pARIS-htt: an optimised expression platform to study huntingtin reveals functional domains required for vesicular trafficking

Abstract

Background: Huntingtin (htt) is a multi-domain protein of 350 kDa that is mutated in Huntington's disease (HD) but whose function is yet to be fully understood. This absence of information is due in part to the difficulty of manipulating large DNA fragments by using conventional molecular cloning techniques. Consequently, few studies have addressed the cellular function(s) of full-length htt and its dysfunction(s) associated with the disease.

Results: We describe a flexible synthetic vector encoding full-length htt called pARIS-htt (Adaptable, RNAi Insensitive &Synthetic). It includes synthetic cDNA coding for full-length human htt modified so that: 1) it is improved for codon usage, 2) it is insensitive to four different siRNAs allowing gene replacement studies, 3) it contains unique restriction sites (URSs) dispersed throughout the entire sequence without modifying the translated amino acid sequence, 4) it contains multiple cloning sites at the N and C-ter ends and 5) it is Gateway compatible. These modifications facilitate mutagenesis, tagging and cloning into diverse expression plasmids. Htt regulates dynein/dynactin-dependent trafficking of vesicles, such as brain-derived neurotrophic factor (BDNF)-containing vesicles, and of organelles, including reforming and maintenance of the Golgi near the cell centre. We used tests of these trafficking functions to validate various pARIS-htt constructs. We demonstrated, after silencing of endogenous htt, that full-length htt expressed from pARIS-htt rescues Golgi apparatus reformation following reversible microtubule disruption. A mutant form of htt that contains a 100Q expansion and a htt form devoid of either HAP1 or dynein interaction domains are both unable to rescue loss of endogenous htt. These mutants have also an impaired capacity to promote BDNF vesicular trafficking in neuronal cells.

Conclusion: We report the validation of a synthetic gene encoding full-length htt protein that will facilitate analyses of its structure/function. This may help provide relevant information about the cellular dysfunctions operating during the disease. As proof of principle, we show that either polyQ expansion or deletion of key interacting domains within full-length htt protein impairs its function in transport indicating that HD mutation induces defects on intrinsic properties of the protein and further demonstrating the importance of studying htt in its full-length context.

Figure 1

pARIS-htt an Adaptable, RNAi Insensitive & Synthetic construct encoding human huntingtin. A) Schematic representation of pARIS-htt. The entire coding sequence is divided into 8 different fragments, each fragment being flanked by unique restriction sites every 1-1.5 kbp and cloned independently into a modified pUC19 backbone. A multi-cassette full-length htt plasmid (pARIS-htt) was generated by assembly of these 8 individual fragments. pARIS-htt construct was tagged with 6×His followed by a mCherry on the amino terminus. The carboxy-terminal part contains HA and tetracysteine (TC) tags. The synthetic construct is fully compatible with the Gateway technology thanks to the introduction of flanking attL sites. (B) pARIS-htt triggers the expression of full-length htt in HEK cells. Cells mock transfected and transfected with pARIS-mCherry-httQ23 or pARIS-mCherry-httQ100 were analyzed by western blot using antibodies raised against different regions of htt: the amino-terminal part (htt-4C8), the carboxy-terminal part (htt-2C1) or the pathogenic polyQ stretch (1C2). The exact epitopes for these antibodies are illustrated in (A). Expression of the different constructs was detected using a high affinity anti-HA antibody. (C) Confocal images of Cos7 mock transfected cells and cells transiently transfected with pARIS-mCherry-httQ23 or pARIS-mCherry-httQ100 constructs. Expression of pARIS-htt is detected as a cytosolic mCherry fluorescent signal which codistributes with htt-4C8 antibody staining. Scale bar 10 μm.

Figure 2

Huntingtin depletion impairs Golgi reformation after microtubule disruption. A) HeLa cells were sequentially transfected with scRNA or siRNA-htt and pARIS-mCherry-httQ23/Q100 and finally analyzed by western blot using antibodies that recognize either endogenous and exogenous htt (htt-4C8) or only exogenous htt (HA). Treatment with siRNA-htt (second lane) results in the complete silencing of endogenous htt. Compared to endogenous htt, pARIS-htt displays lower mobility due to fusion with tags. Note that expression levels of pARIS-mCherry-httQ23/Q100 are not modified by siRNA-htt treatment (lanes 4 and 6). α-tubulin is used as a protein loading control. (B) HeLa cells were transfected with scRNA or siRNA-htt, fixed and processed for staining of a Golgi marker (Ctr 433) and α-tubulin. Unlike scRNA-treated cells, cells silenced for endogenous htt display a dispersed Golgi phenotype but an intact MT network. (C) α-tubulin staining before and after nocodazole (NZ) treatment reveals that MT network is entirely reformed 120 min after NZ removal in HeLa cells. (D) A schematic description of the transfection protocol is summarized. (E) HeLa cells stably expressing GFP-mannosidase II were transfected with scRNA or siRNA-htt and treated with NZ for 120 min to allow a complete MT depolymerization. Golgi reformation was monitored 120 min after NZ washout. In scRNA-treated cells the GA becomes again centrally organized. However, cells depleted from endogenous htt still present a dispersed GA at the same time point. Scale bars 10 μm.

Figure 3

pARIS-mCherry-httQ23 but not pARIS-mCherry-httQ100 restores Golgi reassembly after endogenous huntingtin depletion. A) Gene replacement experiments and Golgi reformation assays were performed adding back pARIS-mCherry-httQ23/Q100 in cells depleted from endogenous htt following the protocol indicated in the scheme. (B) Representative images of cells expressing pARIS-mCherry-httQ23 (upper pannel) or pARIS-mCherry-httQ100 (lower panel) at t = 0 and t = 120 after NZ washout. While cells expressing pARIS-mCherry-httQ23 completely reassemble the GA into tight stacks, cells expressing pARIS-mCherry-httQ100 display scattered Golgi fragments that are unable to reassemble in the perinuclear region. (C) Quantification of the GA reassembly is presented as an analysis of mean Golgi particle volume (μm³) before and after NZ washout for different treatments. Results were obtained from 3 independent experiments in which 280 cells were analyzed. One way ANOVA followed by Fisher's Post-hoc test: ***p < 0.0001; NS non significant. All comparisons are t = 0 vs t = 120; scRNA: 0.283 ± 0.044 vs 4.126 ± 0.771; siRNA-htt 0.073 ± 0.008 vs 0.158 ± 0.06; siRNA-htt + pARIS-mCherry-httQ23: 0.222 ± 0.035 vs 3.763 ± 0.712; siRNA-htt + pARIS-mCherry-httQ100: 0.062 ± 0.006 vs 0.171 ± 0.013.

Figure 4

Htt requires dynein interacting domain to facilitate the transport of Golgi-derived vesicles. A) HEK cells were treated with scRNA or siRNA-htt prior transfection with pARIS-mCherry-httQ23. Cellular lysates were immunoprecipitated using htt-4C8 or anti-dynein (DIC) antibodies and immunocomplexes were subjected to SDS-PAGE to detect either htt or dynein. Dynein co-precipitates with htt when htt-4C8 antibody is used to pull-down endogenous and exogenous htt (Upper panel). Conversely, immunoprecipitation of dynein (lower panel) pulls down both endogenous and exogenous htt (indicated by arrows, lower mobility band corresponding to pARIS-mCherry-httQ23). The same amount of mouse or rabbit IgG's were used as internal immunoprecipitation controls. SN stands for supernatant; IP denotes immunoprecipitation. B) A deletion mutant lacking the minimal dynein interaction domain, denoted as pARIS-mCherry-httQ23-Δdyn, is unable to bind to endogenous dynein (lane 11). (C) Golgi reassembly was monitored in HeLa cells stably expressing GFP-mannosidase II, silenced for endogenous htt and expressing pARIS-mCherry-httQ23-Δdyn as the only cellular source of htt. Most of the cells expressing pARIS-mCherry-httQ23-Δdyn failed to reassemble the GA after NZ washout, suggesting that htt-dynein interaction is required to transport retrogradely Golgi-derived vesicles. Scale bar 10 μm. (D) Quantification of the Golgi dispersion as the mean volume per Golgi particle (μm³) before and after after NZ washout for the different treatments. Results were obtained from 3 independent experiments in which 192 cells were scored. One way ANOVA followed by Fisher's Post-hoc test: ***p < 0.0001; NS non significant. All comparisons t = 0 vs t = 120; siRNA-htt 0.073 ± 0.008 vs 0.158 ± 0.06; siRNA-htt + pARIS-mCherry-httQ23: 0.222 ± 0.035 vs 3.763 ± 0.712; siRNA-htt + pARIS-mCherry-httQ23-Δdyn: 0.073 ± 0.010 vs 0.244 ± 0.072.

Figure 5

Htt requires HAP1 interacting domain to facilitate the transport of Golgi-derived vesicles. A) HEK cells were transfected with pARIS-mCherry-httQ23 or a deletion mutant for the minimal HAP1 interaction domain (denoted as pARIS-mCherry-httQ23-ΔHAP1) in the absence or presence of HAP1-GFP. Exogenous htt was immunoprecipitated (IP) from cell lysates using a HA antibody and immunocomplexes were analyzed for the presence of HAP1-GFP. Immunoprecipitations with mouse IgGs were used as a specificity control. (B) Golgi reformation assays were done in HeLa cells stably expressing GFP-mannosidase II as described previously. Representative image of a pARIS-mCherry-httQ23-ΔHAP1 expressing cell failing to reconstitute the GA after NZ washout. Scale bar 10 μm. (C) Quantification of the Golgi dispersion as the mean volume per Golgi particle (μm³) before and after NZ washout for the different treatments. Results were obtained from 3 independent experiments in which 190 cells were scored. One way ANOVA followed by Fisher's Post Hoc test: ***p < 0.0001. NS, non significant. All comparisons t = 0 vs t = 120; siRNA-htt 0.073 ± 0.008 vs 0.158 ± 0.06; siRNA-htt + pARIS-mCherry-httQ23: 0.222 ± 0.035 vs 3.763 ± 0.712; siRNA-htt + pARIS-mCherry-httQ23-ΔHAP1: 0.073 ± 0.080 vs 0.159 ± 0.060.

Figure 6

pARIS-mCherry-httQ23 facilitates BDNF transport through interaction with dynein and HAP1. A) Fast 3D videomicroscopy was performed to analyze the dynamics of BDNF-eGFP-containing vesicles in mouse neuronal cells expressing BDNF-eGFP alone or cotransfected with pARIS-mCherry-httQ23/Q100 or dynein/HAP1 deletion mutants. Overexpression of pARIS-mCherry-httQ23 recapitulates the transport function of wild-type htt and significantly increases the mean velocity of BDNF-containing vesicles compared to controls values (BDNF transfection alone). Nor the polyQ version neither pARIS-htt deletion mutants are able to stimulate the transport of BDNF containing vesicles. The pausing time of moving vesicles is quantified in (B). Mean overall velocity is indicated as μm/sec. Data were obtained from three independent experiments (control: 4805 tracks from 39 cells; pARIS-mCherry-httQ23: 1970 tracks from 20 cells; pARIS-mCherry-httQ100: 1670 tracks from 18 cells; pARIS-mCherry-httQ23-Δdyn: 4603 tracks from 25 cells; pARIS-mCherry-httQ23-ΔHAP1: 4029 tracks from 20 cells). Fisher's analysis: *P < 0.05; **P < 0.01, NS, non significant.