Proteomic Network Analysis of Alzheimer’s Disease Cerebrospinal Fluid Reveals Alterations Associated with APOE ε4 Genotype and Atomoxetine Treatment

Alzheimer’s disease (AD) is currently defined at the research level by the aggregation of amyloid-β (Aβ) and tau proteins in brain. While biofluid biomarkers are available to measure Aβ and tau pathology, few biomarkers are available to measure the complex pathophysiology that is associated with these two cardinal neuropathologies. Here we describe the proteomic landscape of cerebrospinal fluid (CSF) changes associated with Aβ and tau pathology in 300 individuals as assessed by two different proteomic technologies—tandem mass tag (TMT) mass spectrometry and SomaScan. Harmonization and integration of both data types allowed for generation of a robust protein co-expression network consisting of 34 modules derived from 5242 protein measurements, including disease-relevant modules associated with autophagy, ubiquitination, endocytosis, and glycolysis. Three modules strongly associated with the apolipoprotein E ε4 ( APOE ε4) AD risk genotype mapped to oxidant detoxification, mitogen associated protein kinase (MAPK) signaling, neddylation, and mitochondrial biology, and overlapped with a previously described lipoprotein module in serum. Neddylation and oxidant detoxification/MAPK signaling modules had a negative association with APOE ε4 whereas the mitochondrion module had a positive association with APOE ε4. The directions of association were consistent between CSF and blood in two independent longitudinal cohorts, and altered levels of all three modules in blood were associated with dementia over 20 years prior to diagnosis. Dual-proteomic platform analysis of CSF samples from an AD phase 2 clinical trial of atomoxetine (ATX) demonstrated that abnormal elevations in the glycolysis CSF module—the network module most strongly correlated to cognitive function—were reduced by ATX treatment. Individuals who had more severe glycolytic changes at baseline responded better to ATX. Clustering of individuals based on their CSF proteomic network profiles revealed ten groups that did not cleanly stratify by Aβ and tau status, underscoring the heterogeneity of pathological changes not fully reflected by Aβ and tau. AD biofluid proteomics holds promise for the development of biomarkers that reflect diverse pathologies for use in clinical trials and precision medicine. ### Competing Interest Statement The AGES-Reykjavik proteomics measurements were supported by Novartis Biomedical Research and performed at SomaLogic. L.L.J is an employee and stockholder of Novartis. ### Funding Statement This study was supported by K08AG068604 (E.C.B.J.), the Emory Goizueta Alzheimer's Disease Research Center (P30AG066511, A.I.L.), the Emory Healthy Brain Study (R01AG070937, J.J.L.), the Accelerating Medicines Partnership Program for Alzheimer's Disease (U01AG061357, A.I.L. and N.T.S.), R01AG072120 (A.P.W. and T.S.W.), and R01AG075827 (T.S.W. and A.P.W.). The AGES-Reykjavik study was financed by National Institute on Aging (NIA) contracts N01-AG-12100 and HHSN271201200022C for Vi.G. Icelandic Heart Association (IHA) received a grant from Althingi (the Icelandic Parliament) and Vi.G. received funding from the NIA (1R01AG065596-01A1). ### 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 IRB of Emory University gave ethical approval for this work. 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 Raw data, case traits, and analyses related to this manuscript are available at .