Ciliary neurotrophic factor (CNTF) is present in proopiomelanocortin (POMC) neurons of the rat arcuate nucleus. A–H: POMC and CNTF were detected by multiple immunofluorescence followed by a confocal laser scanning. Sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI). I and J: detection of CNTF immunofluorescence in wild-type (WT) and CNTF-knockout mice (CNTF−/−). A–D: 0.4-μm-thick focal planes. E–H: 1.2-μm-thick stacks of high-magnification scan (×63). I and J: epifluorescence observation. Scale bars = 30 (A–D), 10 (E–H), and 50 μm (I and J).
CNTF receptor subunits are present in POMC neurons of the rat arcuate nucleus. POMC and CNTF receptor-α (CNTFRα; A–C), leukemia inhibitory factor receptor (LIFR; D–F), and glycoprotein 130 kDa (gp130; G–I) were detected by multiple immunofluorescence followed by a confocal laser scanning. Sections were counterstained with DAPI; 0.4-μm-thick focal planes. Scale bar = 15 μm.
CNTF and its receptor subunits are localized in the cytoplasm and the nucleus of arcuate cells. A–F: the distribution pattern of CNTF, CNTFRα, LIFR, gp130, JAK2, and Akt immunoflurescences was reconstructed in 3D with the FreeD software in ethidium homodimer-2 (EtH-2)-counterstained arcuate cells. The plasmic membrane (gray) and nuclear envelope (red) were delineated, and the punctiform staining of the different proteins of interest was represented as green spots. Raw 5-μm-thick stacks (which represent the sum of 25 0.2-μm-thick focal planes) are shown as an inset at the bottom of each reconstruction image. Scale bars = 5 μm. G: the relative density of green spots was quantified in the cytoplasm and the nucleus on 9 distinct cells from 3 different rats. The data are represented as mean percentages of staining ± SE.
CNTF and its receptor subunits are colocalized in the nucleus of rat arcuate cells; transmission electron microscopy analysis. A: deposits of 6- or 10-nm immunogold particles were performed to evaluate particle size segregation. Particle diameters are plotted in a frequency distribution. B: the relative density of gold particles in the cytoplasm, the nucleus, and the extracellular space was quantified and represented as %staining. C–F: CNTF and CNTFRα immunoreactivities were detected in the nucleus with 10-nm gold particles (arrowheads), whereas LIFR and gp130 immunostainings were visualized with 6-nm particles (arrows). Scale bar = 80 nm.
CNTF and its receptor subunits interact in the nucleus of hypothalamic extracts. A: fresh hypothalami were homogenized individually. Then membrane (lane M), cytoplasmic (lane C), and nuclear (lane N) fractions were isolated. Their respective content in CNTF, CNTFRα, LIFR, gp130, JAK2, and Akt was analyzed by Western blot. The purity of the fractions was controlled by detecting cytoplasmic [β-tubulin and glial fibrillary protein (GFAP)] and nuclear [neuronal nuclei (NeuN)] proteins. B: nuclear extracts from rat hypothalamus were immunoprecipitated (IP) with anti-CNTFRα, anti-LIFR, or anti-gp130. The precipitates were then immunoblotted with anti-gp130, CNTFRα, or LIFR. Arrowheads indicate the level of the immunoblotted receptors. The other bands may correspond to immunoglobulins. MW, molecular weight marker; IB, immunoblot.
CNTF specifically stimulates POMC transcription in hypothalamic isolated nuclei. Isolated nuclei were obtained from 5 individual fresh rat hypothalami and subjected to a run-on reaction in the presence or not of CNTF (1 nM) or leptin (10 nM) for 45 min at 22°C. Nascent RNA were reverse transcribed, and quantitative RT-PCR based on nuclear run-on technique was performed. Displayed values are means ± SE. *P < 0.05 compared with control condition with a paired Student t-test; n = 5 rats.
CNTF stimulates Akt signaling in hypothalamic isolated nuclei. Isolated nuclei were obtained from individual fresh rat hypothalami and incubated in the presence or not of ATP (5 μM), CNTF (1 nM), and leptin (10 nM) for 10 min at 37°C. They were then subjected to SDS-PAGE and Western blot analysis. A: montage of nitrocellulose membranes blotted with an anti-phospho (p)-JAK2 or an anti-p-Akt before being stripped and blotted a 2nd time with an anti-total (tot) JAK2 or anti-tot Akt. B: quantification of the blots. The data are presented as the %control condition ± SE. *P < 0.05 and **P < 0.01 compared with control condition with paired Student t-test; n = 10 rats.