Palmitate-activated astrocytes via serine palmitoyltransferase increase BACE1 in primary neurons by sphingomyelinases

Abstract

Astrocytes play a critical role in neurodegenerative diseases, including Alzheimer’s disease (AD). Previously, we showed that saturated free-fatty acid, palmitate (PA), upregulates 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 (N-SMase and A-SMase) in the neurons, to propagate the deleterious effects of palmitate, i.e. BACE1 upregulation. In support of the relevance of SPT in AD, our lab recently measure and found SPT levels to be significantly upregulated in AD brains as compared to controls (Geekiyanage and Chan, 2011). Cytokines, namely TNFα and IL-1β, released into the conditioned media of PA-treated astrocytes activate N-SMase and A-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 TNFα and IL-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.

Rapid Fine Conformational Epitope Mapping Using Comprehensive Mutagenesis and Deep Sequencing

“…The SMARTpoolsiRNAs and the transfection reagent Lipofectamine were diluted with Opti-MEM (Invitrogen) as described (33,34). The diluted SMARTpoolsiRNAs were mixed with RNAiMAX to form siRNA-RNAiMAX complexes.…”

“…Protein samples of 15-30 g were subjected to Western blot analysis as previously described (34,35) using TROP2 antibody (Abcam, Cambridge, MA), ␤-actin (Sigma), anti-mouse, and anti-rabbit HRP-conjugated secondary antibodies (Thermo Scientific) and donkey anti-goat IgG-HRP (Santa Cruz Biotechnology, Dallas, TX). The blots were visualized by SuperSignal West Femto maximum sensitivity substrate (Thermo Scientific).…”

Inflammation in Alzheimer’s Disease and Molecular Genetics: Recent Update

“…The inflammation can be acute or chronic. The inflammatory reaction that involves in most neurodegenerative diseases (Craft et al 2006;Liu et al 2013;Pizza et al 2011;Sudduth et al 2013;Varnum and Ikezu 2012), is often termed ''neuroinflammation''. Microglia, which is supposed to be the resident macrophages of the brain, and atrocities are the main cells that involve in this process.…”

“…Microglia, which is supposed to be the resident macrophages of the brain, and atrocities are the main cells that involve in this process. In the brains of both AD individuals and transgenic animal models, it was found that Ab plaques are surrounded by activated glial cells (Bauer et al 1991;Cagnin et al 2001;Fillit et al 1991;Liu et al 2013;Varnum and Ikezu 2012). Activated microglia and astrocytes strongly secrete inflammatory components such as pro-inflammatory cytokines, chemokines, complement, macrophage inflammatory proteins, monocyte chemoattractant proteins, reactive oxygen species (ROS), nitric oxide (NO) prostaglandins, leukotrienes, thromboxanes and so on (Akiyama et al 2000;Griffin et al 1998;Mrak et al 1995;Town et al 2005;Tuppo and Arias 2005).…”

“…Activated microglia and astrocytes strongly secrete inflammatory components such as pro-inflammatory cytokines, chemokines, complement, macrophage inflammatory proteins, monocyte chemoattractant proteins, reactive oxygen species (ROS), nitric oxide (NO) prostaglandins, leukotrienes, thromboxanes and so on (Akiyama et al 2000;Griffin et al 1998;Mrak et al 1995;Town et al 2005;Tuppo and Arias 2005). The released inflammatory molecules, especially some cytokines such as interleukin (IL)-18, IL-1b and tumor necrosis factor (TNF)-a, impair the balance of normal neurophysiologic condition that correlates with cognition and learning and memory (Fillit et al 1991;Gemma and Bickford 2007;Jankowsky and Patterson 1999;Liu et al 2013;Varnum and Ikezu 2012). These secreted inflammatory mediators, in turn, activate more microglia and astrocytes to produce inflammatory molecules.…”