Huntingtin Associates with Acidic Phospholipids at the Plasma Membrane

We have identified a domain in the N terminus of huntingtin that binds to membranes. A three-dimensional homology model of the structure of the binding domain predicts helical HEAT repeats, which emanate a positive electrostatic potential, consistent with a charge-based mechanism for membrane association. An amphipathic helix capable of inserting into pure lipid bilayers may serve to anchor huntingtin to the membrane. In cells, N-terminal huntingtin fragments targeted to regions of plasma membrane enriched in phosphatidylinositol 4,5-bisphosphate, receptor bound-transferrin, and endogenous huntingtin. N-terminal huntingtin fragments with an expanded polyglutamine tract aberrantly localized to intracellular regions instead of plasma membrane. Our data support a new model in which huntingtin directly binds membranes through electrostatic interactions with acidic phospholipids. We have identified a domain in the N terminus of huntingtin that binds to membranes. A three-dimensional homology model of the structure of the binding domain predicts helical HEAT repeats, which emanate a positive electrostatic potential, consistent with a charge-based mechanism for membrane association. An amphipathic helix capable of inserting into pure lipid bilayers may serve to anchor huntingtin to the membrane. In cells, N-terminal huntingtin fragments targeted to regions of plasma membrane enriched in phosphatidylinositol 4,5-bisphosphate, receptor bound-transferrin, and endogenous huntingtin. N-terminal huntingtin fragments with an expanded polyglutamine tract aberrantly localized to intracellular regions instead of plasma membrane. Our data support a new model in which huntingtin directly binds membranes through electrostatic interactions with acidic phospholipids. Huntingtin (htt) 2The abbreviations used are:htthuntingtinHDHuntington diseaseHIPhuntingtin interactor proteinN-httN-terminal httPHpleckstrin homologyPIphosphatidylinositolPI(4,5)P2phosphatidylinositol 4,5-bisphosphatePLCphospholipase CPR65/Ap65 regulatory A subunitPMplasma membraneaaamino acid(s)Abanti-bodymAbmonoclonal antibodyANOVAanalysis of varianceGSTglutathione S-transferase exists predominantly in the cytoplasm as a soluble protein that associates with the plasma membrane and multiple membranous organelles and vesicles (1DiFiglia M. Sapp E. Chase K. Schwarz C. Meloni A. Young C. Martin E. Vonsattel J.P. Carraway R. Reeves S.A. Boyce F.M. Aronin N. Neuron. 1995; 14: 1075-1081Abstract Full Text PDF PubMed Scopus (619) Google Scholar, 2Sharp A.H. Loev S.J. Schilling G. Li S.H. Li X.J. Bao J. Wagster M.V. Kotzuk J.A. Steiner J.P. Lo A. Hedreen J. Sisodia S. Snyder S.H. Dawson T.M. Ryugo D.K. Ross C.A. Neuron. 1995; 14: 1065-1074Abstract Full Text PDF PubMed Scopus (433) Google Scholar, 3Velier J. Kim M. Schwarz C. Kim T.W. Sapp E. Chase K. Aronin N. DiFiglia M. Exp. Neurol. 1998; 152: 34-40Crossref PubMed Scopus (247) Google Scholar). Many htt binding partners function in membrane trafficking (4Harjes P. Wanker E.E. Trends Biochem. Sci. 2003; 28: 425-433Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar). A specific molecular function for htt at membranes has not been demonstrated. The large size of htt (348 kDa) and interactions with numerous membrane-associated proteins suggest that htt may function as a scaffold. huntingtin Huntington disease huntingtin interactor protein N-terminal htt pleckstrin homology phosphatidylinositol phosphatidylinositol 4,5-bisphosphate phospholipase C p65 regulatory A subunit plasma membrane amino acid(s) anti-body monoclonal antibody analysis of variance glutathione S-transferase A polyglutamine expansion in the N terminus of htt (N-htt) causes neurodegeneration in Huntington disease (HD) and accumulation of htt in neurons. Degradation pathways for htt include endosomal-lysosomal and autophagic pathways and may require targeting to membranes to initiate clearance (5Kegel K.B. Kim M. Sapp E. McIntyre C. Castano J.G. Aronin N. DiFiglia M. J. Neurosci. 2000; 20: 7268-7278Crossref PubMed Google Scholar, 6Qin 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). Therefore, knowledge of the effects of polyglutamine expansion on htt membrane targeting is important for understanding HD pathogenesis. N-htt has a membrane association domain. Membrane fractions prepared from control and HD brains (7Kim Y.J. Yi Y. Sapp E. Wang Y. Cuiffo B. Kegel K.B. Qin Z.H. Aronin N. DiFiglia M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12784-12789Crossref PubMed Scopus (327) Google Scholar) or prepared from cells expressing exogenous htt (5Kegel K.B. Kim M. Sapp E. McIntyre C. Castano J.G. Aronin N. DiFiglia M. J. Neurosci. 2000; 20: 7268-7278Crossref PubMed Google Scholar) contain N-htt fragments (∼440-550 aa). In vitro translated normal and mutant htt (aa 1-548) localized to vesicles in extruded squid axoplasm (8Szebenyi G. Morfini G.A. Babcock A. Gould M. Selkoe K. Stenoien D.L. Young M. Faber P.W. MacDonald M.E. McPhaul M.J. Brady S.T. Neuron. 2003; 40: 41-52Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). The structural features that mediate membrane association of N-htt are unknown and could include a proline-rich Src homology 3-binding domain that lies immediately distal to the polyglutamine stretch and HEAT domains, which are repeated regions of low homology shared by huntingtin with elongation factor 3, the p65 regulatory A subunit of protein phosphatase 2A, and TOR1 and a host of other proteins (9Andrade M.A. Bork P. Nat. Genet. 1995; 11: 115-116Crossref PubMed Scopus (466) Google Scholar). The function of HEAT domains is unknown, but these repeated regions are leucine-rich and predicted to be α-helical in nature, with each repeat consisting of two helices and a short intervening linker. Crystallography studies of the protein phosphatase 2A p65 regulatory subunit (PR65/A) indicate that its multiple HEAT domains create a superhelical structure (10Groves M.R. Hanlon N. Turowski P. Hemmings B.A. Barford D. Cell. 1999; 96: 99-110Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar). htt has three domains at aa 205-329, 745-942, and 1534-1710 containing a total of 10 HEAT repeats (9Andrade M.A. Bork P. Nat. Genet. 1995; 11: 115-116Crossref PubMed Scopus (466) Google Scholar). In this study, we identify sequences in N-htt important for binding to membranes, explore mechanisms by which this binding occurs, and investigate the effect of polyglutamine expansion in N-htt membrane targeting. Cell Culture and Transfections—COS-1 cells (monkey epithelial) and MCF-7 cells (human epithelial) (each obtained from American Type Culture Collection) and X57 (mouse immortalized clonal striatal cells (11Wainwright M.S. Perry B.D. Won L.A. O'Malley K.L. Wang W.Y. Ehrlich M.E. Heller A. J. Neurosci. 1995; 15: 676-688Crossref PubMed Google Scholar)) were cultured and transfected as previously described (5Kegel K.B. Kim M. Sapp E. McIntyre C. Castano J.G. Aronin N. DiFiglia M. J. Neurosci. 2000; 20: 7268-7278Crossref PubMed Google Scholar, 12Kim M. Lee H.S. LaForet G. McIntyre C. Martin E.J. Chang P. Kim T.W. Williams M. Reddy P.H. Tagle D. Boyce F.M. Won L. Heller A. Aronin N. DiFiglia M. J. Neurosci. 1999; 19: 964-973Crossref PubMed Google Scholar). Immunofluorescence, Confocal Analysis, and Cell Counting—Immunostaining and confocal microscopy was performed as previously described using paraformaldehyde fixation (5Kegel K.B. Kim M. Sapp E. McIntyre C. Castano J.G. Aronin N. DiFiglia M. J. Neurosci. 2000; 20: 7268-7278Crossref PubMed Google Scholar, 12Kim M. Lee H.S. LaForet G. McIntyre C. Martin E.J. Chang P. Kim T.W. Williams M. Reddy P.H. Tagle D. Boyce F.M. Won L. Heller A. Aronin N. DiFiglia M. J. Neurosci. 1999; 19: 964-973Crossref PubMed Google Scholar). Individual images for each excitation wavelength (405, 488, or 568 nm) were obtained sequentially using a Bio-Rad Radiance 2100 confocal laser with krypton-argon and blue diode lasers through a 100× Nikon Plan Apo objective (numerical aperture 1.4) with oil immersion on an inverted Nikon Eclipse TE300 fluorescent microscope. Concentrations for primary antibodies were as follows: anti-htt Ab 1 (1 μg/ml) and Ab 585 (1:1000) (1DiFiglia M. Sapp E. Chase K. Schwarz C. Meloni A. Young C. Martin E. Vonsattel J.P. Carraway R. Reeves S.A. Boyce F.M. Aronin N. Neuron. 1995; 14: 1075-1081Abstract Full Text PDF PubMed Scopus (619) Google Scholar); anti-FLAG mAb M5 (10 μg/ml; Sigma). Secondary antibodies were used at 1:1000 and included Cy3 conjugates (Jackson ImmunoResearch, West Grove, PA) and Bodipy FL conjugates (Molecular Probes, Inc., Eugene, OR). Analyses were performed using a ×60 oil objective by an examiner unaware of the experimental conditions. 100 transfected cells per coverslip were counted on three coverslips per condition (n = 3). Transfected cells were scored positive if >50% of the plasma membrane had labeling when viewed through a cross-section containing the nucleus. Statistical significance was determined using ANOVA and a post hoc Bonferroni test using Graphpad InStat software. For neomycin treatment, transfected cells on coverslips were treated with 10 neomycin in for to transfected cells per coverslip were scored for >50% plasma membrane on coverslips per condition (n = For cells were with and with (Molecular in at for were with to fixation with paraformaldehyde and for as described was by cells with the using a oil objective transfected cells and cells per Statistical significance was determined using a a control N-terminal protein which as a for N-terminal and other of protein D.K. Cell 2003; 19: PubMed Scopus Google was proteins in have been previously described (5Kegel K.B. Kim M. Sapp E. McIntyre C. Castano J.G. Aronin N. DiFiglia M. J. Neurosci. 2000; 20: 7268-7278Crossref PubMed Google Scholar, 12Kim M. Lee H.S. LaForet G. McIntyre C. Martin E.J. Chang P. Kim T.W. Williams M. Reddy P.H. Tagle D. Boyce F.M. Won L. Heller A. Aronin N. DiFiglia M. J. Neurosci. 1999; 19: 964-973Crossref PubMed Google Scholar). The of the to the N terminus of htt not the of htt in cells M. Lee H.S. LaForet G. McIntyre C. Martin E.J. Chang P. Kim T.W. Williams M. Reddy P.H. Tagle D. Boyce F.M. Won L. Heller A. Aronin N. DiFiglia M. J. Neurosci. 1999; 19: 964-973Crossref PubMed Google Scholar) or normal htt function in a The of the amino is on htt with new with were using and For an htt was into fluorescent protein The in vitro was used to proteins were using and to of htt by and The of and was by and and analysis were performed as described previously (5Kegel K.B. Kim M. Sapp E. McIntyre C. Castano J.G. Aronin N. DiFiglia M. J. Neurosci. 2000; 20: 7268-7278Crossref PubMed Google Scholar). in 10 1 were at to the and the was at to the membrane and the were with 10 of protein from each of the protein was used to the of For the of antibodies were anti-htt Ab 1 anti-htt mAb and antibodies (Jackson were were using data from at three were using a was at the molecular were Statistical significance was determined using an ANOVA and post hoc Bonferroni were performed as described K.B. Meloni Yi Y. Kim Y.J. E. Cuiffo Sapp E. Wang Y. Qin Z.H. Aronin N. DiFiglia M. J. Full Text Full Text PDF PubMed Scopus Google Scholar) using anti-FLAG mAb were with anti-htt interactor protein 1 monoclonal antibody or anti-htt Ab and of expressing in were in and with protein was using a were into 1 1 and at to in of the proteins was determined by and and were from The experimental of 1 1 and at were prepared by pure or of in lipid vesicles were prepared by a prepared lipid 10 through two of an were at 10 the M. C. E. G. PubMed Scopus Google Scholar). were in a Inc., to the of the at for containing of with or were into the and were performed by the at a of were at The of a and was as the the The was as the at which the specific a the of the to of lipid the of which the and the of the which the were protein was performed as described J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) using Y. S. J. Full Text Full Text PDF PubMed Scopus Google Scholar). from cells expressing htt were prepared in were to or in containing of exogenous htt was by with were at in and as described J. 1998; Full Text Full Text PDF PubMed Scopus Google using anti-htt Ab 1 mAb antibody and using were performed at three 100 of lipid and included phosphatidylinositol 4,5-bisphosphate and aa was to a to sequences capable of lipid binding M. G. E. J. 1995; Full Text PDF PubMed Scopus Google Scholar). htt was the polyglutamine stretch with the The analysis that amphipathic helices and are by regions in positive amphipathic helix of three a that could into membranes and with the of an for and a that be to the of Membrane in studies that normal endogenous and membrane and soluble fractions in clonal striatal cells (5Kegel K.B. Kim M. Sapp E. McIntyre C. Castano J.G. Aronin N. DiFiglia M. J. Neurosci. 2000; 20: 7268-7278Crossref PubMed Google Scholar). We performed of cells expressing a of N-terminal htt from in cells in and in the membrane fractions and had a or were in the in the and fractions In was in the the and analysis of the soluble and membrane fractions a in for with = post hoc Bonferroni and with = post hoc Bonferroni of the and not the of N-htt and suggest that a domain for membrane binding in the N-htt and analysis of N-htt of fractions and from cells expressing huntingtin htt was with antibody Ab 1 for which was with mAb of of in is to the of protein was per and fractions from cells expressing the was with mAb was with Ab for each as the of the to the of and at when with and when with (n = ANOVA and post hoc Bonferroni confocal microscopy of clonal striatal and MCF-7 cells expressing or protein was with anti-FLAG mAb confocal microscopy of MCF-7 cells expressing and protein was with anti-FLAG mAb in confocal of the but through a at the of the nucleus. of X57 cells with >50% by with at (n = 3, ANOVA and post hoc Bonferroni of MCF-7 cells with >50% by with at (n = 3, ANOVA and post hoc Bonferroni of N-htt by Confocal cells expressing or in on the with a of in the cytoplasm for in clonal striatal cells which have a and in MCF-7 cells In was in the in with and was in The of transfected cells with >50% on the was in cells expressing in expressing = 3, post hoc and In with the these suggest that a domain for membrane binding in N-htt a targeting for htt to the may htt and of N-htt to the effects of polyglutamine expansion on the membrane targeting of we mutant and in MCF-7 to the or in the cytoplasm A in cells with >50% was in cells expressing with = 3, post hoc Bonferroni a to and was from the nucleus. A of cells with >50% was with with = 3, post hoc Bonferroni were obtained in clonal striatal cells not data suggest that polyglutamine expansion normal targeting of htt to the htt to htt directly to we performed J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). that endogenous htt and htt and with included with or two in htt an for htt to and to not from cells expressing mutant or a of lipid binding as for A monoclonal antibody specific for expanded polyglutamine mutant N-htt on the of not that and mutant htt with acidic in The acidic is enriched in the M.R. P. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). in cells, the pleckstrin homology domain membrane binding of the protein to binds to on the of the P. J. Cell 1998; PubMed Scopus Google Scholar). with this we that targeted to the plasma membrane in MCF-7 this was by the from to of amino which is for binding to as previously described P. J. Cell 1998; PubMed Scopus Google Scholar) of with in MCF-7 cells of the two proteins that in cells N-htt to plasma membrane domains enriched in and in cells K. M. D. P. J. Cell 2000; PubMed Scopus Google Scholar). of MCF-7 cells expressing in a in the of cells with >50% = these data support the that htt associates with acidic and to domains in Membrane of htt by amino and in htt is in and a of lipid binding proteins as A. A. G. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The aa the positive are in the and htt An analysis of htt using a to identify membrane binding regions M. G. E. J. 1995; Full Text PDF PubMed Scopus Google Scholar) that htt and have a of that could to membranes by electrostatic interactions with phospholipids. The analysis identified an intervening (aa enriched in predicted to an amphipathic a structure capable of membrane The amphipathic helix is in and in and two to and in the and a of an amphipathic In the structure of htt was predicted to be α-helical which membrane htt to the in analysis that was from membranes with of the predicted amphipathic helix (aa and of the (aa in htt test the of the the amphipathic helix in membrane we aa in to the to The of cells with >50% was in cells expressing with cells with = 3, Bonferroni and analysis of the and of (aa which the amphipathic was to not these data suggest that the regions the amphipathic helix a of htt binding to membranes. We that of aa in was to membrane not that sequences htt to membrane Therefore, we aa which the three HEAT repeats (aa a stretch of that a by positive a of (aa and analysis that was to the soluble = Bonferroni membrane binding was to the of positive aa and binding may be to Confocal that was the cytoplasm with to to acidic in a not that regions in htt in aa and and aa to membrane the three HEAT repeats in we a htt to In MCF-7 cells, microscopy that was in the cytoplasm and localized to vesicles and the that htt are for membrane association. A htt into the predicted amphipathic helix (aa was not for membrane its into the membrane could serve to anchor htt to the lipid association. In a in the specific to a of membrane and were and of the of lipid vesicles in a the of or had The for a of the and with protein the protein of the from to The of and the of the by indicate protein that aa in htt into pure lipid bilayers and may anchor htt to membranes. of the htt Membrane is or data of the structure of we to and data for htt in a structural We the three HEAT repeats in htt important for membrane we a three-dimensional structure of htt on and HEAT homology to the determined structure of the protein phosphatase 2A (10Groves M.R. Hanlon N. Turowski P. Hemmings B.A. Barford D. Cell. 1999; 96: 99-110Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar, J. N. 2003; PubMed Scopus Google using multiple to identify an model for the of the binding in htt and these are from the model The model that which we into pure lipid to the of a as as a in in A and and of htt are a structure that may be The predicted helix is amphipathic as for a structure in membrane association A of the electrostatic for the a positive that the predicted membrane binding domain consistent with that htt binds 3). the model amino of the huntingtin structural are consistent with an electrostatic mechanism of binding to of on the is for of and J. Cell 1999; PubMed Scopus Google Scholar, D. Wang Y.J. M. J. Cell 2003; PubMed Scopus Google Scholar). In the domain localized with in domains, which are to be of S. L. R. K. Mol. Cell. PubMed Scopus Google Scholar). with of on the and in at the of cells was with and an control N-terminal protein which in the cytoplasm endogenous htt with in that binding of to the a normal targeting for with by had with cells = that may a of htt capable of normal of htt and htt with is an protein that with htt on vesicles S. E. R. E. R. C. K. Wanker E.E. Hum. Mol. Genet. 2001; PubMed Google is capable of binding D. Ross J. Full Text Full Text PDF PubMed Scopus Google Scholar, Ross L.M. J. 2001; Full Text Full Text PDF PubMed Scopus Google and and to membranes S. E. R. E. R. C. K. Wanker E.E. Hum. Mol. Genet. 2001; PubMed Google Scholar, Ross L.M. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). was identified in a using as htt E.E. C. E. R. S. D. J. Hum. Mol. Genet. PubMed Scopus Google Scholar) or htt M.A. K. K. K. P. C. M. M.R. Nat. Genet. PubMed Scopus Google Scholar). an with was for htt to with membranes, we performed with cells expressing and or with but not with or The suggest that interactions with are not for association of htt with membranes. We have a protein domain in aa important for membrane association and for targeting N-htt to the may htt to the of this are consistent with membrane binding through electrostatic interactions with acidic phospholipids. of htt to at the is consistent with the that htt is in expansion targeting of of Membrane data that a in N-htt containing multiple to membrane association. we that htt binds to in The helical HEAT domain in htt that membrane association is to N-terminal homology domains, which in J. P. J. Cell 2000; PubMed Scopus Google Scholar) and Y. J. J.A. B. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). α-helical superhelical an electrostatic mechanism to in the of an amphipathic helix has been to binding to the membrane M.A. 2003; PubMed Scopus Google Scholar). Our with that the protein the predicted amphipathic helix into membranes. of aa in htt not binding to membranes, of regions on We that htt binds to acidic through an electrostatic using the of positive the amphipathic helix may into the to an lipid binding proteins that electrostatic mechanisms to membranes helices or a and domain into the lipid to an anchor M.A. 2003; PubMed Scopus Google Scholar, Trends Biochem. Sci. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). In the at predicted to an (aa could its into the to an htt is a soluble may a in to a association with the to other proteins S. A. J. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). Our are consistent with binding of N-htt to membranes through electrostatic other mechanisms of membrane binding are for this domain. For the we have could be a that htt to a membrane-associated protein other to and to by that htt associates with with this htt has been on of membranes in cells, each of which contain (1DiFiglia M. Sapp E. Chase K. Schwarz C. Meloni A. Young C. Martin E. Vonsattel J.P. Carraway R. Reeves S.A. Boyce F.M. Aronin N. Neuron. 1995; 14: 1075-1081Abstract Full Text PDF PubMed Scopus (619) Google Scholar, 2Sharp A.H. Loev S.J. Schilling G. Li S.H. Li X.J. Bao J. Wagster M.V. Kotzuk J.A. Steiner J.P. Lo A. Hedreen J. Sisodia S. Snyder S.H. Dawson T.M. Ryugo D.K. Ross C.A. Neuron. 1995; 14: 1065-1074Abstract Full Text PDF PubMed Scopus (433) Google Scholar, 3Velier J. Kim M. Schwarz C. Kim T.W. Sapp E. Chase K. Aronin N. DiFiglia M. Exp. Neurol. 1998; 152: 34-40Crossref PubMed Scopus (247) Google Scholar). For at the is on is and membranes are enriched in M.R. P. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). to other htt may with other proteins to create and binding M.R. P. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). We not an for and for was in the with studies be to htt at with or protein to create specific interactions or binding htt at the with the and with at in the is for plasma membrane of the protein and for J. Cell 1999; PubMed Scopus Google Scholar, D. Wang Y.J. M. J. Cell 2003; PubMed Scopus Google Scholar). htt has been on vesicles the plasma membrane using J. Kim M. Schwarz C. Kim T.W. Sapp E. Chase K. Aronin N. DiFiglia M. Exp. Neurol. 1998; 152: 34-40Crossref PubMed Scopus (247) Google Scholar) and with on vesicles from S. E. R. E. R. C. K. Wanker E.E. Hum. Mol. Genet. 2001; PubMed Google Scholar). on on the in clonal striatal cells (5Kegel K.B. Kim M. Sapp E. McIntyre C. Castano J.G. Aronin N. DiFiglia M. J. Neurosci. 2000; 20: 7268-7278Crossref PubMed Google Scholar). are consistent with the that htt in with htt is to as a for other proteins to membrane or for protein The in with of htt be to an of htt to with htt may for and with other binding proteins for of was in expressing which in large membrane S. L. R. K. Mol. Cell. PubMed Scopus Google Scholar). to that polyglutamine expansion N-htt to the We in binding of or mutant N-htt fragments in lipid that the of mutant N-htt to the on these be of mutant htt with in We be that the soluble used in the the fragments in mutant N-htt may have a for for or binding to an interactor which mutant N-htt of proteins to the plasma membrane. For the protein subunit is through the at the plasma membrane Y. L.A. Lee M.J. J. Full Text Full Text PDF PubMed Scopus Google Scholar). N-terminal htt fragments are through endosomal-lysosomal and autophagic pathways (5Kegel K.B. Kim M. Sapp E. McIntyre C. Castano J.G. Aronin N. DiFiglia M. J. Neurosci. 2000; 20: 7268-7278Crossref PubMed Google Scholar, 6Qin 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). We that htt may require at the plasma membrane to for targeting to clearance targeting of N-terminal mutant htt fragments to the plasma polyglutamine expansion may protein and to HD pathogenesis. We and P. of for normal and mutant and R. J. of for N-terminal protein 1

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