Post-GWAS multiomic functional investigation of the TNIP1 locus in Alzheimer's disease highlights a potential role for GPX3.

INTRODUCTION: Recent genome-wide association studies (GWAS) have reported a genetic association with Alzheimer's disease (AD) at the TNIP1/GPX3 locus, but the mechanism is unclear. METHODS: We used cerebrospinal fluid (CSF) proteomics data to test (n = 137) and replicate (n = 446) the association of glutathione peroxidase 3 (GPX3) with CSF biomarkers (including amyloid and tau) and the GWAS-implicated variants (rs34294852 and rs871269). RESULTS: CSF GPX3 levels decreased with amyloid and tau positivity (analysis of variance P = 1.5 × 10-5) and higher CSF phosphorylated tau (p-tau) levels (P = 9.28 × 10-7). The rs34294852 minor allele was associated with decreased GPX3 (P = 0.041). The replication cohort found associations of GPX3 with amyloid and tau positivity (P = 2.56 × 10-6) and CSF p-tau levels (P = 4.38 × 10-9). DISCUSSION: These results suggest variants in the TNIP1 locus may affect the oxidative stress response in AD via altered GPX3 levels. HIGHLIGHTS: Cerebrospinal fluid (CSF) glutathione peroxidase 3 (GPX3) levels decreased with amyloid and tau positivity and higher CSF phosphorylated tau. The minor allele of rs34294852 was associated with lower CSF GPX3. levels when also controlling for amyloid and tau category. GPX3 transcript levels in the prefrontal cortex were lower in Alzheimer's disease than controls. rs34294852 is an expression quantitative trait locus for GPX3 in blood, neutrophils, and microglia.

Cerebrospinal fluid biomarker panel for synaptic dysfunction in a broad spectrum of neurodegenerative diseases.

Synaptic dysfunction and degeneration is likely the key pathophysiology for the progression of cognitive decline in various dementia disorders. Synaptic status can be monitored by measurement of synaptic proteins in cerebrospinal fluid (CSF). In the current study, the aim was to investigate and compare both known and new synaptic proteins as potential biomarkers of synaptic dysfunction, especially in the context of Alzheimer's disease (AD). Seventeen synaptic proteins were quantified in CSF using two different targeted mass spectrometry assays in the prospective Swedish BioFINDER-2 study. The study included 958 individuals, characterized as having mild cognitive impairment (MCI, n = 205), AD dementia (n = 149), and a spectrum of other neurodegenerative diseases (n = 171), as well as cognitively unimpaired (CU, n = 443). Synaptic protein levels were compared between diagnostic groups and their associations with cognitive decline and key neuroimaging measures (Aß-PET, tau-PET, and cortical thickness) were assessed. Among the 17 synaptic proteins examined, 14 were specifically elevated in the AD continuum. SNAP-25, 14-3-3 zeta/delta, beta-synuclein, and neurogranin exhibited the highest discriminatory accuracy to differentiate AD dementia from controls (AUCs = 0.81-0.93). SNAP-25 and 14-3-3 zeta/delta also had the strongest associations with tau-PET, Aß-PET, and cortical thickness at baseline, and were associated with longitudinal changes in these imaging biomarkers (ß(SE)=-0.056(0.0006) to 0.058(0.005), p < 0.0001). SNAP-25 was the strongest predictor of progression to AD dementia in non-demented individuals (Hazard ratio = 2.11). In contrast, neuronal pentraxins were decreased in all neurodegenerative diseases (except for Parkinson's disease), and NPTX2 showed the strongest associations with subsequent cognitive decline (longitudinal MMSE; ß(SE) = 0.57(0.1), p ≤ 0.0001 and mPACC; ß(SE) = 0.095(0.024), p ≤ 0.001) across the AD continuum. Interestingly, utilizing a ratio of the proteins that displayed higher levels in AD, such as SNAP-25 or 14-3-3 zeta/delta, over NPTX2 improved the biomarkers' association with cognitive decline and brain atrophy. We found that especially 14-3-3 zeta/delta and SNAP-25 are promising synaptic biomarkers of pathophysiological changes in AD. Neuronal pentraxins were identified as general indicators of neurodegeneration and associated with cognitive decline across various neurodegenerative dementias. The ratios of SNAP-25/NPTX2 and 14-3-3 zeta/delta/NPTX2 were found to best predict cognitive decline and brain atrophy.

Profiling amyloid-ß peptides as biomarkers for cerebral amyloid angiopathy.

Brain amyloid-ß (Aß) deposits are key pathological hallmarks of both cerebral amyloid angiopathy (CAA) and Alzheimer's disease (AD). Microvascular deposits in CAA mainly consist of the Aß40 peptide, whereas Aß42 is the predominant variant in parenchymal plaques in AD. The relevance in pathogenesis and diagnostic accuracy of various other Aß isoforms in CAA remain understudied. We aimed to investigate the biomarker potential of various Aß isoforms in cerebrospinal fluid (CSF) to differentiate CAA from AD pathology. We included 25 patients with probable CAA, 50 subjects with a CSF profile indicative of AD pathology (AD-like), and 23 age- and sex-matched controls. CSF levels of Aß1-34 , Aß1-37 , Aß1-38 , Aß1-39 , Aß1-40 , and Aß1-42 were quantified by liquid chromatography mass spectrometry. Lower CSF levels of all six Aß peptides were observed in CAA patients compared with controls (p = 0.0005-0.03). Except for Aß1-42 (p = 1.0), all peptides were decreased in CAA compared with AD-like subjects (p = 0.007-0.03). Besides Aß1-42 , none of the Aß peptides were decreased in AD-like subjects compared with controls. All Aß peptides combined differentiated CAA from AD-like subjects better (area under the curve [AUC] 0.84) than individual peptide levels (AUC 0.51-0.75). Without Aß1-42 in the model (since decreased Aß1-42 served as AD-like selection criterion), the AUC was 0.78 for distinguishing CAA from AD-like subjects. CAA patients and AD-like subjects showed distinct disease-specific CSF Aß profiles. Peptides shorter than Aß1-42 were decreased in CAA patients, but not AD-like subjects, which could suggest different pathological mechanisms between vascular and parenchymal Aß accumulation. This study supports the potential use of this panel of CSF Aß peptides to indicate presence of CAA pathology with high accuracy.

Comparison of immunoassay- with mass spectrometry-derived p-tau quantification for the detection of Alzheimer's disease pathology.

BACKGROUND: Antibody-based immunoassays have enabled quantification of very low concentrations of phosphorylated tau (p-tau) protein forms in cerebrospinal fluid (CSF), aiding in the diagnosis of AD. Mass spectrometry enables absolute quantification of multiple p-tau variants within a single run. The goal of this study was to compare the performance of mass spectrometry assessments of p-tau181, p-tau217 and p-tau231 with established immunoassay techniques. METHODS: We measured p-tau181, p-tau217 and p-tau231 concentrations in CSF from 173 participants from the TRIAD cohort and 394 participants from the BioFINDER-2 cohort using both mass spectrometry and immunoassay methods. All subjects were clinically evaluated by dementia specialists and had amyloid-PET and tau-PET assessments. Bland-Altman analyses evaluated the agreement between immunoassay and mass spectrometry p-tau181, p-tau217 and p-tau231. P-tau associations with amyloid-PET and tau-PET uptake were also compared. Receiver Operating Characteristic (ROC) analyses compared the performance of mass spectrometry and immunoassays p-tau concentrations to identify amyloid-PET positivity. RESULTS: Mass spectrometry and immunoassays of p-tau217 were highly comparable in terms of diagnostic performance, between-group effect sizes and associations with PET biomarkers. In contrast, p-tau181 and p-tau231 concentrations measured using antibody-free mass spectrometry had lower performance compared with immunoassays. CONCLUSIONS: Our results suggest that while similar overall, immunoassay-based p-tau biomarkers are slightly superior to antibody-free mass spectrometry-based p-tau biomarkers. Future work is needed to determine whether the potential to evaluate multiple biomarkers within a single run offsets the slightly lower performance of antibody-free mass spectrometry-based p-tau quantification.

Alzheimer's Disease Biomarker Analysis Using Targeted Mass Spectrometry.

Alzheimer's disease (AD) is characterized by several neuropathological changes, mainly extracellular amyloid aggregates (plaques), intraneuronal inclusions of phosphorylated tau (tangles), as well as neuronal and synaptic degeneration, accompanied by tissue reactions to these processes (astrocytosis and microglial activation) that precede neuronal network disturbances in the symptomatic phase of the disease. A number of biomarkers for these brain tissue changes have been developed, mainly using immunoassays. In this review, we discuss how targeted mass spectrometry (TMS) can be used to validate and further characterize classes of biomarkers reflecting different AD pathologies, such as tau- and amyloid-beta pathologies, synaptic dysfunction, lysosomal dysregulation, and axonal damage, and the prospect of using TMS to measure these proteins in clinical research and diagnosis. TMS advantages and disadvantages in relation to immunoassays are discussed, and complementary aspects of the technologies are discussed.

Differential patterns of lysosomal dysfunction are seen in the clinicopathological forms of primary progressive aphasia.

Increasing evidence implicates endo-lysosomal dysfunction in frontotemporal dementia (FTD). 18 proteins were quantified using a mass spectrometry assay panel in the cerebrospinal fluid of 36 people with the language variant of FTD, primary progressive aphasia (PPA) (including 13 with non-fluent variant (nfvPPA), 11 with semantic variant (svPPA), and 12 with logopenic variant (lvPPA)) and 19 healthy controls. The concentrations of the cathepsins (B, D, F, L1, and Z) as well as AP-2 complex subunit beta, ganglioside GM2 activator, beta-hexosaminidase subunit beta, tissue alpha L-fucosidase, and ubiquitin were decreased in nfvPPA compared with controls. In contrast, the concentrations of amyloid beta A4 protein, cathepsin Z, and dipeptidyl peptidase 2 were decreased in svPPA compared with controls. No proteins were abnormal in lvPPA. These results indicate a differential alteration of lysosomal proteins in the PPA variants, suggesting those with non-Alzheimer's pathologies are more likely to show abnormal lysosomal function.

Optimal blood tau species for the detection of Alzheimer's disease neuropathology: an immunoprecipitation mass spectrometry and autopsy study.

Plasma-to-autopsy studies are essential for validation of blood biomarkers and understanding their relation to Alzheimer's disease (AD) pathology. Few such studies have been done on phosphorylated tau (p-tau) and those that exist have made limited or no comparison of the different p-tau variants. This study is the first to use immunoprecipitation mass spectrometry (IP-MS) to compare the accuracy of eight different plasma tau species in predicting autopsy-confirmed AD. The sample included 123 participants (AD = 69, non-AD = 54) from the Boston University Alzheimer's disease Research Center who had an available ante-mortem plasma sample and donated their brain. Plasma samples proximate to death were analyzed by targeted IP-MS for six different tryptic phosphorylated (p-tau-181, 199, 202, 205, 217, 231), and two non-phosphorylated tau (195-205, 212-221) peptides. NIA-Reagan Institute criteria were used for the neuropathological diagnosis of AD. Binary logistic regressions tested the association between each plasma peptide and autopsy-confirmed AD status. Area under the receiver operating curve (AUC) statistics were generated using predicted probabilities from the logistic regression models. Odds Ratio (OR) was used to study associations between the different plasma tau species and CERAD and Braak classifications. All tau species were increased in AD compared to non-AD, but p-tau217, p-tau205 and p-tau231 showed the highest fold-changes. Plasma p-tau217 (AUC = 89.8), p-tau231 (AUC = 83.4), and p-tau205 (AUC = 81.3) all had excellent accuracy in discriminating AD from non-AD brain donors, even among those with CDR < 1). Furthermore, p-tau217, p-tau205 and p-tau231 showed the highest ORs with both CERAD (ORp-tau217 = 15.29, ORp-tau205 = 5.05 and ORp-tau231 = 3.86) and Braak staging (ORp-tau217 = 14.29, ORp-tau205 = 5.27 and ORp-tau231 = 4.02) but presented increased levels at different amyloid and tau stages determined by neuropathological examination. Our findings support plasma p-tau217 as the most promising p-tau species for detecting AD brain pathology. Plasma p-tau231 and p-tau205 may additionally function as markers for different stages of the disease.

Next-generation proteomics technologies in Alzheimer's disease: from clinical research to routine diagnostics.

INTRODUCTION: Clinical proteomics studies of Alzheimer's disease (AD) research aim to identify biomarkers useful for clinical research, diagnostics, and improve our understanding of the pathological processes involved in the disease. The rapidly increasing performance of proteomics technologies is likely to have great impact on AD research. AREAS COVERED: We review recent proteomics approaches that have advanced the field of clinical AD research. Specifically, we discuss the application of targeted mass spectrometry (MS), labeling-based and label-free MS-based as well as affinity-based proteomics to AD biomarker development, underpinning their importance with the latest impactful clinical studies. We evaluate how proteomics technologies have been adapted to meet current challenges. Finally, we discuss the limitations and potential of proteomics techniques and whether their scope might extend beyond current research-based applications. EXPERT OPINION: To date, proteomics technologies in the AD field have been largely limited to AD biomarker discovery. The recent development of the first successful disease-modifying treatments of AD will further increase the need for blood biomarkers for early, accurate diagnosis, and CSF biomarkers that reflect specific pathological processes. Proteomics has the potential to meet these requirements and to progress into clinical routine practice, provided that current limitations are overcome.

Harmonization and standardization of biofluid-based biomarker measurements for AT(N) classification in Alzheimer's disease.

Fluid biomarkers are currently measured in cerebrospinal fluid and blood for Alzheimer's disease diagnosis and are promising targets for drug development and for patients' follow-up in clinical trials. These biomarkers have been grouped in an unbiased research framework, the amyloid (Aß), tau, and neurodegeneration (AT[N]) biomarker system to aid patients' early diagnosis and stratification. Metrological approaches relying on mass spectrometry have been used for the development of reference materials and reference measurement procedures. Despite their excellent performances as clinical tools, fluid biomarkers often present an important between-laboratory variation. Standardization efforts were carried out on the biomarkers currently included in the AT(N) classification system, involving the collaboration of national metrology institutes, clinicians, researchers, and in vitro diagnostic providers. This article provides an overview of current activities towards standardization. These reference methods and reference materials may be used for recalibration of immunoassays and the establishment of standardized cutoff values allowing a better stratification of Alzheimer's disease patients. Highlights: The AT(N) biomarker system allows stratifying AD patients on the basis of biomarker profiles.Fluid biomarker measurements often present an important between-laboratory variation preventing the establishment of standardized cutoff values.Overview on the standardization initiatives involving the fluid biomarkers currently included in the AT(N) framework.

α-Synuclein seed amplification assay as a diagnostic tool for parkinsonian disorders.

INTRODUCTION: Synucleinopathies such as Parkinson's disease (PD) and multiple system atrophy (MSA) can be challenging to diagnose due to the symptom overlap with, for example, atypical parkinsonisms like progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). Seed amplification assays (SAA), developed for the detection of α-synuclein (αSyn) aggregates in CSF, have been successful when used as a biomarker evaluation for synucleinopathies. In this study, we investigated the potential of this assay to not only detect αSyn seeds in CSF, but also discriminate between movement disorders. METHODS: The αSyn-SAA was tested in a Scandinavian cohort composed of 129 CSF samples from patients with PD (n = 55), MSA (n = 27), CBD (n = 7), and PSP (n = 16), as well as healthy controls (HC, n = 24). RESULTS: The αSyn seed amplification assay (αSyn-SAA) was able to correctly identify all PD samples as positive (sensitivity of 100%) while also discriminating the PD group from HC (70.8% specificity, p < 0.0001) and tauopathies [CBD (71% specificity) and PSP (75% specificity), p < 0.0001)]. The αSyn-SAA was also able to identify almost all MSA samples as positive for αSyn aggregation (sensitivity of 92.6%). In general, this assay is able to discriminate between the synucleinopathies and tauopathies analyzed herein (p < 0.0001) despite the overlapping symptoms in these diseases. CONCLUSION: These findings suggest the αSyn-SAA is a useful diagnostic tool for differentiating between different parkinsonian disorders, although further optimization may be needed.

Novel cerebrospinal fluid biomarkers correlating with shunt responsiveness in patients with idiopathic normal pressure hydrocephalus.

BACKGROUND: Idiopathic Normal pressure hydrocephalus (iNPH) is a form of adult hydrocephalus that is clinically characterized by progressive gait impairment, cognitive dysfunction, and urinary incontinence. The current standard method of treatment involves surgical installation of a CSF diversion shunt. However, only a fraction of patients shows an alleviation of symptoms from shunt surgery. Thus, the purpose of this prospective explorative proteomic study was to identify prognostic CSF biomarkers to predict shunt responsiveness in iNPH patients. Further, we evaluated the ability of the core Alzheimer's disease (AD) CSF biomarkers phosphorylated (p)-tau, total (t)-tau, and amyloid-ß 1-42 (Aß1-42) to serve as predictors of shunt response. METHODS: We conducted a tandem mass tag (TMT) proteomic analysis of lumbar CSF from 68 iNPH patients, sampled pre-shunt surgery. Tryptic digests of CSF samples were labelled with TMTpro reagents. The TMT multiplex samples were fractionated in 24 concatenated fractions by reversed-phase chromatography at basic pH and analysed by liquid chromatography coupled to mass spectrometry (LC-MS) on an Orbitrap Lumos mass spectrometer. The relative abundances of the identified proteins were correlated with (i) iNPH grading scale (iNPHGS) and (ii) gait speed change 1 year after surgery from baseline to identify predictors of shunt responsiveness. RESULTS: We identified four CSF biomarker candidates which correlated most strongly with clinical improvement on the iNPHGS and were significantly changed in shunt-responsive compared to shunt-unresponsive iNPH patients 1 year post-surgery: FABP3 (R = - 0.46, log2(fold change (FC)) = - 0.25, p < 0.001), ANXA4 (R = 0.46, log2(FC) = 0.32, p < 0.001), MIF (R = -0.49, log2(FC) = - 0.20, p < 0.001) and B3GAT2 (R = 0.54, log2(FC) = 0.20, p < 0.001). In addition, five biomarker candidates were selected based on their strong correlation with gait speed change 1 year after shunt installation: ITGB1 (R = - 0.48, p < 0.001), YWHAG (R = - 0.41, p < 0.01), OLFM2 (R = 0.39, p < 0.01), TGFBI (R = - 0.38, p < 0.01), and DSG2 (R = 0.37, p < 0.01). Concentrations of the CSF AD core biomarkers did not differ significantly with shunt responsiveness. CONCLUSION: FABP3, MIF, ANXA4, B3GAT2, ITGB1, YWHAG, OLFM2, TGFBI and DSG2 in CSF are promising prognostic biomarker candidates to predict shunt responsiveness in iNPH patients.

Mass spectrometric simultaneous quantification of tau species in plasma shows differential associations with amyloid and tau pathologies.

Blood phosphorylated tau (p-tau) biomarkers, at differing sites, demonstrate high accuracy to detect Alzheimer's disease (AD). However, knowledge on the optimal marker for disease identification across the AD continuum and the link to pathology is limited. This is partly due to heterogeneity in analytical methods. In this study, we employed an immunoprecipitation mass spectrometry method to simultaneously quantify six phosphorylated (p-tau181, p-tau199, p-tau202, p-tau205, p-tau217 and p-tau231) and two non-phosphorylated plasma tau peptides in a total of 214 participants from the Paris Lariboisière and Translational Biomarkers of Aging and Dementia cohorts. Our results indicate that p-tau217, p-tau231 and p-tau205 are the plasma tau forms that best reflect AD-related brain changes, although with distinct emergences along the disease course and correlations with AD features-amyloid and tau. These findings support the differential association of blood p-tau variants with AD pathology, and our method offers a potential tool for disease staging in clinical trials.

SCRN1: A cerebrospinal fluid biomarker correlating with tau in Alzheimer's disease.

INTRODUCTION: Secernin-1 (SCRN1) is a neuronal protein that co-localizes with neurofibrillary tangles in Alzheimer's disease (AD), but not with tau inclusions in corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), or Pick's disease. METHODS: We measured SCRN1 concentration in cerebrospinal fluid (CSF) using a novel mass spectrometric parallel reaction monitoring method in three clinical cohorts comprising patients with neurochemically characterized AD (n = 25) and controls (n = 28), clinically diagnosed Parkinson's disease (PD; n = 38), multiple system atrophy (MSA; n = 31), PSP (n = 20), CBD (n = 8), healthy controls (n = 37), and neuropathology-confirmed AD (n = 47). RESULTS: CSF SCRN1 was significantly increased in AD (P < 0.01, fold change = 1.4) compared to controls (receiver operating characteristic area under the curve = 0.78) but not in CBD, PSP, PD, or MSA. CSF SCRN1 positively correlated with CSF total tau (R = 0.78, P = 1.1 × 10-13 ), phosphorylated tau181 (R = 0.64, P = 3.2 × 10-8 ), and Braak stage and negatively correlated with Mini-Mental State Examination score. DISCUSSION: CSF SCRN1 is a candidate biomarker of AD, reflecting tau pathology. HIGHLIGHTS: We developed a parallel reaction monitoring assay to measure secernin-1 (SCRN1) in cerebrospinal fluid (CSF). CSF SCRN1 was increased in Alzheimer's disease compared to healthy controls. CSF SCRN1 remained unchanged in Parkinson's disease, multiple system atrophy, progressive supranuclear palsy, or corticobasal degeneration compared to controls. CSF SCRN1 correlated strongly with CSF phosphorylated tau and total tau. CSF SCRN1 increased across Braak stages and negatively correlated with Mini-Mental State Examination score.

Protein disulfide isomerases as CSF biomarkers for the neuronal response to tau pathology.

INTRODUCTION: Cerebrospinal fluid (CSF) biomarkers for specific cellular disease processes are lacking for tauopathies. In this translational study we aimed to identify CSF biomarkers reflecting early tau pathology-associated unfolded protein response (UPR) activation. METHODS: We employed mass spectrometry proteomics and targeted immunoanalysis in a combination of biomarker discovery in primary mouse neurons in vitro and validation in patient CSF from two independent large multicentre cohorts (EMIF-AD MBD, n = 310; PRIDE, n = 771). RESULTS: First, we identify members of the protein disulfide isomerase (PDI) family in the neuronal UPR-activated secretome and validate secretion upon tau aggregation in vitro. Next, we demonstrate that PDIA1 and PDIA3 levels correlate with total- and phosphorylated-tau levels in CSF. PDIA1 levels are increased in CSF from AD patients compared to controls and patients with tau-unrelated frontotemporal and Lewy body dementia (LBD). HIGHLIGHTS: Neuronal unfolded protein response (UPR) activation induces the secretion of protein disulfide isomerases (PDIs) in vitro. PDIA1 is secreted upon tau aggregation in neurons in vitro. PDIA1 and PDIA3 levels correlate with total and phosphorylated tau levels in CSF. PDIA1 levels are increased in CSF from Alzheimer's disease (AD) patients compared to controls. PDIA1 levels are not increased in CSF from tau-unrelated frontotemporal dementia (FTD) and Lewy body dementia (LBD) patients.

Cerebrospinal Fluid Panel of Synaptic Proteins in Cerebral Amyloid Angiopathy and Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA) share pathogenic pathways related to amyloid-ß deposition. Whereas AD is known to affect synaptic function, such an association for CAA remains yet unknown. OBJECTIVE: We therefore aimed to investigate synaptic dysfunction in CAA. METHODS: Multiple reaction monitoring mass spectrometry was used to quantify cerebrospinal fluid (CSF) concentrations of 15 synaptic proteins in CAA and AD patients, and age- and sex-matched cognitively unimpaired controls. RESULTS: We included 25 patients with CAA, 49 patients with AD, and 25 controls. Only neuronal pentraxin-2 levels were decreased in the CSF of CAA patients compared with controls (p = 0.04). CSF concentrations of 12 other synaptic proteins were all increased in AD compared with CAA or controls (all p≤0.01) and were unchanged between CAA and controls. Synaptic protein concentrations in the subgroup of CAA patients positive for AD biomarkers (CAA/ATN+; n = 6) were similar to AD patients, while levels in CAA/ATN- (n = 19) were comparable with those in controls. A regression model including all synaptic proteins differentiated CAA from AD at high accuracy levels (area under the curve 0.987). CONCLUSION: In contrast to AD, synaptic CSF biomarkers were found to be largely unchanged in CAA. Moreover, concomitant AD pathology in CAA is associated with abnormal synaptic protein levels. Impaired synaptic function in AD was confirmed in this independent cohort. Our findings support an apparent differential involvement of synaptic dysfunction in CAA and AD and may reflect distinct pathological mechanisms.

Assessing the commutability of candidate reference materials for the harmonization of neurofilament light measurements in blood.

OBJECTIVES: Neurofilament light chain (NfL) concentration in blood is a biomarker of neuro-axonal injury in the nervous system and there now exist several assays with high enough sensitivity to measure NfL in serum and plasma. There is a need for harmonization with the goal of creating a certified reference material (CRM) for NfL and an early step in such an effort is to determine the best matrix for the CRM. This is done in a commutability study and here the results of the first one for NfL in blood is presented. METHODS: Forty paired individual serum and plasma samples were analyzed for NfL on four different analytical platforms. Neat and differently spiked serum and plasma were evaluated for their suitability as a CRM using the difference in bias approach. RESULTS: The correlation between the different platforms with regards to measured NfL concentrations were very high (Spearman's ρ≥0.96). Samples spiked with cerebrospinal fluid (CSF) showed higher commutability compared to samples spiked with recombinant human NfL protein and serum seems to be a better choice than plasma as the matrix for a CRM. CONCLUSIONS: The results from this first commutability study on NfL in serum/plasma showed that it is feasible to create a CRM for NfL in blood and that spiking should be done using CSF rather than with recombinant human NfL protein.

Cerebrospinal Fluid Biomarkers of Synaptic Dysfunction are Altered in Parkinson's Disease and Related Disorders.

BACKGROUND: Synaptic dysfunction and degeneration are central contributors to the pathogenesis and progression of parkinsonian disorders. Therefore, identification and validation of biomarkers reflecting pathological synaptic alterations are greatly needed and could be used in prognostic assessment and to monitor treatment effects. OBJECTIVE: To explore candidate biomarkers of synaptic dysfunction in Parkinson's disease (PD) and related disorders. METHODS: Mass spectrometry was used to quantify 15 synaptic proteins in two clinical cerebrospinal fluid (CSF) cohorts, including PD (n1  = 51, n2  = 101), corticobasal degeneration (CBD) (n1  = 11, n2  = 3), progressive supranuclear palsy (PSP) (n1  = 22, n2  = 21), multiple system atrophy (MSA) (n1  = 31, n2  = 26), and healthy control (HC) (n1  = 48, n2  = 30) participants, as well as Alzheimer's disease (AD) (n2  = 23) patients in the second cohort. RESULTS: Across both cohorts, lower levels of the neuronal pentraxins (NPTX; 1, 2, and receptor) were found in PD, MSA, and PSP, compared with HC. In MSA and PSP, lower neurogranin, AP2B1, and complexin-2 levels compared with HC were observed. In AD, levels of 14-3-3 zeta/delta, beta- and gamma-synuclein were higher compared with the parkinsonian disorders. Lower pentraxin levels in PD correlated with Mini-Mental State Exam scores and specific cognitive deficits (NPTX2; rho = 0.25-0.32, P < 0.05) and reduced dopaminergic pre-synaptic integrity as measured by DaTSCAN (NPTX2; rho = 0.29, P = 0.023). Additionally, lower levels were associated with the progression of postural imbalance and gait difficulty symptoms (All NPTX; ß-estimate = -0.025 to -0.038, P < 0.05) and cognitive decline (NPTX2; ß-estimate = 0.32, P = 0.021). CONCLUSIONS: These novel findings show different alterations of synaptic proteins in parkinsonian disorders compared with AD and HC. The neuronal pentraxins may serve as prognostic CSF biomarkers for both cognitive and motor symptom progression in PD. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Cerebrospinal fluid biomarker panel of synaptic dysfunction in Alzheimer's disease and other neurodegenerative disorders.

INTRODUCTION: Synaptic degeneration is a key part of the pathophysiology of neurodegenerative diseases, and biomarkers reflecting the pathological alterations are greatly needed. METHOD: Seventeen synaptic proteins were quantified in a pathology-confirmed cerebrospinal fluid cohort of patients with Alzheimer's disease (AD; n = 63), frontotemporal lobar degeneration (FTLD; n = 53), and Lewy body spectrum of disorders (LBD; n = 21), as well as healthy controls (HC; n = 48). RESULTS: Comparisons revealed four distinct patterns: markers decreased across all neurodegenerative conditions compared to HC (the neuronal pentraxins), markers increased across all neurodegenerative conditions (14-3-3 zeta/delta), markers selectively increased in AD compared to other neurodegenerative conditions (neurogranin and beta-synuclein), and markers selectively decreased in LBD and FTLD compared to HC and AD (AP2B1 and syntaxin-1B). DISCUSSION: Several of the synaptic proteins may serve as biomarkers for synaptic dysfunction in AD, LBD, and FTLD. Additionally, differential patterns of synaptic protein alterations seem to be present across neurodegenerative diseases. HIGHLIGHTS: A panel of synaptic proteins were quantified in the cerebrospinal fluid using mass spectrometry. We compared Alzheimer's disease, frontotemporal degeneration, and Lewy body spectrum of disorders. Pathology was confirmed by autopsy or familial mutations. We discovered synaptic biomarkers for synaptic degeneration and cognitive decline. We found differential patterns of synaptic proteins across neurodegenerative diseases.

Antibody-free measurement of cerebrospinal fluid tau phosphorylation across the Alzheimer's disease continuum.

BACKGROUND: Alzheimer's disease is characterized by an abnormal increase of phosphorylated tau (pTau) species in the CSF. It has been suggested that emergence of different pTau forms may parallel disease progression. Therefore, targeting multiple specific pTau forms may allow for a deeper understanding of disease evolution and underlying pathophysiology. Current immunoassays measure pTau epitopes separately and may capture phosphorylated tau fragments of different length depending on the non-pTau antibody used in the assay sandwich pair, which bias the measurement. METHODS: We developed the first antibody-free mass spectrometric method to simultaneously measure multiple phosphorylated epitopes in CSF tau: pT181, pS199, pS202, pT205, pT217, pT231, and pS396. The method was first evaluated in biochemically defined Alzheimer's disease and control CSF samples (n = 38). All seven pTau epitopes clearly separated Alzheimer's disease from non-AD (p < 0.001, AUC = 0.84-0.98). We proceeded with clinical validation of the method in the TRIAD (n = 165) and BioFINDER-2 cohorts (n = 563), consisting of patients across the full Alzheimer's disease continuum, including also young controls (< 40 years), as well as patients with frontotemporal dementia and other neurodegenerative disorders. RESULTS: Increased levels of all phosphorylated epitopes were found in Alzheimer's disease dementia and Aß positron emission tomography-positive patients with mild cognitive impairment compared with Aß-negative controls. For Alzheimer's disease dementia compared with Aß-negative controls, the best biomarker performance was observed for pT231 (TRIAD: AUC = 98.73%, fold change = 7.64; BioFINDER-2: AUC = 91.89%, fold change = 10.65), pT217 (TRIAD: AUC = 99.71%, fold change = 6.33; BioFINDER-2: AUC = 98.12%, fold change = 8.83) and pT205 (TRIAD: AUC = 99.07%, fold change = 5.34; BioFINDER-2: AUC = 93.51%, fold change = 3.92). These phospho-epitopes also discriminated between Aß-positive and Aß-negative cognitively unimpaired individuals: pT217 (TRIAD: AUC = 83.26, fold change = 2.39; BioFINDER-2: AUC = 91.05%, fold change = 3.29), pT231 (TRIAD: AUC = 86.25, fold change = 3.80; BioFINDER-2: AUC = 78.69%, fold change = 3.65) and pT205 (TRIAD: AUC = 71.58, fold change = 1.51; BioFINDER-2: AUC = 71.11%, fold change = 1.70). CONCLUSIONS: While an increase was found for all pTau species examined, the highest fold change in Alzheimer's disease was found for pT231, pT217 and pT205. Simultaneous antibody-free measurement of pTau epitopes by mass spectrometry avoids possible bias caused by differences in antibody affinity for modified or processed forms of tau, provides insights into tau pathophysiology and may facilitate clinical trials on tau-based drug candidates.