Analysis of Seladin-1 role in apoptosis and cholesterol metabolism

Die Alzheimer Krankheit ist eine der häufigsten Formen von seniler Demenz bei älteren Menschen. Sie ist durch den Verlust von Gedächtnis und anderen kognitiven Funktionen gekennzeichnet, welcher wiederum auf den Verfall von Nervenzellen zurückzuführen ist. Bisher gibt es noch keine eindeutige Erklärung für diesen zahlreichen Zerfall der Neurone, doch scheint unter anderem die vermehrte Ablagerung von abnormalen Proteinen die Ursache hierfür zu sein. Diese bestehen teilweise aus extra- und intrazellulärem β-amyloid (Aβ), dem Spaltprodukt des Vorläuferproteins (APP). Aβ reichert sich im Krankheitsfall zu Plaques an und ist schädlich für die Nervenzellen. Zum anderen finden sich in Gehirnen von Alzheimer Patienten intrazelluläre Anreicherungen von so genannten "tangles", das sind hyperphosphorylierte Formen des Zellgerüst-assoziierten Proteins Tau. In ca. 10% der Alzheimer Fälle wird die Krankheit durch Genveränderungen ausgelöst. In dieser familiären Form der Erkrankung treten die klinischen Zeichen und neuropathologischen Veränderungen bereits in jüngeren Jahren auf. Der wichtigste Risikofaktor für die spontane Form der Alzheimer Krankheit ist eine Genvariante des Proteins Apolipoprotein E, welches am Transport von Cholesterin im Blut beteiligt ist. Tierstudien legen ebenfalls einen Zusammenhang zwischen hoher Cholesterin-Aufnahme und der Entwicklung einer Alzheimer Krankheit nahe. Cholesterin ist ein wichtiger Baustein von Zellmembranen. Innerhalb der Membran existieren flossartige Strukturen, die sogenannten "rafts", welche von Cholesterin zusammengehalten werden. Diese "rafts" spielen eine wichtige Rolle bei der Übermittlung von Signalen und werden ausserdem mit der Prozessierung von APP in Zusammenhang gebracht. Seladin-1 ist ein Protein aus der Familie der Flavin-Adenin-Dinukleotid-abhängigen Oxidoreduktasen und ein wesentliches Enzym im Cholesterinstoffwechsel. Seladin-1 wurde vor einigen Jahren im Rahmen einer Studie entdeckt, in welcher erkrankte Hirnregionen von Alzheimer Patienten mit gesunden Bereichen verglichen wurden. In den kranken Regionen ist das Expressionsniveau von Seladin-1 signifikant geringer, weshalb es auch der selektive Alzheimer's Disease Indikator-1 genannt wird. In der vorliegenden Studie wurde die Rolle von Seladin-1 während des programmierten Zelltods (Apoptose) und bezüglich des Cholesterinstoffwechsels näher untersucht. Wir konnten zeigen, dass die Überexpression von Seladin-1 Zellen resistenter gegen Apoptose macht und zu einer Zunahme der Cholesterinkonzentration in der Membran führt. Letzteres resultierte in einer massgeblichen Beeinflussung der Zusammensetzung und Funktion der "rafts". Ebenso konnten wir darlegen, dass höhere Seladin-1- und Cholesterinspiegel die Prozessierung von APP und damit die Aβ Produktion hemmen. Im Gegensatz dazu zeigten Seladin-1 defiziente Mäuse geringere Cholesterinkonzentrationen in der Membran und eine erhöhte Aβ Bildung. Diese Ergebnisse zeigen zum ersten Mal, dass die Modifizierung der Seladin-1 Expression, und damit einer endogenen Komponente, zu einer Veränderung der Cholesterinkonzentration führen kann und dass Seladin-1 ein essentieller Regulator der Zusammensetzung und der Funktion von "rafts" ist. Das Resultat, dass die Erhöhung von Seladin-1 die Aβ Bildung reduziert, weist Seladin-1 als möglichen therapeutischen Angriffspunkt in der Alzheimer Behandlung aus. Accumulation of amyloid-β peptides (Aβ) in the CNS is an invariant feature of the pathophysiology of Alzheimer's disease (AD), the most common form of dementia. Aβ peptides are derived from proteolytic cleavage of the amyloid precursor protein (APP) with the β-secretase cleaving at the N terminus and the γ-secretase(s) at the C terminus of Aβ peptides. Accumulation of Aβ in the brain has a fundamental role in AD-pathology including the induction of tau phosphorylation and neurofibrillary tangle formation, deficits in synaptic transmission and synaptotoxicity, and leads to age-dependent neuronal loss. Given all the above, concentrated effort has been focused on the identification of regulatory mechanisms that control APP cleavage and are involved in Aβ production, on proteases with the capacity to degrade Aβ, as well as on neuroprotective factors. A large body of literature indicates that alterations in cholesterol levels affect APP metabolism. Moreover, cholesterol is the major lipid constituent of the detergent resistant cholesterol-rich membrane domains (DRMs or rafts). The functional significance of DRM domains has been shown in cellular trafficking and in signaling events. The disorganization of DRM due to low cholesterol levels have been described in the hippocampus and cortex of a significant number of AD patients. In addition, DRMs are important to restrict APP β-cleavage and reduce Aβ production in primary neurons in culture. Consistently, these alterations also result in diminished activity of the Aβ degrading enzyme plasmin, which is normally produced in these domains. The majority of studies exploring the biological function of DRMs have been based on pharmacological approaches utilizing drugs that diminish cholesterol or sphingolipids, or based on modifications of DRM proteins in cultured cells. The functions of DRMs in living animals, however, have not yet been shown. It is especially not known how changes in cholesterol levels and distribution affect DRM dependent functions, such as APP processing in vivo. Seladin-1 (the selective Alzheimer's disease indicator-1), encoded by a single gene (DHCR24) on chromosome 1, is an evolutionary conserved gene whose product catalyzes the reduction of the Δ24 double bond of sterol intermediates leading to cholesterol production. It was shown that seladin-1 levels are lower in affected neurons in AD, suggesting that seladin-1 levels may influence the selective vulnerability of neurons in AD. Overexpression of seladin-1 in vitro protected cells from apoptosis induced by oxidative stress and high expression of endogenous seladin-1 was associated with resistance against Aβ-induced toxicity. Moreover, functional expression of seladin-1 resulted in the inhibition of caspase 3 activation after either Aβ- mediated toxicity or oxidative stress and protected the cells from apoptotic cell death. Deficiency in the Dhcr24 gene causes a severe autosomal recessive disorder characterized by elevated levels of the cholesterol precursor desmosterol in plasma. Seladin-1 knock-out mice are viable, although almost no cholesterol was detected in plasma and tissue of these animals. This study gives insight into the role of seladin-1 in neuroprotection, regulation of cholesterol levels and cholesterol-mediated functions in vitro and in vivo. We show that seladin-1 expression modulates APP processing and Aβ generation in vivo. We substantiated the anti-apoptotic function of seladin-1 in human neuroblastoma cells and show for the first time that elevated seladin-1 expression increases the levels of cellular and membrane cholesterol and therefore affects the distribution and function of rafts in these cells. Furthermore, we demonstrate that the overexpression of seladin-1 leads to reduced amyloidogenic APP processing and decreased generation of the Aβ peptide. In contrast, decreased seladin-1 expression in mouse brain resulted in lower loss participates in the pathogenesis of AD. Because of its role in cholesterol synthesis and neuroprotection, increasing seladin-1 activity in CNS neurons may therefore represent a putative therapeutical target for AD treatment

RETRACTED ARTICLE: Age-dependent Increase in Desmosterol Restores DRM Formation and Membrane-related Functions in Cholesterol-free DHCR24−/− Mice

Cholesterol is a prominent modulator of the integrity and functional activity of physiological membranes and the most abundant sterol in the mammalian brain. DHCR24-knock-out mice lack cholesterol and accumulate desmosterol with age. Here we demonstrate that brain cholesterol deficiency in 3-week-old DHCR24−/− mice was associated with altered membrane composition including disrupted detergent-resistant membrane domain (DRM) structure. Furthermore, membrane-related functions differed extensively in the brains of these mice, resulting in lower plasmin activity, decreased β-secretase activity and diminished Aβ generation. Age-dependent accumulation and integration of desmosterol in brain membranes of 16-week-old DHCR24−/− mice led to the formation of desmosterol-containing DRMs and rescued the observed membrane-related functional deficits. Our data provide evidence that an alternate sterol, desmosterol, can facilitate processes that are normally cholesterol-dependent including formation of DRMs from mouse brain extracts, membrane receptor ligand binding and activation, and regulation of membrane protein proteolytic activity. These data indicate that desmosterol can replace cholesterol in membrane-related functions in the DHCR24−/− mous

Age-dependent Increase in Desmosterol Restores DRM Formation and Membrane-related Functions in Cholesterol-free DHCR24−/− Mice

Cholesterol is a prominent modulator of the integrity and functional activity of physiological membranes and the most abundant sterol in the mammalian brain. DHCR24-knock-out mice lack cholesterol and accumulate desmosterol with age. Here we demonstrate that brain cholesterol deficiency in 3-week-old DHCR24−/− mice was associated with altered membrane composition including disrupted detergent-resistant membrane domain (DRM) structure. Furthermore, membrane-related functions differed extensively in the brains of these mice, resulting in lower plasmin activity, decreased β-secretase activity and diminished Aβ generation. Age-dependent accumulation and integration of desmosterol in brain membranes of 16-week-old DHCR24−/− mice led to the formation of desmosterol-containing DRMs and rescued the observed membrane-related functional deficits. Our data provide evidence that an alternate sterol, desmosterol, can facilitate processes that are normally cholesterol-dependent including formation of DRMs from mouse brain extracts, membrane receptor ligand binding and activation, and regulation of membrane protein proteolytic activity. These data indicate that desmosterol can replace cholesterol in membrane-related functions in the DHCR24−/− mous

RETRACTED ARTICLE: Age-dependent Increase in Desmosterol Restores DRM Formation and Membrane-related Functions in Cholesterol-free DHCR24−/− Mice

Cholesterol is a prominent modulator of the integrity and functional activity of physiological membranes and the most abundant sterol in the mammalian brain. DHCR24-knock-out mice lack cholesterol and accumulate desmosterol with age. Here we demonstrate that brain cholesterol deficiency in 3-week-old DHCR24−/− mice was associated with altered membrane composition including disrupted detergent-resistant membrane domain (DRM) structure. Furthermore, membrane-related functions differed extensively in the brains of these mice, resulting in lower plasmin activity, decreased β-secretase activity and diminished Aβ generation. Age-dependent accumulation and integration of desmosterol in brain membranes of 16-week-old DHCR24−/− mice led to the formation of desmosterol-containing DRMs and rescued the observed membrane-related functional deficits. Our data provide evidence that an alternate sterol, desmosterol, can facilitate processes that are normally cholesterol-dependent including formation of DRMs from mouse brain extracts, membrane receptor ligand binding and activation, and regulation of membrane protein proteolytic activity. These data indicate that desmosterol can replace cholesterol in membrane-related functions in the DHCR24−/− mouse

Prosurvival Effect of DHCR24/Seladin-1 in Acute and Chronic Responses to Oxidative Stress▿

DHCR24/seladin-1, a crucial enzyme in sterol synthesis, is of lower abundance in brain areas affected by Alzheimer's disease. While high levels of DHCR24/seladin-1 exert antiapoptotic function by conferring resistance against oxidative stress, the molecular mechanism for this protective effect is not fully understood. Here we show that DHCR24/seladin-1 expression is up-regulated in an acute response and down-regulated in a chronic response to oxidative stress. High levels of DHCR24/seladin-1 were associated with elevated cholesterol concentrations and a general increase in cholesterol biosynthesis upon oxidative stress exposure in neuroblastoma SH-SY5Y cells. DHCR24/seladin-1 overexpression conferred resistance to oxidative stress in a cholesterol-dependent manner. Mutating the reductase activity within DHCR24/seladin-1 abolished this protective effect. Conversely, DHCR24/seladin-1 levels diminished upon chronic exposure to oxidative stress. Low levels of DHCR24/seladin-1 were associated with reduced p53 levels, independent of DHCR24 activity and cholesterol concentrations. Additionally, ablation of DHCR24/seladin-1 prevented apoptosis of primary neurons in a p53-dependent manner and reduced the response of critical p53 targets due to deficient stabilization of p53 and therefore elevated p53 ubiquitination and degradation. Our findings reveal a dual capacity of DHCR24/seladin-1, which appears to be involved in two mechanistically independent prosurvival effects, exerting an acute response and a chronic response to oxidative stress

The role of seladin-1/DHCR24 in cholesterol biosynthesis, APP processing and Aβ generation in vivo

The cholesterol-synthesizing enzyme seladin-1, encoded by the Dhcr24 gene, is a flavin adenine dinucleotide-dependent oxidoreductase and regulates responses to oncogenic and oxidative stimuli. It has a role in neuroprotection and is downregulated in affected neurons in Alzheimer's disease (AD). Here we show that seladin-1-deficient mouse brains had reduced levels of cholesterol and disorganized cholesterol-rich detergent-resistant membrane domains (DRMs). This was associated with inefficient plasminogen binding and plasmin activation, the displacement of β-secretase (BACE) from DRMs to APP-containing membrane fractions, increased β-cleavage of APP and high levels of Aβ peptides. In contrast, overexpression of seladin-1 increased both cholesterol and the recruitment of DRM components into DRM fractions, induced plasmin activation and reduced both BACE processing of APP and Aβ formation. These results establish a role of seladin-1 in the formation of DRMs and suggest that seladin-1-dependent cholesterol synthesis is involved in lowering Aβ levels. Pharmacological enhancement of seladin-1 activity may be a novel Aβ-lowering approach for the treatment of AD