DHCR24 Knockdown Induces Tau Hyperphosphorylation at Thr181, Ser199, Ser262, and Ser396 Sites via Activation of the Lipid Raft-Dependent Ras/MEK/ERK Signaling Pathway in C8D1A Astrocytes

Abstract

The synthetase 3β-hydroxysterol-Δ24 reductase (DHCR24) is a key regulator involved in cholesterol synthesis and homeostasis. A growing body of evidence indicates that DHCR24 is downregulated in the brain of various models of Alzheimer's disease (AD), such as astrocytes isolated from AD mice. For the past decades, astrocytic tau pathology has been found in AD patients, while the origin of phosphorylated tau in astrocytes remains unknown. A previous study suggests that downregulation of DHCR24 is associated with neuronal tau hyperphosphorylation. Herein, the present study is to explore whether DHCR24 deficiency can also affect tau phosphorylation in astrocytes. Here, we showed that DHCR24 knockdown could induce tau hyperphosphorylation at Thr181, Ser199, Thr231, Ser262, and Ser396 sites in C8D1A astrocytes. Meanwhile, we found that DHCR24-silencing cells had reduced the level of free cholesterol in the plasma membrane and intracellular organelles, as well as cholesterol esters. Furthermore, reduced cellular cholesterol level caused a decreased level of the caveolae-associated protein, cavin1, which disrupted lipid rafts/caveolae and activated rafts/caveolae-dependent Ras/MEK/ERK signaling pathway. In contrast, overexpression of DHCR24 prevented the overactivation of Ras/MEK/ERK signaling by increasing cellular cholesterol content, therefore decreasing tau hyperphosphorylation in C8D1A astrocytes. Herein, we firstly found that DHCR24 knockdown can lead to tau hyperphosphorylation in the astrocyte itself by activating lipid raft-dependent Ras/MEK/ERK signaling, which might contribute to the pathogenesis of AD and other degenerative tauopathies.

Keywords: Alzheimer’s disease; Astrocyte; Cholesterol; DHCR24; Tau hyperphosphorylation.

Fig. 1

DHCR24 low expression induced tau hyperphosphorylation at specific sites in C8D1A astrocytes. a, b Expression of DHCR24 protein was measured by Western blot of cell lysates from DHCR24 low expression (LE) and overexpression (OE) group, compared with blank control (Blank, BL) and vector groups (Negative control, NC1 or NC2). c Expression of DHCR24 mRNA was verified by quantitative PCR. The values are shown as the means ± SEM, n = 3 per group. d Representative images of Western blot analysis of phosphorylated tau. Phosphorylation of tau at Thr181, Ser199, Thr231, Ser262, and Ser396 sites was measured among all groups. e, f, g, h, i One-way ANOVA analysis of tau phosphorylation level at Thr181, Ser199, Thr231, Ser262 and Ser396 after DHCR24 knockdown or knock-in. The values are shown as the means ± SEM, n = 4 per group, One-way ANOVA, *P < 0.05; **P < 0.01; compared with blank control (blank, BL) and vector groups (negative control, NC1 or NC2)

Fig. 2

DHCR24 downregulation decreased cellular free cholesterol level in C8D1A astrocytes. a Filipin III staining of whole cell free cholesterol. Cells from DHCR24 low expression (LE) and overexpression (OE) group, also the blank control (Blank, BL) and vector groups (Negative control, NC1 or NC2) were stained with filipin III, which could label whole cell free cholesterol, including cholesterol in the plasma membrane. b Filipin III staining of intracellular pool of free cholesterol. Before the staining, cells were treated with MβCD for 30 min, which could delete plasma membrane cholesterol to allow the intracellular pool of cholesterol visible. Filipin III (Blue), PI (Red), Magnification: 63 × , Scale bar: 10 µm. The images were captured at random and analysis of filipin fluorescent intensity was shown in (c) (whole cell free cholesterol) and (d) (intracellular free cholesterol). One-way ANOVA, *P < 0.05; **P < 0.01; compared with blank control (blank, BL) and vector groups (negative control, NC1 or NC2)

Fig. 3

DHCR24 low expression enhanced the transcription of cholesterol key synthetases in C8D1A astrocytes. a, b, c, d qPCR analysis of the mRNA level of several key cholesterol synthetases, such as SREBP2, SQLE, HMGCR and DHCR7, in DHCR24 low expression (LE) and overexpression (OE) cells. The values are shown as the means ± SEM, n = 3, One-way ANOVA analysis, *P < 0.05; **P < 0.01; compared with control groups (BL, NC1 and NC2)

Fig. 4

DHCR24 downregulation reduced cholesterol esterification and total cholesterol content in C8D1A astrocytes. a Transcription level of ACAT was measured by qPCR analysis. The values are shown as the means ± SEM, n = 3 per group. b Representative images of Oil Red O staining of lipid droplets of astrocytes from blank control group (Blank, BL) and vector groups (Negative control, NC1 or NC2), DHCR24 low expression (LE) and overexpression (OE) groups. Magnification: 60 × , Scale bar: 20 µm. The images were captured at random, and the average number of lipid droplets from each group was analyzed using Image Pro Plus 6.0 version. One-way ANOVA analysis of the results was shown at (c). d Quantification of total cholesterol content. One-way ANOVA analysis, *P < 0.05; **P < 0.01; compared with control groups (BL, NC1 and NC2)

Fig. 5

DHCR24 downregulation disrupted caveolae and activated Ras/MEK/ERK signaling pathway in C8D1A astrocytes. a Expression of cavin1 was measured by Western blot of cell lysates from DHCR24 DHCR24 low expression (LE) and overexpression (OE) groups, n = 4 per group. One-way ANOVA analysis of cavin1 expression level was shown in (b). The values are shown as the means ± SEM, n = 4 for each group. c Ras, total MEK (MEK), phosphorylated MEK (p-MEK), total ERK1/2 (ERK), and phosphorylated ERK1/2 (p-ERK) protein levels were measured by Western blot. One-way ANOVA analysis of their expression among all groups is shown in (d, e, f), n = 4 per group. g Representative images of immunofluorescence staining of p-ERK (Red); Magnification: 20 × , Scale bar: 50 µm. The images were captured at random and analysis of p-ERK fluorescent intensity was shown in (h). One-way ANOVA, *P < 0.05; **P < 0.01; compared with blank control (blank, BL) and vector groups (negative control, NC1 or NC2)

Fig. 6

Inhibition of MEK/ERK signaling reduced tau hyperphosphorylation in DHCR24 knockdown C8D1A astrocytes. a Western blot analysis was applied to assess the protein level of phosphorylated ERK (p-ERK) and phosphorylated tau. DHCR24 knockdown cells were cultured with MEK inhibitor U0126. After 24 h, the phosphorylation level of tau at Thr181, Ser199, Ser262, Thr231, and Ser396 sites were verified. b, c, d, e, f One-way ANOVA analysis of Western blot results of p-ERK and phosphorylated tau. The values are shown as the means ± SEM, n = 4 for each group. *P < 0.05; **P < 0.01; compared with blank control (0 µM)

Fig. 7

Decreased cellular cholesterol by DHCR24 knockdown contributes to astrocytic tau pathology in C8D1A cells. DHCR24 knockdown in C8D1a astrocytes leads to inhibition of cholesterol synthesis and deficiency of cholesterol in the plasma membrane. Meanwhile, reduced membrane cholesterol decreases the cavin-1 expression level, resulting in the disruption of caveolae. Moreover, the impairment of caveolae construction leads to the altered nanoclustering of Ras that locates in this platform and causes its overactivation, then the MAPK/ERK signaling pathway is activated. Eventually, activated ERK1/2 promotes tau hyperphosphorylation