CSF α-synuclein improves diagnostic and prognostic performance of CSF tau and Aβ in Alzheimer’s disease
Jon B. Toledo, AneKorff, Leslie M. Shaw, John Q. Trojanowski, Jing Zhang* for the Alzheimer’s Disease Neuroimaging Initiative and the Parkinson’s Progression Marker Initiative
Supplemental Materials
Methods
Subjects
ADNI was launched in 2004 by the NIA, the National Institute of Biomedical Imaging and Bioengineering (NIBIB), the Food and Drug Administration, private pharmaceutical companies, and non-profit organizations as a public- private partnership. The primary goal of ADNI has been to test whether serial magnetic resonance imaging (MRI) [1,7], positron emission tomography (PET) [2], CSF tau and Aβ, as well as other biological markers [6] and clinical and neuropsychological assessment [5] can be combined to measure the progression of MCI and early AD, as well as the conversion of NC subjects to MCI or AD. The initial ADNI study (ADNI 1) has been renewed (ADNI 2) to continue to 2016 (25). Determination of sensitive and specific markers of very early AD progression is intended to aid researchers and clinicians in developing new treatments and monitor their effectiveness, as well as lessen the time and cost of clinical trials. The Principal Investigator of this initiative is Michael W. Weiner, MD, VA Medical Center and University of California – San Francisco. ADNI is the result of efforts of many co-investigators from a broad range of academic institutions and private corporations.
Recruitment inclusion and exclusion criteria for ADNI 1
Inclusion criteria were as follows: 1) Hachinski Ischemic Score ≤4; 2) Permitted medications stable for 4 weeks prior to screening; 3) Geriatric Depression Scale score < 6; 4) visual and auditory acuity adequate for neuropsychological testing and good general health with no diseases precluding enrollment; 5) 6 grades of education or work history equivalent; 6) Ability to speak English or Spanish fluently; and 7) A study partner with 10 hr per week of contact either in person or on the telephone and who could accompany the participant to the clinical visits.
Criteria for the different diagnostic groups are summarized in supplementary table 1. Groups were age matched. CN subjects could not have any significant cognitive impairment or impaired activities of daily living. AD had mild AD and had to meet the National Institute of Neurological and Communicative Disorders and Stroke–Alzheimer’s Disease and Related Disorders Association criteria for probable AD [4], whereas MCI subjects should not meet these criteria and have largely intact general cognition and functional performance.
Supplementary table 1 ADNI 1 criteria for recruitment of CN, MCI and AD subjects
CN / MCI / ADMemory complaints / Absent / Present / Present
MMSE / 24-30 / 24-30 / 20-26
CDR / 0 / 0.51 / 0.5-1.0
Delayed recall Logical Memory II subscale of WMSR / 16 YoEd: ≥9
8–15 YoEd: ≥5
0-7 YoEd: ≥3 / 16 YoEd: ≤8
8–15 YoEd: ≤4
0-7 YoEd: ≤2 / 16 YoEd: ≤8
8–15 YoEd: ≤4
0-7 YoEd: ≤2
YoEd: years of education
1Mandatory requirement of the memory box score being 0.5 or greater
Selection criteria for subject included in the PPMI
Inclusion criteria at baseline for PD in PPMI is a diagnosis made less than two years before the screening visit, a Hoehn and Yahr (H&Y) stage of I or II, a dopamine transporter deficit on DaTscan imaging, and no previous treatment for parkinsonian signs. Demographically comparable, cognitively normal CN had to be free of a current or active neurological disorder, have no detectable dopamine transporter deficit evidence, no first degree family members with PD, and a MoCA score > 26. Further details have been described elsewhere [3].
CSF collection in the ADNI and PPMI
CSF in the ADNI cohort was collected into polypropylene collection tubes provided to each site, then transferred into polypropylene transfer tubes, frozen on dry ice within 1 hr after collection and shipped overnight on dry ice to the ADNI Biomarker Core laboratory at the Perelman School of Medicine of the University of Pennsylvania. Aliquots (0.5ml) were prepared from these samples after thawing (1 hr) at room temperature and gentle mixing. The aliquots were stored in bar code–labeled polypropylene vials at -80°C.
CSF in the PPMI cohort wascollected in siliconized polypropylene tubes. The first 1–2 mL of CSF were sent for routine testing in the site’s local laboratory and the other 15-20 mL were transferred into 15 mL polypropylene tubes, mixed gently, centrifuged at 2000×g for 10 min at room temperature and transferred into 1.5 mL pre-cooled siliconized polypropylene aliquot tubes followed by immediate freezing on dry ice.Samples were shipped overnight to PPMI Biorepository Core laboratories, where they were thawed,aliquoted into 0.5 mLsiliconized polypropylene tubes, and stored at -80°C.
CSF measurements in the PPMI
In this assay, 200 µL/well of α-syn standards diluted from reconstituted stock (ranging from 6.1 to 1500 pg/mL), duplicate CSF samples (200 µL/well) were added to the capture antibody coated plate after washing the plate 4 times. After overnight incubation at 2–8°C with shaking, 50 µL/well of biotinylated detector antibody was added, followed by incubation for 2 h at room temperature. Diluted streptavidin HRP was added to wells and the plate was incubated at room temperature for an additional 1 h. After washing the plate 4 times, a mixture of 2 different chemiluminescent substrates was added to wells and endpoint luminescence was read with a luminometer (Biotek Synergy2, Winooski, VT). The concentration of α-syn was measured using standard curves with 4-parameter curve fitting. CSF hemoglobin in the PPMI was analyzed at Covance using an ELISA method comprising reagents obtained from Bethyl Laboratories (Montgomery, TX) according to the manufacturer’s instruction.
Results
Supplementary Fig.1Distribution of α-syn levels in the ADNI cohort
Supplementary Fig.2 Distribution of α-syn levels in the in control (red) and PD (blue) subjects of the PPMI cohort
PPMI Study Team Members
Note: Data used in the preparation of this article were obtained from the Parkinson’s Progression Markers Initiative (PPMI) database (March 18, 2012; As such, the investigator-authors within Version 2·02 March 4, 2011 PPMI contributed to the design and implementation of PPMI and/or provided data but did not participate in the analysis or writing of this report. PPMI study team members include (complete listing at PPMI site).
The Parkinson Progression Marker Initiative (PPMI) Executive Steering Committee:
Kenneth Marek, MD, Principal Investigator, Danna Jennings, MD, Shirley Lasch, Institute for Neurodegenerative Disorders, New Haven, CT; Caroline Tanner, MD, PhD (Site Investigator), The Parkinson’s Institute, Sunnyvale, CA; Tanya Simuni, MD (Site Investigator), Northwestern University, Chicago, IL; Christopher S Coffey, PhD, (Statistics Core, PI), University of Iowa, Iowa City, IA, Karl Kieburtz, MD, MPH (Clinical Core, PI), Emily Flagg, (Clinical Core, Project Manager), Clinical Trials Coordination Center, University of Rochester, NY; SohiniChowdhury, MA, Michael J. Fox Foundation, New York, NY
Steering Committee/Cores:
Werner Poewe, MD (Site Investigator), Innsbruck Medical University, Innsbruck, Austria; Brit Mollenhauer, MD (Site Investigator), Paracelsus-Elena Klinik, Kassel, Germany; Todd Sherer, PhD, Mark Frasier, PhD, Claire Meunier, Michael J. Fox Foundation, New York, NY
Michael J. Fox Foundation, New York, NY
Study Cores:
Clinical Coordination Core: Alice Rudolph, PhD, Cindy Casaceli, MBA, Clinical Trials Coordination Center, University of Rochester, NY
Imaging Core: John Seibyl, MD, Principal Investigator, Susan Mendick, MPH, Institute for Neurodegenerative Disorders, New Haven, CT; Norbert Schuff, PhD, University of California, San Francisco, CA
Statistics Core: Chelsea Caspell, MS, Liz Uribe, MS, Jon Yankey, MS, University of Iowa, Iowa City, IA
Bioinformatics Core: Arthur Toga, PhD, Principal Investigator, Karen Crawford, Laboratory of Neuroimaging (LONI), University of California, Los Angeles, CA
BioRepository: Alison Ansbach, MS, Principal Investigator, Coriell Institute for Medical Research, Camden, NJ; Paola Casalin, MD, PhD, FRCP, GuiliaMalferrari, PhD, BioRep, Milan, Italy
Bioanalytics Core: John Trojanowski, MD, PhD, Principal Investigator, Les Shaw, PhD, Principal Investigator, University of Pennsylvania, Philadelphia, PA
Genetics Core: Andrew Singleton, PhD, Principal Investigator, National Institute on Aging, NIH, Bethesda, MD
Neuropsychological and Cognitive Assessments: Keith Hawkins, PsyD, Yale University, New Haven, CT
Site Investigators and Coordinators:
David Russell, MD, PhD, Laura Leary, BS, Institute for Neurodegenerative Disorders, New Haven, CT; Stewart Factor, DO, Barbara Sommerfeld, RN, MSN, Emory University of Medicine, Atlanta, GA; Penelope Hogarth, MD, Emily Pighetti, Oregon Health and Science University, Portland, OR; Karen Williams, Northwestern University, Chicago, IL; David Standaert, MD, PhD, Stephanie Guthrie, MSN, University of Alabama at Birmingham; Robert Hauser, MD, MBA, Laura Murray, LPN, , University of South Florida, Tampa, FL; Joseph Jankovic, MD, Christine Hunter, RN, Baylor College of Medicine, Houston, TX; Matthew Stern, MD, Abigail Darin, University of Pennsylvania, Philadelphia, PA; Jim Leverenz, MD, Marne Baca, University of Washington, Seattle, WA; Sam Frank, MD, Cathi-Ann Thomas, RN, MS, Boston University, Boston, MA; Irene Richard, MD, Cheryl Deeley, MSN, University of Rochester, Rochester, NY; Linda Rees, MPH, The Parkinson’s Institute, Sunnyvale, CA; FabienneSprenger, MD, Innsbruck Medical University, Innsbruck, Austria; Wolfgang Oertel, MD, Diana Willeke, Paracelsus-Elena Klinik, Kassel, Germany; Holly Shill, MD, Lauren Arnieri, BA, Banner Research Institute, Sun City, AZ; Hubert Fernandez, MD, Jennifer Mule, Cleveland Clinic, Cleveland, OH; Daniela Berg, MD, Katharina Gauss, University of Tuebingen, Tubingen, Germany; Douglas Galasko, MD, Deborah Fontaine, BSN, MS, University of California, San Diego; Zoltan Mari, MD, Arita McCoy, RN, Johns Hopkins University, Baltimore, MD; David Brooks, MD, Bina Shah, BSc, Imperial College London, UK; Paolo Barone, MD, PhD, University of Salerno, Salerno, Italy; Stuart Isaacson, MD, Angela James, Parkinson’s Disease and Movement Disorders Center of Boca Raton, Boca Raton, FL; Alberto Espay, MD, MSc, Kristy Espay, University of Cincinnati, Cincinnati, OH; Dominic Rowe, MD, PhD, MadelaineRanola, B.Nursing, Macquerie University, Sydney Australia
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