Sarah Westwood

Blood-Based Proteomic Biomarkers of Alzheimer's Disease Pathology.

The complexity of Alzheimer’s disease (AD) and its long prodromal phase poses challenges for early diagnosis and yet allows for the possibility of the development of disease modifying treatments for secondary prevention. It is, therefore, of importance to develop biomarkers, in particular, in the preclinical or early phases that reflect the pathological characteristics of the disease and, moreover, could be of utility in triaging subjects for preventative therapeutic clinical trials. Much research has sought biomarkers for diagnostic purposes by comparing affected people to unaffected controls. However, given that AD pathology precedes disease onset, a pathology endophenotype design for biomarker discovery creates the opportunity for detection of much earlier markers of disease. Blood-based biomarkers potentially provide a minimally invasive option for this purpose and research in the field has adopted various “omics” approaches in order to achieve this. This review will, therefore, examine the current literature regarding blood-based proteomic biomarkers of AD and its associated pathology.

Blood protein predictors of brain amyloid for enrichment in clinical trials

Measures of neocortical amyloid burden (NAB) identify individuals who are at substantially greater risk of developing Alzheimers disease (AD). Blood‐based biomarkers predicting NAB would have great utility for the enrichment of AD clinical trials, including large‐scale prevention trials.

Blood-Based Biomarker Candidates of Cerebral Amyloid Using PiB PET in Non-Demented Elderly

Increasingly, clinical trials for Alzheimers disease (AD) are being conducted earlier in the disease phase and with biomarker confirmation using in vivo amyloid PET imaging or CSF tau and Aβ measures to quantify pathology. However, making such a pre-clinical AD diagnosis is relatively costly and the screening failure rate is likely to be high. Having a blood-based marker that would reduce such costs and accelerate clinical trials through identifying potential participants with likely pre-clinical AD would be a substantial advance. In order to seek such a candidate biomarker, discovery phase proteomic analyses using 2DGE and gel-free LC-MS/MS for high and low molecular weight analytes were conducted on longitudinal plasma samples collected over a 12-year period from non-demented older individuals who exhibited a range of 11C-PiB PET measures of amyloid load. We then sought to extend our discovery findings by investigating whether our candidate biomarkers were also associated with brain amyloid burden in disease, in an independent cohort. Seven plasma proteins, including A2M, Apo-A1, and multiple complement proteins, were identified as pre-clinical biomarkers of amyloid burden and were consistent across three time points (p <  0.05). Five of these proteins also correlated with brain amyloid measures at different stages of the disease (q <  0.1). Here we show that it is possible to detect a plasma based biomarker signature indicative of AD pathology at a stage long before the onset of clinical disease manifestation. As in previous studies, acute phase reactants and inflammatory markers dominate this signature.

Blood metabolite markers of neocortical amyloid-β burden: discovery and enrichment using candidate proteins.

We believe this is the first study to investigate associations between blood metabolites and neocortical amyloid burden (NAB) in the search for a blood-based biomarker for Alzheimers disease (AD). Further, we present the first multi-modal analysis of blood markers in this field. We used blood plasma samples from 91 subjects enrolled in the University of California, San Francisco Alzheimers Disease Research Centre. Non-targeted metabolomic analysis was used to look for associations with NAB using both single and multiple metabolic feature models. Five metabolic features identified subjects with high NAB, with 72% accuracy. We were able to putatively identify four metabolites from this panel and improve the model further by adding fibrinogen gamma chain protein measures (accuracy=79%). One of the five metabolic features was studied in the Alzheimers Disease Neuroimaging Initiative cohort, but results were inconclusive. If replicated in larger, independent studies, these metabolic features and proteins could form the basis of a blood test with potential for enrichment of amyloid pathology in anti-amyloid trials.

A Decade of Blood Biomarkers for Alzheimer's Disease Research: An Evolving Field, Improving Study Designs, and the Challenge of Replication.

Blood-based biomarkers represent a less invasive and potentially cheaper approach for aiding Alzheimer’s disease (AD) detection compared with cerebrospinal fluid and some neuroimaging biomarkers. Acknowledging that many in the field have made great progress, here we review some of the work that our group has pursued to identify and validate blood-based proteomic biomarkers through both case control and AD pathology endophenotype-based approaches. Our focus is primarily to identify a minimally invasive and hopefully cost-effective blood-based biomarker to reduce screen failure in clinical trials where participants have prodromal or even pre-clinical disease. We summarize some of the key findings and approaches taken in these biomarker studies, while addressing the main challenges, including that of limited replication in the field, and discuss opportunities for biomarker development.

PLASMA PRIMARY FATTY AMIDES ASSOCIATE TO CSF AMYLOID LEVELS AND ALZHEIMER’S DISEASE PROGRESSION IN THE EMIF-AD BIOMARKER DISCOVERY COHORT

with albumin ratio (r1⁄40.28, p1⁄40.006). We observed no independent effects, but a synergistic effect of IL-6 and albumin ratio on Aß (Z1⁄4-2.72, p1⁄40.006). The JN-analyses showed that the association between albumin ratio and Aßwas significant for IL-6 levels 3.48 pg/ml. We found no direct relation between the albumin ratio and p-Tau levels (p1⁄40.91), but mediation analyses indicated that the mediation of Aß,on the association between BBB dysfuction and p-Tau, was contingent on elevated IL-6 levels (indirect effect for IL-6: mean-1s (b1⁄40.026, Z1⁄40.075, p1⁄40.94), mean (b1⁄41.16, Z1⁄41.64, p1⁄40.09), mean+1s (b1⁄43.51, Z1⁄40.197, p1⁄40.049)). There is a negative relationship between Aß and pTau (p<0.001). Conclusions: The results suggest a link between BBB-dysfunction and markers of neurodegeneration via Aßmediated mechanisms in the presence of elevated IL-6 levels, indicating that inflammation and BBB-dysfunction, in addition to Aß are important components in the path to neurodegeneration. These results fit with autopsy studies reporting a common cooccurrence of Aß and vascular pathology in AD-type dementia.

EX-VIVO VALIDATION OF PLASMA PROTEIN FIBRINOGEN GAMMA

established transcranialmagnetic stimulation technique that can assess the level of cholinergic activity in the brain. However, it is not yet known whether SAI is able to distinguish the low cholinergic activity in AD patients compared to unaffected controls and detect the improvement in cholinergic function in response to treatment with ChEI such as donepezil. The objective of this study was to assess whether SAI can be used as a biomarker for increased cholinergic activity, and hence measure potential improvements in response to treatments. Methods: 14 mild-to-moderate AD patients (age 73 +/7.1 years (mean +/-SD), 7 males, MMSE 23.3 +/3.3) on 10 mg donepezil once daily and 20 unaffected control subjects (age 67 +/7.3 years), 9males, meanMMSE 29.8 +/0.5) underwent 4 SAI sessions: 2 sessions per day w4 hours apart, 13.7 +/1.31 days apart. In the AD patient group, the first TMS session of the day was performed prior to the daily dose of donepezil, and the second 3 hours post dose. Results:A mixed analysis of variance revealed no significant difference between the SAI responses between AD patients pre or post donepezil dose and unaffected control subjects (p 1⁄4 0.275). There was also no significant intra-subject difference in the SAI responses preand postdonepezil dose in AD patients (p 1⁄4 0.870). Conclusions: SAI was not able to detect a significant difference in the brain cholinergic state between patients with mild-to-moderate AD and unaffected control subjects. In addition, repeat SAI did not detect intra-subject change in the brain cholinergic state before and 3 hours after administration of donepezil in patients with mild-tomoderate AD.

BLOOD-BASED BIOMARKERS OF ALZHEIMER'S DISEASE PATHOLOGY AND COGNITIVE DECLINE IN NON-DEMENTED ELDERLY

neuron-to-neuron. It is known that a-synuclein oligomers can transfer between cells but not if this requires neurites and synapses. We have previously visualized neuritic neuron-to-neuron transfer of b-amyloid, in an 3D co-culture system with differentiated neuron-like human SH-SY5Y cells. In this study we investigated if a-synuclein is transferred in a similar manner as b-amyloid.We also investigated the intracellular localization and toxicity of the transferred a-synuclein. Methods: Monomeric, oligomeric and fibrillar forms of a -synuclein were labeled with Cy3. SH-SY5Y neuroblastoma cells were extensively differentiated in an ECM gel with the addition of a cocktail of growth-factors and transfected with CellLight endosome-GFP (acceptor cells). Retinoic acid differentiated SH-SY5Y cells were incubated with 1-2 mM monomeric, oligomeric or fibrillary a-synuclein for 3 h (donor cells). Donor cells and acceptor cells were co-cultured for up to 7 days, fixed and analyzed using confocal microscopy. Results:Monomeric, oligomeric and fibrillar a-synuclein forms were taken up by donor cells within 3 hours and remained in the cells for at least 48 hours. After 24 h of co-culture, all tested forms of a-synuclein were transferred from donor cells to acceptor cells if neuritic connections were present. Oligomers were transferred more extensively than monomers. The a-synuclein co-localized with markers for multi vesicular bodies, endosoms and lysosomes. Conclusions: Monomeric, oligomeric and fibrillar forms of a-synuclein were transmitted through neuritic connections from donor to acceptor cells. The transfer of a-synuclein seem to involve multivesicular bodies as well as the endosomal and lysosomal compartments. The high level of differentiation of the acceptor cells, producing neuritic connections and synapses, seemed necessary for the transfer. Thus, we have found many similarities between a-synuclein and b-amyloid transfer.

DISCOVERY AND VALIDATION OF PLASMA BIOMARKERS RELATING TO CSF MEASURES OF ALZHEIMER'S DISEASE PATHOLOGY

BIOMARKERS RELATING TO CSF MEASURES OF ALZHEIMER’S DISEASE PATHOLOGY Alison L. Baird, Nicholas Ashton, Sarah Westwood, Malcolm Ward, Chantal Bazenet, Madhav Thambisetty, Philip Scheltens, Charlotte Teunissen, Simon Lovestone, Kings College London, London, United Kingdom; Kings College London, Institute of Psychiatry, London, United Kingdom; King’s College London, London, United Kingdom; 4 National Institute on Aging, Baltimore, Maryland, United States; 5 VU University Medical Centre, Amsterdam, Netherlands. Contact e-mail: alison.baird@kcl.ac.uk