ABSTRACTHyperphosphorylated isoforms of the microtubule-associated protein tau are the majorcomponents of neurofibrillary lesions in Alzheimer’s disease (AD). Protein phosphatase2A (PP2A) is a major phosphatase implicated in tau dephosphorylation in vitro .Dephosphorylation of tau can be blocked in vivo by okadaic acid, a potent inhibitor ofPP2A. Moreover, activity of PP2A is reduced in AD brains. To elucidate the role ofPP2A in tau phosphorylation and pathogenesis, we expressed a dominant negativemutant form of the catalytic subunit Cα of PP2A, L199P, in mice by using a neuron-specific promoter. We obtained mice with high expression levels of C α L199P incortical, hippocampal and cerebellar neurons. PP2A activity in brain homogenates oftransgenic mice was reduced to 66%. Endogenous tau protein washyperphosphorylated at distinct sites including the AT8 epitope S202/T205, a major AD-associated tau phospho-epitope. AT8-positive tau aggregates accumulated in the somaand dendrites of cortical pyramidal cells and cerebellar Purkinje cells, and colocalizedwith ubiquitin. Our data establish that PP2A plays a crucial role in tau phosphorylation.Our results show also that reduced PP2A activity is associated with alteredcompartmentalization and ubiquitination of tau, resembling a key pathological finding inAD.
Die Alzheimer Krankheit ist eine der haufigsten Formen von seniler Demenz bei alteren Menschen. Sie ist durch den Verlust von Gedachtnis und anderen kognitiven Funktionen gekennzeichnet, welcher wiederum auf den Verfall von Nervenzellen zuruckzufuhren ist. Bisher gibt es noch keine eindeutige Erklarung fur diesen zahlreichen Zerfall der Neurone, doch scheint unter anderem die vermehrte Ablagerung von abnormalen Proteinen die Ursache hierfur zu sein. Diese bestehen teilweise aus extra- und intrazellularem β-amyloid (Aβ), dem Spaltprodukt des Vorlauferproteins (APP). Aβ reichert sich im Krankheitsfall zu Plaques an und ist schadlich fur die Nervenzellen. Zum anderen finden sich in Gehirnen von Alzheimer Patienten intrazellulare Anreicherungen von so genannten tangles, das sind hyperphosphorylierte Formen des Zellgerust-assoziierten Proteins Tau. In ca. 10% der Alzheimer Falle wird die Krankheit durch Genveranderungen ausgelost. In dieser familiaren Form der Erkrankung treten die klinischen Zeichen und neuropathologischen Veranderungen bereits in jungeren Jahren auf. Der wichtigste Risikofaktor fur 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 spielen eine wichtige Rolle bei der Ubermittlung 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-abhangigen 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 wahrend des programmierten Zelltods (Apoptose) und bezuglich des Cholesterinstoffwechsels naher untersucht. Wir konnten zeigen, dass die Uberexpression von Seladin-1 Zellen resistenter gegen Apoptose macht und zu einer Zunahme der Cholesterinkonzentration in der Membran fuhrt. Letzteres resultierte in einer massgeblichen Beeinflussung der Zusammensetzung und Funktion der rafts. Ebenso konnten wir darlegen, dass hohere Seladin-1- und Cholesterinspiegel die Prozessierung von APP und damit die Aβ Produktion hemmen. Im Gegensatz dazu zeigten Seladin-1 defiziente Mause geringere Cholesterinkonzentrationen in der Membran und eine erhohte Aβ Bildung. Diese Ergebnisse zeigen zum ersten Mal, dass die Modifizierung der Seladin-1 Expression, und damit einer endogenen Komponente, zu einer Veranderung der Cholesterinkonzentration fuhren kann und dass Seladin-1 ein essentieller Regulator der Zusammensetzung und der Funktion von ist. Das Resultat, dass die Erhohung von Seladin-1 die Aβ Bildung reduziert, weist Seladin-1 als moglichen 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 D24 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.
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.
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 beta-secretase activity and diminished Abeta 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.
Accumulation in brain of the β‐amyloid peptide (Aβ) is considered as crucial pathogenic event causing Alzheimer's disease (AD). Anti‐Aβ immune therapy is a powerful means for Aβ clearance from the brain. We recently showed that intravenous injections of anti‐Aβ antibodies led to reduction, elevation or no change in brain Aβ 42 concentrations of an AD mouse model. We report here, in a second passive immunization protocol, a different bioactivity of same antibodies to alter brain Aβ 42 concentrations. Comparing the bioactivity of anti‐Aβ antibodies in these two passive immunization paradigms underscores the potential of immune therapy for AD treatment and suggests that both the epitope recognized by the antibody and the mode of antibody administration are crucial for its biological activity.
The accumulation of the β-amyloid peptide (Aβ) is a central event in the pathogenesis of Alzheimer’s disease (AD). Aβ removal from the brain by immune therapy shows promising potential for the treatment of patients with AD, although the mechanisms of the antibody action are incompletely understood. In this study we compared the biological activities of antibodies raised against various Aβ fragments for Aβ reduction in vitro and in vivo. Antibodies against Aβ enhanced the uptake of Aβ<sub>42</sub> aggregates up to 6-fold by primary microglial cells in vitro. The kinetics of Aβ<sub>42</sub> uptake varied considerably among antibodies. Based on the activity to mediate Aβ<sub>42</sub> uptake by microglial cells, we identified a bioactive antibody that significantly reduced Aβ<sub>42</sub> levels in the brains of transgenic mice with neuronal expression of an AD-related mutated amyloid precursor protein. This effect depended on the epitopes recognized by the antibody. Our data suggest that the ability to facilitate Aβ<sub>42</sub> uptake by primary microglia cells in vitro can be used to predict the biological activity of the antibody by passive immunization in vivo. This protocol may prove useful for the rapid validation of the activity of antibodies designed to be used in immune therapy of AD.
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.
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