LIF, a Novel Myokine, Protects Against Amyloid-Beta-Induced Neurotoxicity via Akt-Mediated Autophagy Signaling in Hippocampal Cells

Abstract

Background: Leukemia inhibitory factor, a novel myokine, is known to be associated with neural function, but the underlying molecular mechanism remains unclear.

Methods: HT-22 mouse hippocampal cells, primary hippocampal cells, and Drosophila Alzheimer's disease model were used to determine the effect of leukemia inhibitory factor on neurons. Immunoblot analysis and immunofluorescence method were used to analyze biological mechanism.

Results: Leukemia inhibitory factor increased Akt phosphorylation in a phosphoinositide-3-kinase-dependent manner in hippocampal cells. Leukemia inhibitory factor also increased the phosphorylation of the mammalian target of rapamycin and the downstream S6K. Leukemia inhibitory factor stimulated the phosphorylation of signal transducer and activator of transcription via extracellular signal-regulated kinases. Leukemia inhibitory factor increased c-fos expression through both Akt and extracellular signal-regulated kinases. Leukemia inhibitory factor blocked amyloid β-induced neural viability suppression and inhibited amyloid β-induced glucose uptake impairment through the block of amyloid β-mediated insulin receptor downregulation. Leukemia inhibitory factor blocked amyloid β-mediated induction of the autophagy marker, microtubule-associated protein 1A/1B-light chain 3. Additionally, in primary prepared hippocampal cells, leukemia inhibitory factor stimulated Akt and extracellular signal-regulated kinase, demonstrating that leukemia inhibitory factor has physiological relevance in vivo. Suppression of the autophagy marker, light chain 3II, by leukemia inhibitory factor was observed in a Drosophila model of Alzheimer's disease.

Conclusions: These results demonstrate that leukemia inhibitory factor protects against amyloid β-induced neurotoxicity via Akt/extracellular signal-regulated kinase-mediated c-fos induction, and thus suggest that leukemia inhibitory factor is a potential drug for Alzheimer's disease.

Keywords: Akt; Alzheimer’s disease; LIF; autophagy; mTOR; myokine.

Figure 1.

Leukemia inhibitory factor (LIF) stimulates Akt phosphorylation. (A) HT-22 cells were incubated with LIF (50 ng/mL) for the indicated time periods. Cell lysates were analyzed through western blot using antibodies against phospho-Akt (Ser473), with Akt serving as a control. (B) HT-22 cells were stimulated for 1 hour with several concentrations of LIF. The cell lysates were analyzed through western blot using an antibody against phospho-Akt (Ser473), with Akt serving as a control. (C) HT-22 cells were pretreated with the phosphatidylinositide 3-kinase (PI3K) inhibitor, LY294002 (10 μM, for a 30-min incubation) and then incubated with LIF for 1 hour. Cell lysates were analyzed through western blot using an antibody against phospho-Akt (Ser473), with Akt serving as a control. Experiments were performed in triplicate and repeated 3 times. Representative data are shown.

Figure 2.

Leukemia inhibitory factor (LIF) stimulates the mTOR signaling pathway. (A) HT-22 cells were incubated with LIF (50 ng/mL) for the indicated times. Cell lysates were analyzed through western blot using antibodies against phospho-mTOR (Ser2448), with mTOR serving as a control. (B) HT-22 cells were pretreated with the PI3K inhibitor, LY294002 (10 μM), and then incubated with LIF for 1 hour. Cell lysates were analyzed through western blot using an antibody against phospho-mTOR (Ser2448), with mTOR serving as a control. (C) HT-22 cells were incubated with LIF (50 ng/mL) for the indicated times. Cell lysates were analyzed through western blot using antibodies against phospho-S6K (Thr389), with S6K serving as a control. (D) HT-22 cells were pretreated with the mTOR inhibitor, rapamycin (10 nM, for a 24-hour incubation) and then incubated with LIF for 1 hour. Cell lysates were analyzed through western blot using an antibody against phospho-S6K(Thr389), with S6K serving as a control. Experiments were performed in triplicate and repeated 3 times. Representative data are shown.

Figure 3.

Leukemia inhibitory factor (LIF) is involved in neuronal activity through ERK phosphorylation. (A) HT-22 cells were incubated with LIF (50 ng/mL) for the indicated times. Lysates were analyzed through western blot using an antibody against phospho-ERK, with ERK serving as a control. (B) HT-22 cells were pretreated with the MEK inhibitor, PD98059 (50 mM, for a 30-minute incubation), and then incubated with LIF (50 ng/mL) for 3 minutes. Cell lysates were analyzed through western blot using an antibody against phospho-ERK, with ERK serving as a control. (C) HT-22 cells were incubated with LIF (50 ng/mL) for the indicated times. Lysates were analyzed through western blot using an antibody against phospho-STAT (Tyr705), with STAT serving as a control. (D) HT-22 cells were pretreated with the MEK inhibitor, PD98059 (50 mM, 30 minutes) and then incubated with LIF (50 ng/mL) for 1 hour. Cell lysates were analyzed through western blot using an antibody against phosphor-STAT(Tyr705), with STAT serving as a control. Experiments were performed in triplicate and repeated 3 times. Representative data are shown.

Figure 4.

Leukemia inhibitory factor (LIF) stimulates neuronal activity through c-fos. (A) HT-22 cells were incubated with LIF (50 ng/mL) for the indicated times. Lysates were analyzed through western blot using an antibody against c-fos, with β-actin incubation alone serving as a control. (B) HT-22 cells were pretreated with the phosphatidylinositide 3-kinase (PI3K) inhibitor, LY294002 (10 mM, for a 30-minute incubation) and then incubated with LIF (50 ng/mL) for 15 minutes. Cell lysates were analyzed through western blot using an antibody against c-fos, with β-actin serving as a control. (C) HT-22 cells were pretreated with the MEK inhibitor, PD98059 (50 mM, for a 30-minute incubation) and then incubated with LIF (50 ng/mL) for 15 minutes. Cell lysates were analyzed through western blot using an antibody against c-fos, with β-actin serving as a control. (D) Increase in c-fos activation after LIF treatment. HT-22 cells were treated with LIF (50 ng/mL) for 15 minutes. Images were captured using a confocal microscope. Experiments were performed in triplicate and repeated 3 times. Representative data are shown.

Figure 5.

Leukemia inhibitory factor (LIF) increases cell viability by reducing cytotoxicity of amyloid β. (A) HT-22 cells were incubated with amyloid β at the indicated doses (for 48 hours) and then cell viability was measured. (B) HT-22 cells were incubated with LIF (50 ng/mL) for 48 hours before cell viability was measured. *P < .05 vs basal conditions. (C) HT-22 cells were pretreated with LIF for 1 hour, then incubated with amyloid β (10 µg/mL) for 48 hours, before cell viability was measured. *P < .05 vs amyloid β-treated conditions. (D) HT-22 cells were transiently transfected with c-fos siRNA (50 nM) for 48 hours. The cell lysates were analyzed through western blotting using antibody against c-fos, with β-actin serving as a control. (E) HT-22 cells were transiently transfected with c-fos siRNA (50 nM) for 48 hours and then incubated with LIF (5 ng/mL) for a further 48 hours before cell viability was measured. *P < .05 vs LIF-treated conditions. (F) Primary hippocampal cells were pretreated with LY294002 and PD98059, then incubated with indicated conditions, before cell viability was measured. **P < .01 vs amyloid β-treated conditions. Experiments were performed in triplicate and repeated 3 times. Representative data are shown.

Figure 6.

Leukemia inhibitory factor (LIF) enhances glucose utilization through regulation of the insulin signaling pathway (Insulin receptor, Akt, GLUT3). (A) HT-22 cells were incubated with the amyloid β at the indicated doses (for 48 hours) and then cell viability was measured. (B) HT-22 cells were pretreated with LIF for 1 hour, then incubated with amyloid β (10 µg/mL) for 48 hours, before cell viability was measured. (C) HT-22 cells were incubated with amyloid β (10 µg/mL) for 48 hours and treated with insulin (200 nM) for 1 minute. Cell lysates were analyzed through western blot using an antibody against phospho-insulin receptor, phospho-Akt (Ser473), with β-actin serving as a control. (D) To prepare mRNA, HT-22 cells were pretreated with LIF for 1 hour then incubated with amyloid β (10 µg/mL) for 48 hours, and reverse transcription-polymerase chain reaction was conducted using specific insulin receptor, GLUT3 primers. PCR products were separated on 1.5% agarose gels and visualized under ultraviolet light, with β-actin serving as a control. (E) HT-22 cells were pretreated with LIF (50 ng/mL) then incubated with amyloid β (10 µg/mL) for 48 hours and treated with insulin (200 nM) for 1 minute. Cell lysates were analyzed through western blot using an antibody against phospho-Akt (Ser473), with Akt serving as a control. *P < .05 vs amyloid beta-treated condition. Experiments were performed in triplicate and repeated 3 times. Representative data are shown.

Figure 7.

Leukemia inhibitory factor (LIF) prevents the cellular autophagy induced by amyloid β. (A) HT-22 cells were incubated with amyloid β (10 µg/mL) for the indicated times. Lysates were analyzed through western blot using an antibody against LC3II, with β-actin serving as a control. (B) HT-22 cells were pretreated with LIF for 1 hour, then incubated with amyloid β (10 µg/mL) for 48 hours. Lysates were analyzed through western blot using an antibody against LC3II, with β-actin serving as a control. (C) Amyloid β induces expression of intracellular autophagy genes. LIF prevents the expression of autophagy genes induced by amyloid β. HT-22 cells were pretreated with LIF for 1 hour then incubated with amyloid β (10 µg/mL) for 48 hours. Images were captured using a confocal microscope. Experiments were performed in triplicate and repeated 3 times. Representative data are shown.

Figure 8.

Leukemia inhibitory factor (LIF) activates the Akt, mTOR, and ERK signaling pathway, increases cell viability, and prevents cellular autophagy in primary hippocampal cells. (A) Culture images of primary hippocampal cells. Cells were imaged on day 1 and day 14 of culture, when cells were sufficiently differentiated. (B) HT-22 cells were incubated with LIF (50 ng/mL) for the indicated times. Lysates were analyzed through western blot using an antibody against phospho-Akt (Ser473) and phospho-S6K (Thr389), with Akt and S6K serving as controls, respectively. (C) HT-22 cells were incubated with LIF (50 ng/mL) for the indicated times. Lysates were analyzed through western blot using an antibody against phospho-ERK, with ERK serving as a control. (D) HT-22 cells were pretreated with LIF (50 ng/mL) for 1 hour, then incubated with amyloid β (10 µg/mL) for 48 hours, before cell viability was measured. (E) HT-22 cells were pretreated with the PI3K inhibitor, LY294002 (10 mM, for a 30-minute incubation), and then incubated with LIF (50 ng/mL) for 15 minutes. Cell lysates were analyzed through western blot using an antibody against c-fos, with β-actin serving as a control. (F) HT-22 cells were pretreated with the MEK inhibitor, PD98059 (50 mM, 30 minutes) and then incubated with LIF (50 ng/mL) for 15 minutes. Cell lysates were analyzed through western blot using an antibody against c-fos, with β-actin serving as a control. (G) HT-22 cells were pretreated with LIF (50 ng/mL) for 1 hour then incubated with amyloid β (10 µg/mL) for 48 hours. Lysates were analyzed through western blot using an antibody against LC3II, with β-actin serving as a control. *P < .05 vs amyloid beta-treated condition.

Figure 9.

Leukemia inhibitory factor (LIF) reduces autophagy gene expression in a Drosophila model of Alzheimer’s disease. (A) Amyloid β expressing Drosophila were fed LIF (500 or 1500 ng/mL) for 14 days. Fly head lysates were then analyzed through western blot using an antibody against LIF, with β-actin serving as a control. *P < .05 vs basal condition. (B) Decreased autophagy gene expression was observed in the LIF-fed group. Amyloid β-expressing flies were fed LIF (500 or 1500 ng/mL) for 14 days. Images were captured using a confocal microscope. *P < .05 vs basal condition. (C) Amyloid β-expressing flies were fed LIF (500 or 1500 ng/mL) for 14 days. Fly head lysates were then analyzed through western blot using an antibody against LC3II, with β-actin serving as a control. *P < .05 vs basal condition (scale bar = 50 µm).