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• W4283762010 startingPage "102219" @default.
• W4283762010 abstract "Recent studies demonstrated that the Golgi reassembly stacking proteins (GRASPs), especially GRASP55, regulate Golgi-independent unconventional secretion of certain cytosolic and transmembrane cargoes; however, the underlying mechanism remains unknown. Here, we surveyed several neurodegenerative disease–related proteins, including mutant huntingtin (Htt-Q74), superoxide dismutase 1 (SOD1), tau, and TAR DNA–binding protein 43 (TDP-43), for unconventional secretion; our results show that Htt-Q74 is most robustly secreted in a GRASP55-dependent manner. Using Htt-Q74 as a model system, we demonstrate that unconventional secretion of Htt is GRASP55 and autophagy dependent and is enhanced under stress conditions such as starvation and endoplasmic reticulum stress. Mechanistically, we show that GRASP55 facilitates Htt secretion by tethering autophagosomes to lysosomes to promote autophagosome maturation and subsequent lysosome secretion and by stabilizing p23/TMED10, a channel for translocation of cytoplasmic proteins into the lumen of the endoplasmic reticulum–Golgi intermediate compartment. Moreover, we found that GRASP55 levels are upregulated by various stresses to facilitate unconventional secretion, whereas inhibition of Htt-Q74 secretion by GRASP55 KO enhances Htt aggregation and toxicity. Finally, comprehensive secretomic analysis identified novel cytosolic cargoes secreted by the same unconventional pathway, including transgelin (TAGLN), multifunctional protein ADE2 (PAICS), and peroxiredoxin-1 (PRDX1). In conclusion, this study defines the pathway of GRASP55-mediated unconventional protein secretion and provides important insights into the progression of Huntington’s disease. Recent studies demonstrated that the Golgi reassembly stacking proteins (GRASPs), especially GRASP55, regulate Golgi-independent unconventional secretion of certain cytosolic and transmembrane cargoes; however, the underlying mechanism remains unknown. Here, we surveyed several neurodegenerative disease–related proteins, including mutant huntingtin (Htt-Q74), superoxide dismutase 1 (SOD1), tau, and TAR DNA–binding protein 43 (TDP-43), for unconventional secretion; our results show that Htt-Q74 is most robustly secreted in a GRASP55-dependent manner. Using Htt-Q74 as a model system, we demonstrate that unconventional secretion of Htt is GRASP55 and autophagy dependent and is enhanced under stress conditions such as starvation and endoplasmic reticulum stress. Mechanistically, we show that GRASP55 facilitates Htt secretion by tethering autophagosomes to lysosomes to promote autophagosome maturation and subsequent lysosome secretion and by stabilizing p23/TMED10, a channel for translocation of cytoplasmic proteins into the lumen of the endoplasmic reticulum–Golgi intermediate compartment. Moreover, we found that GRASP55 levels are upregulated by various stresses to facilitate unconventional secretion, whereas inhibition of Htt-Q74 secretion by GRASP55 KO enhances Htt aggregation and toxicity. Finally, comprehensive secretomic analysis identified novel cytosolic cargoes secreted by the same unconventional pathway, including transgelin (TAGLN), multifunctional protein ADE2 (PAICS), and peroxiredoxin-1 (PRDX1). In conclusion, this study defines the pathway of GRASP55-mediated unconventional protein secretion and provides important insights into the progression of Huntington’s disease. Neurodegenerative diseases are characterized by increased neuronal cell death and decreased cognitive abilities of patients. Different diseases, such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease (HD), and amyotrophic lateral sclerosis, are caused by different protein-based aberrations that affect different pathways; yet all cause neuronal cell death by forming inclusion bodies (1Lee H.J. Patel S. Lee S.J. Intravesicular localization and exocytosis of alpha-synuclein and its aggregates.J. Neurosci. 2005; 25: 6016-6024Crossref PubMed Scopus (618) Google Scholar, 2Umeda T. Maekawa S. Kimura T. Takashima A. Tomiyama T. Mori H. Neurofibrillary tangle formation by introducing wild-type human tau into APP transgenic mice.Acta Neuropathol. 2014; 127: 685-698Crossref PubMed Scopus (43) Google Scholar, 3Arrasate M. Finkbeiner S. Protein aggregates in Huntington's disease.Exp. Neurol. 2012; 238: 1-11Crossref PubMed Scopus (237) Google Scholar). These include protein aggregates formed by hyperphosphorylated tau in Alzheimer’s disease (2Umeda T. Maekawa S. Kimura T. Takashima A. Tomiyama T. Mori H. Neurofibrillary tangle formation by introducing wild-type human tau into APP transgenic mice.Acta Neuropathol. 2014; 127: 685-698Crossref PubMed Scopus (43) Google Scholar), mutant superoxide dismutase 1 (SOD1) (4Dangoumau A. Verschueren A. Hammouche E. Papon M.A. Blasco H. Cherpi-Antar C. et al.Novel SOD1 mutation p.V31A identified with a slowly progressive form of amyotrophic lateral sclerosis.Neurobiol. Aging. 2014; 35: 266.e1-e4Crossref Scopus (13) Google Scholar), and TAR DNA–binding protein 43 (TDP-43) in amyotrophic lateral sclerosis (5Johnson B.S. Snead D. Lee J.J. McCaffery J.M. Shorter J. Gitler A.D. TDP-43 is intrinsically aggregation-prone, and amyotrophic lateral sclerosis-linked mutations accelerate aggregation and increase toxicity.J. Biol. Chem. 2009; 284: 20329-20339Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar), and mutant huntingtin (Htt) in HD (6Bates G.P. Dorsey R. Gusella J.F. Hayden M.R. Kay C. Leavitt B.R. et al.Huntington disease.Nat. Rev. Dis. Primers. 2015; 115005Crossref PubMed Scopus (747) Google Scholar, 7Saudou F. Humbert S. The Biology of huntingtin.Neuron. 2016; 89: 910-926Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar). Many of these neurotoxic proteins have been detected in the cerebrospinal fluid (CSF) of patients with diseases, indicating that these proteins could be secreted from cells (8Wild E.J. Boggio R. Langbehn D. Robertson N. Haider S. Miller J.R. et al.Quantification of mutant huntingtin protein in cerebrospinal fluid from Huntington's disease patients.J. Clin. Invest. 2015; 125: 1979-1986Crossref PubMed Scopus (144) Google Scholar, 9Mecocci P. Cherubini A. Bregnocchi M. Chionne F. Cecchetti R. Lowenthal D.T. et al.Tau protein in cerebrospinal fluid: a new diagnostic and prognostic marker in Alzheimer disease?.Alzheimer Dis. Assoc. Disord. 1998; 12: 211-214Crossref PubMed Google Scholar, 10Winer L. Srinivasan D. Chun S. Lacomis D. Jaffa M. Fagan A. et al.SOD1 in cerebral spinal fluid as a pharmacodynamic marker for antisense oligonucleotide therapy.JAMA Neurol. 2013; 70: 201-207Crossref PubMed Scopus (79) Google Scholar). How the secretion affects protein aggregation and toxicity in neurons, and whether secretion facilitates the spreading of toxic proteins to neighboring cells, remain elusive. In the conventional secretory pathway, transmembrane and secretory proteins, such as the amyloid precursor protein, are synthesized in the endoplasmic reticulum (ER) and transported to the Golgi, from where they are targeted to their final destinations. However, many neurotoxic proteins, such as Htt, SOD1, α-synuclein, tau, and TDP-43, are synthesized as cytosolic proteins. Secretion of these proteins is independent of the traditional secretory pathway in which the Golgi functions as a trafficking center. For example, mutant Htt exists in two forms, an intracellular form in patient neurons and an extracellular form in CSF (8Wild E.J. Boggio R. Langbehn D. Robertson N. Haider S. Miller J.R. et al.Quantification of mutant huntingtin protein in cerebrospinal fluid from Huntington's disease patients.J. Clin. Invest. 2015; 125: 1979-1986Crossref PubMed Scopus (144) Google Scholar) exported through a not well-characterized unconventional secretory pathway (11Trajkovic K. Jeong H. Krainc D. Mutant huntingtin is secreted via a late endosomal/lysosomal unconventional secretory pathway.J. Neurosci. 2017; 37: 9000-9012Crossref PubMed Scopus (39) Google Scholar, 12Caron N.S. Banos R. Yanick C. Aly A.E. Byrne L.M. Smith E.D. et al.Mutant huntingtin is cleared from the brain via active mechanisms in huntington disease.J. Neurosci. 2021; 41: 780-796Crossref PubMed Scopus (16) Google Scholar). Little is known about the machinery that controls unconventional secretion, and the interplay between Golgi-dependent conventional trafficking and Golgi-independent unconventional secretion has not been explored. Although the unconventional pathway is independent of the Golgi, unconventional secretion of numerous cytosolic proteins reported so far requires the Golgi reassembly stacking protein of 55 kDa, GRASP55 (GORASP2) (13Lee J.G. Takahama S. Zhang G. Tomarev S.I. Ye Y. Unconventional secretion of misfolded proteins promotes adaptation to proteasome dysfunction in mammalian cells.Nat. Cell Biol. 2016; 18: 765-776Crossref PubMed Google Scholar, 14Rabouille C. Linstedt A.D. Grasp: a multitasking tether.Front. Cell Dev. Biol. 2016; 4: 1Crossref PubMed Google Scholar, 15Villeneuve J. Bassaganyas L. Lepreux S. Chiritoiu M. Costet P. Ripoche J. et al.Unconventional secretion of FABP4 by endosomes and secretory lysosomes.J. Cell Biol. 2018; 217: 649-665Crossref PubMed Scopus (47) Google Scholar). GRASP55, and its homolog GRASP65 (GORASP1), was first characterized as key Golgi stacking proteins (16Barr F.A. Puype M. Vandekerckhove J. Warren G. GRASP65, a protein involved in the stacking of Golgi cisternae.Cell. 1997; 91: 253-262Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar, 17Wang Y. Seemann J. Pypaert M. Shorter J. Warren G. A direct role for GRASP65 as a mitotically regulated Golgi stacking factor.EMBO J. 2003; 22: 3279-3290Crossref PubMed Scopus (153) Google Scholar). They both form transoligomers to link the Golgi cisternae into the unique stacked architecture (18Xiang Y. Wang Y. GRASP55 and GRASP65 play complementary and essential roles in Golgi cisternal stacking.J. Cell Biol. 2010; 188: 237-251Crossref PubMed Scopus (136) Google Scholar). Knockdown of GRASP55 and GRASP65 by RNAi, or KO of both GRASPs by CRISPR–cas9, results in Golgi fragmentation (18Xiang Y. Wang Y. GRASP55 and GRASP65 play complementary and essential roles in Golgi cisternal stacking.J. Cell Biol. 2010; 188: 237-251Crossref PubMed Scopus (136) Google Scholar, 19Bekier 2nd, M.E. Wang L. Li J. Huang H. Tang D. Zhang X. et al.Knockout of the Golgi stacking proteins GRASP55 and GRASP65 impairs Golgi structure and function.Mol. Biol. Cell. 2017; 28: 2833-2842Crossref PubMed Google Scholar). GRASPs were first linked to unconventional secretion of acyl-CoA binding protein (AcbA/Acb1) in Dictyostelium discoideum and Saccharomyces cerevisiae (20Duran J.M. Anjard C. Stefan C. Loomis W.F. Malhotra V. Unconventional secretion of Acb1 is mediated by autophagosomes.J. Cell Biol. 2010; 188: 527-536Crossref PubMed Scopus (322) Google Scholar, 21Kinseth M.A. Anjard C. Fuller D. Guizzunti G. Loomis W.F. Malhotra V. The Golgi-associated protein GRASP is required for unconventional protein secretion during development.Cell. 2007; 130: 524-534Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). In Drosophila, the GRASP protein is involved in unconventional trafficking of α-integrin at specific stages of fly development (22Schotman H. Karhinen L. Rabouille C. dGRASP-mediated noncanonical integrin secretion is required for Drosophila epithelial remodeling.Dev. Cell. 2008; 14: 171-182Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). In mammalian cells, GRASPs are required for unconventional trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) and the cytokine interleukin-1β (23Gee H.Y. Noh S.H. Tang B.L. Kim K.H. Lee M.G. Rescue of DeltaF508-CFTR trafficking via a GRASP-dependent unconventional secretion pathway.Cell. 2011; 146: 746-760Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 24Dupont N. Jiang S. Pilli M. Ornatowski W. Bhattacharya D. Deretic V. Autophagy-based unconventional secretory pathway for extracellular delivery of IL-1beta.EMBO J. 2011; 30: 4701-4711Crossref PubMed Scopus (656) Google Scholar). How GRASP55 regulates autophagosome-dependent unconventional secretion and whether GRASP55 is required for secretion of neurotoxic proteins such as Htt are unknown. In this study, we report that mutant Htt is unconventionally secreted in a GRASP55-dependent manner. Htt secretion is elevated under stress conditions, including energy deprivation and nutrient starvation, inhibition of proteasomal degradation, and induction of ER stress, all of which also upregulate GRASP55 expression. GRASP55 facilitates Htt secretion through two actions: one is to accelerate autophagic flux by facilitating autophagosome and lysosome fusion, the other is to stabilize p23/TMED10, a protein channel in the ER–Golgi intermediate compartment (ERGIC) that translocates cytosolic proteins into the ERGIC lumen for unconventional secretion. Finally, we performed systematic proteomic and secretomic analysis of WT and GRASP55 KO (55KO) cells and identified a list of cytoplasmic proteins whose secretion depends on GRASP55. To determine if GRASP55 is required for the secretion of cytosolic proteins related to neurodegeneration, we expressed a number of neurotoxic proteins, including Htt exon 1 fragment with a long polyglutamine (Q74) stretch (GFP-Htt-Q74), WT SOD1 (GFP-SOD1), TDP-43 (TDP-43-GFP), and tau (FLAG-tau), in WT and 55KO HeLa cells we established earlier (19Bekier 2nd, M.E. Wang L. Li J. Huang H. Tang D. Zhang X. et al.Knockout of the Golgi stacking proteins GRASP55 and GRASP65 impairs Golgi structure and function.Mol. Biol. Cell. 2017; 28: 2833-2842Crossref PubMed Google Scholar). For Htt, we chose the Htt exon 1 fragment because it has been shown that this fragment is harder to degrade, more toxic than full-length Htt, and detected in the CSF with an elevated concentration in HD (25Sathasivam K. Neueder A. Gipson T.A. Landles C. Benjamin A.C. Bondulich M.K. et al.Aberrant splicing of HTT generates the pathogenic exon 1 protein in Huntington disease.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 2366-2370Crossref PubMed Scopus (309) Google Scholar, 26Chen M. Wolynes P.G. Aggregation landscapes of Huntingtin exon 1 protein fragments and the critical repeat length for the onset of Huntington's disease.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: 4406-4411Crossref PubMed Scopus (44) Google Scholar, 27Fodale V. Boggio R. Daldin M. Cariulo C. Spiezia M.C. Byrne L.M. et al.Validation of ultrasensitive mutant huntingtin detection in human cerebrospinal fluid by single molecule counting immunoassay.J. Huntingtons Dis. 2017; 6: 349-361Crossref PubMed Scopus (32) Google Scholar, 28Gerson J.E. Safren N. Fischer S. Patel R. Crowley E.V. Welday J.P. et al.Ubiquilin-2 differentially regulates polyglutamine disease proteins.Hum. Mol. Genet. 2020; 29: 2596-2610Crossref PubMed Scopus (9) Google Scholar). At 48 h post-transfection, we cultured the cells in a serum-free medium for 4 h and determined the level of the corresponding cargo in the conditioned media by Western blot. A conventional secretory pathway cargo, clusterin, was used as a control. All these proteins were found in the conditioned media (Fig. S1, A–D), but Htt-Q74 secretion was the most robust and GRASP55 dependent (Fig. S1A). Therefore, we used GFP-Htt-Q74 as a main marker to study the mechanism of GRASP55-dependent unconventional secretion. Since both GRASP55 and GRASP65 have been shown to be involved in unconventional secretion (29Kim J. Noh S.H. Piao H. Kim D.H. Kim K. Cha J.S. et al.Monomerization and ER relocalization of GRASP is a requisite for unconventional secretion of CFTR.Traffic. 2016; 17: 733-753Crossref PubMed Google Scholar), we expressed GFP-Htt-Q74 in WT, 55KO, 65KO, or double KO (both GRASP55 and GRASP65) HeLa cells. At 48 h post-transfection, about 90 to 95% cells contained soluble Htt in the cytosol, as indicated by the diffuse GFP signal. Another 5 to 10% of cells contained Htt aggregates that appeared as bright dots in the cytoplasm (Fig. 1A). Interestingly, these Htt aggregates were often found in the perinuclear region where the Golgi was localized. This prompted us to look at the effect of Htt expression on Golgi morphology. While soluble Htt did not seem to affect Golgi morphology, Htt aggregates induced Golgi defects as previously reported (30Sbodio J.I. Snyder S.H. Paul B.D. Golgi stress response reprograms cysteine metabolism to confer cytoprotection in Huntington's disease.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: 780-785Crossref PubMed Scopus (52) Google Scholar, 31Brandstaetter H. Kruppa A.J. Buss F. Huntingtin is required for ER-to-Golgi transport and for secretory vesicle fusion at the plasma membrane.Dis. Model. Mech. 2014; 7: 1335-1340Crossref PubMed Scopus (33) Google Scholar), and the effect was more apparent in 55KO cells (Fig. 1A). For comparison, we also tested GFP-Htt-Q23 in parallel, which did not form aggregates or significantly affect the Golgi morphology (Fig. S1E). Similarly, expression of SOD1, TDP-43, or tau in WT cells had no significant impact on the Golgi structure (Fig. S1F). Next, we tested whether Htt secretion is GRASP dependent. After transfection of GFP-Htt-Q74 in WT, 55KO, 65KO, and double KO cells for 24 h, we cultured the cells in serum-free medium for 4 h and determined the level of Htt in the conditioned media by Western blot. Htt was readily detectable in the conditioned media, and the level was significantly reduced in 55KO but not 65KO cells (Fig. 1, B and C), indicating that Htt secretion depends on GRASP55 but not GRASP65. Secretion and processing of clusterin, a cargo molecule secreted by the conventional secretory pathway, were not affected by GRASP KO (Fig. 1B). In this assay, actin and two additional cytosolic proteins, hexokinase (HK1) and glucokinase (GCK), were not detected in the conditioned media (Figs. 1B and S1G). In addition, GFP-Htt-Q74 expression did not cause cell death as determined by the lactate dehydrogenase (LDH) cytotoxicity assay (Fig. S1H). These results indicate that Htt was secreted rather than nonselectively released from dead cells. Taken together, GFP-Htt-Q74 expression causes Golgi defects especially in 55KO cells, whereas 55KO reduces GFP-Htt-Q74 secretion. To confirm that Htt is secreted through an unconventional Golgi-independent pathway, we blocked ER-to-Golgi trafficking by brefeldin A (BFA) treatment. As shown in Figure 1, D and E, BFA treatment diminished clusterin in the conditioned media, indicating a block of conventional secretion. Htt secretion was unaffected by BFA treatment, confirming that it is secreted via a Golgi-independent unconventional secretory pathway. The toxicity of Htt is associated with the length of the polyQ tract within the exon 1 fragment; Htt with more than ∼35 glutamines in the stretch is regarded as a disease mutant (6Bates G.P. Dorsey R. Gusella J.F. Hayden M.R. Kay C. Leavitt B.R. et al.Huntington disease.Nat. Rev. Dis. Primers. 2015; 115005Crossref PubMed Scopus (747) Google Scholar, 7Saudou F. Humbert S. The Biology of huntingtin.Neuron. 2016; 89: 910-926Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar). To determine whether the length of the polyQ tract affects Htt secretion, we coexpressed GFP-Htt-Q74 and GFP-Htt-Q23 in the same cells and compared their secretion. As shown in Figure 1, F and G, when GFP-Htt-Q74 and GFP-Htt-Q23 were coexpressed, the level of GFP-Htt-Q74 in the conditioned media was higher than that of GFP-Htt-Q23. This indicates that the selectivity for GFP-Htt-Q74 secretion relies on the polyQ tract. Indeed, when Q80-GFP (in the absence of Htt exon 1) was expressed in HeLa cells, it was secreted at a comparable rate as GFP-Htt-Q74 (∼10%), and Q80-GFP secretion was also robustly reduced in 55KO cells (Fig. S2, A and B). Interestingly, Htt exhibited two bands, and only the lower band was detected in the conditioned media; we were curious about the difference between the two bands. Htt is highly modified by post-translational modifications, such as phosphorylation, acetylation, and cleavage (32Arbez N. Ratovitski T. Roby E. Chighladze E. Stewart J.C. Ren M. et al.Post-translational modifications clustering within proteolytic domains decrease mutant huntingtin toxicity.J. Biol. Chem. 2017; 292: 19238-19249Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Among these, phosphorylation at S13 and S16 by TANK-binding kinase 1 or IκB kinase has been shown to reduce Htt aggregation and toxicity (33Thompson L.M. Aiken C.T. Kaltenbach L.S. Agrawal N. Illes K. Khoshnan A. et al.IKK phosphorylates Huntingtin and targets it for degradation by the proteasome and lysosome.J. Cell Biol. 2009; 187: 1083-1099Crossref PubMed Scopus (288) Google Scholar, 34Hegde R.N. Chiki A. Petricca L. Martufi P. Arbez N. Mouchiroud L. et al.TBK1 phosphorylates mutant Huntingtin and suppresses its aggregation and toxicity in Huntington's disease models.EMBO J. 2020; 39e104671Crossref PubMed Scopus (16) Google Scholar). To determine whether the two bands represent different phosphorylated forms of Htt, we treated cells with a general serine/threonine kinase inhibitor, staurosporine (STS), or a general phosphatase inhibitor, okadaic acid (OA). STS treatment increased the intensity of the lower band, whereas OA treatment accumulated the upper band, suggesting that the upper band represents a phosphorylated form, whereas the lower band is dephosphorylated. This conclusion was confirmed using a phospho-specific antibody pS13 for Htt (34Hegde R.N. Chiki A. Petricca L. Martufi P. Arbez N. Mouchiroud L. et al.TBK1 phosphorylates mutant Huntingtin and suppresses its aggregation and toxicity in Huntington's disease models.EMBO J. 2020; 39e104671Crossref PubMed Scopus (16) Google Scholar), which recognized only the upper band (Fig. 1, H and I). More importantly, STS treatment increased Htt secretion, whereas OA treatment reduced its secretion (Fig. 1, J and K), indicating that the dephosphorylated aggregate-prone form of Htt is preferentially secreted compared with the phosphorylated soluble form of Htt. Taken together, these results demonstrate that unconventional secretion is selective, at least to a certain degree, and may serve as a clearing mechanism for cells to expel toxic proteins. It has been demonstrated that unconventional secretion is elevated under different stress conditions when the autophagy level is high (35Giuliani F. Grieve A. Rabouille C. Unconventional secretion: a stress on GRASP.Curr. Opin. Cell Biol. 2011; 23: 498-504Crossref PubMed Scopus (89) Google Scholar), and that unconventional secretion of CFTR and Acb1 depends on autophagy or autophagy genes (36Noh S.H. Gee H.Y. Kim Y. Piao H. Kim J. Kang C.M. et al.Specific autophagy and ESCRT components participate in the unconventional secretion of CFTR.Autophagy. 2018; 14: 1761-1778Crossref PubMed Scopus (35) Google Scholar, 37Bruns C. McCaffery J.M. Curwin A.J. Duran J.M. Malhotra V. Biogenesis of a novel compartment for autophagosome-mediated unconventional protein secretion.J. Cell Biol. 2011; 195: 979-992Crossref PubMed Scopus (134) Google Scholar). In addition, we discovered that GRASP55 functions as an energy sensor through O-GlcNAcylation to regulate autophagosome maturation. Under growth conditions, GRASP55 is O-GlcNAcylated and concentrated on the Golgi. Upon glucose starvation, GRASP55 is de-O-GlcNAcylated and relocated from the Golgi to the interface between autophagosomes and lysosomes, where it interacts with LC3 on autophagosomes and lysosomal membrane–associated protein 2 (LAMP2) on lysosomes and functions as a membrane tether to facilitate autophagosome–lysosome fusion (38Zhang X. Wang L. Lak B. Li J. Jokitalo E. Wang Y. GRASP55 senses glucose deprivation through O-GlcNAcylation to promote autophagosome-lysosome fusion.Dev. Cell. 2018; 45: 245-261.e6Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Similar responses of GRASP55 occur under amino acid starvation or when mammalian target of rapamycin activity is inhibited by Torin 1 (38Zhang X. Wang L. Lak B. Li J. Jokitalo E. Wang Y. GRASP55 senses glucose deprivation through O-GlcNAcylation to promote autophagosome-lysosome fusion.Dev. Cell. 2018; 45: 245-261.e6Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 39Zhang X. Wang L. Ireland S.C. Ahat E. Li J. Bekier 2nd, M.E. et al.GORASP2/GRASP55 collaborates with the PtdIns3K UVRAG complex to facilitate autophagosome-lysosome fusion.Autophagy. 2019; 15: 1787-1800Crossref PubMed Scopus (27) Google Scholar, 40Zhang X. Wang Y. GRASP55 facilitates autophagosome maturation under glucose deprivation.Mol. Cell Oncol. 2018; 5e1494948Google Scholar). In addition, GRASP55 also interacts with Beclin-1 to facilitate the assembly and membrane association of the UVRAG phosphatidylinositol 3-kinase (PtdIns3K) complex, and thereby facilitates autophagosome maturation (39Zhang X. Wang L. Ireland S.C. Ahat E. Li J. Bekier 2nd, M.E. et al.GORASP2/GRASP55 collaborates with the PtdIns3K UVRAG complex to facilitate autophagosome-lysosome fusion.Autophagy. 2019; 15: 1787-1800Crossref PubMed Scopus (27) Google Scholar). These observations prompted us to test the effect of energy deprivation and nutrient starvation on Htt secretion. As expected, both glucose starvation and amino acid deprivation (E, by incubating cells in Earle′s balanced salt solution [EBSS]) increased the level of Htt in the conditioned media compared with Dulbecco's modified Eagle's medium (DMEM) (D) (Fig. 2, A and B). A similar effect was observed in 55KO cells, although the overall secretion was significantly lower than WT cells. In addition to starvation, inhibiting proteasomal degradation with MG132, or inducing ER stress with tunicamycin (Tu), both enhanced Htt secretion (Fig. 2, C–F). Under these experimental conditions, no cell death was detected by the LDH cytotoxicity assay (Fig. S2C). These results confirm that Htt secretion is elevated under stress conditions. The fact that GRASP55 localization and function are regulated by cellular stressors suggests that GRASP55 may serve as both a stress sensor and an effector (41Zhang X. Wang Y. Nonredundant roles of GRASP55 and GRASP65 in the Golgi apparatus and beyond.Trends Biochem. Sci. 2020; 45: 1065-1079Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). In addition to its relocalization from the Golgi to autophagosomes under starvation conditions (38Zhang X. Wang L. Lak B. Li J. Jokitalo E. Wang Y. GRASP55 senses glucose deprivation through O-GlcNAcylation to promote autophagosome-lysosome fusion.Dev. Cell. 2018; 45: 245-261.e6Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), we wondered whether GRASP55 level might also be upregulated to meet the high demand for its roles in Golgi structure formation, autophagy, and unconventional secretion in response to stress. To test this hypothesis, we incubated WT HeLa cells in EBSS for 0, 2, 4, and 8 h and measured GRASP55 level by Western blot. Indeed, EBSS treatment increased GRASP55 expression but had no significant effect on other Golgi proteins we tested, including GRASP65, Golgin-160, Golgin-97, and GCC88 (Fig. 3, A and B). The elevation of GRASP55 expression was not limited to amino acid starvation, as it also applied to ER stress. When cells were treated with ER stress inducers, including Tu, thapsigargin, and DTT, the level of GRASP55 but not that of other Golgi proteins increased (Fig. 3, C and D). Since mutant Htt forms aggregates that could induce proteostasis stress, we tested the effect of Htt-Q23 and Htt-Q74 on GRASP55 expression. Both Htt-Q23 and Htt-Q74 expression increased GRASP55 level in cells, although Htt-Q74 exhibited a stronger effect (Fig. S2, D and E). To validate this observation in mice, we took advantage of a previously established knock-in mouse model that expresses full-length Htt-Q200 in the brain (28Gerson J.E. Safren N. Fischer S. Patel R. Crowley E.V. Welday J.P. et al.Ubiquilin-2 differentially regulates polyglutamine disease proteins.Hum. Mol. Genet. 2020; 29: 2596-2610Crossref PubMed Scopus (9) Google Scholar, 42Heng M.Y. Duong D.K. Albin R.L. Tallaksen-Greene S.J. Hunter J.M. Lesort M.J. et al.Early autophagic response in a novel knock-in model of Huntington disease.Hum. Mol. Genet. 2010; 19: 3702-3720Crossref PubMed Scopus (99) Google Scholar). These studies have shown that expression of Htt-Q200 upregulated both LC3-II and p62 levels compared with control, indicating an enhanced autophagosome formation but reduced lysosomal degradation. This conclusion was validated in our Htt mice and cell samples, as indicated by p62 accumulation (Figs. 3, E and F, S2, D and E) (42Heng M.Y. Duong D.K. Albin R.L. Tallaksen-Greene S.J. Hunter J.M. Lesort M.J. et al.Early autophagic response in a novel knock-in model of Huntington disease.Hum. Mol. Genet. 2010; 19: 3702-3720Crossref PubMed Scopus (99) Google Scholar). In response to the proteostasis stress, GRASP55 level was also elevated in the brain tissues, whereas other proteins such as GRASP65 and GM130 did not change (Fig. 3, E and F). Taken together, GRASP55 level is upregulated by cellular stress and Htt expression, possibly to meet the high demand of unconventional secretion. The fact that GRASP55 expression, autophagy, and unconventional secretion are all elevated under stress conditions implies that these events are related. To test if Htt secretion depends on autophagy, we expressed GFP-Htt-Q74 in the ATG7 KO mouse embryonic fibroblast (MEF) cell line established by Dr Masaaki Komatsu (43Komatsu M. Waguri S. Ueno T. Iwata J. Murata S. Tanida I. et al.Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice.J. Cell Biol. 2005; 169: 425-434Crossref PubMed Scopus (1900) Google Scholar) in which autoph" @default.
• W4283762010 created "2022-07-02" @default.
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• W4283762010 date "2022-08-01" @default.
• W4283762010 modified "2023-10-14" @default.
• W4283762010 title "GRASP55 regulates the unconventional secretion and aggregation of mutant huntingtin" @default.
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