Proteomic Characterization of Postmortem Amyloid Plaques Isolated by Laser Capture Microdissection

The presence of amyloid plaques in the brain is one of the pathological hallmarks of Alzheimer's disease (AD). We report here a comprehensive proteomic analysis of senile plaques from postmortem AD brain tissues. Senile plaques labeled with thioflavin-S were procured by laser capture microdissection, and their protein components were analyzed by liquid chromatography coupled with tandem mass spectrometry. We identified a total of 488 proteins coisolated with the plaques, and we found multiple phosphorylation sites on the neurofilament intermediate chain, implicating the complexity and diversity of cellular processes involved in the plaque formation. More significantly, we identified 26 proteins enriched in the plaques of two AD cases by quantitative comparison with surrounding non-plaque tissues. The localization of several proteins in the plaques was further confirmed by the approach of immunohistochemistry. In addition to previously identified plaque constituents, we discovered novel association of dynein heavy chain with the plaques in human postmortem brain and in a double transgenic AD mouse model, suggesting that neuronal transport may play a role in neuritic degeneration. Overall, our results revealed for the first time the sub-proteome of amyloid plaques that is important for further studies on disease biomarker identification and molecular mechanisms of AD pathogenesis. The presence of amyloid plaques in the brain is one of the pathological hallmarks of Alzheimer's disease (AD). We report here a comprehensive proteomic analysis of senile plaques from postmortem AD brain tissues. Senile plaques labeled with thioflavin-S were procured by laser capture microdissection, and their protein components were analyzed by liquid chromatography coupled with tandem mass spectrometry. We identified a total of 488 proteins coisolated with the plaques, and we found multiple phosphorylation sites on the neurofilament intermediate chain, implicating the complexity and diversity of cellular processes involved in the plaque formation. More significantly, we identified 26 proteins enriched in the plaques of two AD cases by quantitative comparison with surrounding non-plaque tissues. The localization of several proteins in the plaques was further confirmed by the approach of immunohistochemistry. In addition to previously identified plaque constituents, we discovered novel association of dynein heavy chain with the plaques in human postmortem brain and in a double transgenic AD mouse model, suggesting that neuronal transport may play a role in neuritic degeneration. Overall, our results revealed for the first time the sub-proteome of amyloid plaques that is important for further studies on disease biomarker identification and molecular mechanisms of AD pathogenesis. Alzheimer's disease (AD) 1The abbreviations used are: AD, Alzheimer's disease; LC-MS/MS, liquid chromatography coupled with tandem mass spectrometry; LCM, laser capture microdissection; Aβ, amyloid β-peptide; APP, Aβ precursor protein. is a devastating neurological disorder that impairs cognitive function and disturbs emotion and personality. Histopathologically, AD is manifested by the extracellular aggregation of amyloid plaques and the intraneuronal neurofibrillary tangles. Although current amyloid cascade hypothesis (1Hardy J. Selkoe D.J. Science. 2002; 297: 353-356Crossref PubMed Scopus (11025) Google Scholar) or tau hypothesis (2Lee V.M. Goedert M. Trojanowski J.Q. Annu. Rev. Neurosci. 2001; 24: 1121-1159Crossref PubMed Scopus (2145) Google Scholar) provides a framework for studying AD pathogenesis, the detailed molecular mechanisms that translate amyloid or tau accumulation into neuronal damage and functional brain impairments are largely unknown. In addition, there are numerous, complex pathological changes in AD brain that contribute to neuronal and synaptic degeneration, including mitochondrial dysfunction, oxidative damage, and inflammation (3Blass J.P. J. Neurosci. Res. 2001; 66: 851-856Crossref PubMed Scopus (138) Google Scholar, 4Nunomura A. Perry G. Aliev G. Hirai K. Takeda A. Balraj E.K. Jones P.K. Ghanbari H. Wataya T. Shimohama S. Chiba S. Atwood C.S. Petersen R.B. Smith M.A. J. Neuropathol. Exp. Neurol. 2001; 60: 759-767Crossref PubMed Scopus (1592) Google Scholar, 5Akiyama H. Barger S. Barnum S. Bradt B. Bauer J. Cole G.M. Cooper N.R. Eikelenboom P. Emmerling M. Fiebich B.L. Finch C.E. Frautschy S. Griffin W.S. Hampel H. Hull M. Landreth G. Lue L. Mrak R. Mackenzie I.R. McGeer P.L. O'Banion M.K. Pachter J. Pasinetti G. Plata-Salaman C. Rogers J. Rydel R. Shen Y. Streit W. Strohm-eyer R. Tooyoma I. Van Muiswinkel F.L. Veerhuis R. Walker D. Webster S. Wegrzyniak B. Wenk G. Wyss-Coray T. Neurobiol. Aging. 2000; 21: 383-421Crossref PubMed Scopus (3705) Google Scholar). The first major breakthrough in understanding the molecular pathogenesis of AD came from the biochemical purification of amyloid β-peptide (Aβ) from senile plaques, as described by Glenner and Wong (6Glenner G.G. Wong C.W. Biochem. Biophys. Res. Commun. 1984; 120: 885-890Crossref PubMed Scopus (4225) Google Scholar), and the subsequent sequencing and identification of the Aβ precursor protein (APP) gene. Although the major insoluble component of plaques has been identified as Aβ (6Glenner G.G. Wong C.W. Biochem. Biophys. Res. Commun. 1984; 120: 885-890Crossref PubMed Scopus (4225) Google Scholar, 7Masters C.L. Simms G. Weinman N.A. Multhaup G. McDonald B.L. Beyreuther K. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4245-4249Crossref PubMed Scopus (3668) Google Scholar), the entire molecular composition of the plaques is not known. The plaques are highly complex structures with a variety of neural and glial elements (8Terry R.D. Gonatas N.K. Weiss M. Am. J. Pathol. 1964; 44: 269-297PubMed Google Scholar), and many proteins have been localized to these structures by immunohistochemistry. However, biochemical verification of the plaque components has been scarce. Moreover, biochemical approaches previously applied to purify plaque components generally use very stringent extraction conditions (e.g. high concentration of salt, urea, and/or protease treatment) that may remove Aβ-associated proteins. The identities and roles of other plaque proteins that may act synergistically or competitively with Aβ in aggregation and deposition are also incomplete. A systematic analysis of plaque proteins should shed light on the underlying molecular processes that govern the plaque formation. Traditionally, proteomic analysis is performed by comparing samples between AD cases and normal controls in two-dimensional gels and identifying proteins of interest by mass spectrometry (9Butterfield D.A. Boyd-Kimball D. Castegna A. J. Neurochem. 2003; 86: 1313-1327Crossref PubMed Scopus (164) Google Scholar). Several groups (10Tsuji T. Shiozaki A. Kohno R. Yoshizato K. Shimohama S. Neurochem. Res. 2002; 27: 1245-1253Crossref PubMed Scopus (91) Google Scholar, 11Schonberger S.J. Edgar P.F. Kydd R. Faull R.L. Cooper G.J. Proteomics. 2001; 1: 1519-1528Crossref PubMed Scopus (173) Google Scholar) have tried this strategy and identified some proteins that are altered in the expression levels. However, the two-dimensional gel method is generally incompatible with proteins of extreme size, pI, and/or hydrophobicity, and it is tedious to determine the identity of hundreds of protein spots displayed on a two-dimensional gel. Many of the limitations can be overcome by the development of liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) (12Aebersold R. Mann M. Nature. 2003; 422: 198-207Crossref PubMed Scopus (5602) Google Scholar, 13Washburn M.P. Wolters D. Yates III, J.R. Nat. Biotechnol. 2001; 19: 242-247Crossref PubMed Scopus (4091) Google Scholar). It is now possible to analyze hundreds to thousands of proteins directly from a complex protein mixture, and the sensitivity can reach low femtomole and even subfemtomole levels. The power of has been by proteomic analysis of structures in as C.E. H. Mann M. 2002; PubMed Scopus Google Scholar), T. P. Mann M. Nature. 2003; PubMed Scopus Google Scholar), L. T. Yates III, J.R. L. Science. 2003; PubMed Scopus Google Scholar), J. M. J.R. S. M. Mann M. 2003; PubMed Scopus Google Scholar), and J. D. M. J. PubMed Scopus Google Scholar). The has also been used to changes in protein as by the identification of protein components in the B. I. M. Mann M. Nat. Biotechnol. 2003; 21: PubMed Scopus Google Scholar) and the functional of the Y. S. R. J. 2002; 21: PubMed Scopus Google Scholar). spectrometry has also the analysis of M. Nat. Biotechnol. 2003; 21: PubMed Scopus Google Scholar, J. D. D. G. J. D. Nat. Biotechnol. 2003; 21: PubMed Scopus Google Scholar). it may be to plaques to by biochemical the of laser capture a as as in PubMed Scopus Google Scholar). The has been used to for and expression analysis the PubMed Scopus Google Scholar). More in has to be G. H. D. S. H. Y. M. III, Y. Proteomics. 2002; 1: PubMed Google Scholar, H. H. Y. III, Proteomics. 2002; PubMed Scopus Google Scholar). It is possible to use to capture structures with high the of samples by is proteomic analysis by two-dimensional limitations can be by the of with the highly we the of postmortem amyloid plaques labeled with thioflavin-S by the approach and the systematic identification of proteins by The entire was performed with samples from two AD proteins were in the plaque and 26 proteins were to be enriched in the plaques in comparison with the surrounding The presence of proteins in the plaques was confirmed by and dynein was found to be a novel component of amyloid plaques in human and in AD mouse studies a approach for comprehensive analysis of the sub-proteome of of and were from a and a cases were as AD to A. D. J.P. G. L. PubMed Google Scholar). The brain were in on and brain were by a and on and of plaques by was performed on the previously Smith Weiss Science. PubMed Scopus Google Scholar). The brain were with for with thioflavin-S for in for and to in a of the were in for and and was performed the a to a laser capture with the laser and laser size, A total of amyloid plaques were procured from in surrounding the plaques were as a was used for the capture of plaques or from one and by were with for The was from with the addition of and protease the the samples were with in the for The total of proteins in the samples was on a gel to a protein with mass spectrometry proteins in were on a gel and with The entire was into by A. M. Mann M. PubMed Scopus Google Scholar). The from gel were in A A was used to a as described J. J. 2001; PubMed Scopus Google Scholar). were a from to were high and in from to mass with by mass in a on mass for J. Yates III, J.R. J. Am. PubMed Scopus Google Scholar) was for the human The were to mass to be and to the and were to the of the in and and We used stringent described previously J. G.J. Yates III, J.R. Nat. Biotechnol. PubMed Scopus Google Scholar, J. J. Res. 2003; PubMed Scopus Google were is and should be or for of we proteins by were found proteins identified by J. J. Res. 2003; PubMed Scopus Google Scholar). were with high The from the identified to proteins was by the presence of for the protein and/or by the of several proteins (e.g. protein J. Mann M. Biochem. Sci. 2002; 27: PubMed Scopus Google Scholar). we proteins and the we proteins identified by one as and were we the of the plaque samples from two by protein comparison between the plaques and the non-plaque was in two The first was on the of identified for of protein We proteins that were identified by in the in the plaques from AD The was on current of in T. P. Mann M. Nature. 2003; PubMed Scopus Google Scholar, W. H. H. S. S. P. M. 2003; PubMed Scopus Google Scholar, D. T. G. Shen 2003; PubMed Scopus Google Scholar). The of of was in the of that were by of the of gel as We analyzed of samples in two on the and found that the quantitative was in by a for as we used as the for protein in the The was also used to the and to the time of between the of It should be in to the identification that was from the on the the of many of the were for sequencing by However, the other on be for a was by analysis in the plaque not in the it is possible to and the in of the to and the not be in the we that the was we proteins that were found to be enriched in AD and of Aβ by comparison of that a with is in the plaques in the non-plaque A is for the entire is from to of the precursor to the identification of a from between the the mass of as the of the Aβ to from the non-plaque and the plaque the The was on the In the in are current by the time of and the the entire A was a of the time of in and double transgenic the with the double and a functional T. J. M.K. N.A. 19: PubMed Scopus Google Scholar, H. Nat. Neurosci. 2002; PubMed Scopus Google Scholar), were were with and and were and in for to of from with AD and two normal controls were the were with for were with a The brain were in in was performed by with one of the or Aβ with heavy chain a Aβ was the were and with and with for The was further by complex and The were with a on a of in AD by plaques in AD brain were with thioflavin-S and by some on the postmortem the plaques were the plaques from one AD brain were to of total protein extraction with The non-plaque from the brain were also procured as a The total protein samples from the plaques and the non-plaque were in on gel a protein composition in the two The proteins in the entire gel were analyzed by as described and were for and a human protein by the J. Yates III, J.R. J. Am. PubMed Scopus Google Scholar). The were further and to the identification of proteins in the plaques and proteins in the non-plaque of the in this AD the plaques are complex and structures and are to human the protein components may in AD We the entire proteomic analysis by samples from the AD and identified proteins in the plaques and proteins in non-plaque The from the two AD cases were combined to in a of 488 proteins in the plaque samples The current proteins in the plaques, of proteins were found in our many of these proteins were also found in the the plaques, some normal cellular elements are components of the plaques, including and these proteins may the capture of normal cellular elements with the determine of the proteins were enriched in the plaque we a strategy in the plaque proteins in AD cases were to the Although it is to protein directly from the of identified for protein is with the of the protein the protein is J. A. 2002; PubMed Scopus Google Scholar, J. U. Mann M. Res. 2002; PubMed Scopus Google Scholar). on this we the proteins that were identified in the controls in the plaques to the and and we the proteins for further quantitative More several groups T. P. Mann M. Nature. 2003; PubMed Scopus Google Scholar, W. H. H. S. S. P. M. 2003; PubMed Scopus Google Scholar, D. T. G. Shen 2003; PubMed Scopus Google Scholar) a method current of in We used this method to the proteins by of the the Aβ was in and by in the plaque and the The current for the is in for in It is that a was used as to the and to the time of The of Aβ was to be of the of Aβ in the The of other proteins was in a in the of 26 proteins enriched in the plaques of AD cases However, the of previously identified plaque proteins is not on this some proteins (e.g. and are not in the and some low proteins (e.g. and were in one AD to sensitivity of the the of 26 proteins that were identified and in the plaque in AD cases are to be plaque of 26 proteins enriched in amyloid plaques as with non-plaque the of that a protein in the samples of non-plaque the of that a protein in the plaque the of proteins from the plaque samples non-plaque protein protein and heavy heavy the of that a protein in the samples of non-plaque the of that a protein in the plaque the of proteins from the plaque samples non-plaque in a of the proteins identified in the plaques from AD postmortem we functional in and and the of some enriched plaque proteins in AD pathogenesis. and and of have been to be localized in amyloid plaques in AD C.S. Smith M.A. Perry G. 2002; PubMed Scopus Google Scholar). We found that and were in the In addition to extracellular of the is important for neuronal as of synaptic components R.D. J. Scholar). In our major of and were identified with high of that are in the plaques as as in non-plaque we identified one in the role in pathogenesis enriched in plaques is the protein tau that is to be with neurofibrillary and A. M. H. H. I. S. M. J. Am. J. Pathol. PubMed Scopus Google Scholar) and the component in the The identification of a variety of protein some of are to be to AD, that these elements may be involved in plaque and impairments may to a in and to neuritic proteins involved in and protein were revealed to be in the plaques by the mass spectrometry as heavy chain, and dynein heavy chain that the AD plaque some proteins to the protein that is for normal synaptic and is that the senile plaque is by and PubMed Google Scholar, L. P. G. Neurobiol. Aging. 2001; PubMed Scopus Google Scholar), and the proteins high expression in in in senile plaques K. G.J. M. PubMed Scopus Google Scholar). we identified many proteins and found that was enriched in the plaques We also identified glial protein and with high of are the major components of intermediate in glial and of is to contribute to AD as by tau in the neurofibrillary tangles. We identified multiple of were enriched in the plaque a of in the studies have that proteins are in neurofibrillary R. J. A. J. M. Neurosci. PubMed Scopus (164) Google Scholar), and one protein has further been to be of tau phosphorylation M. K. J. 2000; PubMed Scopus Google Scholar). Moreover, we to protein phosphorylation sites in the plaque samples by tandem mass and we two in neurofilament and with sites in with The first phosphorylation was in tandem in the neurofilament and was in the AD brain S. K. I. Neurosci. 2002; PubMed Scopus Google Scholar). The a phosphorylation on that we for the first phosphorylation may play a role in the of surrounding the plaque a role in the of proteins and of G. L. I. S. R. A. G. A. M.A. C. M. M. A. Neurobiol. Aging. 2002; PubMed Scopus Google Scholar). identified the enriched in the More of and were found to be in the plaques, suggesting the high in the plaques non-plaque Moreover, we found in the plaques and C. is a and has been to be in AD and with Aβ M. A. G. P. B. J. Neuropathol. Exp. Neurol. 2001; 60: PubMed Scopus Google Scholar, A. Am. J. Pathol. 2001; PubMed Scopus Google Scholar). is also as a for AD M. R. K. D. R. R. 2000; PubMed Scopus Google Scholar). Overall, the of and in multiple cellular the of protein mechanisms and the between and the plaque formation. of further the localization of some of the proteins we analysis on postmortem brain for their with In labeled surrounding the plaque that was by Aβ and some processes were into the plaque a in the plaque Neurobiol. Aging. PubMed Scopus (173) Google Scholar, Landreth J. Neurosci. 2000; PubMed Google Scholar). the and complex in in the plaque with our proteomic More dynein heavy chain was as and structures in many plaques labeled by Aβ suggesting that dynein heavy chain is a of plaque that dynein heavy chain was enriched in the with the surrounding The of dynein with amyloid plaque was in AD model, double transgenic T. J. M.K. N.A. 19: PubMed Scopus Google Scholar, H. Nat. Neurosci. 2002; PubMed Scopus Google Scholar). plaque was in and dynein heavy chain labeled the and in the and In the brain in AD manifested high of amyloid plaques, the of is by dynein structures that may in dynein heavy chain have been to and the of M. R. C. A. A. S. G. H. S. R. S. S. J. T. A. Y. M. D. S. P. C. S. G. A. M. J. H. M. G. G. Science. 2003; PubMed Scopus Google Scholar). It has also been previously K. J.P. Proc. Exp. PubMed Scopus Google Scholar) that dynein in AD brain is with normal that dynein is for transport of in the R.B. J. PubMed Scopus Google Scholar), the of dynein the plaque to neuritic studies revealed a total of 488 proteins in amyloid plaques, the plaque glial and proteins are from and/or glial surrounding the plaque extracellular proteins may be components of the plaque It is also possible that some proteins can damage neuronal degeneration. quantitative mass we further identified 26 proteins enriched in the plaques comparing with the non-plaque from the AD comparison is samples were from the postmortem the of many (e.g. postmortem that are in human results confirmed the presence of proteins that were previously in the plaques by C.S. Smith M.A. Perry G. 2002; PubMed Scopus Google Scholar). The plaque components identified in this Aβ, Selkoe D.J. H. PubMed Scopus Google Scholar), Y. M. H. K. Res. PubMed Scopus Google Scholar), T. T. A. H. R. A. I. T. K. Mann T. J. 2002; 21: PubMed Scopus Google Scholar), Am. J. Pathol. Google Scholar), A. M. K. T. Am. J. Pathol. Google Scholar), H. D. K. M. S. J. Am. J. Pathol. Google Scholar), and M. J. Finch C.E. PubMed Scopus Google Scholar). the other a proteins to be amyloid plaque components were in this C.S. Smith M.A. Perry G. 2002; PubMed Scopus Google Scholar), H. Barger S. Barnum S. Bradt B. Bauer J. Cole G.M. Cooper N.R. Eikelenboom P. Emmerling M. Fiebich B.L. Finch C.E. Frautschy S. Griffin W.S. Hampel H. Hull M. Landreth G. Lue L. Mrak R. Mackenzie I.R. McGeer P.L. O'Banion M.K. Pachter J. Pasinetti G. Plata-Salaman C. Rogers J. Rydel R. Shen Y. Streit W. Strohm-eyer R. Tooyoma I. Van Muiswinkel F.L. Veerhuis R. Walker D. Webster S. Wegrzyniak B. Wenk G. Wyss-Coray T. Neurobiol. Aging. 2000; 21: 383-421Crossref PubMed Scopus (3705) Google Scholar), M. G. J. Neurochem. PubMed Scopus Google Scholar), A. Am. J. Pathol. Google Scholar), M.A. P.L. D. G.J. B. Petersen R.B. Perry G. Am. J. Pathol. Google Scholar), M.A. N.K. Am. J. Pathol. Google Scholar), and I. Perry G. PubMed Scopus Google Scholar). The of these proteins in the plaques in our be to the low of the proteins and/or of with the (12Aebersold R. Mann M. Nature. 2003; 422: 198-207Crossref PubMed Scopus (5602) Google Scholar). results from two studies of AD cases revealed that of the proteins were identified in The between the two may be by the in the and the of as a of was and by mass spectrometry a complex was analyzed by the approach J. J. 2001; PubMed Scopus Google Scholar). was as approach to the proteomic proteins and were largely on the of the of are by in the plaque as by dynein glial and not proteins and a are not in the plaques not and are to be plaque the of 26 proteins to quantitative analysis should plaque Aβ is as the major component of the amyloid plaque our mass spectrometry analysis identified Aβ in plaque and non-plaque it to be enriched in the other proteins were found to a suggesting that Aβ is the major protein in the plaque Although Aβ are to extraction by many for C.L. Simms G. Weinman N.A. Multhaup G. McDonald B.L. Beyreuther K. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4245-4249Crossref PubMed Scopus (3668) Google Scholar), in to other proteins of we several extraction conditions this to the of total proteins. is Aβ C.L. Simms G. Weinman N.A. Multhaup G. McDonald B.L. Beyreuther K. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4245-4249Crossref PubMed Scopus (3668) Google Scholar), and we identified Aβ in the plaque not in the non-plaque by not However, the of Aβ in the that many other major protein in the plaques major with this of analysis is the of the plaque can protein with high We used the approach for the of amyloid plaques from postmortem by proteins with structures that are highly enriched in senile the it is to senile plaques from the surrounding and from other labeled structures as neurofibrillary and amyloid in and subsequent capture of the plaques highly senile plaques are structures that may in their protein one important is the proteins identified are in senile plaques or the diversity is by senile the two identified in neurofilament one the that is to neurofilament heavy chain and tau B. J. B. C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). of neurofilament as as tau may to and of transport M.K. R.D. A. S. T. A. R.B. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar, B. A. J. Sci. Google Scholar), in neuritic the pathological plaque We to with from other proteins as in the with were for the of from a complex was by the mass a of the phosphorylation in the plaques, of the of be The of laser capture with provides a method a cellular approach with biochemical protein the sequencing of proteins in a with high as by the identification of several hundreds of proteins from of total plaque proteins. that other of plaques as and plaques, this can be further to determine proteins involved in the of aggregation and the molecular that the plaque formation. approach can also be applied to plaque in the transgenic mouse it is possible to use this for the analysis of other pathological structures as in protein in or in the other the approach has been used as a to highly protein identification and to protein by the current of More of proteins be by as strategy B. R. Nat. Biotechnol. PubMed Scopus Google Scholar). our this is the first analysis of proteins from AD amyloid The results of this that the protein in amyloid plaques are highly complex and implicating the of many cellular in disease The plaque identified in our be for subsequent on disease biomarker identification and molecular of Alzheimer's We and for their in We also and for their in for the for mouse brain In addition, we are to and Smith for with