Systematic Uncovering of Multiple Pathways Underlying the Pathology of Huntington Disease by an Acid-cleavable Isotope-coded Affinity Tag Approach

Huntington disease (HD) is an autosomal dominant neurodegenerative disease that results from a CAG (glutamine) trinucleotide expansion in exon 1 of huntingtin (Htt). The aggregation of mutant Htt has been implicated in the progression of HD. The earliest degeneration occurs in the striatum. To identify proteins critical for the progression of HD, we applied acid-cleavable ICAT technology to quantitatively determine changes in protein expressions in the striatum of a transgenic HD mouse model (R6/2). The cysteine residues of striatal proteins from HD and wild-type mice were labeled, respectively, with the heavy and light forms of the ICAT reagents. Samples were trypsinized, uncovered by avidin affinity chromatography, and analyzed by nano-LC-MS/MS. Western blot analyses were used to confirm and to calibrate the ICAT ratios. Linear regression was used to uncover a group of proteins that exhibited consistent changes. In two independent ICAT experiments, we identified 427 cysteine-containing striatal proteins among which ∼66% (203 proteins) were detected in both ICAT experiments. Approximately two-thirds of proteins identified in each ICAT experiment were detected in both ICAT experiments. In total, 68 proteins with altered expressions in HD mice were identified. Elevated expressions of two down-regulated proteins (14-3-3ς and FKBP12) effectively reduced Htt aggregates in a striatal cell line, supporting the functional relevance of the above findings. Collectively by using a well defined protocol for data analysis, large scale comparisons of protein expressions by ICAT can be reliable and can provide valuable clues for identifying proteins critical for pathophysiological functions. Huntington disease (HD) is an autosomal dominant neurodegenerative disease that results from a CAG (glutamine) trinucleotide expansion in exon 1 of huntingtin (Htt). The aggregation of mutant Htt has been implicated in the progression of HD. The earliest degeneration occurs in the striatum. To identify proteins critical for the progression of HD, we applied acid-cleavable ICAT technology to quantitatively determine changes in protein expressions in the striatum of a transgenic HD mouse model (R6/2). The cysteine residues of striatal proteins from HD and wild-type mice were labeled, respectively, with the heavy and light forms of the ICAT reagents. Samples were trypsinized, uncovered by avidin affinity chromatography, and analyzed by nano-LC-MS/MS. Western blot analyses were used to confirm and to calibrate the ICAT ratios. Linear regression was used to uncover a group of proteins that exhibited consistent changes. In two independent ICAT experiments, we identified 427 cysteine-containing striatal proteins among which ∼66% (203 proteins) were detected in both ICAT experiments. Approximately two-thirds of proteins identified in each ICAT experiment were detected in both ICAT experiments. In total, 68 proteins with altered expressions in HD mice were identified. Elevated expressions of two down-regulated proteins (14-3-3ς and FKBP12) effectively reduced Htt aggregates in a striatal cell line, supporting the functional relevance of the above findings. Collectively by using a well defined protocol for data analysis, large scale comparisons of protein expressions by ICAT can be reliable and can provide valuable clues for identifying proteins critical for pathophysiological functions. Huntington disease (HD) 1The abbreviations used are: HD, Huntington disease; Htt, Huntingtin; PKC, protein kinase C; FKBP12, FK506-binding protein, 12 kDa; PrxV, peroxiredoxin 5; CSPG, chondroitin sulfate proteoglycan protein; CREB, cAMP-response element-binding protein; WT, wild-type; RSD, relative standard deviation; hrGFP, humanized Renilla green fluorescent protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; 2DGE, two-dimensional gel electrophoresis; PDE, phosphodiesterase. is a hereditary neurodegenerative disease characterized by dementia, chorea, and psychiatric symptoms. The causative mutation is a CAG (glutamine) trinucleotide expansion in exon 1 of the huntingtin (Htt) gene. The normal Htt gene has 35 or fewer repeats in the N-terminal region, whereas the appearance of neurological symptoms is associated with 36 or more CAG repeats (1The Huntington’s Disease Collaborative Research GroupA novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington’s disease chromosomes. The Huntington’s Disease Collaborative Research Group.Cell. 1993; 72: 971-983Abstract Full Text PDF PubMed Scopus (7118) Google Scholar). The major characteristic of HD is regional degeneration of neurons in the striatum and cortex that leads to movement disorders and dementia (2Martin J.B. Gusella J.F. Huntington’s disease. Pathogenesis and management.N. Engl. J. Med. 1986; 315: 1267-1276Crossref PubMed Scopus (17) Google Scholar, 3Vonsattel J.P. Myers R.H. Stevens T.J. Ferrante R.J. Bird E.D. Richardson Jr., E.P. Neuropathological classification of Huntington’s disease.J. Neuropathol. Exp. Neurol. 1985; 44: 559-577Crossref PubMed Scopus (2093) Google Scholar). The toxicity of Htt in specific neurons correlates with the length of polyglutamine expansion (4Davies 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. Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation.Cell. 1997; 90: 537Abstract Full Text Full Text PDF PubMed Scopus (1918) Google Scholar). The of mutant Htt with expansion a of M. in the of Huntington’s Full Text Full Text PDF PubMed Scopus Google Scholar, M. E. S.W. Bates G.P. J.P. of huntingtin in neuronal intranuclear inclusions and in 1997; PubMed Scopus Google Scholar). protein has been a major Scherzinger E. Wanker E.E. of mutant huntingtin in a of protein PubMed Scopus Google Scholar). Htt aggregates were to of protein and and of the and proteins M. of and with N-terminal in of aggregation and PubMed Scopus Google Scholar, J. J. J. M. and gene changes to cell in an cell model of Huntington’s PubMed Google Scholar, J. J. J. of protein and on protein aggregation in of Huntington’s PubMed Scopus Google Scholar, J.P. of proteins in aggregates from with polyglutamine PubMed Scopus Google Scholar). In dysfunction by mutant Htt is critical for polyglutamine a PubMed Scopus Google Scholar, M. M. M. mutant and and cell Full Text Full Text PDF PubMed Scopus Google Scholar). Htt with expansion was to with proteins for the CREB, protein, and M. Wanker E.E. Bates G.P. The Huntington’s disease protein with and protein and PubMed Scopus Google Scholar, Jr., M. M. J. Ross C.A. by huntingtin and with to PubMed Scopus Google Scholar, and in Huntington’s PubMed Scopus Google Scholar, E. E. DiFiglia M. is in the with the protein, and Full Text Full Text PDF PubMed Scopus Google Scholar, M. of Huntington disease protein with PubMed Scopus Google Scholar, cAMP-response element-binding protein to of the by mutant huntingtin with expanded polyglutamine Full Text Full Text PDF PubMed Scopus Google Scholar). changes specific analyses that expressions of a of altered Ross C.A. J. of striatal in a mouse model of Huntington’s PubMed Scopus Google Scholar). be that the the toxicity by mutant Htt L. of huntingtin and polyglutamine and of PubMed Scopus Google Scholar). aggregation is with HD and M. M. M. in a mouse model of Huntington Med. PubMed Scopus Google Scholar, symptoms of Huntington’s disease in a transgenic mouse PubMed Scopus Google from that the toxicity by mutant Htt with expansion M. of mutant huntingtin and the of neuronal PubMed Scopus Google Scholar, M. J.P. J. and of neurons by Full Text Full Text PDF PubMed Scopus Google Scholar). with the above M. toxicity of polyglutamine expansion of Full Text Full Text PDF PubMed Scopus Google that or of mutant Htt were to protein, an Collectively the of Htt aggregates of HD is and to be large scale analyses of changes in HD been the of a of analyses of proteins using two-dimensional gel been on HD M. J.B. of protein and in transgenic a model of Huntington Full Text Full Text PDF PubMed Scopus Google Scholar). to the protein in the striatum of HD mice using a two for of protein using affinity PubMed Scopus Google Scholar, J. E. of protein using PubMed Scopus Google Scholar). an acid-cleavable ICAT has to be used that of the ICAT R.J. J. of protein using affinity and Full Text Full Text PDF PubMed Scopus Google Scholar, of in PubMed Scopus Google Scholar). In the 427 cysteine-containing striatal proteins were detected from two independent ICAT experiments. Western blot analyses were used to confirm and to calibrate the ICAT data by a regression to the In total, changes in the expressions of 68 proteins were was that proteins) were whereas proteins) were disease of proteins in the striatum of HD mice were using Western blot The functional relevance of two down-regulated proteins (14-3-3ς and FKBP12) was by to Htt that large scale using ICAT is a reliable for identifying proteins critical for the of HD. were from and were from mice and were from The and were to mice were identified by a symptoms of Huntington’s disease in a transgenic mouse PubMed Scopus Google Scholar). In total, transgenic mice and wild-type mice were used in were the of a were using by the and In total, transgenic mice and mice were used in each striatal from two to mice were in of 1 and and in were for 1 to the and for to the the and the the The were in of 1 and and for on were by for to the The protein was using the protein protocol was from using the mouse model of HD disease E. Turmaine M. Mangiarini L. Bates G.P. S.W. Wanker E.E. polyglutamine protein aggregates in and in 1997; 90: Full Text Full Text PDF PubMed Scopus Google or cell with large aggregates cAMP-response element-binding protein to of the by mutant huntingtin with expanded polyglutamine Full Text Full Text PDF PubMed Scopus Google Scholar). The of Htt aggregates were detected in the in the or the and In Western blot analyses using a and a the of the of the was striatal of of proteins a of was In total, 12 mice and mice were used in two independent ICAT The protein were above and with ICAT the striatal proteins from and HD mice were labeled, respectively, with light and heavy ICAT reagents. The were and with using an of The was by Samples were using a a of were with and for by a from to and were and to affinity using a avidin The were with to the and by a of The was analyzed by of the on an with PubMed Scopus Google Scholar). by was The of the and were and by was in using the were analyzed using in to data of with in a protein PubMed Scopus Google Scholar). The of the and were the The was from the of the for and analyzed by E. model to the of by and PubMed Scopus Google J. of proteins using affinity and PubMed Scopus Google and E. model for identifying proteins by PubMed Scopus Google Scholar). was used to the of and was used to the of the The of and were The for both ICAT was was using of protein from data by and PubMed Scopus Google Scholar). The above were by for The of proteins were for and protein the were the to the protein which were to the using that effectively of the proteins were to or changes in In the detected were in of protein were The of the proteins in and to the that in a and in a the of for PubMed Scopus Google a of detected protein is in using In ICAT cysteine was used to the of proteins were identified by is consistent with using and the of of the proteins for which was detected in each ICAT experiment in green and of proteins to be using a and of striatal proteins or down-regulated in HD mice with mice by ICAT in a protein by Western blot with a relative standard of were used to calibrate the ICAT in in the of of were using In total, mice and were used in the Western blot were and mice in the that were and to independent of proteins for each were from two to striatal above and used for the Western blot of protein were by using to the of of proteins the of the of PubMed Scopus Google Scholar). The proteins were were with in and with an kinase and by the for 1 were detected by and using for the relative were by the of the protein with that of the and of that of were by from to independent using the The an N-terminal of Htt with the of residues to were cAMP-response element-binding protein to of the by mutant huntingtin with expanded polyglutamine Full Text Full Text PDF PubMed Scopus Google Scholar). of and were from a mouse by using in and the using a The striatal cell was a from E. of and was in an with and L. M. L. E. of a Neurol. PubMed Scopus Google Scholar). The were a a of were in for and to a of of and of the for in were with by more with normal and were above for and of mutant Htt aggregates were E.E. Scherzinger E. for of protein PubMed Scopus Google Scholar). or striatal were in 1 1 1 and and for 1 the was and for The was in of and with of and for proteins were applied to and in a were with in and with an by the for 1 were detected by and using was using the Research with to the of and using was using a on a using The of used for and for and and for and were cAMP-response element-binding protein to of the by mutant huntingtin with expanded polyglutamine Full Text Full Text PDF PubMed Scopus Google Scholar). The relative of the which was using standard of was to that of of the To identify proteins for the disease progression of HD, we applied ICAT technology to quantitatively the protein in the striatum of HD an which symptoms of HD in and Htt symptoms of Huntington’s disease in a transgenic mouse PubMed Scopus Google Scholar). using two-dimensional gel that the of proteins in the of HD mice M. J.B. of protein and in transgenic a model of Huntington Full Text Full Text PDF PubMed Scopus Google Scholar). To the and the ICAT J. of the in PubMed Google we to determine the of the striatum. The used in the has been used in of HD mice a of disease progression (4Davies 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. Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation.Cell. 1997; 90: 537Abstract Full Text Full Text PDF PubMed Scopus (1918) Google and for cell with large aggregates cAMP-response element-binding protein to of the by mutant huntingtin with expanded polyglutamine Full Text Full Text PDF PubMed Scopus Google Scholar). Western blot analyses using a and a of the by the was Western blot analyses that was in the protein of in the striatum of mice of in from independent we used an for the Western blot independent ICAT analyses of the striatal proteins from and wild-type mice were Approximately proteins were identified in each two-thirds of the identified proteins in each ICAT experiment were in both In total, proteins were identified. to of in affinity chromatography, of of the identified The of cysteine-containing proteins identified in and were and was for proteins containing cysteine two-thirds (203 of cysteine-containing proteins were detected in both and were analyzed using an of protein from data by and PubMed Scopus Google to a for each protein The of the for experiment and experiment was critical for To the the the on the that the major protein in a is which by the of the to 1 in more the of proteins be altered disease the is to be in by the of the to 1 above be and the to be To for the to we Western blot analyses of proteins specific were to determine and the The Western blot was an to the of the ICAT is and in To a of proteins with an of were used to calibrate the The was in was used the to a the of the of the changes. In the for proteins were to the a in To the of for disease in which the of a of proteins and in be that the of changes for down-regulated proteins FKBP12, and were with ICAT analyses of the proteins the of the we applied to cysteine-containing proteins detected in both ICAT and used the for of the ICAT using results from Western blot analyses in a that the of each protein is of the with in the protein on the of proteins in the the the of a protein the of the the data the of each protein the independent ICAT experiments, and with the of the of in experiment and experiment were and were been a the of protein in HD mice from both ICAT were well we that be a protein for The Western blot of results that of ICAT analyses is and that two of ICAT data provide an to the a regression of the of experiment and experiment The was and the was The of the two independent ICAT were The from was that results from experiment and experiment a regression on the of both was the and were proteins exhibited in the two ICAT experiments, proteins in ICAT whereas the proteins were the in both ICAT experiments. The that the proteins to be is in the two ICAT in been to in the of the the data and a the ICAT To the of identifying proteins with the that the of in the two ICAT were for that proteins a of with the proteins that were by the regression we analyzed the proteins that in the in the two independent ICAT for changes in on the results of Western blot analyses of we that a was an to a proteins with a of or in with of in the and that been to from were from the the 68 proteins were whereas the proteins were down-regulated in the striatum of HD To the of of protein kinase and been implicated in HD M. J.B. of protein and in transgenic a model of Huntington Full Text Full Text PDF PubMed Scopus Google Scholar, and protein of in HD transgenic of on PubMed Scopus Google Scholar, kinase in the striatum and cortex of transgenic Huntington’s disease Google Scholar, J. Ferrante R.J. and in Huntington’s Med. PubMed Scopus Google Scholar, J. of in the of mutation PubMed Google Scholar). on from protein of and a for the of proteins and Full Text Full Text PDF PubMed Scopus Google and PubMed the proteins proteins In total, down-regulated proteins and proteins were in the striatum of HD is consistent with an using a in which the of down-regulated Ross C.A. J. of striatal in a mouse model of Huntington’s PubMed Scopus Google Scholar). results the that the of HD, in protein expressions the of The major in the down-regulated were and in and In a by M. J.B. of protein and in transgenic a model of Huntington Full Text Full Text PDF PubMed Scopus Google proteins from the striatal of HD mice were identified using a of and proteins were and of proteins and were to be reduced in with that ICAT is of identifying more proteins gel electrophoresis; a in the PubMed Scopus Google we more striatal proteins in the using ICAT (203 In the of striatal proteins with changes was 68 and was that in the using proteins gel electrophoresis; a in the PubMed Scopus Google that the of the HD mice in the was that used in the and that both and that from identifying proteins In the be to two and identify more proteins changes HD we Western blot analyses of proteins to and calibrate the by results from both ICAT and Western blot analyses that and were whereas FKBP12, and kinase were down-regulated in the striatum of HD mice changes in two proteins and were detected using Western blot analyses were in the ICAT analyses a was used the to a that the ICAT analyzed and is for the of proteins changes. the functional relevance of the above with Western blot analyses for FKBP12, changes in of the proteins in the striatum HD mice were the of symptoms of in HD mice was and was reduced to of that of mice the of of proteins in the striatum to be associated with disease progression of HD. is of the is a that with more proteins for the cell and proteins in the PubMed Scopus Google Scholar, J. of with in Full Text Full Text PDF PubMed Scopus Google Scholar). of in the in mutant was to be associated with proteins J. The protein forms a with protein and Neuropathol. Exp. Neurol. PubMed Scopus Google Scholar, L. M. Richardson protein J. PubMed Scopus Google Scholar, protein kinase and 1 Full Text Full Text PDF PubMed Scopus Google Scholar). of proteins has been to the aggregation and toxicity of mutant Htt M. of and with N-terminal in of aggregation and PubMed Scopus Google Scholar, J. protein polyglutamine toxicity and the of by PubMed Scopus Google Scholar). that the of an in the of Htt results of the that of reduced the aggregation of mutant Htt with expanded and and to on HD in a by and The of a of proteins in disease PubMed Scopus Google was and proteins and can be used to toxicity by protein that was degeneration in the striatum of HD mice was detected is a protein of that is to neurons and an J.F. novel and Neurol. PubMed Scopus Google Scholar, L. of in a model of cell of and protein 12 PubMed Scopus Google Scholar). In and a and the expressions of proteins of the in of 1 in PubMed Scopus Google Scholar). To functional we in a striatal cell a mutant Htt with expanded Elevated reduced the of mutant Htt aggregates and In of for M. The protein gene in the and a protein PubMed Scopus Google Htt The used by and to aggregates and was the of the above that the of by ICAT is to be and provide valuable for the of HD the functional of and FKBP12, the ICAT analyses a of proteins that critical in the of HD. the reduced of protein by ICAT is in with that the of is reduced in the striatum of both HD and HD mice kinase in the striatum and cortex of transgenic Huntington’s disease Google Scholar, The of kinase in the striatum from with Huntington’s PubMed Scopus Google Scholar). in and in in HD Myers in mice the Huntington’s disease PubMed Scopus Google Scholar, E. M. striatal for the neuronal in Huntington’s PubMed Scopus Google Scholar). was the reduced of which a critical in of the and The and major in PubMed Scopus Google Scholar, the PubMed Scopus (17) Google Scholar). In HD, is a that of dysfunction cell toxicity E. J. and to toxicity in Huntington’s PubMed Scopus Google Scholar, of huntingtin a of to an with relevance to Huntington’s disease.J. Google Scholar). with the of the and results of ICAT analyses that the expressions of proteins were reduced that proteins M. a for and affinity of repeats and Full Text Full Text PDF PubMed Scopus Google Scholar, neuronal in and the to the Full Text Full Text PDF PubMed Scopus Google Scholar, the protein PubMed Scopus Google and that the by mutant In the of in the striatum of HD mice was reduced of has been implicated in the of and of proteins J. of Full Text Full Text PDF PubMed Scopus Google Scholar). to the of proteins in HD and in the novel and Neurol. PubMed Scopus Google Scholar, R.J. J. by the neurodegenerative in Huntington’s disease PubMed Google Scholar). The in is of the of and of and in HD was and protein reduced in Huntington’s disease transgenic mice to the of PubMed Scopus Google Scholar). of in HD is to dysfunction of and functions. of that dysfunction in HD. expressions of proteins in were in proteins in and the and and M. J.B. of protein and in transgenic a model of Huntington Full Text Full Text PDF PubMed Scopus Google Scholar, J. of in the of mutation PubMed Google Scholar, of Full Text Full Text PDF PubMed Scopus Google Scholar). The of and to to the neurodegenerative of HD. The in the of a protein to glyceraldehyde-3-phosphate was was the protein and of striatal Ferrante R.J. striatal 1997; PubMed Scopus Google Scholar, Jr., of neuronal intranuclear inclusions by of and toxicity a of polyglutamine PubMed Google Scholar). that to of the occurs of glyceraldehyde-3-phosphate in Huntington’s disease PubMed Scopus Google Scholar). In of the and toxicity of Ross C.A. and by PubMed Scopus Google Scholar). The protein is with which in to the of the protein and the functional of in HD Collectively ICAT provide valuable for functional that of mutant Htt results in by aggregates or with a of or M. Wanker E.E. Bates G.P. The Huntington’s disease protein with and protein and PubMed Scopus Google Scholar, Jr., M. M. J. Ross C.A. by huntingtin and with to PubMed Scopus Google Scholar, and in Huntington’s PubMed Scopus Google Scholar, E. E. DiFiglia M. is in the with the protein, and Full Text Full Text PDF PubMed Scopus Google Scholar, M. of Huntington disease protein with PubMed Scopus Google Scholar, cAMP-response element-binding protein to of the by mutant huntingtin with expanded polyglutamine Full Text Full Text PDF PubMed Scopus Google Scholar). results of the analyses that the of and were down-regulated in the striatum of mice that of gene to the reduced protein in the with the protein of and in the were reduced an in protein ICAT with an in which analyses of the striatum of and mice were with of a reduced of in the striatum of mice and the of was reduced in the striatum of HD In the of proteins kinase kinase and that down-regulated in the ICAT were altered Ross C.A. J. of striatal in a mouse model of Huntington’s PubMed Scopus Google Scholar). a in a protein by Htt and of the reduced of proteins the of or more is a for by we that more of the identified whereas with The of with been to the of in the affinity In the length of identified by ICAT in The of identified by ICAT were and in experiment and experiment Approximately of the identified were in the of The length of is to the length of of the the of with by and PubMed Scopus Google Scholar). specific for protein and can be in the using the for in is and results in for The that ICAT containing cysteine and that were in the in the of ICAT In to the of and the of of and in on the was detected in the In and has two that can be in a in the we using ICAT in to ICAT has been to be proteins E.E. of and using affinity for PubMed Scopus Google protein, and to be from the ICAT of PubMed Scopus Google the is to the the 68 proteins with altered expressions by PrxV, and were in the striatum of whereas the proteins were the of proteins and down-regulated be to the degeneration the of HD, and in the HD has been to of neurons the and M. of of a chondroitin sulfate and of and protein kinase in Full Text Full Text PDF PubMed Scopus Google Scholar). in the striatum of HD mice a in protein, PrxV, has been to an and is to and of peroxiredoxin in of on and by Med. PubMed Scopus Google Scholar, B.A. of peroxiredoxin of PubMed Scopus Google Scholar). of in the striatum of HD mice disease progression is to be of a in HD was to that and the Western blot and in the striatum of HD protein in the and of proteins been implicated in the of and L. The in a for the of J. 1997; PubMed Scopus Google Scholar, L. G.P. protein with the and in the PubMed Scopus Google Scholar). of the striatum with an of in the of striatal be of to the of protein in the striatum of HD In we the ICAT of the striatum of HD using results from Western a regression of two independent ICAT the data analyses a of proteins altered by mutant Htt and to the of HD J. Huntington’s disease. and PubMed Scopus Google Scholar, J. L. of for of Huntington’s disease using PubMed Scopus Google Scholar). that of with to be for HD The of the HD and to the of on to and for in the ICAT and for the ICAT experiment analysis, and for analysis, and for the with

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.