Ablation of Proliferating Cells in the CNS Exacerbates Motor Neuron Disease Caused by Mutant Superoxide Dismutase

Proliferation of glia and immune cells is a common pathological feature of many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Here, to investigate the role of proliferating cells in motor neuron disease, SOD1G93A transgenic mice were treated intracerebroventicularly (ICV) with the anti-mitotic drug cytosine arabinoside (Ara-C). ICV delivery of Ara-C accelerated disease progression in SOD1G93A mouse model of ALS. Ara-C treatment caused substantial decreases in the number of microglia, NG2+ progenitors, Olig2+ cells and CD3+ T cells in the lumbar spinal cord of symptomatic SOD1G93A transgenic mice. Exacerbation of disease was also associated with significant alterations in the expression inflammatory molecules IL-1β, IL-6, TGF-β and the growth factor IGF-1

Nov/Ccn3, a novel transcriptional target of FoxO1, impairs pancreatic β-cell function.

Type 2 diabetes is characterized by both insulin resistance and progressive deterioration of β-cell function. The forkhead transcription factor FoxO1 is a prominent mediator of insulin signaling in β-cells. We reasoned that identification of FoxO1 target genes in β-cells could reveal mechanisms linking β-cell dysfunction to insulin resistance. In this study, we report the characterization of Nov/Ccn3 as a novel transcriptional target of FoxO1 in pancreatic β-cells. FoxO1 binds to an evolutionarily conserved response element in the Ccn3 promoter to regulate its expression. Accordingly, CCN3 levels are elevated in pancreatic islets of mice with overexpression of a constitutively active form of FoxO1 or insulin resistance. Our functional studies reveal that CCN3 impairs β-cell proliferation concomitantly with a reduction in cAMP levels. Moreover, CCN3 decreases glucose oxidation, which translates into inhibition of glucose-stimulated Ca(2+) entry and insulin secretion. Our results identify CCN3, a novel transcriptional target of FoxO1 in pancreatic β-cells, as a potential target for therapeutic intervention in the treatment of diabetes

CCN3 modifies the expression of several genes involved in the control of cell cycle progression.

<p>A) mRNA levels of cells treated with or without CCN3 for 24 h. B) Reported changes in gene expression following CCN3 treatment. Results represent means +/– SEM of 3 separate experiments. *, p<0.05; **, p<0.01; ns  =  not significant.</p

CCN3 impairs glucose-stimulated insulin secretion.

<p>A) We evaluated the effects of <i>Ccn3</i> overexpression on insulin secretion by the hGH co-transfection system. In brief, we measured the hGH levels in the media of INS832/13 cells co-transduced with hGH and CCN3 or the empty plasmid and incubated at 2.8 mM (G2.8) or 16 mM glucose (G16). KCl (35 mM) was used as control. B) We studied the effects of CCN3 protein on insulin secretion. Insulin released in the culture medium was measured following incubation of INS832/13 cells in 2.8 mM or 16 mM glucose/KRBH medium or in the presence of 35 mM KCl. Secreted insulin levels were normalized to protein content. C) Measurements of intracellular calcium in living INS832/13 cells treated as described in (B) using the Fura-2 dye. We measured the ratio of fluorescence signals produced by excitation of 340 nm and 380 nm and detected at 510 nm to determine intracellular calcium concentrations. D) Glucose oxidation was measured as ¹⁴CO₂ production from [U-¹⁴C]-glucose in cells treated as described in (A) and the results were normalized to protein content. E) Cellular insulin content was measured by Elisa after lysis of the cells in acidic ethanol. Results represent mean ± SEM of three separate experiments carried out in triplicate. *, p<0.05.</p

<i>Ccn3</i> is a transcriptional target of FoxO1 in β-cells.

<p>A) <i>Ccn3</i> expression by quantitative real-time qPCR in INS832/13 cells transduced with either Ad-β-Gal or Ad-CN-FoxO1 and cultured for 24 h. Results are means +/– SEM of 4 separate experiments. B) <i>Ccn3 </i>mRNA levels in INS832/13 cells treated with or without 10% serum and LY294002 (50 µM) for 4 h. Results represent means +/– SEM of 3 separate experiments. C) <i>Ccn3</i> expression in isolated islets from WT and transgenic mice with CAFoxO1 overexpression in their β-cells (called “305 mice”) (n = 5 for each). *, p<0.05.</p

β INS832/13 cells secrete CCN3 protein.

<p>A–B) Western blot of CCN3 proteins in the media (A) or whole cell extracts (B) of either AdGFP or AdCCN3 infected cells and control uninfected cells. In (B) the top arrow indicate full-length CCN3 at a molecular weight of 47 KDa, whereas the bottom arrow indicate a CCN3 fragment of 35 KDa that have both been described in the literature. C) Immunohistochemical analysis of endogenous CCN3 protein localization in INS832/13 cells. We performed triple immunohistochemistry with CCN3 (blue), insulin (green) and Vamp/synaptobrevin (red) antibodies. D) Simple immunohistochemistry for CCN3 combined with DAPI staining demonstrate both nuclear and cytoplasmic localization of CCN3 in untransfected cells (Control, top) and cells transfected with a scrambled siRNAs (SiCont, middle). No CCN3 expression could be detected in cells transfected with Ccn3 siRNAs after 48 h (SiCcn3, bottom). Representative images of 3 separate experiments are shown.</p

CCN3 is up-regulated in animal models of insulin resistance.

<p>A) We determined CCN3 protein levels in different genetic models of insulin resistance. We prepared paraffin sections from WT, CAFoxO1 transgenics (305), <i>db/db</i>, and <i>Irs2^–/–</i> mice and performed CCN3 immunostaining (at least n = 3 for each). Representative images are shown. B) <i>Ccn3</i> expression in both the exocrine and the endocrine fraction was determined by PCR in 2 months old male animals. Amylase and insulin were used as specific exocrine and endocrine markers. Actin was used as a control in each sample. Representative images of 3 separate experiments are shown.</p

CCN3 protein decreases β-cell proliferation.

<p>A) The effect of CCN3 protein (1 nM) on β-cell proliferation was evaluated by BrdU incorporation in INS832/13 cells incubated at 5 mM glucose (G5), 25 mM glucose (G25) or in the presence of 10% serum. B) We studied the effects of 1 nM CCN3 protein on cAMP levels in INS cells by ELISA. C–D) Dose-dependent effects of CCN3 on cAMP levels in isolated rat islets (C) and INS cells (D). E) We measured <i>Ccn3</i> expression in cells transduced with increasing concentrations of siRNA specifically targeting <i>Ccn3</i> mRNA or scrambled siRNA as control by qPCR. F) Proliferation of INS832/13 cells transduced with either 50 pmol CCN3 or control siRNA. Results represent mean ± SEM of three separate experiments carried out at least in duplicate. *, p<0.05; **, p<0.01.</p