Neuroprotective action of α-Klotho against LPS-activated glia conditioned medium in primary neuronal culture

Abstract

The α-Klotho is an anti-aging protein that, when overexpressed, extends the life span in humans and mice. It has an anti-inflammatory and protective action on renal cells by inhibiting NF-κB activation and production of inflammatory cytokines in response to TNF-α. Furthermore, studies have shown the neuroprotective effect of α-Klotho against neuroinflammation on different conditions, such as aging, animal models of neurodegenerative diseases, and ischemic brain injury. This work aimed to evaluate the effects of α-Klotho protein on primary glial cell culture against the proinflammatory challenge with LPS and how this could interfere with neuronal health. Cortical mixed glial cells and purified astrocytes were pretreated with α- α-Klotho and stimulated with LPS followed by TNFα, IL-1β, IL-6, IFN-γ levels, and NF-κB activity analysis. Conditioned medium from cortical mixed glia culture treated with LPS (glia conditioned medium (GCM) was used to induce neuronal death of primary cortical neuronal culture and evaluate if GCM-KL (medium from glia culture pretreated α-Klotho followed by LPS stimulation) or GCM + LPS in the presence of KL can reverse the effect. LPS treatment in glial cells induced an increase in proinflammatory mediators such as TNF-α, IL-1β, IL-6, and IFN-γ, and activation of astrocyte NF-κB. GCM treated-cortical neuronal culture induced a concentration-dependent neuronal death. Pretreatment with α-Klotho decreased TNF-α and IL-6 production, reverted NF-κB activation, and decreased neuronal death induced by GCM. In addition, KL incubation together with GCM + LPS completely reverts the neuronal toxicity induced by low concentration of GCM-LPS. These data suggest an anti-inflammatory and neuroprotective effect of α-Klotho protein in the CNS. This work demonstrated the therapeutic potential of α-Klotho in pathological processes which involves a neuroinflammatory component.

Figure 1

Effect of α-Klotho (A,B) and LPS (C,D) treatment on cell viability (A,C) and cytotoxicity (B,D) of mouse glial cells. The primary culture of glial cells was subjected to 24-h treatment with LPS in different concentrations. Cell viability and cytotoxicity were assessed by MTT and LDH assays, respectively. One-way ANOVA analysis, followed by Tukey's post-test, *p < 0.05. Results are presented as mean ± SEM of 7 independent experiments.

Figure 2

Effect of α-Klotho on LPS-induced TNF-α secretion in mouse glial cells. The glial culture was pre-treated with serum-free medium (control) or with α-Klotho protein in different concentrations (0.1, 0.5, 1, and 2 nM) and times 1 (A), 4 (B), and 24 (C) hours, and then challenged with 1 µg/mL/mL LPS for 8 h. The supernatant was collected to measure TNF-α levels. One-way ANOVA analysis, followed by Tukey's post-test, *p < 0.05, **p < 0.01. Results are presented as mean ± SEM of 7 independent experiments.

Figure 3

Effect of α-Klotho on IL-1β, IL-6, and IFN-γ levels in mouse cell glia challenged with LPS. The glial culture was pretreated with serum-free medium (control) or with 1 nM α-Klotho for 24 h, and then challenged with 1 µg/mL/mL LPS for 8 h. The supernatant was collected to measure the levels of IL-1β (A), IL-6 (B), and IFN-γ (C). One-way ANOVA analysis, followed by Tukey's post-test, *p < 0.05, **p < 0.01, **** < 0.0001. Results are presented as mean ± SEM of 7 independent experiments.

Figure 4

α-Klotho rescue the NF-κB activation induced by LPS in the nuclear fraction. (A) Purified astrocytes from cortical glial cells were treated for 24 h with vehicle (control) (PBS) or 1 nM α-Klotho. Inflammatory stimulation with LPS 1 μg/ml was then performed. (B) The RelA (p65)-positive nuclei were counted and divided by the total number of nuclei, and the graph shows the comparison between the LPS group and for α-Klotho + LPS group expressed by the ratio in arbitrary units of RelA (p65) translocated to the nucleus over the total amount of RelA (p65) (n = 5). One-way ANOVA analysis, followed by Tukey's post-test, **p < 0,01; ***p < 0,001; ****p < 0,0001. (C,D) Effect of α-Klotho on p65 subunit NFkB translocation in astrocytes cells. Cytosol (20 mg) proteins were extracted from primary cultured cells: (C) Representative Western blotting autoradiographs of RELA (p65) cytosolic and β-actin; (D) Densitometric analysis (arbitrary units, A.U.) of p65 cytolosic/β-actin ratios of groups presented in the panel (n = 5). One-way ANOVA analysis, followed by Tukey's post-test, **p < 0,01; ***p < 0,001; ****p < 0,0001. (E) Nuclear fraction was used to perform the EMSA assay to measure NF-kB activity. (F) Densitometric analysis comparing NF-κB activity of control, α-Klotho, LPS, and α-Klotho–LPS groups (n = 5). One-way ANOVA analysis, followed by Tukey's post-test,***p < 0,0001. (G) A super-shift was also performed to show which NF-κB subunits are involved in this phenomenon.

Figure 5

Effects of GCM -induced cytotoxicity of mouse neurons. The primary embryonic culture of neurons was subjected to 24-h treatment with GCM in different concentrations (25% and 50%). Cytotoxicity was assessed by the LDH assay. One-way ANOVA analysis, followed by Tukey's post-test, *p < 0.05. Results are presented as mean ± SEM of 5 independent experiments.

Figure 6

Effects of GCM and GCM-KL -induced cytotoxicity of primary cortical mouse neurons. The primary embryonic culture of neurons was submitted to 24-h treatment with GCM or GCM-KL in different concentrations, 25% (A) and 50% (B). Cytotoxicity was assessed by the LDH assay. One-way ANOVA analysis, followed by Tukey's post-test, *p < 0.05, **p < 0.01. Results are presented as mean ± SEM of 5 independent experiments. (C) Representative schedule of the anti-inflammatory and neuroprotective effect of α-Klotho protein. LPS induces GCM to produce pro-inflammatory mediators that can lead to neuronal death (A). α-Klotho protein decreases the production of pro-inflammatory mediators induced by LPS in GCM (B), and it can have a protective effect on neurons from the neurotoxic effects of LPS induced by GCM. The figure was “Created with BioRender.com—Agreement number FH248VCXX8 to Scientific Reports”.

Figure 7

Effects of Klotho on GCM -induced cytotoxicity of primary cortical mouse neurons. The primary embryonic culture of neurons was submitted to 24-h treatment with GCM in different concentrations, 25% (A) and 50% (B) in the presence or absence of KL. Cytotoxicity was assessed by the LDH assay. One-way ANOVA analysis, followed by Tukey's post-test, *p < 0.05, Results are presented as mean ± SEM of 5 independent experiments. (C) Representative schedule of the neuroprotective effect of α-Klotho protein on GMP-induced cytotoxicity of neurons. LPS induces GCM to produce pro-inflammatory mediators that can lead to neuronal death (A). α-Klotho protein decreases the production of pro-inflammatory mediators induced by LPS in GCM (B), and it can have a protective effect on neurons from the neurotoxic effects of LPS induced by GCM. The figure was “Created with BioRender.com—Agreement number FH248VCXX8 to Scientific Reports”.