Palmitoylation of huntingtin by HIP14 is essential for its trafficking and function

Abstract

Post-translational modification by the lipid palmitate is crucial for the correct targeting and function of many proteins. Here we show that huntingtin (htt) is normally palmitoylated at cysteine 214, which is essential for its trafficking and function. The palmitoylation and distribution of htt are regulated by the palmitoyl transferase huntingtin interacting protein 14 (HIP14). Expansion of the polyglutamine tract of htt, which causes Huntington disease, results in reduced interaction between mutant htt and HIP14 and consequently in a marked reduction in palmitoylation. Mutation of the palmitoylation site of htt, making it palmitoylation resistant, accelerates inclusion formation and increases neuronal toxicity. Downregulation of HIP14 in mouse neurons expressing wild-type and mutant htt increases inclusion formation, whereas overexpression of HIP14 substantially reduces inclusions. These results suggest that the expansion of the polyglutamine tract in htt results in decreased palmitoylation, which contributes to the formation of inclusion bodies and enhanced neuronal toxicity.

Figure 1

Huntingtin is palmitoylated in neurons and COS cells. Palmitoylation is modulated by CAG size. (a) Rat cortical neurons were treated (+) with 100 μM NMDA for 10 min, followed by metabolic labeling. Huntingtin (htt) was immunoprecipitated (IP) and visualized by western blotting and fluorography. A [³H]palmitate band was detected in untreated cells. However, after NMDA treatment, a palmitoylated fragment of htt, detected by the N terminus–specific antibody BKP1, was generated (right). (b) COS cells transfected with fragments of htt (N548, N224) were metabolically labeled and processed as described above. The smallest palmitoylated htt fragment contained six cysteine residues. (c) COS cells were transfected with N548-15 and metabolically labeled. Immunoprecipitates were treated with or without 1 M NH₂OH and processed as previously described. Palmitate was removed from htt after NH₂OH treatment (right), indicating that it is coupled to htt through a thioester bond. (d,e) COS cells were transfected with plasmids encoding either (i) a GFP-tagged htt fragment containing six cysteines (Htt 79–224), three N-terminal cysteines (Htt 79–149) or three C-terminal cysteines (Htt 141–224), (ii) an N548 htt fragment for the C152S and C204S substitutions, or (iii) a full-length htt construct with and without C214S (Supplementary Fig. 1). Htt from labeled cells was analyzed as described above. Substituting C214 with serine (C214S) abolished the palmitoylation of Htt 79–224 and Htt 141–224 (d), and full-length htt (e). (f) COS cells were transfected with N548, containing 15 or 128Q, and metabolically labeled. Htt from labeled cells was analyzed as described above. (g) Results of four independent experiments were adjusted for protein levels and demonstrated a reduction of ~50% in the palmitoylation of mutant htt (P = 0.001).

Figure 2

Increased inclusions of palmitoylation-resistant mutant htt in COS cells. (a–d) COS cells were transfected with N548-128, with or without the C214S substitution, and stained with htt antibody. (a) Inclusion bodies (arrows) were occasionally seen with the expression of N548-128. However, a C214S substitution altered mutant htt distribution and increased the formation of inclusions. Scale bar, 10 μm. (b) The percentage of cells containing inclusions increased in COS cells expressing N548-128 (mean ± s.e.m.: 6.7 ± 0.46%) and was maximal in cells expressing N548-128(C214S) (19.4 ±± 0.58%). This effect was not observed with N548-128(C204S) (8 ± 0.68%). (c) Time-lapse images captured over 2 h revealed that inclusions developed at an accelerated rate in the presence of the C214S substitution in mutant htt (bottom). Scale bar, 10 μm. (d) The rate of inclusion formation in cells transfected with N548-128(C214S) was significantly (P = 0.03) faster than in cells transfected with N548-128.

Figure 3

Altered distribution of palmitoylation-resistant mutant htt in COS cells and neurons. (a,b) COS cells transfected with N548-128 (C214S) were immunolabeled with antibodies detecting htt and several markers. Htt inclusions colocalized with the centrosome marker γ-tubulin, the proteasome markers ubiquitin and Lmp2, and misfolded protein marker Hsp70, but not with the Golgi marker GM-130, the lysosomal marker Lysotracker or the early endosome marker EEA1. Scale bar: 10 μm in a, 5 μm in b. (c) Rat cortical neurons transfected with N548-15-GFP or N548-128(C214S)-GFP were immunolabeled with antibodies detecting Hsp70 (red). Mutant htt with the C214S substitution accelerated the formation of inclusions that colabeled with Hsp70. Scale bar, 10 μm. (d) Representative images showing that the inclusions formed in cortical neurons expressing N548-128(C214S) also colocalized with Lmp2. Scale bar, 10 μm.

Figure 4

Enhanced toxicity of palmitoylation-resistant mutant htt. (a,b) COS cells transfected with N548-128 and N548-128(C214S) were scored for toxicity by analyzing nuclei for changes associated with cell death. Cell death was significantly (P = 10⁻⁵) induced by the expression of N548-128(C214S). Representative images show a normal nucleus and an apoptotic nucleus (arrowhead). (c–e) Rat cortical neurons were transfected with truncated htt (N548) or full-length htt (flhtt) constructs as indicated, and stained with htt antibody. (c) The percentage of neurons containing inclusions increased in neurons expressing N548-15(C214S) (2.5 ± 0.35%), increased still further in neurons expressing N548-128 (15.17 ± 1.3%) and was maximal in neurons expressing N548-128(C214S) (25.08 ± 3.1%). (d) Representative images showing neurons expressing the indicated flhtt constructs. Scale bar, 5 μm. (e) The percentage of neurons containing inclusions increased in neurons expressing flhtt-15Q(C214S) (0.5 ± 0.27%), increased still further in neurons expressing flhtt-128Q (1 ± 0.41%) and was maximal in neurons expressing flhtt-128Q(C214S) (2.1 ± 0.44%). There was a significant (P = 10⁻⁵) increase in the percentage of cells with nuclear inclusions in neurons expressing flhtt-128Q(C214S). (f) Rat cortical neurons transfected with the indicated htt constructs were treated with NMDA for 10 min and processed for the TUNEL assay. The percentage of cell death was significantly greater in transfected cells expressing N548-15Q (C214S) (23.16 ± 5.2; P = 0.01) and N548-128Q(C214S) (27.75% ± 4.7; P = 0.03), demonstrating that the loss of palmitoylation significantly reduces cellular viability.

Figure 5

HIP14 influences the distribution and catalyzes the palmitoylation of htt in neurons. (a) Cortical neurons transfected with control vector (left) or with FLAG-tagged HIP14 were labeled with the appropriate antibodies. The overexpression of HIP14 resulted in marked redistribution of endogenous htt to the Golgi. Scale bar, 5 μm. (b,c) Cortical neurons transfected with N548-128Q-GFP or N548-128Q(C214S)-GFP alone (left) or with HIP14-FLAG were labeled with the appropriate antibodies. Scale bar, 5 μm. (b) Partial redistribution of N548-128Q-GFP was observed in the presence of HIP14. (c) No effect in the distribution of palmitoylation-resistant htt (N548-128Q(C214S)-GFP) was observed in the presence of HIP14. (d) COS cells transfected with N548-15Q or N548-128Q, and with a control vector or HIP14, were metabolically labeled. Immunoprecipitated htt was analyzed as described above. (e) Palmitoylation was significantly (P = 0.03) increased in cells coexpressing wild-type htt and HIP14. In addition, HIP14 significantly (P = 0.02) increased palmitoylation of mutant htt. However, in the presence of HIP14, mutant htt was still significantly (P = 0.03) less palmitoylated than wild-type htt. (f) Cortical neurons transfected with GFP-tagged N548-128Q or N548-128Q(C214S), and with a control vector or FLAG-tagged HIP14, were labeled with the appropriate antibodies. The percentage of cells containing htt inclusions was significantly (P = 0.005) lower in the presence of HIP14 whereas no change was detected in the number of C214S inclusions, emphasizing the importance of HIP14 palmitoylation of htt in its distribution.

Figure 6

HIP14 associates less with mutant htt, and mutant htt is less palmitoylated in vivo. (a) Coimmunoprecipitation of HIP14 and htt from brains of YAC18 and YAC128 mice demonstrated a weaker interaction between mutant htt and HIP14. (b) Immunoprecipitated htt from YAC18 and YAC128 mouse brain lysate was incubated in the presence or absence of 1 M NH₂OH and labeled with Btn-BMCC sulfhydryl-specific reagent. Western blots were probed with streptavidin-conjugated HRP (top) to detect biotin-labeled htt or probed with htt antibody (bottom) to detect total immunoprecipitated htt. (c) Palmitoylation was significantly (P = 0.01) reduced in htt with an expanded polyglutamine tract.

Figure 7

HIP14 regulates the palmitoylation and distribution of huntingtin in vivo. (a–f) Downregulation of HIP14 expression altered the trafficking of wild-type and mutant htt and increased perinuclear accumulation of Lmp2. Cortical neurons (6 DIV) from YAC18 and YAC128 mice were transfected with GFP and either control siRNA or HIP14 siRNA. Arrowheads in panels a,b and c indicate transfected cells. Scale bar, 5 μm. Representative images show an increase in the cytoplasmic accumulation of wild-type htt in YAC18 neurons (a), and mutant htt (b) and Lmp2 (c) in neurons from YAC128 mice expressing HIP14 siRNA. Quantification of htt (d,e) and Lmp2 (f) staining intensity in the cytoplasm of neurons expressing control or HIP14 siRNA demonstrated that htt and Lmp2 intensities were substantially higher in cells expressing HIP14 siRNA. (g) Downregulation of HIP14 in neurons transfected with N548-128 significantly (P = 0.025) increased the percentage of neurons with inclusions. (h) Downregulation of HIP14 in neurons decreased cellular viability following NMDA-induced cell death. Cortical neurons (8 DIV) were infected with lentiviruses expressing either control or HIP14 siRNA for 3 d, followed by NMDA treatment (Methods). The percentage of cell death in neurons infected with HIP14 siRNA virus was significantly (P = 0.002) higher, demonstrating that decreased expression of HIP14 significantly increases cell death.