Polysialic acid blocks mononuclear phagocyte reactivity, inhibits complement activation, and protects from vascular damage in the retina.

Age-related macular degeneration (AMD) is a major cause of blindness in the elderly population. Its pathophysiology is linked to reactive oxygen species (ROS) and activation of the complement system. Sialic acid polymers prevent ROS production of human mononuclear phagocytes via the inhibitory sialic acid-binding immunoglobulin-like lectin-11 (SIGLEC11) receptor. Here, we show that low-dose intravitreal injection of low molecular weight polysialic acid with average degree of polymerization 20 (polySia avDP20) in humanized transgenic mice expressing SIGLEC11 on mononuclear phagocytes reduced their reactivity and vascular leakage induced by laser coagulation. Furthermore, polySia avDP20 prevented deposition of the membrane attack complex in both SIGLEC11 transgenic and wild-type animals. In vitro, polySia avDP20 showed two independent, but synergistic effects on the innate immune system. First, polySia avDP20 prevented tumor necrosis factor-α, vascular endothelial growth factor A, and superoxide production by SIGLEC11-positive phagocytes. Second, polySia avDP20 directly interfered with complement activation. Our data provide evidence that polySia avDP20 ameliorates laser-induced damage in the retina and thus is a promising candidate to prevent AMD-related inflammation and angiogenesis.

Huperzine A Alleviates Mechanical Allodynia but Not Spontaneous Pain via Muscarinic Acetylcholine Receptors in Mice.

Chronic pain is a major health issue and most patients suffer from spontaneous pain. Previous studies suggest that Huperzine A (Hup A), an alkaloid isolated from the Chinese herb Huperzia serrata, is a potent analgesic with few side effects. However, whether it alleviates spontaneous pain is unclear. We evaluated the effects of Hup A on spontaneous pain in mice using the conditioned place preference (CPP) behavioral assay and found that application of Hup A attenuated the mechanical allodynia induced by peripheral nerve injury or inflammation. This effect was blocked by atropine. However, clonidine but not Hup A induced preference for the drug-paired chamber in CPP. The same effects occurred when Hup A was infused into the anterior cingulate cortex. Furthermore, ambenonium chloride, a competitive inhibitor of acetylcholinesterase, also increased the paw-withdrawal threshold but failed to induce place preference in CPP. Therefore, our data suggest that acetylcholinesterase in both the peripheral and central nervous systems is involved in the regulation of mechanical allodynia but not the spontaneous pain.

Microglia activity modulated by T cell Ig and mucin domain protein 3 (Tim-3).

Microglia are the main innate immune cells in the central nervous system that are actively involved in maintaining brain homeostasis and diseases. T cell Ig and mucin domain protein 3 (Tim-3) plays critical roles in both the adaptive and the innate immune system and is an emerging therapeutic target for treatment of various disorders. In the brain Tim-3 is specifically expressed on microglia but its functional role is unclear. Here, we showed that Tim-3 was up-regulated on microglia by ATP or LPS stimulation. Tim-3 activation with antibodies increased microglia expression of TGF-ß, TNF-α and IL-1ß. Blocking of Tim-3 with antibodies decreased the microglial phagocytosis of apoptotic neurons. Tim-3 blocking alleviated the detrimental effect of microglia on neurons and promoted NG2 cell differentiation in co-cultures. Finally, MAPKs namely ERK1/2 and JNK proteins were phosphorylated upon Tim-3 activation in microglia. Data indicated that Tim-3 modulates microglia activity and regulates the interaction of microglia-neural cells.

Neurodegeneration by activation of the microglial complement-phagosome pathway.

Systemic inflammatory reactions have been postulated to exacerbate neurodegenerative diseases via microglial activation. We now demonstrate in vivo that repeated systemic challenge of mice over four consecutive days with bacterial LPS maintained an elevated microglial inflammatory phenotype and induced loss of dopaminergic neurons in the substantia nigra. The same total cumulative LPS dose given within a single application did not induce neurodegeneration. Whole-genome transcriptome analysis of the brain demonstrated that repeated systemic LPS application induced an activation pattern involving the classical complement system and its associated phagosome pathway. Loss of dopaminergic neurons induced by repeated systemic LPS application was rescued in complement C3-deficient mice, confirming the involvement of the complement system in neurodegeneration. Our data demonstrate that a phagosomal inflammatory response of microglia is leading to complement-mediated loss of dopaminergic neurons.

Astrocyte-restricted disruption of connexin-43 impairs neuronal plasticity in mouse barrel cortex.

There is intensive gap-junctional coupling between glial processes, but their significance in sensory functions remains unknown. Connexin-43 (Cx43), a major component of astrocytic gap-junction channels, is abundantly expressed in astrocytes. To investigate the role of Cx43-mediated gap junctions between astrocytes in sensory functions, we generated Cx43 knockout (KO) mice with a mouse line carrying loxP sites flanking exon 2 of the Cx43 gene and the transgenic line expressing Cre recombinase under control of the glial fibrillary acidic protein promoter, which exhibited a significant loss of Cx43 in astrocytes in the barrel cortex. Although Cx43 expression between the astrocytes measured by immunohistochemistry was virtually abolished in Cx43 KO mice, they had normal architecture in the barrel cortex but the intensity of cytochrome oxide histochemistry decreased significantly. In vivo electrophysiological analysis revealed that the long-term potentiation of the vibrissal evoked responses in the barrel cortex evoked by high-frequency rhythmic vibrissal stimuli (100 Hz, 1 s) was abolished in Cx43 KO mice. Current source density analysis also revealed that astrocytic Cx43 was important to the flow of excitation within the laminar connections in barrel cortex. Behavioral tests showed that the ability of Cx43 KO mice to sense the environment with their whiskers decreased. Even so, the jump-stand experiment showed that they could still discriminate rough from smooth surfaces. Our findings suggest that Cx43-mediated gap-junctional coupling between astrocytes is important in the neuron-glia interactions required for whisker-related sensory functions and plasticity.

Simplified protocol for isolation of multipotential NG2 cells from postnatal mouse.

BACKGROUND: The functions of NG2 cells have attracted much attention since they were identified. At present, our understanding of their properties and functions is still limited due to the lack of an easy protocol for isolating them from mice. NEW METHOD: In the present study, in postnatal mouse cortical tissue cultures, cell confluence was achieved at DIV 6-8 by frequently changing the medium in the absence of viable neurons, and abundant NG2 cells grew on top of the astrocyte layer before microglia started to thrive. Thus, we developed a simple protocol to separate mouse NG2 cells by shaking the cultures on an orbital shaker at 37 °C for only 3-4h. RESULTS: The yield and purity of NG2 cells were sufficiently high, and the cells displayed immunological and electrophysiological phenotypes typical of NG2 cells. They expressed a large delayed-rectifier K+ current (ID) and a transient A-type K+ current (IA) that were electrophysiologically different from astrocytes and neurons. They showed significantly enhanced chemo-attractive migration after application of GABA. They also showed properties of multipotential neuronal precursor cells and were capable of generating oligodendrocytes (54.2±8.1%), neurons (up to 13.3±6.8%), and astrocytes (93.9±4.3%) under defined conditions. Comparison with Existing Method(s): When compared to other methods available for the isolation of mouse NG2 cells, the procedure we present is simple, relatively fast, and economical. CONCLUSIONS: Overall, we present evidence that this new method for isolating NG2 cells from postnatal mice is simple, economical, and effective.

Microglial migration mediated by ATP-induced ATP release from lysosomes.

Microglia are highly motile cells that act as the main form of active immune defense in the central nervous system. Attracted by factors released from damaged cells, microglia are recruited towards the damaged or infected site, where they are involved in degenerative and regenerative responses and phagocytotic clearance of cell debris. ATP release from damaged neural tissues has been suggested to mediate the rapid extension of microglial process towards the site of injury. However, the mechanisms of the long-range migration of microglia remain to be clarified. Here, we found that lysosomes in microglia contain abundant ATP and exhibit Ca(2+)-dependent exocytosis in response to various stimuli. By establishing an efficient in vitro chemotaxis assay, we demonstrated that endogenously-released ATP from microglia triggered by local microinjection of ATPγS is critical for the long-range chemotaxis of microglia, a response that was significantly inhibited in microglia treated with an agent inducing lysosome osmodialysis or in cells derived from mice deficient in Rab 27a (ashen mice), a small GTPase required for the trafficking and exocytosis of secretory lysosomes. These results suggest that microglia respond to extracellular ATP by releasing ATP themselves through lysosomal exocytosis, thereby providing a positive feedback mechanism to generate a long-range extracellular signal for attracting distant microglia to migrate towards and accumulate at the site of injury.

Microglial immunoreceptor tyrosine-based activation and inhibition motif signaling in neuroinflammation.

Elimination of extracellular aggregates and apoptotic neural membranes without inflammation is crucial for brain tissue homeostasis. In the mammalian central nervous system, essential molecules in this process are the Fc receptors and the DAP12-associated receptors which both trigger the microglial immunoreceptor tyrosine-based activation motif- (ITAM-) Syk-signaling cascade. Microglial triggering receptor expressed on myeloid cells-2 (TREM2), signal regulatory protein-beta1, and complement receptor-3 (CD11b/CD18) signal via the adaptor protein DAP12 and activate phagocytic activity of microglia. Microglial ITAM-signaling receptors are counter-regulated by immunoreceptor tyrosine-based inhibition motif- (ITIM-) signaling molecules such as sialic acid-binding immunoglobulin superfamily lectins (Siglecs). Siglecs can suppress the proinflammatory and phagocytic activity of microglia via ITIM signaling. Moreover, microglial neurotoxicity is alleviated via interaction of Siglec-11 with sialic acids on the neuronal glycocalyx. Thus, ITAM- and ITIM-signaling receptors modulate microglial phagocytosis and cytokine expression during neuroinflammatory processes. Their dysfunction could lead to impaired phagocytic clearance and neurodegeneration triggered by chronic inflammation.

Spatially pathogenic forms of tau detected in Alzheimer's disease brain tissue by fluorescence lifetime-based Förster resonance energy transfer.

In tauopathies including Alzheimer's disease (AD) tau molecules have lost their normal spatial distance to each other and appear in oligomeric or aggregated forms. Conventional immunostaining methods allow detection of abnormally phosphorylated or conformationally altered aggregated tau proteins, but fail to visualize oligomeric forms of tau. Here we show that tau molecules that lost their normal spatial localization can be detected on a subcellular level in postmortem central nervous system (CNS) tissue sections of AD patients by fluorescence lifetime-based Förster resonance energy transfer (FRET). Paraffin sections were co-immunostained with two tau-specific monoclonal antibodies recognizing the same epitope, but labeled with distinct fluorescence dyes suitable for spatial resolution at a nanometer scale by lifetime-based FRET. A FRET signal was detected in neuritic plaques and neurofibrillary tangles of CNS tissue sections of AD patients, showing associated tau proteins typically reflecting either fibrillary, oligomeric or aggregated tau. The 'pretangle-like' structures within the neuronal perikarya did not contain spatially pathogenic forms of tau accordingly to this method. Data demonstrate that fluorescence lifetime-based FRET can be applied to human brain tissue sections to detect pathogenic forms of tau molecules that lost their normal spatial distance.

Alleviation of neurotoxicity by microglial human Siglec-11.

Sialic acid-binding Ig superfamily lectins (Siglecs) are members of the Ig superfamily that recognize sialic acid residues of glycoproteins. Siglec-11 is a recently identified human-specific CD33-related Siglec that binds to alpha2,8-linked polysialic acids and is expressed on microglia, the brain resident innate immune cells. Polysialylated neuronal cell adhesion molecule (PSA-NCAM) is a putative ligand of Siglec-11. We observed gene transcription and protein expression of Siglec-11 splice variant 2 in human brain tissue samples by RT-PCR and Western blot analysis. Siglec-11 was detected on microglia in human brain tissue by immunohistochemistry. Human Siglec-11 splice variant 2 was ectopically expressed by a lentiviral vector system in cultured murine microglial cells. Stimulation of Siglec-11 by cross-linking suppressed the lipopolysaccharides (LPS)-induced gene transcription of the proinflammatory mediators interleukin-1beta and nitric oxide synthase-2 in microglia. Furthermore, phagocytosis of apoptotic neuronal material was reduced in Siglec-11 transduced microglia. Expression of PSA-NCAM was detected on microglia and neurons by immunohistochemistry and RT-PCR. Coculture of microglia transduced with Siglec-11 and neurons demonstrated neuroprotective function of Siglec-11. The neuroprotective effect of Siglec-11 was dependent on polysialic acid (PSA) residues on neurons, but independent on PSA on microglia. Thus, data demonstrate that human Siglec-11 ectopically expressed on murine microglia interacts with PSA on neurons, reduces LPS-induced gene transcription of proinflammatory mediators, impairs phagocytosis and alleviates microglial neurotoxicity.

Immune-mediated CNS damage.

Multiple sclerosis (MS) is a demyelinating autoimmune disease. However, the persisting neurological deficits in MS patients result from acute axonal injury and chronic neurodegeneration, which are both triggered by the autoreactive immune response. Innate immunity, mainly mediated by activated microglial cells and invading macrophages, appears to contribute to chronic neurodegeneration. Activated microglia produce several reactive oxygen species and proinflammatory cytokines which affect neuronal function, integrity and survival. Adaptive immunity, particularly in cytotoxic CD8+ T cells, participates in acute demyelination and axonal injury by directly attacking oligodendrocytes and possibly neurons as well. Understanding the mechanisms of immune-mediated neuronal damage might help to design novel therapy strategies for MS.

Signal regulatory protein-beta1: a microglial modulator of phagocytosis in Alzheimer's disease.

The signal regulatory protein-beta1 (SIRPbeta1) is a DAP12-associated transmembrane receptor expressed in a subset of hematopoietic cells. Recently, it was shown that peritoneal macrophages express SIRPbeta1, which positively regulated phagocytosis. Here, we found that SIRPbeta1 was up-regulated and acted as a phagocytic receptor on microglia in amyloid precursor protein J20 (APP/J20) transgenic mice and in Alzheimer's disease (AD) patients. Interferon (IFN)-gamma and IFN-beta stimulated gene transcription of SIRPbeta1 in cultured microglia. Activation of SIRPbeta1 on cultured microglia by cross-linking antibodies induced reorganization of the cytoskeleton protein beta-actin and suppressed lipopolysaccharide-induced gene transcription of tumor necrosis factor-alpha and nitric oxide synthase-2. Furthermore, activation of SIRPbeta1 increased phagocytosis of microsphere beads, neural debris, and fibrillary amyloid-beta (Abeta). Phagocytosis of neural cell debris and Abeta was impaired after lentiviral knockdown of SIRPbeta1 in primary microglial cells. Thus, SIRPbeta1 is a novel IFN-induced microglial receptor that supports clearance of neural debris and Abeta aggregates by stimulating phagocytosis.

Growth inhibition of mesenchymal stem cells by aspirin: involvement of the WNT/beta-catenin signal pathway.

1. Mesenchymal stem cell (MSC) therapy is drawing increasing attention in cardiology. However, the effect of aspirin, an assistant medication used extensively in the treatment of cardiovascular diseases, on MSC is not clear. 2. In the present study, we investigated the effect of aspirin on the growth of MSC in vitro and the underlying mechanism of its action. 3. The 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay revealed that 1, 5 and 10 mmol/L aspirin inhibited the growth of MSC by 18, 37 and 62%, respectively. DNA synthesis of MSC was inhibited by 25, 57 and 90% following treatment with 1, 5 and 10 mmol/L aspirin, respectively, as determined by the tritiated thymidine incorporation assay. No cytotoxicity was observed based on Trypan blue dye exclusion and cell morphological observations. Western blot analysis demonstrated that the protein level of phosphorylated beta-catenin increased, whereas that of cyclin D1 decreased, after treatment of MSC with aspirin. Cell cycle analysis showed that aspirin failed to significantly alter the proportion of cells in different stages of the cell cycle. 4. These observations indicate that aspirin inhibits MSC proliferation and that the downregulation of the wnt/beta-catenin signal pathway may be involved in the growth inhibition of MSC by aspirin.