40 research outputs found
LIF-induced expression of iNOS expression was LIFR-dependent.
<p><b>A.</b> Cells treated with LIF in presence and absence of a blocking antibody to LIFR which reduced immunofluorescence for LIFR (green) and iNOS (red). Blue: nuclear staining with DAPI. Scale bar: 50 µm. <b>B</b>.-Quantification of the average iNOS fluorescence intensity shown in A normalized respect to the control group in three independent experiments. * p<0.05.</p
Primary cultures of olfactory epithelial cells expressed iNOS and LIFR.
<p><b>A</b>. iNOS Immunofluorescence in cells grown for 24 h in control medium and in the same medium containing LIF. Blue: nuclei stained with DAPI. Red: Immunoreactivity to iNOS. Scale bar: 50 µm. <b>B</b>. Co-localization of iNOS (red) and LIFR (green) in cells grown in LIF. Blue: DAPI stained nuclei. Scale bar: 50 µm. <b>C</b>. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed mRNA for iNOS (upper panel) and YWHAZ, as a loading control (lower panel) in rat olfactory mucosal cells grown in control medium (CM), and in medium containing EGF, LIF and L-NIL. The negative control contained no mRNA. MW: DNA size markers; the predicted band for iNOS is 370 bp. <b>D</b>: Quantification of Brdu immunofluorescence of cell grown in control medium (CM), LIF containing medium and L-NIL containing medium. Data were expressed as mean of % Brdu positive cells ± SEM. *p<0.05 with respect to the control group.</p
Olfactory neurosphere-derived cells expressed LIF.
<p><b>A.</b> iNOS immunofluorescence (red) in a neurosphere. Scale bar: 20 µm. <b>B</b>. Co-localization of LIFR (green) and iNOS (red) in cells treated with LIF. DAPI-stained nuclei are blue. Bar: 25 µm. <b>C</b>. Immunofluorescence for iNOS in cells grown in control medium (a), EGF-containing medium (b) and LIF-containing medium (c). Scale bar: 50 µm. <b>D</b>. Flow cytometric analysis showing that LIF increased the iNOS expression measured by the mean intensity of fluorescence of the cells in each group. * p<0.05 respect to the control groups. <b>E</b>. qPCR revealed iNOS mRNA in rat olfactory neurosphere cells grown in control medium (CM), EGF-containing medium and LIF containing medium expressed as the mean ± SEM of the iNOS expression increment fold respect to the control group (CM). *p<0.05 respect to the control groups.</p
Temporal course of LIF-induced iNOS expression.
<p><b>A.</b> Immunofluorescence of iNOS in cells treated with LIF for 0 (a), 6 (b), 12 (c) and 24 h (d). Blue: DAPI nuclear staining. Scale bar: 50 µm. <b>B.</b> qPCR iNOS mRNA treated as in A of three independent experiments expressed as the mean ± SEM of the iNOS expression increment fold respect to the control group (CM). * p<0.05.</p
LIF-induced proliferation depends on iNOS.
<p><b>A</b>. Immunofluorescence for BrdU (green) in cells grown in control medium (CM, a), EGF-containing medium (b) and LIF-containing medium (c). Blue: nuclear staining with DAPI. Scale bar:100 µm. <b>B</b>. BrdU immunofluorescence (green) in the presence of the LIFR blocking antibody (d), the LIFR blocking antibody plus the NO donor SNAP (e), the iNOS inhibitor L-NIL (f) and L-NIL plus SNAP (g). Blue, nuclear staining with DAPI. Scale bar: 100 µm. <b>C.</b> Quantification of data shown in A and B. ≠* p<0.05 between the groups.</p
Image_6_Reduced acetylated α-tubulin in SPAST hereditary spastic paraplegia patient PBMCs.tif
HSP-SPAST is the most common form of hereditary spastic paraplegia (HSP), a neurodegenerative disease causing lower limb spasticity. Previous studies using HSP-SPAST patient-derived induced pluripotent stem cell cortical neurons have shown that patient neurons have reduced levels of acetylated α-tubulin, a form of stabilized microtubules, leading to a chain of downstream effects eventuating in increased vulnerability to axonal degeneration. Noscapine treatment rescued these downstream effects by restoring the levels of acetylated α-tubulin in patient neurons. Here we show that HSP-SPAST patient non-neuronal cells, peripheral blood mononuclear cells (PBMCs), also have the disease-associated effect of reduced levels of acetylated α-tubulin. Evaluation of multiple PBMC subtypes showed that patient T cell lymphocytes had reduced levels of acetylated α-tubulin. T cells make up to 80% of all PBMCs and likely contributed to the effect of reduced acetylated α-tubulin levels seen in overall PBMCs. We further showed that mouse administered orally with increasing concentrations of noscapine exhibited a dose-dependent increase of noscapine levels and acetylated α-tubulin in the brain. A similar effect of noscapine treatment is anticipated in HSP-SPAST patients. To measure acetylated α-tubulin levels, we used a homogeneous time resolved fluorescence technology-based assay. This assay was sensitive to noscapine-induced changes in acetylated α-tubulin levels in multiple sample types. The assay is high throughput and uses nano-molar protein concentrations, making it an ideal assay for evaluation of noscapine-induced changes in acetylated α-tubulin levels. This study shows that HSP-SPAST patient PBMCs exhibit disease-associated effects. This finding can help expedite the drug discovery and testing process.</p
Image_3_Reduced acetylated α-tubulin in SPAST hereditary spastic paraplegia patient PBMCs.TIF
HSP-SPAST is the most common form of hereditary spastic paraplegia (HSP), a neurodegenerative disease causing lower limb spasticity. Previous studies using HSP-SPAST patient-derived induced pluripotent stem cell cortical neurons have shown that patient neurons have reduced levels of acetylated α-tubulin, a form of stabilized microtubules, leading to a chain of downstream effects eventuating in increased vulnerability to axonal degeneration. Noscapine treatment rescued these downstream effects by restoring the levels of acetylated α-tubulin in patient neurons. Here we show that HSP-SPAST patient non-neuronal cells, peripheral blood mononuclear cells (PBMCs), also have the disease-associated effect of reduced levels of acetylated α-tubulin. Evaluation of multiple PBMC subtypes showed that patient T cell lymphocytes had reduced levels of acetylated α-tubulin. T cells make up to 80% of all PBMCs and likely contributed to the effect of reduced acetylated α-tubulin levels seen in overall PBMCs. We further showed that mouse administered orally with increasing concentrations of noscapine exhibited a dose-dependent increase of noscapine levels and acetylated α-tubulin in the brain. A similar effect of noscapine treatment is anticipated in HSP-SPAST patients. To measure acetylated α-tubulin levels, we used a homogeneous time resolved fluorescence technology-based assay. This assay was sensitive to noscapine-induced changes in acetylated α-tubulin levels in multiple sample types. The assay is high throughput and uses nano-molar protein concentrations, making it an ideal assay for evaluation of noscapine-induced changes in acetylated α-tubulin levels. This study shows that HSP-SPAST patient PBMCs exhibit disease-associated effects. This finding can help expedite the drug discovery and testing process.</p
Loss of <i>Usp9x</i> disrupts TGF-β signalling in hippocampal neurons.
<p>(a–b) TGF-β luciferase reporter assays conducted in either wildtype (Nes-<i>Usp9x<sup>+/Y</sup></i>) or null (Nes-<i>Usp9x<sup>−/Y</sup></i>) embryonic hippocampal neuronal cultures. Hippocampal neurons were isolated and transfected with both renilla control and pGL3-TGF-β luciferase reporter plasmids. (a) Cells were grown for 3 days before analysis using dual-luciferase reporter assays and data normalised relative to wildtype readings. (b) Luciferase reporter activity in response to increasing concentrations of TGF-β. Data normalised to controls in the absence of TGF-β. All luciferase data from 6 biological replicates (i.e. cultures isolated from 6 <i>Usp9x<sup>+/y</sup></i> and 6 <i>Usp9x<sup>−/Y</sup></i> embryos). (c) Response of established TGFβ target genes in presence or absence of <i>Usp9x</i>, analysed by RT-qPCR. Isolated hippocampal neurons grown for 2 days prior to the addition of 1 ng/ml TGF-β. (d–e). Morphological analysis of hippocampal neurons exposed to 1 ng/ml TGF-β in the presence or absence of Usp9x. (d) Comparison of mean primary axonal length. (e). Comparison of number of neurite termini.</p
Loss of <i>Usp9x</i> disrupts the architecture of the embryonic neocortex.
<p>The Nestin-cre mediated deletion of Usp9x (B,D) results in loss of demarcation between the cells of the ventricular and sub-ventricular zones (VZ/SVZ), the more disperse cellular density of the intermediate zone (IZ) and the neurons of the cortical plate (CP) seen in control littermates (A,C). C and D are higher magnification images of A and B, respectively. Nestin (E,F) and BLBP (G,H) staining in E18.5 embryos indicated that neural progenitors were more loosely organized in the VZ/SVZ. Neurons of the cortical plate were disorganized in the absence of <i>Usp9x</i> (J) compared with littermates (I). Nissl stain of <i>Usp9x<sup>+/Y</sup></i> (A,C,G) and <i>Usp9x<sup>−/Y</sup></i> (B,D,H) in E16.5 embryos (A–D, G–H). V = ventricle. Scale bar = 100 µm (A), 50 µm (C), 40 µm (E), 100 µm (G), 40 µm (I).</p
<i>Usp9x</i> loss affects neuronal and astrocytic projections.
<p>NF160 antibodies decorate axonal projection from the neocortex (Nc) to the hippocampus (Hp) in E18.5 Nes-<i>Usp9x<sup>+/Y</sup></i> mice (A). These projections were absent in Nes-<i>Usp9x<sup>−/Y</sup></i> mice (B). GFAP staining is reduced in both the hippocampus and neocortex of E18.5 <i>Usp9x <sup>−/Y</sup></i> embryos (D) compared with littermate controls (C). In the hippocampus GFAP-labeled projections extended toward the CA3 region in control embryos (arrowhead in E) but not in the absence of <i>Usp9x</i> (F). Scale bar = 20 µm (A,B), 160×µm (C,D), 80×µm (E,F).</p