PIKFYVE inhibition mitigates disease in models of diverse forms of ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that results from many diverse genetic causes. Although therapeutics specifically targeting known causal mutations may rescue individual types of ALS, these approaches cannot treat most cases since they have unknown genetic etiology. Thus, there is a pressing need for therapeutic strategies that rescue multiple forms of ALS. Here, we show that pharmacological inhibition of PIKFYVE kinase activates an unconventional protein clearance mechanism involving exocytosis of aggregation-prone proteins. Reducing PIKFYVE activity ameliorates ALS pathology and extends survival of animal models and patient-derived motor neurons representing diverse forms of ALS including C9ORF72, TARDBP, FUS, and sporadic. These findings highlight a potential approach for mitigating ALS pathogenesis that does not require stimulating macroautophagy or the ubiquitin-proteosome system.

Human antigen R regulates hypoxia-induced mitophagy in renal tubular cells through PARKIN/BNIP3L expressions.

Mitochondrial dysfunction contributes to the pathophysiology of acute kidney injury (AKI). Mitophagy selectively degrades damaged mitochondria and thereby regulates cellular homeostasis. RNA-binding proteins (RBPs) regulate RNA processing at multiple levels and thereby control cellular function. In this study, we aimed to understand the role of human antigen R (HuR) in hypoxia-induced mitophagy process in the renal tubular cells. Mitophagy marker expressions (PARKIN, p-PARKIN, PINK1, BNIP3L, BNIP3, LC3) were determined by western blot analysis. Immunofluorescence studies were performed to analyze mitophagosome, mitolysosome, co-localization of p-PARKIN/TOMM20 and BNIP3L/TOMM20. HuR-mediated regulation of PARKIN/BNIP3L expressions was determined by RNA-immunoprecipitation analysis and RNA stability experiments. Hypoxia induced mitochondrial dysfunction by increased ROS, decline in membrane potential and activated mitophagy through up-regulated PARKIN, PINK1, BNIP3 and BNIP3L expressions. HuR knockdown studies revealed that HuR regulates hypoxia-induced mitophagosome and mitolysosome formation. HuR was significantly bound to PARKIN and BNIP3L mRNA under hypoxia and thereby up-regulated their expressions through mRNA stability. Altogether, our data highlight the importance of HuR in mitophagy regulation through up-regulating PARKIN/BNIP3L expressions in renal tubular cells.

Transport Characteristics of CJMAED™ Homogeneous Anion Exchange Membranes in Sodium Chloride and Sodium Sulfate Solutions.

The interplay between the ion exchange capacity, water content and concentration dependences of conductivity, diffusion permeability, and counterion transport numbers (counterion permselectivity) of CJMA-3, CJMA-6 and CJMA-7 (Hefei Chemjoy Polymer Materials Co. Ltd., China) anion-exchange membranes (AEMs) is analyzed using the application of the microheterogeneous model to experimental data. The structure-properties relationship for these membranes is examined when they are bathed by NaCl and Na2SO4 solutions. These results are compared with the characteristics of the well-studied homogenous Neosepta AMX (ASTOM Corporation, Japan) and heterogeneous AMH-PES (Mega a.s., Czech Republic) anion-exchange membranes. It is found that the CJMA-6 membrane has the highest counterion permselectivity (chlorides, sulfates) among the CJMAED series membranes, very close to that of the AMX membrane. The CJMA-3 membrane has the transport characteristics close to the AMH-PES membrane. The CJMA-7 membrane has the lowest exchange capacity and the highest volume fraction of the intergel spaces filled with an equilibrium electroneutral solution. These properties predetermine the lowest counterion transport number in CJMA-7 among other investigated AEMs, which nevertheless does not fall below 0.87 even in 1.0 eq L-1 solutions of NaCl or Na2SO4. One of the reasons for the decrease in the permselectivity of CJMAED membranes is the extended macropores, which are localized at the ion-exchange material/reinforcing cloth boundaries. In relatively concentrated solutions, the electric current prefers to pass through these well-conductive but nonselective macropores rather than the highly selective but low-conductive elements of the gel phase. It is shown that the counterion permselectivity of the CJMA-7 membrane can be significantly improved by coating its surface with a dense homogeneous ion-exchange film.

PAR1 biased signaling is required for activated protein C in vivo benefits in sepsis and stroke.

Activated protein C (APC) cleaves protease-activated receptor 1 (PAR1) in vitro at R46 to initiate beneficial cell signaling; however, thrombin and APC can cleave at R41. To elucidate PAR1-dependent aspects of the pharmacologic in vivo mechanisms of APC, we generated C57BL/6 mouse strains carrying QQ41 or QQ46 point mutations in PAR1 (F2r gene). Using these strains, we determined whether or not recombinant murine signaling-selective APC mutants would reduce septic death or provide neuroprotection against ischemic stroke when mice carried PAR1-homozygous mutations that prevent cleavage at either R41 or R46. Intercrossing PAR1+/R46Q mice generated expected numbers of PAR1+/+, PAR1+/R46Q, and R46Q/R46Q offspring whereas intercrossing PAR1+/R41Q mice gave decreased R41Q/R41Q homozygotes (resembling intercrossing PAR1+/PAR1-knockout mice). QQ41-PAR1 and QQ46-PAR1 brain endothelial cells showed the predicted retention or loss of cellular responses to thrombin receptor-activating peptide, thrombin, or APC for each PAR1 mutation. In sepsis studies, exogenous APC reduced mortality from 50% to 10% in Escherichia coli-induced pneumonia for wild-type (Wt) PAR1 and QQ41-PAR1 mice (P < .01) but had no benefit for QQ46-PAR1 mice. In transient distal middle cerebral artery occlusion stroke studies, exogenous APC significantly reduced infarct size, edema, and neuronal apoptosis for Wt mice and QQ41-PAR1 mice but had no detectable benefits for mice carrying QQ46-PAR1. In functional studies of forelimb-asymmetry and foot-fault tests at 24 hours after stroke induction, signaling-selective APC was beneficial for Wt and QQ41-PAR1 mice but not QQ46-PAR1 mice. These results support the concept that APC-induced, PAR1-dependent biased signaling following R46 cleavage is central to the in vivo benefits of APC.

A Novel Anion Exchange Membrane for Bisulfite Anion Separation by Grafting a Quaternized Moiety through BPPO via Thermal-Induced Phase Separation.

Ion-exchange membranes are the core elements for an electrodialysis (ED) separation process. Phase inversion is an effective method, particularly for commercial membrane production. It introduces two different mechanisms, i.e., thermal induced phase separation (TIPS) and diffusion induced phase separation (DIPS). In this study, anion exchange membranes (AEMs) were prepared by grafting a quaternized moiety (QM,2-[dimethylaminomethyl]naphthalen-1-ol) through brominated poly (2,6-dimethyl-1,4-phenylene oxide) (BPPO) via the TIPS method. Those membranes were applied for selective bisulfite (HSO3-) anion separation using ED. The membrane surface morphology was characterized by SEM, and the compositions were magnified using a high-resolution transmission electron microscope (HRTEM). Notably, the membranes showed excellent substance stability in an alkali medium and in grafting tests performed in a QM-soluble solvent. The ED experiment indicated that the as-prepared membrane exhibited better HSO3- separation performance than the state-of-the-art commercial Neosepta AMX (ASTOM, Japan) membrane.

Biomimetic Nanocones that Enable High Ion Permselectivity.

Artificial counterparts of conical-shaped transmembrane protein channels are of interest in biomedical sciences for biomolecule detection and selective ion permeation based on ionic size and/or charge differences. However, industrial-scale applications such as seawater desalination, separation of mono- from divalent cations, and treatment of highly-saline industrial waste effluents are still big challenges for such biomimetic channels. A simple monomer seeding experimental approach is used to grow ionically conductive biomimetic charged nanocone pores at the surface of an acid-functionalized membrane. These readily scalable nanocone membranes enable ultra-fast cation permeation (Na+ =8.4× vs. Mg2+ =1.4×) and high ion charge selectivity (Na+ /Mg2+ =6×) compared to the commercial state-of-the-art permselective membrane (CSO, Selemion, Japan) owing to negligible surface resistance and positively charged conical pore walls.

Blood-spinal cord barrier disruption contributes to early motor-neuron degeneration in ALS-model mice.

Humans with ALS and transgenic rodents expressing ALS-associated superoxide dismutase (SOD1) mutations develop spontaneous blood-spinal cord barrier (BSCB) breakdown, causing microvascular spinal-cord lesions. The role of BSCB breakdown in ALS disease pathogenesis in humans and mice remains, however, unclear, although chronic blood-brain barrier opening has been shown to facilitate accumulation of toxic blood-derived products in the central nervous system, resulting in secondary neurodegenerative changes. By repairing the BSCB and/or removing the BSCB-derived injurious stimuli, we now identify that accumulation of blood-derived neurotoxic hemoglobin and iron in the spinal cord leads to early motor-neuron degeneration in SOD1(G93A) mice at least in part through iron-dependent oxidant stress. Using spontaneous or warfarin-accelerated microvascular lesions, motor-neuron dysfunction and injury were found to be proportional to the degree of BSCB disruption at early disease stages in SOD1(G93A) mice. Early treatment with an activated protein C analog restored BSCB integrity that developed from spontaneous or warfarin-accelerated microvascular lesions in SOD1(G93A) mice and eliminated neurotoxic hemoglobin and iron deposits. Restoration of BSCB integrity delayed onset of motor-neuron impairment and degeneration. Early chelation of blood-derived iron and antioxidant treatment mitigated early motor-neuronal injury. Our data suggest that BSCB breakdown contributes to early motor-neuron degeneration in ALS mice and that restoring BSCB integrity during an early disease phase retards the disease process.

Synthesis, Crystal Structure, and Photoelectric Properties of a New Layered Bismuth Oxysulfide.

[Bi2O2]-containing tetragonal compounds have received enormous attention due to unique functions including ferroelectricity, photocatalysis, and superconductivity. Here, a new layered compound Bi9O7.5S6 was synthesized via a facile hydrothermal route. The compound, belonging to a new structure type crystallizes in a rhombohedral system with space group R3̅m (a = 4.0685(1) Å, c = 31.029(5) Å, V = 444.8(1) Å(3), Z = 1). The overall crystal structure consists of alternatively packed unique [Bi2O2] and [BiS2] layers along [001] which are combined with each other by van der Waals interaction. The phase purity of the product is confirmed by powder X-ray diffraction. XPS analyses indicate +3 for Bi and -2 for S atoms. The temperature dependence of resistivity ρ(T) indicates that the semiconducting sample follows the mechanisms of variable range hopping (VRH) and adiabatic small polaron hopping (SPH). The direct-transition band gap, Eg = 1.27 eV derived from optical absorption spectrum, falls in the optimal region of solar absorber materials. Accordingly, the photoelectric measurement demonstrates the potential for applications for photovoltaic devices.

CuIn(S,Se)(2) thin films prepared from a novel thioacetic acid-based solution and their photovoltaic application.

Low-cost and high-yield preparation of CuInSe2 films is the bottleneck for promising CuInSe2-based thin film solar cells. Here, we developed a simple, safe and cost-effective method using thioacetic acid to fabricate the absorber films of CuIn(S,Se)2 (CISSe). Dissolution of Cu2O and In(OH)3 in thioacetic acid was attributed to the strong coordination ability of S. The adhesive precursor solution can be prepared without any heating, centrifugation and inert gas protection, superior to the previously reported methods. The precursor CISSe layer was easily deposited in air by spin coating to ensure low cost. Uniform and compact CISSe thin films with well-crystallized and pure-phased CISSe grains were obtained after one step annealing. The as-prepared CISSe thin films were successfully applied to solar cells and a energy conversion efficiency of 6.75% was achieved. This facile preparation provides a low-cost and easy method to fabricate Cu-based thin film solar cells.

High-Entropy Oxide of (BiZrMoWCeLa)O2 as a Novel Catalyst for Vanadium Redox Flow Batteries.

In this study, new fluorite high-entropy oxide (HEO), (BiZrMoWCeLa)O2, nanoparticles were produced using a surfactant-assisted hydrothermal technique followed by calcination and were used as novel catalytic materials for vanadium redox flow batteries (VRFBs). The HEO calcined at 750 °C (HEO-750) demonstrates superior electrocatalytic activity toward V3+/V2+ and VO2+/VO2+ redox couples compared to those of cells assembled with other samples. The charge-discharge tests further confirm that VRFBs using the HEO-750 catalyst demonstrate excellent Coulombic efficiency, voltage efficiency, and energy efficiency of 97.22, 87.47, and 85.04% at a current density of 80 mA cm-2 and 98.10, 74.76, and 73.34% at a higher current density of 160 mA cm-2, respectively. Moreover, with 500 charge-discharge cycles, there is no discernible degradation. These results are attributed to the calcination heat treatment, which induces the formation of a new single-phase fluorite structure, which facilitates the redox reactions of the vanadium redox couples. Furthermore, a high surface area, wettability, and plenty of oxygen vacancies can give more surface electroactive sites, improving the electrochemical performance, the charge transfer of the redox processes, and the stability of the VRFBs' electrode. This is the first report on the development of fluorite structure HEO nanoparticles in VRFBs, and it opens the door to further research into other HEOs.

Activated protein C analog protects from ischemic stroke and extends the therapeutic window of tissue-type plasminogen activator in aged female mice and hypertensive rats.

BACKGROUND AND PURPOSE: 3K3A-activated protein C (APC) protects young, healthy male rodents after ischemic stroke. 3K3A-APC is currently under development as a neuroprotectant for acute ischemic stroke in humans. Stroke Therapy Academic Industry Roundtable recommends that after initial studies in young, healthy male animals, further studies should be performed in females, aged animals, and animals with comorbid conditions. Here, we studied the effects of delayed 3KA-APC therapy alone and with tissue-type plasminogen activator (tPA) in aged female mice and spontaneously hypertensive rats. METHODS: We used Stroke Therapy Academic Industry Roundtable recommendations for ensuring good scientific inquiry. Murine recombinant 3K3A-APC (0.2 mg/kg) alone or with recombinant tPA (10 mg/kg) was given intravenously 4 hours after transient middle cerebral artery occlusion in aged female mice and rats and after embolic stroke in spontaneously hypertensive rat. 3K3A-APC was additionally administered within 3 to 7 days after stroke. The neuropathological analysis and neurological scores, foot-fault, forelimb asymmetry, and adhesive removal tests were performed within 7 and 28 days of stroke. RESULTS: In all models, tPA alone had no effects on the infarct volume or behavior. 3K3A-APC alone or with tPA reduced the infarct volume 7 days after the middle cerebral artery occlusion in aged female mice and embolic stroke in spontaneously hypertensive rat by 62% to 66% and 50% to 53%, respectively, significantly improved (P<0.05) behavior, and eliminated tPA-induced intracerebral microhemorrhages. In aged female mice, 3K3A-APC was protective within 4 weeks of stroke. CONCLUSIONS: 3K3A-APC protects from ischemic stroke and extends the therapeutic window of tPA in aged female mice and in spontaneously hypertensive rat with a comorbid condition.

Neurotoxicity of the anticoagulant-selective E149A-activated protein C variant after focal ischemic stroke in mice.

Wild type (WT) activated protein C (APC) and cytoprotective-selective APC variants such as 3K3A-APC (<10% anticoagulant but normal cytoprotective activity) are neuroprotective in murine focal ischemic stroke models. Here we compared the neuroprotective effects of the anticoagulant-selective E149A-APC variant (>3-fold increased anticoagulant activity but defective cytoprotective activities) to those of the cytoprotective-selective 5A-APC variant (<10% anticoagulant activity). After transient distal middle cerebral artery occlusion, mice received a vehicle, E149A-APC or 5A-APC at 0.2mg/kg at 4h after stroke. Treatment with 5A-APC was neuroprotective, as it improved performance on forelimb use asymmetry test and foot fault test (P<0.05), reduced by 48% and 50% the infarct and edema volumes, respectively (P<0.05), and was not associated with an increased risk of bleeding as indicated by normal hemoglobin levels in the ischemic brain at day 7. In contrast, E149A-APC treatment worsened neurological outcome determined by foot fault tests and forelimb use asymmetry tests, and increased significantly by 44% and 60% infarct and edema volume, respectively (P<0.05). At 7days after treatment, E149A-APC compared to vehicle or 5A-APC notably increased by ~5-fold the hemoglobin level in the ischemic hemisphere suggesting it provoked significant intracerebral bleeding. Thus, the enhanced anticoagulant activity of E149A-APC increased post-ischemic accumulation of neurotoxic erythrocyte-derived hemoglobin which likely worsened the neurological and neuropathological outcomes after stroke. Our data emphasize that APC's cytoprotective activities, but not its anticoagulant activity, are key for APC neuroprotection after transient ischemic stroke.

Preparation of monodispersed CuInS2 nanopompons and nanoflake films and application in dye-sensitized solar cells.

Monodispersed nanopompons and thin films of CuInS2 with hierarchical nanostructure are obtained via a solvothermal method. The hierarchical thin film of CuInS2 has been reported for the first time to display excellent catalytic activity as a counter electrode in dye-sensitized solar cells, and a power conversion efficiency of 4.8% is achieved.

A cascade electro-dehydration process for simultaneous extraction and enrichment of uranium from simulated seawater.

Uranium extraction from seawater has become a crucial issue that has raised tremendous attention. The transport of water molecules along with salt ions through an ion-exchange membrane is a common phenomenon for typical electro-membrane processes such as selective electrodialysis (SED). In this study, a cascade electro-dehydration process was proposed for the simultaneous extraction and enrichment of uranium from simulated seawater by taking advantage of water transport through ion-exchange membranes and the high permselectivity of membranes for monovalent ions against uranate ions. The results indicated that the electro-dehydration effect in SED allowed 1.8 times the concentration of uranium with a loose structure CJMC-5 cation-exchange membrane at a current density of 4 mA/cm2. Thereafter, a cascade electro-dehydration by a combination of SED with conventional electrodialysis (CED) enabled approximately 7.5 times uranium concentration with the extraction yield rate reaching over 80% and simultaneously desalting the majority of salts. Overall, a cascade electro-dehydration is a viable approach, creating a novel route for highly effective uranium extraction and enrichment from seawater.

Bipolar Membrane Electrodialysis for Cleaner Production of Diprotic Malic Acid: Separation Mechanism and Performance Evaluation.

Bipolar membrane electrodialysis (BMED) is a promising process for the cleaner production of organic acid. In this study, the separation mechanism of BMED with different cell configurations, i.e., BP-A, BP-A-C, and BP-C (BP, bipolar membrane; A, anion exchange membrane; C, cation exchange membrane), to produce diprotic malic acid from sodium malate was compared in consideration of the conversion ratio, current efficiency and energy consumption. Additionally, the current density and feed concentration were investigated to optimize the BMED performance. Results indicate that the conversion ratio follows BP-C > BP-A-C > BP-A, the current efficiency follows BP-A-C > BP-C > BP-A, and the energy consumption follows BP-C < BP-A-C < BP-A. For the optimized BP-C configuration, the current density was optimized as 40 mA/cm2 in consideration of low total process cost; high feed concentration (0.5-1.0 mol/L) is more feasible to produce diprotic malic acid due to the high conversion ratio (73.4-76.2%), high current efficiency (88.6-90.7%), low energy consumption (0.66-0.71 kWh/kg) and low process cost (0.58-0.59 USD/kg). Moreover, a high concentration of by-product NaOH (1.3497 mol/L) can be directly recycled to the upstream process. Therefore, BMED is a cleaner, high-efficient, low energy consumption and environmentally friendly process to produce diprotic malic acid.

How Chemical Nature of Fixed Groups of Anion-Exchange Membranes Affects the Performance of Electrodialysis of Phosphate-Containing Solutions?

Innovative ion exchange membranes have become commercially available in recent years. However, information about their structural and transport characteristics is often extremely insufficient. To address this issue, homogeneous anion exchange membranes with the trade names ASE, CJMA-3 and CJMA-6 have been investigated in NaxH(3-x)PO4 solutions with pH 4.4 ± 0.1, 6.6 and 10.0 ± 0.2, as well as NaCl solutions with pH 5.5 ± 0.1. Using IR spectroscopy and processing the concentration dependences of the electrical conductivity of these membranes in NaCl solutions, it was shown that ASE has a highly cross-linked aromatic matrix and mainly contains quaternary ammonium groups. Other membranes have a less cross-linked aliphatic matrix based on polyvinylidene fluoride (CJMA-3) or polyolefin (CJMA-6) and contain quaternary amines (CJMA-3) or a mixture of strongly basic (quaternary) and weakly basic (secondary) amines (CJMA-6). As expected, in dilute solutions of NaCl, the conductivity of membranes increases with an increase in their ion-exchange capacity: CJMA-6 < CJMA-3 << ASE. Weakly basic amines appear to form bound species with proton-containing phosphoric acid anions. This phenomenon causes a decrease in the electrical conductivity of CJMA-6 membranes compared to other studied membranes in phosphate-containing solutions. In addition, the formation of the neutral and negatively charged bound species suppresses the generation of protons by the "acid dissociation" mechanism. Moreover, when the membrane is operated in overlimiting current modes and/or in alkaline solutions, a bipolar junction is formed at the CJMA- 6/depleted solution interface. The CJMA-6 current-voltage curve becomes similar to the well-known curves for bipolar membranes, and water splitting intensifies in underlimiting and overlimiting modes. As a result, energy consumption for electrodialysis recovery of phosphates from aqueous solutions almost doubles when using the CJMA-6 membrane compared to the CJMA-3 membrane.

Anti-malaria drug artesunate prevents development of amyloid-ß pathology in mice by upregulating PICALM at the blood-brain barrier.

BACKGROUND: PICALM is one of the most significant susceptibility factors for Alzheimer's disease (AD). In humans and mice, PICALM is highly expressed in brain endothelium. PICALM endothelial levels are reduced in AD brains. PICALM controls several steps in Aß transcytosis across the blood-brain barrier (BBB). Its loss from brain endothelium in mice diminishes Aß clearance at the BBB, which worsens Aß pathology, but is reversible by endothelial PICALM re-expression. Thus, increasing PICALM at the BBB holds potential to slow down development of Aß pathology. METHODS: To identify a drug that could increase PICALM expression, we screened a library of 2007 FDA-approved drugs in HEK293t cells expressing luciferase driven by a human PICALM promoter, followed by a secondary mRNA screen in human Eahy926 endothelial cell line. In vivo studies with the lead hit were carried out in Picalm-deficient (Picalm+/-) mice, Picalm+/-; 5XFAD mice and Picalmlox/lox; Cdh5-Cre; 5XFAD mice with endothelial-specific Picalm knockout. We studied PICALM expression at the BBB, Aß pathology and clearance from brain to blood, cerebral blood flow (CBF) responses, BBB integrity and behavior. RESULTS: Our screen identified anti-malaria drug artesunate as the lead hit. Artesunate elevated PICALM mRNA and protein levels in Eahy926 endothelial cells and in vivo in brain capillaries of Picalm+/- mice by 2-3-fold. Artesunate treatment (32 mg/kg/day for 2 months) of 3-month old Picalm+/-; 5XFAD mice compared to vehicle increased brain capillary PICALM levels by 2-fold, and reduced Aß42 and Aß40 levels and Aß and thioflavin S-load in the cortex and hippocampus, and vascular Aß load by 34-51%. Artesunate also increased circulating Aß42 and Aß40 levels by 2-fold confirming accelerated Aß clearance from brain to blood. Consistent with reduced Aß pathology, treatment of Picalm+/-; 5XFAD mice with artesunate improved CBF responses, BBB integrity and behavior on novel object location and recognition, burrowing and nesting. Endothelial-specific knockout of PICALM abolished all beneficial effects of artesunate in 5XFAD mice indicating that endothelial PICALM is required for its therapeutic effects. CONCLUSIONS: Artesunate increases PICALM levels and Aß clearance at the BBB which prevents development of Aß pathology and functional deficits in mice and holds potential for translation to human AD.

An activated protein C analog with reduced anticoagulant activity extends the therapeutic window of tissue plasminogen activator for ischemic stroke in rodents.

BACKGROUND AND PURPOSE: Tissue plasminogen activator (tPA) is the only approved therapy for acute ischemic stroke. However, tPA has a brief therapeutic window. Its side effects include intracerebral bleeding and neurotoxicity. Therefore, a combination therapy with tPA and agents that can extend the therapeutic window of tPA and/or counteract its side effects are warranted. Here, we studied whether 3K3A-APC, a neuroprotective analog of activated protein C with reduced anticoagulant activity, can enhance the therapeutic effects of tPA in models of ischemic stroke in rodents. METHODS: Human recombinant tPA (10 mg/kg), alone or in combination with human recombinant 3K3A-APC (2 mg/kg), was administered intravenously 4 hours after proximal or distal transient middle cerebral artery occlusion in mice and embolic stroke in rats. The 3K3A-APC was additionally administered for 3 to 4 consecutive days after stroke. The neuropathological and neurological analyses were performed at 1 to 7 days after stroke. RESULTS: In all models, tPA alone had no effects on the infarct volume or behavior (ie, neurological score, foot-fault, forelimb asymmetry, adhesive removal) compared with vehicle. The tPA and 3K3A-APC combination therapy reduced the infarct volume 24 hours and 7 days after proximal or distal transient middle cerebral artery occlusion in mice and 7 days after embolic stroke in rats by 65%, 63%, and 52%, respectively, significantly (P<0.05) improved behavior and eliminated tPA-induced intracerebral microhemorrhages. CONCLUSIONS: The 3K3A-APC extends the therapeutic window of tPA for ischemic stroke in rodents. Therefore, this combination therapy also should be considered for treating stroke in humans.

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.

3K3A-Activated Protein C Protects the Blood-Brain Barrier and Neurons From Accelerated Ischemic Injury Caused by Pericyte Deficiency in Mice.

Pericytes, mural cells of brain capillaries, maintain the blood-brain barrier (BBB), regulate cerebral blood flow (CBF), and protect neurons against ischemic damage. To further investigate the role of pericytes in ischemia, we induced stroke by 45-min transient middle cerebral artery occlusion (tMCAo) in 6-month-old pericyte-deficient Pdgfrb + ⁣/- mice and control Pdgfrb+/+ littermates. Compared to controls, Pdgfrb + ⁣/- mice showed a 26% greater loss of CBF during early reperfusion, and 40-50% increase in the infarct and edema volumes and motor neurological score 24 h after tMCAo. These changes were accompanied by 50% increase in both immunoglobulin G and fibrinogen pericapillary deposits in the ischemic cortex 8 h after tMCAo indicating an accelerated BBB breakdown, and 35 and 55% greater losses of pericyte coverage and number of degenerating neurons 24 h after tMCAo, respectively. Treatment of Pdgfrb + ⁣/- mice with 3K3A-activated protein C (APC), a cell-signaling analog of plasma protease APC, administered intravenously 10 min and 4 h after tMCAo normalized CBF during the early reperfusion phase and reduced infarct and edema volume and motor neurological score by 55-60%, with similar reductions in BBB breakdown and number of degenerating neurons. Our data suggest that pericyte deficiency results in greater brain injury, BBB breakdown, and neuronal degeneration in stroked mice and that 3K3A-APC protects the brain from accelerated injury caused by pericyte deficiency. These findings may have implications for treatment of ischemic brain injury in neurological conditions associated with pericyte loss such as those seen during normal aging and in neurodegenerative disorders such as Alzheimer's disease.