Multiplexed Immunoassay Panel Identifies Novel CSF Biomarkers for Alzheimer's Disease Diagnosis and Prognosis

Background Clinicopathological studies suggest that Alzheimer's disease (AD) pathology begins ∼10–15 years before the resulting cognitive impairment draws medical attention. Biomarkers that can detect AD pathology in its early stages and predict dementia onset would, therefore, be invaluable for patient care and efficient clinical trial design. We utilized a targeted proteomics approach to discover novel cerebrospinal fluid (CSF) biomarkers that can augment the diagnostic and prognostic accuracy of current leading CSF biomarkers (Aβ42, tau, p-tau181). Methods and Findings Using a multiplexed Luminex platform, 190 analytes were measured in 333 CSF samples from cognitively normal (Clinical Dementia Rating [CDR] 0), very mildly demented (CDR 0.5), and mildly demented (CDR 1) individuals. Mean levels of 37 analytes (12 after Bonferroni correction) were found to differ between CDR 0 and CDR>0 groups. Receiver-operating characteristic curve analyses revealed that small combinations of a subset of these markers (cystatin C, VEGF, TRAIL-R3, PAI-1, PP, NT-proBNP, MMP-10, MIF, GRO-α, fibrinogen, FAS, eotaxin-3) enhanced the ability of the best-performing established CSF biomarker, the tau/Aβ42 ratio, to discriminate CDR>0 from CDR 0 individuals. Multiple machine learning algorithms likewise showed that the novel biomarker panels improved the diagnostic performance of the current leading biomarkers. Importantly, most of the markers that best discriminated CDR 0 from CDR>0 individuals in the more targeted ROC analyses were also identified as top predictors in the machine learning models, reconfirming their potential as biomarkers for early-stage AD. Cox proportional hazards models demonstrated that an optimal panel of markers for predicting risk of developing cognitive impairment (CDR 0 to CDR>0 conversion) consisted of calbindin, Aβ42, and age. Conclusions/Significance Using a targeted proteomic screen, we identified novel candidate biomarkers that complement the best current CSF biomarkers for distinguishing very mildly/mildly demented from cognitively normal individuals. Additionally, we identified a novel biomarker (calbindin) with significant prognostic potential.

[1]  M. Folstein,et al.  Clinical diagnosis of Alzheimer's disease , 1984, Neurology.

[2]  M. Greenberg,et al.  β-Amyloid Induces Neuronal Apoptosis Via a Mechanism that Involves the c-Jun N-Terminal Kinase Pathway and the Induction of Fas Ligand , 2001, The Journal of Neuroscience.

[3]  Reinhard Schliebs,et al.  Vascular endothelial growth factor (VEGF) affects processing of amyloid precursor protein and β-amyloidogenesis in brain slice cultures derived from transgenic Tg2576 mouse brain , 2009, International Journal of Developmental Neuroscience.

[4]  K. Blennow,et al.  Intra-individual stability of CSF biomarkers for Alzheimer's disease over two years. , 2007, Journal of Alzheimer's disease : JAD.

[5]  J. Melchor,et al.  The tissue plasminogen activator-plasminogen proteolytic cascade accelerates amyloid-beta (Abeta) degradation and inhibits Abeta-induced neurodegeneration. , 2003, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[6]  S. Strickland,et al.  Fibrinogen and β-Amyloid Association Alters Thrombosis and Fibrinolysis: A Possible Contributing Factor to Alzheimer's Disease , 2010, Neuron.

[7]  S. Christakos,et al.  Specific reduction of calcium-binding protein (28-kilodalton calbindin-D) gene expression in aging and neurodegenerative diseases. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[8]  G. Waldemar,et al.  A Novel Panel of Cerebrospinal Fluid Biomarkers for the Differential Diagnosis of Alzheimer’s Disease versus Normal Aging and Frontotemporal Dementia , 2007, Dementia and Geriatric Cognitive Disorders.

[9]  R. Pearson,et al.  Accumulation of calbindin in cortical pyramidal cells with ageing; a putative protective mechanism which fails in Alzheimer's disease , 2001, Neuropathology and applied neurobiology.

[10]  W. Sheu,et al.  Genotype and Plasma Concentration of Cystatin C in Patients with Late-Onset Alzheimer Disease , 2007, Dementia and Geriatric Cognitive Disorders.

[11]  J. Friedman.  Greedy function approximation: A gradient boosting machine. , 2001 .

[12]  Brian J Cummings,et al.  The induction of the TNFalpha death domain signaling pathway in Alzheimer's disease brain. , 2003, Neurochemical research.

[13]  Gunhild Waldemar,et al.  Novel panel of cerebrospinal fluid biomarkers for the prediction of progression to Alzheimer dementia in patients with mild cognitive impairment. , 2007, Archives of neurology.

[14]  M. Folstein,et al.  Clinical diagnosis of Alzheimer's disease: Report of the NINCDS—ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer's Disease , 2011, Neurology.

[15]  J. Melchor,et al.  Fibrin deposition accelerates neurovascular damage and neuroinflammation in mouse models of Alzheimer's disease. , 2007 .

[16]  Eric R. Ziegel,et al.  The Elements of Statistical Learning , 2003, Technometrics.

[17]  Jing Zhang,et al.  Quantitative proteomics of cerebrospinal fluid from patients with Alzheimer disease. , 2005, Journal of Alzheimer's disease : JAD.

[18]  N. Seeds,et al.  Plasminogen activator activity is inhibited while neuroserpin is up‐regulated in the Alzheimer disease brain , 2009, Journal of neurochemistry.

[19]  A. Burns.  Clinical diagnosis of Alzheimer's disease , 1991 .

[20]  O. Combarros,et al.  Low serum VEGF levels are associated with Alzheimer's disease , 2007, Acta neurologica Scandinavica.

[21]  S. Schmidt,et al.  Binding of cystatin C to Alzheimer’s amyloid β inhibits in vitro amyloid fibril formation , 2004, Neurobiology of Aging.

[22]  A. Fagan,et al.  Cerebrospinal fluid tau and ptau181 increase with cortical amyloid deposition in cognitively normal individuals: Implications for future clinical trials of Alzheimer's disease , 2009, EMBO molecular medicine.

[23]  Rena Li,et al.  Differential activation of tumor necrosis factor receptors distinguishes between brains from Alzheimer's disease and non-demented patients. , 2010, Journal of Alzheimer's disease : JAD.

[24]  K. Titani,et al.  Glutamine synthetase, hemoglobin alpha-chain, and macrophage migration inhibitory factor binding to amyloid beta-protein: their identification in rat brain by a novel affinity chromatography and in Alzheimer's disease brain by immunoprecipitation. , 2000, Biochimica et biophysica acta.

[25]  J. Melchor,et al.  The Tissue Plasminogen Activator-Plasminogen Proteolytic Cascade Accelerates Amyloid-β (Aβ) Degradation and Inhibits Aβ-Induced Neurodegeneration , 2003, The Journal of Neuroscience.

[26]  E. Tangalos,et al.  Mild Cognitive Impairment Clinical Characterization and Outcome , 1999 .

[27]  D. Green,et al.  Metalloproteinase Shedding of Fas Ligand Regulates β-Amyloid Neurotoxicity , 2002, Current Biology.

[28]  P. Carmeliet,et al.  Ageing and amyloid-beta peptide deposition contribute to an impaired brain tissue plasminogen activator activity by different mechanisms , 2007, Neurobiology of Disease.

[29]  A. Fagan,et al.  Cerebrospinal fluid tau/beta-amyloid(42) ratio as a prediction of cognitive decline in nondemented older adults. , 2007, Archives of neurology.

[30]  Christian Humpel,et al.  Chromogranin peptides in Alzheimer's disease , 2004, Experimental Gerontology.

[31]  Henrik Zetterberg,et al.  Longitudinal Study of CSF Biomarkers in Patients with Alzheimer's Disease , 2009, PloS one.

[32]  D. Hochstrasser,et al.  A panel of cerebrospinal fluid potential biomarkers for the diagnosis of Alzheimer's disease , 2003, Proteomics.

[33]  W. Klunk,et al.  Imaging brain amyloid in Alzheimer's disease with Pittsburgh Compound‐B , 2004, Annals of neurology.

[34]  A. Grubb,et al.  Cystatin C reduces the in vitro formation of soluble Aβ1‐42 oligomers and protofibrils , 2007, Scandinavian journal of clinical and laboratory investigation.

[35]  J Philip Miller,et al.  Longitudinal course and neuropathologic outcomes in original vs revised MCI and in pre-MCI , 2006, Neurology.

[36]  H. Hinterhuber,et al.  Measurement of Thirteen Biological Markers in CSF of Patients with Alzheimer’s Disease and Other Dementias , 2005, Dementia and Geriatric Cognitive Disorders.

[37]  David M Holtzman,et al.  Identification and validation of novel CSF biomarkers for early stages of Alzheimer's disease , 2007, Proteomics. Clinical applications.

[38]  M. Weiner,et al.  Relationships between biomarkers in aging and dementia , 2009, Neurology.

[39]  Bruce R. Kowalski,et al.  Partial least-squares path modelling with latent variables , 1979 .

[40]  Der-Zen Liu,et al.  Hemorheological Mechanisms in Alzheimer's Disease , 2007, Microcirculation.

[41]  K. Blennow,et al.  Cerebrospinal fluid beta-amyloid(1-42) in Alzheimer disease: differences between early- and late-onset Alzheimer disease and stability during the course of disease. , 1999, Archives of neurology.

[42]  C. Cotman,et al.  Fas and Fas Ligand are associated with neuritic degeneration in the AD brain and participate in β-amyloid-induced neuronal death , 2003, Neurobiology of Disease.

[43]  B. Hyman,et al.  Serum cystatin C and the risk of Alzheimer disease in elderly men , 2008, Neurology.

[44]  V. Mathura,et al.  Alzheimer’s β‐amyloid peptide blocks vascular endothelial growth factor mediated signaling via direct interaction with VEGFR‐2 , 2010, Journal of neurochemistry.

[45]  Murray Grossman,et al.  Novel CSF biomarkers for Alzheimer’s disease and mild cognitive impairment , 2010, Acta Neuropathologica.

[46]  Barbara Borroni,et al.  VEGF gene and phenotype relation with Alzheimer's disease and mild cognitive impairment. , 2006, Rejuvenation research.

[47]  M. Mattson,et al.  Calbindin D28k blocks the proapoptotic actions of mutant presenilin 1: reduced oxidative stress and preserved mitochondrial function. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[48]  A. Smith,et al.  Longitudinal study of inflammatory factors in serum, cerebrospinal fluid, and brain tissue in Alzheimer disease: interleukin-1beta, interleukin-6, interleukin-1 receptor antagonist, tumor necrosis factor-alpha, the soluble tumor necrosis factor receptors I and II, and alpha1-antichymotrypsin. , 1998, Alzheimer disease and associated disorders.

[49]  N. Bresolin,et al.  VEGF genetic variability is associated with increased risk of developing Alzheimer's disease , 2009, Journal of the Neurological Sciences.

[50]  K. Blennow,et al.  Local and systemic GM‐CSF increase in Alzheimer's disease and vascular dementia , 2001, Acta neurologica Scandinavica.

[51]  J. Philip Miller,et al.  Combining Correlated Diagnostic Tests: Application to Neuropathologic Diagnosis of Alzheimer’s Disease , 2004, Medical decision making : an international journal of the Society for Medical Decision Making.

[52]  Norman Relkin,et al.  Cerebrospinal fluid proteomic biomarkers for Alzheimer's disease , 2007, Annals of neurology.

[53]  M. Staufenbiel,et al.  Cystatin C modulates cerebral beta-amyloidosis. , 2007, Nature genetics.

[54]  Kaj Blennow,et al.  Proteomic studies of potential cerebrospinal fluid protein markers for Alzheimer's disease. , 2003, Brain research. Molecular brain research.

[55]  D. Green,et al.  Metalloproteinase shedding of Fas ligand regulates beta-amyloid neurotoxicity. , 2002, Current biology : CB.

[56]  R. S. Sloviter,et al.  Calbindin-D28k immunoreactivity and selective vulnerability to ischemia in the dentate gyrus of the developing rat , 1993, Brain Research.

[57]  S. Keleş,et al.  Sparse partial least squares regression for simultaneous dimension reduction and variable selection , 2010, Journal of the Royal Statistical Society. Series B, Statistical methodology.

[58]  D. Radvinsky,et al.  Cystatin C inhibits amyloid-beta deposition in Alzheimer's disease mouse models. , 2007, Nature genetics.

[59]  A. Fagan,et al.  Multimodal techniques for diagnosis and prognosis of Alzheimer's disease , 2009, Nature.

[60]  C. Jack,et al.  Hypothetical model of dynamic biomarkers of the Alzheimer's pathological cascade , 2010, The Lancet Neurology.

[61]  Anders Wallin,et al.  Increased intrathecal levels of the angiogenic factors VEGF and TGF-β in Alzheimer’s disease and vascular dementia , 2002, Neurobiology of Aging.

[62]  M. Riepe,et al.  Meningeal derived cerebrospinal fluid proteins in different forms of dementia: is a meningopathy involved in normal pressure hydrocephalus? , 2004, Journal of Neurology, Neurosurgery & Psychiatry.

[63]  M. Mattson,et al.  Evidence for calcium-reducing and excitoprotective roles for the calcium-binding protein calbindin-1328k in cultured hippocampal neurons , 1991, Neuron.

[64]  John Calvin Reed,et al.  Protective effect of transforming growth factor-beta 1 on beta-amyloid neurotoxicity in rat hippocampal neurons. , 1996, Molecular pharmacology.

[65]  Trevor Hastie,et al.  Class Prediction by Nearest Shrunken Centroids, with Applications to DNA Microarrays , 2003 .

[66]  Y. Nakano,et al.  Increased plasma urotensin-II and carotid atherosclerosis are associated with vascular dementia. , 2009, Journal of atherosclerosis and thrombosis.

[67]  S. Ha,et al.  Fibrinogen gamma-A chain precursor in CSF: a candidate biomarker for Alzheimer's disease , 2007, BMC neurology.

[68]  Albert Hofman,et al.  Fibrinogen Is Associated With an Increased Risk of Alzheimer Disease and Vascular Dementia , 2005, Stroke.

[69]  K. Blennow,et al.  CSF markers for incipient Alzheimer's disease , 2003, The Lancet Neurology.

[70]  J. Becker,et al.  Plasma amyloid levels and the risk of AD in normal subjects in the Cardiovascular Health Study , 2008, Neurology.

[71]  G. Brewer,et al.  Age-related differences in NFκB translocation and Bcl-2/Bax ratio caused by TNFα and Abeta42 promote survival in middle-age neurons and death in old neurons , 2008, Experimental Neurology.

[72]  C. Laske,et al.  Increase of SCF plasma concentration during donepezil treatment of patients with early Alzheimer's disease. , 2009, The international journal of neuropsychopharmacology.

[73]  F. Jessen,et al.  The Role of Macrophage Migration Inhibitory Factor in Alzheimer’s Disease , 2010, Molecular medicine.

[74]  Kaj Blennow,et al.  Proteome analysis of cerebrospinal fluid proteins in Alzheimer patients , 2002, Neuroreport.

[75]  Yan Liu,et al.  Detection of biomarkers with a multiplex quantitative proteomic platform in cerebrospinal fluid of patients with neurodegenerative disorders. , 2006, Journal of Alzheimer's disease : JAD.

[76]  Chengjie Xiong,et al.  YKL-40: A Novel Prognostic Fluid Biomarker for Preclinical Alzheimer's Disease , 2010, Biological Psychiatry.

[77]  R. Tibshirani,et al.  Flexible Discriminant Analysis by Optimal Scoring , 1994 .

[78]  Brian J Cummings,et al.  The Induction of the TNFα Death Domain Signaling Pathway in Alzheimer's Disease Brain , 2003, Neurochemical Research.

[79]  Á. Hernanz,et al.  Increased cerebrospinal fluid Fas (Apo-1) levels in Alzheimer’s disease Relationship with IL-6 concentrations , 2000, Brain Research.

[80]  Gina N. LaRossa,et al.  [11C]PIB in a nondemented population , 2006, Neurology.

[81]  E. Levy,et al.  Cystatin C protects neuronal cells from amyloid-beta-induced toxicity. , 2010, Journal of Alzheimer's disease : JAD.

[82]  S. Ghezzi,et al.  Vascular endothelial growth factor gene variability is associated with increased risk for AD , 2005, Journal of the Neurological Sciences.

[83]  Leo Breiman,et al.  Random Forests , 2001, Machine Learning.

[84]  M. Sugishita,et al.  [Clinical Dementia Rating (CDR)]. , 2011, Nihon rinsho. Japanese journal of clinical medicine.

[85]  P. Emson,et al.  Relationship of calbindin D28K‐immunoreactive cells and neuropathological changes in the hippocampal formation of Alzheimer's disease , 2001, Neuropathology : official journal of the Japanese Society of Neuropathology.

[86]  Z. Janka,et al.  Serum and cerebrospinal fluid cystatin C levels in vascular and Alzheimer's dementia. , 2000, Acta neurologica Scandinavica.

[87]  J. Kaye,et al.  The FAS gene, brain volume, and disease progression in Alzheimer's disease , 2010, Alzheimer's & Dementia.

[88]  K. Blennow,et al.  Association between CSF biomarkers and incipient Alzheimer's disease in patients with mild cognitive impairment: a follow-up study , 2006, The Lancet Neurology.

[89]  J. Morris.  The Clinical Dementia Rating (CDR) , 1993, Neurology.

[90]  I. Sokal,et al.  CSF tau/Aβ42 ratio for increased risk of mild cognitive impairment , 2007, Neurology.

[91]  K. Blennow,et al.  Increasing CSF phospho-tau levels during cognitive decline and progression to dementia , 2008, Neurobiology of Aging.

[92]  L. Berg.  Clinical Dementia Rating (CDR). , 1988, Psychopharmacology bulletin.

[93]  Bernhard E. Boser,et al.  A training algorithm for optimal margin classifiers , 1992, COLT '92.

[94]  P. Matarrese,et al.  Oxidative imbalance and cathepsin D changes as peripheral blood biomarkers of Alzheimer disease: A pilot study , 2005, FEBS letters.

[95]  F. Jessen,et al.  Macrophage migration inhibitory factor in mild cognitive impairment and Alzheimer's disease. , 2009, Journal of psychiatric research.

[96]  Anders Wallin,et al.  Reduced levels of amyloid-beta-binding proteins in cerebrospinal fluid from Alzheimer's disease patients. , 2009, Journal of Alzheimer's disease : JAD.

[97]  Trey Sunderland,et al.  Decreased beta-amyloid1-42 and increased tau levels in cerebrospinal fluid of patients with Alzheimer disease. , 2003, JAMA.

[98]  Gina N. LaRossa,et al.  Inverse relation between in vivo amyloid imaging load and cerebrospinal fluid Aβ42 in humans , 2006, Annals of neurology.