Shabnam Baig

Abeta-degrading enzymes in Alzheimer's disease.

In Alzheimers disease (AD) Aβ accumulates because of imbalance between the production of Aβ and its removal from the brain. There is increasing evidence that in most sporadic forms of AD, the accumulation of Aβ is partly, if not in some cases solely, because of defects in its removal—mediated through a combination of diffusion along perivascular extracellular matrix, transport across vessel walls into the blood stream and enzymatic degradation. Multiple enzymes within the central nervous system (CNS) are capable of degrading Aβ. Most are produced by neurons or glia, but some are expressed in the cerebral vasculature, where reduced Aβ‐degrading activity may contribute to the development of cerebral amyloid angiopathy (CAA). Neprilysin and insulin‐degrading enzyme (IDE), which have been most extensively studied, are expressed both neuronally and within the vasculature. The levels of both of these enzymes are reduced in AD although the correlation with enzyme activity is still not entirely clear. Other enzymes shown capable of degrading Aβin vitro or in animal studies include plasmin; endothelin‐converting enzymes ECE‐1 and ‐2; matrix metalloproteinases MMP‐2, ‐3 and ‐9; and angiotensin‐converting enzyme (ACE). The levels of plasmin and plasminogen activators (uPA and tPA) and ECE‐2 are reported to be reduced in AD. Reductions in neprilysin, IDE and plasmin in AD have been associated with possession of APOEε4. We found no change in the level or activity of MMP‐2, ‐3 or ‐9 in AD. The level and activity of ACE are increased, the level being directly related to Aβ plaque load. Up‐regulation of some Aβ‐degrading enzymes may initially compensate for declining activity of others, but as age, genetic factors and diseases such as hypertension and diabetes diminish the effectiveness of other Aβ‐clearance pathways, reductions in the activity of particular Aβ‐degrading enzymes may become critical, leading to the development of AD and CAA.

Distribution and expression of picalm in Alzheimer disease.

PICALM, the gene encoding phosphatidylinositol-binding clathrin assembly (picalm) protein, was recently shown to be associated with risk of Alzheimer disease (AD). Picalm is a key component of clathrin-mediated endocytosis. It recruits clathrin and adaptor protein 2 (AP-2) to the plasma membrane and, along with, AP-2 recognizes target proteins. The attached clathrin triskelions cause membrane deformation around the target proteins enclosing them within clathrin-coated vesicles to be processed in lysosomes or endosomes. We examined the distribution of picalm in control and AD brain tissue and measured levels of picalm messenger RNA (mRNA) by real-time polymerase chain reaction. Immunolabeling of brain tissue showed that picalm is predominately present in endothelial cells. This was further supported by the demonstration of picalm in human cerebral microvascular cells grown in culture. Picalm mRNA was elevated in relation to glyceraldehyde-3-phosphate dehydrogenase but not factor VIII-related antigen or CD31 mRNA in the frontal cortex in AD. No change was seen in the temporal cortex or thalamus. The transport of A&bgr; across vessel walls and into the bloodstream is a major pathway of A&bgr; removal from the brain and picalm is ideally situated within endothelial cells to participate in this process. Further research is needed to determine whether PICALM expression is influenced by A&bgr; levels and whether it affects A&bgr; uptake and transport by endothelial cells.

Neprilysin and insulin-degrading enzyme levels are increased in Alzheimer disease in relation to disease severity

Experimental reduction of neprilysin (NEP) or insulin-degrading enzyme (IDE) in vivo exacerbates &bgr;-amyloid accumulation in the brain. The level of these enzymes is reportedly reduced during aging and in postmortem brains of patients with sporadic Alzheimer disease (AD). To distinguish between primary decreases in NEP and IDE activity that might contribute to &bgr;-amyloid accumulation and decreases secondary to neurodegenerative changes in AD, we measured NEP and IDE levels by indirect sandwich ELISA and enzyme activities by immunocapture-based fluorogenic assays in postmortem frontal cortex from patients of different ages and at different pathological stages of AD, as indicated by Braak tangle stage. The ELISA measurements of neuron-specific enolase were used to adjust for neuronal loss. Both unadjusted and neuron-specific enolase-adjusted NEP levels and activity were significantly increased in AD and positively correlated with Braak stage but negatively with age in AD patients. Insulin-degrading enzyme activity was higher in AD than controls; this was significant after adjustment for neuron-specific enolase level; unadjusted IDE protein level was decreased in AD but not after adjustment. Our findings suggest that reduction in NEP and IDE activity is not the primary cause of &bgr;-amyloid accumulation in AD, but rather a late-stage phenomenon secondary to neurodegeneration.

Loss of perineuronal net N-acetylgalactosamine in Alzheimer’s disease

The perineuronal net (PN), a specialised region of extracellular matrix, is interposed between the neuronal cell surface and astrocytic processes. It is involved in the buffering of ions, in the development, stabilisation and remodelling of synapses and in the regulating the neuronal microenvironment particularly around the parvalbumin-positive GABAergic neurons. We have investigated the relative preservation of Wisteria floribunda agglutinin (WFA)-positive PNs and parvalbumin-positive neurons in Alzheimer’s disease (AD), and the relationship of WFA-positive PNs to parenchymal tau, amyloid β-peptide (Aβ) and MHC class II antigen (a marker of activated microglia), in paraffin sections of 100 cases with AD and 45 controls. The density of PNs that could be labelled with WFA, which binds to the N-acetylgalactosamine (GalNAc) residues of chondroitin sulphate proteoglycans, was reduced by about 2/3 in AD (P<0.001). In contrast, the density of parvalbumin-positive neurons did not differ significantly between AD and controls. Combined fluorescence imaging showed granular disintegration of WFA labelling around some parvalbumin-positive neurons. There was no significant difference in the amount of phosphorylated tau, Aβ or MHC class II antigen in areas with and without WFA-positive PNs. In AD, there is marked loss of PN GalNAc that is not topographically related to neurofibrillary pathology, parenchymal Aβ load or activated microglia. Although the parvalbumin-positive neurons themselves are relatively spared, the loss of PN GalNAc, which maintains a polyanionic microenvironment around neurons, is likely to impair the function of these inhibitory interneurons. This could in turn lead to increased activity of the glutamatergic and other neurons onto which they synapse.

MMP‐2, ‐3 and ‐9 levels and activity are not related to Aβ load in the frontal cortex in Alzheimer's disease

Matrix metalloproteinases (MMPs) ‐2, ‐3 and ‐9 are up‐regulated in several cell types on exposure to amyloid β peptide (Aβ) and have Aβ‐degrading activity in vitro. The aims of this study were to determine (i) the distribution of MMP‐2, ‐3 and ‐9 in the cerebral cortex in Alzheimers disease (AD) and control brains; (ii) whether the levels and activity of these proteases are increased in AD; and (iii) whether their activity is related to Aβ load. In addition, we examined whether promoter polymorphisms in the MMP‐3 and ‐9 genes are associated with AD in the study cohort. Paraffin sections of frontal lobe from AD and control cases were immunostained for MMP‐2, ‐3 and ‐9 and tissue homogenates used for MMP activity assays. DNA from these cases was genotyped for the MMP‐3 5A/6A (‐1171) and MMP‐9 C‐1562T promoter polymorphisms. Immunohistochemistry revealed MMP‐3 in plaques and both MMP‐3 and ‐9 around scattered neurones. The levels and activity of all three MMPs were similar in AD and control brains and bore no relationship to Aβ load. Analysis of MMP‐3 ‐1171 5A/6A allele frequencies showed that the 6A allele (with reduced promoter activity) was associated with AD; the MMP‐9 C‐1562T polymorphism was not. The levels and activities of MMP‐2, ‐3 and ‐9 are not increased in the frontal cortex in AD and are not related to Aβ load. Our findings suggest that altered expression of these proteases does not make a significant contribution to the accumulation of Aβ in AD.

Tau hyperphosphorylation affects Smad 2/3 translocation

Transforming growth factors beta (TGFbeta) regulate multiple biological activities. TGFbeta activation of the Smad pathway results in activation of genes encoding extracellular matrix molecules, proteases, protease activators and protease inhibitors. In Alzheimers disease (AD), TGFbeta protein and mRNA levels are raised, which would be expected to be neuroprotective. However, recent observations suggest that TGFbeta-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 A beta(1-42), a more physiologically relevant model of AD. Our findings suggest that despite the increased level of TGFbeta in AD, the TGFbeta-Smad signalling pathway is impeded within neurones due to sequestration of pSmad 2/3 by hyperphosphorylated tau. This may compromise neuroprotective actions of TGFbeta and contribute to neurodegeneration in AD.

Clusterin mRNA and protein in Alzheimer's disease

Clusterin, a multifunctional lipoprotein is expressed in a number of tissues but expression is particularly high in the brain, where it binds to amyloid-β (Aβ), possibly facilitating Aβ transport into the bloodstream. Its concentration in peripheral blood was identified as a potential biomarker for Alzheimers disease (AD) and predicted retention of (11)C-Pittsburgh Compound B in the temporal lobe. Single-nucleotide polymorphisms in the clusterin gene, CLU, are associated with the risk of developing AD. We measured clusterin mRNA levels in control and AD brains and investigated the relationship of the clusterin protein to soluble, insoluble, and plaque-associated Aβ. Clusterin mRNA levels were unchanged when normalized to GAPDH but modestly increased in the frontal and temporal cortex in AD in relation to NSE and MAP-2. Levels of NSE and MAP-2 mRNA were reduced in the AD frontal cortex. Clusterin protein concentration was unchanged and did not correlate with the amount of Aβ present. In the frontal cortex, clusterin concentration was higher in APOE ε4-negative brains but no effect of APOE was detected in the temporal cortex or thalamus. Overall clusterin mRNA and protein levels are unaltered in the neocortex in AD and clusterin concentration does not reflect Aβ content. The increase in clusterin noted in peripheral blood in AD may reflect increased passage of this chaperone protein across the blood-brain barrier but further work is needed to determine how CLU variants influence the development of AD.

SYMPOSIUM: Clearance of Aβ from the Brain in Alzheimer's Disease: Aβ‐Degrading Enzymes in Alzheimer's Disease

In Alzheimers disease (AD) Aβ accumulates because of imbalance between the production of Aβ and its removal from the brain. There is increasing evidence that in most sporadic forms of AD, the accumulation of Aβ is partly, if not in some cases solely, because of defects in its removal—mediated through a combination of diffusion along perivascular extracellular matrix, transport across vessel walls into the blood stream and enzymatic degradation. Multiple enzymes within the central nervous system (CNS) are capable of degrading Aβ. Most are produced by neurons or glia, but some are expressed in the cerebral vasculature, where reduced Aβ‐degrading activity may contribute to the development of cerebral amyloid angiopathy (CAA). Neprilysin and insulin‐degrading enzyme (IDE), which have been most extensively studied, are expressed both neuronally and within the vasculature. The levels of both of these enzymes are reduced in AD although the correlation with enzyme activity is still not entirely clear. Other enzymes shown capable of degrading Aβin vitro or in animal studies include plasmin; endothelin‐converting enzymes ECE‐1 and ‐2; matrix metalloproteinases MMP‐2, ‐3 and ‐9; and angiotensin‐converting enzyme (ACE). The levels of plasmin and plasminogen activators (uPA and tPA) and ECE‐2 are reported to be reduced in AD. Reductions in neprilysin, IDE and plasmin in AD have been associated with possession of APOEε4. We found no change in the level or activity of MMP‐2, ‐3 or ‐9 in AD. The level and activity of ACE are increased, the level being directly related to Aβ plaque load. Up‐regulation of some Aβ‐degrading enzymes may initially compensate for declining activity of others, but as age, genetic factors and diseases such as hypertension and diabetes diminish the effectiveness of other Aβ‐clearance pathways, reductions in the activity of particular Aβ‐degrading enzymes may become critical, leading to the development of AD and CAA.

SYMPOSIUM: Clearance of Aβ from the Brain in Alzheimer's Disease: Aβ-Degrading Enzymes in Alzheimer's Disease: Aβ-Degrading Enzymes in Alzheimer's Disease

In Alzheimers disease (AD) Aβ accumulates because of imbalance between the production of Aβ and its removal from the brain. There is increasing evidence that in most sporadic forms of AD, the accumulation of Aβ is partly, if not in some cases solely, because of defects in its removal—mediated through a combination of diffusion along perivascular extracellular matrix, transport across vessel walls into the blood stream and enzymatic degradation. Multiple enzymes within the central nervous system (CNS) are capable of degrading Aβ. Most are produced by neurons or glia, but some are expressed in the cerebral vasculature, where reduced Aβ‐degrading activity may contribute to the development of cerebral amyloid angiopathy (CAA). Neprilysin and insulin‐degrading enzyme (IDE), which have been most extensively studied, are expressed both neuronally and within the vasculature. The levels of both of these enzymes are reduced in AD although the correlation with enzyme activity is still not entirely clear. Other enzymes shown capable of degrading Aβin vitro or in animal studies include plasmin; endothelin‐converting enzymes ECE‐1 and ‐2; matrix metalloproteinases MMP‐2, ‐3 and ‐9; and angiotensin‐converting enzyme (ACE). The levels of plasmin and plasminogen activators (uPA and tPA) and ECE‐2 are reported to be reduced in AD. Reductions in neprilysin, IDE and plasmin in AD have been associated with possession of APOEε4. We found no change in the level or activity of MMP‐2, ‐3 or ‐9 in AD. The level and activity of ACE are increased, the level being directly related to Aβ plaque load. Up‐regulation of some Aβ‐degrading enzymes may initially compensate for declining activity of others, but as age, genetic factors and diseases such as hypertension and diabetes diminish the effectiveness of other Aβ‐clearance pathways, reductions in the activity of particular Aβ‐degrading enzymes may become critical, leading to the development of AD and CAA.