Die Alzheimer Krankheit ist die häufigste Altersdemenz. Ein spezifisches pathologisches Merkmal der Alzheimer Krankheit ist die Amyloid-Ablagerung im Gehirn. Die Hauptkomponente der so genannten Amyloid-Plaques ist das Amyloid beta-Peptid (A-beta). A-beta entsteht durch sequenzielle proteolytische Spaltung aus einem membrangebundenen Vorläuferprotein, dem beta-APP (betaamyloid precursor protein). Die kürzlich identifizierte beta-Sekretase (BACE, beta-site APPcleaving enzyme) generiert den Schnitt am N-Terminus von A-beta. Es entsteht ein C-terminales, membrangebundenes beta-APP-Fragment, das beta-APP-CTF. Beta-APP-CTF ist das direkte Substrat für die gamma-Sekretase, die innerhalb der Membrandomäne schneidet, wodurch A-beta freigesetzt wird. In der vorliegenden Arbeit kann erstmalig gezeigt werden, dass BACE auf dem sekretorischen Transportweg aus dem Endoplasmatischen Retikulum (ER), über den Golgi-Apparat zur Zelloberfläche transportiert wird. Auf dem Transport wird BACE durch N-Glycosylierung und Propeptidabspaltung posttranslational modifiziert. BACE wird im ER N-glycosyliert und die mannosereichen Zucker werden auf dem Transport durch den Golgi-Apparat in Endoglycosidase H resistente Zucker des komplexen Typs modifiziert. Die Propeptidabspaltung, durch Furin oder furinähnliche Propeptidkonvertasen, findet unmittelbar vor dem Aufbau der komplexen Zucker statt. Ferner konnte gezeigt werden, dass der Transport von BACE die A-beta-Entstehung limitieren kann. In polarisierten Madin-Darby canine kidney (MDCK) Zellen wird BACE überwiegend zur apikalen Plasmamembran transportiert und damit entgegengesetzt zu seinem Substrat beta-APP. Der gegensätzliche Transport von BACE und beta-APP begrenzt die A-beta Entstehung. Wird der apikale Transport von beta-APP durch Deletion seines basolateralen Sortierungssignals erhöht, entsteht vermehrt A-beta. Der differenzielle Transport von BACE und beta-APP könnte ein Hinweis darauf sein, dass beta-APP nicht das physiologische Substrat von BACE ist. / Alzheimer`s disease is the most common cause of progressive cognitive decline in the aged population. Pathologically Alzheimer`s disease is characterized by the invariant accumulation of senile plaques. Senile plaques are predominantly composed of the amyloid beta-peptide (A-beta), which is derived from the membrane bound beta-amyloid precursor protein (beta-APP) by sequential proteolytic cleavage. The recently identified beta-secretase (BACE) is responsible for the cleavage at the N-terminus of the A-beta domain. This cleavage generates membrane-bound beta-APP-Cterminal fragments (beta-APP-CTF) which are the immediate precursor for gamma-secretase cleavage and therefore for liberation of A-beta. The present work shows that BACE moves along the secretory pathway, while it undergoes post-translational modifications, which can be monitored by a significant increase in the molecular mass and cleavage of its pro-peptide. BACE becomes N-glycosylated within the ER and the increase in molecular mass is caused by complex N-glycosylation. The mature form of BACE is resistant to endoglycosidase H treatment; this indicates that BACE traffics through the Golgi. Furthermore the mature form of BACE does not contain the pro-peptide anymore. Pro-BACE is predominantly located within the endoplasmic reticulum. Pro-peptide cleavage occurs immediately before full maturation by furin or a furin-like proprotein convertase. Moreover traffic of BACE can limit A-beta generation. In the well established model system of polarized Madin-Darby canine kidney (MDCK) cells, the majority of BACE is sorted to the apical domain. Interestingly it has been shown previously that the substrate of BACE, beta-APP is transported to the basolateral surface of MCDK cells. Therefore, substantial amounts of BACE are targeted away from beta-APP to a non-amyloidogenic compartment, a cellular mechanism that limits A-beta generation. Upon deletion of the basolateral sorting signal of beta-APP, apically missorted beta-APP is processed by BACE. The differential targeting of BACE and its substrate beta-APP suggest that beta-APP might not be the major physiological substrate of BACE.

Proteolytic processing of the amyloid precursor protein (APP) may lead to the formation of the Abeta peptide, the major constituent of amyloid plaques in Alzheimer`s disease. The full-length APP is a substrate for at least 2 different (alpha and beta) proteases ("secretases"). The beta-secretase, BACE, cleaves APP in the first step of processing leading to the formation of the neurotoxic Abeta. BACE competes for APP with alpha-secretase, which cleaves APP within its Abeta sequence, thus precluding Abeta formation. It is thus important to understand how is the access of the alpha- and beta-secretase to APP regulated and how are the individual activities of these secretases modulated. Both these regulatory mechanisms, access to substrate and direct activity modulation, can be determined by the lipid composition of the membrane. Integral membrane proteins (like APP and BACE), can be viewed as solutes in a two-dimensional liquid membrane, and as such their state, and biological activity, critically depend on the physico-chemical character (fluidity, curvature, surface charge distribution, lateral domain heterogeneity etc.) of the lipid bilayer. These collective membrane properties will influence the activity of embedded membrane proteins. In addition, activity regulation may involve a direct interaction with a specific lipid (cofactor or co-structure function). Interactions of membrane proteins are furthermore affected by lateral domain organization of the membrane. Previous results had suggested that the regulation of the activity of the alpha- and beta-secretases and of their access to APP is lipid dependent, and involves lipid rafts. Using the baculovirus expression system, we have purified recombinant human full-length APP and BACE to homogeneity, and reconstituted them in large (~100nm, LUVs) and giant (10-150microm, GUVs) unilamellar vesicles. Using a soluble peptide substrate mimicking the beta-cleavage site of APP, we have examined the involvement of individual lipid species in modulating BACE activity in LUVs of various lipid compositions. We have identified 3 groups of lipids that stimulate proteolytic activity of BACE: 1.cerebrosides, 2.anionic glycerophospholipids, 3. cholesterol. Furthermore, we have co-reconstituted APP and BACE together in LUVs and demonstrated that BACE cleaves APP at the correct site, generating the beta-cleaved ectodomain identical to that from cells. We have developed an assay to quantitatively follow the beta-cleavage in proteoliposomes, and we have shown that the rate of cleavage in total brain lipid proteoliposomes is higher than in phosphatidylcholine vesicles. We have also studied partitioning of APP and BACE in GUVs between liquid ordered (lo) and liquid disordered (ld) phases. In this system, significant part of the BACE pool (about 20%) partitions into the lo phase, and its partitioning into lo phase can be further enhanced by cross-linking of membrane components. Only negligible fraction of APP can be found in the lo phase. We continue to study the behavior of co-reconstituted APP and BACE in GUVs The work presented in this thesis has yielded some interesting results and raised further questions. One of the important assignments of this project will in the next stage be the characterization of the impact of membrane domain organization on the beta-cleavage. Different domain arrangements that can be hypothesized in cell membranes can be modeled by varying the degree of phase fragmentation in proteoliposomes comprising reconstituted APP and BACE.

BACE1, die beta-Sekretase, spielt eine zentrale Rolle bei der Entstehung von Amyloid, einem charakteristischen histopathologischen Merkmal der Alzheimer-Demenz. Die physiologische Funktion von BACE1 ist unklar. Neuere Studien zeigten eine Rolle bei der Myelinisierung. Die vorliegende Arbeit untersucht die Rolle von BACE1 bei der Myelinisierung im Zentralen Nervensystem. Zusammenfassend zeigt die Studie keinen Einfluss einer BACE1-Inhibiton auf die primäre Ausprägung der Myelinscheiden im Corpus callosum. Sie widerspricht damit der Hypothese, dass BACE1 via Neuregulin-1-Prozessierung notwendig für die Myelinisierung im ZNS ist. Ob es sich dabei um lokale Differenzen einzelner anatomischer Regionen handelt muss in weiteren Studien untersucht werden. Zudem zeigt diese Arbeit einen kleinen, aber signifikanten Einfluss von BACE1 bei der Remyelinisierung im Corpus callosum nach Cuprizonebehandlung auf.

Systematic variation of membrane anchor, spacer and pharmacophore building blocks leads to an optimisation of the inhibitory effect of tripartite structures towards BACE1-induced cleavage of the amyloid precursor protein (APP). / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Systematic variation of membrane anchor, spacer and pharmacophore building blocks leads to an optimisation of the inhibitory effect of tripartite structures towards BACE1-induced cleavage of the amyloid precursor protein (APP). / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Proteolytic processing of the amyloid precursor protein (APP) may lead to the formation of the Abeta peptide, the major constituent of amyloid plaques in Alzheimer`s disease. The full-length APP is a substrate for at least 2 different (alpha and beta) proteases ("secretases"). The beta-secretase, BACE, cleaves APP in the first step of processing leading to the formation of the neurotoxic Abeta. BACE competes for APP with alpha-secretase, which cleaves APP within its Abeta sequence, thus precluding Abeta formation. It is thus important to understand how is the access of the alpha- and beta-secretase to APP regulated and how are the individual activities of these secretases modulated. Both these regulatory mechanisms, access to substrate and direct activity modulation, can be determined by the lipid composition of the membrane. Integral membrane proteins (like APP and BACE), can be viewed as solutes in a two-dimensional liquid membrane, and as such their state, and biological activity, critically depend on the physico-chemical character (fluidity, curvature, surface charge distribution, lateral domain heterogeneity etc.) of the lipid bilayer. These collective membrane properties will influence the activity of embedded membrane proteins. In addition, activity regulation may involve a direct interaction with a specific lipid (cofactor or co-structure function). Interactions of membrane proteins are furthermore affected by lateral domain organization of the membrane. Previous results had suggested that the regulation of the activity of the alpha- and beta-secretases and of their access to APP is lipid dependent, and involves lipid rafts. Using the baculovirus expression system, we have purified recombinant human full-length APP and BACE to homogeneity, and reconstituted them in large (~100nm, LUVs) and giant (10-150microm, GUVs) unilamellar vesicles. Using a soluble peptide substrate mimicking the beta-cleavage site of APP, we have examined the involvement of individual lipid species in modulating BACE activity in LUVs of various lipid compositions. We have identified 3 groups of lipids that stimulate proteolytic activity of BACE: 1.cerebrosides, 2.anionic glycerophospholipids, 3. cholesterol. Furthermore, we have co-reconstituted APP and BACE together in LUVs and demonstrated that BACE cleaves APP at the correct site, generating the beta-cleaved ectodomain identical to that from cells. We have developed an assay to quantitatively follow the beta-cleavage in proteoliposomes, and we have shown that the rate of cleavage in total brain lipid proteoliposomes is higher than in phosphatidylcholine vesicles. We have also studied partitioning of APP and BACE in GUVs between liquid ordered (lo) and liquid disordered (ld) phases. In this system, significant part of the BACE pool (about 20%) partitions into the lo phase, and its partitioning into lo phase can be further enhanced by cross-linking of membrane components. Only negligible fraction of APP can be found in the lo phase. We continue to study the behavior of co-reconstituted APP and BACE in GUVs The work presented in this thesis has yielded some interesting results and raised further questions. One of the important assignments of this project will in the next stage be the characterization of the impact of membrane domain organization on the beta-cleavage. Different domain arrangements that can be hypothesized in cell membranes can be modeled by varying the degree of phase fragmentation in proteoliposomes comprising reconstituted APP and BACE.