Human cerebral vascular amyloid contains both antiparallel and parallel in-register Aβ40 fibrils

The accumulation of fibrillar amyloid-β (Aβ) peptides alongside or within the cerebral vasculature is the hallmark of cerebral amyloid angiopathy (CAA). This condition commonly co-occurs with Alzheimer''s disease (AD) and leads to cerebral microbleeds, intracranial hemorrhages, and stroke. CAA also occurs sporadically in an age-dependent fashion and can be accelerated by the presence of familial Aβ mutant peptides. Recent studies using Fourier transform infrared (FTIR) spectroscopy of vascular Aβ fibrils derived from rodents containing the double E22Q/D23N mutations indicated the presence of a novel antiparallel β-sheet structure. To address whether this structure is associated solely with the familial mutations or is a common feature of CAA, we propagated Aβ fibrils from human brain vascular tissue of patients diagnosed with nonfamilial CAA. Aβ fibrils were isolated from cerebral blood vessels using laser capture microdissection in which specific amyloid deposits were removed from thin slices of the brain tissue. Transmission electron microscopy revealed that these deposits were organized into a tight meshwork of fibrils, which FTIR measurements showed could serve as seeds to propagate the growth of Aβ40 fibrils for structural studies. Solid-state NMR measurements of the fibrils propagated from vascular amyloid showed they contained a mixture of parallel, in-register, and antiparallel β-sheet structures. The presence of fibrils with antiparallel structure derived from vascular amyloid is distinct from the typical parallel, in-register β-sheet structure that appears in fibrils derived from parenchymal amyloid in AD. These observations reveal that different microenvironments influence the structures of Aβ fibrils in the human brain.     

Inhibition of familial cerebral amyloid angiopathy mutant amyloid beta-protein fibril assembly by myelin basic protein

Deposition of fibrillar amyloid beta-protein (Abeta) in the brain is a prominent pathological feature of Alzheimer disease and related disorders, including familial forms of cerebral amyloid angiopathy (CAA). Mutant forms of Abeta, including Dutch- and Iowa-type Abeta, which are responsible for familial CAA, deposit primarily as fibrillar amyloid along the cerebral vasculature and are either absent or present only as diffuse non-fibrillar plaques in the brain parenchyma. Despite the lack of parenchymal fibril formation in vivo, these CAA mutant Abeta peptides exhibit a markedly increased rate and extent of fibril formation in vitro compared with wild-type Abeta. Based on these conflicting observations, we sought to determine whether brain parenchymal factors that selectively interact with and modulate CAA mutant Abeta fibril assembly exist. Using a combination of immunoaffinity chromatography and mass spectrometry, we identified myelin basic protein (MBP) as a prominent brain parenchymal factor that preferentially binds to CAA mutant Abeta compared with wild-type Abeta. Surface plasmon resonance measurements confirmed that MBP bound more tightly to Dutch/Iowa CAA double mutant Abeta than to wild-type Abeta. Using a combination of biochemical and ultrastructural techniques, we found that MBP inhibited the fibril assembly of CAA mutant Abeta. Together, these findings suggest a possible role for MBP in regulating parenchymal fibrillar Abeta deposition in familial CAA.     

Comparison of Sirius red and Congo red as stains for amyloid in animal tissues.

Sirius red and Congo red were compared for specificity and sensitivity of amyloid staining in animal and human material. Previously described advantages of Sirius red as an amyloid dye were confirmed, as well as its disadvantage of lack of ultraviolet fluorescence. Two further disadvantages of Sirius red were discovered, both relating to animal material: (a) its unexpectedly weak staining of early experimentally induced amyloid deposits and (b) frequent uncontrollable nonspecific staining of fibrous tissues. It is therefore concluded that, overall, Congo red used by the improved alkaline technique of Puchtler, Sweat and Levine (1962) remains the best single method for demonstration of amyloid in both human and animal tissues.     

Methods for staining amyloid in tissues: a review

The traditional way of identifying amyloid in tissue sections has been staining with Congo red and demonstration of green birefringence under crossed polarizers. The original method of Congo red staining, described by Bennhold in 1922, has undergone several modifications to improve its sensitivity, specificity, and reliability. The most common modification is the alkaline Congo red method described by Puchtler and co-workers in 1962. Specificity is improved by using freshly prepared stain and a staining solution fully saturated with sodium chloride. Amyloid proteins can be further distinguished by autoclaving or by treating the tissue with potassium permanganate or alkaline guanidine. Autoclaving the tissues at 120 C for 30 min causes protein AA to lose its affinity for Congo red. Prolongation of autoclaving to 120 min abolishes the Congophilia of protein AL, but prealbumin-related amyloid shows little or no change. Treatment of the tissue with potassium permanganate causes protein AA and B2-microglobulin amyloid to lose their affinity to Congo red. Protein AA fails to stain with Congo red after treatment with alkaline guanidine for 1 min and protein AL and systemic senile amyloid protein (SSA) after 2 hr. Familial amyloid protein (FAP), prealbumin type, can stand 2 hr of alkaline guanidine treatment without losing its ability to stain with Congo red. Other methods of detection of amyloid include fluorescent stains, e.g., thioflavin T or S, and metachromatic stains such as crystal violet. Immunofluorescence and immunoperoxidase methods are used to identify and classify amyloid proteins in tissues. Antibodies against the P component, proteins AA and AL and FAP have been used with great precision. Due to cross-reactivity, these methods do not differentiate between some types of familial and senile systemic amyloidosis.     

Cerebral beta-amyloid angiopathy in aged squirrel monkeys

Cerebral beta-amyloid angiopathy (CAA) is an age-related disorder of the brain vasculature that is involved in up to 20% of non-traumatic cerebral hemorrhage in humans. CAA is a risk factor for cognitive decline, and may exacerbate the dementia of Alzheimer's disease. Progress in discovering the cause and potential therapies for this disorder has been hindered by the paucity of animal models, particularly models of idiopathic CAA. The squirrel monkey (Saimiri spp) develops significant CAA in the natural course of aging. To evaluate the suitability of Saimiri as a model of human CAA, we studied the distribution and composition of Abeta subtypes in CAA and parenchymal (senile plaque) deposits in the brains of aged squirrel monkeys, as well as the relationship between vascular beta-amyloid deposition and comorbid vasculopathies that occur in aged humans. Our findings show that: 1) CAA consists ultrastructurally of classical amyloid fibrils and is the principal type of cerebral beta-amyloidosis in squirrel monkeys; 2) The two primary isoforms of Abeta (Abeta40 and Abeta42) coexist in most microvascular and parenchymal lesions of Saimiri, although Abeta40 tends to predominate in larger arterioles; 3) CAA and parenchymal plaques overlap to a considerable degree in most affected brain areas, and are distributed symmetrically in the two hemispheres; 4) Both CAA and plaques are particularly abundant in rostral regions and comparatively sparse in the occipital lobe; 5) Capillaries are especially vulnerable to CAA in squirrel monkeys; and 6) When CAA is severe, it is associated with a small, but significant, increase in other vasculopathies, including microhemorrhage, fibrinoid extravasation and focal gliosis. These findings, in the context of genetic, vascular and immunologic similarities between squirrel monkeys and humans, support the squirrel monkey as a biologically advantageous model for studying the basic biology of idiopathic, age-related CAA, and for testing emerging therapies for human beta-amyloidoses such as Alzheimer's disease.     

X-34, a fluorescent derivative of Congo red: a novel histochemical stain for Alzheimer's disease pathology.

X-34, a lipophilic, highly fluorescent derivative of Congo red, was examined as a histochemical stain for pathological changes in Alzheimer's disease (AD). X-34 intensely stained neuritic and diffuse plaques, neurofibrillary tangles (NFTs), neuropil threads, and cerebrovascular amyloid. Comparison to standard methods of demonstrating AD pathology showed that X-34 correlated well with Bielschowsky and thioflavin-S staining. X-34 staining of NFTs correlated closely with anti-TAU antibody staining. A 1:1 correspondence of X-34 and anti-A beta antibody staining of plaques and cerebrovascular amyloid was observed. Both X-34 and thioflavin-S staining were eliminated by formic acid pretreatment, suggesting that beta-sheet secondary protein structure is a necessary determinant of staining. X-34 may be a general amyloid stain, like Congo red, because it also stains systemic amyloid deposits due to lambda-light chain monoclonal gammopathy. In conclusion, X-34 is a highly fluorescent marker for beta-sheet structures and intensely labels amyloid plaques, NFTs, neuropil threads, and vascular amyloid in AD brains. It can be used with both paraffin-embedded and frozen tissues as well as in combination with immunohistochemistry for double labeling. The intensity of staining and the simplicity and reproducibility of the technique suggest that it may be a useful addition to the standard techniques for evaluation of AD neuropathology. (J Histochem Cytochem 48:1223-1232, 2000)     

Unwinding fibril formation of medin, the peptide of the most common form of human amyloid

Medin amyloid affects the medial layer of the thoracic aorta of most people above 50 years of age. The consequences of this amyloid are not completely known but the deposits may contribute to diseases such as thoracic aortic aneurysm and dissection or to the general diminished elasticity of blood vessels seen in elderly people. We show that the 50-amino acid residue peptide medin forms amyloid-like fibrils in vitro. With the use of Congo red staining, Thioflavin T fluorescence, electron microscopy, and a solid-phase binding assay on different synthetic peptides, we identified the last 18-19 amino acid residues to constitute the amyloid-promoting region of medin. We also demonstrate that the two C-terminal phenylalanines, previously suggested to be of importance for amyloid formation, are not required for medin amyloid formation.     

Early-onset and robust cerebral microvascular accumulation of amyloid beta-protein in transgenic mice expressing low levels of a vasculotropic Dutch/Iowa mutant form of amyloid beta-protein precursor

Cerebrovascular deposition of amyloid beta-protein (Abeta) is a common pathological feature of Alzheimer's disease and related disorders. In particular, the Dutch E22Q and Iowa D23N mutations in Abeta cause familial cerebrovascular amyloidosis with abundant diffuse amyloid plaque deposits. Both of these charge-altering mutations enhance the fibrillogenic and pathogenic properties of Abeta in vitro. Here, we describe the generation of several transgenic mouse lines (Tg-SwDI) expressing human neuronal Abeta precursor protein (AbetaPP) harboring the Swedish K670N/M671L and vasculotropic Dutch/Iowa E693Q/D694N mutations under the control of the mouse Thy1.2 promoter. Tg-SwDI mice expressed transgenic human AbetaPP only in the brain, but at levels below those of endogenous mouse AbetaPP. Despite the paucity of human AbetaPP expression, quantitative enzyme-linked immunosorbent assay measurements revealed that Tg-SwDI mice developed early-onset and robust accumulation of Abeta in the brain with high association with isolated cerebral microvessels. Tg-SwDI mice exhibited striking perivascular/vascular Abeta deposits that markedly increased with age. The vascular Abeta accumulations were fibrillar, exhibiting strong thioflavin S staining, and occasionally presented signs of microhemorrhage. In addition, numerous largely diffuse, plaque-like structures were observed starting at 3 months of age. In vivo transport studies demonstrated that Dutch/Iowa mutant Abeta was more readily retained in the brain compared with wild-type Abeta. These results with Tg-SwDI mice demonstrate that overexpression of human AbetaPP is not required for early-onset and robust accumulation of both vascular and parenchymal Abeta in mouse brain.     

Deposition of amyloid of unknown origin in articular cartilage.

Articular cartilage, obtained from the large toe during hallux valgus operations in 37 patients, was investigated for the presence of amyloid by using the Congo red staining method. Amyloid deposits were demonstrated, particularly in the superficial layer of the cartilage, in 30 cases. This amyloid did not react immunohistochemically with any of the antibodies against the known five major amyloid types (AA, A lambda, A kappa, AF, AB). From these data it is concluded that hyaline cartilage in older individuals is prone to infiltration by an amyloid of a hitherto unidentified class. From the morphological observations there seems to be no correlation between amyloid deposits and the development of osteoarthrosis.     

The cause of the green polarization color of amyloid stained with Congo red

Summary Experiments done with Congo red crystals and with Congo red deposits polished in a single direction by a glass wheel have shown that the appearance of green polarization color primarily depends on near-perfect parallel alignment of the dye particles. The green polarization color was seen only in the deposits which showed a clear transition from red to colorless when examined for dichroism. Another factor was found to be the thickness of the object, as the green polarization color was not present in too thick or too thin sections of amyloid-containing tissues stained with Congo red.The phenomena can be explained by the assumption that the green polarization color is due to interference between the red ray and the red component of the white ray whenever the retardation by the object approximates half the wavelength of red light.The findings indicate that amyloid differs from other materials which are stained by Congo red in that amyloid deposits bind the dye molecules in a more orderly and parallel fashion. It is suggested that minimal amounts of amyloid which are not visible in Congo red stained sections with ordinary light microscopy and which do not give the green polarization color can best be detected by examination for dichroism in ultraviolet light after having been stained with fluorescent dyes.     

Early-onset Formation of Parenchymal Plaque Amyloid Abrogates Cerebral Microvascular Amyloid Accumulation in Transgenic Mice

The fibrillar assembly and deposition of amyloid β (Aβ) protein, a key pathology of Alzheimer disease, can occur in the form of parenchymal amyloid plaques and cerebral amyloid angiopathy (CAA). Familial forms of CAA exist in the absence of appreciable parenchymal amyloid pathology. The molecular interplay between parenchymal amyloid plaques and CAA is unclear. Here we investigated how early-onset parenchymal amyloid plaques impact the development of microvascular amyloid in transgenic mice. Tg-5xFAD mice, which produce non-mutated human Aβ and develop early-onset parenchymal amyloid plaques, were bred to Tg-SwDI mice, which produce familial CAA mutant human Aβ and develop cerebral microvascular amyloid. The bigenic mice presented with an elevated accumulation of Aβ and fibrillar amyloid in the brain compared with either single transgenic line. Tg-SwDI/Tg-5xFAD mice were devoid of microvascular amyloid, the prominent pathology of Tg-SwDI mice, but exhibited larger parenchymal amyloid plaques compared with Tg-5xFAD mice. The larger parenchymal amyloid deposits were associated with a higher loss of cortical neurons and elevated activated microglia in the bigenic Tg-SwDI/Tg-5xFAD mice. The periphery of parenchymal amyloid plaques was largely composed of CAA mutant Aβ. Non-mutated Aβ fibril seeds promoted CAA mutant Aβ fibril formation in vitro. Further, intrahippocampal administration of biotin-labeled CAA mutant Aβ peptide accumulated on and adjacent to pre-existing parenchymal amyloid plaques in Tg-5xFAD mice. These findings indicate that early-onset parenchymal amyloid plaques can serve as a scaffold to capture CAA mutant Aβ peptides and prevent their accumulation in cerebral microvessels.     

Microvascular pathology and vascular basement membrane components in Alzheimer's disease

Several factors have highlighted the vasculature in Alzheimer's disease (AD): Cerebral amyloid angiopathy (CAA) is common, amyloid fibrils emanate from the vascular basement membrane (VBM), and similar forms of β-amyloid are found in vascular and parenchymal amyloid accumulations. The present article discusses the presence of microvascular pathology in AD. Microangiopathy, in addition to neurofibrillary tangles, senile plaques, and CAA, is a common pathologic hallmark of AD. VBM components are associated with amyloid plaques, and nonamyloidotic alterations of the VBM occur in brain regions susceptible to AD lesions. Also, intra-VBM perivascular cells (traditionally called pericytes), a subset of which share the immunophenotype of microglia and other mononuclear phagocytic system (MPS) cells, have been implicated in vascular alterations and cerebrovascular amyloid deposition. Perivascular and parenchymal MPS cells have access to several sources of the β-amyloid protein precursor, including platelets, circulating white cells, and neurons. MPS cells would thus be ideally situated to uptake and process the precursor, and deposit β-amyloid in a fashion analogous to that seen in other forms of systemic and cerebral amyloidoses.     

Abeta42 is essential for parenchymal and vascular amyloid deposition in mice

Considerable circumstantial evidence suggests that Abeta42 is the initiating molecule in Alzheimer's disease (AD) pathogenesis. However, the absolute requirement for Abeta42 for amyloid deposition has never been demonstrated in vivo. We have addressed this by developing transgenic models that express Abeta1-40 or Abeta1-42 in the absence of human amyloid beta protein precursor (APP) overexpression. Mice expressing high levels of Abeta1-40 do not develop overt amyloid pathology. In contrast, mice expressing lower levels of Abeta1-42 accumulate insoluble Abeta1-42 and develop compact amyloid plaques, congophilic amyloid angiopathy (CAA), and diffuse Abeta deposits. When mice expressing Abeta1-42 are crossed with mutant APP (Tg2576) mice, there is also a massive increase in amyloid deposition. These data establish that Abeta1-42 is essential for amyloid deposition in the parenchyma and also in vessels.     

Review: history of the amyloid fibril

Rudolph Virchow, in 1854, introduced and popularized the term amyloid to denote a macroscopic tissue abnormality that exhibited a positive iodine staining reaction. Subsequent light microscopic studies with polarizing optics demonstrated the inherent birefringence of amyloid deposits, a property that increased intensely after staining with Congo red dye. In 1959, electron microscopic examination of ultrathin sections of amyloidotic tissues revealed the presence of fibrils, indeterminate in length and, invariably, 80 to 100 A in width. Using the criteria of Congophilia and fibrillar morphology, 20 or more biochemically distinct forms of amyloid have been identified throughout the animal kingdom; each is specifically associated with a unique clinical syndrome. Fibrils, also 80 to 100 A in width, have been isolated from tissue homogenates using differential sedimentation or solubility. X-ray diffraction analysis revealed the fibrils to be ordered in the beta pleated sheet conformation, with the direction of the polypeptide backbone perpendicular to the fibril axis (cross beta structure). Because of the similar dimensions and tinctorial properties of the fibrils extracted from amyloid-laden tissues and amyloid fibrils in tissue sections, they have been assumed to be identical. However, the spatial relationship of proteoglycans and amyloid P component (AP), common to all forms of amyloid, to the putative protein only fibrils in tissues, has been unclear. Recently, it has been suggested that, in situ, amyloid fibrils are composed of proteoglycans and AP as well as amyloid proteins and thus resemble connective tissue microfibrils. Chemical and physical definition of the fibrils in tissues will be needed to relate the in vitro properties of amyloid protein fibrils to the pathogenesis of amyloid fibril formation in vivo.     

Endothelial expression of human amyloid precursor protein leads to amyloid β in the blood and induces cerebral amyloid angiopathy in knock-in mice

The deposition of amyloid β (Aβ) in blood vessels of the brain, known as cerebral amyloid angiopathy (CAA), is observed in most patients with Alzheimer’s disease (AD). Compared with the pathology of CAA in humans, the pathology in most mouse models of AD is not as evident, making it difficult to examine the contribution of CAA to the pathogenesis of AD. On the basis of biochemical analyses that showed blood levels of soluble amyloid precursor protein (APP) in rats and mice were markedly lower than those measured in human samples, we hypothesized that endothelial APP expression would be markedly lower in rodents and subsequently generated mice that specifically express human WT APP (APP770) in endothelial cells (ECs). The resulting EC-APP770⁺ mice exhibited increased levels of serum Aβ and soluble APP, indicating that endothelial APP makes a critical contribution to blood Aβ levels. Even though aged EC-APP770⁺ mice did not exhibit Aβ deposition in the cortical blood vessels, crossing these animals with APP knock-in mice (App^NL-F/NL-F) led to an expanded CAA pathology, as evidenced by increased amounts of amyloid accumulated in the cortical blood vessels. These results highlight an overlooked interplay between neuronal and endothelial APP in brain vascular Aβ deposition. We propose that these EC-APP770⁺:App^NL-F/NL-F mice may be useful to study the basic molecular mechanisms behind the possible breakdown of the blood–brain barrier upon administration of anti-Aβ antibodies.     

阿尔茨海默症转基因动物模型脑组织病理学及免疫组化研究

日的通过组织病理学和免疫组织化学方法,验证阿尔茨海默症转基因动物模型。方法取转基因小鼠脑组织,冠状切面中１／３部位,脱水、包埋,进行组织病理学观察,并对相关抗体进行免疫组织化学研究。结果免疫组化显示在大脑皮层、小脑及海马的神经细胞有Ａβ沉淀形成。ＡＰＰ转基因鼠早老素的阳性细胞数及表达量多于对照鼠。刚果红染色可见大脑皮层间有淀粉样物质形成。结论从病理学角度验证此模型的表型与人类病变的相似性,并证明早老素－１可加速淀粉样沉淀的形成,二者的作用是相互的。     

Modulation of microglial activation state following passive immunization in amyloid depositing transgenic mice

Alzheimer's disease is a large and growing health problem. Several lines of transgenic mice overexpressing the amyloid precursor protein (APP) develop both diffuse and compacted amyloid deposits which increase in size and number with age. In the vicinity of compacted deposits, these mice develop neuritic dystrophy and activation of glia. Ultimately, these mice also develop memory deficits. Immunotherapy against the Abeta peptide has been effective in both clearing amyloid deposits from the brain, and improving the mnemonic performance of the transgenic mice. Associated with these actions, are changes in the expression of microglial markers. In some cases, the glial activation markers decline, consistent with reduced provocation from amyloid deposits. However, in a time course study, we found that some markers of microglial activation increase transiently once the immunotherapy is initiated. Still another marker continues to rise for up to 3 months of treatment, and remains elevated even after the parenchymal amyloid deposits are largely removed. These changes are consistent with a shift in the microglial phenotype, transitioning from a condition associated with inflammation and ineffective in clearing Abeta deposits to one with reduced inflammation, and capable of clearing deposited amyloid.     

Insulin-degrading enzyme in brain microvessels: proteolysis of amyloid {beta} vasculotropic variants and reduced activity in cerebral amyloid angiopathy

The accumulation of amyloid beta (Abeta) in the walls of small vessels in the cerebral cortex is associated with diseases characterized by dementia or stroke. These include Alzheimer's disease, Down syndrome, and sporadic and hereditary cerebral amyloid angiopathies (CAAs) related to mutations within the Abeta sequence. A higher tendency of Abeta to aggregate, a defective clearance to the systemic circulation, and insufficient proteolytic removal have been proposed as mechanisms that lead to Abeta accumulation in the brain. By using immunoprecipitation and mass spectrometry, we show that insulin-degrading enzyme (IDE) from isolated human brain microvessels was capable of degrading (125)I-insulin and cleaved Abeta-(1-40) wild type and the genetic variants Abeta A21G (Flemish), Abeta E22Q (Dutch), and Abeta E22K (Italian) at the predicted sites. In microvessels from Alzheimer's disease cases with CAA, IDE protein levels showed a 44% increase as determined by sandwich enzyme-linked immunosorbent assay and Western blot. However, the activity of IDE upon radiolabeled insulin was significantly reduced in CAA as compared with age-matched controls. These results support the notion that a defect in Abeta proteolysis by IDE contributes to the accumulation of this peptide in the cortical microvasculature. Moreover they raise the possibility that IDE inhibition or inactivation is a pathogenic mechanism that may open novel strategies for the treatment of cerebrovascular Abeta amyloidoses.     

Fibrillar amyloid beta-protein binds protease nexin-2/amyloid beta-protein precursor: stimulation of its inhibition of coagulation factor XIa

Cerebrovascular deposition of fibrillar 39-42 amino acid amyloid beta-protein (Abeta), a condition known as cerebral amyloid angiopathy (CAA), is a key pathological feature of Alzheimer's disease and related disorders including hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D). Severe cases of CAA, particularly in HCHWA-D, lead to recurrent and often fatal hemorrhagic strokes. Although the reasons for this pathological consequence remain unclear, alterations in proteolytic hemostasis mechanisms have been implicated. For example, the Abeta parent molecule protease nexin-2/amyloid beta-protein precursor (PN-2/AbetaPP), which is elevated in HCHWA-D cerebral vessels with Abeta deposits, is a potent inhibitor of coagulation factor XIa (FXIa). Here we show that fibrillar HCHWA-D Abeta binds PN-2/AbetaPP, but not its isolated Kunitz-type proteinase inhibitor (KPI) domain, in a saturable, dose-dependent manner with a K(d) of approximately 28 nM. Neither PN-2/AbetaPP nor its KPI domain bound to nonfibrillar HCHWA-D Abeta. The fibrillar Abeta binding domain on PN-2/AbetaPP was localized to residues 18-119. PN-2/AbetaPP that bound to fibrillar HCHWA-D Abeta immobilized either in plastic wells or on the surface of cultured cerebrovascular smooth muscle cells was active in inhibiting FXIa. Quantitative kinetic measurements revealed that fibrillar HCHWA-D Abeta caused a >5-fold enhancement of FXIa inhibition by PN-2/AbetaPP. Similar stimulatory effects on FXIa inhibition by PN-2/AbetaPP were also observed with fibrillar wild-type Abeta. However, fibrillar Abeta had no effect on the inhibition of trypsin by PN-2/AbetaPP. These findings suggest that fibrillar Abeta deposits in cerebral vessels can effectively localize and enhance the anticoagulant functions of PN-2/AbetaPP, thereby contributing to a microenvironment conducive to hemorrhaging.     

Matrix metalloproteinase inhibition reduces oxidative stress associated with cerebral amyloid angiopathy in vivo in transgenic mice

Cerebral amyloid angiopathy (CAA), characterized by extracellular β-amyloid peptide (Aβ) deposits in vessel walls, is present in the majority of cases of Alzheimer's disease and is a major cause of hemorrhagic stroke. Although the molecular pathways activated by vascular Aβ are poorly understood, extracellular matrix metalloproteinases (MMP) and Aβ-induced oxidative stress appear to play important roles. We adapted fluorogenic assays for MMP activity and reactive oxygen species generation for use in vivo . Using multiphoton microcopy in APPswe/PS1dE9 and Tg-2576 transgenic mice, we observed strong associations between MMP activation, oxidative stress, and CAA deposition in leptomeningeal vessels. Antioxidant treatment with α-phenyl- N -tert-butyl-nitrone reduced oxidative stress associated with CAA (∼50% reduction) without affecting MMP activation. Conversely, a selection of agents that inhibit MMP by different mechanisms of action, including minocycline, simvastatin, and GM6001, reduced not only CAA-associated MMP activation (∼30–40% reduction) but also oxidative stress (∼40% reduction). The inhibitors of MMP did not have direct antioxidant effects. Treatment of animals with α-phenyl- N -tert-butyl-nitrone or minocycline did not have a significant effect on CAA progression rates. These data suggest a close association between Aβ-related MMP activation and oxidative stress in vivo and raise the possibility that treatment with MMP inhibitors may have beneficial effects by indirectly reducing the oxidative stress associated with CAA.