![Index [] · for optimal power flow problem, 197–198 outer approximation technique, 170–171, 198–202, 277 piecewise-linear, 283–284 for pooling problem, 213–214 power, 446](https://static.fdocument.org/doc/165x107/5f2e37a71f0f5041eb09ed7c/index-for-optimal-power-iow-problem-197a198-outer-approximation-technique.jpg)
P4-198: Molecular studies of the presenilin protein interactions to γ-secretase inhibitors and...
1
P4-196 PRESENILIN IN NEURONAL SURVIVAL AND NEURODEGENERATION Verena Kallhoff-Munoz, Lingyun Hu, Robia Pautler, Hui Zheng, Baylor College of Medicine, Houston, TX, USA. Contact e-mail: [email protected] Background: Presenilins (PS) have been shown to exhibit -secretase dependent and independent activities, the former requires the D257 site and the latter is mediated through the hydrophilic loop domain encoded by exon 10. Mutations in presenilin genes (PS1 and PS2) lead to early-onset of Alzheimer’s disease (AD), and increasing evidence indicates that these mutations lead to a partial loss of function for its physiological activities. Methods: We are using PS conditional double knockout (PScDKO) mice in which both presenilin genes are postnatally deleted in the forebrain to gain insight into the pathophysiology of AD. We are investigating the -secretase dependent and independent functions in adult CNS by genetic rescue using mice containing the PS1D257A mutation or exon 10 loop deletion (PS1E10), respectively. The in vivo analysis was complemented by primary neuronal culture studies. We examined neuronal cell cycle markers using immunohistochemical methods. Results: The PScDKO mice are viable and exhibit age-related neurodegenerative phenotypes. Cyclin D1 activation and neuronal cell cycle re-entry is one of the earliest hallmarks seen in PScDKO animals and in PS1 null neurons, and this is largely mediated through the PS1 loop domain. However, neurons marked with BrdU do not directly succumb to apoptosis, and -secretase defective mutant PS1D257A leads to neuronal cell death independent of cell cycle re-entry. Nevertheless, cell cycle abnormality enhances the neuronal vul- nerability under stress conditions, and our data reveal p53 as a downstream effecter. Conclusions: The results support a model whereby the PS -secretase activity, in a cell cycle independent manner, plays a predom- inant role in neuronal homeostasis. The PS1 loop domain, through cyclin D1 and p53-dependent mechanisms, facilitates PS1 neuronal function, particularly under stress conditions. P4-197 REGULATION OF HtrA2/OMI ACTIVITY BY THE MITOCHONDRIAL -SECRETASE Pavel F. Pavlov, Karolinska Institutet, Huddinge, Sweden. Contact e- mail: [email protected] Background: Altered metabolism of amyloid- precursor protein (APP) is the molecular hallmark of Alzheimer disease (AD). It has been recently shown that APP as well as amyloid- peptides are accumulated in mito- chondria of AD patients affecting mitochondrial physiology and energy production. Methods: HtrA2/Omi, a serine protease located in the mito- chondrial inter-membrane space is able to cleave APP accumulated in mitochondria. Results: Here we show that HtrA2/Omi interacts with active -secretase in mitochondria via C-terminal part of presenilin, a catalytic subunit of -secretase. This interaction results in stimulation of HtrA2/Omi proteolytic activity and degradation of a subset of mitochondrial proteins. Inhibition of HtrA2/Omi proteolytic activity in APP over-producing cell resulted in increased rate of cell death in comparison to control cell lines. Conclusions: Our results indicate that HtrA2/Omi participates in main- taining of mitochondrial homeostasis and offer the possibility of regulation of mitochondrial function under pathological conditions of AD. P4-198 MOLECULAR STUDIES OF THE PRESENILIN PROTEIN INTERACTIONS TO -SECRETASE INHIBITORS AND MODULATORS Johanna Hultstro ¨m 1 , Bengt Winblad 1 , Johan Lundkvist 2 , Jan Na ¨slund 2 , Helena Karlstro ¨m 1 , 1 Department of NVS, KI-Alzheimer’s Disease Research Center, Karolinska Institutet, Stockholm, Sweden; 2 Department of Molecular Pharmacology, CNS/Pain, AstraZeneca, So ¨derta ¨lje, Sweden. Contact e-mail: [email protected] Background: Processing of the Alzheimer amyloid beta-precursor protein (APP) into the amyloid beta-peptide (A) is a proteolytic event mediated by sequential cleavage by the -secretase and -secretase. -Secretase cleavage is performed by an enzyme complex composed of presenilin (PS), nicastrin, Aph-1 and Pen-2. Identification of two critical aspartate residues in PS1 (Asp-257 and Asp-385) suggests that PS is an unusual aspartyl protease that provides the catalytic core of the enzyme complex. PS is subjected to an endoproteolyitc cleavage, generating a heterodimer com- posed of an N-terminal fragment and a C-terminal fragment. -Secretase cleaves APP in two different sites, -site and the -site, generating the toxic A-peptide and the APP intracellular domain (AICD). -Secretase has been shown to process several type I membrane bound proteins within or just close to the transmembrane domain among them is the Notch receptor. The -secretase processes Notch in at least two different posi- tions, the S3 and the S4 site, generating the Notch intracellular domain (NICD) and Notch beta-peptide (N). We are currently localizing func- tionally important domains in PS1 regulating AICD signalling and A38/ 40/42 formation using truncated and mutated PS1 proteins. We are also examining whether processing of APP and Notch is affected in a similar manner by the same PS1 mutation/alteration and the effect of -secretase inhibitors and modulators. Methods: -Secretase-mediated production of A 38/40/42 and N peptides from APP and Notch, respectively, are monitored by ELISA and mass-spectrometry using the cultured medium from PS1/2 -/- cells. A luciferase reporter gene assay is used to monitor AICD and NICD generation. Results: We are in the process of investigat- ing the importance of certain domains in the PS1 molecule for the pro- cessing of APP and Notch and also for the binding of specific non- transition state -secretase inhibitors and -secretase modulators and the results from these experiments will be presented. Conclusions: Identifying domains in the PS protein that discriminate between APP and Notch intramembrane processing will give us more insight in the mechanism behind -secretase biology. Moreover, small molecules could be designed which inhibit the -site processing of APP without affecting the -cleavage and also the Notch signalling pathway. P4-199 ROLE OF SECRETASE IN THE CELLULAR MEMBRANE LIPID COMPOSITION Jessica Hommes, Jochen Walter, Bernadette Breiden, Irfan Y. Tamboli, Gu ¨nter Schwarzmann, University of Bonn, Bonn, Germany. Contact e- mail: [email protected] Background: Alzheimer’s disease (AD) is the most common cause of dementia and characterized by deposition of the amyloid -peptide (A) in the brain. Presenilins (PS) are genetically associated with AD and so far more than 150 mutations have been characterized, which lead to early-onset AD. PS are critical for the generation of A, because they form the catalytic subunit of the -secretase complex, which contains three additional components; Nicastrin, Aph-1 and Pen-2. Due to it’s ability cleaving a lot of type I transmembrane proteins, thereby PS are involved in many biological processes. Besides amyloid precursor pro- tein (APP) more than three dozen substrates of the -secretase have been identified. The activity of -secretase is influenced by the com- position of the cellular membrane. In turn, -secretase activity has been shown to affect membrane-lipid metabolism, including that of choles- terol and sphingolipids. Several studies indicated a link between lipid metabolism and AD. Here, we sought to analyze the role of -secretase in the membrane-lipid metabolism. Methods: We analyzed cholesterol and ganglioside levels in mouse embryonic fibroblasts (Mefs) of wild- type and PS double-knockout mice (Mef PS1/2 -/-) by using metabolic labeling and thin layer chromatography technique. -secretase was also inhibited pharmacologically by N-[N-(3,5-Difluorophenacetyl)-L-ala- nyl]-S-phenylglycine t-butyl ester (DAPT) and the cholesterol and ganglioside levels in Mefs were analyzed. Results: We found that cellular cholesterol levels were increased in PS deficient fibroblasts as compared to wildtype cells. In addition, pharmacological inhibition of the -secretase led to increased cholesterol levels. Levels of certain glycosphingolipids were also increased in PS deficient cells. Radiola- beling experiments indicate an impaired metabolism upon inhibition of T729 Poster Presentations P4:
Transcript of P4-198: Molecular studies of the presenilin protein interactions to γ-secretase inhibitors and...
P4-196 PRESENILIN IN NEURONAL SURVIVAL ANDNEURODEGENERATION
Verena Kallhoff-Munoz, Lingyun Hu, Robia Pautler, Hui Zheng,Baylor College of Medicine, Houston, TX, USA. Contact e-mail:[email protected]
Background: Presenilins (PS) have been shown to exhibit �-secretasedependent and independent activities, the former requires the D257 site andthe latter is mediated through the hydrophilic loop domain encoded byexon 10. Mutations in presenilin genes (PS1 and PS2) lead to early-onsetof Alzheimer’s disease (AD), and increasing evidence indicates that thesemutations lead to a partial loss of function for its physiological activities.Methods: We are using PS conditional double knockout (PScDKO) micein which both presenilin genes are postnatally deleted in the forebrain togain insight into the pathophysiology of AD. We are investigating the�-secretase dependent and independent functions in adult CNS by geneticrescue using mice containing the PS1D257A mutation or exon 10 loopdeletion (PS1�E10), respectively. The in vivo analysis was complementedby primary neuronal culture studies. We examined neuronal cell cyclemarkers using immunohistochemical methods. Results: The PScDKOmice are viable and exhibit age-related neurodegenerative phenotypes.Cyclin D1 activation and neuronal cell cycle re-entry is one of the earliesthallmarks seen in PScDKO animals and in PS1 null neurons, and this islargely mediated through the PS1 loop domain. However, neurons markedwith BrdU do not directly succumb to apoptosis, and �-secretase defectivemutant PS1D257A leads to neuronal cell death independent of cell cyclere-entry. Nevertheless, cell cycle abnormality enhances the neuronal vul-nerability under stress conditions, and our data reveal p53 as a downstreameffecter. Conclusions: The results support a model whereby the PS�-secretase activity, in a cell cycle independent manner, plays a predom-inant role in neuronal homeostasis. The PS1 loop domain, through cyclinD1 and p53-dependent mechanisms, facilitates PS1 neuronal function,particularly under stress conditions.
P4-197 REGULATION OF HtrA2/OMI ACTIVITY BY THEMITOCHONDRIAL �-SECRETASE
Pavel F. Pavlov, Karolinska Institutet, Huddinge, Sweden. Contact e-mail: [email protected]
Background: Altered metabolism of amyloid-� precursor protein (APP) isthe molecular hallmark of Alzheimer disease (AD). It has been recentlyshown that APP as well as amyloid-� peptides are accumulated in mito-chondria of AD patients affecting mitochondrial physiology and energyproduction. Methods: HtrA2/Omi, a serine protease located in the mito-chondrial inter-membrane space is able to cleave APP accumulated inmitochondria. Results: Here we show that HtrA2/Omi interacts with active�-secretase in mitochondria via C-terminal part of presenilin, a catalyticsubunit of �-secretase. This interaction results in stimulation of HtrA2/Omiproteolytic activity and degradation of a subset of mitochondrial proteins.Inhibition of HtrA2/Omi proteolytic activity in APP over-producing cellresulted in increased rate of cell death in comparison to control cell lines.Conclusions: Our results indicate that HtrA2/Omi participates in main-taining of mitochondrial homeostasis and offer the possibility of regulationof mitochondrial function under pathological conditions of AD.
P4-198 MOLECULAR STUDIES OF THE PRESENILINPROTEIN INTERACTIONS TO �-SECRETASEINHIBITORS AND MODULATORS
Johanna Hultstrom1, Bengt Winblad1, Johan Lundkvist2, Jan Naslund2,Helena Karlstrom1, 1Department of NVS, KI-Alzheimer’s DiseaseResearch Center, Karolinska Institutet, Stockholm, Sweden; 2Departmentof Molecular Pharmacology, CNS/Pain, AstraZeneca, Sodertalje,Sweden. Contact e-mail: [email protected]
Background: Processing of the Alzheimer amyloid beta-precursor protein(APP) into the amyloid beta-peptide (A�) is a proteolytic event mediated
by sequential cleavage by the �-secretase and �-secretase. �-Secretasecleavage is performed by an enzyme complex composed of presenilin (PS),nicastrin, Aph-1 and Pen-2. Identification of two critical aspartate residuesin PS1 (Asp-257 and Asp-385) suggests that PS is an unusual aspartylprotease that provides the catalytic core of the enzyme complex. PS issubjected to an endoproteolyitc cleavage, generating a heterodimer com-posed of an N-terminal fragment and a C-terminal fragment. �-Secretasecleaves APP in two different sites, �-site and the �-site, generating thetoxic A�-peptide and the APP intracellular domain (AICD). �-Secretasehas been shown to process several type I membrane bound proteins withinor just close to the transmembrane domain among them is the Notchreceptor. The �-secretase processes Notch in at least two different posi-tions, the S3 and the S4 site, generating the Notch intracellular domain(NICD) and Notch beta-peptide (N�). We are currently localizing func-tionally important domains in PS1 regulating AICD signalling and A�38/40/42 formation using truncated and mutated PS1 proteins. We are alsoexamining whether processing of APP and Notch is affected in a similarmanner by the same PS1 mutation/alteration and the effect of �-secretaseinhibitors and modulators. Methods: �-Secretase-mediated production ofA� 38/40/42 and N� peptides from APP and Notch, respectively, aremonitored by ELISA and mass-spectrometry using the cultured mediumfrom PS1/2 -/- cells. A luciferase reporter gene assay is used to monitorAICD and NICD generation. Results: We are in the process of investigat-ing the importance of certain domains in the PS1 molecule for the pro-cessing of APP and Notch and also for the binding of specific non-transition state �-secretase inhibitors and �-secretase modulators and theresults from these experiments will be presented. Conclusions: Identifyingdomains in the PS protein that discriminate between APP and Notchintramembrane processing will give us more insight in the mechanismbehind �-secretase biology. Moreover, small molecules could be designedwhich inhibit the �-site processing of APP without affecting the �-cleavageand also the Notch signalling pathway.
P4-199 ROLE OF � SECRETASE IN THE CELLULARMEMBRANE LIPID COMPOSITION
Jessica Hommes, Jochen Walter, Bernadette Breiden, Irfan Y. Tamboli,Gunter Schwarzmann, University of Bonn, Bonn, Germany. Contact e-mail: [email protected]
Background: Alzheimer’s disease (AD) is the most common cause ofdementia and characterized by deposition of the amyloid �-peptide(A�) in the brain. Presenilins (PS) are genetically associated with ADand so far more than 150 mutations have been characterized, which leadto early-onset AD. PS are critical for the generation of A�, because theyform the catalytic subunit of the �-secretase complex, which containsthree additional components; Nicastrin, Aph-1 and Pen-2. Due to it’sability cleaving a lot of type I transmembrane proteins, thereby PS areinvolved in many biological processes. Besides amyloid precursor pro-tein (APP) more than three dozen substrates of the �-secretase havebeen identified. The activity of �-secretase is influenced by the com-position of the cellular membrane. In turn, �-secretase activity has beenshown to affect membrane-lipid metabolism, including that of choles-terol and sphingolipids. Several studies indicated a link between lipidmetabolism and AD. Here, we sought to analyze the role of �-secretasein the membrane-lipid metabolism. Methods: We analyzed cholesteroland ganglioside levels in mouse embryonic fibroblasts (Mefs) of wild-type and PS double-knockout mice (Mef PS1/2 -/-) by using metaboliclabeling and thin layer chromatography technique. �-secretase was alsoinhibited pharmacologically by N-[N-(3,5-Difluorophenacetyl)-L-ala-nyl]-S-phenylglycine t-butyl ester (DAPT) and the cholesterol andganglioside levels in Mefs were analyzed. Results: We found thatcellular cholesterol levels were increased in PS deficient fibroblasts ascompared to wildtype cells. In addition, pharmacological inhibition ofthe �-secretase led to increased cholesterol levels. Levels of certainglycosphingolipids were also increased in PS deficient cells. Radiola-beling experiments indicate an impaired metabolism upon inhibition of
T729Poster Presentations P4:
![ЕВРИСТИЧНІ АЛГОРИТМИ САМОВІДНОВЛЕННЯ …lib.chdu.edu.ua/pdf/naukpraci/technogen/2013/210-198-24.pdf · кого ευρίσκω (heuristiko) – знахідка[1].](https://static.fdocument.org/doc/165x107/5a7cdbbb7f8b9a72118d10dd/-libchdueduuapdfnaukpracitechnogen2013210-198-24pdf.jpg)
