Palmitate Increases β-site AβPP-Cleavage Enzyme 1 Activity and Amyloid-β Genesis by Evoking Endoplasmic Reticulum Stress and Subsequent C/EBP Homologous Protein Activation

Abstract

Epidemiological studies implicate diets rich in saturated free fatty acids (sFFA) as a potential risk factor for developing Alzheimer's disease (AD). In particular, high plasma levels of the sFFA palmitic acid (palmitate) were shown to inversely correlate with cognitive function. However, the cellular mechanisms by which sFFA may increase the risk for AD are not well known. Endoplasmic reticulum (ER) stress has emerged as one of the signaling pathways initiating and fostering the neurodegenerative changes in AD by increasing the aspartyl protease β-site AβPP cleaving enzyme 1 (BACE1) and amyloid-β (Aβ) genesis. In this study, we determined the extent to which palmitate increases BACE1 and Aβ levels in vitro and in vivo as well as the potential role of ER stress as cellular mechanism underlying palmitate effects. We demonstrate, in palmitate-treated SH-SY5Y neuroblastoma cells and in the hippocampi of palmitate-enriched diet-fed mice, that palmitate evokes the activation of the C/EBP Homologous Protein (CHOP), a transcription factor that is specifically responsive to ER stress. Induction of CHOP expression is associated with increased BACE1 mRNA, protein and activity levels, and subsequent enhanced amyloidogenic processing of amyloid-β protein precursor (AβPP) that culminates in a substantial increase in Aβ genesis. We further show that CHOP is an indispensable molecular mediator of palmitate-induced upregulation in BACE1 activity and Aβ genesis. Indeed, we show that Chop-/- mice and CHOP knocked-down SH-SY5Y neuroblastoma cells do not exhibit the same commensurate degree of palmitate-induced increase in BACE1 expression levels and Aβ genesis.

Keywords: Alzheimer’s disease; C/EBP homologous protein; amyloid-β; endoplasmic reticulum stress; palmitic acid; saturated free fatty acids; β-site AβPP cleaving enzyme 1.

Fig.1

Dose response effects of the saturated fatty acids, palmitate and stearate, and their respective MUFA, palmitoleate and oleate, on the expression of ER stress markers and cell death in human neuroblastoma SH-SY5Y cells. A-D) Representative western blots show that treatment with exogenous palmitate (A) and stearate (B) at a concentration >100 μM for 24 h, while treatment with exogenous palmitoleate (C) and oleate (D) only at a concentration of >375 μM for 24 h, significantly increases the expression of ER stress markers - GRP78, GRP94, and CHOP in whole cell lysates from SH-SY5Y-APP_Swe cells. E) Cell death assessed by the release of LDH in the conditioned medium shows that treatment with exogenous palmitate and stearate at a concentration >250 μM for 24 h, while treatment with exogenous palmitoleate and oleate only at a concentration of 500 μM for 24 h, evoked significant cell death in SH-SY5Y-APP_Swe cells. Data is expressed as Mean±S.D and includes determination made in four (n = 4) separate cell culture experiments. ^***p < 0.001 versus BSA-treated cells.

Fig.2

Exogenous palmitate treatment and a palmitate-enriched diet evoke ER stress in human neuroblastoma SH-SY5Y cells and the mouse hippocampus, respectively. A) Representative western blots show that palmitate treatment (100 μM for 24 h) of SH-SY5Y-APP_Swe cells and feeding C57BL/6J wild-type mice a palmitate-enriched diet for three months, results in the activation of the three arms of ER stress signaling - IRE1α, PERK, and ATF6 pathway as assessed by an increase in the phosphorylation of IRE1α and PERK as well as the augmentation in the nuclear translocation of ATF3, ATF4, ATF6, and CHOP. B) Palmitate treatment (100 μM for 24 h) of SH-SY5Y-APP_Swe cells and feeding C57BL/6J wild-type mice a palmitate-enriched diet for three months, also increases the transcriptional activities of the six transcription factors measured in the hippocampus. Data is expressed as Mean±S.D and includes determination made in three (n = 3) separate cell culture experiments and six (n = 6) different animals from each group. ^***p < 0.001 versus BSA-treated control cells or C57BL/6J wild-type mice fed a control chow diet. PA, palmitic acid.

Fig.3

BACE1 expression and subsequent Aβ genesis is induced by the sFFA, palmitate and stearate, but not by their MUFA counterparts, palmitoleate and oleate. A) Representative western blots show that exogenous palmitate and stearate treatment (100 μM for 24 h), but not palmitoleate and oleate treatment (100 μM for 24 h), significantly increases BACE1 protein levels accompanied by an increase in the amyloidogenic processing of AβPP as evidenced by an increase in sAβPPβ and CTFβ levels concomitant with a decrease in sAβPPα and CTFα levels in the whole cell homogenates from SH-SY5Y-APP_Swe cells. B, C) Exogenous palmitate and stearate treatment, but not palmitoleate and oleate treatment, significantly increases BACE1 mRNA expression (B) and BACE1 activity (C) in SH-SY5Y-APP_Swe cells. D) ELISA immunoassays show that exogenous palmitate and stearate treatment, but not palmitoleate and oleate treatment, significantly increases the levels of the intracellular Aβ_1 - 42 species in the whole cell lysates and secreted Aβ_1 - 42 species in the conditioned media, from SH-SY5Y-APP_Swe cells. The ER stress inducer, Tunicamycin, also increased the following - BACE1 protein levels (A), BACE1 mRNA expression (B) and BACE1 activity (C), and the ensuing levels of intracellular as well as secreted Aβ_1 - 42 species (D) in SH-SY5Y-APP_Swe cells. Data is expressed as Mean±S.D and includes determination made in four (n = 4) separate cell culture experiments. ^***p < 0.001 versus BSA-treated cells.

Fig.4

Palmitate induces BACE1 expression and subsequent Aβ genesis by inducing ER stress. A) Representative western blots show that pretreatment (for 2 h) of the human neuroblastoma cells with the molecular chaperone 4-PBA significantly precludes the palmitate-induced increase in BACE1 protein levels accompanied by a decrease in the amyloidogenic processing of AβPP as evidenced by a decrease in the palmitate-induced increase in sAβPPβ and CTFβ levels concomitant with an increase in the palmitate-induced decrease in sAβPPα and CTFα levels in the whole cell homogenates from SH-SY5Y-APP_Swe cells. B, C) Pretreatment with 4-PBA attenuates the palmitate-induced increase in BACE1 mRNA expression (B) and BACE1 activity (C) in SH-SY5Y-APP_Swe cells. D) ELISA immunoassays show that pretreatment with 4-PBA significantly attenuates the exogenous palmitate treatment-induced increase in the levels of the intracellular Aβ_1 - 42 species in the whole cell lysates and secreted Aβ_1 - 42 species in the conditioned media, from SH-SY5Y-APP_Swe cells. Pretreatment (for 2 h) with the molecular chaperone 4-PBA also significantly precludes the Tunicamycin-induced increase in the following - BACE1 protein levels (A), BACE1 mRNA expression (B) and BACE1 activity (C), and intracellular as well as secreted Aβ_1 - 42 species (D), in SH-SY5Y-APP_Swe cells. Data is expressed as Mean±S.D and includes determination made in four (n = 4) separate cell culture experiments. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 versus BSA-treated cells; ^††p < 0.01, ^†††p < 0.001, versus palmitate-treated cells or Tunicamycin-treated cells.

Fig.5

CHOP mediates the palmitate-induced increase in BACE1 expression and subsequent Aβ genesis. A) Representative western blots show that knocking-down CHOP expression using a RNAi approach significantly attenuates the palmitate-induced increase in BACE1 protein levels accompanied by a decrease in the amyloidogenic processing of AβPP as evidenced by a decrease in the palmitate-induced increase in sAβPPβ and CTFβ levels concomitant with an increase in the palmitate-induced decrease in sAβPPα and CTFα levels in the whole cell homogenates from SH-SY5Y-APP_Swe cells. B, C) Knocking-down CHOP expression attenuates the palmitate-induced increase in BACE1 mRNA expression (B) and BACE1 activity (C) in SH-SY5Y-APP_Swe cells. D) ELISA immunoassays show that knocking-down CHOP expression significantly mitigates the exogenous palmitate treatment-induced increase in the levels of the intracellular Aβ_1 - 42 species in the whole cell lysates and secreted Aβ_1 - 42 species in the conditioned media, from SH-SY5Y-APP_Swe cells. Data is expressed as Mean±S.D and includes determination made in four (n = 4) separate cell culture experiments. ^**p < 0.01, ^***p < 0.001 versus BSA-treated GFP knock-down cells or BSA-treated scrambled siRNA transfected cells; ^†p < 0.05, ^††p < 0.01, versus palmitate-treated GFP knock-down cells or palmitate-treated scrambled siRNA transfected cells. PA, palmitic acid.

Fig.6

Chop^–/– mice are significantly protected from the palmitate-enriched diet-induced increase in BACE1 expression and ensuing Aβ genesis. A) Representative western blots show that nine-month-old Chop^–/– mice fed a palmitate-enriched diet for three months, do not exhibit the increase in BACE1 protein levels as well as the accompanying increase in sAβPPβ and CTFβ levels concomitant with a decrease in sAβPPα and CTFα levels, to the same degree in the hippocampal region compared to the C57BL/6J wild-type mice fed a palmitate-enriched diet. B, C) Chop^–/– mice fed a palmitate-enriched diet do not exhibit the increase in BACE1 mRNA expression (B) and BACE1 activity (C), to the same degree in the hippocampal region compared to the C57BL/6J wild-type mice fed a palmitate-enriched diet. D) ELISA immunoassays show that the Chop^–/– mice fed a palmitate-enriched diet have significantly lower levels of total formic acid-soluble Aβ_1 - 40 and Aβ_1 - 42 species in the hippocampus, compared to the C57BL/6J wild-type mice fed a palmitate-enriched diet. Data is expressed as Mean±S.D and includes determination made in six (n = 6) different animals from each group. ^*p < 0.05, ^***p < 0.001 versus C57BL/6J wild-type mice fed a control chow diet; ^†p < 0.05, ^††p < 0.01, versus C57BL/6J wild-type mice fed a palmitate-enriched diet; ^Δp < 0.05 versus Chop^–/– mice fed a control chow diet. PA, palmitic acid.

Fig.7

Ectopic overexpression of native CHOP, but not the transcriptionally dead leucine zipper domain deficient mutant CHOP (CHOP LZ^–), evokes an increase in BACE1 expression and subsequent Aβ genesis. A) Representative western blots show that the leucine zipper domain of CHOP that is responsible for its transcriptional activity, is necessary to induce BACE1 protein levels as well as the accompanying increase in sAβPPβ and CTFβ levels concomitant with a decrease in sAβPPα and CTFα levels in SH-SY5Y-APP_Swe cells. C, D) Ectopic overexpression of native CHOP (wt CHOP), but not the transcriptionally dead leucine zipper domain deficient mutant CHOP (CHOP LZ^–), elicits an increase in BACE1 mRNA expression (B) and BACE1 activity (C) in SH-SY5Y-APP_Swe cells. D) ELISA immunoassays show that the ectopic overexpression of native CHOP (wt CHOP), but not the transcriptionally dead leucine zipper domain deficient mutant CHOP (CHOP LZ^–), increases the levels of the intracellular Aβ_1 - 42 species in the whole cell lysates and secreted Aβ_1 - 42 species in the conditioned media, from SH-SY5Y-APP_Swe cells. Data is expressed as Mean±S.D and includes determination made in three (n = 3) separate cell culture experiments. ^***p < 0.001 versus empty vector (EV)-transfected cells.