Aβ34 is a BACE1-derived degradation intermediate associated with amyloid clearance and Alzheimer's disease progression.
ABSTRACT: The beta-site APP cleaving enzyme 1 (BACE1) is known primarily for its initial cleavage of the amyloid precursor protein (APP), which ultimately leads to the generation of Aβ peptides. Here, we provide evidence that altered BACE1 levels and activity impact the degradation of Aβ40 and Aβ42 into a common Aβ34 intermediate. Using human cerebrospinal fluid (CSF) samples from the Amsterdam Dementia Cohort, we show that Aβ34 is elevated in individuals with mild cognitive impairment who later progressed to dementia. Furthermore, Aβ34 levels correlate with the overall Aβ clearance rates in amyloid positive individuals. Using CSF samples from the PREVENT-AD cohort (cognitively normal individuals at risk for Alzheimer's disease), we further demonstrate that the Aβ34/Aβ42 ratio, representing Aβ degradation and cortical deposition, associates with pre-clinical markers of neurodegeneration. We propose that Aβ34 represents a marker of amyloid clearance and may be helpful for the characterization of Aβ turnover in clinical samples.
Project description:BACKGROUND:One of the major challenges in diagnosing dementia with Lewy bodies (DLB) is the common co-morbid presence of amyloid pathology. To understand the putative role of altered amyloid-β (Aβ) metabolism in dementia with DLB, we analyzed levels of different cerebrospinal fluid (CSF) Aβ peptides (Aβ38, Aβ40, Aβ42) in DLB, Alzheimer's disease (AD), and cognitively normal controls. METHODS:CSF from patients with DLB (n = 72; age 68 ± 6 years; 10%F; Mini-mental State examination (MMSE) 23 ± 4), AD (n = 38; age 68 ± 6 years; 8%F; MMSE 22 ± 5), and cognitively normal controls (n = 38; age 67 ± 7 years; 13%F; MMSE 29 ± 2) was analyzed using the Meso Scale Discovery assay for human Aβ peptides. We performed general linear models to compare CSF Aβ peptide levels between groups. Associations between CSF Aβ peptides and MMSE score at baseline and longitudinal changes over time were assessed with linear mixed models. RESULTS:For all three CSF Aβ peptides and compared to controls (Aβ38 2676 ± 703 pg/ml, Aβ40 6243 ± 1500 pg/ml, and Aβ42 692 ± 205 pg/ml), we observed lower levels in DLB (Aβ38 2247 ± 638, Aβ40 5432 ± 1340, and Aβ42 441 ± 185, p < 0.05), whereas AD patients showed only lower Aβ42 levels (304 ± 71, p < 0.001). The observed differences in Aβ38 and Aβ40 were independent of co-morbid AD pathology (CSF tau/Aβ42 > 0.52) and APOE genotype. Finally, lower Aβ peptide levels were associated with lower MMSE score (β = 1.02-1.11, p < 0.05). CONCLUSION:We demonstrated different profiles of CSF Aβ reduction in DLB and AD. In particular, while AD is characterized by an isolated drop in Aβ42, DLB comes with reductions in Aβ38, Aβ40, and Aβ42. This suggests that amyloid metabolism is affected in DLB, even in the absence of co-morbid AD pathology.
Project description:Increased APP (amyloid precursor protein) processing causes β-amyloid (Aβ) accumulation in autosomal dominant Alzheimer's disease (AD), but it is unclear if it also affects sporadic Aβ accumulation. We tested healthy controls and patients with mild cognitive symptoms (N=331) in the BioFINDER study, using cerebrospinal fluid (CSF) Aβ40 as a surrogate for amyloidogenic APP processing. We find that levels of brain Aβ fibrils (measured by 18F-flutemetamol PET) are independently associated with high CSF Aβ40 (P<0.001) and APOE ɛ4 (P<0.001). The association between CSF Aβ40 and brain Aβ is stronger in APOE ɛ4-negative than in positive people (P=0.0080). The results are similar for CSF Aβ38 and for a combination of CSF Aβ38 and CSF Aβ40. In conclusion, sporadic Aβ accumulation may be partly associated with increased amyloidogenic APP production, especially in APOE ɛ4-negative subjects. The risk for sporadic AD may consequently depend on increased Aβ production, in addition to decreased Aβ clearance.
Project description:Alzheimer's disease is pathologically defined by accumulation of extracellular amyloid-β (Aβ). Approximately 25 mutations in β-amyloid precursor protein (APP) are pathogenic and cause autosomal dominant Alzheimer's disease. To date, the mechanism underlying the effect of APP mutation on Aβ generation is unclear. Therefore, investigating the mechanism of APP mutation on Alzheimer's disease may help understanding of disease pathogenesis. Thus, APP mutations (A673T, A673V, E682K, E693G, and E693Q) were transiently co-transfected into human embryonic kidney cells. Western blot assay was used to detect expression levels of APP, beta-secretase 1, and presenilin 1 in cells. Enzyme-linked immunosorbent assay was performed to determine Aβ1-40 and Aβ1-42 levels. Liquid chromatography-tandem mass chromatography was used to examine VVIAT, FLF, ITL, VIV, IAT, VIT, TVI, and VVIA peptide levels. Immunofluorescence staining was performed to measure APP and early endosome antigen 1 immunoreactivity. Our results show that the protective A673T mutation decreases Aβ42/Aβ40 rate by downregulating IAT and upregulating VVIA levels. Pathogenic A673V, E682K, and E693Q mutations promote Aβ42/Aβ40 rate by increasing levels of CTF99, Aβ42, Aβ40, and IAT, and decreasing VVIA levels. Pathogenic E693G mutation shows no significant change in Aβ42/Aβ40 ratio because of inhibition of γ-secretase activity. APP mutations can change location from the cell surface to early endosomes. Our findings confirm that certain APP mutations accelerate Aβ generation by affecting the long Aβ cleavage pathway and increasing Aβ42/40 rate, thereby resulting in Alzheimer's disease.
Project description:Impaired brain clearance of amyloid-beta peptides (Aβ) 40 and 42 across the blood-brain barrier (BBB) is believed to be one of the pathways responsible for Alzheimer's disease (AD) pathogenesis. Hyperinsulinemia prevalent in type II diabetes was shown to damage cerebral vasculature and increase Aβ accumulation in AD brain. However, there is no clarity on how aberrations in peripheral insulin levels affect Aβ accumulation in the brain. This study describes, for the first time, an intricate relation between plasma insulin and Aβ transport at the BBB. Upon peripheral insulin administration in wild-type mice: the plasma clearance of Aβ40 increased, but Aβ42 clearance reduced; the plasma-to-brain influx of Aβ40 increased, and that of Aβ42 reduced; and the clearance of intracerebrally injected Aβ40 decreased, whereas Aβ42 clearance increased. In hCMEC/D3 monolayers (in vitro BBB model) exposed to insulin, the luminal uptake and luminal-to-abluminal permeability of Aβ40 increased and that of Aβ42 reduced; the abluminal-to-luminal permeability of Aβ40 decreased, whereas Aβ42 permeability increased. Moreover, Aβ cellular trafficking machinery was altered. In summary, Aβ40 and Aβ42 demonstrated distinct distribution kinetics in plasma and brain compartments, and insulin differentially modulated their distribution. Cerebrovascular disease and metabolic disorders may disrupt this intricate homeostasis and aggravate AD pathology.
Project description:Widespread implementation of cerebrospinal fluid (CSF) biomarkers of Alzheimer's disease (AD) in clinical settings requires improved accuracy for diagnosis of prodromal disease and for distinguishing AD from non-AD dementias. Novel and promising CSF biomarkers include neurogranin, a marker of synaptic degeneration, and YKL-40, a marker of neuroinflammation.CSF neurogranin and YKL-40 were measured in a cohort of 338 individuals including cognitively healthy controls and patients with stable mild cognitive impairment (sMCI), MCI who later developed AD (MCI-AD), AD dementia, Parkinson's disease dementia (PDD), dementia with Lewy bodies (DLB), vascular dementia (VaD), and frontotemporal dementia (FTD). The diagnostic accuracy of neurogranin and YKL-40 were compared with the core AD biomarkers, β-amyloid (Aβ42 and Aβ40) and tau.Neurogranin levels were increased in AD and decreased in non-AD dementia compared with healthy controls. As a result, AD patients showed considerably higher CSF levels of neurogranin than DLB/PDD, VaD and FTD patients. CSF YKL-40 levels were increased in AD compared with DLB/PDD but not with VaD or FTD. Neither CSF neurogranin nor YKL-40 levels differed significantly between sMCI patients and MCI-AD patients. Both biomarkers correlated positively with CSF Aβ40 and tau. CSF neurogranin and YKL-40 could separate AD dementia from non-AD dementias (neurogranin, area under the curve [AUC] = 0.761; YKL-40, AUC = 0.604; Aβ42/neurogranin, AUC = 0.849; Aβ42/YKL-40, AUC = 0.785), but the diagnostic accuracy was not better compared to CSF Aβ and tau (Aβ42, AUC = 0.755; tau AUC = 0.858; Aβ42/tau, AUC = 0.895; Aβ42/Aβ40, AUC = 0.881). Similar results were obtained when separating sMCI from MCI-AD cases.CSF neurogranin and YKL-40 do not improve the diagnostic accuracy of either prodromal AD or AD dementia when compared to the core CSF AD biomarkers. Nevertheless, the CSF level of neurogranin is selectively increased in AD dementia, whereas YKL-40 is increased in both AD and FTD suggesting that synaptic degeneration and glial activation may be important in these neurodegenerative conditions.
Project description:Objective:Despite the critical importance of pathologically confirmed samples for biomarker validation, only a few studies have correlated CSF Aβ42 values in vivo with postmortem Alzheimer's disease (AD) pathology, while none evaluated the CSF Aβ42/Aβ40 ratio. We compared CSF Aβ42 and Aβ42/Aβ40 ratio as biomarkers predicting AD neuropathological changes in patients with a short interval between lumbar puncture and death. Methods:We measured CSF Aβ40 and Aβ42 and assessed AD pathology in 211 subjects with rapidly progressive dementia (RPD) and a definite postmortem diagnosis of Creutzfeldt-Jakob disease (n = 159), AD (n = 12), dementia with Lewy bodies (DLB, n = 4), AD/DLB mixed pathologies (n = 5), and various other pathologies (n = 31). Results:The score reflecting the severity of Aβ pathology showed a better correlation with ln(Aβ42/Aβ40) (R 2 = 0.506, β = -0.713, P < 0.001) than with ln(Aβ42) (R 2 = 0.206, β = -0.458, P < 0.001), which was confirmed after adjusting for covariates. Aβ42/Aβ40 ratio showed significantly higher accuracy than Aβ42 in the distinction between cases with or without AD pathology (AUC 0.818 ± 0.028 vs. 0.643 ± 0.039), especially in patients with Aβ42 levels ≤495 pg/mL (AUC 0.888 ± 0.032 vs. 0.518 ± 0.064). Using a cut-off value of 0.810, the analysis of Aβ42/Aβ40 ratio yielded 87.0% sensitivity, 88.2% specificity in the distinction between cases with an intermediate-high level of AD pathology and those with low level or no AD pathology. Interpretation:The present data support the use of CSF Aβ42/Aβ40 ratio as a biomarker of AD pathophysiology and noninvasive screener for Aβ pathology burden, and its introduction in the research diagnostic criteria for AD.
Project description:Processing of amyloid-β (Aβ) precursor protein (APP) by γ-secretase produces multiple species of Aβ: Aβ40, short Aβ peptides (Aβ37-39), and longer Aβ peptides (Aβ42-43). γ-Secretase modulators, a class of Alzheimer's disease therapeutics, reduce production of the pathogenic Aβ42 but increase the relative abundance of short Aβ peptides. To evaluate the pathological relevance of these peptides, we expressed Aβ36-40 and Aβ42-43 in Drosophila melanogaster to evaluate inherent toxicity and potential modulatory effects on Aβ42 toxicity. In contrast to Aβ42, the short Aβ peptides were not toxic and, when coexpressed with Aβ42, were protective in a dose-dependent fashion. In parallel, we explored the effects of recombinant adeno-associated virus-mediated expression of Aβ38 and Aβ40 in mice. When expressed in nontransgenic mice at levels sufficient to drive Aβ42 deposition, Aβ38 and Aβ40 did not deposit or cause behavioral alterations. These studies indicate that treatments that lower Aβ42 by raising the levels of short Aβ peptides could attenuate the toxic effects of Aβ42.
Project description:BACKGROUND:The β-secretase, BACE1, cleaves APP to initiate generation of the β-amyloid peptide, Aβ, that comprises amyloid plaques in Alzheimer's disease (AD). Reducing BACE1 activity is an attractive therapeutic approach to AD, but complete inhibition of BACE1 could have mechanism-based side-effects as BACE1-/- mice show deficits in axon guidance, myelination, memory, and other neurological processes. Since BACE1+/- mice appear normal there is interest in determining whether 50% reduction in BACE1 is potentially effective in preventing or treating AD. APP transgenic mice heterozygous for BACE1 have decreased Aβ but the extent of reduction varies greatly from study to study. Here we assess the effects of 50% BACE1 reduction on the widely used 5XFAD mouse model of AD. RESULTS:50% BACE1 reduction reduces Aβ42, plaques, and BACE1-cleaved APP fragments in female, but not in male, 5XFAD/BACE1+/- mice. 5XFAD/BACE1+/+ females have higher levels of Aβ42 and steady-state transgenic APP than males, likely caused by an estrogen response element in the transgene Thy-1 promoter. We hypothesize that higher transgenic APP level in female 5XFAD mice causes BACE1 to no longer be in excess over APP so that 50% BACE1 reduction has a significant Aβ42 lowering effect. In contrast, the lower APP level in 5XFAD males allows BACE1 to be in excess over APP even at 50% BACE1 reduction, preventing lowering of Aβ42 in 5XFAD/BACE1+/- males. We also developed and validated a dot blot assay with an Aβ42-selective antibody as an accurate and cost-effective alternative to ELISA for measuring cerebral Aβ42 levels. CONCLUSIONS:50% BACE1 reduction lowers Aβ42 in female 5XFAD mice only, potentially because BACE1 is not in excess over APP in 5XFAD females with higher transgene expression, while BACE1 is in excess over APP in 5XFAD males with lower transgene expression. Our results suggest that greater than 50% BACE1 inhibition might be necessary to significantly lower Aβ, given that BACE1 is likely to be in excess over APP in the human brain. Additionally, in experiments using the 5XFAD mouse model, or other Thy-1 promoter transgenic mice, equal numbers of male and female mice should be used, in order to avoid artifactual gender-related differences.
Project description:BACKGROUND:Widespread implementation of Alzheimer's disease biomarkers in routine clinical practice requires the establishment of standard operating procedures for pre-analytical handling of cerebrospinal fluid (CSF). METHODS:Here, CSF collection and storage protocols were optimized for measurements of β-amyloid (Aβ). We investigated the effects of (1) storage temperature, (2) storage time, (3) centrifugation, (4) sample mixing, (5) blood contamination, and (6) collection gradient on CSF levels of Aβ. For each study participant, we used fresh CSF directly collected into a protein low binding (LoB) tube that was analyzed within hours after lumbar puncture (LP) as standard of truth. Aβ42 and Aβ40 were measured in de-identified CSF samples using EUROIMMUN and Mesoscale discovery assays. RESULTS:CSF Aβ42 and Aβ40 were stable for at least 72 h at room temperature (RT), 1 week at 4 °C, and 2 weeks at - 20 °C and - 80 °C. Centrifugation of non-blood-contaminated CSF or mixing of samples before the analysis did not affect Aβ levels. Addition of 0.1-10% blood to CSF that was stored at RT without centrifugation led to a dose- and time-dependent decrease in Aβ42 and Aβ40, while Aβ42/Aβ40 did not change. The effects of blood contamination were mitigated by centrifugation and/or storage at 4 °C or - 20 °C. Aβ levels did not differ between the first to fourth 5-ml portions of CSF. CONCLUSIONS:CSF can be stored for up to 72 h at RT, 1 week at 4 °C, or at least 2 weeks at either - 20 °C or - 80 °C before Aβ measurements. Centrifugation of fresh non-blood-contaminated CSF after LP, or mixing before analysis, is not required. In case of visible blood contamination, centrifugation and storage at 4 °C or - 20 °C is recommended. After discarding the first 2 ml, any portion of up to 20 ml of CSF is suitable for Aβ analysis. These findings will be important for the development of a clinical routine protocol for pre-analytical handling of CSF.
Project description:In this study of preclinical Alzheimer's disease (AD) we assessed the added diagnostic value of using cerebrospinal fluid (CSF) Aβ ratios rather than Aβ42 in isolation for detecting individuals who are positive on amyloid positron emission tomography (PET).Thirty-eight community-recruited cognitively intact older adults (mean age 73, range 65-80 years) underwent (18)F-flutemetamol PET and CSF measurement of Aβ1-42, Aβ1-40, Aβ1-38, and total tau (ttau). (18)F-flutemetamol retention was quantified using standardized uptake value ratios in a composite cortical region (SUVRcomp) with reference to cerebellar grey matter. Based on a prior autopsy validation study, the SUVRcomp cut-off was 1.57. Sensitivities, specificities and cut-offs were defined based on receiver operating characteristic analysis with CSF analytes as variables of interest and (18)F-flutemetamol positivity as the classifier. We also determined sensitivities and CSF cut-off values at fixed specificities of 90 % and 95 %.Seven out of 38 subjects (18 %) were positive on amyloid PET. Aβ42/ttau, Aβ42/Aβ40, Aβ42/Aβ38, and Aβ42 had the highest accuracy to identify amyloid-positive subjects (area under the curve (AUC) ≥ 0.908). Aβ40 and Aβ38 had significantly lower discriminative power (AUC = 0.571). When specificity was fixed at 90 % and 95 %, Aβ42/ttau had the highest sensitivity among the different CSF markers (85.71 % and 71.43 %, respectively). Sensitivity of Aβ42 alone was significantly lower under these conditions (57.14 % and 42.86 %, respectively).For the CSF-based definition of preclinical AD, if a high specificity is required, our data support the use of Aβ42/ttau rather than using Aβ42 in isolation.