Cerebral Amyloid Angiopathy Formation Research Articles

Astrocytic APOE4 removal confers cerebrovascular protection despite increased cerebral amyloid angiopathy

BackgroundAlzheimer Disease (AD) and cerebral amyloid angiopathy (CAA) are both characterized by amyloid-β (Aβ) accumulation in the brain, although Aβ deposits mostly in the brain parenchyma in AD and in the cerebrovasculature in CAA. The presence of CAA can exacerbate clinical outcomes of AD patients by promoting spontaneous intracerebral hemorrhage and ischemia leading to CAA-associated cognitive decline. Genetically, AD and CAA share the ε4 allele of the apolipoprotein E (APOE) gene as the strongest genetic risk factor. Although tremendous efforts have focused on uncovering the role of APOE4 on parenchymal plaque pathogenesis in AD, mechanistic studies investigating the role of APOE4 on CAA are still lacking. Here, we addressed whether abolishing APOE4 generated by astrocytes, the major producers of APOE, is sufficient to ameliorate CAA and CAA-associated vessel damage.MethodsWe generated transgenic mice that deposited both CAA and plaques in which APOE4 expression can be selectively suppressed in astrocytes. At 2-months-of-age, a timepoint preceding CAA and plaque formation, APOE4 was removed from astrocytes of 5XFAD APOE4 knock-in mice. Mice were assessed at 10-months-of-age for Aβ plaque and CAA pathology, gliosis, and vascular integrity.ResultsReducing the levels of APOE4 in astrocytes shifted the deposition of fibrillar Aβ from the brain parenchyma to the cerebrovasculature. However, despite increased CAA, astrocytic APOE4 removal reduced overall Aβ-mediated gliosis and also led to increased cerebrovascular integrity and function in vessels containing CAA.ConclusionIn a mouse model of CAA, the reduction of APOE4 derived specifically from astrocytes, despite increased fibrillar Aβ deposition in the vasculature, is sufficient to reduce Aβ-mediated gliosis and cerebrovascular dysfunction.

Fruquintinib/HMPL-013 ameliorates cognitive impairments and pathology in a mouse model of cerebral amyloid angiopathy (CAA)

Cerebral amyloid angiopathy (CAA) is characterized by the cerebrovascular amyloid-β (Aβ) accumulation, and always accompanied by Alzheimer's disease (AD). The mechanisms revealing CAA pathogenesis are still unclear, and it is challenging to develop an efficient therapeutic strategy for its treatment. Vascular endothelial growth factor (VEGF) and its receptors including VEGFR-1,-2,-3 activation are involved in Aβ processing, and modulate numerous cellular events associated with central nervous system (CNS) diseases. In the present study, we attempted to explore the regulatory function of fruquintinib (also named as HMPL-013), a highly selective inhibitor of VEGFR-1,-2,-3 tyrosine kinases, on CAA progression in Tg-SwDI mice. Here, we found that HMPL-013-rich diet consumption for 12 months significantly improved the behavioral performances and cerebral blood flow (CBF) of Tg-SwDI mice compared with the vehicle group. Importantly, HMPL-013 administration considerably reduced Aβ1-40 and Aβ1-42 burden in cortex and hippocampus of Tg-SwDI mice through regulating Aβ metabolism process. Congo red staining confirmed Aβ deposition in vessel walls, reflecting CAA formation, which was, however, strongly ameliorated after HMPL-013 treatment. Neuron death, aberrant glial activation and pro-inflammatory response in brain tissues of Tg-SwDI mice were dramatically alleviated after HMPL-013 consumption. More studies showed that the protective effects of HMPL-013 against CAA might be partially attributed to its regulation on the expression of genes associated with blood vasculature. Intriguingly, VEGF and phosphorylated VEGFR-1,-2 protein expression levels were remarkably decreased by HMPL-013 in cortex and hippocampus of Tg-SwDI mice, which were validated in HMPL-013-treated brain vascular endothelial cells (BVECs) under hypoxia. Finally, we found that VEGF-induced human umbilical vein endothelial cells (HUVEC) proliferation and tube formation were strongly abolished upon HMPL-013 exposure. Collectively, all these findings demonstrated that oral administration of HMPL-013 had therapeutic potential against CAA by reducing Aβ deposition, inflammation and neuron death via suppressing VEGF/VEGFR-1,-2 signaling.

Microglia-specific ApoE knock-out does not alter Alzheimer's disease plaque pathogenesis or gene expression.

Previous studies suggest that microglial-expressed Apolipoprotein E (ApoE) is necessary to shift microglia into a neurodegenerative transcriptional state in Alzheimer's disease (AD) mouse models. On the other hand, elimination of microglia shifts amyloid beta (Aβ) accumulation from parenchymal plaques to cerebral amyloid angiopathy (CAA), mimicking the effects of global APOE*4 knock-in. Here, we specifically knock-out microglial-expressed ApoE while keeping astrocytic-expressed ApoE intact. When microglial-specific ApoE is knocked-out of a 5xFAD mouse model of AD, we found a ~35% increase in average Aβ plaque size, but no changes in plaque load, microglial number, microglial clustering around Aβ plaques, nor the formation of CAA. Immunostaining revealed ApoE protein present in plaque-associated microglia in 5xFAD mice with microglial-specific ApoE knockout, suggesting that microglia can take up ApoE from other cellular sources. Mice with Apoe knocked-out of microglia had lower synaptic protein levels than control mice, indicating that microglial-expressed ApoE may have a role in synapse maintenance. Surprisingly, microglial-specific ApoE knock-out resulted in few differentially expressed genes in both 5xFAD and control mice; however, some rescue of 5xFAD associated neuronal networks may occur with microglial-specific ApoE knock-out as shown by weighted gene co-expression analysis. Altogether, our data indicates that microglial-expressed ApoE may not be necessary for plaque formation or for the microglial transcriptional shift into a Disease Associated Microglia state that is associated with reactivity to plaques but may be necessary for plaque homeostasis in disease and synaptic maintenance under normal conditions.

Impact of Amyloid-β on Platelet Mitochondrial Function and Platelet-Mediated Amyloid Aggregation in Alzheimer's Disease.

Background: Alzheimer’s disease (AD) is characterized by an accumulation of amyloid β (Aβ) peptides in the brain and mitochondrial dysfunction. Platelet activation is enhanced in AD and platelets contribute to AD pathology by their ability to facilitate soluble Aβ to form Aβ aggregates. Thus, anti-platelet therapy reduces the formation of cerebral amyloid angiopathy in AD transgenic mice. Platelet mitochondrial dysfunction plays a regulatory role in thrombotic response, but its significance in AD is unknown and explored herein. Methods: The effects of Aβ-mediated mitochondrial dysfunction in platelets were investigated in vitro. Results: Aβ40 stimulation of human platelets led to elevated reactive oxygen species (ROS) and superoxide production, while reduced mitochondrial membrane potential and oxygen consumption rate. Enhanced mitochondrial dysfunction triggered platelet-mediated Aβ40 aggregate formation through GPVI-mediated ROS production, leading to enhanced integrin αIIbβ3 activation during synergistic stimulation from ADP and Aβ40. Aβ40 aggregate formation of human and murine (APP23) platelets were comparable to controls and could be reduced by the antioxidant vitamin C. Conclusions: Mitochondrial dysfunction contributes to platelet-mediated Aβ aggregate formation and might be a promising target to limit platelet activation exaggerated pathological manifestations in AD.

Efficacy and Safety of Direct Oral Anticoagulant for Treatment of Atrial Fibrillation in Cerebral Amyloid Angiopathy

A 75-year-old man with a history of atrial fibrillation (AF) and anticoagulant therapy presented with a headache. Cerebral amyloid angiopathy (CAA) was diagnosed after MRI of the brain revealed cortical superficial siderosis, lobar intracerebral hemorrhage, and lobar microbleeds. Anticoagulant therapy was carefully discontinued. Several years later, he was admitted with sudden onset left upper-extremity weakness. In addition to CAA bleeding lesions, a diffusion-weighted brain MRI showed multiple infarct lesions of high signal intensity. The administration of edoxaban 7.5 mg/day (later increased up to 30 mg/day) prevented ischemic stroke recurrence without exacerbation of cerebral bleeding. This could indicate that CAA patients with AF who had previous adverse effects from warfarin can safely use newer direct oral anticoagulants, such as edoxaban, to prevent ischemic stroke without danger of cerebral hemorrhage. The superiority of edoxaban as compared with warfarin might be due to its antioxidant effect because vascular oxidative stress plays a causal role in CAA-induced cerebrovascular dysfunction, CAA-induced cerebral hemorrhage, and CAA formation itself. We explained the beneficial effect of edoxaban for CAA by the mechanism of oxidative stress in the paper.

Intracisternal injection of beta-amyloid seeds promotes cerebral amyloid angiopathy

Beta amyloid (Aβ) is a key component of parenchymal Aβ plaques and vascular Aβ fibrils, which lead to cerebral amyloid angiopathy (CAA) in Alzheimer’s disease (AD). Recent studies have revealed that Aβ contained in the cerebrospinal fluid (CSF) can re-enter into brain through paravascular spaces. However, whether Aβ in CSF may act as a constant source of pathogenic Aβ in AD is still unclear. This study aimed to examine whether Aβ pathology could be worsened when CSF Aβ level was enhanced by intra-cisternal infusion of aged brain extract containing abundant Aβ in TgCRND8 host mice. TgCRND8 mouse is an AD animal model which develops predominant parenchymal Aβ plaques in the brain at as early as 3 months of age. Here, we showed that single intracisternal injection of Aβ seeds into TgCRND8 mice before the presence of Aβ pathology induced robust prion-like propagation of CAA within 90 days. The induced CAA is mainly distributed in the cerebral cortex, hippocampus and thalamus of TgCRND8 mice. Surprisingly, despite the robust increase in CAA levels, the TgCRND8 mice had a marked decrease in parenchymal Aβ plaques and the plaques related neuroinflammation in the brains compared with the control mice. These results amply indicate that Aβ in CSF may act as a source of Aβ contributing to the growth of vascular Aβ deposits in CAA. Our findings provide experimental evidence to unravel the mechanisms of CAA formation and the potential of targeting CSF Aβ for CAA.

Cerebral amyloid angiopathy-linked β-amyloid mutations promote cerebral fibrin deposits via increased binding affinity for fibrinogen

Cerebral amyloid angiopathy (CAA), where beta-amyloid (Aβ) deposits around cerebral blood vessels, is a major contributor of vascular dysfunction in Alzheimer's disease (AD) patients. However, the molecular mechanism underlying CAA formation and CAA-induced cerebrovascular pathology is unclear. Hereditary cerebral amyloid angiopathy (HCAA) is a rare familial form of CAA in which mutations within the (Aβ) peptide cause an increase in vascular deposits. Since the interaction between Aβ and fibrinogen increases CAA and plays an important role in cerebrovascular damage in AD, we investigated the role of the Aβ-fibrinogen interaction in HCAA pathology. Our work revealed the most common forms of HCAA-linked mutations, Dutch (E22Q) and Iowa (D23N), resulted in up to a 50-fold stronger binding affinity of Aβ for fibrinogen. In addition, the stronger interaction between fibrinogen and mutant Aβs led to a dramatic perturbation of clot structure and delayed fibrinolysis. Immunofluorescence analysis of the occipital cortex showed an increase of fibrin(ogen)/Aβ codeposition, as well as fibrin deposits in HCAA patients, compared to early-onset AD patients and nondemented individuals. Our results suggest the HCAA-type Dutch and Iowa mutations increase the interaction between fibrinogen and Aβ, which might be central to cerebrovascular pathologies observed in HCAA.

Origins of Beta Amyloid Differ Between Vascular Amyloid Deposition and Parenchymal Amyloid Plaques in the Spinal Cord of a Mouse Model of Alzheimer's Disease.

Cerebral amyloid angiopathy (CAA) refers to pathological changes occurring in cerebral blood vessels caused by deposition of beta amyloid (Aβ) protein. However, the mechanisms involved in the origin of Aβ for the formation of CAA and its link to parenchymal amyloid depositions remained to be unraveled. Here, we found CAA and parenchymal plaques distributed separately instead of mingling with each other in the spinal cord of TgCRND8 mice. Parenchymal plaques predominantly located in the dorsal horn whereas CAA distributed in the ventral horn. We further found that the ratio of Aβ40/Aβ42 was significantly higher in the ventral than that in the dorsal by ELISA assay, suggesting that origin of Aβ forming parenchymal plaques may be different from that of CAA in the spinal cord. This hypothesis was further demonstrated by the surgical methods which indicated eliminating parenchymal plaques did not alter CAA in the affected spinal cord. We also examined the ratio of Aβ40/Aβ42 in the cerebral spinal fluid (CSF) in order to identify the origin of the CAA formation, and found the Aβ40/Aβ42 ratio was similar to that of CAA formation in the ventral horn. We further demonstrated that CSF tracer distributed along ventral horn vessels, in exactly the same pattern as Aβ deposition in CAA in ventral part of spinal cord. These findings verified the concept that CSF influx may act as a constant source for delivering Aβ, and contribute to the growth of paraarterial deposits in CAA. Taken together, the results of the present study highlight the important role of the Aβ40/Aβ42 ratio in determining vascular versus parenchymal amyloid deposition. Unlike parenchymal plaques, Aβ of CAA comes from CSF; thus, manipulation of CSF Aβ could represent a novel strategy to treat CAA.

Drusen in the Peripheral Retina of the Alzheimer's Eye.

Recent work on Alzheimer's disease (AD) diagnosis focuses on neuroimaging modalities; however, these methods are expensive, invasive, and not available to all patients. Ocular imaging of biomarkers, such as drusen in the peripheral retina, could provide an alternative method to diagnose AD. This study compares macular and peripheral drusen load in control and AD eyes. Postmortem eye tissues were obtained from donors with a neuropathological diagnosis of AD. Retina from normal donors were processed and categorized into younger (<55 years) and older (>55 years) groups. After fixation and dissection, 3-6 mm punches of RPE/choroid were taken in macular and peripheral (temporal, superior, and inferior) retinal regions. Oil red O positive drusen were counted and grouped into two size categories: small (<63 μm) and intermediate (63-125 μm). There was a significant increase in the total number of macular and peripheral hard drusen in older, compared to younger, normal eyes (p<0.05). Intermediate hard drusen were more commonly found in the temporal region of AD eyes compared to older normal eyes, even after controlling for age (p<0.05). Among the brain and eye tissues from AD donors, there was a significant relationship between cerebral amyloid angiopathy (CAA) severity and number of temporal intermediate hard drusen (r=0.78, p<0.05). Imaging temporal drusen in the eye may have benefit for diagnosing and monitoring progression of AD. Our results on CAA severity and temporal intermediate drusen in the AD eye are novel. Future studies are needed to further understand the interactions among CAA and drusen formation.

Murine versus human apolipoprotein E4: differential facilitation of and co-localization in cerebral amyloid angiopathy and amyloid plaques in APP transgenic mouse models.

IntroductionAmyloid β (Aβ) accumulates in the extracellular space as diffuse and neuritic plaques in Alzheimer’s disease (AD). Aβ also deposits on the walls of arterioles as cerebral amyloid angiopathy (CAA) in most cases of AD and sometimes independently of AD. Apolipoprotein E (apoE) ɛ4 is associated with increases in both Aβ plaques and CAA in humans. Studies in mouse models that develop Aβ deposition have shown that murine apoE and human apoE4 have different abilities to facilitate plaque or CAA formation when studied independently. To better understand and compare the effects of murine apoE and human apoE4, we bred 5XFAD (line 7031) transgenic mice so that they expressed one copy of murine apoE and one copy of human apoE4 under the control of the normal murine apoE regulatory elements (5XFAD/apoEm/4). ResultsThe 5XFAD/apoEm/4 mice contained levels of parenchymal CAA that were intermediate between 5XFAD/apoEm/m and 5XFAD/apoE4/4 mice. In 5XFAD/apoEm/4 mice, we found that Aβ parenchymal plaques co-localized with much more apoE than did parenchymal CAA, suggesting differential co-aggregation of apoE with Aβ in plaques versus CAA. More importantly, within the brain parenchyma of the 5XFAD/apoEm/4 mice, plaques contained more murine apoE, which on its own results in more pronounced and earlier plaque formation, while CAA contained more human apoE4 which on its own results in more pronounced CAA formation. We further confirmed the co-aggregation of mouse apoE with Aβ in plaques by showing a strong correlation between insoluble mouse apoE and insoluble Aβ in PS1APP-21/apoEm/4 mice which develop plaques without CAA.ConclusionsThese studies suggest that both murine apoE and human apoE4 facilitate differential opposing effects in influencing Aβ plaques versus CAA via different co-aggregation with these two amyloid lesions and set the stage for understanding these effects at a molecular level.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-015-0250-y) contains supplementary material, which is available to authorized users.

Contribution of reactive oxygen species to cerebral amyloid angiopathy, vasomotor dysfunction, and microhemorrhage in aged Tg2576 mice

Cerebral amyloid angiopathy (CAA) is characterized by deposition of amyloid β peptide (Aβ) within walls of cerebral arteries and is an important cause of intracerebral hemorrhage, ischemic stroke, and cognitive dysfunction in elderly patients with and without Alzheimer's Disease (AD). NADPH oxidase-derived oxidative stress plays a key role in soluble Aβ-induced vessel dysfunction, but the mechanisms by which insoluble Aβ in the form of CAA causes cerebrovascular (CV) dysfunction are not clear. Here, we demonstrate evidence that reactive oxygen species (ROS) and, in particular, NADPH oxidase-derived ROS are a key mediator of CAA-induced CV deficits. First, the NADPH oxidase inhibitor, apocynin, and the nonspecific ROS scavenger, tempol, are shown to reduce oxidative stress and improve CV reactivity in aged Tg2576 mice. Second, the observed improvement in CV function is attributed both to a reduction in CAA formation and a decrease in CAA-induced vasomotor impairment. Third, anti-ROS therapy attenuates CAA-related microhemorrhage. A potential mechanism by which ROS contribute to CAA pathogenesis is also identified because apocynin substantially reduces expression levels of ApoE-a factor known to promote CAA formation. In total, these data indicate that ROS are a key contributor to CAA formation, CAA-induced vessel dysfunction, and CAA-related microhemorrhage. Thus, ROS and, in particular, NADPH oxidase-derived ROS are a promising therapeutic target for patients with CAA and AD.

Contribution of blood platelets to vascular pathology in Alzheimer&amp;#39;s disease

Cerebral amyloid angiopathy (CAA) is a critical factor in the pathogenesis of Alzheimer’s disease (AD). In the clinical setting, nearly 98% AD patients have CAA, and 75% of these patients are rated as severe CAA. It is characterized by the deposition of the β-amyloid peptide (mainly Aβ40) in the walls of cerebral vessels, which induces the degeneration of vessel wall components, reduces cerebral blood flow, and aggravates cognitive decline. Platelets are anuclear cell fragments from bone marrow megakaryocytes and their function in hemostasis and thrombosis has long been recognized. Recently, increasing evidence suggests that platelet activation can also mediate the onset and development of CAA. First, platelet activation and adhesion to a vessel wall is the initial step of vascular injury. Activated platelets contribute to more than 90% circulating Aβ (mainly Aβ1-40), which in turn activates platelets and results in the vicious cycle of Aβ overproduction in damaged vessel. Second, the uncontrolled activation of platelets leads to a chronic inflammatory reaction by secretion of chemokines (eg, platelet factor 4 [PF4], regulated upon activation normal T-cell expressed and presumably secreted [RANTES], and macrophage inflammatory protein [MIP-1α]), interleukins (IL-1β, IL-7, and IL-8), prostaglandins, and CD40 ligand (CD40L). The interaction of these biological response modulators with platelets, endothelial cells, and leukocytes establishes a localized inflammatory response that contributes to CAA formation. Finally, activated platelets are the upholder of fibrin clots, which are structurally abnormal and resistant to degradation in the presence of Aβ42. Thus, opinion has emerged that targeting blood platelets may provide a new avenue for anti-AD therapy.

Modeling Alzheimer's disease using iPS cells reveals stress phenotypes associated with intracellular beta-amyloid oligomer and differential drug responsiveness

Background:Aberrant metabolism of amyloid b peptide (A b) results in the formation of amyloid plaques (APs), which are pathological hallmarks of Alzheimer’s disease (AD). Most cases of AD are also associated with cerebral amyloid angiopathy (CAA) which is characterized by A b deposition around brain blood microvessels. The molecular and cellular mechanism underlying the formation of APs and CAA remain unclear. In particular, the neural cell(s) responsible for the formation of these amyloid deposits has not yet been identified, although neurons are currently believed to be the major source of A b. However, this view lacks concrete evidence. In fact, reactive astrocytes have been shown to be very active for the production of A b, and brain vascular microvessel endothelial cells (ECs) and brain vascular smooth muscle cells (BVSMCs) can produce A b. In this study, we further characterized the A b production in ECs in terms of the pathogenesis of CAA. Methods: Human umbilical vein endothelial cells were used as a model of ECs. A b is adhesive and can bind to the extracellular matrix (EM). As the EM is more abundant at a high cell density, the level of free A b released from ECs may be affected by the cell density.To test this hypothesis, ECs were cultured at different cell densities, and the level of A b in the culture medium was analyzed by ELISA. Results: The Ab production in ECs was 2-4-fold higher at a low cell density than at a high cell density, where A b degrading activity was detected in the culture medium. Conclusions: The A b production in ECs was dependent on the cell density and it was suppressed at a high cell density, where physiologically-relevant cell-cell interactions in bloodmicrovessels may occur. Even under such conditions, the A b production of ECs was more than 100-fold higher than that of human BVSMCs cultured under similar conditions. These findings suggest that ECs, rather than the BVSMCs, play a major causative role in CAA, although the contribution of pericytes and astroglial cells, which are other components of the brain microvessels, remains unclear.

Extracellular matrix modulator lysyl oxidase colocalizes with amyloid-beta pathology in Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis—Dutch type

Accumulation of amyloid-beta (Aβ) in brain vessel walls and parenchyma, known as cerebral amyloid angiopathy (CAA) and senile plaques (SPs), respectively, plays a key role in Alzheimer's disease (AD) and hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D) pathogenesis. Although the mechanisms underlying CAA and SP formation remain largely unknown, evidence is mounting that local alterations of the extracellular matrix (ECM) in the brain vessel wall and/or parenchyma play an important role. Lysyl oxidase (LOX, E.C. 1.4.3.13) is an inducible amine oxidase that modulates the ECM by catalyzing the formation of molecular covalent cross-links in ECM proteins. The aim of this study is to investigate the association of LOX with CAA and with classic and diffuse SPs in both AD and HCHWA-D cases. We observed an association of LOX with Aβ in CAA and with Aβ in both classic and diffuse SPs in AD and HCHWA-D cases. In addition, LOX staining was observed in reactive astrocytes associated with these lesions. We conclude that the ECM modulating enzyme LOX is associated with the Aβ-related pathological hallmarks of both AD and HCHWA-D, and that our findings provide additional insights into the mechanisms underlying the formation of these lesions.

Pathophysiology of the Vascular Wall and its Relevance for Cerebrovascular Disorders in Aged Rodents

Chronic hypertension and cerebral amyloid angiopathy (CAA) are the main pathologies which can induce the rupture of cerebral vessels and intracerebral hemorrhages, as a result of degenerative changes in the vascular wall. A lot of progress has been made in this direction since the successful creation of the first mouse model for the study of Alzheimer's disease (AD), as the spectrum of AD pathology includes a plethora of changes found in pure cerebrovascular diseases. We describe here some of these mouse models having important vascular changes that parallel human AD pathology, and more importantly, we show how these models have helped us understand more about the mechanisms that lead to CAA formation. An important cellular event associated with reduced structural and functional recovery after stroke in aged animals is the early formation of a scar in the infarcted region that impairs subsequent neural recovery and repair. We review recent evidence showing that the rapid formation of the glial scar following stroke in aged rats is associated with premature cellular proliferation that originates primarily from the walls of capillaries in the corpus callosum adjacent to the infarcted region. After stroke several vascular mechanisms are turned-on immediately to protect the brain from further damage and help subsequent neuroregeneration and functional recovery. Although does occur after stroke, vasculogenesis is overshadowed in its protective/restorative role by the angiogenesis and arteriogenesis. Understanding the basic mechanisms underlying functional recovery after cerebral stroke in aging subjects is likely to yield new insights into the treatment of brain injury in the clinic.

Apolipoprotein E protects cultured pericytes and astrocytes from D-Aβ 1–40-mediated cell death

Cerebral amyloid angiopathy (CAA) is a common pathological finding in Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis of the Dutch type; in this latter condition it is caused by deposition of mutated amyloid β protein (Aβ Glu22Gln; D-Aβ 1–40). Previously, we found a dependence of the Aβ-mediated toxicity and apolipoprotein E (apoE) production by cultured pericytes on apoE genotype. Given their close association with the cerebrovascular wall both astrocytes and pericytes may be involved in CAA development, a process that includes Aβ deposition and clearance and that may be affected by interaction with locally produced apolipoprotein E (apoE). Although astrocytes are regarded as the major source of apolipoprotein E (apoE) in the brain, also pericytes produce apoE. In this study we compared the apoE production capacity, the effects of apoE on D-Aβ 1–40 internalization, D-Aβ 1–40 cell surface accumulation and the vulnerability for D-Aβ 1–40-induced toxicity of either cell type in order to quantify the relative contributions of astrocytes and pericytes in the various processes that contribute to CAA formation. Strikingly, cultured astrocytes produced only 3–10% of the apoE amounts produced by pericytes. Furthermore, pericytes with the apoE ɛ4 allele produced three times less apoE and were more vulnerable to D-Aβ 1–40 treatment than pericytes without an ɛ4 allele. Such relations were not observed with astrocytes in vitro. Both pericytes and astrocytes, however, were protected from Aβ-induced cytotoxicity by high levels of pericyte-derived apoE, but not recombinant apoE. In addition, pericyte-derived apoE dose-dependently decreased both internalization of Aβ and Aβ accumulation at the cell surface in either cell type. The present data suggest that apoE produced by pericytes, rather than astrocyte-produced apoE, modulates Aβ cytotoxicity and Aβ removal near the vasculature in the brain. Furthermore, since apoE production in pericytes is genotype dependent, this may contribute to the apoE genotype-dependent development of CAA in vivo.

Transglutaminases and Transglutaminase‐Catalyzed Cross‐Links Colocalize with the Pathological Lesions in Alzheimer's Disease Brain

Alzheimer's disease (AD) is characterized by pathological lesions, in particular senile plaques (SPs), cerebral amyloid angiopathy (CAA) and neurofibrillary tangles (NFTs), predominantly consisting of self-aggregated proteins amyloid beta (Abeta) and tau, respectively. Transglutaminases (TGs) are inducible enzymes, capable of modifying conformational and/or structural properties of proteins by inducing molecular covalent cross-links. Both Abeta and tau are substrates for TG cross-linking activity, which links TGs to the aggregation process of both proteins in AD brain. The aim of this study was to investigate the association of transglutaminase 1 (TG1), transglutaminase 2 (TG2) and TG-catalyzed cross-links with the pathological lesions of AD using immunohistochemistry. We observed immunoreactivity for TG1, TG2 and TG-catalyzed cross-links in NFTs. In addition, both TG2 and TG-catalyzed cross-links colocalized with Abeta in SPs. Furthermore, both TG2 and TG-catalyzed cross-links were associated with CAA. We conclude that these TGs demonstrate cross-linking activity in AD lesions, which suggests that both TG1 and TG2 are likely involved in the protein aggregation processes underlying the formation of SPs, CAA and/or NFTs in AD brain.

Transcriptomic and genetic studies identify IL-33 as a candidate gene for Alzheimer's disease

The only recognized genetic determinant of the common forms of Alzheimer's disease (AD) is the epsilon 4 allele of the apolipoprotein E gene (APOE). To identify new candidate genes, we recently performed transcriptomic analysis of 2741 genes in chromosomal regions of interest using brain tissue of AD cases and controls. From 82 differentially expressed genes, 1156 polymorphisms were genotyped in two independent discovery subsamples (n=945). Seventeen genes exhibited at least one polymorphism associated with AD risk, and following correction for multiple testing, we retained the interleukin (IL)-33 gene. We first confirmed that the IL-33 expression was decreased in the brain of AD cases compared with that of controls. Further genetic analysis led us to select three polymorphisms within this gene, which we analyzed in three independent case-control studies. These polymorphisms and a resulting protective haplotype were systematically associated with AD risk in non-APOE epsilon 4 carriers. Using a large prospective study, these associations were also detected when analyzing the prevalent and incident AD cases together or the incident AD cases alone. These polymorphisms were also associated with less cerebral amyloid angiopathy (CAA) in the brain of non-APOE epsilon 4 AD cases. Immunohistochemistry experiments finally indicated that the IL-33 expression was consistently restricted to vascular capillaries in the brain. Moreover, IL-33 overexpression in cellular models led to a specific decrease in secretion of the A beta(40) peptides, the main CAA component. In conclusion, our data suggest that genetic variants in IL-33 gene may be associated with a decrease in AD risk potentially in modulating CAA formation.

P4-160: Transglutaminase 1, Transglutaminase 2 and Transglutaminase-catalyzed cross-links colocalize with the pathological lesions in Alzheimer's disease brain

Alzheimer's disease (AD) is characterized by several pathological lesions, in particular senile plaques (SPs), cerebral amyloid angiopathy (CAA) and neurofibrillary tangles (NFTs), predominantly consisting of self-aggregated proteins amyloid-beta (Aβ) and tau, respectively. Transglutaminases (TGases) are inducible enzymes, capable of modifying the conformational and/or structural properties of proteins, such as Aβ and tau, by inducing intra- and intermolecular covalent cross-links. Both Aβ and tau are substrates for TGase cross-linking activity, which suggests that TGases play a role in the initiation and persistence of the aggregation process of both Aβ and tau in AD brains. The aim of this study was to investigate the association of Transglutaminase 1 (TG1), Transglutaminase 2 (TG2) and TG-catalyzed cross-links with the pathological lesions of AD. For this purpose, a panel of well-characterized antibodies against TG1, TG2 and the TG-catalyzed cross-links was used in immunohistochemistry. We observed the immunoreactivity for TG1, TG2 and TG-catalyzed cross-links in NFTs. In addition, both TG2 and TG-catalyzed cross-links colocalize with Aβ in SPs. Furthermore, staining of TG2 and TG-catalyzed antibodies was also observed in CAA, although specific colocalization with deposited Aβ in CAA was absent. We conclude that both TG1 and TG2 are present in SPs, CAA and NFTs, and that these enzymes display cross-linking activity in AD lesions. We suggest that both TG1 and TG2 are involved in the initiation and/or persistence of the protein aggregation processes underlying the formation of SPs and CAA, and NFTs in AD brain.

Human Apolipoprotein E4 Alters the Amyloid-β 40:42 Ratio and Promotes the Formation of Cerebral Amyloid Angiopathy in an Amyloid Precursor Protein Transgenic Model

Alzheimer's disease (AD) is characterized by the aggregation and deposition of the normally soluble amyloid-beta (Abeta) peptide in the extracellular spaces of the brain as parenchymal plaques and in the walls of cerebral vessels as cerebral amyloid angiopathy (CAA). CAA is a common cause of brain hemorrhage and is found in most patients with AD. As in AD, the epsilon4 allele of the apolipoprotein E (apoE) gene (APOE) is a risk factor for CAA. To determine the effect of human apoE on CAA in vivo, we bred human APOE3 and APOE4 "knock-in" mice to a transgenic mouse model (Tg2576) that develops amyloid plaques as well as CAA. The expression of both human apoE isoforms resulted in a delay in Abeta deposition of several months relative to murine apoE. Tg2576 mice expressing the more fibrillogenic murine apoE develop parenchymal amyloid plaques and CAA by 9 months of age. At 15 months of age, the expression of human apoE4 led to substantial CAA with very few parenchymal plaques, whereas the expression of human apoE3 resulted in almost no CAA or parenchymal plaques. Additionally, young apoE4-expressing mice had an elevated ratio of Abeta 40:42 in brain extracellular pools and a lower 40:42 ratio in CSF, suggesting that apoE4 results in altered clearance and transport of Abeta species within different brain compartments. These findings demonstrate that, once Abeta fibrillogenesis occurs, apoE4 favors the formation of CAA over parenchymal plaques and suggest that molecules or treatments that increase the ratio of Abeta 40:42 may favor the formation of CAA versus parenchymal plaques.