Chemokines and Inflammatory Mediators Interact to Regulate Adult Murine Neural Precursor Cell Proliferation, Survival and Differentiation

Adult neural precursor cells (NPCs) respond to injury or disease of the CNS by migrating to the site of damage or differentiating locally to replace lost cells. Factors that mediate this injury induced NPC response include chemokines and pro-inflammatory cytokines, such as tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ), which we have shown previously promotes neuronal differentiation. RT-PCR was used to compare expression of chemokines and their receptors in normal adult mouse brain and in cultured NPCs in response to IFNγ and TNFα. Basal expression of many chemokines and their receptors was found in adult brain, predominantly in neurogenic regions, with OB≫SVZ>hippocampus and little or no expression in non-neurogenic regions, such as cortex. Treatment of SVZ-derived NPCs with IFNγ and TNFα (alone and in combination) resulted in significant upregulation of expression of specific chemokines, with CXCL1, CXCL9 and CCL2 most highly upregulated and CCL19 downregulated. Unlike IFNγ, chemokine treatment of NPCs in vitro had little or no effect on survival, proliferation or migration. Neuronal differentiation was promoted by CXCL9, CCL2 and CCL21, while astrocyte and total oligodendrocyte differentiation was not affected. However, IFNγ, CXCL1, CXCL9 and CCL2 promoted oligodendrocyte maturation. Therefore, not only do NPCs express chemokine receptors, they also produce several chemokines, particularly in response to inflammatory mediators. This suggests that autocrine or paracrine production of specific chemokines by NPCs in response to inflammatory mediators may regulate differentiation into mature neural cell types and may alter NPC responsiveness to CNS injury or disease

Explant Methodology for Analyzing Neuroblast Migration

The subventricular zone (SVZ) in the mammalian forebrain contains stem/progenitor cells that migrate through the rostral migratory stream (RMS) to the olfactory bulb throughout adulthood. SVZ-derived explant cultures provide a convenient method to assess factors regulating the intermediary stage of neural stem/progenitor cell migration. Here, we describe the isolation of SVZ-derived RMS explants from the neonatal mouse brain, and the conditions required to culture and evaluate their migration

Primer sequences used for RT-PCR.

<p>Primer sequences used for RT-PCR.</p

Regulation of NPC proliferation survival and migration by chemokines and IFNγ.

<p>(A,B) Effect on neurosphere growth. Neurospheres were grown for 7 days in the presence of chemokines (100 ng/ml) and/or IFNγ (100 U/ml) and neurosphere growth determined by assessment of viable cell number using the MTS assay. Results of a representative experiment are shown in A and the combined results of three independent experiments, which are expressed as a percentage of neurosphere growth under basal conditions are shown in B. Effect on NPC proliferation (C,D), survival (E,F) and migration (G,H): neurospheres were pre-treated for 24 hrs with IFNγ then plated onto fibronectin in proliferation medium alone (IFNγ pre), with IFNγ alone (IFNγ+IFNγ), with chemokines alone or with chemokines plus IFNγ for 24 hrs. Basal controls were not pre-treated with IFNγ nor treated with any factor at the time of plating. Representative results from individual experiments are shown in C, E and G and the combined results of three independent experiments, expressed as a percentage of basal, are shown in D, F and H. (C,D) Effect on proliferation was assessed by counting the percentage of cells immunostained for the proliferation marker Ki67. (E,F) Effect on cell survival was assessed by counting the percentage of cells that were labeled by TUNEL staining. (G,H) Effect on cell migration was assessed by measuring the area of spread of cells from individual neurospheres and dividing by cell number per neurosphere. Results are expressed as mean+/−sem, *<i>p</i><0.05, **<i>p</i><0.01.</p

Primer sequences used for qPCR.

<p>Primer sequences used for qPCR.</p

Confirmation of chemokine induction by qPCR.

<p>qPCR was used to confirm the upregulation or induction of chemokine expression by IFNγ and/or TNFα. Due to large differences in the level of expression of the different chemokines, results are presented using a log₁₀ scale and show mean+/−SEM of 3 independent experiments in which all samples were run together and normalized against GAPDH. CXCL1 and CCL2 were detectable in the neurospheres under basal conditions, therefore their expression following incubation with IFNγ and TNFα was compared to the level of expression under basal conditions (which was set to 1). CXCL9 and CXCL13 were not expressed at detectable levels under basal conditions (C_T≥32). CXCL9 was detectable following addition of TNFα, which was used as the reference condition in this case (and set to 1; denoted by #) for comparison with expression induced by IFNγ and IFNγ+TNFα. CXCL13 was not detectable with TNFα treatment (denoted by 0) but was induced by IFNγ which was used as the reference condition (and set to 1; denoted by #) for comparison with levels of expression induced by TNFα plus IFNγ.</p

Expression of CXCR chemokine receptors in neurogenic versus non-neurogenic regions of adult brain.

<p>qPCR analysis of chemokine receptor CXCR1–7 expression in neurogenic subventricular (SVZ), olfactory bulb, and hippocampus and non-neurogenic cortex. Due to large differences in the level of expression of the different receptors, results are presented using a log₁₀ scale and show mean+/−SEM of 3 independent experiments in which all samples were run together and normalized against GAPDH. All samples were compared to expression of CXCR5 in the olfactory bulb as the reference sample (denoted by #), which was expressed at just detectable levels (C_T<32) and given a value of 1. Receptors that were not expressed (C_T≥32) are denoted by 0 in their respective columns.</p

Summary of chemokine ligand and receptor expression in adult brain.

<p>SVZ, Subventricular zone; OB, olfactory bulb; Hipp, hippocampus; Ctx, cortex.</p><p>Expression levels: − none; +/− very low; + low; ++ moderate, +++ high; ++++ very high.</p

Effect of selected chemokines on NPC differentiation.

<p>(A) The effect of chemokines (10 ng/ml) on neuronal differentiation as assessed by quantifying the relative percentage of cells that expressed the neuronal marker βIII-tubulin at 3 days under treatment conditions compared to the basal control. (B) The effect on astrocyte differentiation was assessed by counting the percentage of cells immunostained for the astrocyte marker GFAP at 7 days, expressed as a relative percentage compared to the basal control. (C) The effect on oligodendrocyte differentiation was assessed by counting the number of cells immunostained for the oligodendrocyte marker O4 at 7 days and expressing this as a percentage relative to the basal control. (D) Representative micrographs of βIII-tubulin, GFAP and DAPI labeling of cultures differentiated for 3 days in the presence of 10 ng/ml CXCL9, CCL2 or CCL21, compared to basal conditions. Results in A–C are expressed as mean+/−sem from at <i>n</i>≥3 independent experiments, *<i>p</i><0.05, **<i>p</i><0.01. Scale bar in D, 50 µm.</p

Effect of selected chemokines+/−IFNγ on oligodendrocyte precursor maturation.

<p>The effect of chemokines (10 ng/ml)+/−IFNγ (100 U/ml) on oligodendrocyte maturation was assessed by quantifying the percentage of O4 expressing cells at 3 days (A) and 7 days (B) that had the morphology of precursor cells, pro-oligodendrocytes or oligodendrocytes (C). Results are expressed as mean+/−sem from <i>n</i> = 3 experiments, *<i>p</i><0.05, **<i>p</i><0.01 ***<i>p</i><0.01. Scale bar in C, 50 µm.</p