Age-dependent neurovascular dysfunction and damage in a mouse model of cerebral amyloid angiopathy.

BACKGROUND AND PURPOSE: Accumulation of amyloid-ß in cerebral blood vessels occurs in familial and sporadic forms of cerebral amyloid angiopathy and is a prominent feature of Alzheimer disease. However, the functional correlates of the vascular pathology induced by cerebral amyloid angiopathy and the mechanisms involved have not been fully established. METHODS: We used male transgenic mice expressing the Swedish, Iowa, and Dutch mutations of the amyloid precursor protein (Tg-SwDI) to examine the effect of cerebral amyloid angiopathy on cerebrovascular structure and function. Somatosensory cortex cerebral blood flow was monitored by laser-Doppler flowmetry in anesthetized Tg-SwDI mice and wild-type littermates equipped with a cranial window. RESULTS: Tg-SwDI mice exhibited reductions in cerebral blood flow responses to whisker stimulation, endothelium-dependent vasodilators, or hypercapnia at 3 months when compared with wild-type mice, whereas the response to adenosine was not attenuated. However, at 18 and 24 months, all cerebrovascular responses were markedly reduced. At this time, there was evidence of cerebrovascular amyloid deposition, smooth muscle fragmentation, and pericyte loss. Neocortical superfusion with the free radical scavenger manganic(I-II)meso-tetrakis(4-benzoic acid) porphyrin rescued endothelium-dependent responses and functional hyperemia completely at 3 months but only partially at 18 months. CONCLUSIONS: Tg-SwDI mice exhibit a profound age-dependent cerebrovascular dysfunction, involving multiple regulatory mechanisms. Early in the disease process, oxidative stress is responsible for most of the vascular dysfunction, but with advancing disease structural alterations of the vasomotor apparatus also play a role. Early therapeutic interventions are likely to have the best chance to counteract the deleterious vascular effects of cerebral amyloid angiopathy.

Scavenger receptor CD36 is essential for the cerebrovascular oxidative stress and neurovascular dysfunction induced by amyloid-beta.

Increasing evidence indicates that cerebrovascular dysfunction plays a pathogenic role in Alzheimer's dementia (AD). Amyloid-ß (Aß), a peptide central to the pathogenesis of AD, has profound vascular effects mediated, for the most part, by reactive oxygen species produced by the enzyme NADPH oxidase. The mechanisms linking Aß to NADPH oxidase-dependent vascular oxidative stress have not been identified, however. We report that the scavenger receptor CD36, a membrane glycoprotein that binds Aß, is essential for the vascular oxidative stress and neurovascular dysfunction induced by Aß1-40. Thus, topical application of Aß1-40 onto the somatosensory cortex attenuates the increase in cerebral blood flow elicited by neural activity or by endothelium-dependent vasodilators in WT mice but not in CD36-null mice (CD36(0/0)). The cerebrovascular effects of infusion of Aß1-40 into cerebral arteries are not observed in mice pretreated with CD36 blocking antibodies or in CD36(0/0) mice. Furthermore, CD36 deficiency prevents the neurovascular dysfunction observed in transgenic mice overexpressing the Swedish mutation of the amyloid precursor protein Tg2576 despite elevated levels of brain Aß1-40. CD36 is also required for the vascular oxidative stress induced by exogenous Aß1-40 or observed in Tg2576 mice. These observations establish CD36 as a key link between Aß1-40 and the NADPH oxidase-dependent vascular oxidative stress underlying the neurovascular dysfunction and suggest that CD36 is a potential therapeutical target to counteract the cerebrovascular dysfunction associated with Aß.

Brain and circulating levels of Aß1-40 differentially contribute to vasomotor dysfunction in the mouse brain.

BACKGROUND AND PURPOSE: Amyloid-ß (Aß), a peptide that accumulates in the brain and circulates in the blood of patients with Alzheimer disease, alters the regulation of cerebral blood flow and may contribute to the brain dysfunction underlying the dementia. However, the contributions of brain and circulating Aß1-40 to the vascular dysfunction have not been elucidated. METHODS: We used transgenic mice overexpressing mutated forms of the amyloid precursor protein in which Aß1-40 is elevated in blood and brain (Tg-2576) or only in brain (Tg-SwDI). Mice were equipped with a cranial window, and the increase in cerebral blood flow induced by neural activity (whisker stimulation), or by topical application of endothelium-dependent vasodilators, was assessed by laser-Doppler flowmetry. RESULTS: The cerebrovascular dysfunction was observed also in Tg-SwDI mice, but despite ≈40% higher levels of brain Aß1-40, the effect was less marked than in Tg-2576 mice. Intravascular administration of Aß1-40 elevated plasma Aß1-40 and enhanced the dysfunction in Tg-SwDI mice, but not in Tg-2576 mice. CONCLUSIONS: The results provide evidence that Aß1-40 acts on distinct luminal and abluminal vascular targets, the deleterious cerebrovascular effects of which are additive. Furthermore, the findings highlight the importance of circulating Aß1-40 in the cerebrovascular dysfunction and may provide insight into the cerebrovascular alterations in conditions in which elevations in plasma Aß1-40 occur.

A novel operant testing regimen for multi-construct cognitive characterization of a murine model of Alzheimer's amyloid-related behavioral impairment.

A common method for modeling pathological and behavioral aspects of Alzheimer's disease (AD) is the transgenic mouse. While transgenic strains are often well characterized pathologically, behavioral studies of cognitive deficits often employ a limited set of aversively motivated, spatial learning and memory tests, under brief testing periods. Here we illustrate an alternative operant behavioral methodology to provide a comprehensive characterization under repetitive testing conditions, and with appetitive motivation. In this study, we employed the commonly used Tg2576 murine model of Alzheimer's disease amyloid pathology, since it has been the subject of many previous behavioral studies. In these mice, we compared the learning of simple and complex, as well as spatial and non-spatial rules. The mice were assessed on a progressively more complex and interlocking battery of operant tasks, ranging from simple rule learning to delayed recall, as well as tests of motor and sensory ability. In general, as compared to wild type control mice, within-group variability was high in the Tg2576 mice, and deficits were most apparent in more complex discrimination tasks. Furthermore, a consistent decrease in the rate at which Tg2576 mice completed testing trials was observed, pointing to a potential motivation difference or speed-accuracy tradeoffs as a defining characteristic of this strain under these test conditions. Using sensitive adjusting retention interval procedures, it was also possible to isolate a difference in retention interval and separate it from non-mnemonic processes. Overall, these experiments demonstrate the utility of this novel operant approach for characterizing the cognitive deficits of transgenic murine models of dementia.

AbetaPP/APLP2 family of Kunitz serine proteinase inhibitors regulate cerebral thrombosis.

The amyloid beta-protein precursor (AbetaPP) is best recognized as the precursor to the Abeta peptide that accumulates in the brains of patients with Alzheimer's disease, but less is known about its physiological functions. Isoforms of AbetaPP that contain a Kunitz-type serine proteinase inhibitor (KPI) domain are expressed in brain and, outside the CNS, in circulating blood platelets. Recently, we showed that KPI-containing forms of AbetaPP regulates cerebral thrombosis in vivo (Xu et al., 2005, 2007). Amyloid precursor like protein-2 (APLP2), a closely related homolog to AbetaPP, also possesses a highly conserved KPI domain. Virtually nothing is known of its function. Here, we show that APLP2 also regulates cerebral thrombosis risk. Recombinant purified KPI domains of AbetaPP and APLP2 both inhibit the plasma clotting in vitro. In a carotid artery thrombosis model, both AbetaPP(-/-) and APLP2(-/-) mice exhibit similar significantly shorter times to vessel occlusion compared with wild-type mice indicating a prothrombotic phenotype. Similarly, in an experimental model of intracerebral hemorrhage, both AbetaPP(-/-) and APLP2(-/-) mice produce significantly smaller hematomas with reduced brain hemoglobin content compared with wild-type mice. Together, these results indicate that AbetaPP and APLP2 share overlapping anticoagulant functions with regard to regulating thrombosis after cerebral vascular injury.

Human apolipoprotein E redistributes fibrillar amyloid deposition in Tg-SwDI mice.

Human apolipoprotein (ApoE) genotype influences the development of Alzheimer's disease and cerebral amyloid angiopathy (CAA). Specific mutations within the amyloid-beta protein (Abeta) peptide have been identified that cause familial forms of CAA. However, the effect of APOE genotype on accumulation of CAA mutant Abeta in brain is not well understood. In the present study, we determined how human ApoE3 or ApoE4 influence cerebral Abeta accumulation in transgenic mice (Tg-SwDI) that accumulate human Dutch/Iowa (E22Q/D23N) CAA mutant Abeta in brain, primarily in the form of fibrillar cerebral microvascular amyloid. Using Tg-SwDI mice bred onto a human APOE3/3 or human APOE4/4 background, we found that both human ApoE3 and ApoE4 proteins led to a strong reduction in the amount of cerebral microvascular amyloid with an unexpected concomitant appearance of extensive fibrillar parenchymal plaque amyloid. There was strong colocalization of all ApoE proteins with fibrillar amyloid deposits in the mice. In Tg-SwDI/hAPOE3/3 and Tg-SwDI/hAPOE4/4 mice, there was no change in the levels of total Abeta(40) and Abeta(42) or in the amounts of soluble and insoluble Abeta in brain compared with Tg-SwDI mice on the endogenous mouse APOE background. The shift from primarily cerebral microvascular amyloid to parenchymal plaque amyloid in Tg-SwDI/hAPOE3/3 and Tg-SwDI/hAPOE4/4 mice resulted in a parallel shift in the association of activated microglia. These findings indicate that human ApoE has a strong influence on the spatial development of human Dutch/Iowa CAA mutant amyloid accumulation in mouse brain and that microglial activation is in response to the spatial accumulation of fibrillar amyloid.

Progression of amyloid pathology to Alzheimer's disease pathology in an amyloid precursor protein transgenic mouse model by removal of nitric oxide synthase 2.

Alzheimer's disease (AD) is characterized by three primary pathologies in the brain: amyloid plaques, neurofibrillary tangles, and neuron loss. Mouse models have been useful for studying components of AD but are limited in their ability to fully recapitulate all pathologies. We crossed the APPSwDI transgenic mouse, which develops amyloid beta (Abeta)-protein deposits only, with a nitric oxide synthase 2 (NOS2) knock-out mouse, which develops no AD-like pathology. APPSwDI/NOS2(-/-) mice displayed impaired spatial memory compared with the APPSwDI mice, yet they have unaltered levels of Abeta. APPSwDI mice do not show tau pathology, whereas APPSwDI/NOS2(-/-) mice displayed extensive tau pathology associated with regions of dense microvascular amyloid deposition. Also, APPSwDI mice do not have any neuron loss, whereas the APPSwDI/NOS2(-/-) mice have significant neuron loss in the hippocampus and subiculum. Neuropeptide Y neurons have been shown to be particularly vulnerable in AD. These neurons appear to be particularly vulnerable in the APPSwDI/NOS2(-/-) mice as we observe a dramatic reduction in the number of NPY neurons in the hippocampus and subiculum. These data show that removal of NOS2 from an APP transgenic mouse results in development of a much greater spectrum of AD-like pathology and behavioral impairments.

Experimental investigation of antibody-mediated clearance mechanisms of amyloid-beta in CNS of Tg-SwDI transgenic mice.

Novel amyloid precursor protein transgenic mice, which contain the Swedish as well as the vasculotropic Dutch and Iowa mutations (Tg-SwDI), were used to investigate the mechanisms of antibody-mediated clearance of amyloid-beta (Abeta) from the brain. Export of the Abeta-DI peptide across the blood-brain barrier is severely reduced because of the vasculotropic mutations. Therefore, antibody-mediated clearance of Abeta-DI is dependent on antibodies entering the brain. In this report, we immunized Tg-SwDI mice with various peptide antigens, including Abeta40-DI, Abeta42, and an Abeta epitope vaccine. Immunization of Tg-SwDI mice with substantial cortical diffuse and vascular fibrillar deposits failed to promote clearance of parenchymal or vascular amyloid deposits. We then immunized young Tg-SwDI mice before the accumulation of Abeta and saw no evidence that anti-Abeta antibodies could diminish deposition of parenchymal or vascular amyloid deposits. However, injection of anti-Abeta antibodies, affinity-purified from immunized Tg-SwDI mice, into the hippocampus induced a rapid clearance of diffuse Abeta deposits but not vascular amyloid deposits. These results further support the "peripheral sink hypothesis" as a legitimate mechanism of antibody-mediated clearance of Abeta when the blood-brain barrier remains intact. Thus, approaches that deliver immunotherapy to the brain may be more effective at clearing Abeta than immunization strategies in which the majority of the antibodies are in the periphery.

Minocycline reduces microglial activation and improves behavioral deficits in a transgenic model of cerebral microvascular amyloid.

Cerebral microvascular amyloid beta protein (Abeta) deposition and associated neuroinflammation is increasingly recognized as an important component leading to cognitive impairment in Alzheimer's disease and related cerebral amyloid angiopathy disorders. Transgenic mice expressing the vasculotropic Dutch/Iowa (E693Q/D694N) mutant human Abeta precursor protein in brain (Tg-SwDI) accumulate abundant cerebral microvascular fibrillar amyloid deposits and exhibit robust neuroinflammation. In the present study, we investigated the effect of the anti-inflammatory drug minocycline on Abeta accumulation, neuroinflammation, and behavioral deficits in Tg-SwDI mice. Twelve-month-old mice were treated with saline or minocycline by intraperitoneal injection every other day for a total of 4 weeks. During the final week of treatment, the mice were tested for impaired learning and memory. Brains were then harvested for biochemical and immunohistochemical analysis. Minocycline treatment did not alter the cerebral deposition of Abeta or the restriction of fibrillar amyloid to the cerebral microvasculature. Similarly, minocycline-treated Tg-SwDI mice exhibited no change in the levels of total Abeta, the ratios of Abeta40 and Abeta42, or the amounts of soluble, insoluble, or oligomeric Abeta compared with the saline-treated control Tg-SwDI mice. In contrast, the numbers of activated microglia and levels of interleukin-6 were significantly reduced in minocycline-treated Tg-SwDI mice compared with saline-treated Tg-SwDI mice. In addition, there was a significant improvement in behavioral performance of the minocycline-treated Tg-SwDI mice. These finding suggest that anti-inflammatory treatment targeted for cerebral microvascular amyloid-induced microglial activation can improve cognitive deficits without altering the accumulation and distribution of Abeta.

Increased severity of hemorrhage in transgenic mice expressing cerebral protease nexin-2/amyloid beta-protein precursor.

BACKGROUND AND PURPOSE: Secreted isoforms of amyloid beta-protein precursor (AbetaPP) that contain the Kunitz proteinase inhibitor domain, also known as protease nexin-2 (PN2), are enriched in brain. Although little is known of its physiological function, the potent inhibition of certain prothrombotic proteinases by PN2/AbetaPP suggests that it may function to regulate cerebral thrombosis during vascular injury events. METHODS: To examine the antithrombotic function of cerebral PN2/AbetaPP in vivo, we performed measurements of carotid artery thrombosis and experimental intracerebral hemorrhage in transgenic mice with specific and modest overexpression of PN2/AbetaPP in brain. Comparisons were made with wild-type mice and Tg-rPF4/APP mice, a model that possesses specific and modest overexpression of PN2/AbetaPP in platelets and exhibits reduced thrombosis in vivo. RESULTS: Modest overexpression of PN2/AbetaPP in transgenic mouse brain had no effect on intraluminal carotid arterial thrombosis but resulted in larger hematoma volumes and hemoglobin levels (23.1+/-2.7 mm(3) [n=6; P<0.01] and 1411+/-202 microg/hemisphere [n=12; P<0.01], respectively), compared with wild-type mice (15.9+/-2.2 mm(3) [n=6] and 935+/-418 microg/hemisphere [n=12], respectively). CONCLUSIONS: These findings indicate that cerebral PN2/AbetaPP plays a significant role in regulating thrombosis in brain and that modest age-related increases in the cerebral levels of this protein could markedly enhance the extent of cerebral hemorrhage.

Reducing cerebral microvascular amyloid-beta protein deposition diminishes regional neuroinflammation in vasculotropic mutant amyloid precursor protein transgenic mice.

Cerebral microvascular amyloid-beta (Abeta) protein deposition is emerging as an important contributory factor to neuroinflammation and dementia in Alzheimer's disease and related familial cerebral amyloid angiopathy disorders. In particular, cerebral microvascular amyloid deposition, but not parenchymal amyloid, is more often correlated with dementia. Recently, we generated transgenic mice (Tg-SwDI) expressing the vasculotropic Dutch (E693Q)/Iowa (D694N) mutant human Abeta precursor protein in brain that accumulate abundant cerebral microvascular fibrillar amyloid deposits. In the present study, our aim was to assess how the presence or absence of fibrillar Abeta deposition in the cerebral microvasculature affects neuroinflammation in Tg-SwDI mice. Using Tg-SwDI mice bred onto an apolipoprotein E gene knock-out background, we found a strong reduction of fibrillar cerebral microvascular Abeta deposition, which was accompanied by a sharp decrease in microvascular-associated neuroinflammatory cells and interleukin-1beta levels. Quantitative immunochemical measurements showed that this reduction of the neuroinflammation occurred in the absence of lowering the levels of total Abeta40/Abeta42 or soluble Abeta oligomers in brain. These findings suggest that specifically reducing cerebral microvascular fibrillar Abeta deposition, in the absence of lowering either the total amount of Abeta or soluble Abeta oligomers in brain, may be sufficient to ameliorate microvascular amyloid-associated neuroinflammation.

Deficient cerebral clearance of vasculotropic mutant Dutch/Iowa Double A beta in human A betaPP transgenic mice.

Cerebral amyloid angiopathy (CAA) is a prominent pathological feature of Alzheimer's disease and related familial CAA disorders. However, the mechanisms that account for the cerebral vascular accumulation of amyloid beta-peptide (A beta) have not been defined. Recently, we reported novel transgenic mice (Tg-SwDI) expressing neuronally derived Swedish/Dutch/Iowa vasculotropic mutant human A beta precursor (A betaPP) that develop early-onset and robust accumulation of fibrillar cerebral microvascular A beta. Deficient clearance of Dutch/Iowa mutant A beta from brain across the capillary blood-brain barrier into the circulation may contribute to its potent cerebral accumulation. To further evaluate this theory, we generated a new transgenic mouse (Tg-Sw) that is nearly identical to Tg-SwDI, except lacking the Dutch/Iowa A beta mutations. Tg-Sw and Tg-SwDI mice expressed comparable levels of human A betaPP in brain and not in peripheral tissues. However, Tg-SwDI mice strongly accumulated Dutch/Iowa mutant A beta in brain, particularly in the cerebral microvasculature, whereas Tg-Sw mice exhibited no accumulations of wild-type A beta. Conversely, Tg-SwDI mice had no detectable Dutch/Iowa mutant A beta in plasma whereas Tg-Sw mice exhibited consistent levels of human wild-type A beta in plasma. Together, these findings suggest that while wild-type A beta is readily transported out of brain into plasma, Dutch/Iowa mutant A beta is deficient in this clearance process, likely contributing to its robust accumulation in the cerebral vasculature.

Dexamethasone diminishes the pro-inflammatory and cytotoxic effects of amyloid beta-protein in cerebrovascular smooth muscle cells.

BACKGROUND: Cerebrovascular deposition of fibrillar amyloid beta-protein (Abeta), a condition known as cerebral amyloid angiopathy (CAA), is a prominent pathological feature of Alzheimer's disease (AD) and related disorders. Accumulation of cerebral vascular fibrillar Abeta is implicated in promoting local neuroinflammation, causes marked degeneration of smooth muscle cells, and can lead to loss of vessel wall integrity with hemorrhage. However, the relationship between cerebral vascular fibrillar Abeta-induced inflammatory responses and localized cytotoxicity in the vessel wall remains unclear.Steroidal-based anti-inflammatory agents, such as dexamethasone, have been reported to reduce neuroinflammation and hemorrhage associated with CAA. Nevertheless, the basis for the beneficial effects of steroidal anti-inflammatory drug treatment with respect to local inflammation and hemorrhage in CAA is unknown. The cultured human cerebrovascular smooth muscle (HCSM) cell system is a useful in vitro model to study the pathogenic effects of Abeta in CAA. To examine the possibility that dexamethasone may influence CAA-induced cellular pathology, we investigated the effect of this anti-inflammatory agent on inflammatory and cytotoxic responses to Abeta by HCSM cells. METHODS: Primary cultures of HCSM cells were treated with or without pathogenic Abeta in the presence or absence of the steroidal anti-inflammatory agent dexamethasone or the non-steroidal anti-inflammatory drugs indomethacin or ibuprofen. Cell viability was measured using a fluorescent live cell/dead cell assay. Quantitative immunoblotting was performed to determine the amount of cell surface Abeta and amyloid beta-protein precursor (AbetaPP) accumulation and loss of vascular smooth cell alpha actin. To assess the extent of inflammation secreted interleukin-6 (IL-6) levels were measured by ELISA and active matrix metalloproteinase-2 (MMP-2) levels were evaluated by gelatin zymography. RESULTS: Pathogenic Abeta-induced HCSM cell death was markedly reduced by dexamethasone but was unaffected by ibuprofen or indomethacin. Dexamethasone had no effect on the initial pathogenic effects of Abeta including HCSM cell surface binding, cell surface fibril-like assembly, and accumulation of cell surface AbetaPP. However, later stage pathological consequences of Abeta treatment associated with inflammation and cell degeneration including increased levels of IL-6, activation of MMP-2, and loss of HCSM alpha actin were significantly diminished by dexamethasone but not by indomethacin or ibuprofen. CONCLUSION: Our results suggest that although dexamethasone has no appreciable consequence on HCSM cell surface fibrillar Abeta accumulation it effectively reduces the subsequent pathologic responses including elevated levels of IL-6, MMP-2 activation, and depletion of HCSM alpha actin. Dexamethasone, unlike indomethacin or ibuprofen, may diminish these pathological processes that likely contribute to inflammation and loss of vessel wall integrity leading to hemorrhage in CAA.

Human apolipoprotein E2 promotes parenchymal amyloid deposition and neuronal loss in vasculotropic mutant amyloid-ß protein Tg-SwDI mice.

Human apolipoprotein (ApoE) genotype influences the development of Alzheimer's disease and cerebral amyloid angiopathy (CAA), where the ε4 allele increases and the ε2 allele decreases the risk for developing disease. Specific mutations within the amyloid-ß (Aß) peptide have been identified that cause familial forms of CAA. However, the influence of APOE genotype on accumulation of CAA mutant Aß in brain is not well understood. Earlier, we showed that human ApoE4 redistributes fibrillar amyloid deposition from the cerebral microvasculature to parenchymal plaques in Tg-SwDI mice, a model that accumulates human Dutch/Iowa (E22Q/D23N) CAA mutant Aß in brain (Xu et al., J Neurosci 28, 5312-5320, 2008). Human ApoE2 can reduce Aß pathology in transgenic models of parenchymal plaques. Here we determined if human ApoE2 can influence the location and severity of amyloid pathology in Tg-SwDI mice. Comparing Tg-SwDI mice bred onto a human APOE2/2 or human APOE4/4 background, we found there was no change in the brain levels of total Aß(40) and Aß(42) compared to mice on the endogenous mouse APOE background. In Tg-SwDI mice on either human APOE background, there was a similarly strong reduction in the levels of microvascular CAA and emergence of extensive parenchymal plaque amyloid. In both Tg-SwDI-hAPOE2/2 and Tg-SwDI-hAPOE4/4 mice, the distribution of ApoE proteins and neuronal loss were associated with parenchymal amyloid plaques. These findings suggest that compared with human ApoE4, human ApoE2 does not beneficially influence the quantitative or spatial accumulation of human Dutch/Iowa CAA mutant amyloid or associated pathology in transgenic mice.

Protease nexin-2/amyloid beta-protein precursor limits cerebral thrombosis.

The amyloid beta-protein precursor (AbetaPP) is best known as the parent molecule to the amyloid beta-peptide that accumulates in the brains of patients with Alzheimer's disease. Secreted isoforms of AbetaPP that contain the Kunitz proteinase inhibitor domain are analogous to the previously identified cell-secreted proteinase inhibitor known as protease nexin-2 (PN2). Although PN2/AbetaPP is enriched in brain and in circulating blood platelets, little is understood of its physiological function and potential role in disease processes outside of amyloid beta-peptide generation. We hypothesized that the potent inhibition of certain procoagulant proteinases by PN2/AbetaPP, coupled with its abundance in platelets and brain, indicate that it may function to regulate cerebral thrombosis. Here we show that specific and modest 2-fold overexpression of PN2/AbetaPP in circulating platelets of transgenic mice caused a marked inhibition of thrombosis in vivo. In contrast, deletion of PN2/AbetaPP in AbetaPP gene knockout mice resulted in a significant increase in thrombosis. Similarly, platelet PN2/AbetaPP transgenic mice developed larger hematomas in experimental intracerebral hemorrhage, whereas AbetaPP gene knockout mice exhibited reduced hemorrhage size. These findings indicate that PN2/AbetaPP plays a significant role in regulating cerebral thrombosis and that modest increases in this protein can profoundly enhance cerebral hemorrhage.

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.