Multi-analyte proteomic analysis identifies blood-based neuroinflammation, cerebrovascular and synaptic biomarkers in preclinical Alzheimer’s disease

Background Blood-based biomarkers are gaining grounds for Alzheimer’s disease (AD) detection. However, two key obstacles need to be addressed: the lack of methods for multi-analyte assessments and the need for markers of neuroinflammation, vascular, and synaptic dysfunction. Here, we evaluated a novel multi-analyte biomarker platform, NULISAseq CNS disease panel, a multiplex NUcleic acid-linked Immuno-Sandwich Assay (NULISA) targeting ∼120 analytes, including classical AD biomarkers and key proteins defining various disease hallmarks.

The NULISAseq panel was applied to 176 plasma samples from the MYHAT-NI cohort of cognitively normal participants from an economically underserved region in Western Pennsylvania. Classical AD biomarkers, including p-tau181, p-tau217, p-tau231, GFAP, NEFL, Aβ40, and Aβ42, were also measured using Single Molecule Array (Simoa). Amyloid pathology, tau pathology, and neurodegeneration were evaluated with [11C] PiB PET, [18F]AV-1451 PET, and MRI, respectively. Linear mixed models were used to examine cross-sectional and Wilcoxon rank sum tests for longitudinal associations between NULISA biomarkers and AD pathologies. Spearman correlations were used to compare NULISA and Simoa.

NULISA concurrently measured 116 plasma biomarkers with good technical performance, and good correlation with Simoa measures. Cross-sectionally, p-tau217 was the top hit to identify Aβ pathology, with age, sex, and APOE genotype-adjusted AUC of 0.930 (95%CI: 0.878-0.983). Fourteen markers were significantly decreased in Aβ-PET+ participants, including TIMP3, which regulates brain Aβ production, the neurotrophic factor BDNF, the energy metabolism marker MDH1, and several cytokines. Longitudinally, FGF2, IL4, and IL9 exhibited Aβ PET-dependent yearly increases in Aβ-PET+ participants. Markers with tau PET-dependent longitudinal changes included the microglial activation marker CHIT1, the reactive astrogliosis marker CHI3L1, the synaptic protein NPTX1, and the cerebrovascular markers PGF, PDGFRB, and VEFGA; all previously linked to AD but only reliably measured in cerebrospinal fluid. SQSTM1, the autophagosome cargo protein, exhibited a significant association with neurodegeneration status after adjusting age, sex, and APOE ε4 genotype.

Together, our results demonstrate the feasibility and potential of immunoassay-based multiplexing to provide a comprehensive view of AD-associated proteomic changes. Further validation of the identified inflammation, synaptic, and vascular markers will be important for establishing disease state markers in asymptomatic AD. Competing Interest Statement The authors have declared no competing interest. Funding Statement TKK was supported by the NIH (R01 AG083874, U24 AG082930, P30 AG066468, RF1 AG052525-01A1, R01 AG053952-05, R37 AG023651-17, RF1 AG025516-12A1, R01 AG073267-02, R01 AG075336-01, R01 AG072641-02, P01 AG025204-16) and the Alzheimer;s Association (#AARF-21-850325). MDI was supported by NIH/NIA grants P01AG14449 and P01AG025204. The MYHAT study was supported by R37 AG023651-17 and MYHAT-NI by R01 AG052521. Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The MYHAT-NI study was approved by the University of Pittsburgh Institutional Review Board (STUDY19020264). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data availability De-identified, cohort-level data will be shared at the request of verified investigators to replicate procedures and results reported in this article. Data transfer agreements in accordance with US legislation and the decisions of the University of Pittsburgh’s Institutional Review Board, which covers the jurisdiction of the MYHAT-NI study, may need to be established. Abbreviations - Aβ - Amyloid-beta - AD - Alzheimer’s disease - AUC - Area under Curve - BBB - Blood brain barrier - CDR - Clinical dementia rating - CNS - Central nervous system - CSF - Cerebrospinal fluid - CV - Coefficient of variation - FDR - False discovery rate - IC - Internal control - IQR - Interquartile range - LOD - Limit of detection - MCI - Mild cognitive impairment - MMSE - Mini-Mental State Examination - MRI - Magnetic resonance imaging - MYHAT - Monongahela Youghiogheny Healthy Aging Team - MYHAT - NI Monongahela Youghiogheny Healthy Aging Team-Neuroimaging - NPQ - NULISA protein quantification - NULISA - NUcleic acid-linked Immuno-Sandwich Assay - NULISAseq - NULISA with next-generation sequencing readout - PiB - Pittsburgh compound-B - PLA - Proximity ligation assay - p-tau - Phosphorylated tau - PET - Positron emission tomography - QC - Quality control - ROC - Receiver operating characteristic - ROIs - Regions of interest - SD - Standard deviation - Simoa - Single-molecule array - SUVR - Standardized uptake value ratio