Leukocyte surface biomarkers implicate deficits of innate immunity in sporadic Alzheimer's disease.

INTRODUCTION: Blood-based diagnostics and prognostics in sporadic Alzheimer's disease (AD) are important for identifying at-risk individuals for therapeutic interventions. METHODS: In three stages, a total of 34 leukocyte antigens were examined by flow cytometry immunophenotyping. Data were analyzed by logistic regression and receiver operating characteristic (ROC) analyses. RESULTS: We identified leukocyte markers differentially expressed in the patients with AD. Pathway analysis revealed a complex network involving upregulation of complement inhibition and downregulation of cargo receptor activity and Aß clearance. A proposed panel including four leukocyte markers - CD11c, CD59, CD91, and CD163 - predicts patients' PET Aß status with an area under the curve (AUC) of 0.93 (0.88 to 0.97). CD163 was the top performer in preclinical models. These findings have been validated in two independent cohorts. CONCLUSION: Our finding of changes on peripheral leukocyte surface antigens in AD implicates the deficit in innate immunity. Leukocyte-based biomarkers prove to be both sensitive and practical for AD screening and diagnosis.

Systemic perturbations of the kynurenine pathway precede progression to dementia independently of amyloid-ß.

Increasing evidence suggests that kynurenine pathway (KP) dyshomeostasis may promote disease progression in dementia. Studies in Alzheimer's disease (AD) patients confirm KP dyshomeostasis in plasma and cerebrospinal fluid (CSF) which correlates with amyloid-ß and tau pathology. Herein, we performed the first comprehensive study assessing baseline levels of KP metabolites in participants enrolling in the Australian Imaging Biomarkers Flagship Study of Aging. Our purpose was to test the hypothesis that changes in KP metabolites may be biomarkers of dementia processes that are largely silent. We used a cross-sectional analytical approach to assess non-progressors (N = 73); cognitively normal (CN) or mild cognitive impairment (MCI) participants at baseline and throughout the study, and progressors (N = 166); CN or MCI at baseline but progressing to either MCI or AD during the study. Significant KP changes in progressors included increased 3-hydroxyanthranilic acid (3-HAA) and 3-hydroxyanthranilic acid/anthranilic acid (3-HAA/AA) ratio, the latter having the largest effect on the odds of an individual being a progressor (OR 35.3; 95% CI between 14 and 104). 3-HAA levels were hence surprisingly bi-phasic, high in progressors but low in non-progressors or participants who had already transitioned to MCI or dementia. This is a new, unexpected and interesting result, as most studies of the KP in neurodegenerative disease show reduced 3-HAA/AA ratio after diagnosis. The neuroprotective metabolite picolinic acid was also significantly decreased while the neurotoxic metabolite 3-hydroxykynurenine increased in progressors. These results were significant even after adjustment for confounders. Considering the magnitude of the OR to predict change in cognition, it is important that these findings are replicated in other populations. Independent validation of our findings may confirm the utility of 3-HAA/AA ratio to predict change in cognition leading to dementia in clinical settings.

Analysis of plasma proteins using 2D gels and novel fluorescent probes: in search of blood based biomarkers for Alzheimer's disease.

BACKGROUND: The Australian Imaging and Biomarker Lifestyle (AIBL) study of aging is designed to aid the discovery of biomarkers. The current study aimed to discover differentially expressed plasma proteins that could yield a blood-based screening tool for Alzheimer's disease. METHODS: The concentration of proteins in plasma covers a vast range of 12 orders of magnitude. Therefore, to search for medium to low abundant biomarkers and elucidate mechanisms of AD, we immuno-depleted the most abundant plasma proteins and pre-fractionated the remaining proteins by HPLC, prior to two-dimensional gel electrophoresis. The relative levels of approximately 3400 protein species resolved on the 2D gels were compared using in-gel differential analysis with spectrally resolved fluorescent protein detection dyes (Zdyes™). Here we report on analysis of pooled plasma samples from an initial screen of a sex-matched cohort of 72 probable AD patients and 72 healthy controls from the baseline time point of AIBL. RESULTS: We report significant changes in variants of apolipoprotein E, haptoglobin, α1 anti-trypsin, inter-α trypsin inhibitor, histidine-rich glycoprotein, and a protein of unknown identity. α1 anti-trypsin and α1 anti-chymotrypsin demonstrated plasma concentrations that were dependent on APOE ε4 allele dose. Our analysis also identified an association with the level of Vitamin D binding protein fragments and complement factor I with sex. We then conducted a preliminary validation study, on unique individual samples compared to the discovery cohort, using a targeted LC-MS/MS assay on a subset of discovered biomarkers. We found that targets that displayed a high degree of isoform specific changes in the 2D gels were not changed in the targeted MS assay which reports on the total level of the biomarker. CONCLUSIONS: This demonstrates that further development of mass spectrometry assays is needed to capture the isoform complexity that exists in theses biological samples. However, this study indicates that a peripheral protein signature has potential to aid in the characterization of AD.

Identification of Leukocyte Surface P2X7 as a Biomarker Associated with Alzheimer's Disease.

Alzheimer's disease (AD) has shown altered immune responses in the periphery. We studied P2X7 (a proinflammatory receptor and a scavenger receptor) and two integrins, CD11b and CD11c, on the surface of circulating leukocytes and analysed their associations with Aß-PET, brain atrophy, neuropsychological assessments, and cerebrospinal fluid (CSF) biomarkers. Total 287 age-matched, sex-balanced participants were recruited in a discovery cohort and two validation cohorts through the AIBL study and studied using tri-colour flow cytometry. Our results demonstrated reduced expressions of P2X7, CD11b, and CD11c on leukocytes, particularly monocytes, in Aß +ve cases compared with Aß -ve controls. P2X7 and integrin downregulation was observed at pre-clinical stage of AD and stayed low throughout disease course. We further constructed a polygenic risk score (PRS) model based on 12 P2RX7 risk alleles to assess the genetic impact on P2X7 function in AIBL and ADNI cohorts. No significant association was identified between the P2RX7 gene and AD, indicating that P2X7 downregulation in AD is likely caused by environmental changes rather than genetic factors. In conclusion, the downregulation of P2X7 and integrins at pre-clinical stage of AD indicates altered pro-inflammatory responses, phagocytic functions, and migrating capabilities of circulating monocytes in early AD pathogenesis. Our study not only improves our understanding of peripheral immune involvement in early stage of AD but also provides more insights into novel biomarker development, diagnosis, and prognosis of AD.

Changes in plasma amyloid beta in a longitudinal study of aging and Alzheimer's disease.

BACKGROUND: A practical biomarker is required to facilitate the preclinical diagnosis of Alzheimer's disease (AD). METHODS: Plasma amyloid beta (Aß)1-40, Aß1-42, Aßn-40, and Aßn-42 peptides were measured at baseline and after 18 months in 771 participants from the Australian Imaging Biomarkers and Lifestyle (AIBL) study of aging. Aß peptide levels were compared with clinical pathology, neuroimaging and neuropsychological measurements. RESULTS: Although inflammatory and renal function covariates influenced plasma Aß levels significantly, a decrease in Aß1-42/Aß1-40 was observed in patients with AD, and was also inversely correlated with neocortical amyloid burden. During the 18 months, plasma Aß1-42 decreased in subjects with mild cognitive impairment (MCI) and in those transitioning from healthy to MCI. CONCLUSION: Our findings are consistent with a number of published plasma Aß studies and, although the prognostic value of individual measures in any given subject is limited, the diagnostic contribution of plasma Aß may demonstrate utility when combined with a panel of peripheral biomarkers.

Concordance Between Cerebrospinal Fluid Biomarkers with Alzheimer's Disease Pathology Between Three Independent Assay Platforms.

BACKGROUND: To enhance the accuracy of clinical diagnosis for Alzheimer's disease (AD), pre-mortem biomarkers have become increasingly important for diagnosis and for participant recruitment in disease-specific treatment trials. Cerebrospinal fluid (CSF) biomarkers provide a low-cost alternative to positron emission tomography (PET) imaging for in vivo quantification of different AD pathological hallmarks in the brains of affected subjects; however, consensus around the best platform, most informative biomarker and correlations across different methodologies are controversial. OBJECTIVE: Assessing levels of Aß-amyloid and tau species determined using three different versions of immunoassays, the current study explored the ability of CSF biomarkers to predict PET Aß-amyloid (32 Aß-amyloid-and 45 Aß-amyloid+), as well as concordance between CSF biomarker levels and PET Aß-amyloid imaging. METHODS: Prediction and concordance analyses were performed using a sub-cohort of 77 individuals (48 healthy controls, 15 with mild cognitive impairment, and 14 with AD) from the Australian Imaging Biomarker and Lifestyle study of aging. RESULTS: Across all three platforms, the T-tau/Aß42 ratio biomarker had modestly higher correlation with SUVR/BeCKeT (ρ= 0.69-0.8) as compared with Aß42 alone (ρ= 0.66-0.75). Differences in CSF biomarker levels between the PET Aß-amyloid-and Aß-amyloid+ groups were strongest for the Aß42/Aß40 and T-tau/Aß42 ratios (p < 0.0001); however, comparison of predictive models for PET Aß-amyloid showed no difference between Aß42 alone and the T-tau/Aß42 ratio. CONCLUSION: This study confirms strong concordance between CSF biomarkers and PET Aß-amyloid status is independent of immunoassay platform, supporting their utility as biomarkers in clinical practice for the diagnosis of AD and for participant enrichment in clinical trials.

Peripheral α-defensins 1 and 2 are elevated in Alzheimer's disease.

Biomarkers enabling the preclinical identification of Alzheimer's disease (AD) remain one of the major unmet challenges in the field. The blood cellular fractions offer a viable alternative to current cerebrospinal fluid and neuroimaging modalities. The current study aimed to replicate our earlier reports of altered binding within the AD-affected blood cellular fraction to copper-loaded immobilized metal affinity capture (IMAC) arrays. IMAC and anti-amyloid-ß (Aß) antibody arrays coupled with mass spectrometry were used to analyze blood samples collected from 218 participants from within the AIBL Study of Aging. Peripheral Aß was fragile and prone to degradation in the AIBL samples, even when stored at -80°C. IMAC analysis of the AIBL samples lead to the isolation and identification of alpha-defensins 1 and 2 at elevated levels in the AD periphery, validating earlier findings. Alpha-defensins 1 and 2 were elevated in AD patients indicating that an inflammatory phenotype is present in the AD periphery; however, peripheral Aß levels are required to supplement their prognostic power.

Alzheimer's disease cerebrospinal fluid biomarkers are not influenced by gravity drip or aspiration extraction methodology.

INTRODUCTION: Cerebrospinal fluid (CSF) biomarkers, although of established utility in the diagnostic evaluation of Alzheimer's disease (AD), are known to be sensitive to variation based on pre-analytical sample processing. We assessed whether gravity droplet collection versus syringe aspiration was another factor influencing CSF biomarker analyte concentrations and reproducibility. METHODS: Standardized lumbar puncture using small calibre atraumatic spinal needles and CSF collection using gravity fed collection followed by syringe aspirated extraction was performed in a sample of elderly individuals participating in a large long-term observational research trial. Analyte assay concentrations were compared. RESULTS: For the 44 total paired samples of gravity collection and aspiration, reproducibility was high for biomarker CSF analyte assay concentrations (concordance correlation [95%CI]: beta-amyloid1-42 (Aß42) 0.83 [0.71 - 0.90]), t-tau 0.99 [0.98 - 0.99], and phosphorylated tau (p-tau) 0.82 [95 % CI 0.71 - 0.89]) and Bonferroni corrected paired sample t-tests showed no significant differences (group means (SD): Aß42 366.5 (86.8) vs 354.3 (82.6), p = 0.10; t-tau 83.9 (46.6) vs 84.7 (47.4) p = 0.49; p-tau 43.5 (22.8) vs 40.0 (17.7), p = 0.05). The mean duration of collection was 10.9 minutes for gravity collection and <1 minute for aspiration. CONCLUSIONS: Our results demonstrate that aspiration of CSF is comparable to gravity droplet collection for AD biomarker analyses but could considerably accelerate throughput and improve the procedural tolerability for assessment of CSF biomarkers.

Alzheimer's Disease Normative Cerebrospinal Fluid Biomarkers Validated in PET Amyloid-ß Characterized Subjects from the Australian Imaging, Biomarkers and Lifestyle (AIBL) study.

BACKGROUND: The cerebrospinal fluid (CSF) amyloid-ß (Aß)(1-42), total-tau (T-tau), and phosphorylated-tau (P-tau181P) profile has been established as a valuable biomarker for Alzheimer's disease (AD). OBJECTIVE: The current study aimed to determine CSF biomarker cut-points using positron emission tomography (PET) Aß imaging screened subjects from the Australian Imaging, Biomarkers and Lifestyle (AIBL) study of aging, as well as correlate CSF analyte cut-points across a range of PET Aß amyloid ligands. METHODS: Aß pathology was determined by PET imaging, utilizing ¹¹C-Pittsburgh Compound B, ¹8F-flutemetamol, or ¹8F-florbetapir, in 157 AIBL participants who also underwent CSF collection. Using an INNOTEST assay, cut-points were established (Aß(1-42) >544 ng/L, T-tau <407 ng/L, and P-tau181P <78 ng/L) employing a rank based method to define a "positive" CSF in the sub-cohort of amyloid-PET negative healthy participants (n = 97), and compared with the presence of PET demonstrated AD pathology. RESULTS: CSF Aß(1-42) was the strongest individual biomarker, detecting cognitively impaired PET positive mild cognitive impairment (MCI)/AD with 85% sensitivity and 91% specificity. The ratio of P-tau181P or T-tau to Aß(1-42) provided greater accuracy, predicting MCI/AD with Aß pathology with ≥92% sensitivity and specificity. Cross-validated accuracy, using all three biomarkers or the ratio of P-tau or T-tau to Aß(1-42) to predict MCI/AD, reached ≥92% sensitivity and specificity. CONCLUSIONS: CSF Aß(1-42) levels and analyte combination ratios demonstrated very high correlation with PET Aß imaging. Our study offers additional support for CSF biomarkers in the early and accurate detection of AD pathology, including enrichment of patient cohorts for treatment trials even at the pre-symptomatic stage.

Longitudinal analysis of serum copper and ceruloplasmin in Alzheimer's disease.

BACKGROUND: Several studies have reported that peripheral levels of copper and ceruloplasmin (CP) can differentiate patients with Alzheimer's disease (AD) from non-AD cases. The aim of this study was to determine the diagnostic value of serum copper, CP, and non-CP copper levels in a large cohort of AD subjects. METHODS: Serum copper and CP concentrations were measured at baseline and at 18-months in participants from the Australian Imaging Biomarkers and Lifestyle Study of Ageing. Cross-sectional and longitudinal analyses were conducted using both univariate and multivariate testing adjusting for age, gender, total protein, and ApoE ε4 genotype status. RESULTS: There was no significant difference in levels of serum copper or CP between the AD and healthy control groups, however, we identified a near-significant decrease in non-CP copper in the mild cognitive impairment and AD groups at baseline (p = 0.02) that was significant at 18-months (p = 0.003). CONCLUSION: Our results suggest that there may be decreased non-CP copper levels in mild cognitive impairment and AD, which is consistent with diminished copper-dependent biochemical activities described in AD.

Blood-based protein biomarkers for diagnosis of Alzheimer disease.

OBJECTIVE: To identify plasma biomarkers for the diagnosis of Alzheimer disease (AD). DESIGN: Baseline plasma screening of 151 multiplexed analytes combined with targeted biomarker and clinical pathology data. SETTING: General community-based, prospective, longitudinal study of aging. PARTICIPANTS: A total of 754 healthy individuals serving as controls and 207 participants with AD from the Australian Imaging Biomarker and Lifestyle study (AIBL) cohort with identified biomarkers that were validated in 58 healthy controls and 112 individuals with AD from the Alzheimer Disease Neuroimaging Initiative (ADNI) cohort. RESULTS: A biomarker panel was identified that included markers significantly increased (cortisol, pancreatic polypeptide, insulinlike growth factor binding protein 2, ß(2) microglobulin, vascular cell adhesion molecule 1, carcinoembryonic antigen, matrix metalloprotein 2, CD40, macrophage inflammatory protein 1α, superoxide dismutase, and homocysteine) and decreased (apolipoprotein E, epidermal growth factor receptor, hemoglobin, calcium, zinc, interleukin 17, and albumin) in AD. Cross-validated accuracy measures from the AIBL cohort reached a mean (SD) of 85% (3.0%) for sensitivity and specificity and 93% (3.0) for the area under the receiver operating characteristic curve. A second validation using the ADNI cohort attained accuracy measures of 80% (3.0%) for sensitivity and specificity and 85% (3.0) for area under the receiver operating characteristic curve. CONCLUSIONS: This study identified a panel of plasma biomarkers that distinguish individuals with AD from cognitively healthy control subjects with high sensitivity and specificity. Cross-validation within the AIBL cohort and further validation within the ADNI cohort provides strong evidence that the identified biomarkers are important for AD diagnosis.

Homocysteine, vitamin B12, and folic acid levels in Alzheimer's disease, mild cognitive impairment, and healthy elderly: baseline characteristics in subjects of the Australian Imaging Biomarker Lifestyle study.

There is some debate regarding the differing levels of plasma homocysteine, vitamin B12 and serum folate between healthy controls (HC), mild cognitive impairment (MCI), and Alzheimer's disease (AD). As part of the Australian Imaging Biomarker Lifestyle (AIBL) study of aging cohort, consisting of 1,112 participants (768 HC, 133 MCI patients, and 211 AD patients), plasma homocysteine, vitamin B12, and serum and red cell folate were measured at baseline to investigate their levels, their inter-associations, and their relationships with cognition. The results of this cross-sectional study showed that homocysteine levels were increased in female AD patients compared to female HC subjects (+16%, p-value < 0.001), but not in males. Red cell folate, but not serum folate, was decreased in AD patients compared to HC (-10%, p-value = 0.004). Composite z-scores of short- and long-term episodic memory, total episodic memory, and global cognition all showed significant negative correlations with homocysteine, in all clinical categories. Increasing red cell folate had a U-shaped association with homocysteine, so that high red cell folate levels were associated with worse long-term episodic memory, total episodic memory, and global cognition. These findings underscore the association of plasma homocysteine with cognitive deterioration, although not unique to AD, and identified an unexpected abnormality of red cell folate.

Plasma amyloid-beta as a biomarker in Alzheimer's disease: the AIBL study of aging.

Amyloid-beta (Abeta) plays a central role in the pathogenesis of Alzheimer's disease (AD) and has been postulated as a potential biomarker for AD. However, there is a lack of consensus as to its suitability as an AD biomarker. The objective of this study was to determine the significance of plasma Abeta as an AD biomarker and its relationship with Abeta load and to determine the effect of different assay methods on the interpretation of Abeta levels. Plasma Abeta1-40, Abeta1-42, and N-terminal cleaved fragments were measured using both a commercial multiplex assay and a well-documented ELISA in 1032 individuals drawn from the well-characterized Australian Imaging, Biomarkers and Lifestyle (AIBL) study of aging. Further, Abeta levels were compared to Abeta load derived from positron-emission tomography (PET) with the Pittsburgh compound B (PiB). Lower Abeta1-42 and Abeta1-42/1-40 ratio were observed in patients with AD and inversely correlated with PiB-PET derived Abeta load. However, assay methodology significantly impacted the interpretation of data. The cross-sectional analysis of plasma Abeta isoforms suggests that they may not be sufficient per se to diagnose AD. The value of their measurement in prognosis and monitoring of AD interventions needs further study, in addition to future longitudinal comparisons together with other predictors, which will determine whether plasma Abeta has diagnostic value in a panel of biomarkers.