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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. [ABSTRACT FROM AUTHOR]

Accumulating data suggest that the downregulation of DHCR24 is linked to the pathological risk factors of AD, denoting a potential role of DHCR24 in AD pathogenesis. However, it remains unclear whether the downregulation of DHCR24 affects the abnormal heper-phosphorylation of tau protein, which is involved in tauopathy. In present papers, immunofluorescence and Filipin III fluorescence results showed that DHCR24 knockdown significantly lowered the level of plasma membrane cholesterol and expression level of membrane lipid-raft structural protein caveolin-1; and overexpression of DHCR24 could increase the plasma membrane cholesterol levels and facilitating caveolae structure through increase the expression of caveolin-1. PP2A is the key phosphatase involving in tau phosphorylation, which is localized in cholesterol-dependent caveola/raft lipid domains. Here, the PP2A activity was detected by western blot assay. Interestingly, the level of p-PP2Ac at Y307 (inactive) and p-GSK3β at Y216 (active) in the downstream of the PP2A signal pathway were both significantly increased in silencing DHCR24 SH-SY5Y cells, which denoted an inhibition of the PP2A and activation of GSK3β signaling. Conversely, overexpression of DHCR24 blunted the inhibition effect of PP2A and activation of GSK3β. Besides, in the SH-SY5Y cell lines we demonstrated that DHCR24 knockdown obviously induced hyperphosphorylation of tau at Thr181, Thr231, Ser262, Ser396, and Ser422 Sites. In contrast, DHCR24 overexpression protects neuronal SH-SY5Y cells against the hyperphosphorylation of tau at Thr181, Thr231, Ser262, Ser396, and Ser422 Sites. Furthermore, PP2A activator D-erythro-Sphingosine (DES) also obviously inhibited the hyperphosphorylation of tau induced by DHCR24 knockdown. Collectively, our findings firstly confirmed that DHCR24 knockdown obviously induced abnormal hyperphosphorylation of tau by a novel lipid raft-dependent PP2A signaling. We propose that DHCR24 downregulation led to altered cholesterol synthesis as a potential mechanism in the progression of tau hyperphosphorylation involving in AD and other tauopathies. [ABSTRACT FROM AUTHOR]

Various useful animal models, such as Alzheimer's disease and Niemann-Pick disease, were provided by U18666A. However, the pathogenesis of U18666A-induced diseases, including U18666A-mediated apoptosis, remains incompletely elucidated, and therapeutic strategies are still limited. Dihydrotestosterone (DHT) has been reported to contribute to the prevention and treatment of neurodegenerative disorders. Our study investigated the neuroprotective activity of DHT in U18666A-related diseases. Apoptosis of C6 cells was detected by Hoechst 33258 fluorescent staining and flow cytometry with annexin V-FITC/PI dual staining. Cell viability was assessed using Cell Counting Kit-8. Expression of apoptosis-related proteins, such as Akt, seladin-1, Bcl-2 family proteins, and caspase-3, was determined using Western blot. Our results demonstrated that the apoptotic rate of C6 cells significantly increased after U18666A addition, but was remarkably reduced after DHT treatment. Pretreatment with DHT attenuated U18666A-induced cell viability loss. PI3K inhibitor LY294002 could suppress DHT anti-apoptotic effect. Furthermore, we discovered that U18666A could significantly downregulate seladin-1 expression in a dose-dependent manner, but no significant change was observed in Bcl-xL, Bax, and P-Akt protein expressions. Compared with U18666A-treated group, the expression of P-Akt, seladin-1, and Bcl-xL significantly increased, and the expression of Bax and caspase-3 remarkably reduced after DHT treatment. However, in the presence of LY294002, the effect of DHT was reversed. In conclusion, we found that seladin-1 may take part in U18666A-induced apoptosis. DHT may inhibit U18666A-induced apoptosis by regulating downstream apoptosis-related proteins including seladin-1, caspase-3, Bcl-xL, and Bax through activation of the PI3K/Akt signal pathway. [ABSTRACT FROM AUTHOR]

Previous evidences indicate that androgen is neuroprotective in the brain. However, the underling mechanisms remain to be fully elucidated. Moreover, it is controversial whether dihydrotestosterone (DHT) modulates the expression of apoptosis-related effectors, such as survivin, XIAP, bax, and bcl-xl proteins mediated by the PI3-K/Akt pathway, which contributes to androgen neuroprotection. In this study using a C6 glial cell model, apoptotic cells were detected by flow cytometry. Akt, seladin-1, survivin, XIAP, bcl-xl, and bax protein expression is investigated by Western blot. After amyloid β-protein fragment (Aβ25-35) treatment, apoptotic cells at early (annexin V+, PI−) and late (annexin V+, PI+) stages were significantly increased. Apoptosis at early and late was obviously inhibited in the presence of DHT. The effect of DHT was markedly blocked by PI3-K inhibitor LY294002.To elicit the mechanism of DHT protection, the expression of seladin-1, survivin, XIAP, bax, and bcl-xl protein was determined in C6 cells treated with Aβ25-35, DHT, or LY294002. Aβ25-35 significantly downregulated the expression of seladin-1, survivin, XIAP, bcl-xl protein and upregulated the expression of bax protein. DHT significantly inhibited the expression of bax, seladin-1, survivin, XIAP, and bcl-xl protein induced by Aβ25-35. Further, we found the effect of DHT was significantly inhibited by LY294002. Collectively, in a C6 glial cell model, we firstly found that DHT inhibits Aβ25-35-induced apoptosis by a rapid nongenic PI-3K/Akt activation as well as regulation of seladin-1, survivin, XIAP, bcl-xl, and bax proteins. [ABSTRACT FROM AUTHOR]