Background: Alzheimer’s disease (AD) is a chronic neurodegenerative disorder accounting for more than 50% of all dementia cases. AD neuropathology is characterized by the formation of extracellular plaques and intracellular neurofibrillary tangles consisting of aggregated amyloid-β and tau, respectively. The disease mechanism has only been partially elucidated and is believed to also involve many other proteins.
Objective: This study intended to perform a proteomic profiling of post mortem AD brains and compare it with control brains as well as brains from other neurological diseases to gain insight into the disease pathology.
Methods: Here we used label-free shotgun mass spectrometry to analyze temporal neocortex samples from AD, other neurological disorders, and non-demented controls, in order to identify additional proteins that are altered in AD. The mass spectrometry results were verified by antibody suspension bead arrays.
Results: We found 50 proteins with altered levels between AD and control brains. The majority of these proteins were found at lower levels in AD. Pathway analyses revealed that several of the decreased proteins play a role in exocytic and endocytic pathways, whereas several of the increased proteins are related to extracellular vesicles. Using antibody-based analysis, we verified the mass spectrometry results for five representative proteins from this group of proteins (CD9, HSP72, PI42A, TALDO, and VAMP2) and GFAP, a marker for neuroinflammation.
Conclusions: Several proteins involved in exo-endocytic pathways and extracellular vesicle functions display altered levels in the AD brain. We hypothesize that such changes may result in disturbed cellular clearance and a perturbed cell-to-cell communication that may contribute to neuronal dysfunction and cell death in AD.
EvansDA, FunkensteinHH, AlbertMS, ScherrPA, CookNR, ChownMJ, HebertLE, HennekensCH, TaylorJO (1989) Prevalence of Alzheimer’s disease in a community population of older persons. Higher than previously reported. JAMA262, 2551–2556.
FratiglioniL, De RonchiD, Aguero-TorresH (1999) Worldwide prevalence and incidence of dementia. Drugs Aging15, 365–375.
4.
ForstlH, KurzA (1999) Clinical features of Alzheimer’s disease. Eur Arch Psychiatry Clin Neurosci249, 288–290.
5.
DaviesL, WolskaB, HilbichC, MulthaupG, MartinsR, SimmsG, BeyreutherK, MastersCL (1988) A4 amyloid protein deposition and the diagnosis of Alzheimer’s disease: Prevalence in aged brains determined by immunocytochemistry compared with conventional neuropathologic techniques. Neurology38, 1688–1693.
6.
AndreasenNC (1997) Linking mind and brain in the study of mental illnesses: A project for a scientific psychopathology. Science275, 1586–1593.
7.
HonerWG, FalkaiP, BayerTA, XieJ, HuL, LiHY, ArangoV, MannJJ, DworkAJ, TrimbleWS (2002) Abnormalities of SNARE mechanism proteins in anterior frontal cortex in severe mental illness. Cereb Cortex12, 349–356.
8.
ReddyPH, ManiG, ParkBS, JacquesJ, MurdochG, WhetsellWJr, KayeJ, ManczakM (2005) Differential loss of synaptic proteins in Alzheimer’s disease: Implications for synaptic dysfunction. J Alzheimers Dis7, 103–117; discussion 173-180.
9.
MasliahE, HonerWG, MalloryM, VoigtM, KushnerP, HansenL, TerryR (1994) Topographical distribution of synaptic-associated proteins in the neuritic plaques of Alzheimer’s disease hippocampus. Acta Neuropathol87, 135–142.
10.
SzeCI, TroncosoJC, KawasC, MoutonP, PriceDL, MartinLJ (1997) Loss of the presynaptic vesicle protein synaptophysin in hippocampus correlates with cognitive decline in Alzheimer disease. J Neuropathol Exp Neurol56, 933–944.
11.
SzeCI, BiH, Kleinschmidt-DeMastersBK, FilleyCM, MartinLJ (2000) Selective regional loss of exocytotic presynaptic vesicle proteins in Alzheimer’s disease brains. J Neurol Sci175, 81–90.
12.
SahekiY, De CamilliP (2012) Synaptic vesicle endocytosis. Cold Spring Harb Perspect Biol4, a005645.
13.
HarrisTW, SchuskeK, JorgensenEM (2001) Studies of synaptic vesicle endocytosis in the nematode C. elegans. Traffic2, 597–605.
14.
WuLG, HamidE, ShinW, ChiangHC (2014) Exocytosis and endocytosis: Modes, functions, and coupling mechanisms. Annu Rev Physiol76, 301–331.
15.
MulcahyLA, PinkRC, CarterDR (2014) Routes and mechanisms of extracellular vesicle uptake. J Extracell Vesicles3, 24641.
16.
RajendranL, HonshoM, ZahnTR, KellerP, GeigerKD, VerkadeP, SimonsK (2006) Alzheimer’s disease beta-amyloid peptides are released in association with exosomes. Proc Natl Acad Sci U S A103, 11172–11177.
17.
WangG, DinkinsM, HeQ, ZhuG, PoirierC, CampbellA, Mayer-ProschelM, BieberichE (2012) Astrocytes secrete exosomes enriched with proapoptotic ceramide and prostate apoptosis response 4 (PAR-4): Potential mechanism of apoptosis induction in Alzheimer disease (AD). J Biol Chem287, 21384–21395.
18.
GuptaA, PulliamL (2014) Exosomes as mediators of neuroinflammation. J Neuroinflammation11, 68.
ProperziF, LogozziM, FaisS (2013) Exosomes: The future of biomarkers in medicine. Biomark Med7, 769–778.
21.
FiandacaMS, KapogiannisD, MapstoneM, BoxerA, EitanE, SchwartzJB, AbnerEL, PetersenRC, FederoffHJ, MillerBL, GoetzlEJ (2015) Identification of preclinical Alzheimer’s disease by a profile of pathogenic proteins in neurally derived blood exosomes: A case-control study. Alzheimers Dement11, 600–607.e601.
22.
MirraSS, HeymanA, McKeelD, SumiSM, CrainBJ, BrownleeLM, VogelFS, HughesJP, VanbelleG, BergL (1991) The Consortium to Establish a Registry for Alzheimer’s Disease (CERAD) 2. Standardization of the Neuropathologic Assessment of Alzheimer’s Disease. Neurology41, 479–486.
23.
MastroR, HallM (1999) Protein delipidation and precipitation by tri-n-butylphosphate, acetone, and methanol treatment for isoelectric focusing and two-dimensional gel electrophoresis. Anal Biochem273, 313–315.
24.
WetterhallM, ShevchenkoG, ArtemenkoK, SjodinMOD, BergquistJ (2011) Analysis of membrane and hydrophilic proteins simultaneously derived from the mouse brain using cloud-point extraction. Anal Bioanal Chem400, 2827–2836.
25.
LowryOH, RosebroughNJ, FarrAL, RandallRJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem193, 265–275.
SturmM, BertschA, GroplC, HildebrandtA, HussongR, LangeE, PfeiferN, Schulz-TrieglaffO, ZerckA, ReinertK, KohlbacherO (2008) OpenMS - an open-source software framework for mass spectrometry. BMC Bioinformatics9, 163.
29.
CraigR, BeavisRC (2004) TANDEM: Matching proteins with tandem mass spectra. Bioinformatics20, 1466–1467.
30.
GeerLY, MarkeySP, KowalakJA, WagnerL, XuM, MaynardDM, YangX, ShiW, BryantSH (2004) Open mass spectrometry search algorithm. J Proteome Res3, 958–964.
31.
NahnsenS, BertschA, RahnenführerJ, NordheimA, KohlbacherO (2011) Probabilistic consensus scoring improves tandem mass spectrometry peptide identification. J Proteome Res10, 3332–3343.
32.
KultimaK, NilssonA, ScholzB, RossbachUL, FalthM, AndrenPE (2009) Development and evaluation of normalization methods for label-free relative quantification of endogenous peptides. Mol Cell Proteomics8, 2285–2295.
33.
VizcainoJA, CsordasA, del-ToroN, DianesJA, GrissJ, LavidasI, MayerG, Perez-RiverolY, ReisingerF, TernentT, XuQW, WangR, HermjakobH (2016) 2016 update of the PRIDE database and its related tools. Nucleic Acids Res44, D447–D456.
34.
R Core Team (2013) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL http://www.R-project.org.
35.
SmythGK (2004) Linear models and empirical bayes methods for assessing differential expression in microarray experiments.Article. Stat Appl Genet Mol Biol3, 3.
36.
SmythGK (2005) Limma: Linear models for microarray data. In Bioinformatics and Computational Biology Solutions using R and Bioconductor, GentlemanR, CareyV, DudoitS, IrizarryR, HuberW, eds. Springer, New York, pp. 397–420.
37.
TemplM, KowarikA, FilzmoserP (2011) Iterative stepwise regression imputation using standard and robust methods. Comput Stat Data Anal55, 2793–2806.
38.
SmythGK, MichaudJ, ScottHS (2005) Use of within-array replicate spots for assessing differential expression in microarray experiments. Bioinformatics21, 2067–2075.
39.
SilvaJC, GorensteinMV, LiGZ, VissersJP, GeromanosSJ (2006) Absolute quantification of proteins by LCMSE: A virtue of parallel MS acquisition. Mol Cell Proteomics5, 144–156.
40.
FranceschiniA, SzklarczykD, FrankildS, KuhnM, SimonovicM, RothA, LinJ, MinguezP, BorkP, von MeringC, JensenLJ (2013) STRING v9.1: Protein-protein interaction networks, with increased coverage and integration. Nucleic Acids Res41, D808–D815.
41.
AyogluB, ChaouchA, LochmullerH, PolitanoL, BertiniE, SpitaliP, HillerM, NiksEH, GualandiF, PontenF, BushbyK, Aartsma-RusA, SchwartzE, Le PriolY, StraubV, UhlenM, CirakS, t HoenPA, MuntoniF, FerliniA, SchwenkJM, NilssonP, Al-Khalili SzigyartoC (2014) Affinity proteomics within rare diseases: A BIO-NMD study for blood biomarkers of muscular dystrophies. EMBO Mol Med6, 918–936.
42.
BachmannJ, BurteF, PramanaS, ConteI, BrownBJ, OrimadegunAE, AjetunmobiWA, AfolabiNK, AkinkunmiF, OmokhodionS, AkinbamiFO, ShokunbiWA, KampfC, PawitanY, UhlenM, SodeindeO, SchwenkJM, WahlgrenM, Fernandez-ReyesD, NilssonP (2014) Affinity proteomics reveals elevated muscle proteins in plasma of children with cerebral malaria. PLoS Pathog10, e1004038.
DezfouliM, VickovicS, IglesiasMJ, NilssonP, SchwenkJM, AhmadianA (2014) Magnetic bead assisted labeling of antibodies at nanogram scale. Proteomics14, 14–18.
47.
SchochS, DeakF, KonigstorferA, MozhayevaM, SaraY, SudhofTC, KavalaliET (2001) SNARE function analyzed in synaptobrevin/VAMP knockout mice. Science294, 1117–1122.
48.
UngarD, HughsonFM (2003) SNARE protein structure and function. Annu Rev Cell Dev Biol19, 493–517.
49.
PengL, LiuH, RuanH, TeppWH, StoothoffWH, BrownRH, JohnsonEA, YaoWD, ZhangSC, DongM (2013) Cytotoxicity of botulinum neurotoxins reveals a direct role of syntaxin 1 and SNAP-25 in neuron survival. Nat Commun4, 1472.
50.
JohannesL, DoussauF, ClabecqA, HenryJP, DarchenF, PoulainB (1996) Evidence for a functional link between Rab3 and the SNARE complex. J Cell Sci109(Pt 12), 2875–2884.
51.
BeitesCL, XieH, BowserR, TrimbleWS (1999) The septin CDCrel-1 binds syntaxin and inhibits exocytosis. Nat Neurosci2, 434–439.
52.
ZhouA, BrewerKD, RizoJ (2013) Analysis of SNARE complex/synaptotagmin-1 interactions by one-dimensional NMR spectroscopy. Biochemistry52, 3446–3456.
53.
LeendersAG, Lopes da SilvaFH, GhijsenWE, VerhageM (2001) Rab3a is involved in transport of synaptic vesicles to the active zone in mouse brain nerve terminals. Mol Biol Cell12, 3095–3102.
54.
DulubovaI, LouX, LuJ, HuryevaI, AlamA, SchneggenburgerR, SudhofTC, RizoJ (2005) A Munc13/RIM/Rab3 tripartite complex: From priming to plasticity?EMBO J24, 2839–2850.
55.
VerhageM, MaiaAS, PlompJJ, BrussaardAB, HeeromaJH, VermeerH, ToonenRF, HammerRE, van den BergTK, MisslerM, GeuzeHJ, SudhofTC (2000) Synaptic assembly of the brain in the absence of neurotransmitter secretion. Science287, 864–869.
56.
WangX, ThiagarajanR, WangQ, TewoldeT, RichMM, EngischKL (2008) Regulation of quantal shape by Rab3A: Evidence for a fusion pore-dependent mechanism. J Physiol586, 3949–3962.
57.
ToonenRF, WierdaK, SonsMS, de WitH, CornelisseLN, BrussaardA, PlompJJ, VerhageM (2006) Munc18-1 expression levels control synapse recovery by regulating readily releasable pool size. Proc Natl Acad Sci U S A103, 18332–18337.
58.
HondiusDC, van NieropP, LiKW, HoozemansJJ, van der SchorsRC, van HaastertES, van der ViesSM, RozemullerAJ, SmitAB (2016) Profiling the human hippocampal proteome at all pathologic stages of Alzheimer’s disease. Alzheimers Dement12, 654–668.
59.
MusunuriS, WetterhallM, IngelssonM, LannfeltL, ArtemenkoK, BergquistJ, KultimaK, ShevchenkoG (2014) Quantification of the brain proteome in Alzheimer’s disease using multiplexed mass spectrometry. J Proteome Res13, 2056–2068.
60.
ZelayaMV, Perez-ValderramaE, de MorentinXM, TunonT, FerrerI, LuquinMR, Fernandez-IrigoyenJ, SantamariaE (2015) Olfactory bulb proteome dynamics during the progression of sporadic Alzheimer’s disease: Identification of common and distinct olfactory targets across Alzheimer-related co-pathologies. Oncotarget6,` 39437–39456.
61.
HeuserJ (1980) Three-dimensional visualization of coated vesicle formation in fibroblasts. J Cell Biol84, 560–583.
62.
KasprowiczJ, KuenenS, MiskiewiczK, HabetsRL, SmitzL, VerstrekenP (2008) Inactivation of clathrin heavy chain inhibits synaptic recycling but allows bulk membrane uptake. J Cell Biol182, 1007–1016.
63.
TianY, ChangJC, FanEY, FlajoletM, GreengardP (2013) Adaptor complex AP2/PICALM, through interaction with LC3, targets Alzheimer’s APP-CTF for terminal degradation via autophagy. Proc Natl Acad Sci U S A110, 17071–17076.
64.
OwenDJ, CollinsBM, EvansPR (2004) Adaptors for clathrin coats: Structure and function. Annu Rev Cell Dev Biol20, 153–191.
SunY, CarrollS, KaksonenM, ToshimaJY, DrubinDG (2007) PtdIns(4,5)P2 turnover is required for multiple stages during clathrin- and actin-dependent endocytic internalization. J Cell Biol177, 355–367.
67.
AppelqvistH, WasterP, KagedalK, OllingerK (2013) The lysosome: From waste bag to potential therapeutic target. J Mol Cell Biol5, 214–226.
68.
NixonRA, YangDS, LeeJH (2008) Neurodegenerative lysosomal disorders: A continuum from development to late age. Autophagy4, 590–599.
69.
SollvanderS, NikitidouE, BrolinR, SoderbergL, SehlinD, LannfeltL, ErlandssonA (2016) Accumulation of amyloid-beta by astrocytes result in enlarged endosomes and microvesicle-induced apoptosis of neurons. Mol Neurodegener11, 38.
70.
AnandPK, AnandE, BleckCK, AnesE, GriffithsG (2010) Exosomal Hsp70 induces a pro-inflammatory response to foreign particles including mycobacteria. PLoS One5, e10136.
71.
HeppnerFL, RansohoffRM, BecherB (2015) Immune attack: The role of inflammation in Alzheimer disease. Nat Rev Neurosci16, 358–372.
72.
StinconeA, PrigioneA, CramerT, WamelinkMM, CampbellK, CheungE, Olin-SandovalV, GruningN, KrugerA, Tauqeer AlamM, KellerMA, BreitenbachM, BrindleKM, RabinowitzJD, RalserM (2015) The return of metabolism: Biochemistry and physiology of the pentose phosphate pathway. Biol Rev Camb Philos Soc90, 927–963.
73.
PalmerAM (1999) The activity of the pentose phosphate pathway is increased in response to oxidative stress in Alzheimer’s disease. J Neural Transm (Vienna)106, 317–328.
74.
ChenZ, ZhongC (2013) Decoding Alzheimer’s disease from perturbed cerebral glucose metabolism: Implications for diagnostic and therapeutic strategies. Prog Neurobiol108, 21–43.
75.
PrunottoM, FarinaA, LaneL, PerninA, SchifferliJ, HochstrasserDF, LescuyerP, MollS (2013) Proteomic analysis of podocyte exosome-enriched fraction from normal human urine. J Proteomics82, 193–229.
76.
AlberioT, McMahonK, CuccurulloM, GethingsLA, LawlessC, ZibettiM, LopianoL, VissersJP, FasanoM (2014) Verification of a Parkinson’s disease protein signature in T-lymphocytes by multiple reaction monitoring. J Proteome Res13, 3554–3561.
77.
DimouL, GotzM (2014) Glial cells as progenitors and stem cells: New roles in the healthy and diseased brain. Physiol Rev94, 709–737.
78.
KatoS, GondoT, HoshiiY, TakahashiM, YamadaM, IshiharaT (1998) Confocal observation of senile plaques in Alzheimer’s disease: Senile plaque morphology and relationship between senile plaques and astrocytes. Pathol Int48, 332–340.
79.
Ben HaimL, Carrillo-de SauvageMA, CeyzeriatK, EscartinC (2015) Elusive roles for reactive astrocytes in neurodegenerative diseases. Front Cell Neurosci9, 278.
