title: Identification of novel CSF biomarkers for neurodegeneration and their validation by a high-throughput multiplexed targeted proteomic assay.
abstract: BACKGROUND
  Currently there are no effective treatments for many neurodegenerative diseases. Reliable biomarkers for identifying and stratifying these diseases will be important in the development of future novel therapies. Lewy Body Dementia (LBD) is considered an under diagnosed form of dementia for which markers are needed to discriminate LBD from other forms of dementia such as Alzheimer's Disease (AD). This work describes a Label-Free proteomic profiling analysis of cerebral spinal fluid (CSF) from non-neurodegenerative controls and patients with LBD. Using this technology we identified several potential novel markers for LBD. These were then combined with other biomarkers from previously published studies, to create a 10 min multiplexed targeted and translational MRM-LC-MS/MS assay. This test was used to validate our new assay in a larger cohort of samples including controls and the other neurodegenerative conditions of Alzheimer's and Parkinson's disease (PD).
  
  
  RESULTS
  Thirty eight proteins showed significantly (p < 0.05) altered expression in LBD CSF by proteomic profiling. The targeted MRM-LC-MS/MS assay revealed 4 proteins that were specific for the identification of AD from LBD: ectonucleotide pyrophosphatase/phosphodiesterase 2 (p < 0.0001), lysosome-associated membrane protein 1 (p < 0.0001), pro-orexin (p < 0.0017) and transthyretin (p < 0.0001). Nineteen proteins were elevated significantly in both AD and LBD versus the control group of which 4 proteins are novel (malate dehydrogenase 1, serum amyloid A4, GM2-activator protein, and prosaposin). Protein-DJ1 was only elevated significantly in the PD group and not in either LBD or AD samples. Correlations with Alzheimer-associated amyloid β-42 levels, determined by ELISA, were observed for transthyretin, GM2 activator protein and IGF2 in the AD disease group (r(2) ≥ 0.39, p ≤ 0.012). Cystatin C, ubiquitin and osteopontin showed a strong significant linear relationship (r(2) ≥ 0.4, p ≤ 0.03) with phosphorylated-tau levels in all groups, whilst malate dehydrogenase and apolipoprotein E demonstrated a linear relationship with phosphorylated-tau and total-tau levels in only AD and LBD disease groups.
  
  
  CONCLUSIONS
  Using proteomics we have identified several potential and novel markers of neurodegeneration and subsequently validated them using a rapid, multiplexed mass spectral test. This targeted proteomic platform can measure common markers of neurodegeneration that correlate with existing diagnostic makers as well as some that have potential to show changes between AD from LBD.
authors.affiliation: Centre for Translational Omics, University College London Institute of Child Health, 30 Guilford Street, London, WC1N 1EH, UK. wendy.heywood@ucl.ac.uk.|Neuropediatrics Unit, IRCCS Istituto Neurologico Carlo Besta, Milan, 20133, Italy. wendy.heywood@ucl.ac.uk.
authors.forename: Wendy E
authors.initials: WE
authors.lastname: Heywood
authors.affiliation: Neurology Unit, Department of Pathophysiology and Transplantation, University of Milan, Fondazione Cà Granda, IRCCS Ospedale Policlinico, Via F.Sforza 35, 20122, Milan, Italy. daniela.galimberti@unimi.it.
authors.forename: Daniela
authors.initials: D
authors.lastname: Galimberti
authors.affiliation: Centre for Translational Omics, University College London Institute of Child Health, 30 Guilford Street, London, WC1N 1EH, UK. emily.bliss.10@ucl.ac.uk.
authors.forename: Emily
authors.initials: E
authors.lastname: Bliss
authors.affiliation: Centre for Translational Omics, University College London Institute of Child Health, 30 Guilford Street, London, WC1N 1EH, UK. e.sirka@ucl.ac.uk.
authors.forename: Ernestas
authors.initials: E
authors.lastname: Sirka
authors.affiliation: Dementia Research Centre, University College London Institute of Neurology, London, UK. r.paterson@ucl.ac.uk.
authors.forename: Ross W
authors.initials: RW
authors.lastname: Paterson
authors.affiliation: Reta Lila Weston Institute of Neurological Studies, UCL Institute of Neurology, Queen Square, London, UK. n.magdalinou@ucl.ac.uk.
authors.forename: Nadia K
authors.initials: NK
authors.lastname: Magdalinou
authors.affiliation: Neuropediatrics Unit, IRCCS Istituto Neurologico Carlo Besta, Milan, 20133, Italy. mcarecchio@gmail.com.
authors.forename: Miryam
authors.initials: M
authors.lastname: Carecchio
authors.affiliation: Centre for Translational Omics, University College London Institute of Child Health, 30 Guilford Street, London, WC1N 1EH, UK. emma.reid.09@ucl.ac.uk.
authors.forename: Emma
authors.initials: E
authors.lastname: Reid
authors.affiliation: Dementia Research Centre, University College London Institute of Neurology, London, UK. a.heslegrave@ucl.ac.uk.
authors.forename: Amanda
authors.initials: A
authors.lastname: Heslegrave
authors.affiliation: Neurology Unit, Department of Pathophysiology and Transplantation, University of Milan, Fondazione Cà Granda, IRCCS Ospedale Policlinico, Via F.Sforza 35, 20122, Milan, Italy. chiara.fenoglio@unimi.it.
authors.forename: Chiara
authors.initials: C
authors.lastname: Fenoglio
authors.affiliation: Neurology Unit, Department of Pathophysiology and Transplantation, University of Milan, Fondazione Cà Granda, IRCCS Ospedale Policlinico, Via F.Sforza 35, 20122, Milan, Italy. elio.scarpini@unimi.it.
authors.forename: Elio
authors.initials: E
authors.lastname: Scarpini
authors.affiliation: Dementia Research Centre, University College London Institute of Neurology, London, UK. j.schott@ucl.ac.uk.
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Schott
authors.affiliation: Dementia Research Centre, University College London Institute of Neurology, London, UK. n.fox@ucl.ac.uk.
authors.forename: Nick C
authors.initials: NC
authors.lastname: Fox
authors.affiliation: Dementia Research Centre, University College London Institute of Neurology, London, UK. j.hardy@ucl.ac.uk.
authors.forename: John
authors.initials: J
authors.lastname: Hardy
authors.affiliation: Dementia Research Centre, University College London Institute of Neurology, London, UK. k.bhatia@ion.ucl.ac.uk.
authors.forename: Kailiash
authors.initials: K
authors.lastname: Bhatia
authors.affiliation: Dementia Research Centre, University College London Institute of Neurology, London, UK. k.bhatia@ion.ucl.ac.uk.
authors.forename: Kailash
authors.initials: K
authors.lastname: Bahtia
authors.affiliation: Centre for Translational Omics, University College London Institute of Child Health, 30 Guilford Street, London, WC1N 1EH, UK. s.heales@ucl.ac.uk.|Great Ormond Street Hospital for Children, London, WC1N 3JH, UK. s.heales@ucl.ac.uk.
authors.forename: Simon
authors.initials: S
authors.lastname: Heales
authors.affiliation: Great Ormond Street Hospital for Children, London, WC1N 3JH, UK. Neil.Sebire@gosh.nhs.uk.
authors.forename: Neil J
authors.initials: NJ
authors.lastname: Sebire
authors.affiliation: Dementia Research Centre, University College London Institute of Neurology, London, UK. henrik.zetterberg@clinchem.gu.se.
authors.forename: Henrik
authors.initials: H
authors.lastname: Zetterberg
authors.affiliation: Dementia Research Centre, University College London Institute of Neurology, London, UK. henrik.zetterberg@clinchem.gu.se.|Clinical Neurochemistry Laboratory, Institute of Neuroscience and Physiology, Department of Psychiatry and Neurochemistry, The Sahlgrenska Academy, University of Gothenburg, 431 80, Mölndal, Sweden. henrik.zetterberg@clinchem.gu.se.
authors.forename: Henrik
authors.initials: H
authors.lastname: Zetterburg
authors.affiliation: Centre for Translational Omics, University College London Institute of Child Health, 30 Guilford Street, London, WC1N 1EH, UK. kevin.mills@ucl.ac.uk.|Great Ormond Street Hospital for Children, London, WC1N 3JH, UK. kevin.mills@ucl.ac.uk.
authors.forename: Kevin
authors.initials: K
authors.lastname: Mills
mesh_terms: D000368:Aged; D016229:Amyloid beta-Peptides / Q000134:cerebrospinal fluid* / Q000378:metabolism; D015415:Biomarkers / Q000134:cerebrospinal fluid*; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D008875:Middle Aged; D019636:Neurodegenerative Diseases / Q000175:diagnosis*; D040901:Proteomics / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods; D016875:tau Proteins / Q000134:cerebrospinal fluid* / Q000378:metabolism
chemical_list: D016229:Amyloid beta-Peptides; D015415:Biomarkers; D016875:tau Proteins