using-vivo-tissue-cell-explant-approaches-to-study-morphogenesis

Using <em>In Vivo</em> and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic ...

Yale University School of Medicine

Shanghai Jiaotong University School of Medicine

Ngb (neuroglobin) is a newly discovered hexaco-ordinate globin that is expressed in vertebrate brain and peripheral nervous systems. Expression of Ngb increases in response to oxygen deprivation and protects neurons from hypoxia in vitro and in vivo. However, the lack of its transduction ability into cells resulted in limited neuroprotection. To educe its neuroprotection under hypoxia, a cell-permeable Ngb fusion protein was generated. A rat brain Ngb gene was cloned and fused with a gene fragment encoding the nine-amino-acid TAT PTD (transactivator-of-transcription protein-transduction domain; RKKRRQRRR) of HIV-1 in a prokaryotic expression vector to generate a genetic in-frame N-terminal hexahistidine-tagged) TAT PTD-Ngb fusion protein. It was expressed in soluble form in Escherichia coli BL21(DE3)plysS and purified with Ni(2+)-affinity chromatography. The results showed that the purified fusion protein TAT PTD-Ngb can enter into the primary cultured cortical neurons in a dose-dependent manner when added exogenously to the culture media and can be detected in cells within 48 h. The cell viability under hypoxia was increased and apoptosis induced by hypoxia was decreased after TAT PTD-Ngb was transduced into cortical neurons. The results provide a clue for the research of Ngb and suggest that transduction of TAT PTD-Ngb may be one of the ways for the therapy of CNS (central nervous system) diseases, especially cerebrovascular diseases and neurodegenerative diseases.

Translocation and neuroprotective properties of transactivator-of-transcription protein-transduction domain-neuroglobin fusion protein in primary cultured cortical neurons.

During immune-mediated demyelinating lesions, the endocannabinoid system is involved in the pathogenesis of both neuroinflammation and neurodegeneration through different mechanisms. Here we explored the cellular distribution of the CB1 receptor (CB1R) in the central nervous system (CNS) and detected the level of CB1R expression during experimental autoimmune encephalomyelitis (EAE) by RT-qPCR, Western blotting, and immunostaining. Expression of CB1R was observed in neurons and microglia/macrophages but was barely detected in astrocytes. During EAE, the expression of CB1R in spinal cords was reduced at days 9, 17, and 28 postimmunization (p.i.), but the level of CB1R expression in spleens did not show a significant difference compared with complete Freund's adjuvant (CFA)-immunized mice. A selective CB1R antagonist (SR141716A) increased EAE clinical score, accompanied by weight loss. Unexpectedly, SR141716A inhibited the expression of CB1R but increased the expression of CB2R in brains, spinal cords, and spleens simultaneously. The administration of SR141716A increased interferon-γ, interleukin-17 (IL-17), and inflammatory cytokines such as IL-1β, IL-6, and tumor necrosis factor-α in brains and/or spinal cords. A similar increase was observed in spontaneous and specific antigen-stimulated splenic mononuclear cells compared with vehicle controls. Interestingly, the expression of CX3CL1 was increased in brains and spinal cords but declined in spleens of EAE mice treated with SR141716A. These results indicate that manipulation of the CB1R may have therapeutic value in MS, but its complexity remains to be carefully considered and studied in further clinical application.

Immunoregulation of experimental autoimmune encephalomyelitis by the selective CB1 receptor antagonist.

During immune mediated demyelinating lesions, the endocannabinoid system is involved in the pathogenesis of both neuroinflammation and neurodegeneration through different mechanisms. Here, we explored the cellular distribution of cannabinoid 2 receptor (CB(2)R) in the central nervous system (CNS) and detected the level of CB(2)R expression during experimental autoimmune encephalomyelitis (EAE) by RT-PCR, Western blot and immunostaining. Our results show that CB(2)R was expressed in neurons, microglia and astrocytes. During EAE, the expression of CB(2)R in spinal cord rose slowly at days 9 and 17 post immunization (p.i.), and elevated rapidly at day 28 p.i., while the expression of CB(2)R in spleen elevated rapidly and got a plateau at days 17 and 28 p.i. Only the increase of CB(2)R expression in spinal cord demonstrated a significant difference when compared to control mice immunized with complete Freund's adjuvant (CFA). The selective CB(2)R antagonist (SR144528) exacerbated EAE clinical severity accompanied by weight loss. SR144528 inhibited the expression of CB(2)R, but increased the expression of CB(1)R in brain, spinal cord and spleen. The administration of SR144528 declined interferon-γ, IL-17, IL-4, IL-10, IL-1β, IL-6 and tumor necrosis factor-α, but increased CX3CL1 in brain and/or spinal cord. In contrast, IL-17 and MCP-1 were increased, while CX3CL1 was decreased in splenic mononuclear cells as compared to vehicle controls. These results indicate that manipulation of CB(2)R may have therapeutic value in MS, but its complexity remains to be considered and studied for further clinical application.

Targeting CB(2) receptor as a neuroinflammatory modulator in experimental autoimmune encephalomyelitis.

During immuno-mediated demyelinating lesions, endocannabinoid system participates in both inflammatory and neurodegenerative damage through several mechanisms that involve neuronal and immune cells. Here, we constructed lentiviral vector to upregulate CB1 receptor (CB1R) in the lumbar spinal cord 5-6 region and observe the effect of clinical score and possible mechanism on the occurrence and development of experimental autoimmune encephalomyelitis (EAE). The results show that overexpression of CB1R delayed the onset of clinical signs and ameliorated the severity of disease. Overexpression of CB1R significantly inhibited the expression of NF-kB/p65 and TLR-4 as well as levels of IL-1β, IL-6, and TNF-α, followed by a decrease of IL-17 and an increase of IL-10 in the spinal cord of mice. The percentage of M1 marker CD11b(+)CD16/32(+) cells was decreased, while the percentage of M2 marker CD11b(+)CD206(+) and CD11b(+)IL-10(+) cells was elevated in splenic mononuclear cells (MNCs) of mice with overexpression of CB1R. Interestingly, overexpression of CB1R dramatically enhanced the expression of neurotrophic NT-3, BDNF, and GDNF in the spinal cord. These results indicate that local overexpression of CB1R in the spinal cord exhibited neuroprotective effects in EAE, mainly suppressing inflammatory microenvironment and elevating neurotrophic factors, slightly declining IL-1β and IL-17 in the spleen, and increased IL-10 in the brain. Its complexity remains to be carefully considered and further studied in further investigation.

Zhi-Yin Lou

Department of Neurology

Yale University School of Medicine

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

Zhi-Yin Lou

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Neurology

Yale University School of Medicine

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

Department of Neurology