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.

Cerebrospinal fluid biomarkers distinguish postmortem-confirmed Alzheimer's disease from other dementias and healthy controls in the OPTIMA cohort.

Cerebrospinal fluid (CSF) amyloid-ß (Aß) and tau have been studied as markers of Alzheimer's disease (AD). Combined Aß42 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 Aß42 and higher t-tau, p-tau, t-tau/Aß42, and t-tau/Aß40 compared to CTLs, with MCI intermediate. CTL and MCI participants who progressed to AD demonstrated more AD-like profiles. Aß40, sAßPPα, and sAßPPß were lower in AD compared to CTL. High-level discriminators of AD from CTL were t-tau/Aß40 (AUROC 0.986, sens/spec of 92%/94%), p-tau/Aß42 (AUROC 0.972, sens/spec of 94%/90%), and Aß42 (AUROC 0.941, sens/spec of 88%). For discriminating AD from ODS, p-tau/Aß42 demonstrated sens/spec of 88%/100% (95%/86% at the AD versus CTL cutoff) and Aß42 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 Aß 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.

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, Alzheimer's 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.

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

Alzheimer's 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.

Cerebral subcortical small vessel disease in subjects with pathologically confirmed Alzheimer disease: a clinicopathologic study in the Oxford Project to Investigate Memory and Ageing (OPTIMA).

The understanding of how cerebrovascular disease (CVD) contributes to dementia is hampered by a lack of agreed and validated pathologic methods to accord weight to the contribution of different aspects of CVD to dementia. A previous study from the Oxford Project to Investigate Memory and Ageing (OPTIMA) validated a scheme for assessing the contribution of subcortical small vessel disease (SVD) toward dementia in the elderly by showing a significant inverse relationship between the severity of SVD and cognition in subjects without any other dementia pathology using this method. In the present paper, the method has been used to assess severity of SVD in 161 cases of neuropathologically confirmed Alzheimer disease. The results showed there was no relationship between the SVD score and cognitive scores acquired in the last 2 years of life. SVD scores were significantly related to age (P<0.0017) and were slightly but significantly higher in females than males (P<0.049). SVD scores were not related to blood pressure at entry to OPTIMA and were significantly lower when compared with the cohort of OPTIMA cases with only CVD (mean 5.06 ± 1.85 vs. 5.9 ± 2.67; P<0.0065). We conclude that when Alzheimer disease pathology is present in elderly subjects, it overwhelms the modest contribution that SVD makes to cognitive impairment.

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).

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.

Haplotype-specific expression of exon 10 at the human MAPT locus.

Neurofibrillary tangles composed of exon 10+ microtubule associated protein tau (MAPT) deposits are the characteristic feature of the neurodegenerative diseases progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). PSP, CBD and more recently Alzheimer's disease and Parkinson's disease, are associated with the MAPT H1 haplotype, but the relationship between genotype and disease remains unclear. Here, we investigate the hypothesis that H1 expresses more exon 10+ MAPT mRNA compared to the other haplotype, H2, leading to a greater susceptibility to neurodegeneration in H1 carriers. We performed allele-specific gene expression on two H1/H2 heterozygous human neuronal cell lines, and 14 H1/H2 heterozygous control individual post-mortem brain tissue from two brain regions. In both tissue culture and post-mortem brain tissue, we show that the MAPT H1 haplotype expresses significantly more exon 10+ MAPT mRNA than H2. In post-mortem brain tissue, we show that the total level of MAPT expression from H1 and H2 is not significantly different, but that the H1 chromosome expresses up to 1.43-fold more exon 10+ MAPT mRNA than H2 in the globus pallidus, a brain region highly affected by tauopathy (maximum exon 10+ MAPT H1:H2 transcript ratio=1.425, SD=0.205, P<0.0001), and up to 1.29-fold more exon 10+ MAPT mRNA than H2 in the frontal cortex (maximum exon 10+ MAPT H1:H2 transcript ratio=1.291, SD=0.315, P=0.006). These data may explain the increased susceptibility of H1 carriers to neurodegeneration and suggest a potential mechanism between MAPT genetic variability and the pathogenesis of neurodegenerative disease.