Brigit E. Riley

CNS neurons are endowed with the ability to recover from cytotoxic insults associated with the accumulation of proteinaceous polyglutamine aggregates via a process that appears to involve capture and degradation of aggregates by autophagy. The ubiquitin-proteasome system protects cells against proteotoxicity by degrading soluble monomeric misfolded aggregation-prone proteins but is ineffective against, and impaired by, non-native protein oligomers. Here we show that autophagy is induced in response to impaired ubiquitin proteasome system activity. We show that ATG proteins, molecular determinants of autophagic vacuole formation, and lysosomes are recruited to pericentriolar cytoplasmic inclusion bodies by a process requiring an intact microtubule cytoskeleton and the cytoplasmic deacetylase HDAC6. These data suggest that HDAC6-dependent retrograde transport on microtubules is used by cells to increase the efficiency and selectivity of autophagic degradation. CNS neurons are endowed with the ability to recover from cytotoxic insults associated with the accumulation of proteinaceous polyglutamine aggregates via a process that appears to involve capture and degradation of aggregates by autophagy. The ubiquitin-proteasome system protects cells against proteotoxicity by degrading soluble monomeric misfolded aggregation-prone proteins but is ineffective against, and impaired by, non-native protein oligomers. Here we show that autophagy is induced in response to impaired ubiquitin proteasome system activity. We show that ATG proteins, molecular determinants of autophagic vacuole formation, and lysosomes are recruited to pericentriolar cytoplasmic inclusion bodies by a process requiring an intact microtubule cytoskeleton and the cytoplasmic deacetylase HDAC6. These data suggest that HDAC6-dependent retrograde transport on microtubules is used by cells to increase the efficiency and selectivity of autophagic degradation. Accumulation of protein aggregates within intracellular inclusion bodies (IB) 3The abbreviations used are: IBintracellular inclusion bodyHDHuntington diseasemTORmammalian target of rapamycinRNAiRNA interferenceCFTRcystic fibrosis transmembrane receptorGFPgreen fluorescent proteinCFPcyan fluorescent proteinALLNN-acetyl-Leu-Leu-norleucinalshRNAshort hairpin RNA. is a pathological hallmark of most neurodegenerative diseases. In Huntington disease (HD) and related dominantly inherited, late-onset neurodegenerative diseases, pathology is the direct consequence of expansion of CAG triplet repeats that encode homopolymeric tracts of glutamine (polyQ) within the mutant gene product (1Zoghbi H.Y. Orr H.T. Annu. Rev. Neurosci. 2000; 23: 217-247Crossref PubMed Scopus (1103) Google Scholar). In HD, these polyQ tracts typically consist of greater than 39 glutamines in the huntingtin protein (Htt), while Htt with fewer than ∼30 glutamines is not associated with disease (2Huntington's Disease Collaborative Research Group Cell. 1993; 72: 971-983Abstract Full Text PDF PubMed Scopus (7080) Google Scholar). This CAG length dependence of disease onset and severity in HD and in related CAG expansion disorders (1Zoghbi H.Y. Orr H.T. Annu. Rev. Neurosci. 2000; 23: 217-247Crossref PubMed Scopus (1103) Google Scholar) correlates strongly with the propensity of expanded polyQ proteins to aggregate in vitro, in cell culture (3Scherzinger E. Lurz R. Turmaine M. Mangiarini L. Hollenbach B. Hasenbank R. Bates G.P. Davies S.W. Lehrach H. Wanker E.E. Cell. 1997; 90: 549-558Abstract Full Text Full Text PDF PubMed Scopus (1084) Google Scholar). or in mouse (4Menalled L.B. Chesselet M.F. Trends Pharmacol. Sci. 2002; 23: 32-39Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). or Drosophila (5Jackson G.R. Salecker I. Dong X. Yao X. Arnheim N. Faber P.W. MacDonald M.E. Zipursky S.L. Neuron. 1998; 21: 633-642Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar) models of HD. There is compelling data in support of the hypothesis that CAG expansion and other neurodegenerative diseases are disorders of protein conformation, in which normally well behaved proteins adopt non-native toxic oligomeric conformations (6Taylor J.P. Hardy J. Fischbeck K.H. Science. 2002; 296: 1991-1995Crossref PubMed Scopus (1016) Google Scholar, 7Horwich A.L. Weissman J.S. Cell. 1997; 89: 499-510Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 8Kopito R.R. Ron D. Nat. Cell Biol. 2000; 2: E207-E209Crossref PubMed Scopus (321) Google Scholar). Aggregated forms of polyQ-expanded Htt can disrupt cellular function in a variety of ways including specific co-aggregation with and inactivation of nuclear transcription factors (9Nucifora Jr., F.C. Sasaki M. Peters M.F. Huang H. Cooper J.K. Yamada M. Takahashi H. Tsuji S. Troncoso J. Dawson V.L. Dawson T.M. Ross C.A. Science. 2001; 291: 2423-2428Crossref PubMed Scopus (944) Google Scholar, 10Steffan J.S. Kazantsev A. Spasic-Boskovic O. Greenwald M. Zhu Y.Z. Gohler H. Wanker E.E. Bates G.P. Housman D.E. Thompson L.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6763-6768Crossref PubMed Scopus (878) Google Scholar), interference with axonal transport (11Gunawardena S. Her L.S. Brusch R.G. Laymon R.A. Niesman I.R. GordeskyGold B. Sintasath L. Bonini N.M. Goldstein L.S. Neuron. 2003; 40: 25-40Abstract Full Text Full Text PDF PubMed Scopus (530) Google Scholar), damage to mitochondrial or cellular membranes (12Hashimoto M. Rockenstein E. Crews L. Masliah E. Neuromolecular Med. 2003; 4: 21-36Crossref PubMed Scopus (358) Google Scholar), and impairment of the ubiquitin-proteasome system (13Bence N. Sampat R. Kopito R.R. Science. 2001; 292: 1552-1555Crossref PubMed Scopus (1821) Google Scholar, 14Jana N.R. Zemskov E.A. Wang G. Nukina N. Hum. Mol. Genet. 2001; 10: 1049-1059Crossref PubMed Scopus (384) Google Scholar). Recent studies have suggested that cellular toxicity (15Caughey B. Lansbury P.T. Annu. Rev. Neurosci. 2003; 26: 267-298Crossref PubMed Scopus (1450) Google Scholar, 16Ross C.A. Poirier M.A. Nat. Med. 2004; 10: S10-S17Crossref PubMed Scopus (2475) Google Scholar) and ubiquitin-proteasome system impairment (17Bennett E.J. Bence N.F. Jayakumar R. Kopito R.R. Mol. Cell. 2005; 17: 351-365Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar) is associated with non-native soluble oligomeric or protofibrillar forms of aggregation-prone proteins such as Htt, whereas large, microscopically detectable, IB may be cytoprotective (18Arrasate M. Mitra S. Schweitzer E.S. Segal M.R. Finkbeiner S. Nature. 2004; 431: 805-810Crossref PubMed Scopus (1614) Google Scholar). intracellular inclusion body Huntington disease mammalian target of rapamycin RNA interference cystic fibrosis transmembrane receptor green fluorescent protein cyan fluorescent protein N-acetyl-Leu-Leu-norleucinal short hairpin RNA. Because polyglutamine aggregates are largely insoluble, refractory to chemical denaturation (19Hazeki N. Tukamoto T. Goto J. Kanazawa I. Biochem. Biophys. Res. Commun. 2000; 277: 386-393Crossref PubMed Scopus (80) Google Scholar), and because they accumulate in inclusion bodies (IB) (20Davies S.W. Turmaine M. Cozens B.A. DiFiglia M. Sharp A.H. Ross C.A. Scherzinger E. Wanker E.E. Mangiarini L. Bates G.P. Cell. 1997; 90: 537-548Abstract Full Text Full Text PDF PubMed Scopus (1912) Google Scholar), it is widely assumed that cells have little or no capacity to eliminate them. However, several recent studies have established that neurons in conditional transgenic mouse models of polyQ disease have the capacity to recover from the toxicity of transient expression of aggregation prone proteins containing expanded polyglutamine tracts (21Yamamoto A. Lucas J.J. Hen R. Cell. 2000; 101: 57-66Abstract Full Text Full Text PDF PubMed Scopus (911) Google Scholar, 22Zu T. Duvick L.A. Kaytor M.D. Berlinger M.S. Zoghbi H.Y. Clark H.B. Orr H.T. J. Neurosci. 2004; 24: 8853-8861Crossref PubMed Scopus (228) Google Scholar). Thus, cellular mechanisms must exist to suppress the toxicity associated with intracellular accumulation of conformationally defective mutant proteins like Htt and ataxin-1. Elucidating the molecular processes underlying these cytoprotective mechanisms is clearly important to the development of therapeutic approaches to the treatment of Huntington and other neurodegenerative diseases. A growing body of evidence supports the hypothesis that autophagy is a primary mechanism through which mammalian cells can capture and degrade protein aggregates. In classic autophagy, cytoplasmic contents, including ribosomes, soluble proteins and organelles, are captured into bilamellar autophagosomes, which, upon fusion with lysosomes, mature into degradative autolysosomes (23Mortimore G.E. Miotto G. Venerando R. Kadowaki M. Subcell Biochem. 1996; 27: 93-135Crossref PubMed Scopus (86) Google Scholar). In yeast these events require the function of ATG genes, which encode proteins called Atgs (24Klionsky D.J. Cregg J.M. Dunn Jr., W.A. Emr S.D. Sakai Y. Sandoval I.V. Sibirny A. Subramani S. Thumm M. Veenhuis M. Ohsumi Y. Dev Cell. 2003; 5: 539-545Abstract Full Text Full Text PDF PubMed Scopus (1020) Google Scholar, 25Klionsky D.J. Ohsumi Y. Annu. Rev. Cell Dev. Biol. 1999; 15: 1-32Crossref PubMed Scopus (391) Google Scholar). A functional role for autophagy in clearance of aggregated Htt is suggested by the observation that 3-methyladenine, an inhibitor of type III phosphoinositide 3-kinases required for many vesicular trafficking events, including autophagosome formation (26Petiot A. Ogier-Denis E. Blommaart E.F. Meijer A.J. Codogno P. J. Biol. Chem. 2000; 275: 992-998Abstract Full Text Full Text PDF PubMed Scopus (1035) Google Scholar), increases levels of polyQ-expanded Htt inclusions and decreases the clearance of aggregates from mammalian cells expressing these constructs (27Ravikumar B. Stewart A. Kita H. Kato K. Duden R. Rubinsztein D.C. Hum. Mol. Genet. 2003; 12: 985-994Crossref PubMed Scopus (108) Google Scholar, 28Qin Z.H. Wang Y. Kegel K.B. Kazantsev A. Apostol B.L. Thompson L.M. Yoder J. Aronin N. DiFiglia M. Hum. Mol. Genet. 2003; 12: 3231-3244Crossref PubMed Scopus (234) Google Scholar). Pharmacological activation of mTOR, a protein kinase that regulates a myriad of cellular responses to changes in nutrient and hormonal status, including autophagy (29Hay N. Sonenberg N. Genes Dev. 2004; 18: 1926-1945Crossref PubMed Scopus (3461) Google Scholar), delays the neurotoxicity associated with mutant Htt in cellular (30Ravikumar B. Duden R. Rubinsztein D.C. Hum. Mol. Genet. 2002; 11: 1107-1117Crossref PubMed Scopus (938) Google Scholar) and animal (31Ravikumar B. Vacher C. Berger Z. Davies J.E. Luo S. Oroz L.G. Scaravilli F. Easton D.F. Duden R. O'Kane C.J. Rubinsztein D.C. Nat. Genet. 2004; 36: 585-595Crossref PubMed Scopus (1990) Google Scholar) models of HD. Ultrastructural evidence suggesting a proliferation of lysosomes and autophagic bodies in striatal neurons expressing mutant Htt (32Kegel K.B. Kim M. Sapp E. McIntyre C. Castano J.G. Aronin N. DiFiglia M. J. Neurosci. 2000; 20: 7268-7278Crossref PubMed Google Scholar) and in Huntington (33Sapp E. Schwarz C. Chase K. Bhide P.G. Young A.B. Penney J. Vonsattel J.P. Aronin N. DiFiglia M. Ann. Neurol. 1997; 42: 604-612Crossref PubMed Scopus (298) Google Scholar) and Alzheimer disease brains (34Nixon R.A. Cataldo A.M. Mathews P.M. Neurochem. Res. 2000; 25: 1161-1172Crossref PubMed Scopus (280) Google Scholar), further supports a linkage between degeneration and autophagy (35Larsen K.E. Sulzer D. Histol. Histopathol. 2002; 17: 897-908PubMed Google Scholar), although these morphological studies cannot distinguish between a role for autophagy in cytoprotection or cell death. Recently, we reported that RNAi-mediated Atg knockdown leads to increased steady-state levels of Htt and completely prevents clearance of cytoplasmic polyglutamine aggregates from cell models of HD (36Iwata A. Christianson J. Bucci M. Ellerby L. Nukina N. Forno L. Kopito R. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13135-13140Crossref PubMed Scopus (273) Google Scholar). We also observed that cytoplasmic IB are consistently labeled with antibodies to endogenous Atgs, irrespective of the particular aggregating species, suggesting that recruitment of autophagocytic machinery to sites of IB formation may be a general feature of the cellular defense against protein aggregation (36Iwata A. Christianson J. Bucci M. Ellerby L. Nukina N. Forno L. Kopito R. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13135-13140Crossref PubMed Scopus (273) Google Scholar). Aggregated proteins in the mammalian cytoplasm are sequestered into pericentriolar IB called “aggresomes” (37Johnston J.A. Ward C.L. Kopito R.R. J. Cell Biol. 1998; 143: 1883-1898Crossref PubMed Scopus (1778) Google Scholar). Aggresomes form when the ubiquitin proteasome system is overwhelmed with aggregationprone protein by a process in which small protein aggregates are actively transported on microtubules by a process requiring dynein/dynactin motors (38Kopito R.R. Trends Cell Biol. 2000; 10: 524-530Abstract Full Text Full Text PDF PubMed Scopus (1603) Google Scholar) and the tubulin deacetylase HDAC6 (39Kawaguchi Y. Kovacs J.J. McLaurin A. Vance J.M. Ito A. Yao T.P. Cell. 2003; 115: 727-738Abstract Full Text Full Text PDF PubMed Scopus (1202) Google Scholar). The recent demonstration that neurons expressing polyglutamine-expanded Htt survive better if they can form aggresomes (18Arrasate M. Mitra S. Schweitzer E.S. Segal M.R. Finkbeiner S. Nature. 2004; 431: 805-810Crossref PubMed Scopus (1614) Google Scholar), together with the finding that polyglutamine toxicity is enhanced by microtubule disrupting agents that prevent aggresome formation (40Muchowski P.J. Ning K. D'Souza-Schorey C. Fields S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 727-732Crossref PubMed Scopus (117) Google Scholar, 41Taylor J.P. Tanaka F. Robitschek J. Sandoval C.M. Taye A. Markovic-Plese S. Fischbeck K.H. Hum. Mol. Genet. 2003; 12: 749-757Crossref PubMed Scopus (364) Google Scholar), strongly supports the hypothesis that aggresome formation is a cytoprotective mechanism. One way that aggresome formation could be cytoprotective would be to facilitate the delivery of dispersed protein aggregates to the autophagy pathway (38Kopito R.R. Trends Cell Biol. 2000; 10: 524-530Abstract Full Text Full Text PDF PubMed Scopus (1603) Google Scholar). Indeed evidence for such a mechanism is suggested by the demonstration that pharmacological inhibition of autophagy slows the clearance of aggresomes composed of mutant peripheral myelin protein PMP22, a Schwann cell protein associated with a host of demyelinating neuropathies (42Fortun J. Dunn Jr., W.A. Joy S. Li J. Notterpek L. J. Neurosci. 2003; 23: 10672-10680Crossref PubMed Google Scholar). Autophagy has been primarily considered to be a bulk, non-selective pathway by which cells scavenge cytoplasmic proteins and organelles in response to nutrient deprivation (43Kamada Y. Sekito T. Ohsumi Y. Curr. Top. Microbiol. Immunol. 2004; 279: 73-84Crossref PubMed Google Scholar). However, efficient degradation of protein aggregates by autophagy requires that they be concentrated in nascent autophagic structures. Previous investigators have reported that microtubule disruption interferes with autophagy by impairing autolysosome formation (44Aplin A. Jasionowski T. Tuttle D.L. Lenk S.E. Dunn Jr., W.A. J. Cell. Physiol. 1992; 152: 458-466Crossref PubMed Scopus (152) Google Scholar, 45Webb J.L. Ravikumar B. Rubinsztein D.C. Int. J. Biochem. Cell Biol. 2004; 36: 2541-2550Crossref PubMed Scopus (79) Google Scholar). Here we show that autophagosome formation is strongly activated by acute proteasome impairment. Because proteasome impairment inevitably accompanies the production and/or accumulation of aggregated proteins (46Liu C.W. Giasson B.I. Lewis K.A. Lee V.M. Demartino G.N. Thomas P.J. J. Biol. Chem. 2005; 280: 22670-22678Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 47Park Y. Hong S. Kim S.J. Kang S. Mol. Cells. 2005; 19: 23-30PubMed Google Scholar), this finding suggests a mechanism by which protein aggregation could induce an autophagic response. We find that recruitment of Atgs, aggregates, and lysosomes to aggresomes requires both an intact microtubule cytoskeleton and the tubulin deacetylase, HDAC6. These data suggest that minus-end-directed transport on microtubules is a mechanism used by cells to enhance the efficiency and selectivity of autophagic degradation of aggregated proteins. Thus, the aggresome and autophagy pathways operate cooperatively to eliminate aggregation-prone proteins that escape surveillance by the ubiquitin proteasome system. Cell Lines Cell Culture and Transfection—Human embryonic kidney HEK-293 cells, HeLa cells, and neuro2a (N2Aa) cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics at 37 °C in 95 and 5% CO2. Plasmid transfection was done using Lipofectamine 2000 (Invitrogen) following the manufacturer's protocol. Most of the analyses were done after 48 h of transfection unless otherwise noted. For immunoblotting, cells were lysed in 50 mm Tris, pH 7.5, 100 mm NaCl, 0.5% Triton X-100, 2.5 mm MgCl2 containing 1× protease inhibitors (Roche Applied Science) and phosphatase inhibitors. Extracts were passaged ten times with a 21-gauge needle followed by passage through a 25-gauge needle four times. Extracts were centrifuged at 14,000 rpm for 10 min at 4 °C, and supernatants were quantified using Bio-Rad protein assay (Bio-Rad). Protein was electrophoresed through 18% Tris-glycine gels for LC3-CFP and transferred to polyvinylidene difluoride (Bio-Rad). Membranes were probed overnight at 4 °C with antibody. Protein was visualized with enhanced chemiluminescence (ECL) plus (Amersham Biosciences) and quantified using the Typhoon Imager (Amersham Biosciences) and ImageQuaNT software. Clonal cell lines were selected and maintained at 800 μg/ml G418 (Invitrogen). The Neuro2a huntingtin-inducible cell line was a generous gift from Dr. Nobuyuki Nukina (Wako-shi, Saitama, Japan) (48Wang G.H. Mitsui K. Kotliarova S. Yamashita A. Nagao Y. Tokuhiro S. Iwatsubo T. Kanazawa I. Nukina N. Neuroreporô. 1999; 10: 2435-2438Crossref PubMed Scopus (82) Google Scholar). Plasmid Constructs—CFTR (37Johnston J.A. Ward C.L. Kopito R.R. J. Cell Biol. 1998; 143: 1883-1898Crossref PubMed Scopus (1778) Google Scholar) and GFP-Q25 and GFP-Q103 (13Bence N. Sampat R. Kopito R.R. Science. 2001; 292: 1552-1555Crossref PubMed Scopus (1821) Google Scholar) huntingtin constructs were described elsewhere. Human Atg8/LC3 cDNAs were previously described (36Iwata A. Christianson J. Bucci M. Ellerby L. Nukina N. Forno L. Kopito R. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13135-13140Crossref PubMed Scopus (273) Google Scholar). Antibodies and Reagents—Anti-Atg8/LC3B antibody was generated against full length recombinant human 6His-LC3-I produced in E. Coli or N terminus 15 peptides from human LC3B sequence. Anti-HDAC6 antibody was purchased from Calbiochem (La Jolla, CA). Anti-GFP antibody was purchased from Roche (Mannheim, Germany). Anti-actin and anti-ubiquitin monoclonal antibodies were from Chemicon International (Temecula, CA). Anti-myc 9E10 antibody was purchased from Invitrogen. γ-Tubulin antibody was from Sigma. Antibodies to LAMP-1 and LAMP-2 (H4B4) and a-tubulin (DM1a) were obtained from the University of Iowa Hybridoma Bank. Tubacin and niltubacin were a gift from S. Schreiber and J. Bradner (49Haggarty S.J. Koeller K.M. Wong J.C. Grozinger C.M. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4389-4394Crossref PubMed Scopus (901) Google Scholar). Microscopy—Cells were grown on glass coverslips coated with poly-l-lysine and collagen, fixed in 4% paraformaldehyde, permeabilized by 0.25%Triton X-100, and blocked with 5% bovine serum albumin in phosphate-buffered saline. Primary antibody incubation was done at 4 °C for overnight followed by 1-h incubation at room temperature with Alexa 488 or 546 labeled secondary antibodies (Molecular Probes, Eugene, OR) and 50 μg/ml bisbenzimide (Sigma) for nuclear staining. Conventional epifluorescence micrographs were obtained on a Zeiss Axiovert 200M microscope with a 100× oil lens (NA1.4, Zeiss). Digital (12-bit) images were acquired with a cooled charge-coupled device (Roper Scientific, Trenton, NJ) and processed by using Metamorph software (Universal Imaging, Media, PA). The excitation filters used for conventional microscopy were 365WB50 (bisbenzimide), 440AF21 (CFP), 500AF25 (GFP, Alexa488), and 560AF55 (Alexa546). Emission filters were 450DF65 (bisbenzimide), 480AF30 (CFP), 545AF35 (GFP, alexa488), and 645DF55 (Alexa546). The dichroics were: 400DCLP (bisbenzimide), 455DRLP (CFP), 525DRLP (GFP, Alexa488), and 595DRLP (Alexa546). Confocal images were obtained using a Leica LSM 510 with a63× oil lens (numerical aperture 1.2, Leica). Transmission Electron Microscopy—Cells were gently scraped, pelleted, and fixed in 3% paraformaldehyde, 1.5% glutaraldehyde, and 5% sucrose in 0.1 m sodium cacodylate buffer for 3 h at room temperature. Cells were washed briefly in 0.1 m cacodylate buffer, pH 7.4, and post-fixed using 1% osmium tetroxide and 0.5% potassium ferrocyanide in 0.1 m sodium cacodylate buffer for 1 h at room temperature. The cells were washed with 0.05 m cacodylate buffer, en bloc stained with 1% uranyl acetate in 10% ethanol for 1 h and rapidly dehydrated through a series of graded ethanol followed by propylene oxide. The cells were placed in a 1:1 mixture of propylene oxide and LX-112 resin (Ladd Research Industries, Williston, VT) overnight, immersed in two changes of LX-112 resin (2 h each), and polymerized at 60 °C for 24-48 h. 60-nm ultrathin sections were contrasted with 2% uranyl acetate for 10 min at room temperature, followed by lead citrate for 2 min at room temperature. Grids were viewed and photographed using a JEOL 1200EX II transmission electron microscope (JEOL, Peabody, MA). RNA Interference—For HDAC6 knock-down, sequence from 117-136 bp of its open reading frame was subcloned to pSUPER vector (Oligoengine, Seattle, WA). Filter Trap Assay—A polyglutamine filter trap assay was done following the published protocol (3Scherzinger E. Lurz R. Turmaine M. Mangiarini L. Hollenbach B. Hasenbank R. Bates G.P. Davies S.W. Lehrach H. Wanker E.E. Cell. 1997; 90: 549-558Abstract Full Text Full Text PDF PubMed Scopus (1084) Google Scholar, 50Wanker E.E. Scherzinger E. Heiser V. Sittler A. Eickhoff H. Lehrach H. Methods Enzymol. 1999; 309: 375-386Crossref PubMed Scopus (201) Google Scholar) using anti-GFP. The protein amount was normalized to equal the amount of soluble fraction. Promoter Shutdown Experiment—Neuro2a huntingtin-inducible cells, expressing GFP-Htt Q150 under control of an ecdysone promoter (48Wang G.H. Mitsui K. Kotliarova S. Yamashita A. Nagao Y. Tokuhiro S. Iwatsubo T. Kanazawa I. Nukina N. Neuroreporô. 1999; 10: 2435-2438Crossref PubMed Scopus (82) Google Scholar) were induced with 1 μm ponasterone A for 72 h after which the medium was replaced with Dulbecco's modified Eagle's medium lacking ponasterone A. 5 mm dibutyryl cAMP (N6,2′-O-dibutyryladenosine-3′,5′-cyclic monophosphate sodium salt, Sigma) was present throughout the experiment. Transcription Analysis—Cells were preincubated with methionine, cysteine-free medium for 30 min, then incubated with 10 mCi of [35S]methionine (MP Biomedicals, Irvine, CA). The cells were incubated for 15 min and harvested in phosphate-buffered saline supplemented with 2 mm methionine. Then the cells were lysed and immunoprecipitated by anti-GFP antibody. The amount of protein synthesized during the chase period was analyzed by SDS-PAGE and autoradiography. Data Analysis—Image analysis was done by Image J version 1.30 (National Institutes of Health). Statistical analyses were done by Statview version 5.0 (SAS institute, Cary, NC) using Student's t test. ImageJ Plug-ins Analyze particles and cell counter were used to quantify “autophagosomes” as defined by a threshold value of 159-254 and a particle size of 5. Proteasome Inhibition Induces Autophagy—In yeast and mammalian cells, autophagy is under strict metabolic control, sensing nutrient insufficiency via the inhibitory TOR (mTOR) signaling pathway, and non-selectively breaking down cellular proteins in response to nutrient availability (25Klionsky D.J. Ohsumi Y. Annu. Rev. Cell Dev. Biol. 1999; 15: 1-32Crossref PubMed Scopus (391) Google Scholar, 51Yoshimori T. Biochem. Biophys. Res. Commun. 2004; 313: 453-458Crossref PubMed Scopus (466) Google Scholar). To test whether autophagy is also activated by intracellular protein aggregation, steady-state levels of the lipid-conjugated form of Atg8/LC3, LC3-II, an established indicator of autophagic activation (52Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5468) Google Scholar), were assessed in cells exposed to the proteasome inhibitor ALLN (Fig. 1). LC3-II levels increased with time following a 1-2 h lag, in response to exposure to ALLN (Fig. 1A) or other proteasome inhibitors (data not shown). Similarly, CFP-LC3-II levels increased in response to proteasome inhibition in cells stably expressing a CFP-tagged form of LC3 (data not shown). The increase in Atg8/LC3-II levels corresponded to the appearance of fluorescent puncta in HEK-293 cells stably expressing CFP-Atg8/LC3 treated with the proteasome inhibitors MG132 (Fig. 1B, bottom panel), ALLN or lactacystin (data not shown), that were indistinguishable in appearance from autophagosomes formed in response to the classic inducer of macroautophagy: amino acid starvation (Fig. 1B, middle panel). Similar punctate structures were observed in untransfected HEK-293 cells labeled with antibodies to endogenous Atg8/LC3 and Atg12 (data not shown; see Fig. 2) but not in cells expressing CFP-Atg8/LC3G120A, a conjugation-defective Atg8/LC3 mutant (Fig. 1B, right panels). Quantification of LC3 puncta formation in HEK-293 cells exposed to ALLN (Fig. 1C), revealed that, at rest, the vast majority of cells had ≤1 LC3-positive puncta, whereas the number of cells with multiple puncta increased by 2 h after administration of the drug. Atg8/LC3 remained soluble following extraction of MG132-treated cells with non-ionic detergent and did not appear to form covalent conjugates with ubiquitin (supplemental Fig. S1). These data demonstrate that autophagy is rapidly induced in response to impaired proteasome activity.FIGURE 2Accumulation of autophagic vacuoles around aggresomes. A, HeLa cells were cultured in Dulbecco's modified Eagle's medium (a-c), Hanks' balanced salt solution (HBSS) for 4 h (d-f), Dulbecco's modified Eagle's medium plus lactacystin (10 μm) for 18 h (g-i) or Dulbecco's modified Eagle's medium plus lactacystin (10 μm) with 1 μg/ml nocodazole for 18 h (j-l). Cells were fixed and stained with anti-ubiquitin (a, d, g, and j) and Atg8/LC3 antibodies (b, e, h, and k). Arrows indicate aggresomes. Bar = 2 μm. B, transmission electron microscopy of HEK-293 cells exposed to ALLN for 0 h (a), 3 h (b), or 21 h (c). Typical autophagic structures were absent from untreated cells (a) and clearly evident in ALLN-treated cells (b and c) and increased in abundance with time of exposure to the drug. Note clustering of electron-dense autophagic bodies at juxtanuclear sites (lower right of panel c). C, accumulation of autophagosomes near pericentriolar aggresome. a, electron micrograph of HEK-293 cells, transiently expressing the ΔF508 mutant of CFTR, were treated overnight with ALLN. Boxes denote regions shown at higher magnification in panels b-d. b, higher magnification micrograph showing pair of centrioles in the center of the electron dense ΔF508-CFTR aggresome. c, asterisks denote late degradative autophagosomes. d, high magnification view of autophagosome from panel c. Scale bars, 1 μm. Abbreviations: ag, aggresome; c, centriole; n, nucleus; m, mitochondrion; and pm, plasma membrane.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Clearance of Protein Aggregates Requires Microtubule-dependent Transport—To assess how proteasome inhibition influences the subcellular distribution of autophagosomes, endogenous Atg8/LC3 was localized in HeLa cells by immunofluorescence microscopy and transmission electron microscopy (Fig. 2). In unperturbed cells (Fig. 2A, panels a-c), Atg8/LC3 was present in numerous very fine cytoplasmic puncta that were slightly enriched in the Golgi region. In amino acid-starved cells (Fig. 2A, panels d-f), Atg8/LC3 antibody labeled discrete autophagic structures that were distributed randomly throughout the cytoplasm. No correlation was observed between the distribution of Atg8/LC3 and that of ubiquitin, which maintained a uniformly diffuse distribution in both resting and starved cells. By contrast, overnight exposure to lactacystin (Fig. 2A; g-i) or other proteasome inhibitors (data not shown) resulted in a dramatic redistribution of both ubiquitin and Atg8/LC3 immunoreactivity to a discrete juxtanuclear region, where the two antigens appeared to be extensively colocalized. Juxtanuclear concentration and colocalization of ubiquitin and Atg8/LC3 immunoreactive structures was abrogated by treatment with nocodazole (Fig. 2A, panels j-l). The appearance of LC3-immunopositive puncta in response to proteasome inhibition correlated with the appearance, by transmission electron microscopy (Fig. 2B), of multilamellar, darkly stained structures that could be identified as degradative autophagosomes by the presence of membrane-enclo

Parkin is a RING-between-RING E3 ligase that functions in the covalent attachment of ubiquitin to specific substrates, and mutations in Parkin are linked to Parkinson's disease, cancer and mycobacterial infection. The RING-between-RING family of E3 ligases are suggested to function with a canonical RING domain and a catalytic cysteine residue usually restricted to HECT E3 ligases, thus termed 'RING/HECT hybrid' enzymes. Here we present the 1.58 Å structure of Parkin-R0RBR, revealing the fold architecture for the four RING domains, and several unpredicted interfaces. Examination of the Parkin active site suggests a catalytic network consisting of C431 and H433. In cells, mutation of C431 eliminates Parkin-catalysed degradation of mitochondria, and capture of an ubiquitin oxyester confirms C431 as Parkin's cellular active site. Our data confirm that Parkin is a RING/HECT hybrid, and provide the first crystal structure of an RING-between-RING E3 ligase at atomic resolution, providing insight into this disease-related protein.

The protein ubiquitin is an important post-translational modifier that regulates a wide variety of biological processes. In cells, ubiquitin is apportioned among distinct pools, which include a variety of free and conjugated species. Although maintenance of a dynamic and complex equilibrium among ubiquitin pools is crucial for cell survival, the tools necessary to quantify each cellular ubiquitin pool have been limited. We have developed a quantitative mass spectrometry approach to measure cellular concentrations of ubiquitin species using isotope-labeled protein standards and applied it to characterize ubiquitin pools in cells and tissues. Our method is convenient, adaptable and should be a valuable tool to facilitate our understanding of this important signaling molecule.

Pathognomonic accumulation of ubiquitin (Ub) conjugates in human neurodegenerative diseases, such as Huntington's disease, suggests that highly aggregated proteins interfere with 26S proteasome activity. In this paper, we examine possible mechanisms by which an N-terminal fragment of mutant huntingtin (htt; N-htt) inhibits 26S function. We show that ubiquitinated N-htt-whether aggregated or not-did not choke or clog the proteasome. Both Ub-dependent and Ub-independent proteasome reporters accumulated when the concentration of mutant N-htt exceeded a solubility threshold, indicating that stabilization of 26S substrates is not linked to impaired Ub conjugation. Above this solubility threshold, mutant N-htt was rapidly recruited to cytoplasmic inclusions that were initially devoid of Ub. Although synthetically polyubiquitinated N-htt competed with other Ub conjugates for access to the proteasome, the vast majority of mutant N-htt in cells was not Ub conjugated. Our data confirm that proteasomes are not directly impaired by aggregated N-terminal fragments of htt; instead, our data suggest that Ub accumulation is linked to impaired function of the cellular proteostasis network.

Genetic ablation of autophagy in mice leads to liver and brain degeneration accompanied by the appearance of ubiquitin (Ub) inclusions, which has been considered to support the hypothesis that ubiquitination serves as a cis-acting signal for selective autophagy. We show that tissue-specific disruption of the essential autophagy genes Atg5 and Atg7 leads to the accumulation of all detectable Ub-Ub topologies, arguing against the hypothesis that any particular Ub linkage serves as a specific autophagy signal. The increase in Ub conjugates in Atg7(-/-) liver and brain is completely suppressed by simultaneous knockout of either p62 or Nrf2. We exploit a novel assay for selective autophagy in cell culture, which shows that inactivation of Atg5 leads to the selective accumulation of aggregation-prone proteins, and this does not correlate with an increase in substrate ubiquitination. We propose that protein oligomerization drives autophagic substrate selection and that the accumulation of poly-Ub chains in autophagy-deficient circumstances is an indirect consequence of activation of Nrf2-dependent stress response pathways.

The polyglutamine disorders are a class of nine neuro-degenerative disorders that are inherited gain-of-function diseases caused by expansion of a translated CAG repeat. Even though the disease-causing proteins are widely expressed, specific collections of neurons are more susceptible in each disease, resulting in characteristic patterns of pathology and clinical symptoms. One hypothesis poses that altered protein function is fundamental to pathogenesis, with protein context of the expanded polyglutamine having key roles in disease-specific processes. This review will focus on the role of the disease-causing polyglutamine proteins in gene transcription and the extent to which the mutant proteins induce disruption of transcription.

Detailed analysis of disease-affected tissue provides insight into molecular mechanisms contributing to pathogenesis. Substantia nigra, striatum, and cortex are functionally connected with increasing degrees of alpha-synuclein pathology in Parkinson's disease. We undertook functional and causal pathway analysis of gene expression and proteomic alterations in these three regions, and the data revealed pathways that correlated with disease progression. In addition, microarray and RNAseq experiments revealed previously unidentified causal changes related to oligodendrocyte function and synaptic vesicle release, and these and other changes were reflected across all brain regions. Importantly, subsets of these changes were replicated in Parkinson's disease blood; suggesting peripheral tissue may provide important avenues for understanding and measuring disease status and progression. Proteomic assessment revealed alterations in mitochondria and vesicular transport proteins that preceded gene expression changes indicating defects in translation and/or protein turnover. Our combined approach of proteomics, RNAseq and microarray analyses provides a comprehensive view of the molecular changes that accompany functional loss and alpha-synuclein pathology in Parkinson's disease, and may be instrumental to understand, diagnose and follow Parkinson's disease progression.

Neuronal tau reduction confers resilience against β-amyloid and tau-related neurotoxicity in vitro and in vivo. Here, we introduce a novel translational approach to lower expression of the tau gene MAPT at the transcriptional level using gene-silencing zinc finger protein transcription factors (ZFP-TFs). Following a single administration of adeno-associated virus (AAV), either locally into the hippocampus or intravenously to enable whole-brain transduction, we selectively reduced tau messenger RNA and protein by 50 to 80% out to 11 months, the longest time point studied. Sustained tau lowering was achieved without detectable off-target effects, overt histopathological changes, or molecular alterations. Tau reduction with AAV ZFP-TFs was able to rescue neuronal damage around amyloid plaques in a mouse model of Alzheimer's disease (APP/PS1 line). The highly specific, durable, and controlled knockdown of endogenous tau makes AAV-delivered ZFP-TFs a promising approach for the treatment of tau-related human brain diseases.

SUMO (small ubiquitin-like modifier) is a member of the ubiquitin family of proteins. SUMO targets include proteins involved in numerous roles including nuclear transport and transcriptional regulation. The previous finding that mutant ataxin-1[82Q] disrupted promyelocytic leukemia (PML) oncogenic domains prompted us to determine whether ataxin-1 disrupts another component of PML oncogenic domains, Sp100 (100-kDa Speckled protein). Similar to the PML protein, mutant ataxin-1[82Q] redistributed Sp100 to mutant ataxin-1[82Q] nuclear inclusions. Based on the ability of PML and Sp100 to be covalently modified by SUMO, we investigated the ability of ataxin-1 to be SUMOylated. SUMO-1 was found to covalently modify the polyglutamine repeat protein ataxin-1. There was a decrease in ataxin-1 SUMOylation in the presence of the expanded polyglutamine tract, ataxin-1[82Q]. The phospho-mutant, ataxin-1[82Q]-S776A, restored SUMO levels to those of wild-type ataxin-1[30Q]. SUMOylation of ataxin-1 was dependent on a functional nuclear localization signal. Ataxin-1 SUMOylation was mapped to at least five lysine residues. Lys(16), Lys(194) preceding the polyglutamine tract, Lys(610)/Lys(697) in the C-terminal ataxin high mobility group domain, and Lys(746) all contribute to ataxin-1 SUMOylation.

Clinical gene therapy has been practiced for more than a quarter century and the first products are finally gaining regulatory/marketing approval. As of 2016, there have been 11 haemophilia gene therapy clinical trials of which six are currently open. Each of the ongoing phase 1/2 trials is testing a variation of a liver-directed adeno-associated viral (AAV) vector encoding either factor VIII (FVIII) or factor IX (FIX) . As summarized herein, the clinical results to date have been mixed with some perceived success and a clear recognition of the immune response to AAV as an obstacle to therapeutic success. We also attempt to highlight promising late-stage preclinical activities for AAV-FVIII where, due to inherent challenges with manufacture, delivery and transgene product biosynthesis, more technological development has been necessary to achieve results comparable to what has been observed previously for AAV-FIX. Finally, we describe the development of a stem cell-based lentiviral vector gene therapy product that has the potential to provide lifelong production of FVIII and provide a functional 'cure' for haemophilia A. Integral to this program has been the incorporation of a blood cell-specific gene expression element driving the production of a bioengineered FVIII designed for optimal efficiency. As clearly outlined herein, haemophilia remains at the forefront of the rapidly advancing clinical gene therapy field where there exists a shared expectation that transformational advances are on the horizon.

The ataxin-1 interacting ubiquitin-like protein (A1Up) contains an amino-terminal ubiquitin-like (UbL) region, four stress-inducible, heat shock chaperonin-binding motifs (STI1), and an ubiquitin-associated domain (UBA) at the carboxyl terminus of A1Up. Although proteins that have both an UbL and UBA domain are thought to play a crucial role in proteasome-mediated activities, few are characterized, except for hHR23A/B. Similar to other UbL-containing proteins, the UbL of A1Up is essential for the interaction of A1Up with the S5a subunit of the 19S proteasome. Importantly, the interaction with the 19S proteasome was disrupted in the presence of the polyglutamine repeat protein, ataxin-1. The UbL domain of A1Up is ubiquitinated by both Lys⁴⁸-linked and Lys⁶³-linked chains. Intact A1Up is stable, suggesting that ubiquitination of A1Up is important for degradation-independent targeting of A1Up to the 19S proteasome. The UBA domain of A1Up binds polyubiquitin chains and has a role in the stability of A1Up and in the subcellular localization of A1Up. When the UBA domain was deleted, the localization of A1Up was entirely cytoplasmic, and it co-localized with the proteasome. Interestingly, the interaction between A1Up and mutant ataxin-1-(82Q) increased the half-life of A1Up, whereas nonpathogenic wild-type ataxin-1-(30Q) or ataxin-1-(82Q)-A776 did not.

Genetic inactivation of autophagy in liver or brain leads to the appearance of ubiquitin- and p62-positive inclusions coincident with liver dysfunction and neurodegeneration, respectively. In our recent study we measured the abundance of polyubiquitin species in autophagy-deficient tissues and demonstrated that a specific polyubiquitin chain linkage is not the decisive autophagic substrate-targeting signal. Instead our data suggest that aggregation or oligomerization of a misfolded protein, in the absence of detectable polyubiquitin modification, is an important signal for autophagic degradation. We determined that the ubiquitin accumulation observed upon autophagy inhibition is caused by p62-mediated activation of Nrf2 resulting in global transcriptional changes to ubiquitin-associated genes. Thus, substrate polyubiquitination does not appear to be the major autophagy substrate-targeting signal and the primary role of p62 appears to be Nrf2 activation, not ubiquitin-dependent substrate degradation. Selective targeting of proteins to distinct subcellular machineries is fundamental to the regulation of cellular decisions between catabolism and anabolism. In particular, ubiquitin (Ub)-mediated targeting of proteins is essential in maintaining cellular homeostasis, and has been predominantly associated with nonlysosomal-mediated degradation. However, liver- and brain-specific autophagy knockout mice exhibit accumulation of Ub- and p62-positive inclusions suggesting that Ub-modification targets cargo for selective autophagic degradation. Subsequent reports have established the requirement of p62 oligomerization for the appearance of Ub-positive inclusions and suggest that p62 is a selective ‘autophagy adaptor’ for recognition and delivery of Ub-modified cargo to autophagosomes. Autophagy contributes to the detoxification of misfolded, aggregated proteins commonly associated with neurodegenerative disorders (Alzheimer disease, Parkinson disease, Huntington disease, and Lou Gehrig's disease [amyotrophic lateral sclerosis] and prion encephalophathies) and the presence of these misfolded proteins also correlates with the appearance of Ub- and p62-positive inclusions. Revealing how proteins are ‘marked’ for selective recognition by the autophagy machinery is essential. To determine whether specific polyubiquitin linkages target substrates for selective autophagic degradation we used Ub absolute quantification (AQUA) mass spectrometry to measure the amount of Ub that accumulates in both liver- and brain-specific autophagy knockout mouse models. We observed a global increase in all Ub isopeptide and non-isopeptide species and the results were similar between two different autophagy-deficient models in two separate tissues. Moreover the increased levels of Ub conjugates observed in samples of Atg7-null tissues were suppressed when this mutation was combined with deletions of p62 or Nrf2. These results are significant for two reasons. First, Nrf2 controls expression levels of detoxification enzymes by regulating genes that contain an antioxidant response element (ARE). If substrate polyubiquitination was the major autophagy targeting signal then we would not expect Ub accumulation in autophagy-deficient tissue to depend upon the presence of this transcription factor. Second, whereas p62 has been hypothesized to be an adaptor facilitating the autophagic degradation of ubiquitinated substrates, it has been previously shown that loss of p62 protects tissues from autophagy deficiency by preventing Nrf2 activation. Our observations are consistent with a role for p62 in controlling Nrf2—and not with a role as a simple Ub-dependent autophagy adaptor. Our findings suggest that accumulation of Ub during autophagy deficiency is a result of Nrf2 stress signaling downstream of the multifunctional scaffolding protein p62 rather than polyubiquitin functioning as the substrate targeting signal for selective autophagy. To identify Ub-related Nrf2 target genes that might explain the Ub dysregulation observed in autophagy-deficient tissues, we used bioinformatics, functional genomics and RT-PCR. Nrf2 (and its heterodimeric binding partner Maf) transcription factor-binding sites were found to be enriched among Ub-associated genes and the autophagy network. Within the autophagy network, there are 35 Ub-associated Nrf2 targets, and 22 are differentially affected in Atg7-/- mice compared to wild-type mice. Importantly, these changes are reversed in the Atg7-/Nrf2-double knockout mice confirming Nrf2-dependent regulation. Our overall interpretation of these data is that autophagy deficiency results in a stress response that activates Nrf2, globally affecting numerous Ub-related proteins. It is important to note that upregulation of ARE-containing genes has been reported for numerous neurodegenerative disorders. Understanding the mechanistic details of how Nrf2 binds ARE elements within Ub-associated genes, with what binding partner it binds, and Nrf2 nuclear/cytoplasmic trafficking in response to autophagy inhibition/p62 accumulation will reveal the physiological relevance of Nrf2-p62 Ub-associated signaling in neurodegenerative disorders. Since our results from mouse indicate that polyubiquitination is not the major autophagy substrate targeting signal, we used quantitative mass spectrometry and flow cytometry to measure Ub-modification of a selective autophagy substrate in an autophagy-regulatable stable cell line. Ub has been implicated to function as a signal in several forms of selective autophagy such as pexophagy, mitophagy and xenophagy. However, in these studies, the dependence of p62 and Ub on autophagic clearance was shown by immunofluorescence studies in which the absence of p62 results in the loss of substrate puncta formation or Ub colocalization with the substrate. Colocalization, though indicative of a signaling role, does not demonstrate covalent Ub modification of substrates. Our work addresses directly whether substrate modification by polyubiquitin chains targets proteins for selective autophagy and enables us to identify additional features of selective substrates. We developed a flow cytometry cell-based assay to measure selective autophagy using an autophagy-regulatable cell line stably expressing a bicistronic reporter construct containing both the misfolded, aggregation-prone protein huntingtin with an expanded polyglutamine tract (htt(Q47)) fused to GFP (green fluorescent protein), and the non-aggregation-prone protein cherry chFP (cherry fluorescent protein). Following autophagy shut-off, the reporter cell lines provide the ability to quantify and compare the relative accumulation of the two different fluorescent reporters, where autophagic selectivity is indicated by a ratio of greater than one. Using our flow cytometry assay we measured the selective accumulation of the aggregation-prone protein htt(Q47) compared to the non-aggregation prone protein chFP and although there is global accumulation of polyubiquitin chains following autophagy shut-off there is no increase in polyubiquitin-modified htt(Q47). Overall, these data demonstrate that aggregation or oligomerization of a misfolded protein, in the absence of detectable Ub-modification, results in selective accumulation following genetic ablation of autophagy. Based on these observations the major conclusion of our manuscript is that oligomerization targets proteins for selective autophagy in mammalian cells, and that polyubiquitination does not appear to be the major autophagy targeting signal; the primary role of p62 in autophagy deficiency appears to be Nrf2 activation, not Ub-dependent substrate degradation. Moreover, our data also demonstrate the broad importance of Nrf2-driven Ub signaling as an important cellular detoxification mechanism acting in addition to the arsenal of Nrf2-oxidative stress genes, and suggest that sustained activation could be detrimental to the cell (Fig. 1). Figure 1 Schematic of Ub-associated signaling regulated by the Nrf2-p62 axis. The Keap1-Cul3-Rbx1 E3 Ub ligase maintains low levels of Nrf2 in the cell. In response to oxidative insult, accumulation of misfolded, aggregation-prone proteins or additional unknown ...

Surface Plasmon Resonance (SPR) is rarely used as a primary High-throughput Screening (HTS) tool in fragment-based approaches. With SPR instruments becoming increasingly high-throughput it is now possible to use SPR as a primary tool for fragment finding. SPR becomes, therefore, a valuable tool in the screening of difficult targets such as the ubiquitin E3 ligase Parkin. As a prerequisite for the screen, a large number of SPR tests were performed to characterize and validate the active form of Parkin. A set of compounds was designed and used to define optimal SPR assay conditions for this fragment screen. Using these conditions, more than 5000 pre-selected fragments from our in-house library were screened for binding to Parkin. Additionally, all fragments were simultaneously screened for binding to two off target proteins to exclude promiscuous binding compounds. A low hit rate was observed that is in line with hit rates usually obtained by other HTS screening assays. All hits were further tested in dose responses on the target protein by SPR for confirmation before channeling the hits into Nuclear Magnetic Resonance (NMR) and other hit-confirmation assays.

Mutations in the E3 ubiquitin ligase Parkin or the mitochondrial kinase PINK1 cause autosomal recessive forms of Parkinson’s disease [1, 2]. Genetic and cell biological studies have implicated PINK1 and Parkin as critical elements in mitophagy, a mitochondrial quality control pathway that involves the ubiquitin-proteasome system (UPS) and the autophagy-lysosomal system [1, 2]. Under basal conditions, PINK1 is processed by mitochondrial proteases and targeted for degradation by the UPS [1, 2]. Following persistent mitochondrial damage (e.g., treatment with the mitochondrial uncoupling agent CCCP) PINK1 is stabilized and accumulates in an active form on the outer mitochondrial membrane [1, 2]. Although PINK1 activity is essential for the mitochondrial recruitment of cytoplasmic Parkin and for the subsequent ubiquitin-dependent clearance of damaged mitochondria, the mode of Parkin activation and recruitment has been elusive [1, 2]. A series of recent papers indicates that PINK1 initiates mitophagy by a two-pronged mechanism involving direct phosphorylation of ubiquitin at serine 65 [3–5] and the ubiquitin-like domain (UbL) of Parkin, also, at serine 65 [6–8] (Fig. 1A, steps 1–2). Biochemical and structural analyses of Parkin demonstrated that the unique Parkin domain (UPD):Rcat interface (previously termed RING0:RING2), the repressor element of Parkin (REP):RING1 interface, and potentially the UbL:RING1 interface mediate autoinhibition of Parkin under steady state conditions in the cell [9–14]. It is tempting to speculate that PINK1 phosphorylation of the Parkin UbL and/or the binding of phosphorylated ubiquitin releases the autoinhibitory elements to allow E2~Ub binding or facilitates conformational rearrangements to confer E2~Ub discharge, ultimately leading to exposure of an optimally aligned Parkin active site. Although the studies on PINK1 phosphorylation of ubiquitin [3–5] and Parkin [6–8] suggest a novel mechanism for PINK1 activation of Parkin, the expression of phosphomimetics of ubiquitin and Parkin was insufficient to promote the mitochondrial recruitment of Parkin [3, 8]. Thus the mechanism underlying PINK1-mediated Parkin recruitment remained a mystery. Figure 1 Damage-induced feedforward and positive feedback loops mediate the cellular decision to destroy mitochondria. In December’s issue of PLOS Genetics, Shiba-Fukushima et al. provide compelling data indicating that PINK1 directly phosphorylates polyubiquitin chains to mediate the mitochondrial recruitment and activation of Parkin (Fig. 1A, steps 3–4) [15]. In vitro, affinity purified Parkin from cells bound purified polyubiquitin chains phosphorylated by recombinant PINK1 with a preference for long K63-linked polyubiquitin over K48-linked polyubiquitin chains, although this preference was not recapitulated with Parkin purified from bacteria. To simulate mitochondrial linked polyubiquitin chains, the authors expressed four tandem copies of ubiquitin G76V fused to the mitochondrial targeting sequence of Tom70 [15]. Phosphomimetic (S65E) versions of the tandem polyubiquitin chains were bound by Parkin through its RING1-BRcat domains (previously termed RING1:IBR) and were able to promote stable mitochondrial association of Parkin even in the absence of mitochondrial damage [15]. Both cytosolic phosphomimetic ubiquitin and mitochondrially targeted phosphomimetic tandem polyubiquitin chains were sufficient to activate Parkin, as evidenced by increased Parkin C431S-ubiquitin oxyester formation, but only when the Parkin UbL phosphomimetic was used [15]. To further understand the physiological importance of their findings, Shiba-Fukushima et al. employed PINK1-/- and Parkin -/- Drosophila models, which are associated with severe mitochondrial swelling and matrix disorganization and age-dependent motor defects. Strikingly, the expression of mitochondrially targeted phosphomimetic tandem polyubiquitin chains significantly improve both mitochondrial morphology and motor function in PINK1-/- flies, with little effect on mitochondrial morphology in Parkin -/- flies [15]. These results are in excellent agreement with a recent publication that employed quantitative proteomics to study PINK1-stimulated Parkin polyubiquitination [16]. Together, these two publications [15, 16] suggest that PINK1 phosphorylation of polyubiquitin is the rate-limiting event required for the mitochondrial recruitment, and potentially also the activation, of Parkin. The destruction of mitochondria represents an irreversible cellular decision with significant consequences for cellular physiology. The emerging data support a model in which the decision to degrade damaged mitochondria is controlled by dual coherent feedforward loops that precede a positive feedback loop (Fig. 1B). In a feedfoward loop, two input factors, one of which controls the other, jointly regulate a third target factor. In the PINK1-Parkin pathway, the first feedforward loop mediates maximal activation of Parkin by PINK1 phosphorylation of the Parkin UbL and of ubiquitin. The second feedforward loop involves the generation of mitochondrial polyubiquitin chains by PINK1-activated Parkin, and/or another mitochondrial E3 ligase, and their subsequent phosphorylation by PINK1. These phosphorylated polyubiquitin chains appear to be capable of initiating a self-propagating positive feedback loop, recruiting Parkin to the mitochondria and presumably stimulating polyubiquitination of mitochondrial substrates, which can then be phosphorylated by PINK1 and recruit additional Parkin. The organization of these network motifs predicts beneficial features with respect to the decision to degrade mitochondria, including an initial delay period and a mechanism to detect the persistence of mitochondrial damage (Fig. 1C). During the delay period (i.e., low levels of phosphorylated polyubiquitin), the decision to commit to mitophagy would be rapidly reversible, providing a useful means of filtering out brief, low levels of mitochondrial damage signals and preventing unwarranted mitochondrial destruction (Fig. 1C, blue line). Only a persistent damage stimulus that overcomes a specific threshold would be sufficient to initiate the positive feedback loop and commit mitochondria for mitophagy (Fig. 1C, red line). The actions of putative unidentified ubiquitin and Parkin UbL phosphatases, or of mitochondrial deubiquitinating enzymes USP30 [17], USP15 [18], or USP8 [19], which antagonize Parkin-mediated polyubiquitination, would be predicted to regulate the extent of the delay period and the precise commitment threshold. Interestingly, USP30 has been reported to be targeted for UPS degradation by Parkin [17], providing an elegant mechanism to gradually reduce the magnitude of UPS30’s influence during persistent mitochondrial damage. The new study from Shiba-Fukushima et al. [15] contributes an intriguing model for the role of PINK1 in Parkin recruitment to damaged mitochondria and raises several interesting questions for future investigation. Is an unidentified Parkin UbL and/or ubiquitin phosphatase involved in mitochondrial quality control? The mitochondrial phosphatase PGAM5, which functions downstream of PINK1 [20], is a logical candidate. If polyubiquitination induces the proteasomal degradation of tagged substrates, how does the phospho-polyubiquitin mitochondrial signal persist, are they shielded by phospho-polyubiquitin binding domain-containing proteins or not efficiently recognized by the proteasome? Does Parkin self-association [21] amplify the feedforward loop? How is the binding and exchange of substrates, phospho-polyubiquitin chains and/or phospho-ubiquitin by Parkin coordinated to control the timing of substrate degradation? Finally, it will be imperative to determine the therapeutic potential of small molecule mimetics of phosphorylated ubiquitin (or Parkin UbL) and regulators of the PINK1-Parkin mitochondrial quality control pathway in the search for cures of idiopathic Parkinson’s disease.

Blue cone monochromacy (BCM) is a rare X-linked retinal disease characterized by the absence of L- and M-opsin in cone photoreceptors, considered a potential gene therapy candidate. However, most experimental ocular gene therapies utilize subretinal vector injection which would pose a risk to the fragile central retinal structure of BCM patients. Here we describe the use of ADVM-062, a vector optimized for cone-specific expression of human L-opsin and administered using a single intravitreal (IVT) injection. Pharmacological activity of ADVM-062 was established in gerbils, whose cone-rich retina naturally lacks L-opsin. A single IVT administration dose of ADVM-062 effectively transduced gerbil cone photoreceptors and produced a de novo response to long-wavelength stimuli. To identify potential first-in-human doses we evaluated ADVM-062 in non-human primates. Cone-specific expression of ADVM-062 in primates was confirmed using ADVM-062.myc, a vector engineered with the same regulatory elements as ADVM-062. Enumeration of human OPN1LW.myc-positive cones demonstrated that doses ≥3 × 1010 vg/eye resulted in transduction of 18%-85% of foveal cones. A Good Laboratory Practice (GLP) toxicology study established that IVT administration of ADVM-062 was well tolerated at doses that could potentially achieve clinically meaningful effect, thus supporting the potential of ADVM-062 as a one-time IVT gene therapy for BCM.

Abstract Hemophilia A, which is caused by a mutation in the Factor 8 (F8) gene resulting in a deficiency or lack of the Factor VIII (FVIII) protein, is the most common inherited bleeding disorder in humans with an estimated worldwide incidence of half a million people. The disorder is X-linked and occurs in approximately 1 in 5,000 males; however there is also a growing appreciation of the impact on carrier females having a single mutant allele, with at least 10% of hemophilia A female carriers having less than normal clotting activity. Even modest increases in Factor V III activity (>1% of normal) can have a positive impact on patient lives, thus making the disease an ideal candidate for liver-directed gene therapy. Recombinant AAV (rAAV) has been used extensively for nearly 20 years as a gene therapy vector in preclinical and clinical studies where rAAV delivery to non-dividing tissues such as liver, brain and muscle affords stable, long-term transgene expression. However, there has been a lag in the clinical translation of a rAAV gene therapy approach for Hemophilia A/human F8 (hF8) compared to Hemophilia B/human Factor 9 due to poor yields of rAAV encoding a F8 transgene at clinical scale, and a requirement for large doses of rAAV F8 vector to achieve therapeutically relevant levels of circulating human FVIII (hFVIII), with the attendant risk of inducing an AAV-directed immune response requiring transient immunosuppression. To address these issues we optimized a rAAV F8 cDNA vector cassette to improve both virus yields and liver-specific hFVIII expression. The rAAV F8 cDNA vector cassette optimization required multi-factorial modifications to the liver-specific promoter module, hF8 transgene, synthetic polyadenylation signal and vector backbone sequence. This iterative process resulted in improved vector yields at research scale and greater than five-fold improvement in vector yields at clinical scale using our proven manufacturing process. Administration of the optimized rAAV hF8 cDNA packaged in serotype AAV2/6 at a dose of ~7.2E+12 vg/kg to both wild type and Hemophilia A mice resulted in robust circulating hFVIII levels and activity (levels in wild type mice were 241.6% of normal, and activity in Hemophilia A mice were 330.9% of normal). An analysis of hF8 mRNA levels in different tissues following dosage with our optimized vector demonstrated that hF8 expression from the modified promoter module was restricted to the liver. Notably there was a striking impact on hemostasis in the Hemophilia A mice treated with the optimized rAAV hF8 cDNA, with a reduction in bleeding time from 38.3 minutes to 2.5 minutes in treated mice (n = 12, p-value < 0.0001), which is in line with bleeding times in wild type mice. Initial studies in non-human primates (NHPs) resulted in supraphysiological levels of circulating hFVIII with mean peak values of 400-600% of normal levels. A follow up dose-ranging study was performed in NHPs with a rAAV2/6 F8 cDNA vector manufactured using our GMP clinical manufacturing process. Administration of vector doses ranging from 6E+11 vg/kg to 6E+12 vg/kg resulted in therapeutic circulating levels of hFVIII that were 5% - 229% of normal levels. The peak circulating hFVIII levels achieved in this dose-ranging study using GMP clinical-scale vector exceeds the levels previously reported in NHPs by several fold on an AAV vector dose basis. The high potency of this enhanced rAAV F8 cDNA cassette could significantly reduce the dose required to achieve therapeutically relevant levels in human subjects and reduce the potential of developing immune responses to AAV capsid requiring immunosuppression. Disclosures Riley: Sangamo BioSciences Inc: Employment. Boonsripisal:Sangamo BioSciences Inc: Employment. Goodwin:Sangamo BioSciences Inc: Employment. Garces:Sangamo BioSciences Inc: Employment. Ballaron:Sangamo BioSciences Inc: Employment. Tran:Sangamo BioSciences Inc: Employment. Kang:Sangamo BioSciences Inc: Employment. Zhang:Sangamo BioSciences Inc: Employment. Meyer:Sangamo BioSciences Inc: Employment. Greengard:Sangamo BioSciences Inc: Employment. Surosky:Sangamo BioSciences Inc: Employment. Ando:Sangamo BioSciences Inc: Employment. Lillicrap:bayer: Research Funding; biogen: Research Funding; CSL: Research Funding; Octapharma: Research Funding; Sangamo Biosciences Inc: Research Funding. Nichol:Sangamo BioSciences Inc: Employment. Holmes:Sangamo BioSciences Inc: Employment.

Hemophilia A is the most common inherited bleeding disorder in humans and is caused by a deficiency of blood coagulation factor VIII (FVIII). This disease is an ideal candidate for liver-directed gene therapy, as even modest increases in FVIII activity (>1% of normal) can ameliorate the severe bleeding phenotype. Adeno-associated viral (AAV) vectors have shown great promise in both preclinical and clinical trials to efficiently deliver therapeutic transgenes to the liver. Suboptimal virus yields of AAV comprising human FVIII (hFVIII) for use in human therapy have hampered clinical scale manufacturing, both from mammalian (HEK293) and insect cells (Baculovirus system). We optimized an hFVIII AAV vector cassette to improve both virus yields and liver-specific hFVIII expression. Compared to historical hFVIII AAV vector cassettes that routinely produce 20% of standard, non-hFVIII containing AAV2/6 virus preparations, the improved hFVIII AAV vector cassette produced yields 100% of standard AAV2/6, both from HEK293 cells at the cell factory (CF) scale, but also in the Baculovirus system at large scale. Improved yields were also observed using other AAV serotypes including hFVIII AAV2/8 and AAV2/9. Using virus produced from the improved hFVIII AAV cassette we transduced mice at 6E+10 vg/mouse (~2E+12 vg/kg). Peak levels of hFVIII protein were achieved in mouse plasma at day 14 and represented supraphysiological levels of normal hFVIII in humans with serotypes AAV2/8 and AAV2/9; up to 337% and 516% of normal hFVIII plasma levels respectively (1U = 200 ng/mL = 100%). Transducing mice with serotype AAV2/6, known to be inefficient at transducing mouse liver, at 6E+10 vg/mouse (~2E+12 vg/kg) we achieved a mean peak value of 91.9 % +/- 15.5 SEM (n=6) of normal hFVIII plasma levels in humans. At a higher dose representing ~6E+12 vg/kg we achieved a mean peak value of hFVIII plasma over six independent in vivo mouse studies of 169.2 % +/- 10.1 SEM (n = 36). Supraphysiological hFVIII plasma levels were also achieved in non-human primates at a dose of 2E+12 vg/kg using serotype AAV2/6, representing up to 840% of normal hFVIII levels (8.4U). The robust production of hFVIII from the enhanced hFVIII AAV vector could significantly reduce the dose required to achieve therapeutic levels in human subjects. Additionally the improved hFVIII AAV vector cassette will enable clinical scale manufacturing.

Surface Plasmon Resonance (SPR) is rarely used as a primary High-throughput Screening (HTS) tool in fragment-based approaches.With SPR instruments becoming increasingly high-throughput it is now possible to use SPR as a primary tool for fragment finding.SPR becomes, therefore, a valuable tool in the screening of difficult targets such as the ubiquitin E3 ligase Parkin.As a prerequisite for the screen, a large number of SPR tests were performed to characterize and validate the active form of Parkin.A set of compounds was designed and used to define optimal SPR assay conditions for this fragment screen.Using these conditions, more than 5000 pre-selected fragments from our in-house library were screened for binding to Parkin.Additionally, all fragments were simultaneously screened for binding to two off target proteins to exclude promiscuous binding compounds.A low hit rate was observed that is in line with hit rates usually obtained by other HTS screening assays.All hits were further tested in dose responses on the target protein by SPR for confirmation before channeling the hits into Nuclear Magnetic Resonance (NMR) and other hit-confirmation assays.