Bruton's tyrosine kinase drives neuroinflammation and anxiogenic behavior in mouse models of stress.

BACKGROUND: Current therapies targeting several neurotransmitter systems are only able to partially mitigate the symptoms of stress- and trauma-related disorder. Stress and trauma-related disorders lead to a prominent inflammatory response in humans, and in pre-clinical models. However, mechanisms underlying the induction of neuroinflammatory response in PTSD and anxiety disorders are not clearly understood. The present study investigated the mechanism underlying the activation of proinflammatory NLRP3 inflammasome and IL1ß in mouse models of stress. METHODS: We used two mouse models of stress, i.e., mice subjected to physical restraint stress with brief underwater submersion, and predator odor stress. Mice were injected with MCC950, a small molecule specific inhibitor of NLRP3 activation. To pharmacologically inhibit BTK, a specific inhibitor ibrutinib was used. To validate the observation from ibrutinib studies, a separate group of mice was injected with another BTK-specific inhibitor LFM-A13. Seven days after the induction of stress, mice were examined for anxious behavior using open field test (OFT), light-dark test (LDT), and elevated plus maze test (EPM). Following the behavior tests, hippocampus and amygdale were extracted and analyzed for various components of NLRP3-caspase 1-IL1ß pathway. Plasma and peripheral blood mononuclear cells were also used to assess the induction of NLRP3-Caspase 1-IL-1ß pathway in stressed mice. RESULTS: Using two different pre-clinical models of stress, we demonstrate heightened anxious behavior in female mice as compared to their male counterparts. Stressed animals exhibited upregulation of proinflammatory IL1ß, IL-6, Caspase 1 activity and NLRP3 inflammasome activation in brain, which were significantly higher in female mice. Pharmacological inhibition of NLRP3 inflammasome activation led to anxiolysis as well as attenuated neuroinflammatory response. Further, we observed induction of activated Bruton's tyrosine kinase (BTK), an upstream positive-regulator of NLRP3 inflammasome activation, in hippocampus and amygdala of stressed mice. Next, we conducted proof-of-concept pharmacological BTK inhibitor studies with ibrutinib and LFM-A13. In both sets of experiments, we found BTK inhibition led to anxiolysis and attenuated neuroinflammation, as indicated by significant reduction of NLRP3 inflammasome and proinflammatory IL-1ß in hippocampus and amygdala. Analysis of plasma and peripheral blood mononuclear cells indicated peripheral induction of NLRP3-caspase 1-IL1ß pathway in stressed mice. CONCLUSION: Our study identified BTK as a key upstream regulator of neuroinflammation, which drives anxiogenic behavior in mouse model of stress. Further, we demonstrated the sexually divergent activation of BTK, providing a clue to heightened neuroinflammation and anxiogenic response to stress in females as compared to their male counterparts. Our data from the pharmacological inhibition studies suggest BTK as a novel target for the development of potential clinical treatment of PTSD and anxiety disorders. Induction of pBTK and NLRP3 in peripheral blood mononuclear cells of stressed mice suggest the potential effect of stress on systemic inflammation.

STAT3 inhibitor mitigates cerebral amyloid angiopathy and parenchymal amyloid plaques while improving cognitive functions and brain networks.

Previous reports indicate a potential role for signal transducer and activator of transcription 3 (STAT3) in amyloid-ß (Aß) processing and neuritic plaque pathogenesis. In the present study, the impact of STAT3 inhibition on cognition, cerebrovascular function, amyloid pathology, oxidative stress, and neuroinflammation was studied using in vitro and in vivo models of Alzheimer's disease (AD)-related pathology. For in vitro experiments, human brain vascular smooth muscle cells (HBVSMC) and human brain microvascular endothelial cells (HBMEC) were used, and these cultured cells were exposed to Aß peptides followed by measurement of activated forms of STAT3 expression and reactive oxygen species (ROS) generation. Further, 6 months old 5XFAD/APOE4 (5XE4) mice and age-matched negative littermates were used for in vivo experiments. These mice were treated with STAT3 specific inhibitor, LLL-12 for 2 months followed by neurobehavioral and histopathological assessment. In vitro experiments showed exposure of cerebrovascular cells to Aß peptides upregulated activated forms of STAT3 and produced STAT3-mediated vascular oxidative stress. 5XE4 mice treated with the STAT3-specific inhibitor (LLL-12) improved cognitive functions and functional connectivity and augmented cerebral blood flow. These functional improvements were associated with a reduction in neuritic plaques, cerebral amyloid angiopathy (CAA), oxidative stress, and neuroinflammation. Reduction in amyloid precursor protein (APP) processing and attenuation of oxidative modification of lipoprotein receptor related protein-1 (LRP-1) were identified as potential underlying mechanisms. These results demonstrate the broad impact of STAT3 on cognitive functions, parenchymal and vascular amyloid pathology and highlight the therapeutic potential of STAT3 specific inhibition for treatment of AD and CAA.

cAMP regulated EPAC1 supports microvascular density, angiogenic and metastatic properties in a model of triple negative breast cancer.

Breast cancer is a leading cause of cancer-related mortality in women. Triple-negative breast cancer (TNBC; HER2-, ER-/PR-) is an aggressive subtype prone to drug resistance and metastasis, which is characterized by high intratumor microvascular density (iMVD) resulting from angiogenesis. However, the mechanisms contributing to the aggressive phenotypes of TNBC remain elusive. We recently reported that down-regulation of exchange factor directly activated by cyclic AMP (cAMP), also known as EPAC1, leads to a reduction in metastatic properties including proliferation and cell migration in TNBC cell lines. Here, we report that EPAC1 supports TNBC-induced angiogenesis, tumor cell migration and invasiveness as well as pro-metastatic phenotypes in endothelial cells induced through the tumor secretome. Using an approach that integrates proteomics with bioinformatics and gene ontologies, we elucidate that EPAC1 supports a tumor-secreted network of angiogenic, cell adhesion and cell migratory pathways. Using confocal microscopy, we show that signaling molecules involved in focal adhesion, including Paxillin and MENA, are down-regulated in the absence of EPAC1, and electric cell substrate impedance sensing technique confirmed a role for EPAC1 on TNBC-induced endothelial cell permeability. Finally, to provide a translational bridge, we studied iMVD and therapy response using a primary human tumor explant assay, CANscriptTM, which suggests a link between therapy-modulated neovascularization and drug sensitivity. These data provide mechanistic insight into the role of EPAC1 in regulating the tumor microenvironment, iMVD and cancer cell-induced angiogenesis, a dynamic mechanism under drug pressure that may associate to treatment failure.

A novel fluorescent imaging technique for assessment of cerebral vasospasm after experimental subarachnoid hemorrhage.

Various techniques have been developed to study changes in the cerebral vasculature in numerous neuropathological processes including subarachnoid hemorrhage (SAH). One of the most widely employed techniques uses India ink-gelatin casting, which presents numerous challenges due to its high viscosity, rapid solidification, and its impact on immunohistochemical analysis. To overcome these limitations, we developed a novel technique for assessing cerebral vasospasm using cerebrovascular perfusion with ROX, SE (5-Carboxy-X-Rhodamine, Succinimidyl Ester), a fluorescent labeling dye. We found that ROX SE perfusion achieves excellent delineation of the cerebral vasculature, was qualitatively and quantitatively superior to India ink-gelatin casting for the assessment of cerebral vasospasm, permits outstanding immunohistochemical examination of non-vasospasm components of secondary brain injury, and is a more efficient and cost-effective experimental technique. ROX SE perfusion is therefore a novel and highly useful technique for studying cerebrovascular pathology following experimental SAH.

Minocycline protects against delayed cerebral ischemia after subarachnoid hemorrhage via matrix metalloproteinase-9 inhibition.

Objective: Delayed cerebral ischemia (DCI) is an independent risk factor for poor outcome after aneurysmal subarachnoid hemorrhage (SAH) and is multifactorial in etiology. While prior studies have suggested a role for matrix metalloproteinase-9 (MMP-9) in early brain injury after SAH, its contribution to the pathophysiology of DCI is unclear. Methods: In the first experiment, wild-type (WT) and MMP-9-/- mice were subjected to sham or endovascular perforation SAH surgery. In separate experiments, WT and MMP-9-/-mice were administered vehicle or minocycline either pre- or post-SAH. All mice underwent assessment of multiple components of DCI including vasospasm, neurobehavioral function, and microvessel thrombosis. In another experiment, rabbits were subjected to sham or cisterna magna injection SAH surgery, and administered vehicle or minocycline followed by vasospasm assessment. Results: MMP-9 expression and activity was increased after SAH. Genetic (MMP-9-/- mice) and pharmacological (pre-SAH minocycline administration) inhibition of MMP-9 resulted in decreased vasospasm and neurobehavioral deficits. A therapeutically feasible strategy of post-SAH administration of minocycline resulted in attenuation of multiple components of DCI. Minocycline administration to MMP-9-/- mice did not yield additional protection. Consistent with experiments in mice, both pre- and post-SAH administration of minocycline attenuated SAH-induced vasospasm in rabbits. Interpretation: MMP-9 is a key player in the pathogenesis of DCI. The consistent attenuation of multiple components of DCI with both pre- and post-SAH administration of minocycline across different species and experimental models of SAH, combined with the excellent safety profile of minocycline in humans suggest that a clinical trial in SAH patients is warranted.

Heparan sulfate proteoglycans mediate Aß-induced oxidative stress and hypercontractility in cultured vascular smooth muscle cells.

BACKGROUND: Substantial evidence suggests that amyloid-ß (Aß) species induce oxidative stress and cerebrovascular (CV) dysfunction in Alzheimer's disease (AD), potentially contributing to the progressive dementia of this disease. The upstream molecular pathways governing this process, however, are poorly understood. In this report, we examine the role of heparan sulfate proteoglycans (HSPG) in Aß-induced vascular smooth muscle cell (VSMC) dysfunction in vitro. RESULTS: Our results demonstrate that pharmacological depletion of HSPG (by enzymatic degradation with active, but not heat-inactivated, heparinase) in primary human cerebral and transformed rat VSMC mitigates Aß(1-40⁻) and Aß(1-42⁻)induced oxidative stress. This inhibitory effect is specific for HSPG depletion and does not occur with pharmacological depletion of other glycosaminoglycan (GAG) family members. We also found that Aß(1-40) (but not Aß(1-42)) causes a hypercontractile phenotype in transformed rat cerebral VSMC that likely results from a HSPG-mediated augmentation in intracellular Ca(2+) activity, as both Aß(1-40⁻)induced VSMC hypercontractility and increased Ca(2+) influx are inhibited by pharmacological HSPG depletion. Moreover, chelation of extracellular Ca(2+) with ethylene glycol tetraacetic acid (EGTA) does not prevent the production of Aß(1-40⁻) or Aß(1-42⁻)mediated reactive oxygen species (ROS), suggesting that Aß-induced ROS and VSMC hypercontractility occur through different molecular pathways. CONCLUSIONS: Taken together, our data indicate that HSPG are critical mediators of Aß-induced oxidative stress and Aß(1-40⁻)induced VSMC dysfunction.

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 ß peptide (Aß) 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 Aß-induced vessel dysfunction, but the mechanisms by which insoluble Aß 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.

Impairment of glymphatic pathway function promotes tau pathology after traumatic brain injury.

Traumatic brain injury (TBI) is an established risk factor for the early development of dementia, including Alzheimer's disease, and the post-traumatic brain frequently exhibits neurofibrillary tangles comprised of aggregates of the protein tau. We have recently defined a brain-wide network of paravascular channels, termed the "glymphatic" pathway, along which CSF moves into and through the brain parenchyma, facilitating the clearance of interstitial solutes, including amyloid-ß, from the brain. Here we demonstrate in mice that extracellular tau is cleared from the brain along these paravascular pathways. After TBI, glymphatic pathway function was reduced by ∼60%, with this impairment persisting for at least 1 month post injury. Genetic knock-out of the gene encoding the astroglial water channel aquaporin-4, which is importantly involved in paravascular interstitial solute clearance, exacerbated glymphatic pathway dysfunction after TBI and promoted the development of neurofibrillary pathology and neurodegeneration in the post-traumatic brain. These findings suggest that chronic impairment of glymphatic pathway function after TBI may be a key factor that renders the post-traumatic brain vulnerable to tau aggregation and the onset of neurodegeneration.

Low levels of copper disrupt brain amyloid-ß homeostasis by altering its production and clearance.

Whereas amyloid-ß (Aß) accumulates in the brain of normal animals dosed with low levels of copper (Cu), the mechanism is not completely known. Cu could contribute to Aß accumulation by altering its clearance and/or its production. Because Cu homeostasis is altered in transgenic mice overexpressing Aß precursor protein (APP), the objective of this study was to elucidate the mechanism of Cu-induced Aß accumulation in brains of normal mice and then to explore Cu's effects in a mouse model of Alzheimer's disease. In aging mice, accumulation of Cu in brain capillaries was associated with its reduction in low-density lipoprotein receptor-related protein 1 (LRP1), an Aß transporter, and higher brain Aß levels. These effects were reproduced by chronic dosing with low levels of Cu via drinking water without changes in Aß synthesis or degradation. In human brain endothelial cells, Cu, at its normal labile levels, caused LRP1-specific down-regulation by inducing its nitrotyrosination and subsequent proteosomal-dependent degradation due in part to Cu/cellular prion protein/LRP1 interaction. In APP(sw/0) mice, Cu not only down-regulated LRP1 in brain capillaries but also increased Aß production and neuroinflammation because Cu accumulated in brain capillaries and, unlike in control mice, in the parenchyma. Thus, we have demonstrated that Cu's effect on brain Aß homeostasis depends on whether it is accumulated in the capillaries or in the parenchyma. These findings should provide unique insights into preventative and/or therapeutic approaches to control neurotoxic Aß levels in the aging brain.

A lipoprotein receptor cluster IV mutant preferentially binds amyloid-ß and regulates its clearance from the mouse brain.

Soluble low density lipoprotein receptor-related protein-1 (sLRP1) binds ~70% of amyloid ß-peptide (Aß) in human plasma. In Alzheimer disease (AD) and individuals with mild cognitive impairment converting to AD, plasma sLRP1 levels are reduced and sLRP1 is oxidized, which results in diminished Aß peripheral binding and higher levels of free Aß in plasma. Experimental studies have shown that free circulating Aß re-enters the brain and that sLRP1 and/or its recombinant wild type cluster IV (WT-LRPIV) prevent Aß from entering the brain. Treatment of Alzheimer APPsw(+/0) mice with WT-LRPIV has been shown to reduce brain Aß pathology. In addition to Aß, LRPIV binds multiple ligands. To enhance LRPIV binding for Aß relative to other LRP1 ligands, we generated a library of LRPIV-derived fragments and full-length LRPIV variants with glycine replacing aspartic acid residues 3394, 3556, and 3674 in the calcium binding sites. Compared with WT-LRPIV, a lead LRPIV-D3674G mutant had 1.6- and 2.7-fold higher binding affinity for Aß40 and Aß42 in vitro, respectively, and a lower binding affinity for other LRP1 ligands (e.g. apolipoprotein E2, E3, and E4 (1.3-1.8-fold), tissue plasminogen activator (2.7-fold), matrix metalloproteinase-9 (4.1-fold), and Factor Xa (3.8-fold)). LRPIV-D3674G cleared mouse endogenous brain Aß40 and Aß42 25-27% better than WT-LRPIV. A 3-month subcutaneous treatment of APPsw(+/0) mice with LRPIV-D3674G (40 µg/kg/day) reduced Aß40 and Αß42 levels in the hippocampus, cortex, and cerebrospinal fluid by 60-80% and improved cerebral blood flow responses and hippocampal function at 9 months of age. Thus, LRPIV-D3674G is an efficient new Aß clearance therapy.

Apolipoprotein E controls cerebrovascular integrity via cyclophilin A.

Human apolipoprotein E has three isoforms: APOE2, APOE3 and APOE4. APOE4 is a major genetic risk factor for Alzheimer's disease and is associated with Down's syndrome dementia and poor neurological outcome after traumatic brain injury and haemorrhage. Neurovascular dysfunction is present in normal APOE4 carriers and individuals with APOE4-associated disorders. In mice, lack of Apoe leads to blood-brain barrier (BBB) breakdown, whereas APOE4 increases BBB susceptibility to injury. How APOE genotype affects brain microcirculation remains elusive. Using different APOE transgenic mice, including mice with ablation and/or inhibition of cyclophilin A (CypA), here we show that expression of APOE4 and lack of murine Apoe, but not APOE2 and APOE3, leads to BBB breakdown by activating a proinflammatory CypA-nuclear factor-κB-matrix-metalloproteinase-9 pathway in pericytes. This, in turn, leads to neuronal uptake of multiple blood-derived neurotoxic proteins, and microvascular and cerebral blood flow reductions. We show that the vascular defects in Apoe-deficient and APOE4-expressing mice precede neuronal dysfunction and can initiate neurodegenerative changes. Astrocyte-secreted APOE3, but not APOE4, suppressed the CypA-nuclear factor-κB-matrix-metalloproteinase-9 pathway in pericytes through a lipoprotein receptor. Our data suggest that CypA is a key target for treating APOE4-mediated neurovascular injury and the resulting neuronal dysfunction and degeneration.

A multimodal RAGE-specific inhibitor reduces amyloid ß-mediated brain disorder in a mouse model of Alzheimer disease.

In Alzheimer disease (AD), amyloid ß peptide (Aß) accumulates in plaques in the brain. Receptor for advanced glycation end products (RAGE) mediates Aß-induced perturbations in cerebral vessels, neurons, and microglia in AD. Here, we identified a high-affinity RAGE-specific inhibitor (FPS-ZM1) that blocked Aß binding to the V domain of RAGE and inhibited Aß40- and Aß42-induced cellular stress in RAGE-expressing cells in vitro and in the mouse brain in vivo. FPS-ZM1 was nontoxic to mice and readily crossed the blood-brain barrier (BBB). In aged APPsw/0 mice overexpressing human Aß-precursor protein, a transgenic mouse model of AD with established Aß pathology, FPS-ZM1 inhibited RAGE-mediated influx of circulating Aß40 and Aß42 into the brain. In brain, FPS-ZM1 bound exclusively to RAGE, which inhibited ß-secretase activity and Aß production and suppressed microglia activation and the neuroinflammatory response. Blockade of RAGE actions at the BBB and in the brain reduced Aß40 and Aß42 levels in brain markedly and normalized cognitive performance and cerebral blood flow responses in aged APPsw/0 mice. Our data suggest that FPS-ZM1 is a potent multimodal RAGE blocker that effectively controls progression of Aß-mediated brain disorder and that it may have the potential to be a disease-modifying agent for AD.

Pericytes control key neurovascular functions and neuronal phenotype in the adult brain and during brain aging.

Pericytes play a key role in the development of cerebral microcirculation. The exact role of pericytes in the neurovascular unit in the adult brain and during brain aging remains, however, elusive. Using adult viable pericyte-deficient mice, we show that pericyte loss leads to brain vascular damage by two parallel pathways: (1) reduction in brain microcirculation causing diminished brain capillary perfusion, cerebral blood flow, and cerebral blood flow responses to brain activation that ultimately mediates chronic perfusion stress and hypoxia, and (2) blood-brain barrier breakdown associated with brain accumulation of serum proteins and several vasculotoxic and/or neurotoxic macromolecules ultimately leading to secondary neuronal degenerative changes. We show that age-dependent vascular damage in pericyte-deficient mice precedes neuronal degenerative changes, learning and memory impairment, and the neuroinflammatory response. Thus, pericytes control key neurovascular functions that are necessary for proper neuronal structure and function, and pericyte loss results in a progressive age-dependent vascular-mediated neurodegeneration.

Protein S controls hypoxic/ischemic blood-brain barrier disruption through the TAM receptor Tyro3 and sphingosine 1-phosphate receptor.

The anticoagulant factor protein S (PS) has direct cellular activities. Lack of PS in mice causes lethal coagulopathy, ischemic/thrombotic injuries, vascular dysgenesis, and blood-brain barrier (BBB) disruption with intracerebral hemorrhages. Thus, we hypothesized that PS maintains and/or enhances the BBB integrity. Using a BBB model with human brain endothelial cells, we show PS inhibits time- and dose-dependently (half maximal effective concentration [EC(50)] = 27 +/- 3 nM) oxygen/glucose deprivation-induced BBB breakdown, as demonstrated by measurements of the transmonolayer electrical resistance, permeability of endothelial monolayers to dextran (40 kDa), and rearrangement of F-actin toward the cortical cytoskeletal ring. Using Tyro-3, Axl, and Mer (TAM) receptor, tyrosine kinase silencing through RNA interference, specific N-terminus-blocking antibodies, Tyro3 phosphorylation, and Tyro3-, Axl- and Mer-deficient mouse brain endothelial cells, we show that Tyro3 mediates PS vasculoprotection. After Tyro3 ligation, PS activated sphingosine 1-phosphate receptor (S1P(1)), resulting in Rac1-dependent BBB protection. Using 2-photon in vivo imaging, we show that PS blocks postischemic BBB disruption in Tyro3(+/+), Axl(-/-), and Mer(-/-) mice, but not in Tyro3(-/-) mice or Tyro3(+/+) mice receiving low-dose W146, a S1P(1)-specific antagonist. Our findings indicate that PS protects the BBB integrity via Tyro3 and S1P(1), suggesting potentially novel treatments for neurovascular dysfunction resulting from hypoxic/ischemic BBB damage.

Species-specific anticoagulant and mitogenic activities of murine protein S.

BACKGROUND: The protein C pathway down-regulates thrombin generation and promotes cytoprotection during inflammation and stress. In preclinical studies using models of murine injury (e.g., sepsis and ischemic stroke), murine protein S may be required because of restrictive species specificity. DESIGN AND METHODS: We prepared and characterized recombinant murine protein S using novel coagulation assays, immunoassays, and cell proliferation assays. RESULTS: Purified murine protein S had good anticoagulant co-factor activity for murine activated protein C, but not for human activated protein C, in mouse or rat plasma. In human plasma, murine protein S was a poor co-factor for murine activated protein C and had no anticoagulant effect with human activated protein C, suggesting protein S species specificity for factor V in addition to activated protein C. We estimated that mouse plasma contains 22+/-1 microg/mL protein S and developed assays to measure activated protein C co-factor activity of the protein S in murine plasma. Activated protein C-independent anticoagulant activity of murine protein S was demonstrable and quantifiable in mouse plasma, and this activity was enhanced by exogenous murine protein S. Murine protein S promoted the proliferation of mouse and human smooth muscle cells. The potency of murine protein S was higher for mouse cells than for human cells and similarly, human protein S was more potent for human cells than for mouse cells. CONCLUSIONS: The spectrum of bioactivities of recombinant murine protein S with mouse plasma and smooth muscle cells is similar to that of human protein S. However, in vitro and in vivo studies of the protein C pathway in murine disease models are more appropriately performed using murine protein S. This study extends previous observations regarding the remarkable species specificity of protein S to the mouse.

Activated protein C therapy slows ALS-like disease in mice by transcriptionally inhibiting SOD1 in motor neurons and microglia cells.

Activated protein C (APC) is a signaling protease with anticoagulant activity. Here, we have used mice expressing a mutation in superoxide dismutase-1 (SOD1) that is linked to amyotrophic lateral sclerosis (ALS) to show that administration of APC or APC analogs with reduced anticoagulant activity after disease onset slows disease progression and extends survival. A proteolytically inactive form of APC with reduced anticoagulant activity provided no benefit. APC crossed the blood-spinal cord barrier in mice via endothelial protein C receptor. When administered after disease onset, APC eliminated leakage of hemoglobin-derived products across the blood-spinal cord barrier and delayed microglial activation. In microvessels, motor neurons, and microglial cells from SOD1-mutant mice and in cultured neuronal cells, APC transcriptionally downregulated SOD1. Inhibition of SOD1 synthesis in neuronal cells by APC required protease-activated receptor-1 (PAR1) and PAR3, which inhibited nuclear transport of the Sp1 transcription factor. Diminished mutant SOD1 synthesis by selective gene excision within endothelial cells did not alter disease progression, which suggests that diminished mutant SOD1 synthesis in other cells, including motor neurons and microglia, caused the APC-mediated slowing of disease. The delayed disease progression in mice after APC administration suggests that this approach may be of benefit to patients with familial, and possibly sporadic, ALS.

Neuroprotective activities of activated protein C mutant with reduced anticoagulant activity.

The anticoagulant activated protein C (APC) protects neurons and endothelium via protease activated receptor (PAR)1, PAR3 and endothelial protein C receptor. APC is neuroprotective in stroke models. Bleeding complications may limit the pharmacologic utility of APC. Here, we compared the 3K3A-APC mutant with 80% reduced anticoagulant activity and wild-type (wt)-APC. Murine 3K3A-APC compared with wt-APC protected mouse cortical neurons from N-methyl-D-aspartate-induced apoptosis with twofold greater efficacy and more potently reduced N-methyl-D-aspartate excitotoxic lesions in vivo. Human 3K3A-APC protected human brain endothelial cells (BECs) from oxygen/glucose deprivation with 1.7-fold greater efficacy than wt-APC. 3K3A-APC neuronal protection required PAR1 and PAR3, as shown by using PAR-specific blocking antibodies and PAR1- and PAR3-deficient cells and mice. BEC protection required endothelial protein C receptor and PAR1. In neurons and BECs, 3K3A-APC blocked caspase-9 and -3 activation and induction of p53, and decreased the Bax/Bcl-2 pro-apoptotic ratio. After distal middle cerebral artery occlusion (dMCAO) in mice, murine 3K3A-APC compared with vehicle given 4:00 h after dMCAO improved the functional outcome and reduced the infarction volume by 50% within 3 days. 3K3A-APC compared with wt-APC multi-dosing therapy at 12:00 h, 1, 3, 5 and 7 days after dMCAO significantly improved functional recovery and reduced the infarction volume by 75% and 38%, respectively, within 7 days. The wt-APC, but not 3K3A-APC, significantly increased the risk of intracerebral bleeding as indicated by a 50% increase in hemoglobin levels in the ischemic hemisphere. Thus, 3K3A-APC offers a new approach for safer and more efficacious treatments of neurodegenerative disorders and stroke with APC.

Species-dependent neuroprotection by activated protein C mutants with reduced anticoagulant activity.

Activated protein C (APC) is a protease with anticoagulant and cytoprotective activities. APC is neuroprotective in rodent models of stroke. But, an APC variant with reduced anticoagulant activity, 3K3A-APC, compared to wild-type APC shows greater neuroprotection with no risk for bleeding in stroke models. To determine whether 3K3A-APC exhibits species-dependent neuroprotection similar to that as seen with wild-type APC, we studied murine and human recombinant 3K3A-APC mutants which show approximately 80% reduced anticoagulant activity. Murine 3K3A-APC (0.2 mg/kg i.v.) administered at 4 h after embolic stroke improved substantially functional outcome and reduced by 80% the infract volume 7 days after stroke. Human 3K3A-APC was neuroprotective after embolic stroke in mice, but at significantly higher concentrations (i.e. 2 mg/kg i.v.). Species-dependent neuroprotection, i.e. murine > human 3K3A-APC, was confirmed in a mouse model of permanent middle cerebral artery occlusion. Human 3K3A-APC had by fivefold greater cytoprotective activity than murine 3K3A-APC in oxygen-glucose deprivation model in human brain endothelial cells, whereas murine 3K3A-APC was by 2.5-fold more potent than human 3K3A-APC in a mouse model of NMDA-induced neuronal apoptosis. Thus, 3K3A-APC exhibits species-dependent neuroprotection which should be taken into account when designing human trials for ischemic stroke with APC mutants.