BackgroundUsing antibody‐based Olink proteomics, we recently established that Thimet Oligopeptidase (THOP1) is a potential cerebrospinal fluid (CSF) biomarker for Alzheimer’s Disease (AD). THOP1 is a neuropeptidase able to degradate Aβ peptides. Here, we aimed to develop specific THOP1 immunoassays for further clinical validation and to enable future implementation of this biomarker.MethodWe developed and compared novel immunoassays for CSF THOP1 analysis on automated Ella™ and Simoa™ platforms. After analytical validation, THOP1 levels were measured in a selection of CSF samples from the original discovery study (24 cognitively unimpaired controls, 24 AD and 24 dementia with Lewy bodies (DLB) patients) on both platforms. Passing bablok‐regression analysis showed that platforms were comparable and thus we selected the Ella platform for further validation in two independent cohorts (Amsterdam Dementia cohort (ADC) including 56 controls, 45 mild cognitive impairment with amyloid pathology (MCI‐Aβ+), 55 AD and 55 DLB patients; Sant Pau Initiative on Neurodegeneration (SPIN) cohort including 53 controls, 50 MCI‐Aβ+, 47 AD and 50 DLB patients). Differences in THOP1 levels between diagnostic groups and correlations to the AD CSF biomarkers were tested by ANCOVA (corrected for sex) and Spearman correlation analyses.ResultTHOP1 levels moderately correlated between proteomics analysis and the novel assays (Olink‐Ella: rho=0.720; Olink‐Simoa: rho=0.584; Ella‐Simoa rho=0.713; figure 1). Consistent with results obtained on the Olink proteomics platform, THOP1 levels were significantly increased in AD compared to DLB patients (Ella: 1.6‐fold; Simoa: 1.7‐fold) and controls (Ella: 1.8‐fold; Simoa: p>0.05). We next showed increased THOP1 levels in both MCI‐Aβ+ (ADC: 1.2‐fold, p<0.05; SPIN: 1.4‐fold, p<0.001) and AD patients (ADC: 1.03‐fold, p>0.05; SPIN: 1.4‐fold, p<0.001) compared to controls (Figure 2). THOP1 levels correlated moderately with levels of t‐Tau (ADC: rho=0.644; SPIN: rho=0.721), p‐Tau (ADC: rho=0.480; SPIN: rho=0.712) and Aβ40 (ADC: not available; SPIN: rho=0.625) but not with Aβ42 (ADC: rho=0.110; SPIN: rho=‐0.09).ConclusionWe show increased CSF THOP1 levels in amyloid positive AD and MCI patients and correlation with CSF Aβ40, which together with literature showing colocalization of THOP1 with Aβ plaques suggests a relationship with Aβ‐production. The association of THOP1 to tau pathologies highlights the potential of this protein as a disease monitoring biomarker.
INTRODUCTIONOur previous antibody‐based cerebrospinal fluid (CSF) proteomics study showed that Thimet oligopeptidase (THOP1), an amyloid beta (Aβ) neuropeptidase, was increased in mild cognitive impairment with amyloid pathology (MCI‐Aβ+) and Alzheimer's disease (AD) dementia compared with controls and dementia with Lewy bodies (DLB), highlighting the potential of CSF THOP1 as an early specific biomarker for AD. We aimed to develop THOP1 immunoassays for large‐scale analysis and validate our proteomics findings in two independent cohorts.METHODSWe developed in‐house CSF THOP1 immunoassays on automated Ella and Simoa platforms. The performance of the different assays were compared using Passing–Bablok regression analysis in a subset of CSF samples from the discovery cohort (n = 72). Clinical validation was performed in two independent cohorts (cohort 1: n = 200; cohort 2: n = 165) using the Ella platform.RESULTSTHOP1 concentrations moderately correlated between proteomics analysis and our novel assays (Rho > 0.580). In both validation cohorts, CSF THOP1 was increased in MCI‐Aβ+ (>1.3‐fold) and AD (>1.2‐fold) compared with controls; and between MCI‐Aβ+ and DLB (>1.2‐fold). Higher THOP1 concentrations were detected in AD compared with DLB only when both cohorts were analyzed together. In both cohorts, THOP1 correlated with CSF total tau (t‐tau), phosphorylated tau (p‐tau), and Aβ40 (Rho > 0.540) but not Aβ42.DISCUSSIONValidation of our proteomics findings underpins the potential of CSF THOP1 as an early specific biomarker associated with AD pathology. The use of antibody‐based platforms in both the discovery and validation phases facilitated the translation of proteomics findings, providing an additional workflow that may accelerate the development of biofluid‐based biomarkers.
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