochondria in WT neurons transfected with the Drp1.K38A mutation.
Figure 11.
Figure 11.
Mutant Htt aggregates co-localize with oligomers in BACHD primary neurons. (A) Immunoreactivity mutant Htt oligomers (immunolabeled with the A11 antibody). (B) Mutant Htt. (C) Co-localization of mutant aggregates and oligomers.
Figure 12.
Figure 12.
Double-labeling analysis of synaptophysin and MAP2 in BACHD and WT primary neurons. Upper panel shows immunoreactivity of synaptophysin (A), MAP2 (B), DAPI (C) and merged (D) in WT neurons. Lower panel shows immunoreactivity of synaptophysin (E), MAP2 (F), DAPI (G) and merged (H) in WT neurons. Images are taken with a confocal microscope using a 60× objective. (B) Quantification of synaptophysin in BACHD and WT neurons. Significantly decreased synaptophysin was found in BACHD neurons.
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