Catharine Joachim

Methylomic profiling implicates cortical deregulation of ANK1 in Alzheimer's disease

Alzheimer’s disease (AD) is a chronic neurodegenerative disorder characterized by progressive neuropathology and cognitive decline. We describe a cross-tissue analysis of methylomic variation in AD using samples from three independent human post-mortem brain cohorts. We identify a differentially methylated region in the ankyrin 1 (ANK1) gene that is associated with neuropathology in the entorhinal cortex, a primary site of AD manifestation. This region was confirmed as significantly hypermethylated in two other cortical regions (superior temporal gyrus and prefrontal cortex) but not in the cerebellum, a region largely protected from neurodegeneration in AD, nor whole blood obtained pre-mortem, from the same individuals. Neuropathology-associated ANK1 hypermethylation was subsequently confirmed in cortical samples from three independent brain cohorts. This study represents the first epigenome-wide association study (EWAS) of AD employing a sequential replication design across multiple tissues, and highlights the power of this approach for identifying methylomic variation associated with complex disease.Alzheimers disease (AD) is a chronic neurodegenerative disorder that is characterized by progressive neuropathology and cognitive decline. We performed a cross-tissue analysis of methylomic variation in AD using samples from four independent human post-mortem brain cohorts. We identified a differentially methylated region in the ankyrin 1 (ANK1) gene that was associated with neuropathology in the entorhinal cortex, a primary site of AD manifestation. This region was confirmed as being substantially hypermethylated in two other cortical regions (superior temporal gyrus and prefrontal cortex), but not in the cerebellum, a region largely protected from neurodegeneration in AD, or whole blood obtained pre-mortem from the same individuals. Neuropathology-associated ANK1 hypermethylation was subsequently confirmed in cortical samples from three independent brain cohorts. This study represents, to the best of our knowledge, the first epigenome-wide association study of AD employing a sequential replication design across multiple tissues and highlights the power of this approach for identifying methylomic variation associated with complex disease.

Haplotype-specific expression of the N-terminal exons 2 and 3 at the human MAPT locus

The microtubule-associated protein tau (MAPT) H1 haplotype shows a strong association to the sporadic neurodegenerative diseases, progressive supranuclear palsy and corticobasal degeneration. The functional biological mechanisms behind the genetic association have started to emerge with differences recently shown in haplotype splicing of the neuropathologically relevant exon 10. Here we investigate the hypothesis that expression of the alternatively spliced N-terminal exons also differs between the two MAPT haplotypes. We performed allele-specific gene expression analysis on a H1/H2 heterozygous human neuronal cell line model and 14 H1/H2 heterozygous human post-mortem brain tissues from two brain regions. In both cell culture and post-mortem brain tissue, we show that the protective MAPT H2 haplotype significantly expresses two-fold more 2N (exons 2+3+) MAPT transcripts than the disease-associated H1 haplotype. We suggest that inclusion of exon 3 in MAPT transcripts may contribute to protecting H2 carries from neurodegeneration.

Cerebral amyloid angiopathy, subcortical white matter disease and dementia: literature review and study in OPTIMA.

Cerebral amyloid angiopathy (CAA) is of increasing clinical and research interest as the ability to detect it and its consequences by neuroimaging in living subjects has advanced. There is also increasing interest in understanding its possible role in the development of intracerebral hemorrhage, Alzheimers disease (AD) and vascular dementia. In this article, the literature on this subject is reviewed and novel findings relating CAA to subcortical white matter damage in 224 subjects in the Oxford project to Investigate Memory and Ageing (OPTIMA) are reported. The relationship between CAA and subcortical tissue damage in the OPTIMA subjects was found to be critically dependent on ApoE genotype, there being a positive relationship between measures of CAA and subcortical small vessel disease in ApoEε4 carriers and a significant negative relationship in ApoEε2 carriers. These findings draw attention, as have many other studies, to the importance of ApoE genotype as a major risk factor not only for dementia but also for damage to blood vessels in the aging brain.

Ubiquitinated inclusions in inclusion-body myositis patients are immunoreactive for cathepsin D but not β-amyloid

The nature of the inclusions in the human muscle disease inclusion-body myositis (IBM) has been the subject of debate. Parallels with Alzheimers disease have been drawn after these inclusions were found to be ubiquitinated, and immunoreactive with antibodies to beta-amyloid (A beta) and certain amyloid-associated proteins. We have used a battery of antibodies against A beta and associated proteins to immunostain muscle biopsies from patients with IBM. Although the inclusions are ubiquitinated, we could not show immunoreactivity for A beta or the associated proteins investigated. We did, however, find that the ubiquitinated inclusions colocalised with the lysosomal marker, cathepsin D.

Cerebrospinal Fluid Biomarkers Distinguish Postmortem-Confirmed Alzheimer's Disease from Other Dementias and Healthy Controls in the OPTIMA Cohort

Cerebrospinal fluid (CSF) amyloid-β (Aβ) and tau have been studied as markers of Alzheimers disease (AD). Combined Aβ42 and t-tau distinguishes AD from healthy controls with a sensitivity and specificity (sens/spec) near 89% across studies. This study examined these markers in the homogeneous OPTIMA cohort, using extensive longitudinal follow up and postmortem evaluation to confirm clinicopathological status. Baseline CSF was analyzed from 227 participants with AD (97% autopsy-confirmed), mild cognitive impairment (MCI; 73% confirmed), other dementia syndrome (ODS; 100% confirmed), and controls (CTL; 27% confirmed, follow up approximately 9-13 years). Biomarker concentrations were analyzed using validated ELISAs. AD patients had lower CSF Aβ42 and higher t-tau, p-tau, t-tau/Aβ42, and t-tau/Aβ40 compared to CTLs, with MCI intermediate. CTL and MCI participants who progressed to AD demonstrated more AD-like profiles. Aβ40, sAβPPα, and sAβPPβ were lower in AD compared to CTL. High-level discriminators of AD from CTL were t-tau/Aβ40 (AUROC 0.986, sens/spec of 92%/94%), p-tau/Aβ42 (AUROC 0.972, sens/spec of 94%/90%), and Aβ42 (AUROC 0.941, sens/spec of 88%). For discriminating AD from ODS, p-tau/Aβ42 demonstrated sens/spec of 88%/100% (95%/86% at the AD versus CTL cutoff) and Aβ42 demonstrated sens/spec of 84%/100% (88%/100% at the AD versus CTL cutoff). In a well-characterized, homogeneous population, a single cutoff for baseline CSF Aβ and tau markers can distinguish AD with a high level of sens/spec compared to other studies. It may be important to characterize sources of demographic and biological variability to support the effective use of CSF diagnostic assays in the broader AD population.

High Resolution Discovery Proteomics Reveals Candidate Disease Progression Markers of Alzheimer's Disease in Human Cerebrospinal Fluid.

Disease modifying treatments for Alzheimer’s disease (AD) constitute a major goal in medicine. Current trends suggest that biomarkers reflective of AD neuropathology and modifiable by treatment would provide supportive evidence for disease modification. Nevertheless, a lack of quantitative tools to assess disease modifying treatment effects remains a major hurdle. Cerebrospinal fluid (CSF) biochemical markers such as total tau, p-tau and Ab42 are well established markers of AD; however, global quantitative biochemical changes in CSF in AD disease progression remain largely uncharacterized. Here we applied a high resolution open discovery platform, dMS, to profile a cross-sectional cohort of lumbar CSF from post-mortem diagnosed AD patients versus those from non-AD/non-demented (control) patients. Multiple markers were identified to be statistically significant in the cohort tested. We selected two markers SME-1 (p<0.0001) and SME-2 (p = 0.0004) for evaluation in a second independent longitudinal cohort of human CSF from post-mortem diagnosed AD patients and age-matched and case-matched control patients. In cohort-2, SME-1, identified as neuronal secretory protein VGF, and SME-2, identified as neuronal pentraxin receptor-1 (NPTXR), in AD were 21% (p = 0.039) and 17% (p = 0.026) lower, at baseline, respectively, than in controls. Linear mixed model analysis in the longitudinal cohort estimate a decrease in the levels of VGF and NPTXR at the rate of 10.9% and 6.9% per year in the AD patients, whereas both markers increased in controls. Because these markers are detected by mass spectrometry without the need for antibody reagents, targeted MS based assays provide a clear translation path for evaluating selected AD disease-progression markers with high analytical precision in the clinic.

Butyrylcholinesterase K variant associated with higher enzyme activity in the temporal cortex of elderly patients

There is evidence to suggest an involvement of the K variant of the butyrylcholinesterase gene (BCHE) in dementia. We have examined the relationship between BCHE genotype and butyrylcholinesterase (BuChE) activity in autopsy brain tissue. We studied 164 autopsy cases, 144 with dementia and 20 controls, including 13 K homozygotes and 48 K heterozygotes, from three centres: Newcastle, Oxford and London. Mean BuChE activity in temporal cortex was 37% higher in K homozygotes than in wild-type homozygotes. Linear regression analysis, controlling for gender, diagnosis, age at death and study centre, showed that the number of BCHE-K alleles was associated with increasing BuChE activity (p=0.009).

P3-092: Prediction of short-term and long-term cognitive decline in moderate Alzheimer's disease patients in the Oxford project to investigate memory and aging (optima) cohort using CSF biomarkers

Background: Assessment of changes in the rate of cognitive decline of patients with AD is a primary objective of AD clinical trials. However, the heterogeneity in the presentation of the disease reduces the sensitivity of cognitive endpoints for evaluating drug efficacy. Consequently, the potential for CSF markers to predict the rate of cognitive decline was examined as a strategy for mitigating the effects of such variance. Methods: The expression of amyloid (A 40, A 42, sAPP , sAPP ) and tau (t-tau, p-tau-181) markers was measured in baseline visit CSF collected from patients with clinicallydiagnosed AD (n 15), pathologically-confirmed AD (n 29), and agematched controls (n 37) who were part of the OPTIMA natural history cohort. The relationship between CSF marker expression and decline in CAMCOG and MMSE scores over the subsequent year was explored using linear regression analysis, and the relationship to long-term ( 10 years) decline in CAMCOG scores explored using nonlinear mixed effects modeling. Results: The AD regression modeling suggested that high baseline t-tau (log2 pg/ml) was associated with more rapid decline in CAMCOG scores within the first year from baseline (%R 10, m [95%CI] -4.5 [-8.4, -0.7], p 0.023). The ratio of t-tau/A 42 was also associated with short-term CAMCOG decline (%R 21, m [95%CI] -5.7 [-8.8, -2.6], p 0.00067). Examination of other tau/amyloid ratios, and of MMSE decline, produced similar results. By comparison, common demographic variables, as well as CSF A 42/A 40, were not associated with decline in the first year. The nonlinear modeling suggested that high baseline tau was also associated with more rapid long-term CAMCOG decline. The CAMCOG half-life was reduced by approximately 50% for patients with baseline tau levels at the 97.5% quantile compared to those at the 2.5% quantile. Conclusions: These findings indicate that CSF biomarkers can account for some variance in the analysis of cognitive decline in AD and that tau/amyloid expression ratios are stronger predictors of short-term decline than the individual markers. This could be due to a more complete representation of the disease process (pathogenesis by amyloid and degeneration by tau) by the ratios. The tau marker alone, however, appears to be a strong predictor of both short-term and long-term decline.

P3-094: Three novel CSF assays expand a core panel of CSF biomarkers: BACE activity, oligomeric Aβ and homocysteine assessed in pathologically confirmed Alzheimer subjects within the Oxford project to investigate memory and aging (Optima) cohort

Background: A key goal within the Alzheimer’s disease (AD) research community is the early clinical detection and diagnosis of AD in patients who are at risk, or present with symptoms, of the disease. In addition to the standard clinical criteria, much effort in the field is aimed at identifying biochemical and imaging biomarkers. Methods: In a collaborative effort to discover early biomarkers of AD between OPTIMA and Merck Research Laboratories, we have expanded a panel of six previously validated and commercially available CSF biochemical assays for APP catabolites (A 40, A 42, sAPP , sAPP ) and tau (tTau, pTau-181). Three new assays were developed, optimized and analytically validated for (1) CSF BACE activity, (2) CSF oligomeric A , and (3) CSF Homocysteine (HCY) levels. These biochemical markers were measured in the CSF of pathologically confirmed AD (N 27-30) and clinically evaluated control cases (N 29). Results: Uni variate results exhibited an age-dependent increase in CSF BACE activity ( 1.0 pM/yr, p 0.05). In AD subjects, a modest but significant decline in age-adjusted CSF BACE activity was observed compared to controls (50% reduction, p 0.02). Regarding oligomeric A , in a commercially purchased sample set of human CSF, we found AD patients have 82 % higher oligomeric A levels than their age-matched non-demented controls (p 0.01). When oligomeric A was assessed in the post-mortem confirmed OPTIMA samples, there did not appear to be a significant difference between AD and CTL. One can’t be sure if there is an assay sensitivity issue or the demographics of the sample sets were different. Finally regarding CSF Homocysteine, we observed meaningful age dependence in CSF Homocysteine levels (slope 0.016 per year, p 0.0003). Differences between AD and CTL after age-adjustment are significant in females (11.2% greater in AD v CTL females, p 0.049). Conclusions: Taken together, these findings obtained from a post-mortem confirmed case analysis, enhance the putative value and use of specific CSF markers in support of the detection and diagnosis of Alzheimer’s disease. P3-095 LEVELS OF CSF BIOMARKERS PREDICT RATE OF COGNITIVE DECLINE IN INDIVIDUALS WITH VERY MILD DEMENTIA OF THE ALZHEIMER’S TYPE