Involvement of astroglial ceramide in palmitic acid-induced Alzheimer-like changes in primary neurons

Abstract

A high-fat diet has been shown to significantly increase the risk of the development of Alzheimer's disease (AD), a neurodegenerative disease histochemically characterized by the accumulation of amyloid beta (Abeta) protein in senile plaques and hyperphosphorylated tau in neurofibrillary tangles. Previously, we have shown that saturated free fatty acids (FFAs), palmitic and stearic acids, caused increased amyloidogenesis and tau hyperphosphorylaion in primary rat cortical neurons. These FFA-induced effects observed in neurons were found to be mediated by astroglial FFA metabolism. Therefore, in the present study we investigated the basic mechanism relating astroglial FFA metabolism and AD-like changes observed in neurons. We found that palmitic acid significantly increased de-novo synthesis of ceramide in astroglia, which in turn was involved in inducing both increased production of the Abeta protein and hyperphosphorylation of the tau protein. Increased amyloidogenesis and hyperphoshorylation of tau lead to formation of the two most important pathophysiological characteristics associated with AD, Abeta or senile plaques and neurofibrillary tangles, respectively. In addition to these pathophysiological changes, AD is also characterized by certain metabolic changes; abnormal cerebral glucose metabolism is one of the distinct characteristics of AD. In this context, we found that palmitic acid significantly decreased the levels of astroglial glucose transporter (GLUT1) and down-regulated glucose uptake and lactate release by astroglia. Our present data establish an underlying mechanism by which saturated fatty acids induce AD-associated pathophysiological as well as metabolic changes, placing 'astroglial fatty acid metabolism' at the center of the pathogenic cascade in AD.

Fig. 1

Palmitic acid (PA)-induced, de-novo synthesis of ceramide in astroglia. Astroglia were treated for 24 h with 0.2 mM PA or 4% bovine serum albumin [control (C)], after which cellular lipids were extracted for ceramide determination by high-performance liquid chromatography. PA significantly increased ceramide synthesis in astroglia, which was completely inhibited by treatment of astroglia with 2 mM L-cycloserine (L-CS), an inhibitor of de-novo synthesis of ceramide. Data are taken from three different experiments and are expressed as mean ± SD. One-way ANOVA with Tukey’s post-hoc method was used for analysing the differences between treatment groups. *P < 0.05 compared with control; #P < 0.05 compared with PA treatment.

Fig. 2

Immunostaining of intracellular reactive oxygen species (ROS) in astroglia. Astroglia were treated for 24 h with 0.2 mM of palmitic acid (PA) or 4% bovine serum albumin (control) and then stained with 5-(6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate for intracellular ROS detection and examined with confocal fluorescence microscopy (Zeiss LSM, 5 Pa). Objective lens magnification, 40×.

Fig. 3

Measurement of lactate dehydrogenase (LDH) release from astroglia treated with palmitic acid (PA). Treatment for 24 h with 0.2 mM PA failed to liberate LDH from astroglia as compared with controls. In contrast, 1 h treatment of astroglia with 300 mM H₂O₂ (positive control) induced significant LDH liberation after 24 h as compared with both control and PA-treated cells. Data are taken from three different experiments and are expressed as mean ± SD. One-way ANOVA with Tukey’s post-hoc method was used for analysing the differences between treatment groups. *P < 0.05 compared with control.

Fig. 4

Involvement of astroglial ceramide in palmitic acid (PA)-astroglia-induced BACE1 up-regulation and amyloid beta (Aβ) protein production in neurons. Astroglia were treated with 0.2 mM of PA or 4% bovine serum albumin [control (C)] for 24 h, followed by transfer of the astroglia-conditioned media to neurons (24 h treatment). (A) Immunoblot shows increased level of BACE1 in neurons treated with PA-astroglia-conditioned media compared with controls and the abnormal protein elevation was inhibited by treatment of astroglia with 2 mM L-cycloserine (L-CS). Histogram corresponding to BACE1 blot represents quantitative determinations of intensities of the relevant bands normalized to actin. (B) PA-astroglia-treated neurons show increased production of Aβ40 and Aβ42 as compared with controls, which was inhibited by treatment of astroglia with 2 mM L-CS. Data represent mean ± SD of three independent experiments. One-way ANOVA with Tukey’s post-hoc method was used for analysing the differences between treatment groups. *P < 0.05 compared with control; #P < 0.05 compared with PA treatment.

Fig. 5

Involvement of astroglial ceramide in palmitic acid (PA)-astroglia-induced tau hyperphosphorylation in neurons. In neurons treated with conditioned media from PA-treated astroglia, tau was found pathologically hyperphosphorylated as shown by immunoblotting with PHF-1 and AT8 antibodies. Tau-1 detects dephosphorylated tau, thus showing decreased levels in PA-astroglia-treated neurons. These PA-astroglia-induced tau abnormalities were blocked by inhibiting astroglial ceramide synthesis with 2 mM L-cycloserine (L-CS). Histograms corresponding to PHF-1 and AT8 blots represent quantitative determinations of intensities of the relevant bands normalized with actin. Data represent mean ± SD of three independent experiments. One-way ANOVA with Tukey’s post-hoc method was used for analysing the differences between treatment groups. *P < 0.05 compared with control (C); #P < 0.05 compared with PA treatment.

Fig. 6

Palmitic acid (PA)-astroglia-induced activation of Alzheimer’s disease-specific kinases in neurons is mediated by astroglial ceramide. Conditioned media from PA-treated astroglia activated (A) glycogen synthase kinase 3 (GSK-3) [increased levels of phosphorylated GSK-3 (P-GSK-3)] and (B) cyclin-dependent kinase 5 (cdk5) (increased cleavage of p35 to p25) but not (C) MAP Erk1/2 [no change in the levels of phosphorylated MAP Erk1/2 (P-MAP Erk1/2)]. The treatment of astroglia with 2 mM L-cycloserine (L-CS) inhibited PA-astroglia-induced activation of both GSK-3 and cdk5. Data are representative of three different experiments. C, control.

Fig. 7

Glycogen synthase kinase 3 (GSK-3) is involved in palmitic acid (PA)-astroglia-induced tau hyperphosphorylation in neurons. Astroglia-conditioned media were transferred to neurons, with or without various kinase inhibitors, i.e. 10 mM LiCl₂ (GSK-3 inhibitor), 10 mM roscovitine (cyclin-dependent kinase 5 inhibitor) and 30 mM PD98059 (mitogen-activated protein kinase inhibitor). Immunoblot analysis with PHF-1 and AT8 antibodies shows that only LiCl₂ inhibited the observed PA-induced tau hyperphosphorylation in neurons. Histogram data represent mean ± SD of three independent experiments. One-way ANOVA and Tukey’s post-hoc method was used for analysing the differences between treatment groups. *P < 0.05 compared with control; #P < 0.05 compared with PA treatment. C, control.

Fig. 8

Palmitic acid (PA) down-regulates glucose uptake and lactate release by astroglia. The cortical neurons and astroglia were treated for 24 h with 0.2 mM of PA or 4% bovine serum albumin. (A) In neurons, PA treatment did not change glucose uptake and lactate production. (B) PA treatment significantly decreased glucose uptake and lactate production by astroglia. Data represent mean ± SD of six experiments. Student’s t-test was used for analysing the differences between the two treatment groups. *P < 0.05 compared with respective control. C, control.

Fig. 9

Palmitic acid (PA) down-regulates GLUT1 level in astroglia. Astroglia were treated for 24 h with 0.2 mM of PA or 4% bovine serum albumin. The immunoblot analysis shows that PA treatment significantly decreased the levels of GLUT1 as compared with the untreated astroglia. β-actin is shown as a marker for protein loading. The histogram represents quantitative determinations of intensities of the relative bands normalized with actin. Data represent mean ± SD of three independent experiments. Student’s t-test was used for analysing the differences between the two treatment groups. *P < 0.05 compared with respective control (C).

Fig. 10

Measurement of intracellular ATP in astroglia. The cortical astroglia were treated for 24 h with 0.2 mM of palmitic acid (PA) or 4% bovine serum albumin. PA treatment increased cellular ATP production in astroglia. Data represent mean ± SD of three experiments. Student’s t-test was used for analysing the differences between the two treatment groups. *P < 0.05 compared with respective control (C).

Fig. 11

Proposed cellular mechanism by which astroglial free fatty acid (FFA) metabolism leads to Alzheimer’s disease (AD)-like physiological and metabolic changes. The elevated levels of saturated FFAs associated with various risk factors for AD down-regulate glucose metabolism in astroglia (decrease in GLUT1 levels, glucose uptake and lactate production in astroglia). Furthermore, FFA-treated astroglia secrete factor(s) that induce reactive oxygen species (ROS) production in neurons. The increased ROS production leads to up-regulation of BACE1 levels and glycogen synthase kinase 3 (GSK-3) activity, which in turn cause amyloidogenic processing of amyloid precursor protein (APP) and hyperphosphorylation of tau, respectively. The FFA-induced ceramide synthesis in astroglia plays a central role in causing the above-mentioned AD-like pathophysiological changes in neurons.