Memantine, a Noncompetitive N-Methyl-D-Aspartate Receptor Antagonist, Attenuates Cerebral Amyloid Angiopathy by Increasing Insulin-Degrading Enzyme Expression.

Sporadic cerebral amyloid angiopathy (CAA) is characterized by cerebrovascular amyloid beta (Aß) deposits and causes cerebral hemorrhages and dementia in elderly people. Memantine is used in Alzheimer's disease to inhibit the glutamatergic system by blocking N-methyl-D-aspartate receptors. Its therapeutic effects in CAA are unclear, however. Here, we used APP23 transgenic mice (CAA model) to investigate whether memantine has direct therapeutic effects on cerebrovascular Aß deposits. We treated APP23 mice and age-matched wild-type littermates with memantine at ages 6-18 months. We counted the numbers of vessels with Aß and hemosiderin deposits. We measured soluble and insoluble Aß40 and Aß42 levels and levels of amyloid precursor protein (APP), APP-processing enzymes (α-, ß-, γ-secretase), and Aß-degrading enzymes (insulin-degrading enzyme [IDE], neprilysin). Memantine reduced cerebrovascular Aß and hemosiderin deposits in APP23 mice. Compared with controls, memantine-treated APP23 mice had reduced Aß40 levels and increased levels of hippocampal and vascular IDE. Our results suggest that memantine reduces cerebrovascular Aß deposits by enhancing Aß-cleaving IDE expression. The clinical availability of memantine may allow its use as a novel therapeutic agent in CAA.

Occludin deficiency with BACE1 elevation in cerebral amyloid angiopathy.

OBJECTIVE: A significant cause of spontaneous hemorrhages in the elderly is cerebral amyloid angiopathy (CAA), which causes degeneration of cerebral vessels, but the mechanisms are unclear. METHODS: We isolated leptomeningeal vessels from rapidly autopsied brains (the average of postmortem intervals was 3.28 hours) from 9 patients with CAA and 10 age-matched controls, and used molecular, cell biology, and immunohistochemical approaches to examine ß-site APP-cleaving enzyme 1 (BACE1) protein expression and enzymatic activities as well as tight junction molecular components in small- and medium-sized arteries of the cerebral cortex and leptomeninges. RESULTS: We not only identified that the cerebral vessels, including leptomeningeal and cortical vessels, synthesize and express BACE1, but also found a significant elevation of both BACE1 protein levels and enzymatic activities in leptomeningeal vessels from patients with CAA. Moreover, overexpression of BACE1 in endothelial cells resulted in a significant reduction of occludin, a tight junction protein in blood vessels. CONCLUSION: These findings suggest that in addition to neurons, cerebral vascular cells express functional BACE1. Moreover, elevated vascular BACE1 may contribute to deficiency of occludin in cerebral vessels, which ultimately has a critical role in pathogenesis of CAA and its related hemorrhage.

Wild-type amyloid beta 1-40 peptide induces vascular smooth muscle cell death independently from matrix metalloprotease activity.

Cerebral amyloid angiopathy (CAA) is an important cause of intracerebral hemorrhages in the elderly, characterized by amyloid-ß (Aß) peptide accumulating in central nervous system blood vessels. Within the vessel walls, Aß-peptide deposits [composed mainly of wild-type (WT) Aß(1-40) peptide in sporadic forms] induce impaired adhesion of vascular smooth muscle cells (VSMCs) to the extracellular matrix (ECM) associated with their degeneration. This process often results in a loss of blood vessel wall integrity and ultimately translates into cerebral ischemia and microhemorrhages, both clinical features of CAA. In this study, we decipher the molecular mechanism of matrix metalloprotease (MMP)-2 activation in WT-Aß(1-40) -treated VSMC and provide evidence that MMP activity, although playing a critical role in cell detachment disrupting ECM components, is not involved in the WT-Aß(1-40) -induced degeneration of VSMCs. Indeed, whereas this peptide clearly induced VSMC apoptosis, neither preventing MMP-2 activity nor hampering the expression of membrane type1-MMP, or preventing tissue inhibitors of MMPs-2 (TIMP-2) recruitment (two proteins evidenced here as involved in MMP-2 activation), reduced the number of dead cells. Even the use of broad-range MMP inhibitors (GM6001 and Batimastat) did not affect WT-Aß(1-40) -induced cell apoptosis. Our results, in contrast to those obtained using the Aß(1-40) Dutch variant suggesting a link between MMP-2 activity, VSMC mortality and degradation of specific matrix components, indicate that the ontogenesis of the Dutch familial and sporadic forms of CAAs is different. ECM degradation and VSMC degeneration would be tightly connected in the Dutch familial form while being two independent processes in sporadic forms of CAA.

MMP-2/MMP-9 plasma level and brain expression in cerebral amyloid angiopathy-associated hemorrhagic stroke.

Cerebral amyloid angiopathy (CAA) is one of the main causes of intracerebral hemorrhage (ICH) in the elderly. Matrix metalloproteinases (MMPs) have been implicated in blood-brain barrier disruption and ICH pathogenesis. In this study, we determined the levels MMP-2 and MMP-9 in plasma and their brain expression in CAA-associated hemorrhagic stroke. Although MMP-2 and MMP-9 plasma levels did not differ among patients and controls, their brain expression was increased in perihematoma areas of CAA-related hemorrhagic strokes compared with contralateral areas and nonhemorrhagic brains. In addition, MMP-2 reactivity was found in ß-amyloid (Aß)-damaged vessels located far from the acute ICH and in chronic microbleeds. MMP-2 expression was associated to endothelial cells, histiocytes and reactive astrocytes, whereas MMP-9 expression was restricted to inflammatory cells. In summary, MMP-2 expression within and around Aß-compromised vessels might contribute to the vasculature fatal fate, triggering an eventual bleeding.

Matrix metalloproteinase-ß19 expressed in cerebral amyloid angiopathy.

BACKGROUND: Cerebral amyloid angiopathy (CAA) is a frequent finding in the brains of patients with Alzheimer's disease (AD). CAA may be complicated with CAA-associated intracerebral haemorrhage (CAAH). Previous studies have revealed matrix metalloproteinase (MMP) expression in a mouse model of CAA and in human intracerebral haemorrhage. Here we studied the involvement of MMPs in human CAA and CAAH. MATERIAL AND METHODS: To investigate the putative expression of MMPs in human CAA and CAAH (Step 1), immunohistochemistry (IHC) against MMPs-1, -2, -7, -9, -19 and -26 was applied on tissue microarray (TMA) constructed of cerebral samples from 29 individuals with AD, 15 with CAAH and 2 controls. The findings in TMA were confirmed (Step 2) in tissue samples from 64 individuals, 45 presenting with CAA (including 36 with CAAH) and 19 without CAA (including 11 with hypertensive cerebral haemorrhage). RESULTS: In Step 1, immunoreactivity against MMPs-19 and -26 was detected in cerebral blood vessels in CAA. The results were confirmed in Step 2, where CAA (p<0.001) and intracerebral haemorrhage (p=0.045) were associated with vascular immunoreactivity against MMP-19. Multivariate analysis showed that the association between vascular MMP-19 and intracerebral haemorrhage was dependent from CAA. MMP-26 associated with CAA (p=0.021) but not with intracerebral haemorrhage. CONCLUSION: This is the first human study showing local MMP-19 immunoreactivity in the Aß-amyloid-laden blood vessels in CAA, suggesting that MMPs may be involved in CAA.

Differential cerebral deposition of IDE and NEP in sporadic and familial Alzheimer's disease.

Alzheimer's disease (AD) is characterized by amyloid beta (A beta) accumulation in the brain and is classified as familial early-onset (FAD) or sporadic late-onset (SAD). Evidences suggest that deficits in the brain expression of insulin degrading enzyme (IDE) and neprilysin (NEP), both proteases involved in amyloid degradation, may promote A beta deposition in SAD. We studied by immunohistochemistry IDE and NEP cortical expression in SAD and FAD samples carrying the E280A presenilin-1 missense mutation. We showed that IDE, a soluble peptidase, is linked with aggregated A beta 40 isoform while NEP, a membrane-bound protease, negatively correlates with amyloid angiopathy and its expression in the senile plaques is independent of aggregated amyloid and restricted to SAD cases. NEP, but not IDE, is over-expressed in dystrophic neurites, both proteases are immunoreactive in activated astrocytes but not in microglia and IDE was the only one detected in astrocytes of white matter from FAD cases. Collectively, our results support the notion that gross conformational changes involved in the modification from "natively folded-active" to "aggregated-inactive" IDE and NEP may be a relevant pathogenic mechanism in SAD.

The contribution of cerebral vascular semicarbazide-sensitive amine oxidase to cerebral amyloid angiopathy in Alzheimer's disease.

Semicarbazide-sensitive amine oxidase (SSAO) catalyses the oxidative deamination of a variety of endogenous substrates, such as methylamine and aminoacetone, to produce highly reactive aldehydes, which are capable of inducing protein cross-linkage, beta amyloid (Abeta) aggregation and advanced glycation end-product formation. In the brain, SSAO is exclusively located on the blood vessels. Deposits of Abeta, the hallmark of Alzheimer's disease (AD), are closely associated with cerebral blood vessels, that is, cerebral amyloid angiopathy (CAA). In the present study, we examined whether SSAO-mediated deamination contributes to CAA in AD. We employed immunohistochemistry to examine the colocalization of SSAO and Abeta in post mortem brains of AD patients. To assess the role of SSAO-mediated deamination in the deposition of Abeta on blood vessel walls, we developed an in vitro blood vessel model using sections of human umbilical cord. We found a strong expression of SSAO colocalized with Abeta deposits on the blood vessels in AD brains. We also demonstrated that SSAO-mediated deamination increases the deposition of Abeta onto blood vessel walls. Our results support the hypothesis that cerebral vascular SSAO-catalysed deamination contributes to CAA in AD brains.

Angiotensin-converting enzyme (ACE) levels and activity in Alzheimer's disease, and relationship of perivascular ACE-1 to cerebral amyloid angiopathy.

AIMS: Several observations point to the involvement of angiotensin-converting enzyme-1 (ACE-1) in Alzheimer's disease (AD): ACE-1 cleaves amyloid-beta peptide (Abeta) 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 Abeta 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 Abeta 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 Abeta 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 Abeta, 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 Abeta 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.

Oxidative damage of 14-3-3 zeta and gamma isoforms in Alzheimer's disease and cerebral amyloid angiopathy.

Previous studies have shown oxidative damage resulting from amyloid Abeta exposure to cultured cells and in murine models. A target of oxidation is 14-3-3 which comprises a group of proteins involved in kinase activation and chaperone activity. The present study shows glycoxidative damage, as revealed with mono and bi-dimensional gel electrophoresis and Western blotting, followed by in-gel digestion and mass spectrometry, in the frontal cortex in Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA), a neurodegenerative disease with deposition of Abeta in cerebral blood vessels and in diffuse plaques unaccompanied by intraneuronal hyper-phosphorylated tau deposition. malondialdehyde-lysine (MDA-Lys)-, but not 4-hydroxy-2-nonenal (HNE)-immunoreactive adducts, and N-carboxyethyl-lysine (CEL), but not N-carboxymethyl-lysine (CML)-products, were present in 14-3-3 involving zeta and gamma isoforms in both AD and CAA. These findings demonstrate that 14-3-3 glyco- and lipoxidation occurs in AD and CAA, probably as a direct consequence of Abeta deposition.

Semicarbazide-sensitive amine oxidase (SSAO) and its possible contribution to vascular damage in Alzheimer's disease.

One of the key pathological features of the progressive neurodegenerative disorder Alzheimer's disease (AD) is cerebral amyloid angiopathy (CAA). CAA is present in most cases of AD, and it is characterized by the deposition of beta-amyloid (Abeta) in brain vessels, inducing the degeneration of vascular smooth muscle cells and endothelial cells. Herein we report that semicarbazide-sensitive amine oxidase (SSAO) is overexpressed in cerebrovascular tissue of patients with AD-CAA, and that it colocalizes with beta-amyloid deposits. This over-expression correlates with high SSAO activity in plasma of severe AD patients. In addition, we have observed that the catalytic activity of SSAO is able to induce apoptosis in smooth muscle cells in vitro. Taken together, these results allow us to postulate that SSAO may contribute to the vascular damage associated to AD.

Matrix metalloproteinase-9 and spontaneous hemorrhage in an animal model of cerebral amyloid angiopathy.

We examined the potential role of the extra-cellular matrix-degrading enzyme, matrix metalloproteinase-9 (MMP-9), in the pathogenesis of cerebral amyloid angiopathy (CAA)-induced spontaneous hemorrhage. The amyloid-beta peptide (Abeta) induced the synthesis, release and activation of MMP-9 in murine cerebral endothelial cells, resulting in increased extracellular matrix degradation. Furthermore, extensive MMP-9 immunoreactivity was observed in CAA-vessels with evidence of microhemorrhage in aged APPsw transgenic mice, but not detected in aged wild type or young APPsw mice. These results suggest that increased vascular MMP-9 expression, stimulated by Abeta, may play a role in the pathogenesis of spontaneous intracerebral hemorrhage in patients with CAA.

Pathogenic A beta induces the expression and activation of matrix metalloproteinase-2 in human cerebrovascular smooth muscle cells.

Cerebral amyloid angiopathy (CAA) is a major pathological feature of Alzheimer's disease and related disorders. Human cerebrovascular smooth muscle (HCSM) cells, which are intimately associated with CAA, have been used as an in vitro model system to investigate pathologic interactions with amyloid beta protein (A beta). Previously we have shown that pathogenic forms of A beta induce several pathologic responses in HCSM cells including fibril assembly at the cell surface, increase in the levels of A beta precursor, and apoptotic cell death. Here we show that pathogenic A beta stimulates the expression and activation of matrix metalloproteinase-2 (MMP-2). Furthermore, we demonstrate that the increase in MMP-2 activation is largely caused by increased expression of membrane type-1 (MT1)-MMP expression, the primary MMP-2 activator. Finally, treatment with MMP-2 inhibitors resulted in increased HCSM cell viability in the presence of pathogenic A beta. Our findings suggest that increased expression and activation of MMP-2 may contribute to HCSM cell death in response to pathogenic A beta. In addition, these activities may also contribute to loss of vessel wall integrity in CAA resulting in hemorrhagic stroke. Therefore, further understanding into the role of MMPs in HCSM cell degeneration may facilitate designing therapeutic strategies to treat CAA found in AD and related disorders.

Association of neprilysin polymorphism with cerebral amyloid angiopathy.

OBJECTIVES: The risk of sporadic cerebral amyloid angiopathy (CAA) may be associated with genetic polymorphisms of molecules related to anabolism or catabolism of amyloid beta protein (Abeta). The authors investigated whether a polymorphism of the gene (NEP) coding for neprilysin, an enzyme catabolising Abeta, is associated with CAA. METHODS: The study analysed the GT repeat polymorphism in the enhancer/promoter region of NEP and severity of CAA in 164 necropsied elderly Japanese subjects. RESULTS: The subjects had NEP polymorphisms with 19 to 23 GT repeats and were classified into nine genotypes. CAA severity was significantly higher in the subjects with up to 40 repeats in total than those with more than 40 repeats (p=0.005). There was a significant correlation between the number of the shorter alleles (19 or 20 repeats) and CAA severity (p=0.024). In addition, there was no interaction between the NEP polymorphism and apolipoprotein E genotype. CONCLUSIONS: These results suggest the association between the NEP polymorphism and the risk of CAA. Further study using more samples from populations with different ethnic backgrounds is necessary.

Declining expression of neprilysin in Alzheimer disease vasculature: possible involvement in cerebral amyloid angiopathy.

Molecular, genetic, and pharmacological studies have shown that neprilysin (also called NEP) catabolizes amyloid beta peptides (A beta) in healthy conditions. However, in Alzheimer disease (AD), A beta accumulates forming senile plaques in brain parenchyma and amyloid deposition around blood vessels. In this study, we tested at cellular level the relationship between neprilysin and A beta in human healthy and AD brain. Our results provided evidence for declining levels of neprilysin in AD brains as compared to healthy controls in parallel with increasing deposition of A beta. In hippocampus of AD individuals we observed a significant down-regulation of neprilysin expression in pyramidal neurons, consistent with the possibility that neprilysin controls the level of A beta accumulation and plaque formation in this area. In the cortex and leptomeninges, neprilysin was expressed in the smooth muscle cells of blood vessels. In sections from AD patients we observed a clear inverse relationship between neprilysin and A beta peptide levels in the vasculature, implicating neprilysin in cerebral amyloid angiopathy.

No association of paraoxonase genotype or atherosclerosis with cerebral amyloid angiopathy.

BACKGROUND AND PURPOSE: Both cerebral amyloid angiopathy (CAA) and paraoxonase have been reported to be related to lipid metabolism and atherosclerosis. We investigated whether the paraoxonase gene (PON1) polymorphism and atherosclerosis are associated with risk of CAA. METHODS: Associations of the PON1 polymorphism and atherosclerosis of the aorta and coronary and cerebral arteries with the severity of CAA were investigated in 154 elderly Japanese individuals, including 47 patients with Alzheimer's disease. RESULTS: The PON1 polymorphism or severity of atherosclerosis of the arteries was not associated with the severity of CAA. CONCLUSIONS: The PON1 polymorphism and atherosclerosis would not appear to be associated with risk of CAA in the elderly, although further study with larger samples is necessary for confirmation.

Butyrylcholinesterase K variant and cerebral amyloid angiopathy.

BACKGROUND AND PURPOSE: Cholinesterases are found histochemically in the vessels affected with cerebral amyloid angiopathy (CAA). A gene for the K variant of butyrylcholinesterase (BCHE-K) may be associated with late-onset Alzheimer's disease (AD). In search of genetic risk factors for CAA, we investigated the association of BCHE-K with CAA. METHODS: The association between the severity of CAA and BCHE-K was investigated in 155 autopsy cases of the elderly, including 48 patients with AD. RESULTS: There was no significant association of BCHE-K with the severity of CAA in the total, AD, or non-AD cases. Status of the epsilon4 allele of apolipoprotein E gene did not influence the results. CONCLUSIONS: Our results may suggest that BCHE-K is not a definitive risk factor for CAA in the elderly, although further study with larger samples is necessary to confirm this.

Cholinesterase activity in the plaques, tangles and angiopathy of Alzheimer's disease does not emanate from amyloid.

Previous histochemical observations in our laboratory have demonstrated the presence of butyrylcholinesterase and an enzymatically altered form of acetylcholinesterase activity in the plaques, tangles and amyloid-containing vessels of Alzheimer's disease. These findings suggested possible interactions between amyloid and cholinesterases. In this study we employed a cholinesterase biochemical assay to determine whether the amyloid precursor protein either had cholinesterase activity itself or influenced the enzymatic activity of cholinesterases. None of the three amyloid precursor sequences used (695, 751, 770, up to 16 micrograms/ml) exhibited any acetylcholinesterase or butyrylcholinesterase activity that could be detected by our method. In addition, none of the amyloid precursor proteins influenced the enzymatic activity of purified acetylcholinesterase or butyrylcholinesterase in a specific manner. It is therefore quite unlikely that amyloid can, by itself, account for the intense cholinesterase activity associated with the pathological lesions of AD.