Aims: Several observations point to the involvement of angiotensin‐converting enzyme‐1 (ACE‐1) in Alzheimer's disease (AD): ACE‐1 cleaves amyloid‐β peptide (Aβ) in vitro, the level and activity of ...ACE‐1 are reportedly increased in AD, and variations in the ACE‐1 gene are associated with AD. We analysed ACE‐1 activity and expression in AD and control brains, particularly in relation to Aβ load and cerebral amyloid angiopathy (CAA). Methods: ACE‐1 activity was measured in the frontal cortex from 58 control and 114 AD cases of known Aβ load and CAA severity. The distribution of ACE‐1 was examined immunohistochemically. In five AD cases with absent or mild CAA, five with moderate to severe CAA and five controls with absent or mild CAA, levels of vascular ACE‐1 were assessed by quantitative immunofluorescence. Results: ACE‐1 activity was increased in AD (P < 0.001) and correlated directly with parenchymal Aβ load (P = 0.05). Immunohistochemistry revealed ACE‐1 in neurones and cortical blood vessels – in the intima but most abundant perivascularly. Cases with moderate to severe CAA had significantly more vessel‐associated ACE‐1 than did those with little or no CAA. Perivascular ACE‐1 did not colocalize with Aβ, smooth muscle actin, glial fibrillary acidic protein, collagen IV, vimentin or laminin, but was similarly distributed to extracellular matrix (ECM) proteins fibronectin and decorin. Conclusions: Our findings indicate that ACE‐1 activity is increased in AD, in direct relationship to parenchymal Aβ load. Increased ACE‐1, probably of neuronal origin, accumulates perivascularly in severe CAA and colocalizes with vascular ECM. The possible relationship of ACE‐1 to the deposition of perivascular ECM remains to be determined.
Abstract Transforming growth factors β (TGFβ) regulate multiple biological activities. TGFβ activation of the Smad pathway results in activation of genes encoding extracellular matrix molecules, ...proteases, protease activators and protease inhibitors. In Alzheimer's disease (AD), TGFβ protein and mRNA levels are raised, which would be expected to be neuroprotective. However, recent observations suggest that TGFβ-Smad signalling is disrupted by the hyperphosphorylation of tau, the primary component of neurofibrillary tangles: phosphorylated Smad2/3 (pSmad 2/3) co-localises with phosphorylated tau in the neuronal cytoplasm and levels are reduced in the nucleus. We have investigated whether in vitro induction of tau hyperphosphorylation influences pSmad 2/3 localisation in rat primary cortical cells. Treatment with okadaic acid, a protein phosphatase 1 and 2A inhibitor caused hyperphosphorylation of tau at epitopes hyperphosphorylated in AD and disrupted pSmad 2/3 translocation into the nucleus. The disruptive effect of tau phosphorylation on pSmad 2/3 translocation was confirmed by treatment of primary cortical cells with synthetic oligomeric Aβ1-42 , a more physiologically relevant model of AD. Our findings suggest that despite the increased level of TGFβ in AD, the TGFβ-Smad signalling pathway is impeded within neurones due to sequestration of pSmad 2/3 by hyperphosphorylated tau. This may compromise neuroprotective actions of TGFβ and contribute to neurodegeneration in AD.
Cerebral amyloid angiopathy (CAA) affects over 90% of patients with Alzheimer's disease (AD) and increases the risk of cerebral haemorrhage and infarction. Caveolae – cholesterol‐enriched ...plasmalemmal microinvaginations – are implicated in the production of amyloid β peptide (Aβ). Caveolin‐1 (CAV‐1) is essential for the formation of caveolae. Caveolin‐2 (CAV‐2) is expressed at the plasma membrane only when in a stable hetero‐oligomeric complex with CAV‐1. CAV‐1 and CAV‐2 are highly co‐expressed by endothelium and smooth muscle. Recent studies suggest that down‐regulation of CAV‐1 causes a reduction in α‐secretase activity and consequent accumulation of Aβ. We have used quantitative immunohistochemical techniques to assess the relationship between CAV‐1 and CAV‐2 with respect to Aβ accumulation in the cerebral vasculature in a series of post mortem brains. CAV‐1 and CAV‐2 were co‐expressed within the tunica media and endothelium of cerebral blood vessels. There were regional differences in CAV‐1 immunolabelling, which was significantly greater in the frontal cortex and white matter than in the parietal lobe (in both control and AD cases) or the temporal lobe (in AD alone). However, CAV‐1 labelling in AD did not differ from that in controls in any of the three lobes examined. Assessment of CAV‐1 labelling in relation to the severity of CAA showed CAV‐1 to be significantly increased in the frontal white matter in a subgroup of AD cases with absent/mild CAA compared with controls with absent/mild CAA and to AD cases with moderate/severe CAA, but the latter groups did not show significant differences from one another. CAV‐1 labelling did not vary with age, gender, APOE genotype, post mortem delay or brain weight. Only segments of blood vessels with particularly abundant Aβ and extensive loss of smooth muscle actin showed loss of CAV‐1 and CAV‐2 from the tunica media. Within these vessels endothelial CAV‐1 was preserved and discontinuous CAV‐2 labelling was noted along the outer aspect of the vessel wall. Our findings suggest that alterations in the expression of vascular CAV‐1 and CAV‐2 are unlikely to play a role in the development of CAA in AD.
Neprilysin (NEP) degrades amyloid-β (Aβ) 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 Aβ-free and Aβ-laden vessels, strongly suggesting that the reduction in NEP is not simply secondary to CAA. Possession of APOE ε4 was associated with significantly lower levels of both parenchymal and vascular NEP. Colinearity of ε4 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.
Includes bibliographical references (leaves. 23-28).
The frequency and geographical range of hannful algal blooms (HABs) are believed to be on the increase, with adverse affects on marine and human ...health making the implementation of stringent controls governmg monitoring programmes commonplace. The South African monitoring programme was established in 1989 and relies upon microscopic identification of HAB species. Microscopic identification is labour-intensive, requiring a high level of taxonomic expertise, and could be considered impractical for routine monitoring where analysis of large numbers of samples is required. Novel monitoring techniques, focusing mainly on probe technology, are being developed for rapid, unequivocal identification and enumeration of HAB species. In this study, a triplex peR assay, incorporating a genus-specific ribosomal DNA primer designed from phylogenetic studies on local Alexandrium populations, was optimised for application to environmental samples and tested against natural assemblages containing Alexandrium minutum. Specific positive results were consistently generated for samples containing A. minutum. Samples absent of A. minuturn cells did not generate the Alexandrium-specific amplicon. The absolute detection limit of 440 A. minutum cells r1 for this assay was established. Effects of non-target cells on the sensitivity of the assay were also investigated: although a decrease in sensitivity was found, A. minutum cells could still be detected in the presence of 100 times more non-target cells. This assay has been shown to be a useful tool for unequivocal identification of A. minutum cells within local environmental samples.
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