Lecanemab blocks the effects of the Aß/fibrinogen complex on blood clots and synapse toxicity in organotypic culture.

Proteinaceous brain inclusions, neuroinflammation, and vascular dysfunction are common pathologies in Alzheimer's disease (AD). Vascular deficits include a compromised blood-brain barrier, which can lead to extravasation of blood proteins like fibrinogen into the brain. Fibrinogen's interaction with the amyloid-beta (Aß) peptide is known to worsen thrombotic and cerebrovascular pathways in AD. Lecanemab, an FDA-approved antibody therapy for AD, clears Aß plaque from the brain and slows cognitive decline. Here, we show that lecanemab blocks fibrinogen's binding to Aß protofibrils, preventing Aß/fibrinogen-mediated delayed fibrinolysis and clot abnormalities in vitro and in human plasma. Additionally, we show that lecanemab dissociates the Aß/fibrinogen complex and prevents fibrinogen from exacerbating Aß-induced synaptotoxicity in mouse organotypic hippocampal cultures. These findings reveal a possible protective mechanism by which lecanemab may slow disease progression in AD.

A possible mechanism for the enhanced toxicity of beta-amyloid protofibrils in Alzheimer's disease.

The amyloid-beta peptide (Aß) is a driver of Alzheimer's disease (AD). Aß monomers can aggregate and form larger soluble (oligomers/protofibrils) and insoluble (fibrils) forms. There is evidence that Aß protofibrils are the most toxic form, but the reasons are not known. Consistent with a critical role for this form of Aß in AD, a recently FDA-approved therapeutic antibody targeted against protofibrils, lecanemab, slows the progression of AD in patients. The plasma contact system, which can promote coagulation and inflammation, has been implicated in AD pathogenesis. This system is activated by Aß which could lead to vascular and inflammatory pathologies associated with AD. We show here that the contact system is preferentially activated by protofibrils of Aß. Aß protofibrils bind to coagulation factor XII and high molecular weight kininogen and accelerate the activation of the system. Furthermore, lecanemab blocks Aß protofibril activation of the contact system. This work provides a possible mechanism for Aß protofibril toxicity in AD and why lecanemab is therapeutically effective.

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

Cerebral amyloid angiopathy (CAA), where beta-amyloid (Aß) 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 (Aß) peptide cause an increase in vascular deposits. Since the interaction between Aß and fibrinogen increases CAA and plays an important role in cerebrovascular damage in AD, we investigated the role of the Aß-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 Aß for fibrinogen. In addition, the stronger interaction between fibrinogen and mutant Aßs led to a dramatic perturbation of clot structure and delayed fibrinolysis. Immunofluorescence analysis of the occipital cortex showed an increase of fibrin(ogen)/Aß 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 Aß, which might be central to cerebrovascular pathologies observed in HCAA.

Vascular Dysfunction in Alzheimer's Disease: Alterations in the Plasma Contact and Fibrinolytic Systems.

Alzheimer's disease (AD) is the most common neurodegenerative disease, affecting millions of people worldwide. The classical hallmarks of AD include extracellular beta-amyloid (Aß) plaques and neurofibrillary tau tangles, although they are often accompanied by various vascular defects. These changes include damage to the vasculature, a decrease in cerebral blood flow, and accumulation of Aß along vessels, among others. Vascular dysfunction begins early in disease pathogenesis and may contribute to disease progression and cognitive dysfunction. In addition, patients with AD exhibit alterations in the plasma contact system and the fibrinolytic system, two pathways in the blood that regulate clotting and inflammation. Here, we explain the clinical manifestations of vascular deficits in AD. Further, we describe how changes in plasma contact activation and the fibrinolytic system may contribute to vascular dysfunction, inflammation, coagulation, and cognitive impairment in AD. Given this evidence, we propose novel therapies that may, alone or in combination, ameliorate AD progression in patients.

An antibody against HK blocks Alzheimer's disease peptide ß-amyloid-induced bradykinin release in human plasma.

Bradykinin is a proinflammatory factor that mediates angioedema and inflammation in many diseases. It is a key player in some types of hereditary angioedema and is involved in septic shock, traumatic injury, Alzheimer's disease (AD), and stroke, among others. Activation of the plasma contact system leads to elevated levels of plasma kallikrein, which cleaves high molecular weight kininogen (HK) to release bradykinin. Drug development for bradykinin-meditated pathologies has focused on designing inhibitors to the enzymes that cleave HK (to prevent bradykinin release) or antagonists of endothelial bradykinin receptors (to prevent downstream bradykinin action). Here we show a strategy to block bradykinin generation by using an HK antibody that binds to HK, preventing its cleavage and subsequent bradykinin release. We show that this antibody blocks dextran sodium sulfate-induced HK cleavage and bradykinin production. Moreover, while the pathogenic AD peptide ß-amyloid (Aß)42 cleaves HK and induces a dramatic increase in bradykinin production, our HK antibody blocked these events from occurring. These results may provide strategies for developing treatments for bradykinin-driven pathologies.

Blood-derived plasminogen drives brain inflammation and plaque deposition in a mouse model of Alzheimer's disease.

Two of the most predominant features of the Alzheimer's disease (AD) brain are deposition of ß-amyloid (Aß) plaques and inflammation. The mechanism behind these pathologies remains unknown, but there is evidence to suggest that inflammation may predate the deposition of Aß. Furthermore, immune activation is increasingly being recognized as a major contributor to the pathogenesis of the disease, and disorders involving systemic inflammation, such as infection, aging, obesity, atherosclerosis, diabetes, and depression are risk factors for the development of AD. Plasminogen (PLG) is primarily a blood protein synthesized in the liver, which when cleaved into its active form, plasmin (PL), plays roles in fibrinolysis, wound healing, cell signaling, and inflammatory regulation. Here we show that PL in the blood is a regulator of brain inflammatory action and AD pathology. Depletion of PLG in the plasma of an AD mouse model through antisense oligonucleotide technology dramatically improved AD pathology and decreased glial cell activation in the brain, whereas an increase in PL activity through α-2-antiplasmin (A2AP) antisense oligonucleotide treatment exacerbated the brain's immune response and plaque deposition. These studies suggest a crucial role for peripheral PL in mediating neuroimmune cell activation and AD progression and could provide a link to systemic inflammatory risk factors that are known to be associated with AD development.

Increased plasma bradykinin level is associated with cognitive impairment in Alzheimer's patients.

Alzheimer's disease (AD) is characterized by the presence of proteinaceous brain deposits, brain atrophy, vascular dysfunction, and chronic inflammation. Along with cerebral inflammation, peripheral inflammation is also evident in many AD patients. Bradykinin, a proinflammatory plasma peptide, is also linked to AD pathology. For example, bradykinin infusion into the hippocampus causes learning and memory deficits in rats, and blockade of the bradykinin receptor lessens cognitive impairment in AD mouse models. Even though it has been hypothesized that plasma bradykinin could contribute to inflammation in AD, the level of plasma bradykinin and its association with beta-amyloid (Aß) pathology in AD patients had not been explored. Here, we assessed plasma bradykinin levels in AD patients and age-matched non-demented (ND) control individuals. We found significantly elevated plasma bradykinin levels in AD patients compared to ND subjects. Additionally, changes in plasma bradykinin levels were more profound in many AD patients with severe cognitive impairment, suggesting that peripheral bradykinin could play a role in dementia most likely via inflammation. Bradykinin levels in the cerebrospinal fluid (CSF) were reduced in AD patients and exhibited an inverse correlation with the CSF Aß40/Aß42 ratio. We also report that bradykinin interacts with the fibrillar form of Aß and co-localizes with Aß plaques in the post-mortem human AD brain. These findings connect the peripheral inflammatory pathway to cerebral abnormalities and identify a novel mechanism of inflammatory pathology in AD.

Knockdown of circulating C1 inhibitor induces neurovascular impairment, glial cell activation, neuroinflammation, and behavioral deficits.

The cross-talk between blood proteins, immune cells, and brain function involves complex mechanisms. Plasma protein C1 inhibitor (C1INH) is an inhibitor of vascular inflammation that is induced by activation of the kallikrein-kinin system (KKS) and the complement system. Knockout of C1INH was previously correlated with peripheral vascular permeability via the bradykinin pathway, yet there was no evidence of its correlation with blood-brain barrier (BBB) integrity and brain function. In order to understand the effect of plasma C1INH on brain pathology via the vascular system, we knocked down circulating C1INH in wild-type (WT) mice using an antisense oligonucleotide (ASO), without affecting C1INH expression in peripheral immune cells or the brain, and examined brain pathology. Long-term elimination of endogenous C1INH in the plasma induced the activation of the KKS and peritoneal macrophages but did not activate the complement system. Bradykinin pathway proteins were elevated in the periphery and the brain, resulting in hypotension. BBB permeability, extravasation of plasma proteins into the brain parenchyma, activation of glial cells, and elevation of pro-inflammatory response mediators were detected. Furthermore, infiltrating innate immune cells were observed entering the brain through the lateral ventricle walls and the neurovascular unit. Mice showed normal locomotion function, yet cognition was impaired and depressive-like behavior was evident. In conclusion, our results highlight the important role of regulated plasma C1INH as it acts as a gatekeeper to the brain via the neurovascular system. Thus, manipulation of C1INH in neurovascular disorders might be therapeutically beneficial.

Plasminogen mediates communication between the peripheral and central immune systems during systemic immune challenge with lipopolysaccharide.

BACKGROUND: Systemic inflammation has been implicated in the progression of many neurodegenerative diseases and may be an important driver of the disease. Dementia and cognitive decline progress more rapidly following acute systemic infection, and systemic inflammation midlife is predictive of the degree of cognitive decline. Plasmin, the active form of the serine protease plasminogen (PLG), is a blood protein that plays physiological roles in fibrinolysis, wound healing, cell signaling, extracellular matrix degradation, and inflammatory regulation. METHODS: Mice were treated with an antisense oligonucleotide to deplete liver-produced PLG prior to systemic challenge with lipopolysaccharide (LPS), a major component of the outer membrane of gram-negative bacteria, known to induce a strong immune response in animals. Following treatment, the innate immune response in the brains of these animals was examined. RESULTS: Mice that were PLG-deficient had dramatically reduced microgliosis and astrogliosis in their brains after LPS injection. We found that blood PLG regulates the brain's innate immune response to systemic inflammatory signaling, affecting the migration of perivascular macrophages into the brain after challenge with LPS. CONCLUSIONS: Depletion of plasma PLG with an antisense oligonucleotide dramatically reduced glial cell activation and perivascular macrophage migration into the brain following LPS injection. This study suggests a critical role for PLG in mediating communication between systemic inflammatory mediators and the brain.

Depletion of coagulation factor XII ameliorates brain pathology and cognitive impairment in Alzheimer disease mice.

Vascular abnormalities and inflammation are found in many Alzheimer disease (AD) patients, but whether these changes play a causative role in AD is not clear. The factor XII (FXII) -initiated contact system can trigger both vascular pathology and inflammation and is activated in AD patients and AD mice. We have investigated the role of the contact system in AD pathogenesis. Cleavage of high-molecular-weight kininogen (HK), a marker for activation of the inflammatory arm of the contact system, is increased in a mouse model of AD, and this cleavage is temporally correlated with the onset of brain inflammation. Depletion of FXII in AD mice inhibited HK cleavage in plasma and reduced neuroinflammation, fibrinogen deposition, and neurodegeneration in the brain. Moreover, FXII-depleted AD mice showed better cognitive function than untreated AD mice. These results indicate that FXII-mediated contact system activation contributes to AD pathogenesis, and therefore this system may offer novel targets for AD treatment.

Aminopyrimidine Class Aggregation Inhibitor Effectively Blocks Aß-Fibrinogen Interaction and Aß-Induced Contact System Activation.

Accumulating evidence suggests that fibrinogen, a key protein in the coagulation cascade, plays an important role in circulatory dysfunction in Alzheimer's disease (AD). Previous work has shown that the interaction between fibrinogen and ß-amyloid (Aß), a hallmark pathological protein in AD, induces plasmin-resistant abnormal blood clots, delays fibrinolysis, increases inflammation, and aggravates cognitive function in mouse models of AD. Since Aß oligomers have a much stronger affinity for fibrinogen than Aß monomers, we tested whether amyloid aggregation inhibitors could block the Aß-fibrinogen interaction and found that some Aß aggregation inhibitors showed moderate inhibitory efficacy against this interaction. We then modified a hit compound so that it not only showed a strong inhibitory efficacy toward the Aß-fibrinogen interaction but also retained its potency toward the Aß42 aggregation inhibition process. Furthermore, our best hit compound, TDI-2760, modulated Aß42-induced contact system activation, a pathological condition observed in some AD patients, in addition to inhibiting the Aß-fibrinogen interaction and Aß aggregation. Thus, TDI-2760 has the potential to lessen vascular abnormalities as well as Aß aggregation-driven pathology in AD.

Interactions of ß-amyloid peptide with fibrinogen and coagulation factor XII may contribute to Alzheimer's disease.

PURPOSE OF REVIEW: To review the evidence that the Alzheimer peptide ß-amyloid interacts with the blood coagulation system and influences the pathophysiology of the disease. RECENT FINDINGS: ß-amyloid can interact with fibrinogen and blood coagulation factor XII and trigger ischemia and inflammation. SUMMARY: ß-amyloid interacts with fibrinogen and factor XII. These interactions can lead to increased clotting, abnormal clot formation, persistent fibrin deposition, and generation of proinflammatory molecules. These events can damage neurons and could contribute to the cognitive decline in Alzheimer's disease patients.

The cellular origin of laminin determines its role in blood pressure regulation.

Laminin of different cellular sources has distinct functions. In addition to vascular smooth muscle cells (SMCs), aorta also contains a small population of nestin(+) cells, whose function remains unknown. This study investigates the role of SMC- and nestin(+) cell-derived laminin in blood pressure (BP) regulation and SMC contractibility. Using mice with laminin deficiency in SMCs (SKO) or nestin(+) cells (NKO), we examined laminin-dependent changes in BP. Contractile protein expression was reduced in SKO but not NKO mice, consistent with their, respectively, low and normal baseline BP measurements. At the ultrastructural level, SKO SMCs maintained the contractile phenotype with reduced elasticity, whereas NKO SMCs switched to the synthetic phenotype and showed degeneration. Additionally, angiotensin II (Ang II) significantly increased BP in SKO but not NKO mice. It also enhanced contractile proteins to the same levels and induced SMC degeneration in both knockout mice. These data suggest that SMC laminin regulates BP via modulating contractile protein expression, whereas nestin(+) cell-derived laminin contributes to SMC phenotypic switch.

Tissue plasminogen activator is required for the development of fetal alcohol syndrome in mice.

Ethanol exposure during developmental synaptogenesis can lead to brain defects referred to as fetal alcohol syndrome (FAS), which can include mental health problems such as cognitive deficits and mental retardation. In FAS, widespread neuronal death and brain mass loss precedes behavioral and cognitive impairments in adulthood. Because tissue plasminogen activator (tPA) has been implicated in neurodegeneration, we examined whether it mediates FAS. Neonatal WT and tPA-/- mice were injected with ethanol to mimic FAS in humans. In WT mice, ethanol elicited caspase-3 activation, significant forebrain neurodegeneration, and decreased contextual fear conditioning in adults. However, tPA-deficient mice were protected from these neurotoxicities, and this protection could be abrogated by exogenous tPA. Selective pharmacological modulators of NMDA and GABAA receptor pathways revealed that the effects of tPA were mediated by the NR2B subunit of the NMDA receptor. This study identifies tPA as a critical signaling component in FAS.

Lecanemab Blocks the Effects of the Aß/Fibrinogen Complex on Blood Clots and Synapse Toxicity in Organotypic Culture.

Proteinaceous brain inclusions, neuroinflammation, and vascular dysfunction are common pathologies in Alzheimer's disease (AD). Vascular deficits include a compromised blood-brain barrier, which can lead to extravasation of blood proteins like fibrinogen into the brain. Fibrinogen's interaction with the amyloid-beta (Aß) peptide is known to worsen thrombotic and cerebrovascular pathways in AD. Lecanemab, an FDA-approved antibody therapy for AD, shows promising results in facilitating reduction of Aß from the brain and slowing cognitive decline. Here we show that lecanemab blocks fibrinogen's binding to Aß protofibrils, normalizing Aß/fibrinogen-mediated delayed fibrinolysis and clot abnormalities in vitro and in human plasma. Additionally, we show that lecanemab dissociates the Aß/fibrinogen complex and prevents fibrinogen from exacerbating Aß-induced synaptotoxicity in mouse organotypic hippocampal cultures. These findings reveal a possible protective mechanism by which lecanemab may slow disease progression in AD.

Alzheimer's disease peptide beta-amyloid interacts with fibrinogen and induces its oligomerization.

Increasing evidence supports a vascular contribution to Alzheimer's disease (AD), but a direct connection between AD and the circulatory system has not been established. Previous work has shown that blood clots formed in the presence of the ß-amyloid peptide (Aß), which has been implicated in AD, have an abnormal structure and are resistant to degradation in vitro and in vivo. In the present study, we show that Aß specifically interacts with fibrinogen with a K(d) of 26.3 ± 6.7 nM, that the binding site is located near the C terminus of the fibrinogen ß-chain, and that the binding causes fibrinogen to oligomerize. These results suggest that the interaction between Aß and fibrinogen modifies fibrinogen's structure, which may then lead to abnormal fibrin clot formation. Overall, our study indicates that the interaction between Aß and fibrinogen may be an important contributor to the vascular abnormalities found in AD.

Anti-HK antibody inhibits the plasma contact system by blocking prekallikrein and factor XI activation in vivo.

A dysregulated plasma contact system is involved in various pathological conditions, such as hereditary angioedema, Alzheimer disease, and sepsis. We previously showed that the 3E8 anti-high molecular weight kininogen (anti-HK) antibody blocks HK cleavage and bradykinin generation in human plasma ex vivo. Here, we show that 3E8 prevented not only HK cleavage but also factor XI (FXI) and prekallikrein (PK) activation by blocking their binding to HK in mouse plasma in vivo. 3E8 also inhibited contact system-induced bradykinin generation in vivo. Interestingly, FXII activation was also inhibited, likely because of the ability of 3E8 to block the positive feedback activation of FXII by kallikrein (PKa). In human plasma, 3E8 also blocked PK and FXI binding to HK and inhibited both thrombotic (FXI activation) and inflammatory pathways (PK activation and HK cleavage) of the plasma contact system activation ex vivo. Moreover, 3E8 blocked PKa binding to HK and dose-dependently inhibited PKa cleavage of HK. Our results reveal a novel strategy to inhibit contact system activation in vivo, which may provide an effective method to treat human diseases involving contact system dysregulation.

Striatal fibrinogen extravasation and vascular degeneration correlate with motor dysfunction in an aging mouse model of Alzheimer's disease.

Introduction: Alzheimer's Disease (AD) patients exhibit signs of motor dysfunction, including gait, locomotion, and balance deficits. Changes in motor function often precede other symptoms of AD as well as correlate with increased severity and mortality. Despite the frequent occurrence of motor dysfunction in AD patients, little is known about the mechanisms by which this behavior is altered. Methods and Results: In the present study, we investigated the relationship between cerebrovascular impairment and motor dysfunction in a mouse model of AD (Tg6799). We found an age-dependent increase of extravasated fibrinogen deposits in the cortex and striatum of AD mice. Interestingly, there was significantly decreased cerebrovascular density in the striatum of the 15-month-old as compared to 7-month-old AD mice. We also found significant demyelination and axonal damage in the striatum of aged AD mice. We analyzed striatum-related motor function and anxiety levels of AD mice at both ages and found that aged AD mice exhibited significant impairment of motor function but not in the younger AD mice. Discussion: Our finding suggests an enticing correlation between extravasated fibrinogen, cerebrovascular damage of the striatum, and motor dysfunction in an AD mouse model, suggesting a possible mechanism underlying motor dysfunction in AD.

Tissue plasminogen activator modulates the cellular and behavioral response to cocaine.

Cocaine exposure induces long-lasting molecular and structural adaptations in the brain. In this study, we show that tissue plasminogen activator (tPA), an extracellular protease involved in neuronal plasticity, modulates the biochemical and behavioral response to cocaine. When injected in the acute binge paradigm, cocaine enhanced tPA activity in the amygdala, which required activation of corticotropin-releasing factor type-1 (CRF-R1) receptors. Compared with WT mice, tPA-/- mice injected with cocaine displayed attenuated phosphorylation of ERK, cAMP response element binding protein (CREB), and dopamine and cAMP-regulated phosphoprotein 32 kDa (DARPP-32) and blunted induction of immediate early genes (IEGs) c-Fos, Egr-1, and Homer 1a in the amygdala and the nucleus accumbens (NAc). tPA-/- mice also displayed significantly higher basal preprodynorphin (ppDyn) mRNA levels in the NAc in comparison to WT mice, and cocaine decreased ppDyn mRNA levels in tPA-/- mice only. Cocaine-induced locomotor sensitization and conditioned place preference (CPP) were attenuated in tPA-/- mice. Cocaine exposure also had an anxiolytic effect in tPA-/- but not WT mice. These results identify tPA as an important and novel component of the signaling pathway that modulates cocaine-induced changes in neuroadaptation and behavior.