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Vol. 20, Issue 5, 1478-1492, March 1, 2009
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*Departament de Biologia Cel·lular, Immunologia i Neurociències, Facultat de Medicina, IDIBAPS, Universitat de Barcelona, E-08036 Barcelona, Spain; Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Spain; and Institut de Nanotecnologia, Universitat de Barcelona, E-08036 Barcelona, Spain
Submitted July 16, 2008;
Revised December 22, 2008;
Accepted January 6, 2009
Monitoring Editor: Robert G. Parton
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), which produces significant dysfunction and neural death, especially in the medium spiny neurons of the striatum. Htt is a huge protein with a widespread distribution in neurons, including cell bodies, dendrites and axons (Sharp et al., 1995). Detailed subcellular fractionation and microscopic studies have shown that htt is also associated with vesicles and microtubules (Difiglia et al., 1995; Hoffner et al., 2002), where it interacts with molecular motors that promote the transport of vesicles containing brain derived-neurotrophic factor (BDNF) (Gauthier et al., 2004). We previously demonstrated that htt also participates in post-Golgi trafficking of proteins that follow the regulated secretory pathway (del Toro et al., 2006). Thus, mutant htt (mhtt) impairs post-Golgi trafficking, which in turn decreases the number of vesicles containing BDNF, thereby contributing to a reduced trophic support that is essential for striatal neurons during HD progression (Canals et al., 2004). However, the molecular mechanism by which htt participates in post-Golgi trafficking remains unclear. Htt colocalizes with the Golgi apparatus and with exocytic vesicles (Difiglia et al., 1995) and reduced expression of htt by RNA interference (RNAi) disrupts the Golgi apparatus (Caviston et al., 2007). Moreover, several htt-interacting proteins, such as HIP14 (Singaraja et al., 2002) and optineurin (Hattula and Peranen, 2000), contribute to the function of the Golgi apparatus and exocytosis.
Optineurin, a Rab8 effector (Hattula and Peranen, 2000), colocalizes with htt in the Golgi apparatus where it contributes to post-Golgi trafficking and Golgi organization (Sahlender et al., 2005). Rab8 belongs to the family of Rab GTPases, central regulators of vesicle budding, motility, and fusion (for review, see Stenmark and Olkkonen, 2001; Pfeffer, 2003). Optineurin and Rab8 form a complex that participates in the regulation of the post-Golgi transport of proteins, the sorting of which is mediated by the clathrin adaptor complex 1 (AP-1) (Sahlender et al., 2005; Au et al., 2007).
Here, we examined whether htt regulates post-Golgi transport via the optineurin/Rab8 complex in striatal-derived cells that express wild-type (wt) full-length htt (with 7Q, wt cells) or full-length mhtt (with 111Q, mhtt cells). We demonstrate that mhtt cells show reduced levels of htt in the Golgi apparatus, which in turn presents decreased levels of optineurin and Rab8. This combination leads to reduced clathrin-dependent post-Golgi trafficking to lysosomes. Therefore, mhtt affects post-Golgi transport to the plasma membrane (del Toro et al., 2006) and to lysosomes by uncoupling the optineurin/Rab8 complex at late Golgi compartments.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-adaptin antibodies were from BD Biosciences Transduction Laboratories (Lexington, KY), and anti-optineurin and anti-lysosomal–associated membrane protein (Lamp)-1 antibodies were from Abcam (Cambridge, MA). The anti-cathepsin D antibody was a generous gift from Dr. William Brown (Cornell University, Ithaca, NY). Fluorescent Cy3 (anti-rabbit), Cy2 (anti-mouse), and Alexa 647 (anti-mouse and -rabbit) secondary antibodies were from Invitrogen (Carlsbad, CA) and Texas Red-conjugated anti-mouse antibody was from Jackson ImmunoResearch Laboratories (West Grove, PA). Lipofectamine 2000 was from Invitrogen. All other reagents used were from Sigma-Aldrich (St. Louis, MO).
DNA Constructs
Lamp-1 fused to red fluorescent protein (RFP) was kindly supplied by Dr. Walther Mothes (Yale University School of Medicine, New Haven, CT). The mannose-6-phosphate receptor (MPR) fused to green fluorescent protein (GFP) and clathrin light chain B (LcB) fused to yellow fluorescence protein (YFP) were generously supplied by Dr. Juan S Bonifacino (National Institutes of Health, Bethesda, MD). Galactosyltransferase (GT) fused to YFP or cyan fluorescence protein (CFP) was kindly supplied by Dr. Jennifer Lippincott-Schwartz (National Institutes of Health). The Rab8 and Rab8 Q67L construct fused to GFP was supplied by Dr. Ira Mellman (Yale University School of Medicine). Vesicular stomatitis virus glycoprotein (VSV-G) fused to GFP was kindly provided by Kai Simons (Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany). Full-length 17Q htt (FL-17Q-htt) and full-length 75Q htt (FL-75Q-htt) were gifts from Drs. Fréderic Saudou and Sandrine Humbert (Institut Curie, Orsay, France).
Striatal-derived wt and mhtt, M213, Primary Striatal Cultures, and Transfection
To study the role of htt in post-Golgi trafficking, we used the following cell lines: striatal knockin cells stably expressing full-length htt (7Q/7Q, wt cells) or full-length mhtt (111Q/111Q, mhtt cells) established from HdhQ111 knockin mice (Trettel et al., 2000). M213 cells, a conditionally immortalized rat striatal derived neural stem cells (Giordano et al., 1993). Both cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, and 1 mM sodium pyruvate. For primary striatal cultures, certified time pregnant B6CBA mice (Charles River Laboratories, Les Oncins, France) were deeply anesthetized on gestational day 14.5 (E14.5) and fetuses (E0.5, day of vaginal plug) were rapidly removed from the uterus by cesarean section. Fetal brains were then excised and placed in sterile phosphate-buffered saline (PBS), pH 7.4. The striatal primordia were dissected bilaterally, pooled and gently dissociated with a fire-polished Pasteur pipet. Cells were plated onto 24-well plates containing glass coverslips precoated with 0.1 mg/ml poly-D-lysine (Sigma-Aldrich) at a density of 150,000 cells/cm2. We obtained a neuronal culture growing the cells in Eagle's minimum essential medium (Invitrogen) supplemented with B27 (Invitrogen). The plates were placed in an incubator at 37°C in a 5% CO2 atmosphere. Cells were transfected using Lipofectamine 2000, following the manufacturer's instructions.
Full-length htt constructs with 17 or 75 polyQ tracts (FL-17Q-htt and FL-75Q-htt, respectively) were cotransfected (8:1) with GT-YFP, which selectively marks the Golgi apparatus. As a control condition, cells were transfected with GT-YFP alone. Cells transfected with FL-17Q-htt or FL-75Q-htt were selected on the basis of their increased immunoreactivity for anti-htt antibody because there is no tag in these vectors. Rab8 Q67L-GFP was also cotransfected with GT-CFP (8:1), and as a control condition GT-CFP was transfected alone.
Small Interfering RNA (siRNA) Transfection
For silencing htt, RNA duplexes targeted four different regions of mouse htt gene (GenBank accession no. NM_010414): 5'-agagcuaggcacacugcucat-3', 5'-aaaacuacccucuggaugugg-3', 5'-uuaacaaggcuugucccaaag-3', 5'-acaacatctgtacaattgcag-3' as described previously (Caviston et al., 2007). Control cells were transfected with nontargeting RNA duplex oligonucleotides. Twenty-four hours after plating cells on coverslips, cells were cotransfected with 60 nM htt siRNA or control siRNA and GT-YFP or MPR-GFP by using Lipofectamine 2000, following the manufacturer's instructions. All experiments were performed 72 h after transfection.
Electron Microscopy
Striatal knockin cells were rapidly washed in prewarmed (37°C) 0.1 M PIPES buffer, pH 7.4, and then fixed with 1.25% glutaraldehyde in PIPES buffer containing 2% sucrose and 2 mM Mg2SO4 for 1 h at room temperature. They were then gently scraped, pelleted at 100g for 10 min, rinsed three times in PIPES buffer, and postfixed with 1% (wt/vol) OsO4, 1% (wt/vol) K3Fe(CN)6 in PIPES buffer for 1 h in the dark. Cells were then treated for 5 min with 0.1% tannic acid, dehydrated with graded ethanol solutions, and finally embedded in Epon plastic resin. Ultrathin sections (50–70 nm thick) were stained with lead citrate and observed on a JEOL 1010 electron microscope. Micrographs of 20–25 randomly selected fields were obtained with a Bioscan digital camera (Gatan, Munich, Germany). The density of clathrin-coated vesicle (CCV) profiles was measured by dividing the total number of CCV profiles by the cytoplasmic area (square micrometers) of each field. Results are expressed as the mean ± SD.
Immunofluorescence Staining and Confocal Microscopy Analysis
At 24 h posttransfection, cells were fixed in 4% paraformaldehyde for 10 min, 0.2 M glycine for 20 min, and then permeabilized in 0.1% saponin for 10 min. Blocking was done in 1% bovine serum albumin (BSA) in PBS for 1 h. Specimens were incubated with primary antibodies: anti-GM130 (1:250), anti-htt (1:100), anti-optineurin (1:100), anti-rab8 (1:100), anti--adaptin (1:100), anti-Lamp-1 (1:100), and anti-cathepsin D (1:100). Thereafter, samples were incubated with the following secondary antibodies: Cy2-conjugated anti-mouse (1:200), Cy3-conjugated anti-rabbit (1:300), Texas Red-conjugated anti-mouse (1:300), or anti-mouse or anti-rabbit Alexa 647 (1:200). In some cultures, nuclei were counterstained with the fluorescent dye ToPro-3 (Invitrogen).
Double-stained cells were examined by confocal microscopy using a TCS SL laser scanning confocal spectral microscope (Leica Microsystems Heidelberg, Mannheim, Germany) with argon and HeNe lasers attached to a DMIRE2 inverted microscope (Leica Microsystems Heidelberg). Images were taken using a 63x numerical aperture objective with a 4x digital zoom and standard (1 Airy disk) pinhole. For each cell, the entire three-dimensional stack of images from the ventral surface to the top of the cell was obtained by using the Z drive in the TCS SL microscope. The size of the optical image was 0.5 µm. Colocalization was measured by using the "colocalization" plug-in of the freeware ImageJ version 1.33 by Wayne Rasband (National Institutes of Health). Briefly, for each cell stack, the cell area was delineated and the total number of double-positive pixels for htt, optineurin, Rab8, or -adaptin, and a specific subcellular region for each slice was summed. This value was divided by the number of total positive pixels for htt, optineurin, Rab8, or -adaptin in the stack, and multiplied by 100.
For the quantitation of MPR-GFP–containing structures, we measured those fluorescent spots composed of between 2 and 20 pixels (both included, 108-1080 nm). In all cases, 20–30 cells were randomly selected from at least three independent cultures.
Staining of Lysosomes and Autophagic Vacuoles
Lysosomes were labeled by incubating the cells with 1 µM LysoTracker Red DND-99 (Invitrogen) for 30 min at room temperature. Thereafter, cells were washed in PBS and fixed as described above. Given that in our images 1 pixel represents 54 nm, we considered a lysosome to be any fluorescent spot positive for LysoTracker and mad up of between 6 and 40 pixels (from 324 to 2160 nm inclusive). For staining of autophagic vacuoles, a monodansylcadaverine (MDC) stock solution (20 mM) was diluted 1:400 in PBS and applied to the cells for 15 min. Cells were washed with PBS and incubated 30 min with medium containing 1 µM LysoTracker. Thereafter, cells were washed in PBS and fixed as described above.
Inverse Fluorescence Recovery after Photobleaching Analysis Experiments
iFRAP experiments were carried out using the Leica confocal microscope described previously equipped with an incubation system with temperature and CO2 control. Cells (3.5 x 105) were seeded on a 35-mm plates (NUNC A/S, Roskilde, Denmark), each with a glass coverslip of 22 mm (Microcoverglass; Electron Microscopy Sciences, Hatfield, PA). After 24 h, cells were transfected with 4 µg of Lamp-1-RFP, MPR-GFP, LcB-YFP, or VSV-G-GFP constructs. For full-length htt constructs, FL-17Q-htt or FL-75Q-htt constructs were cotransfected (100:1) with MPR-GFP. At 24 h posttransfection, cells were incubated with 0.1 mg/ml cycloheximide in culture medium for 2 h at 20°C. Subsequently, the glass coverslips holding transfected cells was mounted in the videoconfocal chamber, and temperature was warmed to 33°C.
For visualization of RFP, CFP, GFP, and YFP, images were acquired using a 63x oil immersion objective lens (numerical aperture, 1.32), by using either a 514-nm laser line (for RFP), a 488-nm laser line (for GFP/YFP), or a 458-nm laser line (for CFP); excitation beam splitter RSP 500; emission range detection 500–600 nm; and a confocal pinhole set at 2–3 Airy units to minimize changes in fluorescence efficiency caused by fluorescence proteins moving away from the plane of focus.
The whole cytoplasmic area of the transfected cell was photobleached using 80 scans with the 514- or 488-nm laser line at full power. Images were then collected in stream mode every 5 s during 10 min for RFP or 20 min for GFP/YFP.
All iFRAP data were corrected and normalized using the equation described in Dundr et al. (2002) (also see Supplemental Material). iFRAP curves were modeled equally well with a two-phase exponential decay equation (Supplemental Figure 1). Nine to 12 cells were randomly selected from at least three independent transfections. All cells expressed similar levels of tagged proteins to render the data comparable.
Isolation of Golgi Membranes
Golgi fractions from knockin cells were prepared at 4°C as described in Balch et al. (1984), with few modifications (Fernandez-Ulibarri et al., 2007). Briefly, twenty-two 100-mm plates (NUNC A/S) per condition were pelleted and washed twice in cold PBS (10 min at 500 x g) and lysis buffer (10 mM Tris-HCl, 0.25 M sucrose, 1 mM phenylmethylsulfonyl fluoride [PMSF], 10 µg/ml aprotinin, and 1 µg/ml leupeptin, pH 7.4). The cell pellet was then resuspended in 4 volumes of lysis buffer and homogenized using the Ball-Balch homogenizer device. The homogenate was brought to a sucrose concentration of 37% (wt/vol) by adding 62% (wt/vol) sucrose in 10 mM Tris-HCl, pH 7.4, and EDTA (1 mM, final concentration). Fifteen millimeter of this solution was placed at the bottom of a SW 28 ultracentrifuge tube and carefully overlaid with 10 ml of sucrose at 35% (wt/vol) and 10 ml sucrose at 29% (wt/vol) in 10 mM Tris-HCl, pH 7.4. Gradients were centrifuged at 25,000 rpm for 2.5 h. The Golgi-enriched membrane fraction was recovered at the 35–29% sucrose interphase and subsequently stored at –80°C. Protein concentration was determined by detergent-compatible protein assay (Bio-Rad, Hercules, CA).
Western Blot Analysis
Total protein extracts were prepared from striatal cell cultures by homogenization in lysis buffer (50 mM Tris base, 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 1% NP-40, 1 mM PMSF, 10 µg/ml aprotinin, and 1 µg/ml leupeptin, pH 7.4). Samples were centrifuged at 10,000 x g for 10 min, and the protein contents were determined as described above. Proteins from total extracts (20 µg for optineurin or Rab8 and 30 µg for cathepsin D analyses) or proteins from Golgi membranes (15 µg for htt and 10 µg for optineurin or Rab8 analyses) were resuspended in 5x SDS sample buffer (62.5 mM phosphate buffer, 50% glycerol, 12.5% SDS, 16.7 mM dithiothreitol, and 1.2 mM Bromophenol blue, pH 7.0), boiled for 5 min, resolved by 6 or 12% SDS-polyacrylamide gel electrophoresis (PAGE), and transferred to Immobilon-P transfer membranes (Millipore, Billerica, MA). Blots were blocked in 5% nonfat powdered milk and 5% BSA in Tris-buffered saline/Tween 20 (TBS-T: 50 mM Tris-HCl, 150 mM NaCl, pH 7.4, and 0.05% Tween 20) for 1 h at room temperature. The membranes were then incubated overnight at 4°C with anti-htt (1:1000), anti-optineurin (1:500), anti-Rab8 (1:1000), or anti-cathepsin D (1:1000) antibodies. Membranes were then rinsed three times in TBS-T and incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. After washing the membranes for 30 min with TBS-T, they were developed using the enhanced chemiluminescence substrate kit (GE Healthcare, Uppsala, Sweden). The phoretix densitometry program (Phoretix International, Newcastle, United Kingdom) was used to quantify the htt, optineurin, Rab8, and cathepsin D bands. Values are given as the mean ± SEM.
Immunoprecipitation
Immunoprecipitation was performed by incubation of total protein extracts (as described above; 300 µg) with 1 µg of anti-Rab8 antibody overnight at 4°C followed by a 4-h incubation with 50 µl of protein A-Sepharose Cl-4B (Sigma-Aldrich). The beads were washed by centrifugation three times and then boiled for 10 min in 10 µl of 5x SDS sample buffer. The immunocomplexes were resolved on 6 or 12% SDS-PAGE and transferred to Immobilon-P transfer membranes. Western blot analysis was carried out as described above. Blots were incubated with anti-htt (1:1000), anti-optineurin (1:1000), and anti-Rab8 (1:1000) and detected using enhanced chemiluminescent reagents.
Proteolysis Assays
Cells (5 x 104/well) were seeded on a 24-well plate (NUNC A/S) containing a glass coverslip of 12 mm. After 24 h, cells were washed with PBS, and 500 µl per well of medium containing 25 µg/ml the quenched fluorescent substrate DQ-collagen IV (Invitrogen) was added. After 2 h, cells were fixed, and the fluorescent degradation products of DQ-collagen IV were observed in the Leica TCS SL laser scanning confocal spectral microscope, as described above.
Statistical Analysis
GraphPad Prism version 4.0 (GraphPad software, San Diego, CA) software was used to perform unpaired t test or one-way analysis of variance and Bonferroni post hoc test. For iFRAP experiments, the nonparametric t test followed by the Mann–Whitney test was used.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Caviston et al., 2007) but also with exocytic vesicles (Gauthier et al., 2004). To study the mechanism by which mhtt impairs post-Golgi trafficking, we first performed colocalization studies between htt and GT-YFP, which specifically labels the Golgi apparatus, in knockin wt and mhtt cells. As shown in Figure 1, htt localized to the Golgi apparatus in wt cells and also in cytosolic vesicular structures. In contrast, mhtt cells displayed a 65% reduction in the colocalization between GT-YFP and htt compared with wt cells (Figure 1A). To confirm the delocalization of mhtt from the Golgi apparatus, wt cells were cotransfected with full-length htt (FL-17Q-htt) or mhtt (FL-75Q-htt) and GT-YFP. Localization of htt in the Golgi apparatus was observed in cells transfected with FL-17Q-htt. However, cells transfected with FL-75Q-htt showed a diffuse distribution of mhtt, with lower levels at the Golgi apparatus (Figure 1B). This observation was confirmed by Western blot of isolated Golgi membranes, which showed a decrease in htt protein levels in mhtt cells (Figure 1C; wt, 100 ± 26.6%; mhtt, 19.8 ± 4.4%, with respect to wt; p < 0.01). Given that both cell lines express similar levels of htt (Trettel et al., 2000), these findings are not attributable to a decrease in the total levels of htt in mhtt cells.
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). Double immunocytochemistry for optineurin and the Golgi matrix protein GM130 showed that optineurin is mainly localized at the Golgi apparatus in wt cells (Figure 2A). Interestingly, we observed a delocalization of optineurin from the Golgi apparatus in mhtt cells, showing a 21% reduction compared with wt cells (Figure 2A). Consistent with this result, Western blot analysis showed no differences in the levels of total optineurin protein between wt and mhtt cells (Figure 2B; wt, 100 ± 14%; mhtt, 105 ± 9.4%, with respect to wt), whereas the protein levels of optineurin in isolated Golgi membranes were significantly reduced in mhtt cells (Figure 2B; wt, 100 ± 23%; mhtt, 41 ± 17%, with respect to wt; p < 0.01). In addition, double immunocytochemistry for htt and optineurin in wt cells showed colocalization of both proteins at the Golgi apparatus (Figure 2C, arrowheads) and also in vesicular structures (enlarged area shown in Figure 2C). In contrast, mhtt cells showed a punctate distribution of htt and optineurin and lower presence of both proteins at the Golgi apparatus (Figure 2C). In addition, mhtt cells showed a 32% decrease in the colocalization between these two proteins (Figure 2C, enlarged area).
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) (Supplemental Figure 2A). Moreover, optineurin and htt colocalized at the Golgi apparatus (Figure 3B and Supplemental Figure 2B, arrowheads). In addition, both proteins were found in vesicles-like structures throughout the cytoplasm and also close to the Golgi apparatus (enlarged area shown in Figure 3B and Supplemental Figure 2B) in both cells.
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), we observed that wt cells transfected with htt siRNA showed partial Golgi disruption. Interestingly, the colocalization between optineurin and GT-YFP was lower in wt cells with reduced expression of htt with respect to those transfected with control siRNA (Figure 4C), indicating that htt is implicated in the localization of optineurin at the Golgi apparatus.
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), which has been localized to the trans-Golgi network (TGN) (Huber et al., 1993), participating in post-Golgi trafficking (Ang et al., 2003). Thus, we next examined whether mhtt also alters the presence of Rab8 in the Golgi apparatus. Because Rab8 tagged with GFP has been used to determine Rab8 intracellular distribution (Ang et al., 2003; Sahlender et al., 2005), we determined the localization of Rab8 fused with GFP in wt and mhtt cells. Rab8 showed good colocalization around the Golgi apparatus in wt cells (Figure 5A). In contrast, mhtt cells showed lower levels of Rab8 in the Golgi apparatus (Figure 5A). These results were confirmed by Western blot analysis of isolated Golgi membranes. Total levels of Rab8 did not differ between wt and mhtt cells (Figure 5B; wt: 100 ± 8.3%; mhtt: 103 ± 16.8% with respect to wt), but protein levels of Rab8 in isolated Golgi membranes were reduced in the latter (Figure 5B; wt, 100 ± 3.7%; mhtt, 82 ± 1.3%, with respect to wt; p < 0.05).
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To further analyze the role of mhtt in the htt/optineurin/Rab8 complex, we next transfected wt and mhtt cell lines with a constitutively active Rab8-GTP-bound mutant form (Rab8 Q67L) and GT-CFP or GT-CFP alone as a control condition. This mutation alters Rab8-dependent trafficking (Peranen et al., 1996; Ang et al., 2003) by disrupting the localization of proteins involved in this transport such as AP-1B in polarized epithelial cells (Ang et al., 2003) and AP-1 in knockin striatal cells (wt cells; Supplemental Figure 3). Thus, we next examined whether expression of Rab8 Q67L alters the localization of htt and optineurin in both wt and mhtt cells. The wt cells cotransfected with Rab8 Q67L-GFP and GT-CFP showed a 37.5 and 27.8% reduction in the colocalization of optineurin and htt with GT-CFP, respectively (Figure 6). Interestingly, overexpression of Rab8 Q67L in mhtt cells did not result in additive impairment with the htt mutation (Figure 6). The lack of synergic effects suggested that the mutations of htt and Rab8 act in a similar manner on the Golgi apparatus. In agreement with that, expression of Rab8 Q67L (Peranen et al., 1996) or mhtt (Supplemental Figure 4A and Supplemental Movie 1) results in 28–30% reduction in the transport of VSV-G, a glycoprotein from the vesicular stomatitis virus, which is released from the Golgi apparatus to the plasma membrane.
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), by uncoupling the htt/optineurin/Rab8 complex at the Golgi apparatus. To test this hypothesis, we next characterized whether mhtt affects the CCVs in the Golgi apparatus.
mhtt Alters Clathrin-dependent Post-Golgi Trafficking to Lysosomes
Optineurin and Rab8 participate in the regulated post-Golgi transport mediated by the AP-1 (Sahlender et al., 2005; Au et al., 2007). Thus, we examined whether mhtt affects AP-1 localization at the Golgi apparatus. Double immunocytochemistry for -adaptin, a specific subunit of AP-1, and mannosidase 2 (ManII), which marks the Golgi apparatus, showed an 18% higher colocalization in mhtt than in wt cells (Figure 7A). Consistent with this result, Western blot analysis showed no differences in the levels of total -adaptin protein between wt and mhtt cells, whereas the protein levels of -adaptin in isolated Golgi membranes were significantly increased in mhtt cells (Figure 7B). These findings suggest that clathrin-dependent post-Golgi trafficking is impaired, because AP-1 provides the first step of cargo-sequestration specificity in this route (Brodsky et al., 2001; McNiven and Thompson, 2006). Thus, we next examined the ultrastructure of the Golgi apparatus in wt and mhtt cells. The former showed the characteristic Golgi morphology of stacked flattened cisternae (Figure 7C). In contrast, in mhtt cells the Golgi was partially disorganized showing high degree of vesiculation, although some cisternae remained stacked and flattened (Figure 7C). Interestingly, mhtt cells showed an abnormal accumulation of CCV profiles in proximity to Golgi cisternae (Figure 7C; wt, 0.19 ± 0.14 vs. mhtt, 0.87 ± 0.27 CCVs/µm2; p < 0.001). We did not observe clathrin-coated budding profiles in mhtt cells, suggesting that there is not a deficit in CCV budding or fission. Notice that in wt cells, Golgi-associated CCV profiles were almost undetectable (Figure 7C). This result indicates a potential alteration in either CCV-mediated cargo transport or clathrin uncoating in mhtt cells and therefore points out to defects in AP-1–dependent post-Golgi trafficking.
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). mhtt cells showed increased colocalization between Lamp-1 and GT-YFP (Figure 7D). Given that both cell lines express similar levels of Lamp-1 as we observed by Western blot (data not shown), this result indicated an altered post-Golgi transport of Lamp-1. Consistently, iFRAP analysis showed a 37.4% reduction in the exit of Lamp-1 from the TGN in mhtt cells (Figure 7E, Table 1, and Supplemental Movie 2). To confirm the dependence of post-Golgi trafficking to lysosomes on the optineurin/Rab8 complex, we cotransfected Lamp-1 with Rab8 or the Rab8 Q67L mutant form in wt and mhtt cells and carried out iFRAP analysis. Overexpression of Rab8 did not induce significant differences in the post-Golgi transport of Lamp-1 with respect to cells not transfected with Rab8 (Figure 7F and Table 1). In contrast, wt cells expressing Rab8 Q67L showed a 44% reduction in the TGN egress of Lamp-1 compared with wt cells (Figure 7F and Table 1), which is consistent with the delocalization of htt and optineurin from de Golgi apparatus in the presence of Rab8 Q67L (Figure 6). Interestingly, no significant differences were found between mhtt cells, and either mhtt or wt cells expressing Rab8 Q67L (Figure 7F, Table 1, and Supplemental Movie 2). Together, these results suggest that mhtt impairs Rab8-dependent post-Golgi trafficking by means of a defect in the transport of CCVs from the TGN. In agreement with this view, iFRAP studies showed a 20% reduction in the exit of the LcB from the Golgi apparatus in mhtt (Table 1, Supplemental Figure 4B, and Supplemental Movie 3). This observation suggests that the disruption of the htt/optineurin/Rab8 complex by mhtt affects the assembly of CCVs with their motor proteins.
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), is altered by mhtt. Wt cells showed a widespread distribution of MPR in the Golgi apparatus and in cytoplasmatic vesicular structures. In contrast, as shown in Figure 8A, mhtt cells retained the MPR labeling in the Golgi apparatus, displaying a 68% decrease in the number of MPR-containing cytosolic structures and a 260% increase in the colocalization between MPR and GM130 (Figure 8B). This altered subcellular distribution is indicative that post-Golgi transport of MPR is impaired in mhtt cells. In accordance to this result, iFRAP analyses for MPR showed a 30% decrease of TGN exit in mhtt cells (Figure 8C, Table 1, and Supplemental Movie 4). In addition, expression of FL-17Q-htt restored the post-Golgi transport of MPR in mhtt cells (Figure 8D, Table 1, and Supplemental Movie 5). To confirm the involvement of htt in the post-Golgi transport of MPR, we performed iFRAP analysis in wt cells with depletion of htt by siRNA. Wt cells with reduced expression of htt showed a 35% decrease of MPR post-Golgi transport (Figure 8E, Table 1, and Supplemental Movie 6). Consistent with these results, expression of FL-75Q-htt reduced the number MPR-containing cytosolic structures in wt cells in comparison to those transfected with FL-17Q-htt or GT-CFP alone (Figure 8F). Moreover, mhtt cells expressing FL-17Q-htt restored normal levels of MPR-containing cytosolic structures (Figure 8G). Together, these results indicate that, in mhtt cells, the loss of htt function impairs the post-Golgi transport of MPR.
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). Double immunocytochemistry for cathepsin D and LysoTracker showed a 63% decrease in the colocalization of these two markers in mhtt compared with wt cells (Figure 9, A and C). Cathepsin D is synthesized as procathepsin D (52 kDa), which is transported from the Golgi apparatus to lysosomes, in which it is processed to an active enzymatic form (48 kDa; Chinni et al., 1998). Thus, we examined by Western blot the total protein levels as well as the percentage of mature and immature cathepsin D forms versus total cathepsin D. mhtt cells showed a 43% decrease in the total protein levels of cathepsin D. Besides this decrease, the ratio of procathepsin D to total levels of cathepsin D increased 57% in mhtt compared with wt cells. In contrast, the ratio of mature cathepsin D to total levels decreased 14% in mhtt cells (Figure 9D). These results are indicative of altered transport of procathepsin D from the TGN to lysosomes in mhtt cells, which is supported by immunocytochemistry and iFRAP results of Lamp-1 and MPR (Figures 7 and 8).
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). mhtt cells showed fewer fluorescent punctae cytoplasmatic structures than wt cells. This observation indicated a reduced proteolysis of DQ-collagen IV, which implies altered lysosome functionality (Figure 9E).
Altered lysosome activity has been shown to induce autophagic stress (Zhang et al., 2007). Thus, we investigated whether the enlarged LysoTracker-positive structures found in mhtt cells could be in fact autophagic vacuoles. Therefore, we incubated wt and mhtt cells with MDC, which is a marker for autophagic vacuoles (Biederbick et al., 1995) as well as LysoTracker to stain acidic compartments. mhtt cells showed increased staining of MDC-positive structures, which were colocalized with enlarged LysoTracker-positive structures (Figure 10A), suggesting an increase in autophagic vacuoles. To confirm these results we performed electron microscopy studies. We observed that mhtt cells presented autophagic vacuoles possessing part of the cytoplasm or granular deposits. We also found fingerprint profiles (Figure 10B) and structures surrounded by double-layered membranes (Figure 10, C–E). Together, these results indicate that mhtt cells have altered lysosome activity and increased autophagic vacuole-like structures.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). However, the mechanism by which mhtt impairs this regulated exocytic process is not known. Here, we show that mhtt impairs the optineurin/Rab8-dependent secretory pathway, which in turn alters clathrin-mediated post-Golgi exit. Thus, mhtt influences not only the regulated secretion to the plasma membranes (del Toro et al., 2006) but also the lysosomal route, which in turn leads to lysosomal dysfunction.
Our experiments showed that htt is located in the Golgi apparatus and also in transport vesicles, which is in agreement with previous studies (Difiglia et al., 1995; Gauthier et al., 2004). In addition, our results show that mhtt has a reduced localization in the Golgi apparatus with respect to htt. This observation is consistent with reduced interaction between mhtt and HIP14, a palmitoyl transferase that facilitates htt interaction with Golgi membranes (Huang and El-Husseini, 2005; Yanai et al., 2006). Interestingly, we observed that delocalization of mhtt was accompanied with reduced levels of optineurin in the Golgi apparatus. Consistently, expression of full-length htt restored the levels of optineurin in the Golgi of mhtt cells. The fact that htt interacts with optineurin (Hattula and Peranen, 2000) and also with the Golgi membranes, suggest that htt may play a central role in the correct localization and function of optineurin in the Golgi apparatus. This is in agreement with our observation that silencing htt by siRNA delocalized optineurin from the Golgi apparatus.
In addition to optineurin, mhtt also delocalized Rab8 from the Golgi apparatus. Rab8 belongs to the family of Rab GTPases and plays an important role in the post-Golgi transport (Ang et al., 2003). Because htt interacts directly with optineurin but not with Rab8 (Hattula and Peranen, 2000), one possible explanation for the delocalization of Rab8 from the Golgi apparatus in mhtt cells is the decrease of optineurin in this compartment. In keeping with this view, immunoprecipitation studies showed reduced interaction between mhtt and the optineurin/Rab8. However, the optineurin-Rab8 interaction was not affected by the presence of mhtt. Together, these results suggest that htt plays an important role in the localization of optineurin/Rab8 at the Golgi apparatus. Optineurin and Rab8 are essential for post-Golgi trafficking (Ang et al., 2003; Sahlender et al., 2005). In support, depletion of optineurin by RNAi or overexpression of Rab8Q67L result in altered post-Golgi trafficking of the VSV-G (Peranen et al., 1996; Sahlender et al., 2005). Similarly, mhtt affects the clathrin-dependent post-Golgi trafficking (del Toro et al., 2006), including the post-Golgi transport of VSV-G (present study). Therefore, the reduced interaction between mhtt and the optineurin/Rab8 complex together with the mislocalization of these proteins in mhtt cells, perturbs the functionality of the Golgi apparatus.
CCVs emanate from the TGN to deliver cargos to the cell surface or to lysosomes (for review, see Pfeffer, 2003). Electron microscopy images showed partial disruption of the Golgi apparatus that could be related to the reduction of optineurin and htt in this apparatus. In fact, depletion of optineurin or htt results in Golgi disorganization (Sahlender et al., 2005; Caviston et al., 2007). Interestingly, there is an abnormal increase in CCVs in the proximity of the TGN in mhtt cells. This finding suggests that CCVs do not leave the TGN correctly. Supporting this notion is the accumulation of AP-1 a clathrin adaptor complex, in the Golgi apparatus of mhtt cells, which was accompanied by reduced egress of clathrin from the TGN as showed the iFRAP analysis. Thus, it is plausible that alteration of the htt/optineurin/Rab8 complex by mhtt impairs the correct motion of CCVs from the TGN. This hypothesis is supported by the observation that optineurin interacts with myosin VI (Sahlender et al., 2005), which acts as an actin based motor for the transport of vesicles that exit from the TGN (Au et al., 2007). In addition, htt binds to dynein and HAP-1 facilitating the motility of vesicles along microtubules (Caviston et al., 2007). Thus, alteration of the htt/optineurin/Rab8 complex affects the correct motion of CCVs from the TGN because of a lack of interaction with their motors, which would explain the retention of secretory proteins in the TGN of mhtt cells (del Toro et al., 2006; Figure 11).
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). Interestingly, no additive effects were observed when Rab8 Q67L was expressed in mhtt cells. The lack of synergic effects indicates that the mhtt affects lysosomal transport via optineurin/Rab8 complex. MPR transports newly synthesized lysosomal hydrolases bearing mannose 6-phosphate from the TGN to late endosomes or prelysosomes (Dahms et al., 1989). Consistent with altered post-Golgi transport of MPR, mhtt cells showed an incorrect processing of procathepsin D, which in turn reduced the levels of mature cathepsin D in lysosomes. These findings are coincident with the reduction of lysosomal activity observed in mhtt cells. Interestingly, we observed that overexpression of truncated forms of mhtt, such as exon1–103Q-htt, stimulated post-Golgi transport of Lamp-1 and also increased the labeling of cathepsin D in enlarged LysoTracker-positive structures (Supplemental Figure 5), which is in agreement with a previous study (Kegel et al., 2000). This effect could be explained by several protein interactions that are lost in the case of shorter fragments of htt. In fact, shorter fragments of mhtt do not modify intracellular transport (Gauthier et al., 2004; Colin et al., 2008) or post-Golgi trafficking (del Toro et al., 2006).
It has been shown that macroautophagy, a large-scale catabolic mechanism, is required to clear mhtt aggregates (Rubinsztein et al., 2007). In fact, stimulation of autophagy by rapamycin is protective in HD models (Ravikumar et al., 2004) and has been proposed as a therapeutic strategy for this disease (Rubinsztein, 2006). Macroautophagy requires the fusion of autophagosomes with lysosomes to degrade the proteins into the autophagolysosome (Rubinsztein et al., 2007). Therefore, the results reported here could provide a link to explain the reduced lysosome activity with an impaired macroautophagy in HD. Consistently, mhtt cells showed an increase in autophagic vacuoles similar to those seen in cathepsin D-deficient mice, which exhibit dramatic autophagic stress (Koike et al., 2005).
In conclusion, our findings demonstrate that htt participates in the clathrin coated-mediated post-Golgi trafficking through the optineurin/Rab8 complex. mhtt delocalizes the optineurin/Rab8 complex from the Golgi apparatus, thereby impairing post-Golgi trafficking to the lysosomal pathway, which could contribute to defects in lysosome functionality in HD.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Dr. Josep M. Canals (jmcanals{at}ub.edu)
Abbreviations used: AP-1, clathrin adaptor complex 1; CCV, clathrin-coated vesicle; GT, galactosyltransferase; htt, huntingtin; Lamp, lysosome-associated membrane protein; LcB, clathrin light chain B; MPR, mannose-6-phosphate receptor; TGN, trans-Golgi network; VSV-G, vesicular stomatitis virus glycoprotein.
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