Genetic variation in MME in relation to neprilysin protein and enzyme activity, Aß levels, and Alzheimer's disease risk.

Neprilysin (NEP), also known as membrane metalloendopeptidase (MME), is considered amongst the most important ß-amyloid (Aß)-degrading enzymes with regard to prevention of Alzheimer's disease (AD) pathology. Variation in the NEP gene (MME) has been suggested as a risk factor for AD. We conducted a genetic association study of 7MME SNPs - rs1836914, rs989692, rs9827586, rs6797911, rs61760379, rs3736187, rs701109 - with respect to AD risk in a cohort of 1057 probable and confirmed AD cases and 424 age-matched non-demented controls from the United Kingdom, Italy and Sweden. We also examined the association of these MME SNPs with NEP protein level and enzyme activity, and on biochemical measures of Aß accumulation in frontal cortex - levels of total soluble Aß, oligomeric Aß(1-42), and guanidine-extractable (insoluble) Aß - in a sub-group of AD and control cases with post-mortem brain tissue. On multivariate logistic regression analysis one of the MME variants (rs6797911) was associated with AD risk (P = 0.00052, Odds Ratio (O.R. = 1.40, 95% confidence interval (1.16-1.70)). None of the SNPs had any association with Aß levels; however, rs9827586 was significantly associated with NEP protein level (p=0.014) and enzyme activity (p=0.006). Association was also found between rs701109 and NEP protein level (p=0.026) and a marginally non-significant association was found for rs989692 (p=0.055). These data suggest that MME variation may be associated with AD risk but we have not found evidence that this is mediated through modification of NEP protein level or activity.

ACE variants and association with brain Aß levels in Alzheimer's disease.

ACE is a candidate gene for Alzheimer's disease (AD) and associations have been reported between ACE variants and plasma ACE levels, AD risk, AD age at onset of disease and cerebrospinal fluid levels of Aß. Despite evidence that ACE can degrade Aß, the relationships between ACE variants and the levels of different types of Aß in the brain are not known. We have investigated the relationship between AD-associated ACE variants, for which the associations with brain activity of ACE were previously analysed, and brain homogenate levels of soluble, insoluble and oligomeric Aß. Reported AD risk variants in the ACE indel (rs1799752) and its 'proxy' rs4343 were significantly associated with soluble Aß level in AD only (p=0.001), as was rs1800764 but less so (p=0.014). In contrast, insoluble Aß was associated with ACE indel and rs4343 variants in controls only (p < 0.01). No associations were found for oligomeric Aß. These data indicate a complex relationship between ACE and Aß that differs between AD and control brains.

Higher soluble amyloid beta concentration in frontal cortex of young adults than in normal elderly or Alzheimer's disease.

Little is known about the relationship between soluble amyloid beta (Abeta) and age. We have measured soluble and insoluble Abeta by enzyme-linked immunosorbent assay (ELISA) in post-mortem frontal cortex in normal brains (16-95 years) and AD. Insoluble Abeta increased with age, and was significantly higher in Alzheimer's disease (AD) than age-matched controls. However, levels of soluble Abeta declined with age and were significantly greater in younger adults than older adults with or without AD. In AD, insoluble : soluble Abeta ratio was much higher than in age-matched controls. The high levels of soluble Abeta in young adults included oligomeric species of Abeta(1-42). These observations do not preclude Abeta oligomers as neurotoxic mediators of AD but suggest that if they are, the toxicity may be restricted to certain species (eg, beta-pleated protofibrillar species not detected by our assay) or takes decades to manifest. The dramatically increased insoluble : soluble Abeta in AD points to an altered dynamic equilibrium of Abeta in AD, reflecting both enhanced aggregation and continued overproduction or impaired removal of the soluble peptide in older age, when the concentration of this peptide should be declining.

Changes with age in the activities of beta-secretase and the Abeta-degrading enzymes neprilysin, insulin-degrading enzyme and angiotensin-converting enzyme.

We recently found that insoluble Abeta increases, but soluble Abeta decreases with age in normal brains. We now report the changes in activities of beta-secretase (BACE-1) and Abeta-degrading enzymes with age, and their relationships to concentrations of soluble and insoluble Abeta. We measured BACE-1 activity and the levels and activities of neprilysin (NEP), insulin-degrading enzyme (IDE) and angiotensin-converting enzyme (ACE) in normal control brains (16 years-95 years). We also compared the measurements to those in AD. BACE-1 activity correlated closely with age in controls and was significantly higher in AD. In controls, NEP and IDE activities (but not protein levels) increased with age but ACE activity and level did not. BACE-1 activity correlated directly with insoluble but inversely with soluble Abeta. IDE activity correlated directly with insoluble Abeta and NEP activity was inversely related to soluble Abeta. ACE level correlated directly with insoluble and inversely with soluble Abeta in controls but not AD. Both Abeta-synthesizing and -degrading enzyme activities increase with age, coinciding with declining soluble Abeta and increasing insoluble Abeta. Further research is needed to establish whether these changes in enzyme activity and Abeta levels are causally related and if so how.

Oligomeric Abeta in Alzheimer's disease: relationship to plaque and tangle pathology, APOE genotype and cerebral amyloid angiopathy.

Despite accumulating evidence of a central role for oligomeric amyloid beta (Abeta) in the pathogenesis of Alzheimer's Disease (AD), there is scant information on the relationship between the levels and distribution of oligomeric Abeta and those of other neurodegenerative abnormalities in AD. In the present study, we have found oligomeric Abeta to be associated with both diffuse and neuritic plaques (mostly co-localized with Abeta(1-42)) and with cerebrovascular deposits of Abeta in paraffin sections of formalin-fixed human brain tissue. The amount of oligomeric Abeta that was labeled in the sections correlated with total Abeta plaque load, but not phospho-tau load, cerebral amyloid angiopathy (CAA) severity or APOE genotype. Although soluble, oligomeric and insoluble Abeta levels were all significantly increased in AD brain homogenates, case-to-case variation and overlap between AD and controls were considerable. Over the age-range studied (43-98 years), the levels of soluble Abeta, oligomeric Abeta(42), oligomeric Abeta(40) and insoluble Abeta did not vary significantly with age. Oligomeric Abeta(1-42) and insoluble Abeta levels were significantly higher in women. Overall, the level of insoluble Abeta, but neither oligomeric nor soluble Abeta, was associated with Braak stage, CAA severity and APOEepsilon4 frequency, raising questions as to the role of soluble and oligomeric Abeta in the progression of AD.

Characterisation of two antibodies to oligomeric Abeta and their use in ELISAs on human brain tissue homogenates.

Oligomeric forms of Abeta are believed to be the major toxic species of this peptide in Alzheimer's disease (AD). Although the characterisation of oligomer-specific antibodies has been reported, these have not been successfully incorporated into an enzyme-linked immunosorbent assay (ELISA), and measurement of the levels of oligomeric Abeta in brain tissue has remained problematic. We have examined the specificity of two monoclonal antibodies, 7 A1a and 1G5, for synthetic oligomers of Abeta(1-42) and for oligomeric Abeta(1-42) in human brain homogenates, and the utility of these two antibodies for measuring oligomeric Abeta(1-42) by sandwich ELISA. Both antibodies were found to recognise a range of synthetic oligomers of Abeta(1-42) but to cross-react in Western blots with a 34 kDa protein, shown by two-dimensional gel electrophoresis and mass spectrometry to be tropomyosin. However, by using 7A1a and 1 G5 in combination with an Abeta(1-42) capture antibody, we were able specifically to detect and to measure the levels of oligomeric Abeta(1-42) in brain homogenates by ELISA. The development of a simple ELISA for measurement of oligomeric Abeta should facilitate further studies of the role of oligomeric species of Abeta in AD.

Decreased expression and activity of neprilysin in Alzheimer disease are associated with cerebral amyloid angiopathy.

Neprilysin (NEP) degrades amyloid-beta (Abeta) and is thought to contribute to its clearance from the brain. In Alzheimer disease (AD), downregulation of NEP has been suggested to contribute to the development of cerebral amyloid angiopathy (CAA). We examined the relationship among NEP, CAA, and APOE status in AD and elderly control cases. NEP was most abundant in the tunica media of cerebrocortical blood vessels and in pyramidal neurons. In homogenates of the frontal cortex, NEP protein levels were reduced in AD but not significantly; NEP enzymatic activity was significantly reduced in AD. Immunohistochemistry revealed a reduction of both vascular and parenchymal NEP. The loss of vessel-associated NEP in AD was inversely related to the severity of CAA, and analysis of cases with severe CAA showed that levels of vascular NEP were reduced to the same extent in Abeta-free and Abeta-laden vessels, strongly suggesting that the reduction in NEP is not simply secondary to CAA. Possession of APOE epsilon4 was associated with significantly lower levels of both parenchymal and vascular NEP. Colinearity of epsilon4 with the presence of moderate to severe CAA precluded assessment of the independence of this association from NEP levels. However, logistic regression analysis showed low NEP levels to be a significant independent predictor of moderate to severe CAA.