Mutant Huntingtin Is Secreted via a Late Endosomal/Lysosomal Unconventional Secretory Pathway

Abstract

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder caused by the expansion of a CAG triplet in the gene encoding for huntingtin (Htt). The resulting mutant protein (mHtt) with extended polyglutamine (polyQ) sequence at the N terminus leads to neuronal degeneration both in a cell-autonomous and a non-cell-autonomous manner. Recent studies identified mHtt in the extracellular environment and suggested that its spreading contributes to toxicity, but the mechanism of mHtt release from the cell of origin remains unknown. In this study, we performed a comprehensive, unbiased analysis of secretory pathways and identified an unconventional lysosomal pathway as an important mechanism for mHtt secretion in mouse neuroblastoma and striatal cell lines, as well as in primary neurons. mHtt secretion was dependent on synaptotagmin 7, a regulator of lysosomal secretion, and inhibited by chemical ablation of late endosomes/lysosomes, suggesting a lysosomal secretory pattern. mHtt was targeted preferentially to the late endosomes/lysosomes compared with wild-type Htt. Importantly, we found that late endosomal/lysosomal targeting and secretion of mHtt could be inhibited efficiently by the phosphatidylinositol 3-kinase and neutral sphingomyelinase chemical inhibitors, Ly294002 and GW4869, respectively. Together, our data suggest a lysosomal mechanism of mHtt secretion and offer potential strategies for pharmacological modulation of its neuronal secretion.SIGNIFICANCE STATEMENT This is the first study examining the mechanism of mutant huntingtin (mHTT) secretion in an unbiased manner. We found that the protein is secreted via a late endosomal/lysosomal unconventional secretory pathway. Moreover, mHtt secretion can be reduced significantly by phosphatidylinositol 3-kinase and neutral sphingomyelinase inhibitors. Understanding and manipulating the secretion of mHtt is important because of its potentially harmful propagation in the brain.

Keywords: Huntington's disease; late endosome; lysosome; mutant huntingtin; secretion.

Figure 1.

Preferential secretion of mHtt via unconventional secretory pathway. A, mHtt is secreted preferentially compared with wtHtt. Left, Neuro2A-mHtt cells were incubated for the final 16 h in OptiMEM of total 48 h after plating. Equal volumes of the concentrated media (SN) and cell lysates (CL) were analyzed by Western blotting using anti-Htt antibody. Tubulin was used as a loading control. Top right, Ratio of Htt in the media and cell lysates expressed as a percentage of mHtt. n = 3, p = 0.0021. Bottom right, LDH assay was performed on the media before concentrating. n = 3, p = 0.1601. B, mHtt is secreted preferentially from cells coexpressing mHtt and wtHtt. Left, Naive Neuro2A cells were cotransfected with 590aa Htt/myc/His/97Q (mHtt) and 25Q (wtHtt) at the ratios 1:1 (left lanes) and 2:3 (right lanes). Cells were incubated for the final 16 h of total 48 h after transfection in OptiMEM. Concentrated media (SN) and cell lysates (CL) were analyzed by Western blotting using anti-myc antibody. Right, Ratio of Htt in the media and cell lysates expressed as a percentage of mHtt. n = 3, p = 0.0114. C, Secreted Htt is not depleted from the media through endocytosis. Left, Media conditioned overnight on Neuro2A stably expressing mHtt or wtHtt was collected and further incubated in empty wells (/) or with naive Neuro2A cells (cells) for 4 h. Media was then concentrated and analyzed by Western blotting using anti-Htt antibody. Center, Htt in the media incubated with cells expressed as a percentage of Htt in the media incubated in empty wells. n = 3; p = 0.5230 and 0.5183. Right, Media conditioned on Neuro2A cells transiently expressing mHtt-GFP (donor cells) was transferred on naive Neuro2A (acceptor cells) and incubated for 4 h in the presence of 200 nm bafilomycin A1 to prevent degradation of putatively endocytosed Htt. Naive Neuro2A not exposed to Htt-conditioned media was used as a negative control (ctrl). Cells were then fixed, mounted, and analyzed by confocal microscopy. Nuclei were visualized using DAPI staining. n = 3. D, mHtt secretion is temperature dependent. Neuro2A-mHtt cells were incubated at 25°C or 37°C on the benchtop for 4 h in OptiMEM (left) and at 37°C with 10 μg/ml cycloheximide (CHX) or DMSO (ctrl) (center). Concentrated media and cell lysates were analyzed by Western blotting using anti-Htt antibody. Tubulin was used as a loading control. Right, Ratio of mHtt in the media and cell lysates expressed as a percentage of 37°C control. n = 3, p = 0.0009 and 0.957. E, Genetic and pharmacological disruption of the constitutive secretory pathway leads to collapse of the Golgi complex and is nontoxic. Left, Neuro2A-mHtt cells were transfected with HA-tagged Arf1wt or Arf1T31N (Arf1dn) and incubated for the last 16 of total 24 h after transfection in OptiMEM. Right, Cells were treated with DMSO (ctrl) or 5 μg/ml brefeldin A (BFA) in preconditioned OptiMEM for 1 h. Cells were then transduced with CellLight Golgi-GFP for the last 16 h of incubation and analyzed by confocal microscopy upon immunolabeling with anti-HA antibody (left) or without additional staining (right). Nuclei were visualized using DAPI staining. Scale bar, 10 μm. Bottom, LDH assay was performed on 30 μl of the media before concentrating. n = 3, p = 0.2189 and 0.9424. F, mHtt secretion does not follow a constitutive secretion pathway. Top, Cells were treated as in E. Concentrated media and the cell lysates were analyzed by Western blotting using anti-Htt antibody. Tubulin was used as a loading control. Bottom, Ratio of mHtt in the media and cell lysates expressed as a percentage of control. n = 3, p = 0.91 and 0.89. Error bars indicate SD. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., Not significant.

Figure 2.

Intracellular mHtt localizes to LE/Lys vesicles, whereas secreted mHtt exists in a free form. A, Extracellular mHtt is predominantly in a free form. Top, OptiMEM conditioned for 24 h on Neuro2A-mHtt was collected and sequentially centrifuged on 2000 × g to eliminate cell debris, 10,000 × g to pellet ectosomes, and 100,000 × g to pellet exosomes. The remaining supernatant was concentrated. The 10,000 × g pellet, the 100,000 × g pellet, and the concentrated supernatant were analyzed by Western blotting using anti-Htt antibody. Bottom, Relative amount of mHtt in each fraction expressed as a percentage of total mHtt. n = 3. B, Portion of intracellular mHtt is localized in the light membrane fraction. Left, Postnuclear supernatant (PNS) obtained from Neuro2A-mHtt cells was subjected to floatation in the sucrose density gradient and 2 μg of cytosolic (cyt), heavy membrane (HM), and light membrane (LM) fractions were analyzed by Western blotting using anti-Htt antibody. Lamp1 and tubulin were used as light membrane and cytosolic markers, respectively. Right, Relative amount of mHtt in each fraction expressed as a percentage of total mHtt. n = 3. C, mHtt-containing vesicles colocalize with Lamp1-positive compartments. Neuro2A-mHtt cells were transduced with CellLight Golgi-GFP (top left), treated 20 min with 20 μg/ml transferrin-Alexa Fluor 488 (top right), or left untreated (remaining panels). Cells were then pre-permeabilized, fixed, immunolabeled with anti-Htt (all), anti-EEA1 (second raw left), anti-Lamp2 (second row right), or anti-Lamp1 (third row) antibodies; and analyzed by confocal microscopy. D, Primary cortical neurons derived from rat embryos and transduced with Htt571/72Q lentivirus were pre-permeabilized and fixed 7 d after transduction, immunolabeled with anti-Htt and anti-Lamp1 antibodies, and analyzed by confocal microscopy. E, Vesicular mHtt is found within the lumen of Lamp1-positive vesicles. Untreated cells were pre-permeabilized, fixed, immunolabeled with anti-Htt and anti-Lamp1 antibodies, and analyzed by N-SIM. Nuclei were visualized using DAPI staining. Scale bar, 10 μm. Error bars indicate SD.

Figure 3.

LE/Lys secretion of mHtt. A, Disruption of lysosomal exocytosis leads to decreased secretion of mHtt. Left, Neuro2A-mHtt cells were transfected with scrambled shRNA or shRNA against synaptotagmin 7; naive Neuro2A cells with Flag-tagged full-length mHtt (FL Htt) and scrambled or synaptotagmin 7 shRNAs at 3:1 ratio; primary cortical neurons were transduced with Htt571/72Q-Flag and scrambled or synaptotagmin 7 shRNA lentiviruses at 5:3 ratio; and STHdh⁺/Hdh⁺ cells were transfected with GFP-tagged 571 aa mHtt and scrambled or synaptotagmin 7 shRNAs. Silencing was allowed to proceed for 48 h in total for transfected cells or 7 d for transduced neurons and secreted mHtt was collected for the last 16 h. Concentrated media and cell lysates were analyzed by Western blotting using anti-Htt, anti-Flag, or anti-GFP antibodies. Tubulin was used as a loading control. Knock down of syt7 was verified by probing with anti-syt 7 antibody (bottom). Right, Ratio of mHtt in the media and cell lysates expressed as a percentage of control. n = 3, p = 0.0283, 0.0035, 0.0001, and 0.0055. B, Calcium chelation leads to increased Htt punctae. Left, Neuro2A-mHtt cells were treated with DMSO or 0.2 μm BAPTA AM for 4 h. Cells were then pre-permeabilized, fixed, immunolabeled with anti-Htt Ab, and analyzed by confocal microscopy. Nuclei were visualized using DAPI staining. Right, Integrated density of mHtt signal per cell expressed as a percentage of control. Scale bar, 10 μm. n = 3, p = 0.021. C, Ablation of LE/Lys results in reduced secretion of mHtt. Top, Neuro2A-mHtt cells were pulsed with HRP (ablation/abl) or plain OptiMEM (ctrl). HRP was chased for 90, 30, or 0 min to label LE/Lys, intermediate compartments of the endocytic pathway, or early endosomes, respectively. Cells were then treated with DAB, incubated for 30 min in preconditioned OptiMEM and concentrated media, and cell lysates were analyzed by Western blotting using anti-Htt antibody. Tubulin was used as a loading control. Bottom left, Ratio of mHtt in the media and cell lysates expressed as a percentage of control. Control for each time point is assigned a 100% value. n = 4, 3, and 3; p = 0.0014, 0.4754, and 0.9708, respectively. Bottom right, GCase activity was measured in the media conditioned on the cells with and without ablated LE/Lys (90 min chase) and the values were normalized to the total amount of proteins in the cell lysate. n = 4; p = 0.0001. Error bars indicate SD. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., Not significant.

Figure 4.

mHtt is targeted preferentially to LE/Lys compared with wtHtt. A, Enrichment of mHtt over wtHtt in the light membrane fraction. Neuro2A-mHtt and Neuro2A-wtHtt cells were homogenized and subjected to floatation in sucrose density gradients. Then, 2 μg of light membrane (LM) (left) and cytosolic (cyt) proteins (center) were analyzed by Western blotting using anti-Htt antibody. Lamp1 and tubulin were used as loading controls for the light membranes and cytosols, respectively. Right, Ratio of light membrane and cytosolic Htt expressed as a percentage of mHtt. n = 3, p = 0.0222. B, Increased targeting of mHtt over wtHtt to Lamp1-positive vesicles. Left, Neuro2A-mHtt (top) or Neuro2A-wtHtt (bottom) cells were pre-permeabilized, fixed, immunolabeled with anti-Htt and anti-Lamp1 antibodies, and analyzed by confocal microscopy. DAPI was used to visualize nuclei. Scale bar, 10 μm. Top right, Htt integrated density per cell expressed as a percentage of mHtt. n = 4, p = 0.0001. Bottom right, Htt/Lamp1 colocalization. n = 3, p = 0.0036. Error bars indicate SD. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5.

Inhibition of NS reduces the secretion of mHtt without intracellular accumulation of mHtt and increased cytotoxicity. A, Reduced secretion of mHtt by inhibition of NS. Left, Neuro2A-mHtt cells, naive Neuro2A cells transfected with Flag-tagged full-length mHtt (FL Htt), primary cortical neurons 7 d after transduction with Htt571/72Q-Flag lentivirus, and mHtt hetero-(Q111/+) and homozygous (Q111/Q111) striatal cells were treated with DMSO (ctrl) or 5 μm GW4869 (GW) for 16 h. Concentrated media and cell lysates were analyzed by Western blotting using anti-Htt, anti-Flag, or anti-polyQ antibodies. Tubulin was used as a loading control. Bottom left, Ratio of mHtt in the media and cell lysates expressed as a percentage of control. n = 3, p = 0.0001, 0.0001, 0.0001, 0.0001, and 0.0048. Bottom right, LDH assay was performed on 30 μl of the media from mHtt-Neuro2A cells before concentrating. n = 3, p = 0.1021. B, Left, Neuro2A-mHtt cells were treated with DMSO (ctrl) or GW4869 (GW) for the indicated periods of time. Cells were lysed and 10 μg protein equivalents were analyzed by Western blotting. Tubulin was used as a loading control. Right, Ratios of Htt and tubulin expressed as a percentage of 0 h time point value. n = 4. p = 0.7424, 0.4773, 0.2886, 0.5062, and 0.7390. C, LDH cytotoxicity assay was performed on 50 μl of the media for each time point and the values were normalized to the total cellular protein amount. n = 4. p = 0.9564, 0.2814, 0.3196, 0.5858, and 0.5483. Error bars indicate SD. n.s., Not significant.

Figure 6.

Inhibition of PI3K reduces the secretion of mHtt. A, Reduced secretion of mHtt by inhibition of PI3K. Top, Neuro2A-mHtt cells were treated for 3 h with DMSO (ctrl), 10 mm 3-MA, or 25 μm Ly294002 (Ly); naive Neuro2A cells were transfected with FL Htt, primary cortical neurons were transduced with Htt571/72Q-Flag lentivirus and STHdh⁺/Hdh⁺ were transfected with Htt 571/72Q-GFP. Cells were then treated for 3 h with DMSO or Ly294002. Concentrated media and cell lysates were analyzed by Western blotting using anti-Htt, anti-Flag, or anti-GFP antibodies. Tubulin was used as a loading control. Bottom left, Ratio of mHtt in the media and cell lysates expressed as a percentage of control. n = 3, p = 0.0126, 0.0011, 0.0012, 0.0031, and 0.0003. Bottom right, The LDH assay was performed on 30 μl of the media from Neuro2A-mHtt cells before concentrating. n = 3, p = 0.4760 and 0.6717. B, 3-MA or Ly294002 treatment disrupts vesicular targeting of pEGFP-2xFYVE efficiently. Neuro2A-mHtt cells were transfected with pEGFP-2xFYVE. Twenty hours after transfection, the cells were treated for 3 h with DMSO (ctrl), 10 mm 3-MA, or 25 μm Ly294002 (Ly), fixed, and analyzed by confocal microscopy. Nuclei were visualized using DAPI staining. Scale bar, 10 μm. C, Knock down of Vps34 leads to reduced secretion of mHtt. Left, Cells were transfected with scrambled shRNA or shRNAs against Vps34 and incubated for 3–4 d. Media were replaced with OptiMEM 16 h before harvesting of the media and cell lysis. Concentrated media and the cell lysates were analyzed by Western blotting using anti-Htt antibody. Tubulin was used as a loading control. Knock down of Vps34 was verified by probing with anti-Vps34 antibody (bottom). Right, Ratio of mHtt in the media and cell lysates expressed as a percentage of control. n = 3, p = 0.0338, 0.0161, and 0.0170. Error bars indicate SD. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., Not significant.

Figure 7.

NS and PI3K inhibition impair vesicular targeting of mHtt. A, Punctate mHtt is reduced by inhibition of NS or PI3K. Neuro2A-mHtt cells were treated for 16 h with DMSO (ctrl) or 5 μm GW4869 (GW) (left) and for 3 h with DMSO or 25 μm Ly294002 (Ly) (right), followed by pre-permeabilization, fixation, immunolabeling with anti-Htt antibody, and analysis by confocal microscopy. DAPI was used to visualize nuclei. Scale bar, 10 μm. Right, Integrated density of mHtt signal per cell expressed as a percentage of control. n = 3, p = 0.0001 and 0.0067. B, mHtt in the light membrane fraction is reduced by inhibition of NS or PI3K. Left, Neuro2A-mHtt cells were treated for 16 h with DMSO (ctrl) or 5 μm GW4869 (GW) and for 3 h with DMSO (ctrl) or 25 μm Ly294002 (Ly). PNS were subjected to floatation in the sucrose density gradient and 2 μg of cytosolic (cyt) and light membrane (LM) protein equivalents were analyzed by Western blotting using anti-Htt antibody. Lamp1 and tubulin were used as LM and cyt markers, respectively. Center, Ratios of mHtt in the LM and cyt expressed as a percentage of control. n = 3, p = 0.0111 and 0.0001. Right, Ratios of mHtt and Lamp1 in the LM expressed as a percentage of control. n = 3, p = 0.0001 and 0.0001. Error bars indicate SD. *p < 0.05; **p < 0.01; ***p < 0.001.