Aquaporin-4 as an early cerebrospinal fluid biomarker of Alzheimer's disease

Importance: Aquaporin-4 (AQP4) plays a critical role in the glymphatic system, responsible for clearing brain solutes like Abeta peptides. Exploring AQP4 as an Alzheimer's disease (AD) biomarker might aid in the understanding of AD neuropathology and monitor the effects of novel drug candidates on the glymphatic system. Objective: To determine the potential of CSF AQP4 as an early stage AD biomarker using a newly established immunoassay. Design: A discovery cohort (n = 157) (2010-2022), composed by AD patients, other neurodegenerative conditions and controls (CON), was used to assess the diagnostic performance of CSF AQP4. Subsequently, AQP4 concentration across the clinical AD spectrum was analyzed in two independent validation cohorts (n = 176) (2016-2023). Stratified randomization based on diagnosis and blinded analyses were performed. Setting Multicenter study: Ulm University Hospital (discovery), University of Perugia (validation cohort I), University Hospital of Torino (validation cohort II). Participants: Discovery cohort: 38 CON, 40 AD, 21 primary progressive aphasia, 20 behavioural variant frontotemporal dementia, 17 amyotrophic lateral sclerosis (ALS), and 21 Lewy body disease (LBD). Validation cohorts: 55 CON, 14 preclinical AD, 51 AD with mild cognitive impairment (AD-MCI), 39 AD dementia (ADD) and 17 mild cognitive impairment with non-AD pathology (non-AD MCI). The discovery cohort was selected through random sampling, while validation cohort I and II followed a consecutive sampling method. Exposures: CSF AQP4 Main Outcome (s) and Measure (s): AQP4 CSF biomarker detection Results: A total of 333 participants were included in this study. In the discovery cohort, the median (IQR) age was 69 (61-75) years and 46.5% of the cohort were women. CSF AQP4 concentration was increased in AD patients compared to CON (p < 0.001), ALS (p = 0.015), and LBD (p = 0.012) patients. CSF AQP4 in AD patients were further analyzed in validation cohort I (median (IQR) age, 74 (71-77) years; 62.0% women), and II (median (IQR) age, 71 (65-75) years; 58.5% women). When analyzing the different stages of the AD continuum in validation cohort I, AD-MCI (p = 0.011) and ADD (p = 0.002) patients had significantly higher AQP4 concentrations than CON. Similar results were obtained in cohort II, where AQP4 levels were higher in AD-MCI (p < 0.001) and ADD (p = 0.028) patients compared to controls. The AQP4 accuracy (area under the receiver operating characteristic curve [AUC]) to distinguish AD patients from CON was 0.81 (95% CI: 0.71 to 0.90, p <0.001) in the discovery cohort, 0.70 (95% CI: 0.60 to 0.81, p<0.001) in validation cohort I and 0.82 (95% CI 0.71 to 0.94, p <0.001) in II. Moreover, patients with AD-MCI could be distinguished from non-AD MCI with an AUC of 0.79 (95% CI: 0.65 to 0.93, p = 0.002). Conclusions and Relevance: Three independent cohorts consistently showed elevated AQP4 levels in AD (including AD-MCI and ADD) compared to CON and other neurodegenerative conditions, suggesting specificity to AD pathology. These findings contribute to understanding AD neuropathology and propose AQP4 as a potential early biomarker of AD. Further investigations are needed to proof AQP4 as a fluid blood brain barrier damage marker. ### Competing Interest Statement Ms. Gomez de San Jose has no disclosures to report Dr. Halbgebauer has no disclosures to report Prof. Steinacker has no disclosures to report Dr. Anderl-Straub has no disclosures to report Dr. Abu-Rumeileh has no disclosures to report Dr. Barba has no disclosures to report Dr. Oeckl has no disclosures to report Dr. Bellomo has no disclosures to report Dr. Gaetani participated on advisory boards for, and received writing honoraria and travel grants from Almirall, Biogen, Euroimmun, Fujirebio, Lilly, Merck, Mylan, Novartis, Roche, Sanofi, Siemens Healthineers and Teva. Dr. Toja has no disclosures to report Ms. Mravinacova has no disclosures to report Dr. Bergstrom has no disclosures to report Dr. Manberg has no disclosures to report Dr. Grassini has no disclosures to report Prof. Rainero has no disclosures to report Prof. Nilsson has no disclosures to report Prof. Parnetti has no disclosures to report Prof. Otto has no disclosures to report ### Funding Statement MO, LP, PN and GSJN are supported by the Marie Skłodowska-Curie grant agreement No. 860197 - MIRIADE project (European Union's Horizon 2020 research and innovation program) GB is supported by the Postdoctoral Fellowship for Basic Scientists grant of the Parkinson's Foundation (Award ID: PF-PRF-934916). LP and LG are funded by the European Union-Next Generation EU - PNRR M6C2 - Investimento 2.1 Valorizzazione e potenziamento della ricerca biomedica del SSN (PNRR-MAD-2022-12376035). LB is supported by the Medical Faculty of Martin-Luther University Halle Wittenberg (Junior Clinician Scientist Programm No. JCS24/02). SAB received research support from the Medical Faculty of Martin-Luther University Halle-Wittenberg (Clinician Scientist-Programm No. CS22/06). ### 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 Ethics Comittee of the University of Ulm gave ethical approval for this work. The Ethics Comittee of the University of Perugia gave ethical approval for this work. The Ethics Comittee of the University of Turin 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 All data produced in the present study are available upon reasonable request to the authors