A Novel Neurofilament Light Chain ELISA Validated in Patients with Alzheimer's Disease, Frontotemporal Dementia, and Subjective Cognitive Decline, and the Evaluation of Candidate Proteins for Immunoassay Calibration.

Neurofilament light chain (Nf-L) is a well-known biomarker for axonal damage; however, the corresponding circulating Nf-L analyte in cerebrospinal fluid (CSF) is poorly characterized. We therefore isolated new monoclonal antibodies against synthetic peptides, and these monoclonals were characterized for their specificity on brain-specific intermediate filament proteins. Two highly specific antibodies, ADx206 and ADx209, were analytically validated for CSF applications according to well-established criteria. Interestingly, using three different sources of purified Nf-L proteins, a significant impact on interpolated concentrations was observed. With a lower limit of analytical sensitivity of 100 pg/mL using bovine Nf-L as the calibrator, we were able to quantify the Nf-L analyte in each sample, and these Nf-L concentrations were highly correlated to the Uman diagnostics assay (Spearman rho = 0.97, p < 0.001). In the clinical diagnostic groups, the new Nf-L ELISA could discriminate patients with Alzheimer's disease (AD, n = 20) from those with frontotemporal lobe dementia (FTD, n = 20) and control samples with subjective cognitive decline (SCD, n = 20). Henceforth, this novel Nf-L ELISA with well-defined specificity and epitopes can be used to enhance our understanding of harmonizing the use of Nf-L as a clinically relevant marker for neurodegeneration in CSF.

Neurogranin and tau in cerebrospinal fluid and plasma of patients with acute ischemic stroke.

BACKGROUND: While neurogranin has no value as plasma biomarker for Alzheimer's disease, it may be a potential blood biomarker for traumatic brain injury. This evokes the question whether there are changes in neurogranin levels in blood in other conditions of brain injury, such as acute ischemic stroke (AIS). METHODS: We therefore explored neurogranin in paired cerebrospinal fluid (CSF)/plasma samples of AIS patients (n = 50) from a well-described prospective study. In parallel, we investigated another neuronal protein, i.e. tau, which has already been suggested as potential AIS biomarker in CSF and blood. ELISA as well as Single Molecule Array (Simoa) technology were used for the biochemical analyses. Statistical analyses included Shapiro-Wilk testing, Mann-Whitney analyses and Pearson's correlation analysis. RESULTS: In contrast to tau, of which high levels in both CSF and plasma were related to stroke characteristics like severity and long-term outcome, plasma neurogranin levels were only correlated with infarct volume. Likewise, CSF neurogranin levels were significantly higher in patients with an infarct volume > 5 mL than in patients with smaller infarct volumes. Finally, neurogranin and tau were significantly correlated in CSF, whereas a weaker relationship was observed in plasma. CONCLUSIONS: These findings indicate that although plasma and CSF neurogranin may reflect the volume of acute cerebral ischemia, this synaptic protein is less likely to be a potential AIS biomarker. Levels of tau correlated with severity and outcome of stroke in both plasma and CSF, in the present study as well as previous reports, confirming the potential of tau as an AIS biomarker.

Tau monoclonal antibody generation based on humanized yeast models: impact on Tau oligomerization and diagnostics.

A link between Tau phosphorylation and aggregation has been shown in different models for Alzheimer disease, including yeast. We used human Tau purified from yeast models to generate new monoclonal antibodies, of which three were further characterized. The first antibody, ADx201, binds the Tau proline-rich region independently of the phosphorylation status, whereas the second, ADx215, detects an epitope formed by the Tau N terminus when Tau is not phosphorylated at Tyr(18). For the third antibody, ADx210, the binding site could not be determined because its epitope is probably conformational. All three antibodies stained tangle-like structures in different brain sections of THY-Tau22 transgenic mice and Alzheimer patients, and ADx201 and ADx210 also detected neuritic plaques in the cortex of the patient brains. In hippocampal homogenates from THY-Tau22 mice and cortex homogenates obtained from Alzheimer patients, ADx215 consistently stained specific low order Tau oligomers in diseased brain, which in size correspond to Tau dimers. ADx201 and ADx210 additionally reacted to higher order Tau oligomers and presumed prefibrillar structures in the patient samples. Our data further suggest that formation of the low order Tau oligomers marks an early disease stage that is initiated by Tau phosphorylation at N-terminal sites. Formation of higher order oligomers appears to require additional phosphorylation in the C terminus of Tau. When used to assess Tau levels in human cerebrospinal fluid, the antibodies permitted us to discriminate patients with Alzheimer disease or other dementia like vascular dementia, indicative that these antibodies hold promising diagnostic potential.

C-terminal neurogranin is increased in cerebrospinal fluid but unchanged in plasma in Alzheimer's disease.

INTRODUCTION: Biomarkers monitoring synaptic degeneration/loss would be valuable for Alzheimer's disease (AD) diagnosis. Postsynaptic protein neurogranin may be a promising cerebrospinal fluid (CSF) biomarker but has not yet been evaluated as a plasma biomarker. METHODS: Using an in-house designed prototype enzyme-linked immunosorbent assay (ELISA) targeting neurogranin C-terminally, we studied neurogranin in paired CSF/plasma samples of controls (n = 29) versus patients experiencing MCI, or dementia, due to AD (in total n = 59). RESULTS: CSF neurogranin was increased in AD and positively correlated with CSF tau, whereas there was a negative relationship between CSF neurogranin (and tau) and CSF Aß1-42/Aß1-40. No differences were detected in plasma neurogranin between controls and AD. Also, there was no correlation between CSF and plasma neurogranin, excluding confounding effects of the latter. DISCUSSION: This study strengthens the potential of neurogranin as an AD CSF biomarker, which now needs validation in larger studies. As tools, straightforward immunoassays can be used, as demonstrated by the described ELISA.

Integration policies shape ethnic-racial majorities' threat reactions to increasing diversity.

Increasing ethnic and racial diversity often fuels feelings of threat among ethnic-racial majorities (e.g., self-identified white Americans and European nationals). We contend that these threat perceptions depend on the policy context. Across four studies, we test whether more inclusive immigrant integration policies attenuate ethnic-racial majorities' threat reactions. Studies 1 to 3 (n = 469, 733, and 1745, respectively) used experimental methods with white American participants in the United States. Study 4 (n = 499,075) used secondary analysis of survey data comparing attitudes of nationals in 30 European countries and measured the impact of actual changes in diversity and policies over 10 years. Our results show that integration policies shape threat reactions even in those situations when increasing diversity could be seen as the most threatening: when narratives highlight the majority's impending minority position or when diversity suddenly increases. When policies are more inclusive toward immigrants, ethnic-racial majority participants report less threat (or no threat) in response to increasing diversity.

The use of synaptic biomarkers in cerebrospinal fluid to differentiate behavioral variant of frontotemporal dementia from primary psychiatric disorders and Alzheimer's disease.

BACKGROUND: Lack of early molecular biomarkers in sporadic behavioral variants of frontotemporal dementia (bvFTD) and its clinical overlap with primary psychiatric disorders (PPD) hampers its diagnostic distinction. Synaptic dysfunction is an early feature in bvFTD and identification of specific biomarkers might improve its diagnostic accuracy. Our goal was to understand the differential diagnostic potential of cerebrospinal fluid (CSF) synaptic biomarkers in bvFTD versus PPD and their specificity towards bvFTD compared with Alzheimer's disease (AD) and controls. Additionally, we explored the association of CSF synaptic biomarkers with social cognition, cognitive performance, and disease severity in these clinical groups. METHODS: Participants with probable bvFTD (n = 57), PPD (n = 71), AD (n = 60), and cognitively normal controls (n = 39) with available CSF, cognitive tests, and disease severity as frontotemporal lobar degeneration-modified clinical dementia rating scale (FTLD-CDR) were included. In a subset of bvFTD and PPD cases, Ekman 60 faces test scores for social cognition were available. CSF synaptosomal-associated protein 25 (SNAP25), neurogranin (Ng), neuronal pentraxin 2 (NPTX2), and glutamate receptor 4 (GluR4) were measured, along with neurofilament light (NfL), and compared between groups using analysis of covariance (ANCOVA) and logistic regression. Diagnostic accuracy was assessed using ROC analyses, and biomarker panels were selected using Wald's backward selection. Correlations with cognitive measures were performed using Pearson's partial correlation analysis. RESULTS: NPTX2 concentrations were lower in the bvFTD group compared with PPD (p < 0.001) and controls (p = 0.003) but not compared with AD. Concentrations of SNAP25 (p < 0.001) and Ng (p < 0.001) were elevated in patients with AD versus those with bvFTD and controls. The modeled panel for differential diagnosis of bvFTD versus PPD consisted of NfL and NPTX2 (AUC = 0.96, CI: 0.93-0.99, p < 0.001). In bvFTD versus AD, the modeled panel consisted of NfL, SNAP25, Ng, and GluR4 (AUC = 0.86, CI: 0.79-0.92, p < 0.001). In bvFTD, lower NPTX2 (Pearson's r = 0.29, p = 0.036) and GluR4 (Pearson's r = 0.34, p = 0.014) concentrations were weakly associated with worse performance of total cognitive score. Lower GluR4 concentrations were also associated with worse MMSE scores (Pearson's r = 0.41, p = 0.002) as well as with worse executive functioning (Pearson's r = 0.36, p = 0.011) in bvFTD. There were no associations between synaptic markers and social cognition or disease severity in bvFTD. CONCLUSION: Our findings of involvement of NTPX2 in bvFTD but not PPD contribute towards better understanding of bvFTD disease pathology.

Synaptic biomarkers in the cerebrospinal fluid associate differentially with classical neuronal biomarkers in patients with Alzheimer's disease and frontotemporal dementia.

BACKGROUND: Loss of synaptic functionality has been recently identified as an early-stage indicator of neurological diseases. Consequently, monitoring changes in synaptic protein levels may be relevant for observing disease evolution or treatment responses in patients. Here, we have studied the relationship between fluid biomarkers of neurodegeneration and synaptic dysfunction in patients with Alzheimer's disease (AD), frontotemporal dementia (FTD), and subjective cognitive decline (SCD). METHODS: The exploratory cohort consisted of cerebrospinal fluid (CSF) samples (n = 60) from patients diagnosed with AD (n = 20), FTD (n = 20), and SCD (n = 20) from the Amsterdam Dementia Cohort. We developed two novel immunoassays for the synaptic proteins synaptosomal-associated protein-25 (SNAP25) and vesicle-associated membrane protein-2 (VAMP2). We measured the levels of these biomarkers in CSF, in addition to neuronal pentraxin-2 (NPTX2), glutamate ionotropic receptor-4 (GluR4), and neurogranin (Ng) for this cohort. All in-house immunoassays were validated and analytically qualified prior to clinical application. CSF neurogranin (Ng) was measured using a commercially available ELISA. RESULTS: This pilot study indicated that SNAP25, VAMP2, and Ng may not be specific biomarkers for AD as their levels were significantly elevated in patients with both AD and FTD compared to SCD. Moreover, the strength of the correlations between synaptic proteins was lower in the AD and FTD clinical groups compared to SCD. SNAP25, VAMP2, and Ng correlated strongly with each other as well as with total Tau (Tau) and phosphorylated Tau (PTau) in all three clinical groups. However, this correlation was weakened or absent with NPTX2 and GluR4. None of the synaptic proteins correlated to neurofilament light (NfL) in any clinical group. CONCLUSION: The correlation of the synaptic biomarkers with CSF Tau and PTau but the lack thereof with NfL implies that distinct pathological pathways may be involved in synaptic versus axonal degeneration. Our results reflect the diversity of synaptic pathology in neurodegenerative dementias.

A Combination of Neurofilament Light, Glial Fibrillary Acidic Protein, and Neuronal Pentraxin-2 Discriminates Between Frontotemporal Dementia and Other Dementias.

BACKGROUND: The differential diagnosis of frontotemporal dementia (FTD) is still a challenging task due to its symptomatic overlap with other neurological diseases and the lack of biofluid-based biomarkers. OBJECTIVE: To investigate the diagnostic potential of a combination of novel biomarkers in cerebrospinal fluid (CSF) and blood. METHODS: We included 135 patients from the Center for Memory Disturbances, University of Perugia, with the diagnoses FTD (n = 37), mild cognitive impairment due to Alzheimer's disease (MCI-AD, n = 47), Lewy body dementia (PDD/DLB, n = 22), and cognitively unimpaired patients as controls (OND, n = 29). Biomarker levels of neuronal pentraxin-2 (NPTX2), neuronal pentraxin receptor, neurofilament light (NfL) and glial fibrillary acidic protein (GFAP) were measured in CSF, as well as NfL and GFAP in serum. We assessed biomarker differences by analysis of covariance and generalized linear models (GLM). We performed receiver operating characteristics analyses and Spearman correlation to determine biomarker associations. RESULTS: CSF NPTX2 and serum GFAP levels varied most between diagnostic groups. The combination of CSF NPTX2, serum NfL and serum GFAP differentiated FTD from the other groups with good accuracy (FTD versus MCI-AD: area under the curve (AUC) [95% CI] = 0.89 [0.81-0.96]; FTD versus PDD/DLB: AUC = 0.82 [0.71-0.93]; FTD versus OND: AUC = 0.80 [0.70-0.91]). CSF NPTX2 and serum GFAP correlated positively only in PDD/DLB (ρ= 0.56, p < 0.05). NPTX2 and serum NfL did not correlate in any of the diagnostic groups. Serum GFAP and serum NfL correlated positively in all groups (ρ= 0.47-0.74, p < 0.05). CONCLUSION: We show the combined potential of CSF NPTX2, serum NfL, and serum GFAP to differentiate FTD from other neurodegenerative disorders.

Comparing the Clinical Utility and Diagnostic Performance of CSF P-Tau181, P-Tau217, and P-Tau231 Assays.

BACKGROUND AND OBJECTIVES: Phosphorylated tau (p-tau) in CSF is considered an important biomarker in Alzheimer disease (AD) and has been incorporated in recent diagnostic criteria. Several variants exist, including p-tau at threonines 181 (p-tau181), 217 (p-tau217), and 231 (p-tau231). However, no studies have compared their diagnostic performance or association to ß-amyloid (Aß) and tau-PET. Understanding which p-tau variant to use remains an important yet answered question. We aimed to compare the diagnostic accuracy of p-tau181, p-tau217, and p-tau231 in CSF for AD and their association with Aß and tau-PET. METHODS: A total of 629 participants in the Swedish BioFINDER-2 study were included (cognitively unimpaired, n = 334; Aß-positive mild cognitive impairment, n = 84; AD dementia, n = 119; and non-AD disorders, n = 92). In addition to p-tau181 and p-tau217 measured using assays with the same detector antibodies from Eli Lilly (p-tau181Lilly, p-tau217Lilly) and p-tau231, we also included p-tau181 measurements from 2 commonly used assays (Innotest and Elecsys). RESULTS: Although all p-tau variants increased across the AD continuum, p-tau217Lilly showed the greatest dynamic range (13-fold increase vs 1.9-5.4-fold increase for other p-tau variants for AD dementia vs non-AD). P-Tau217Lilly showed stronger correlations with Aß- and tau-PET (p < 0.0001). P-Tau217Lilly exhibited higher accuracy than other p-tau variants for separating AD dementia from non-AD (area under the curve [AUC], 0.98 vs 0.88 [p < 0.0001] - 0.96 [p < 0.05]) and for identifying Aß-PET (AUC, 0.86 vs 0.74 [p < 0.0001] and 0.83 [p < 0.001]) and tau-PET positivity (AUC, 0.94 vs 0.80-0.92, p < 0.0001). Finally, p-Tau181Lilly generally performed better than the other p-tau181 assays (e.g., AD dementia vs non-AD, AUC, 0.96 vs 0.88 [p-tau181Innotest] and 0.89 [p-tau181Elecsys]; p < 0.0001). DISCUSSION: CSF p-tau217Lilly seems to be more useful than other included p-tau assays in the workup of AD. Varied results across p-tau181 assays highlights the importance of anti-tau antibodies for biomarker performance. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that p-tau217 provides higher diagnostic accuracy for diagnosis of AD dementia than p-tau181 or p-tau231.

Synaptic biomarkers in CSF aid in diagnosis, correlate with cognition and predict progression in MCI and Alzheimer's disease.

INTRODUCTION: Amyloid, Tau, and neurodegeneration biomarkers can stage Alzheimer's Disease (AD). Synaptic biomarkers may help track cognition. METHODS: In cognitively normal controls, Mild Cognitive Impairment (MCI) and AD, we investigated CSF biomarkers in relation to cognitive measures and as predictors of cognitive and global decline. RESULTS: There were 90 normal controls (mean age 73.0, 58% women), 57 MCI (mean age 74.3, 35% women), and 46 AD (mean age 70.7, 41% women). CSF Aß1-42 and Neuronal Pentraxin 2 (NPTX2) were decreased, and CSF Tau, neurogranin, and SNAP25 increased in AD versus controls. Aß1-42/Tau or NPTX2/Tau discriminated AD and controls best. NPTX2/Tau correlated strongly with cognition in AD and MCI and predicted a 2-3-year decline. We replicated findings in the ADNI cohort. DISCUSSION: CSF synaptic biomarkers, particularly NPTX2, which regulates synaptic homeostasis, relate to cognition and predict progression in AD beyond Aß1-42 and Tau. This is relevant for prognosis and clinical trials.

Neurogranin and BACE1 in CSF as Potential Biomarkers Differentiating Depression with Cognitive Deficits from Early Alzheimer's Disease: A Pilot Study.

BACKGROUND/AIMS: Major depressive disorder (MDD) can cooccur with early Alzheimer's disease (AD) or may cause memory problems independently of AD. Previous studies have suggested that the AD-related cerebrospinal fluid (CSF) biomarkers tau and Aß(1-42) could help discriminate between early AD and depression unrelated to AD. Moreover, the postsynaptic protein neurogranin and presynaptic BACE1 have increasingly gained attention as potential new AD biomarkers, but they have not yet been investigated concerning depression. METHODS: Using ELISAs, we studied CSF neurogranin and BACE1 levels in patients with mild (n = 21) and moderate (n = 19) AD, as well as in MDD patients with (n = 20) and without (n = 20) cognitive deficits. The clinical examinations included analyses of t-tau, Aß(1-42), and Aß(1-40), besides neuropsychological tests and cranial magnetic resonance imaging. Depressive symptom severity was assessed using the Geriatric Depression Scale (GDS). RESULTS: Along with classic AD biomarkers, neurogranin and BACE1 CSF levels differed between moderate AD and MDD (p ≤ 0.01). MDD associated with cognitive deficits was distinguished from mild AD through the CSF neurogranin/BACE1 ratio (p < 0.05), which was strongly correlated with GDS scores (ρ = -0.656; p < 0.01). CONCLUSION: The neurogranin/BACE1 ratio in CSF can distinguish between depression and AD among patients with similar cognitive deficits, along with the classic AD biomarkers. Further longitudinal studies are ongoing to identify which biomarkers have prognostic value.

The Cerebrospinal Fluid Neurogranin/BACE1 Ratio is a Potential Correlate of Cognitive Decline in Alzheimer's Disease.

BACKGROUND: In diagnosing Alzheimer's disease (AD), ratios of cerebrospinal fluid (CSF) biomarkers, such as CSF Aß1-42/tau, have an improved diagnostic performance compared to the single analytes, yet, still a limited value to predict cognitive decline. Since synaptic dysfunction/loss is closely linked to cognitive impairment, synaptic proteins are investigated as candidate CSF AD progression markers. OBJECTIVE: We studied CSF levels of the postsynaptic protein neurogranin and protein BACE1, predominantly localized presynaptically, and their relation to CSF total-tau, Aß1-42, Aß1-40, and Aß1-38. All six analytes were considered as single parameters as well as ratios. METHODS: Every ELISA involved was based on monoclonal antibodies, including the BACE1 and neurogranin immunoassay. The latter specifically targets neurogranin C-terminally truncated at P75, a more abundant species of the protein in CSF. We studied patients with MCI due to AD (n = 38) and 50 dementia due to AD patients, as well as age-matched cognitively healthy elderly (n = 20). A significant subset of the patients was followed up by clinical and neuropsychological (MMSE) examinations for at least one year. RESULTS: The single analytes showed statistically significant differences between the clinical groups, but the ratios of analytes indeed had a higher diagnostic performance. Furthermore, only the ratio of CSF neurogranin trunc P75/BACE1 was significantly correlated with the yearly decline in MMSE scores in patients with MCI and dementia due to AD, pointing toward the prognostic value of the ratio. CONCLUSION: This is the first study demonstrating that the CSF neurogranin trunc P75/BACE1 ratio, reflecting postsynaptic/presynaptic integrity, is related to cognitive decline.

Inhibition of hepatitis C virus-like particle binding to target cells by antiviral antibodies in acute and chronic hepatitis C.

Hepatitis C virus (HCV) is a leading cause of chronic viral hepatitis worldwide. The study of antibody-mediated virus neutralization has been hampered by the lack of an efficient and high-throughput cell culture system for the study of virus neutralization. The HCV structural proteins have been shown to assemble into noninfectious HCV-like particles (HCV-LPs). Similar to serum-derived virions, HCV-LPs bind and enter human hepatocytes and hepatoma cell lines. In this study, we developed an HCV-LP-based model system for a systematic functional analysis of antiviral antibodies from patients with acute or chronic hepatitis C. We demonstrate that cellular HCV-LP binding was specifically inhibited by antiviral antibodies from patients with acute or chronic hepatitis C in a dose-dependent manner. Using a library of homologous overlapping envelope peptides covering the entire HCV envelope, we identified an epitope in the N-terminal E2 region (SQKIQLVNTNGSWHI; amino acid positions 408 to 422) as one target of human antiviral antibodies inhibiting cellular particle binding. Using a large panel of serum samples from patients with acute and chronic hepatitis C, we demonstrated that the presence of antibodies with inhibition of binding activity was not associated with viral clearance. In conclusion, antibody-mediated inhibition of cellular HCV-LP binding represents a convenient system for the functional characterization of human anti-HCV antibodies, allowing the mapping of envelope neutralization epitopes targeted by naturally occurring antiviral antibodies.