Activated Bone Marrow-Derived Macrophages Eradicate Alzheimer's-Related Aβ42 Oligomers and Protect Synapses.

Impaired synaptic integrity and function due to accumulation of amyloid β-protein (Aβ42) oligomers is thought to be a major contributor to cognitive decline in Alzheimer's disease (AD). However, the exact role of Aβ42 oligomers in synaptotoxicity and the ability of peripheral innate immune cells to rescue synapses remain poorly understood due to the metastable nature of oligomers. Here, we utilized photo-induced cross-linking to stabilize pure oligomers and study their effects vs. fibrils on synapses and protection by Aβ-phagocytic macrophages. We found that cortical neurons were more susceptible to Aβ42 oligomers than fibrils, triggering additional neuritic arborization retraction, functional alterations (hyperactivity and spike waveform), and loss of VGluT1- and PSD95-excitatory synapses. Co-culturing neurons with bone marrow-derived macrophages protected synapses against Aβ42 fibrils; moreover, immune activation with glatiramer acetate (GA) conferred further protection against oligomers. Mechanisms involved increased Aβ42 removal by macrophages, amplified by GA stimulation: fibrils were largely cleared through intracellular CD36/EEA1+-early endosomal proteolysis, while oligomers were primarily removed via extracellular/MMP-9 enzymatic degradation. In vivo studies in GA-immunized or CD115+-monocyte-grafted APPSWE/PS1ΔE9-transgenic mice followed by pre- and postsynaptic analyses of entorhinal cortex and hippocampal substructures corroborated our in vitro findings of macrophage-mediated synaptic preservation. Together, our data demonstrate that activated macrophages effectively clear Aβ42 oligomers and rescue VGluT1/PSD95 synapses, providing rationale for harnessing macrophages to treat AD

Activated Bone Marrow-Derived Macrophages Eradicate Alzheimer's-Related Aβ₄₂ Oligomers and Protect Synapses

Impaired synaptic integrity and function due to accumulation of amyloid β-protein (Aβ₄₂) oligomers is thought to be a major contributor to cognitive decline in Alzheimer's disease (AD). However, the exact role of Aβ₄₂ oligomers in synaptotoxicity and the ability of peripheral innate immune cells to rescue synapses remain poorly understood due to the metastable nature of oligomers. Here, we utilized photo-induced cross-linking to stabilize pure oligomers and study their effects vs. fibrils on synapses and protection by Aβ-phagocytic macrophages. We found that cortical neurons were more susceptible to Aβ₄₂ oligomers than fibrils, triggering additional neuritic arborization retraction, functional alterations (hyperactivity and spike waveform), and loss of VGluT1- and PSD95-excitatory synapses. Co-culturing neurons with bone marrow-derived macrophages protected synapses against Aβ₄₂ fibrils; moreover, immune activation with glatiramer acetate (GA) conferred further protection against oligomers. Mechanisms involved increased Aβ₄₂ removal by macrophages, amplified by GA stimulation: fibrils were largely cleared through intracellular CD36/EEA1⁺-early endosomal proteolysis, while oligomers were primarily removed via extracellular/MMP-9 enzymatic degradation. In vivo studies in GA-immunized or CD115⁺-monocyte-grafted APP_(SWE)/PS1_(ΔE9)-transgenic mice followed by pre- and postsynaptic analyses of entorhinal cortex and hippocampal substructures corroborated our in vitro findings of macrophage-mediated synaptic preservation. Together, our data demonstrate that activated macrophages effectively clear Aβ₄₂ oligomers and rescue VGluT1/PSD95 synapses, providing rationale for harnessing macrophages to treat AD

Vasodilator Phosphostimulated Protein (VASP) Protects Endothelial Barrier Function During Hypoxia

The endothelial barrier controls the passage of solutes from the vascular space. This is achieved through active reorganization of the actin cytoskeleton. A central cytoskeletal protein involved into this is vasodilator-stimulated phosphoprotein (VASP). However, the functional role of endothelial VASP during hypoxia has not been thoroughly elucidated. We determined endothelial VASP expression through real-time PCR (Rt-PCR), immunhistochemistry, and Western blot analysis during hypoxia. VASP promoter studies were performed using a PGL3 firefly luciferase containing plasmid. Following approval by the local authorities, VASP−/− mice and littermate controls were subjected to normobaric hypoxia (8% O2, 92% N2) after intravenous injection of Evans blue dye. In in vitro studies, we found significant VASP repression in human microvascular and human umbilical vein endothelial cells through Rt-PCR, immunhistochemistry, and Western blot analysis. The VASP promoter construct demonstrated significant repression in response to hypoxia, which was abolished when the binding of hypoxia-inducible factor 1 alpha was excluded. Exposure of wild-type (WT) and VASP−/− animals to normobaric hypoxia for 4 h resulted in an increase in Evans blue tissue extravasation that was significantly increased in VASP−/− animals compared to WT controls. In summary, we demonstrate here that endothelial VASP holds significant importance for endothelial barrier properties during hypoxia

Liposome-Mediated Cellular Delivery of Active gp91phox

International audienceBACKGROUND: Gp91(phox) is a transmembrane protein and the catalytic core of the NADPH oxidase complex of neutrophils. Lack of this protein causes chronic granulomatous disease (CGD), a rare genetic disorder characterized by severe and recurrent infections due to the incapacity of phagocytes to kill microorganisms. METHODOLOGY: Here we optimize a prokaryotic cell-free expression system to produce integral mammalian membrane proteins. CONCLUSIONS: Using this system, we over-express truncated forms of the gp91(phox) protein under soluble form in the presence of detergents or lipids resulting in active proteins with a "native-like" conformation. All the proteins exhibit diaphorase activity in the presence of cytosolic factors (p67(phox), p47(phox), p40(phox) and Rac) and arachidonic acid. We also produce proteoliposomes containing gp91(phox) protein and demonstrate that these proteins exhibit activities similar to their cellular counterpart. The proteoliposomes induce rapid cellular delivery and relocation of recombinant gp91(phox) proteins to the plasma membrane. Our data support the concept of cell-free expression technology for producing recombinant proteoliposomes and their use for functional and structural studies or protein therapy by complementing deficient cells in gp91(phox) protein

CD36 and Fyn kinase mediate malaria-induced lung endothelial barrier dysfunction in mice infected with Plasmodium berghei.

PMC3744507Severe malaria can trigger acute lung injury characterized by pulmonary edema resulting from increased endothelial permeability. However, the mechanism through which lung fluid conductance is altered during malaria remains unclear. To define the role that the scavenger receptor CD36 may play in mediating this response, C57BL/6J (WT) and CD36-/- mice were infected with P. berghei ANKA and monitored for changes in pulmonary endothelial barrier function employing an isolated perfused lung system. WT lungs demonstrated a >10-fold increase in two measures of paracellular fluid conductance and a decrease in the albumin reflection coefficient (σalb) compared to control lungs indicating a loss of barrier function. In contrast, malaria-infected CD36-/- mice had near normal fluid conductance but a similar reduction in σalb. In WT mice, lung sequestered iRBCs demonstrated production of reactive oxygen species (ROS). To determine whether knockout of CD36 could protect against ROS-induced endothelial barrier dysfunction, mouse lung microvascular endothelial monolayers (MLMVEC) from WT and CD36-/- mice were exposed to H2O2. Unlike WT monolayers, which showed dose-dependent decreases in transendothelial electrical resistance (TER) from H2O2 indicating loss of barrier function, CD36-/- MLMVEC demonstrated dose-dependent increases in TER. The differences between responses in WT and CD36-/- endothelial cells correlated with important differences in the intracellular compartmentalization of the CD36-associated Fyn kinase. Malaria infection increased total lung Fyn levels in CD36-/- lungs compared to WT, but this increase was due to elevated production of the inactive form of Fyn further suggesting a dysregulation of Fyn-mediated signaling. The importance of Fyn in CD36-dependent endothelial signaling was confirmed using in vitro Fyn knockdown as well as Fyn-/- mice, which were also protected from H2O2- and malaria-induced lung endothelial leak, respectively. Our results demonstrate that CD36 and Fyn kinase are critical mediators of the increased lung endothelial fluid conductance caused by malaria infection.JH Libraries Open Access Fun

Osteopontin depletion in macrophages perturbs proteostasis via regulating UCHL1-UPS axis and mitochondria-mediated apoptosis

IntroductionOsteopontin (OPN; also known as SPP1), an immunomodulatory cytokine highly expressed in bone marrow-derived macrophages (BMMΦ), is known to regulate diverse cellular and molecular immune responses. We previously revealed that glatiramer acetate (GA) stimulation of BMMΦ upregulates OPN expression, promoting an anti-inflammatory, pro-healing phenotype, whereas OPN inhibition triggers a pro-inflammatory phenotype. However, the precise role of OPN in macrophage activation state is unknown.MethodsHere, we applied global proteome profiling via mass spectrometry (MS) analysis to gain a mechanistic understanding of OPN suppression versus induction in primary macrophage cultures. We analyzed protein networks and immune-related functional pathways in BMMΦ either with OPN knockout (OPNKO) or GA-mediated OPN induction compared with wild type (WT) macrophages. The most significant differentially expressed proteins (DEPs) were validated using immunocytochemistry, western blot, and immunoprecipitation assays.Results and discussionWe identified 631 DEPs in OPNKO or GA-stimulated macrophages as compared to WT macrophages. The two topmost downregulated DEPs in OPNKO macrophages were ubiquitin C-terminal hydrolase L1 (UCHL1), a crucial component of the ubiquitin-proteasome system (UPS), and the anti-inflammatory Heme oxygenase 1 (HMOX-1), whereas GA stimulation upregulated their expression. We found that UCHL1, previously described as a neuron-specific protein, is expressed by BMMΦ and its regulation in macrophages was OPN-dependent. Moreover, UCHL1 interacted with OPN in a protein complex. The effects of GA activation on inducing UCHL1 and anti-inflammatory macrophage profiles were mediated by OPN. Functional pathway analyses revealed two inversely regulated pathways in OPN-deficient macrophages: activated oxidative stress and lysosome-mitochondria-mediated apoptosis (e.g., ROS, Lamp1-2, ATP-synthase subunits, cathepsins, and cytochrome C and B subunits) and inhibited translation and proteolytic pathways (e.g., 60S and 40S ribosomal subunits and UPS proteins). In agreement with the proteome-bioinformatics data, western blot and immunocytochemical analyses revealed that OPN deficiency perturbs protein homeostasis in macrophages—inhibiting translation and protein turnover and inducing apoptosis—whereas OPN induction by GA restores cellular proteostasis. Taken together, OPN is essential for macrophage homeostatic balance via the regulation of protein synthesis, UCHL1-UPS axis, and mitochondria-mediated apoptotic processes, indicating its potential application in immune-based therapies

Macrophages in Alzheimer’s disease: the blood-borne identity

Alzheimer’s disease (AD) is a progressive and incurable neurodegenerative disorder clinically characterized by cognitive decline involving loss of memory, reasoning and linguistic ability. The amyloid cascade hypothesis holds that mismetabolism and aggregation of neurotoxic amyloid-β (Aβ) peptides, which are deposited as amyloid plaques, are the central etiological events in AD. Recent evidence from AD mouse models suggests that blood-borne mononuclear phagocytes are capable of infiltrating the brain and restricting β-amyloid plaques, thereby limiting disease progression. These observations raise at least three key questions: (1) what is the cell of origin for macrophages in the AD brain, (2) do blood-borne macrophages impact the pathophysiology of AD and (3) could these enigmatic cells be therapeutically targeted to curb cerebral amyloidosis and thereby slow disease progression? This review begins with a historical perspective of peripheral mononuclear phagocytes in AD, and moves on to critically consider the controversy surrounding their identity as distinct from brain-resident microglia and their potential impact on AD pathology

cGMP increases antioxidant function and attenuates oxidant cell death in mouse lung microvascular endothelial cells by a protein kinase G-dependent mechanism

Increasing evidence suggests that endothelial cytotoxicity from reactive oxygen species (ROS) contributes to the pathogenesis of acute lung injury. Treatments designed to increase intracellular cGMP attenuate ROS-mediated apoptosis and necrosis in several cell types, but the mechanisms are not understood, and the effect of cGMP on pulmonary endothelial cell death remains controversial. In the current study, increasing intracellular cGMP by either 8pCPT-cGMP (50 μM) or atrial natriuretic peptide (10 nM) significantly attenuated cell death in H2O2-challenged mouse lung microvascular (MLMVEC) monolayers. 8pCPT-cGMP also decreased perfusate LDH release in isolated mouse lungs exposed to H2O2 or ischemia-reperfusion. The protective effect of increasing cGMP in MLMVECs was accompanied by enhanced endothelial H2O2 scavenging (measured by H2O2 electrode) and decreased intracellular ROS concentration (measured by 2′,7′-dichlorofluorescin fluorescence) as well as decreased phosphorylation of p38 MAPK and Akt. The cGMP-mediated cytoprotection and increased H2O2 scavenging required >2 h of 8pCPT-cGMP incubation in wild-type MLMVEC and were absent in MLMVEC from protein kinase G (PKGI)−/− mice suggesting a PKGI-mediated effect on gene regulation. Catalase and glutathione peroxidase 1 (Gpx-1) protein were increased by cGMP in wild-type but not PKGI−/− MLMVEC monolayers. Both the cGMP-mediated increases in antioxidant proteins and H2O2 scavenging were prevented by inhibition of translation with cycloheximide. 8pCPT-cGMP had minimal effects on catalase and Gpx-1 mRNA. We conclude that cGMP, through PKGI, attenuated H2O2-induced cytotoxicity in MLMVEC by increasing catalase and Gpx-1 expression through an unknown posttranscriptional effect