Study participants
Six hundred and fifty-one subjects in prodromal stage of AD who were northern Han Chinese in origin were derived from Chinese Alzheimer’s Biomarker and Life style (CABLE) study. CABLE is a large cohort study mainly focusing on Alzheimer’s risk factors and biomarkers in Chinese elderly adults. The samples in CABLE study were recruited at Qingdao Municipal Hospital, consisting of cognitively normal (CN) elders as well as individuals with MCI. All participants were Han Chinese in origin aged 50 to 90 years. Controls had MiniMental State Examination (MMSE) scores of 24 or higher, where lower scores indicate more impairment and higher scores less impairment (range, 0-30), and a Clinical Dementia Rating (CDR) score of 0, where lower scores indicate less impairment and higher scores more impairment (range, 0-3). Patients with MCI had MMSE scores of 24 or higher, objective memory loss tested by delayed recall of the Wechsler Memory Scale (WMS) logical memory II (>1 SD below the normal mean), a CDR score of 0.5, preserved activities of daily living, and absence of dementia. The exclusion criteria were: (1) central nervous system infection, head trauma, epilepsy, multiple sclerosis or other major neurological disorders; (2) major psychological disorders (e.g., depression); (3) severe systemic diseases (e.g., malignant tumors) that may affect CSF or blood levels of AD biomarkers including Aβ and tau; (4) family history of genetic disease. All participants underwent clinical and neuropsychological assessments, biochemical testing, as well as blood and CSF sample collection. Demographic information, AD risk factor profile and medical history were also collected by a comprehensive questionnaire and an electronic medical record system.
Data were obtained from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) database (adni.loni.usc.edu), an independent replication cohort. The ADNI was launched in 2003 as a public-private partnership, led by Principal Investigator Michael W. Weiner, MD. The primary goal of ADNI has been to test whether serial magnetic resonance imaging (MRI), positron emission tomography (PET), other biological markers, as well as clinical and neuropsychological assessment can be combined to measure the progression of mild cognitive impairment (MCI) and early AD. For up-to-date information, see www.adni-info.org.
Our ADNI cohort included all CN controls, MCI patients and AD patients with available baseline CSF α-synuclein samples. Inclusion/exclusion criteria are described at http://www.adni-info.org. In our study, we stratified the MCI group into stable MCI (sMCI) with no progression to AD dementia during at least 2-year follow-up, and progressive MCI (pMCI) with progression to AD dementia during at least 2-year follow-up. Therefore, we included the following 4 groups: CN controls, sMCI group, pMCI group and AD group. As to “ATN” binary (i.e., positive or negative) categories: amyloid positive (A+) and negative (A-) were separated by a cutoff value of 192 pg/ml for CSF Aβ level. Tau pathology positive (T+) and negative (T-) were separated by a cutoff value of 23 pg/ml for CSF phosphorylated tau (p-tau) level.
The CABLE study was approved by the Institutional Ethics Committees of Qingdao Municipal Hospital. Written informed consent was obtained from all study participants directly or from their caregivers. The ADNI study was approved by the Institutional Review Board at each of the participating centers, and all participants provided written informed consent.
CSF Measurements
CSF was taken by lumbar punctures through the L3/L4 interspace and gently mixed to avoid gradient effects. The samples were promptly centrifuged at 2000 g for 10 min to eliminate cells and other insoluble materials, aliquoted in 1 ml portions, snap frozen at –80◦C until use. CSF was sampled between 08:00 and 09:00 in the morning in order to take into account a possible circadian rhythm effect. The CSF samples were stored at –80℃ until further analysis of Aβ and tau.
In CABLE study, CSF Aβ42, total tau (t-tau), p-tau and CSF total α-synuclein concentrations were measured separately using an enzyme-linked immunosorbent assay (ELISA) kit (Fujirebio, Ghent, Belgium). All the ELISA measurements were performed according to the manufacturers’ instructions. The samples and standards were measured in duplicate, and the means of the duplicates were used for the statistical analyses.
In ADNI database, CSF Aβ42, t-tau and p-tau were measured at the ADNI biomarker core (University of Pennsylvania) using the multiplex xMAP Luminex platform (Luminex Corp, Austin, TX, USA) with the INNOBIA AlzBio3 kit (Fujirebio, Ghent, Belgium). Levels of CSF total α-synuclein concentrations in the ADNI cohort were measured by Luminex MicroPlex Microspheres (Luminex Corp, Austin, TX). A biotinylated goat antihuman α-syn antibody (R&D systems, Minneapolis, MN, USA) was used as the detection antibody. The α-synuclein Luminex assay demonstrated low day-to-day and plate-to-plate signal variability. The accuracy of the assay was further determined by the recovery of spiked α-synuclein protein, which was close to 93 %.
Neuroimaging
Structural MRI was performed only for the ADNI subjects using a Siemens Trio 3.0T scanner or Vision 1.5T scanner (GE, Siemens and Philips). Free-surfer software package version 4.3 and 5.1 image processing framework were used to process regional volume estimates for the 1.5 and 3.0T MRI images, respectively. ROIs included the hippocampus and ventricles.
Statistical analyses
We tested associations between CSF α-synuclein and demographic factors using the Mann-Whitney test and the Spearman rank correlation test. We tested the associations of CSF α-synuclein with CSF Aβ42, t-tau, and p-tau levels using linear regression adjusted for age, gender, educational level, diagnosis and APOE ε4 genotype (with CSF α-synuclein as predictor). In the ADNI database, associations between CSF α-synuclein concentrations and the diagnostic groups were tested in an analysis of covariance model adjusted for age, gender, educational level and APOE ε4 genotype. Logistic regression analysis was used to assess the impact of different CSF analytes on the risk of conversion to AD. The receiver-operator curves and the area under the curves were derived from the predictive probabilities of the logistic regression models. We tested the associations of CSF α-synuclein concentrations with longitudinal cognition and brain structure using linear mixed-effects models. These models had random intercepts and slopes for time and an unstructured covariance matrix for the random effects and included the interaction between (continuous) time and CSF α-synuclein as predictor with adjustment for confounders. All tests were 2-sided. Statistical significance was set at P < 0.05. All regression analyses were corrected for age, gender, educational level, diagnosis, and APOE ε4 genotype. The following variables were natural log-transformed to ensure normality: CSF α-synuclein, p-tau, t-tau, Aβ and hippocampus volume. All statistical analyses were performed using a software program (R, version 3.4.0; The R Foundation).