S-nitrosylation of dynamin-related protein 1 mediates mutant huntingtin-induced mitochondrial fragmentation and neuronal injury in Huntington's disease

Abstract

Aims: Dynamin-related protein1 (Drp1) is a large GTPase that mediates mitochondrial fission. We recently reported in Alzheimer's disease (AD) that S-nitrosylation of Drp1 (forming S-nitroso [SNO]-Drp1) results in GTPase hyperactivity and mitochondrial fragmentation, thus impairing bioenergetics and inducing synaptic damage and neuronal loss. Here, since aberrant mitochondrial dynamics are also key features of Huntington's disease (HD), we investigated whether formation of SNO-Drp1 contributes to the pathogenesis of HD in cell-based and animal models.

Results: We found that expression of mutant huntingtin (mutHTT) protein in primary cultured neurons triggers significant production of nitric oxide (NO). Consistent with this result, increased levels of SNO-Drp1 were found in the striatum of a transgenic mouse model of HD as well as in human postmortem brains from HD patients. Using specific fluorescence markers, we found that formation of SNO-Drp1 induced excessive mitochondrial fragmentation followed by loss of dendritic spines, signifying synaptic damage. These neurotoxic events were significantly abrogated after transfection with non-nitrosylatable mutant Drp1(C644A), or by the blocking of NO production using an nitric oxide synthase inhibitor. These findings suggest that SNO-Drp1 is a key mediator of mutHTT toxicity, and, thus, may represent a novel drug target for HD.

Innovation and conclusion: Our findings indicate that aberrant S-nitrosylation of Drp1 is a prominent pathological feature of neurodegenerative diseases such as AD and HD. Moreover, the SNO-Drp1 signaling pathway links mutHTT neurotoxicity to a malfunction in mitochondrial dynamics, resulting in neuronal synaptic damage in HD.

FIG. 1.

MutHTT increases NO production in rat cortical neurons. Neurons at DIV14 were co-transfected with ptdTomato and the N-terminal fragment of either wild-type HTT (wtHTT; top, white arrow) or mutant HTT (mutHTT; bottom, dotted arrow), and stained with the fluorescent NO probe, DAF-FM. (A, D) Fluorescent images of tdTomato show transfected cells. (B, E) Images of DAF-FM fluorescence 7 h after transfection. (C, F) Overlay of the tdTomato and DAF-FM fluorescent images. (G) Quantitative analysis of DAF-FM intensity normalized to control (ctrl; non-transfected cells; *p<0.05 by Student's t-test, n=16 for wtHTT, n=19 for mutHTT). DAF-FM, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate; DIV14, 14 days in vitro; HTT, Huntingtin; NO, nitric oxide. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 2.

S-nitrosylation of Drp1 in the striatum of BACHD mice. (A, C) Striatum and cerebellum of transgenic BACHD mice and wild-type mice (Ctrl) were subjected to the biotin-switch assay to detect SNO-Drp1. Levels of mutHTT and wtHTT were detected by immunoblotting (note the very faint band representing mutHTT in the striatum of the BACHD mouse). (B, D) Relative ratio of SNO-Drp1 to total Drp1 (Drp1) was determined by densitometric quantification of biotin-switch and immunoblot analyses (**p<0.01 by t-test, n=6). Drp1, dynamin-related protein 1; SNO, S-nitrosothiols.

FIG. 3.

Increased SNO-Drp1 in human HD patient brain. (A) Biotin-switch analysis of postmortem brains obtained from human HD patients and patients deceased for non-CNS-related causes (Ctrl, see Table 1). (B) Densitometric quantification of SNO-Drp1 levels in postmortem brains of human HD patients and control patients. Intensity of SNO-Drp1 band normalized to total Drp1 level (*p<0.05 by t-test). CNS, central nervous system; HD, Huntington's disease.

FIG. 4.

S-nitrosylation promotes Drp1 binding to HTT. (A) HTT was detected in Drp1 co-immunoprecipitates (IP) prepared from BACHD mouse striatum (left). The co-immunoprecipitation assay was performed in triplicate. After co-immunoprecipitation, the Drp1-HTT complex was incubated with ascorbate to reduce SNO on Drp1 via denitrosylation. Lack of the HTT and Drp1 interaction in WT mouse striatum (right). (B) HEK293-nNOS cells transfected with either full-length wtHTT or mutHTT were exposed to the physiological NO donor SNOC and assayed for SNO-wtHTT or SNO-mutHTT by the biotin-switch assay. The NOS inhibitor NNA was included in the culture medium to block endogenous NO production. Arrows indicate either mutHtt (transfected) or wtHtt (transfected and endogenous). NNA, N-nitro-l-arginine; NOS, nitric oxide synthase; SNOC, S-nitrosocysteine.

FIG. 5.

SNO-Drp1 mediates mutHTT-induced mitochondrial fragmentation. Representative 3D-deconvolution fluorescent images were obtained 1 day after transfection. Cortical neurons were co-transfected at DIV14 with the mitochondrial marker mito-DsRed2 plus wtHTT and wtDrp1 (A, B), mutHTT and wtDrp1 (C, D, G, H), or mutHTT and S-nitrosylation-resistant mutant Drp1(C644A) (E, F). For some experiments, the NOS inhibitor NNA (1 mM) was included in the culture medium to block NO production (G, H). (B, D, F, H) Magnified insets corresponding to boxed areas in (A, C, E), and (G), respectively. (I) Quantification of mitochondrial fragmentation. Neurons with fragmented mitochondria were scored in a masked fashion (See “Materials and Methods” for details). Data presented as mean±SEM (**p<0.01, n=30–36). SEM, standard error of the means. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 6.

S-nitrosylation of Drp1 decreases relative mitochondrial SA in neurons. Cortical neurons (DIV14) were co-transfected with N-terminal fragments of HTT (wt or mut) and Drp1 (wt or C644A). The ratio of mitochondrial SA (labeled with mito-DsRed2) to neuronal SA (represented by GFP) was calculated for each condition, as described in the “Materials and Methods” section. (A) Fluorescence microscopy images of mito-DsRed2, GFP, and overlay of both fluorescence channels. (B) Quantification of mitochondrial fragmentation by SA analysis 1 day after neuronal transfection. Data presented as mean±SEM. (***p<0.005, n=30–36). GFP, green fluorescent protein; SA, surface area. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 7.

mutHTT significantly decreases dendritic spine density via formation of SNO-Drp1. Cortical neurons were transfected with N-terminal fragments of wtHTT or mutHTT plus either wtDrp1 or mutant Drp1(C644A). Co-transfection with GFP permitted visualization of morphological changes in dendritic spines. (A) Representative fluorescence microscopy images of dendritic spines were obtained from neurons 2 days post transfection of wtHTT plus wtDrp1, mutHTT plus wtDrp1, or mutHTT plus Drp1 C644A. (B) Dendritic spine density in neurons expressing the indicated plasmid constructs. The NOS inhibitor NNA (1 mM) was added to the culture medium to inhibit NO production (***p<0.005, n=4). To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 8.

Proposed mechanism by which SNO-Drp1 mediates mutHTT-induced mitochondrial fragmentation and synaptic injury in HD. Under physiological conditions, Drp1 basal activity contributes to the equilibrium of fission and fusion events in mitochondrial dynamics. The normal mitochondrial fission and fusion cycle is important for maintaining synaptic plasticity via effective distribution of functional, bioenergetically competent mitochondria to synaptic sites. In HD, expansion of the polyQ repeat in mutHTT results in increased NO production. High levels of NO species cause aberrant S-nitrosylation of Drp1 by reacting at Cys644, with resultant increased GTPase activity and excessive mitochondrial fragmentation. In addition, S-nitrosylation increases the binding affinity of Drp1 and HTT, and Drp1 may possibly be transnitrosylated from SNO-mutHTT. Consequently, excessively fragmented mitochondria contribute to the synapse loss and neuronal damage in HD.