Amyloid beta: from pre-analytical factors to disease mechanisms

Background: Biomarkers are powerful tools for interrogating the basic science of disease processes, in the clinical detection of disease states, and as both targets and endpoints in therapeutic strategy. Amyloid beta (Aβ) is a core biomarker for Alzheimer’s disease (AD), but measurement variation between sites and experiments limits its potential. Furthermore, although the role of brain Aβ accumulation early in AD is extremely well attested, the biological mechanisms underlying this remain poorly understood. // Methods: To contribute to the development of treatments for AD patients and those at risk, this thesis set out to identify important pre-analytical confounding factors in Aβ measurement, strategies to mitigate them, and identify disease relevant patterns of Aβ peptide production in human CSF and an induced pluripotent stem cell-derived cortical neuron model of familial AD (fAD). // Results: A series of experiments demonstrated the importance of sample surface exposure to the measurement of Aβ peptides and tau. The volume at which samples are stored and iterative contact with fresh surfaces had profound effect on Aβ, but not tau, with greater surface exposure resulting in depletion of Aβ concentration. The mechanism was demonstrated to be protein surface adsorption. Importantly, the different Aβ peptides did not absorb to polypropylene to the same extent; Aβ42 concentration decreased proportionally more with surface exposure treatment than Aβ40 and Aβ38. It was observed that the addition of a non-ionic surfactant (Tween 20) to samples significantly mitigated the effect of surface exposure treatments on Aβ peptides and tau. However, the use of this additive did not meaningfully improve variability when sample storage conditions were standardised. Furthermore, variances in clinic to laboratory temperature and time interval did not significantly affect Aβ or tau concentration. Validation of an in vitro model of fAD was conducted. Experiment identified the use of Aβ ratios as a robust method for normalising data variability between and within cell lines over extended time periods. Furthermore, comparison of paired CSF, cell media, cell lysates, and post-mortem cortical tissue from the same individual demonstrated physiologically consistent patterns of Aβ ratios across sample types. Finally, comparison of multiple fAD mutation and control cell lines demonstrated quantitative and qualitative differences in secreted Aβ. APP V717I neurons increased secretion of Aβ42 and Aβ38 relative to Aβ43 and Aβ40. PSEN1 mutations increased secretion of longer Aβ peptides relative to shorter Aβ peptides, with mutation specific differences such as greatly increased Aβ43 in PSEN1 R278I.// Conclusions: This work demonstrated several novel considerations in the use of Aβ peptides as biomarkers for AD. Data principally highlight the importance of Aβ ratios to AD biomarker research, the necessity of controlling pre-analytical sample surface exposure intended for the measurement of ‘sticky’ protein biomarkers such as Aβ peptides, and the validity of iPSC-derived neuronal models for exploring the production of Aβ in AD and health

The PSEN1 E280G mutation leads to increased amyloid-β43 production in induced pluripotent stem cell neurons and deposition in brain tissue

Mutations in the presenilin 1 gene, PSEN1, which cause familial Alzheimer’s disease alter processing of amyloid precursor protein, leading to the generation of various amyloid-β peptide species. These species differ in their potential for aggregation. Mutation-specific amyloid-β peptide profiles may thereby influence pathogenicity and clinical heterogeneity. There is particular interest in comparing mutations with typical and atypical clinical presentations, such as E280G. We generated PSEN1 E280G mutation induced pluripotent stem cells from two patients and differentiated them into cortical neurons, along with previously reported PSEN1 M146I, PSEN1 R278I and two control lines. We assessed both the amyloid-β peptide profiles and presenilin 1 protein maturity. We also compared amyloid-β peptide profiles in human post-mortem brain tissue from cases with matched mutations. Amyloid-β ratios significantly differed compared with controls and between different patients, implicating mutation-specific alterations in amyloid-β ratios. Amyloid-β42:40 was increased in the M146I and both E280G lines compared with controls. Amyloid-β42:40 was not increased in the R278I line compared with controls. The amyloid-β43:40 ratio was increased in R278I and both E280G lines compared with controls, but not in M146I cells. Distinct amyloid-β peptide patterns were also observed in human brain tissue from individuals with these mutations, showing some similar patterns to cell line observations. Reduced presenilin 1 maturation was observed in neurons with the PSEN1 R278I and E280G mutations, but not the M146I mutation. These results suggest that mutation location can differentially alter the presenilin 1 protein and affect its autoendoproteolysis and processivity, contributing to the pathological phenotype. Investigating differences in underlying molecular mechanisms of familial Alzheimer’s disease may inform our understanding of clinical heterogeneity

Cerebrospinal fluid in the differential diagnosis of Alzheimer's disease: clinical utility of an extended panel of biomarkers in a specialist cognitive clinic.

BACKGROUND: Cerebrospinal fluid (CSF) biomarkers are increasingly being used to support a diagnosis of Alzheimer's disease (AD). Their clinical utility for differentiating AD from non-AD neurodegenerative dementias, such as dementia with Lewy bodies (DLB) or frontotemporal dementia (FTD), is less well established. We aimed to determine the diagnostic utility of an extended panel of CSF biomarkers to differentiate AD from a range of other neurodegenerative dementias. METHODS: We used immunoassays to measure conventional CSF markers of amyloid and tau pathology (amyloid beta (Aβ)1-42, total tau (T-tau), and phosphorylated tau (P-tau)) as well as amyloid processing (AβX-38, AβX-40, AβX-42, soluble amyloid precursor protein (sAPP)α, and sAPPβ), large fibre axonal degeneration (neurofilament light chain (NFL)), and neuroinflammation (YKL-40) in 245 patients with a variety of dementias and 30 controls. Patients fulfilled consensus criteria for AD (n = 156), DLB (n = 20), behavioural variant frontotemporal dementia (bvFTD; n = 45), progressive non-fluent aphasia (PNFA; n = 17), and semantic dementia (SD; n = 7); approximately 10% were pathology/genetically confirmed (n = 26). Global tests based on generalised least squares regression were used to determine differences between groups. Non-parametric receiver operating characteristic (ROC) curves and area under the curve (AUC) analyses were used to quantify how well each biomarker discriminated AD from each of the other diagnostic groups (or combinations of groups). CSF cut-points for the major biomarkers found to have diagnostic utility were validated using an independent cohort which included causes of AD (n = 104), DLB (n = 5), bvFTD (n = 12), PNFA (n = 3), SD (n = 9), and controls (n = 10). RESULTS: There were significant global differences in Aβ1-42, T-tau, T-tau/Aβ1-42 ratio, P-tau-181, NFL, AβX-42, AβX-42/X-40 ratio, APPα, and APPβ between groups. At a fixed sensitivity of 85%, AβX-42/X-40 could differentiate AD from controls, bvFTD, and SD with specificities of 93%, 85%, and 100%, respectively; for T-tau/Aβ1-42 these specificities were 83%, 70%, and 86%. AβX-42/X-40 had similar or higher specificity than Aβ1-42. No biomarker or ratio could differentiate AD from DLB or PNFA with specificity > 50%. Similar sensitivities and specificities were found in the independent validation cohort for differentiating AD and other dementias and in a pathology/genetically confirmed sub-cohort. CONCLUSIONS: CSF AβX-42/X-40 and T-tau/Aβ1-42 ratios have utility in distinguishing AD from controls, bvFTD, and SD. None of the biomarkers tested had good specificity at distinguishing AD from DLB or PNFA

Generation of twenty four induced pluripotent stem cell lines from twenty four members of the Lothian 4 Birth Cohort 1936

Cognitive decline is among the most feared aspects of ageing. We have generated induced pluripotent stem cells (iPSCs) from 24 people from the Lothian Birth Cohort 1936, whose cognitive ability was tested in childhood and in older age. Peripheral blood mononuclear cells (PBMCs) were reprogrammed using non-integrating oriP/EBNA1 backbone plasmids expressing six iPSC reprogramming factors (OCT3/4 (POU5F1), SOX2, KLF4, L-Myc, shp53, Lin28, SV40LT). All lines demonstrated STR matched karyotype and pluripotency was validated by multiple methods. These iPSC lines are a valuable resource to study molecular mechanisms underlying individual differences in cognitive ageing and resilience to age-related neurodegenerative diseases

No evidence of neuronal damage as measured by neurofilament light chain in a HIV cure study utilising a kick-and-kill approach

Objective HIV-remission strategies including kick-and-kill could induce viral transcription and immune-activation in the central nervous system, potentially causing neuronal injury. We investigated the impact of kick-and-kill on plasma neurofilament light (NfL), a marker of neuro-axonal injury, in RIVER trial participants commencing antiretroviral treatment (ART) during primary infection and randomly allocated to ART-alone or kick-and-kill (ART + vaccination + vorinostat (ART + V + V)). Design Sub-study measuring serial plasma NfL concentrations. Methods Plasma NfL (using Simoa digital immunoassay), plasma HIV-1 RNA (using single-copy assay) and total HIV-1 DNA (using quantitative polymerase chain reaction in peripheral CD4+ T-cells) were measured at randomisation (following ≥22 weeks ART), week 12 (on final intervention day in ART + V + V) and week 18 post-randomisation. HIV-specific T-cells were quantified by intracellular cytokine staining at randomisation and week 12. Differences in plasma NfL longitudinally and by study arm were analysed using mixed models and Student's t-test. Associations with plasma NfL were assessed using linear regression and rank statistics. Results At randomisation, 58 male participants had median age 32 years and CD4+ count 696 cells/μL. No significant difference in plasma NfL was seen longitudinally and by study arm, with median plasma NfL (pg/mL) in ART-only vs ART + V + V: 7.4 vs 6.4, p = 0.16 (randomisation), 8.0 vs 6.9, p = 0.22 (week 12) and 7.1 vs 6.8, p = 0.74 (week 18). Plasma NfL did not significantly correlate with plasma HIV-1 RNA and total HIV-1 DNA concentration in peripheral CD4+ T-cells at any timepoint. While higher HIV-specific T-cell responses were seen at week 12 in ART + V + V, there were no significant correlations with plasma NfL. In multivariate analysis, higher plasma NfL was associated with older age, higher CD8+ count and lower body mass index. Conclusions Despite evidence of vaccine-induced HIV-specific T-cell responses, we observed no evidence of increased neuro-axonal injury using plasma NfL as a biomarker up to 18 weeks following kick-and-kill, compared with ART-only

Amyloid-beta 42 adsorption following serial tube transfer

INTRODUCTION: Cerebrospinal fluid (CSF) amyloid-beta 38 (Aβ38), 40 (Aβ40), 42 (Aβ42) and total tau (T-tau) are finding increasing utility as biomarkers of Alzheimer’s disease (AD). The purpose of this study was to determine whether measured CSF biomarker concentrations were affected by transfer of CSF between tubes, and whether addition of a non-ionic surfactant mitigates any observed effects. METHODS: AD and control CSF was transferred consecutively between polypropylene tubes. Aβ peptides and T-tau were measured with and without addition of Tween 20 (0.05%). RESULTS: Measured concentrations of Aβ42 decreased by approximately 25% with each consecutive transfer. Measured concentrations of Aβ38 and Aβ40 were also observed to decrease significantly with each consecutive transfer (approximately 16% loss per transfer). Measured concentrations of T-tau also decreased significantly, but at much smaller magnitude than the Aβ peptides (approximately 4% loss per transfer). The addition of Tween 20 mitigated this effect in all samples. CONCLUSIONS: Consecutive CSF transfer between tubes has a significant impact on the measured concentration of all Aβ peptides, and significant effect of lesser magnitude on T-tau. This would be sufficient to alter biomarker ratios enough to mislead diagnosis. The introduction of Tween 20 at the initial aliquoting stage was observed to significantly mitigate this effect

Identification of an important potential confound in CSF AD studies: aliquot volume.

BACKGROUND: Cerebrospinal fluid (CSF) amyloid β1-42 (Aβ1-42), total tau (T-tau) and phosphorylated tau181 (P-tau) are finding increasing utility as biomarkers of Alzheimer's disease (AD). The purpose of this study was to determine whether measured CSF biomarker concentrations were affected by aliquot storage volume and whether addition of detergent-containing buffer mitigates any observed effects. METHODS: AD and control CSF was distributed into polypropylene tubes in aliquots of different volumes (50-1500 μL). Aβ1-42, T-tau and P-tau were measured with and without addition of Tween 20 (0.05%). RESULTS: Measured concentrations of Aβ1-42 increased two-fold with aliquot storage volume. A volume increase of 10 µL caused an Aβ1-42 increase of 0.95 pg/mL [95% confidence interval (CI) 0.36-1.50, p=0.02] in controls, and 0.60 pg/mL (CI 0.23-0.98 pg/mL, p=0.003) in AD samples. Following addition of Tween 20, the positive relationship between Aβ1-42 and aliquot volume disappeared. T-tau and P-tau were not significantly affected. CONCLUSIONS: CSF aliquot storage volume has a significant impact on the measured concentration of Aβ1-42. The introduction of a buffer detergent at the initial aliquoting stage may be an effective solution to this problem