Andrew D. Watt

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

A practical biomarker is required to facilitate the preclinical diagnosis of Alzheimers disease (AD).

Oligomers, fact or artefact? SDS-PAGE induces dimerization of β-amyloid in human brain samples

The formation of low-order oligomers of β-amyloid (Aβ) within the brain is widely believed to be a central component of Alzheimer’s disease (AD) pathogenesis. However, despite advances in high-throughput and high-resolution techniques such as xMAP and mass spectrometry (MS), investigations into these oligomeric species have remained reliant on low-resolution Western blots and enzyme-linked immunosorbent assays. The current investigation compared Aβ profiles within human cortical tissue using sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE), xMAP and surface enhanced laser desorption/ionization time-of-flight MS and found that whilst there was significant correlation across the techniques regarding levels of monomeric Aβ, only SDS-PAGE was capable of detecting dimeric isoforms of Aβ. The addition of synthetic di-tyrosine cross-linked Aβ1–40Met35(O) to the AD tissue demonstrated that the MS methodology was capable of observing dimeric Aβ at femto-molar concentrations, with no noticeable effect on monomeric Aβ levels. Focus turned to the association between SDS-PAGE and levels of observable dimeric Aβ within the AD brain tissue. These investigations revealed that increased levels of dimeric Aβ were observed with increasing concentrations of SDS in the sample buffer. This finding was subsequently confirmed using synthetic Aβ1–42 and suggests that SDS was inducing the formation of dimeric Aβ. The findings that SDS promotes Aβ dimerization have significant implications for the putative role of low-order oligomers in AD pathogenesis and draw into question the utility of oligomeric Aβ as a therapeutic target.

Variability in Blood-Based Amyloid-β Assays: The Need for Consensus on Pre-Analytical Processing

Effective therapeutic interventions for Alzheimers disease (AD) will require treatment regimes to move toward the earliest stages of the disease. For this to occur the field has to identify biomarkers that are able to accurately identify individuals at risk for progression toward AD in the presymptomatic stage. One very significant implication is that some form of population-based screening will need to be undertaken in order to identify those at risk. To date, efforts in neuroimaging brain amyloid-β (Aβ) and changes in cerebrospinal fluid Aβ and tau levels shows promise, however, it is questionable as to whether these methods are applicable for screening the general population. The Aβ peptide is also found in blood which is the most economical and efficient biological fluid to analyze. Unfortunately, investigations into blood-based diagnostic markers have produced mixed results. This variability is likely to be the result of differences in the preanalytical processing of samples and as such is delaying progress in the field. Reported preanalytical processing techniques from 87 recent articles focusing on the measurement of Aβ in blood were compared, to investigate whether basic sample-handling techniques were comparable between studies. This comparison revealed that not only is it likely that some of the variability in blood-based results is attributable to discrepancies in preanalytical methodologies but also that the field is failing to adequately report sample processing techniques. This review highlights the current shortcomings in methodological reporting and recommends a standardized blood collection methodology based on the limited consensus of the reviewed articles.

An increased neutrophil-lymphocyte ratio in Alzheimer's disease is a function of age and is weakly correlated with neocortical amyloid accumulation.

Inflammation is a hallmark of Alzheimers disease (AD). Whether directly involved in the pathogenesis, or a downstream consequence of neuronal death, the blood neutrophil-lymphocyte ratio (NLR) is reported to be a putative, non-invasive peripheral biomarker for AD. The aim of this study was to re-evaluate the diagnostic utility of longitudinal measures of the NLR. The NLR was stable across all time-points and weakly correlated with neocortical amyloid burden (R=0.21 at baseline, 0.27 at 18 months, 0.20 at 36 months and 0.10 at 54 months). Cross-sectionally, the NLR was significantly elevated in AD participants as compared to HC participants at baseline (p<0.0001), 18 months (p<0.0001), 36 months (p=0.002) and at 54 months (p=0.007), however only prior to adjustment for age, sex and APOEε4 allele status (p>0.05 at all time-points except for 18 months; p<0.0001). Longitudinally, the NLR was not significantly different between HC and AD participants (p>0.05) adjusted for age, sex and APOEε4 allele status. Comparing the NLR between cognitive transition groups over time (transition towards an AD type dementia), there was no significant difference in the NLR levels between those participants, who did not transition and those participants who did transition, or those in the stable AD group after adjusting for age, sex and APOEε4 allele status (p>0.05). Despite inflammation being a hallmark in AD and previous reports showing that the NLR can discriminate HC from AD patients, our results suggest that the sensitivity of the NLR itself is not robust enough for diagnostic utility. We identified significant relationships cross sectionally (p<0.05 at baseline, 18 months and 36 months) between the NLR and neocortical amyloid burden, but this relationship was lost after longitudinal analyses (p>0.5). The NLR also had limited association with cognitive decline, although in our cohort, the number of participants transitioning was relatively small. In conclusion, the NLR may reflect AD-related inflammatory processes in the periphery, but age and sex are dominant covariates which need to be controlled for in population-based screening.

Plasma Amyloid-β Levels are Significantly Associated with a Transition Toward Alzheimer's Disease as Measured by Cognitive Decline and Change in Neocortical Amyloid Burden

BACKGROUND We evaluated the utility of longitudinal measures of plasma amyloid-β (Aβ) as a means to identify pre-symptomatic cognitive decline in Alzheimers disease (AD) when coupled to neuroimaging and neuropsychological parameters. METHODS Participants from the Australian Imaging, Biomarkers and Lifestyle (AIBL) study were grouped based upon cognitive change and changes in measurable levels of neocortical amyloid over 36 months. Participants were classified as those who transitioned for cognitive decline and change in neocortical amyloid, those healthy controls that did not transition, and stable AD participants over 36 months. RESULTS Comparisons of plasma Aβ levels between the transition and non-transitional groups showed Aβ1-42 and the Aβ1-42/Aβ1-40 ratio were significantly decreased at baseline (p = 0.008 and p = 0.002, respectively) and at 18 months (p = 0.003 and p = 0.004, respectively). Both measures of neocortical amyloid and two previously published composite scores validated the creation of the novel transitional grouping (p < 0.0001). In addition Aβn-42 performed well as a longitudinal prognostic indicator of transition toward cognitive decline, with a significant decrease in the transition group at the 18 month time point (p = 0.01). CONCLUSION We demonstrated that baseline plasma Aβ1-42 and the Aβ1-42/Aβ1-40 ratio were putative biomarkers indicative of cognitive decline and validated this result using 18 month data. We created a novel transitional grouping and validated this measure using published measures of neocortical amyloid and composite memory scores. These findings suggest that longitudinal plasma Aβ could contribute to a pre-symptomatic biomarker panel for AD.

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 Alzheimers 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.

Progress towards a consensus on biomarkers for Alzheimer's disease: a review of peripheral analytes

Alzheimers disease (AD) is the most common cause of dementia in the elderly population and attempts to develop therapies have been unsuccessful because there is no means to target an effective therapeutic window. CNS biomarkers are insightful but impractical for high-throughput population-based screening. Therefore, a peripheral, blood-based biomarker for AD would significantly improve early diagnosis, potentially enable presymptomatic detection and facilitate effective targeting of disease-modifying treatments. The various constituents of blood, including plasma, platelets and cellular fractions, are now being systematically explored as a pool of putative peripheral biomarkers for AD. In this review we cover some less known peripheral biomarkers and highlight the latest developments for their clinical application.

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

Biomarkers enabling the preclinical identification of Alzheimers 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-β (Aβ) antibody arrays coupled with mass spectrometry were used to analyze blood samples collected from 218 participants from within the AIBL Study of Aging. Peripheral Aβ 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 Aβ levels are required to supplement their prognostic power.

Metals, Membranes, and Amyloid-β Oligomers: Key Pieces in the Alzheimer's Disease Puzzle?

Over the past 100 years, there has been an exponential increase in our understanding of the underlying pathology of Alzheimers disease (AD). This growth in knowledge has largely stemmed from the intensification of research into AD which has occurred over the past three decades and the incorporation of the amyloid cascade hypothesis as the generally accepted dogma of AD pathogenesis. While at times contentious, the notion that AD arises from aberrations in amyloid-β (Aβ) production and degradation has led to a number of significant breakthroughs in the way in which AD is currently diagnosed and in the attempts at disease modifying therapies, from investigations into the underlying factors mediating the aggregation of Aβ to the development of therapeutic strategies and measures of neuroimaging allowing Aβ burden to be monitored within the AD-affected brain. This review focuses on some of the recent work we have conducted toward elucidating the role of Aβ in AD.

Anti-Aβ antibody target engagement: a response to Siemers et al.

engagement with brain Aβ, consistent with published clinical data”. To reiterate, we reported data showing that bapineuzumab was capable of binding soluble Aβ with a low nanomolar affinity and demonstrated that the antibody could detect Aβ species in buffer and in brain homogenate and plasma from transgenic animal models of AD. Furthermore, in agreement with the phase 3 clinical data [23], we demonstrated target engagement for bapineuzumab, where its target is brain-derived amyloid-β peptides associated with Alzheimer’s pathology (see, Fig. 3a [28]). Our data also showed that neither solanezumab nor crenezumab effectively engaged this pool of Aβ, again consistent with the publicly available clinical data [9, 10]. We have read with interest the commentary by siemers et al. [28] regarding our paper describing the ability of the three anti-Aβ antibodies, bapineuzumab, crenezumab and solanezumab to engage Aβ in both a synthetic and a biological setting. We appreciate the opportunity to clarify any misunderstandings and here provide a brief response to their concerns. siemers et al. [28] begin their commentary by stating that our findings led to the conclusion “that all three antibodies failed to engage the intended molecular targets”. This statement is wrong; as clearly stated in the abstract of our paper, “Bapineuzumab demonstrated target