Interleukin-1β enhances neuronal vulnerability to proNGF-mediated apoptosis by increasing surface expression of p75(NTR) and sortillin

Abstract

Many types of injury such as seizure, ischemia, and oxidative stress cause upregulation of the p75 neurotrophin receptor (p75(NTR)) in brain neurons, where it promotes apoptosis, however the mechanism by which p75(NTR) is regulated under these conditions is not well understood. Proinflammatory cytokines such as interleukin-1β (IL-1β) are highly produced under these injury conditions and, in particular, are expressed rapidly in the rat hippocampus after seizure. IL-1β is known to increase neuronal vulnerability under many conditions, although it does not directly induce neuronal death. Recently, we have shown that these cytokines regulate p75(NTR) induction both in neurons and astrocytes in vitro. Here, we show that IL-1β infusion into the brain induces p75(NTR) in neurons of the CA1 area of the hippocampus. While IL-1β induction of p75(NTR) is not sufficient to induce cell death, we demonstrate that IL-1β primes the neurons by recruiting p75(NTR) and its coreceptor sortilin to the cell surface, making the neurons more vulnerable to subsequent challenge by proNGF. These results suggest a mechanism by which IL-1β exacerbates neuronal death following injury.

Keywords: CSF; EDTA; IL-1β; NGF; TdT; apoptosis; brain injury; cerebrospinal fluid; ethylenediaminetetraacetic acid; interleukin-1; interleukin-1β; neurotrophin; p75; p75 neurotrophin receptor; p75(NTR); terminal deoxynucleotidyl transferase.

Figure 1. Unilateral IL-1β infusion increases p75^NTR expression in vivo

Rats were cannulated 7 days before infusion with IL-1β (10ng). A. p75^NTR mRNA was induced by IL-1β. Tissue was taken after 4 hr or 1d treatment with IL-1β (mean±SEM, n = 3). Asterisk denotes difference from saline (p<0.05). B. 2 days after the infusion, each hippocampus was taken for Western blot assay. p75^NTR expression was increased with IL-1β infusion. Quantification of blots from three different experiments and densitometric values were normalized to actin and are expressed relative to the saline control (CTRL). Error bars represent SEM. * P<0.05 relative to CTRL, two-tailed t test. C. 2 days after the IL-1β infusion, brains were perfused, sectioned through the hippocampus, immunostained with anti-p75^NTR (green) and anti-NeuN (red). IL-1β infusion increased p75^NTR expression (arrows) in the CA1 region of the hippocampus (right column) compared to saline infusion (left column). Scale bars = 50μm. n=6. D. Counts of p75^NTR-positive cells indicated a 3-fold increase following IL-1β infusion.

Figure 2. IL-1β is not sufficient to mediate cell death in vivo

A. TUNEL staining showed negative labeling with both saline (left column) and IL-1β (right column) infusion, indicating that IL-1β itself does not directly elicit cell death. Double staining for TUNEL (green) and Hoechst (blue). Images are representative of two independent experiments. B. Positive control TUNEL assay with DNase-I. Scale bars = 50μm.

Figure 3. IL-1β induces increased secretion of mature NGF

A. Bars indicate NGF mRNA level (mean±SEM, n = 3) by qPCR after 4 h treatment with IL-1β. Asterisk denotes difference from saline (p<0.05). B. Western blots show cerebrospinal fluid (CSF) collected from IL-1β infused rats compared to saline-infused controls or rats after pilocarpine-induced seizures. A short exposure shows the seizure-induced increase in proNGF without being cleaved, while the longer exposure shows the IL-1β-induced increase in mature NGF in the CSF. Blot is representative of three independent experiments. C. In situ zymogram shows increased tPA activity with IL-1β infusion. Fresh frozen sections were covered with an in situ zymogram assay buffer containing 20 μg/mL plasminogen. Areas of tPA activity are indicated by black spots on a bright background. Images are representative of two independent experiments. Scale bars = 250μm. D. Quantification of the area showing tPA activity relative to the entire hippocampus in the control and IL-1β infused hippocampus. Asterisk indicates significantly different from control by Student’s t-test, p < 0.01.

Figure 4. IL-1β primed neurons are more vulnerable to proNGF than NGF

A. Immunostaining of hippocampal neurons for sortilin and p75^NTR demonstrating colocalization of the receptors. B. Bars show relative cell number (mean±SEM, n = 4 independent experiments) in hippocampal neuronal cultures. Cells were treated for 4–6 h with IL-1β (10ng/mL), followed by overnight treatment with NGF (100ng/mL). IL-1β did not exacerbate NGF-mediated neuronal death. Asterisks denote difference from untreated control (p < 0.05). C. Bars indicate relative cell number (mean±SEM, n = 4 independent experiments) in hippocampal neuronal cultures treated with IL-1β (10 ng/mL) for 4–6 hrs, followed by proNGF (1–10ng/mL). ProNGF elicited more cell death in IL-1β primed neurons. * denotes difference from control and # indicates difference from proNGF alone (p<0.05; one-way ANOVA and Tukey’s post hoc analysis).

Figure 5. IL-1β recruits sortilin and p75^NTR receptors to the plasma membrane

A. Cultured hippocampal neurons were treated with IL-1β for 8h, incubated with biotin for 1h, and lysates were precipitated with streptavidin. Total biotinylated cell surface protein and 20μg of non-biotinylated intracellular proteins were analyzed by Western blot for sortilin, p75^NTR and transferrin receptor. Blots were stripped and re-probed for actin, which was only present in the intracellular fraction. Quantification of sortilin (B) and p75^NTR (C) from three independent blots. Asterisk denotes difference from control (p<0.05; one-way ANOVA and Tukey’s post hoc analysis).