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, 34 (2), 540-50

Palmitate-activated Astrocytes via Serine Palmitoyltransferase Increase BACE1 in Primary Neurons by Sphingomyelinases

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Palmitate-activated Astrocytes via Serine Palmitoyltransferase Increase BACE1 in Primary Neurons by Sphingomyelinases

Li Liu et al. Neurobiol Aging.

Abstract

Astrocytes play a critical role in neurodegenerative diseases, including Alzheimer's disease (AD). Previously, we showed that saturated free fatty acid, palmitic acid (PA), upregulates β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) level and amyloidogenesis in primary rat neurons mediated by astrocytes. However, the molecular mechanisms by which conditioned media from PA-treated astrocytes upregulates BACE1 level in neurons are unknown. This study demonstrates that serine palmitoyltransferase (SPT) in the astrocytes increases ceramide levels, which enhances the release of cytokines that mediate the activation of neural and acidic sphingomyelinase (SMase) in the neurons, to propagate the deleterious effects of PA (i.e., BACE1 upregulation). In support of the relevance of SPT in AD, our laboratory recently measured and found SPT levels to be significantly upregulated in AD brains as compared with controls. Cytokines, namely tumor necrosis factor-α and interleukin-1β, released into the conditioned media of PA-treated astrocytes activate neural and acidic SMase in the neurons. Neutralizing the cytokines in the PA-treated astrocyte conditioned media reduced BACE1 upregulation. However, inhibiting SPT in the astrocytes decreased the levels of both tumor necrosis factor-α and interleukin-1β in the conditioned media, which in turn reduced the SMase activities and BACE1 level in primary neurons. Thus, our results suggest that the activation of the astrocytes by PA is mediated by SPT, and the activated astrocytes increases BACE1 level in the neurons; the latter is mediate by the SMases.

Figures

Fig. 1
Fig. 1. Changes in ceramide levels in primary rat neurons
Primary rat astrocytes were cultured with BSA (B) or palmitate (P) or palmitate plus L-CS (P+LCS) (LCS: SPT inhibitor) for 12hr. The conditioned media (CM-B, CM-P, or CM-P+LCS, respectively) were subsequently used to treat primary rat neurons for 12 or 24hr. Ceramide was detected by LC/MS/MS and normalized to CM-B (ctrl) (n=3) (A, B). (C) Primary rat neurons were treated with CM-B, CM-P, or pre and co-treated with L-CS (CM-P-LCS) for 12hr. Ceramide was detected by LC/MS/MS and normalized to CM-B (ctrl) (n=3). *: p<0.05, **: p<0.01, ***: p<0.001. A line indicates comparison between the two bars connected by the line.
Fig. 2
Fig. 2. Effect of CM on the SMase level in the neurons
(A, B) The conditioned media, CM-B, CM-P or CM-P+LCS, were used to treat neurons for 1, 6, 12 and 24hr and their mRNA levels were measured by real-time PCR. The mRNA of A-SMase (A) and N-SMase (B) were normalized to actin, then normalized to CM-B (ctrl) at each time point (n=3). A-SMase (C) and N-SMase (D) activities in neurons cultured in CM-B (ctrl), CM-P or CM-P+LCS for 12hr (n=3). *: p<0.05, **: p<0.01, ***: p<0.001. A line indicates comparison between the two bars connected by the line.
Fig. 3
Fig. 3. Effect of inhibiting SMase activities on the ceramide levels in the neurons
Ceramide levels of primary rat neurons cultured in CM-B (ctrl), CM-P, CM-P plus desipramine (A-SMase inhibitor, CM-P-A), CM-P plus GW4869 (N-SMase inhibitor, CM-P-N), or CM-P plus desipramine and GW4869 (CM-P-AN) for 12hr. Ceramide was detected by LC/MS/MS (n=3). *: p<0.05, **: p<0.01, ***: p<0.001. A line indicates comparison between the two bars connected by the line.
Fig. 4
Fig. 4. TNFα and IL-1β levels of astrocytes upon PA treatment
Astrocytes were treated with BSA (B), palmitate (P) or palmitate plus L-CS (P+LCS) for indicated time. (A) mRNA levels of TNFα and IL-1β were detected by real-time PCR (n=3). (B) Protein fold-change of TNFα and IL-1β. The protein levels of cytokines in the supernatant were measured by ELISA (n=3). *: p<0.05, **: p<0.01, ***: p<0.001. A line indicates comparison between the two bars connected by the line.
Fig. 5
Fig. 5. Neutralizing TNFα and IL-1β decrease SMase activity
A-SMase (A) or N-SMase (B) activities in neurons upon treatment with CM-B (ctrl), CM-P, CM-P-T (TNFα in CM-P media was neutralized with TNFα antibody), CM-P-I (IL-1β in CM-P media was neutralized with IL-1β antibody), or CM-P-TI (TNFα and IL-1β were neutralized in CM-P media) treatment for 12hr (n=3). *: p<0.05, **: p<0.01, ***: p<0.001. A line indicates comparison between the two bars connected by the line.
Fig. 6
Fig. 6. Effect of CM-P on BACE1 level in the neurons
Primary rat neurons were cultured with CM-B (ctrl) or CM-P for 6, 12 and 24hr. (A) Representative western blot result of BACE1 protein. (B) Quantification of western blot results (n=3). *: p<0.05, **: p<0.01, ***: p<0.001. A line indicates comparison between the two bars connected by the line.
Fig. 7
Fig. 7. Effect of inhibiting SMase on the BACE1 level in neurons upon CM treatment
(A) Representative western blot result of BACE1 protein levels in primary rat neurons cultured in CM-B (ctrl), CM-P, CM-P plus desipramine (A-SMase inhibitor, CM-P-A), CM-P plus GW4869 (N-SMase inhibitor, CM-P-N), or CM-P plus desipramine and GW4869 (CM-P-AN) for 12hr (n=3). (B) Quantification of western blot results (n=3). *: p<0.05, **: p<0.01, ***: p<0.001. A line indicates comparison between the two bars connected by the line.
Fig. 8
Fig. 8. Neutralizing TNFα and IL-1β decrease BACE1 in neurons
(A) BACE1 protein levels in neurons upon treatment with CM-B (ctrl), CM-P, CM-P-T (TNFα in CM-P media was neutralized with TNFα antibody), CM-P-I (IL-1β in CM-P media was neutralized with IL-1β antibody), or CM-P-TI (TNFα and IL-1β in CM-P media were neutralized) treatment for 12hr (n=3). (B) Quantification of western blot results (n=3). *: p<0.05, **: p<0.01, ***: p<0.001. A line indicates comparison between the two bars connected by the line.
Fig. 9
Fig. 9. Proposed cellular mechanism by which palmitic acid metabolism induces amyloidogenesis in primary neurons mediated by astrocytes
The astrocytes in the brain metabolize PA to generate ceramides through the de novo ceramide synthesis pathway by SPT, which initiates the release of soluble molecules, i.e. TNFα and IL-1β, from the astrocytes. In turn, these soluble molecules activate the SMase-ceramide pathway in the neurons to increase ceramide levels. Upregulated ceramide increase BACE1 level and enhance Aβ production (Ko and Puglielli, 2009; Puglielli et al., 2003). The increased extracellular Aβ level may act on both the astrocytes and the neurons to enhance the intracellular ceramide levels. This ceramide-Aβ-ceramide cascade creates a continuing cycle to further induce cell death (Jana and Pahan, 2010; Li et al., 2009).

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