Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CNTF-mediated protection of photoreceptors requires initial activation of the cytokine receptor gp130 in M?ller glial cells

doi: 10.1073/pnas.1303604110

Figure Lengend Snippet: Lentivirus-mediated expression of human CNTF in vitro and in vivo. (A) Schematics of lentiviruses encoding the CMV promoter followed by IRES-GFP (LV-IG) or human CNTF with a signal peptide (SP) and IRES-GFP (LV-hCNTF). (B) Western blot of cell extracts or culture media from HEK 293T cells transfected with LV-IG or LV-hCNTF probed for CNTF. The left lane was loaded with 100 ng of recombinant human CNTF. (C) Immunolabeling of 293T cells infected with LV-IG or LV-hCNTF for 48 h against GFP (green) and CNTF (red). (D) ELISA for hCNTF output from infected ARPE-19 cells (1.5 × 105 cells, between 48 and 72 h after infection) and hCNTF levels in rds/P216L eyes 10 d after subretinal injection with LV-IG or LV-hCNTF at P25. (E and F) Immunolabeling for GFP and CNTF of rds/P216L eyes 5 d after LV-IG or LV-hCNTF injections at P27. [Scale bars, (E) 500 and (F) 50 μm.] gcl, ganglion cell layer; inl, inner nuclear layer; onl, outer nuclear layer; rpe, retinal pigment epithelium.

Article Snippet: The amounts of viral produced hCNTF in vitro and in vivo were determined with a human CNTF Quantikine ELISA kit (R&D Systems) as described in SI Materials and Methods .

Techniques: Expressing, In Vitro, In Vivo, Western Blot, Transfection, Recombinant, Immunolabeling, Infection, Enzyme-linked Immunosorbent Assay, Injection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CNTF-mediated protection of photoreceptors requires initial activation of the cytokine receptor gp130 in M?ller glial cells

doi: 10.1073/pnas.1303604110

Figure Lengend Snippet: Lentiviral-mediated CNTF expression prevents degeneration and improves the morphology of mutant photoreceptors. (A–E) Histological staining by toluidine blue shows degeneration caused by the rds/P216L mutation and the protective effect of LV-hCNTF. Viral infections were performed at P25 and eyes analyzed by P52. (F–I) TEM analysis reveals improved photoreceptor morphology of LV-hCNTF infected rds/P216L retinas. (J) Quantification of the ONL thickness at P45 from the optic nerve head (ONH) to the ciliary margin for the WT and rds/P216L mutant injected with LV-IG or LV-hCNTF at P25 (n = 3 for all). (K) Quantification of IS/OS lengths at P45 for WT and rds/P216L mutant photoreceptors with or without lentivirus infections. Statistically significant sample pairs are indicated. ***P < 0.001. [Scale bars, (A–E) 50 and (F–I) 10 μm.] is, inner segment; olm, outer limiting membrane; onl, outer nuclear layer; os, outer segment; rpe, retinal pigment epithelium.

Article Snippet: The amounts of viral produced hCNTF in vitro and in vivo were determined with a human CNTF Quantikine ELISA kit (R&D Systems) as described in SI Materials and Methods .

Techniques: Expressing, Mutagenesis, Staining, Infection, Injection, Membrane

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CNTF-mediated protection of photoreceptors requires initial activation of the cytokine receptor gp130 in M?ller glial cells

doi: 10.1073/pnas.1303604110

Figure Lengend Snippet: Exogenous CNTF corrects mislocalization of opsins and affects photoreceptor gene expression. (A) A diagram shows where images were obtained from an adult retinal section. (B–G) Confocal micrographs of immunolabeled P45 retinas for rhodopsin (B–D) or M-opsin (E–G) merged with DAPI images. Viral injections for rds retinas were performed at P25. Arrows and arrowheads indicate mislocalized opsins in cell soma and synapses, respectively. (H) A diagram of a flat mount adult retina showing the injection site. (I–P) Images of flat mount rds/P216L retinas injected at P25 with LV-IG or LV-hCNTF and immunolabeled for M-opsin at P45. (Q) Quantification of photoreceptor transcripts by real-time PCR at P52 (n = 3 for all). For statistics, see Table S3. [Scale bars, (B–G) 20 and (I–P) 100 μm.] is, inner segment; onl, outer nuclear layer; os, outer segment; rpe, retinal pigment epithelium.

Article Snippet: The amounts of viral produced hCNTF in vitro and in vivo were determined with a human CNTF Quantikine ELISA kit (R&D Systems) as described in SI Materials and Methods .

Techniques: Gene Expression, Immunolabeling, Injection, Real-time Polymerase Chain Reaction

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CNTF-mediated protection of photoreceptors requires initial activation of the cytokine receptor gp130 in M?ller glial cells

doi: 10.1073/pnas.1303604110

Figure Lengend Snippet: Deletion of gp130 in Müller glia reduces neuroprotection and blocks activation of STAT and ERK by CNTF. (A and B) Immunolabeling for pSTAT3 in rds/P216L mutant retinas 1 h after PBS (A) or CNTF (B) injection at P41. (C) Immunolabeling of ROSA-YFP retinas for GFP and Cyclin D3 at P81 in the presence or absence of GFAP-Cre. (D–K) Confocal images of P216L; ROSA retinas injected with LV-hCNTF at P25 and labeled at P45 for DAPI (D and H), GFP (E and I), pSTAT3 (F and J), and pERK (G and K). (L) Quantification of ONL thickness for the control rds/P216L eyes injected with LV-hCNTF (n = 3) and gp130 Müller cKO eyes injected with LV-IG (n = 2) or LV-hCNTF (n = 3). (M) Western blot of retinal extracts from rds/P216L eyes that were gp130 Müller cKO or gp130+/− injected with PBS or CNTF for 1 h and probed for pSTAT3, STAT3, and γ Tubulin. Except for noninjected WT, extracts from two individual injected eyes are shown. [Scale bars, (A, C, and D), 50 μm.] gcl, ganglion cell layer; inl, inner nuclear layer; onl, outer nuclear layer; rpe, retinal pigment epithelium.

Article Snippet: The amounts of viral produced hCNTF in vitro and in vivo were determined with a human CNTF Quantikine ELISA kit (R&D Systems) as described in SI Materials and Methods .

Techniques: Activation Assay, Immunolabeling, Mutagenesis, Injection, Labeling, Control, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CNTF-mediated protection of photoreceptors requires initial activation of the cytokine receptor gp130 in M?ller glial cells

doi: 10.1073/pnas.1303604110

Figure Lengend Snippet: Deletion of gp130 in rod cells prevents CNTF-mediated rescue of mutant photoreceptors from degeneration. (A and B) Immunolabeling for pSTAT3 in rds/P216L mutant retinas 24 h after CNTF injection at P41. (C and D) Confocal images of P216L;ROSA control gp130+/− (C) and gp130 Rod cKO (D) retinas injected with LV-hCNTF at P25 and labeled for GFP and DAPI at P45. (E–J) Confocal images of control (E, F, H, and I) and gp130 Rod cKO (G and J) P216L retinas injected with LV-IG or LV-hCNTF and labeled for pSTAT3 (E–G) or pERK and GS (H–J). (E′–G″) show magnified images for pSTAT3 (E′–G′) and merged DAPI /pSTAT3 (E″–G″) labeling of the outer nuclear layer from E–G, respectively. (K) Quantification of ONL thickness for the control gp130+/− (n = 6) and gp130 Rod cKO;P216L (n = 5) retinas injected with LV-hCNTF. (Scale bars, 50 μm in all panels.) gcl, ganglion cell layer; inl, inner nuclear layer; onl, outer nuclear layer; rpe, retinal pigment epithelium.

Article Snippet: The amounts of viral produced hCNTF in vitro and in vivo were determined with a human CNTF Quantikine ELISA kit (R&D Systems) as described in SI Materials and Methods .

Techniques: Mutagenesis, Immunolabeling, Injection, Control, Labeling

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CNTF-mediated protection of photoreceptors requires initial activation of the cytokine receptor gp130 in M?ller glial cells

doi: 10.1073/pnas.1303604110

Figure Lengend Snippet: Summary of CNTF triggered protection in the rds/P216L model of photoreceptor degeneration. (A) Real-time PCR quantification of GFAP and growth factor transcripts. WT and P216L are noninjected samples at P52; P216L, LV-IG and P216L, LV-hCNTF were injected with viruses at P25 and analyzed at P52. n = 3 for all. For statistics, see Table S11. (B) Real-time PCR quantification of transcript levels for LIF, CNTF, and CT-1 in rds/P216L retinas. Injections of CNTF or PBS were performed at P25, and RNAs were collected at 3 or 24 h after injections. n = 3 for all. For statistics, see Table S12. (C) Comparison of ONL thickness for WT and rds/P216L mutant retinas under the influence of hCNTF in gp130+/+, gp130 Müller or rod heterozygous, and gp130 Müller or Rod cKO genetic backgrounds. Significant P values (Student t test) are indicated. (D) A proposed indirect model of exogenous CNTF-induced neuroprotection of rod photoreceptors in the rds/peripherin mutant retina. The secreted human CNTF derived from LV-transduced RPE initially activates signaling events in Müller glia through the gp130 cytokine receptor. This Muller glia response triggers the release of other signaling molecules, which subsequently activate gp130 expressed by rod photoreceptors to enhance cell survival. Loss of gp130 in Müller glia prevents the activation of the entire signaling cascade, whereas removal of gp130 from rod cells prevents rod photoreceptor rescue and diminishes sustained activation of Müller glia.

Article Snippet: The amounts of viral produced hCNTF in vitro and in vivo were determined with a human CNTF Quantikine ELISA kit (R&D Systems) as described in SI Materials and Methods .

Techniques: Real-time Polymerase Chain Reaction, Injection, Comparison, Mutagenesis, Derivative Assay, Activation Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Persistent Gliosis Interferes with Neurogenesis in Organotypic Hippocampal Slice Cultures

doi: 10.3389/fncel.2016.00131

Figure Lengend Snippet: Gene expression profile of glial activation markers during OHSC cultivation by RT-qPCR analysis . Initial expression values (0 DIV) are set as 100% and shown as gray columns. (A) Gene expression of markers for astrogliosis (Nestin, CNTF) points to a very fast and strong activation of astrocytes, reaching its maximum within 1–3 DIV. (B) Gene expression of microglial activation markers (MHCIIAβ, IL-1β). Microgliosis follows a slower but exponential increase dynamic, which does not seem to reach a plateau during 15 DIV. For sample comparison, one-way ANOVA with Tukey’s post-test was used. Values are displayed as the mean ± SD. n = 3–5 samples per time point (see Materials and Methods). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: To induce and accelerate astroglial activation, OHSC were treated for the first two DIV with CNTF (20 ng/ml, Cell Concepts GmbH), a potent inducer of reactive gliosis ( ).

Techniques: Gene Expression, Activation Assay, Quantitative RT-PCR, Expressing, Comparison

Journal: Frontiers in Cellular Neuroscience

Article Title: Persistent Gliosis Interferes with Neurogenesis in Organotypic Hippocampal Slice Cultures

doi: 10.3389/fncel.2016.00131

Figure Lengend Snippet: Pro- and anti-inflammatory treatments and their respective influence on neurogenesis. (A) Time course of the POMC-eGFP signal in representative OHSC, treated with substances promoting (CNTF) or counteracting (PSB 0739 and indomethacin) glial activation. (B) Quantification of the eGFP signal intensity during the cultivation period reveals significant differences between the respective treatments. OHSC were treated with CNTF (20 ng/ml) for 48 h (0–2 DIV) or with PSB 0739 (100 nM) or indomethacin (50 μM) for the entire cultivation period. Enhancing astrogliosis by CNTF treatment accelerates the decay of neurogenesis, while the anti-inflammatory treatment with indomethacin exerts potent protective effects on neurogenesis. PSB 0739 does not alter the course of the eGFP signal intensity. For sample comparison, one-way ANOVA with Tukey’s post-test was used. Values are displayed as mean ± SD. n = 9; ∗ p < 0.05.

Article Snippet: To induce and accelerate astroglial activation, OHSC were treated for the first two DIV with CNTF (20 ng/ml, Cell Concepts GmbH), a potent inducer of reactive gliosis ( ).

Techniques: Activation Assay, Comparison

Journal: Frontiers in Cellular Neuroscience

Article Title: Persistent Gliosis Interferes with Neurogenesis in Organotypic Hippocampal Slice Cultures

doi: 10.3389/fncel.2016.00131

Figure Lengend Snippet: Pro- and anti-inflammatory treatments and their respective influence on the gene expression profile . RT-qPCR of markers for neurogenesis (A) and glial activation (B) . Initial expression values (0 DIV) are set as 100% and shown as a gray columns. While CNTF treatment increases the expression of astrogliosis markers (CNTF, B ) and causes the downregulation of DCX (A) , anti-inflammatory treatments with PSB 0739 and indomethacin significantly increase the expression of markers for neurogenesis (Hes5, NeuroD1, DCX), pointing to an efficient protection of OHSC neurogenesis. On the other hand, PSB 0739 and indomethacin only decreased the expression of CNTF at four of five measured time points, whereas MHCIIAβ and IL-1β expressions were mostly unaffected. For sample comparison, one-way ANOVA with Tukey’s post-test was used. Values are displayed as mean ± SD. n = 3–6 samples per time point (see Materials and Methods). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: To induce and accelerate astroglial activation, OHSC were treated for the first two DIV with CNTF (20 ng/ml, Cell Concepts GmbH), a potent inducer of reactive gliosis ( ).

Techniques: Gene Expression, Quantitative RT-PCR, Activation Assay, Expressing, Comparison