Seladin‐1/DHCR24 protects neuroblastoma cells against Aβ toxicity by increasing membrane cholesterol content

Research Center on the Molecular Basis of Neurodegeneration, University of Florence, Florence, Italy
Journal of Cellular and Molecular Medicine (Impact Factor: 4.01). 01/2008; 12(5b):1990 - 2002. DOI: 10.1111/j.1582-4934.2008.00216.x


The role of brain cholesterol in Alzheimer's disease (AD) is currently a matter of debate. Experimental evidence suggests that reducing circulating and brain cholesterol protects against AD, however recent data indicate that low membrane cholesterol results in neurode-generation and that the cholesterol synthesis catalyst seladin-1 is down-regulated in AD-affected brain regions. We previously reported a significant correlation between resistance to amyloid toxicity and content of membrane cholesterol in differing cultured cell types. Here we provide evidence that Aβ42 pre-fibrillar aggregates accumulate more slowly and in reduced amount at the plasma membrane of human SH-SY5Y neuroblastoma cells overexpressing seladin-1 or treated with PEG-cholesterol than at the membrane of control cells. The accumulation was significantly increased in cholesterol-depleted cells following treatment with the specific seladin-1 inhibitor 5,22E-cholestadien-3-ol or with methyl-β-cyclodextrin. The resistance to amyloid toxicity and the early cytosolic Ca2+ rise following exposure to Aβ42 aggregates were increased and prevented, respectively, by increasing membrane cholesterol whereas the opposite effects were found in cholesterol-depleted cells. These results suggest that seladin-1-dependent cholesterol synthesis reduces membrane-aggregate interaction and cell damage associated to amyloid-induced imbalance of cytosolic Ca2+. Our findings extend recently reported data indicating that seladin-1 overexpression directly enhances the resistance to Aβ toxicity featuring seladin-1/DHCR 24 as a possible new susceptibility gene for sporadic AD.

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Available from: Anna Pensalfini, Sep 08, 2014
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    • "Thus, cholesterol in the neuron is responsible for maintenance of neural function [25]. Seladin-1 protects the neuron by increasing the intracellular cholesterol synthesis [21], [22], [26]. In a previous report, TH administration to some neuronal precursor cell lines induced Seladin-1 gene expression [27]. "
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    ABSTRACT: Selective Alzheimer's disease (AD) indicator 1 (Seladin-1) has been identified as a gene down-regulated in the degenerated lesions of AD brain. Up-regulation of Seladin-1 reduces the accumulation of β-amyloid and neuronal death. Thyroid hormone (TH) exerts an important effect on the development and maintenance of central nervous systems. In the current study, we demonstrated that Seladin-1 gene and protein expression in the forebrain was increased in thyrotoxic mice compared with that of euthyroid mice. However, unexpectedly, no significant decrease in the gene and protein expression was observed in hypothyroid mice. Interestingly, an agonist of liver X receptor (LXR), TO901317 (TO) administration in vivo increased Seladin-1 gene and protein expression in the mouse forebrain only in a hypothyroid state and in the presence of mutant TR-β, suggesting that LXR-α would compensate for TR-β function to maintain Seladin-1 gene expression in hypothyroidism and resistance to TH. TH activated the mouse Seladin-1 gene promoter (-1936/+21 bp) and site 2 including canonical TH response element (TRE) half-site in the region between -159 and -154 bp is responsible for the positive regulation. RXR-α/TR-β heterodimerization was identified on site 2 by gel-shift assay, and chromatin immunoprecipitation assay revealed the recruitment of TR-β to site 2 and the recruitment was increased upon TH administration. On the other hand, LXR-α utilizes a distinct region from site 2 (-120 to -102 bp) to activate the mouse Seladin-1 gene promoter. Taking these findings together, we concluded that TH up-regulates Seladin-1 gene expression at the transcriptional level and LXR-α maintains the gene expression.
    PLoS ONE 01/2013; 8(1):e54901. DOI:10.1371/journal.pone.0054901 · 3.23 Impact Factor
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    • "Allegedly, this specific binding to ganglioside results in the alteration of Aβ secondary structure, leading to the formation of amyloid fibrils [15, 35]. The assembly of higher Aβ aggregates to the formation of amyloid fibrils in these microdomains has been subjected to intense studies [12, 13, 35–37] which suggest that lipid molecules in the surface membrane, such as cholesterol and sphingomyelin, are involved in the process [12, 15]. The fluorescent analysis performed in this investigation reveals the positive impact of the concentration of cholesterol in the membrane on the extent of Aβ binding to the membrane surface region. "
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    ABSTRACT: We performed a fluorescent analysis of the binding of Aβ to the surface membrane of different types of cells lines such as PC12, GT1-7, and ex vivo neurons. Analyses were performed on sorted cells with membrane bound Aβ Competitive binding between Aβ phosphatidyl serine- (PtdSer-) specific binder annexin V and an anti-PtdSer antibody provided compelling data confirming the involvement of PtdSer as one of the surface membrane signal molecules for Aβ. We found that populations of cells that exhibited high surface membrane binding affinity for Aβ also show higher membrane cholesterol levels compared to cells that did not bind Aβ. This direct relationship was upheld in cholesterol-enriched or cholesterol-depleted cell membranes. We conclude that the initial process for the cell-selective binding by Aβ, to later conversion of elemental Aβ units into larger structures such as fibrils or to the potentially toxic ion channel aggregates, is highly influenced by the membrane content of PtdSer and cholesterol in the cell surface membrane.
    International Journal of Alzheimer's Disease 06/2011; 2011(7):917629. DOI:10.4061/2011/917629
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    • "At various time intervals aliquots were centrifuged, dried under N2 to remove TFE or TFA when necessary, dissolved in DMEM without phenol red, and immediately added to SH-SY5Y cell culture media for 24 h at a final YHKB concentration of 2 µM. Aggregate cytotoxicity was assessed in 96-well plates by the MTT assay (Sigma, Milan, Italy), as previously reported [26]. Briefly, after the exposure to 2 µM YHKB aggregates for 24 h at 37°C, the cell cultures were incubated with 0.5 mg/ml MTT solution at 37°C for 4 h and with cell lysis buffer (20% SDS, 50% N,N-dimethylformamide, pH 4.7) for 3 hours. "
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    ABSTRACT: The assembly of soluble proteins into ordered fibrillar aggregates with cross-β structure is an essential event of many human diseases. The polypeptides undergoing aggregation are generally small in size. To explore if the small size is a primary determinant for the formation of amyloids under pathological conditions we have created two databases of proteins, forming amyloid-related and non-amyloid deposits in human diseases, respectively. The size distributions of the two protein populations are well separated, with the systems forming non-amyloid deposits appearing significantly larger. We have then investigated the propensity of the 486-residue hexokinase-B from Saccharomyces cerevisiae (YHKB) to form amyloid-like fibrils in vitro. This size is intermediate between the size distributions of amyloid and non-amyloid forming proteins. Aggregation was induced under conditions known to be most effective for amyloid formation by normally globular proteins: (i) low pH with salts, (ii) pH 5.5 with trifluoroethanol. In both situations YHKB aggregated very rapidly into species with significant β-sheet structure, as detected using circular dichroism and X-ray diffraction, but a weak Thioflavin T and Congo red binding. Moreover, atomic force microscopy indicated a morphology distinct from typical amyloid fibrils. Both types of aggregates were cytotoxic to human neuroblastoma cells, as indicated by the MTT assay. This analysis indicates that large proteins have a high tendency to form toxic aggregates, but low propensity to form regular amyloid in vivo and that such a behavior is intrinsically determined by the size of the protein, as suggested by the in vitro analysis of our sample protein.
    PLoS ONE 01/2011; 6(1):e16075. DOI:10.1371/journal.pone.0016075 · 3.23 Impact Factor
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