Increased astrocyte expression of IL-6 or CCL2 in transgenic mice alters levels of hippocampal and cerebellar proteins

Abstract

Emerging research has identified that neuroimmune factors are produced by cells of the central nervous system (CNS) and play critical roles as regulators of CNS function, directors of neurodevelopment and responders to pathological processes. A wide range of neuroimmune factors are produced by CNS cells, primarily the glial cells, but the role of specific neuroimmune factors and their glial cell sources in CNS biology and pathology have yet to be fully elucidated. We have used transgenic mice that express elevated levels of a specific neuroimmune factor, the cytokine IL-6 or the chemokine CCL2, through genetic modification of astrocyte expression to identify targets of astrocyte produced IL-6 or CCL2 at the protein level. We found that in non-transgenic mice constitutive expression of IL-6 and CCL2 occurs in the two CNS regions studied, the hippocampus and cerebellum, as measured by ELISA. In the CCL2 transgenic mice elevated levels of CCL2 were evident in the hippocampus and cerebellum, whereas in the IL-6 transgenic mice, elevated levels of IL-6 were only evident in the cerebellum. Western blot analysis of the cellular and synaptic proteins in the hippocampus and cerebellum of the transgenic mice showed that the elevated levels of CCL2 or IL-6 resulted in alterations in the levels of specific proteins and that these actions differed for the two neuroimmune factors and for the two brain regions. These results are consistent with cell specific profiles of action for IL-6 and CCL2, actions that may be an important aspect of their respective roles in CNS physiology and pathophysiology.

Transgenic mice with increased astrocyte expression of IL-6 show altered effects of acute ethanol on synaptic function

“…As reported previously, levels of IL-6 in slices studied in vitro were higher than in samples from whole hippocampus immediately snap frozen after dissection (Gruol et al, 2014). In addition, levels of activated p42/44 MAPK were not higher in the IL-6 tg slices compared with the non-tg slices, as was observed for the samples from whole hippocampus (Nelson et al, 2012).…”

“…The alterations in GABAergic interneurons that express PAV observed in the hippocampus and cortex of IL-6 tg mice are consistent with altered local circuit activity in the IL-6 tg hippocampus (Campbell et al, 1993; Heyser et al, 1997; Samland et al, 2003). Also, our Western blot analysis of the level of GAD65/67, the synthetic enzyme for GABA, showed reduced levels in the IL-6-tg hippocampus compared to the non-tg hippocampus, although no genotypic difference was observed in PAV levels (Gruol et al, 2014; Nelson et al, 2012). Thus, effects on inhibitory interneurons could contribute to the increased EEG power in the gamma frequency range in the IL-6 tg mice.…”

“…Our previous studies identified neuroadaptive changes in the hippocampus of IL-6 tg mice that enhanced the fEPSP and the PS (Gruol et al, 2014; Nelson et al, 2012). Here we show that neuroadaptive changes in the IL-6 tg hippocampus also alter the effects of acute ethanol on these synaptic responses.…”

“…Immunohistochemical studies showed increased levels of activated STAT3 (i.e., pSTAT3), primarily in astrocytes and microglia of the IL-6 tg CNS, indicative of activation of IL-6R and consistent with elevated levels of IL-6 in the IL-6 tg CNS (Sanz et al, 2008). The IL-6 tg CNS shows increase expression of IL-6 regulated genes (e.g., GFAP, eb22, Socs3), increased levels of GFAP, and increased levels of activated STAT3 (pSTAT3), the signal transduction molecule through which IL-6 acts to increase GFAP (Gruol et al, 2014; Herrmann et al, 2008; Nelson et al, 2012; Sanz et al, 2008; Shu et al, 2011). The increased sensitivity to seizure activity observed in the IL-6 tg mice was specific for IL-6 in that a similar sensitivity was not observed in transgenic mice that express elevated levels of TNF-α in the CNS through astrocyte expression (Samland et al, 2003).…”

“…Protein samples for ELISA or Western blot were prepared from the IL-6 tg and non-tg hippocampal slices following protocols described previously (Gruol et al, 2014; Nelson et al, 2012). Proteins were extracted from the hippocampal samples by sonication in cold lysis buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.5% NP-40, a Complete Protease Inhibitor Cocktail Tablet (Roche Diagnostics, Mannheim, Germany), and a cocktail of phosphatase inhibitors (Na + pyrophosphate, β-glycerophosphate, NaF, Na + orthovanadate; all from Sigma-Aldrich).…”