The large hydrophilic loop of presenilin 1 is important for regulating gamma-secretase complex assembly and dictating the amyloid beta peptide (Abeta) Profile without affecting Notch processing.

Gamma-secretase is an enzyme complex that mediates both Notch signaling and beta-amyloid precursor protein (APP) processing, resulting in the generation of Notch intracellular domain, APP intracellular domain, and the amyloid beta peptide (Abeta), the latter playing a central role in Alzheimer disease (AD). By a hitherto undefined mechanism, the activity of gamma-secretase gives rise to Abeta peptides of different lengths, where Abeta42 is considered to play a particular role in AD. In this study we have examined the role of the large hydrophilic loop (amino acids 320-374, encoded by exon 10) of presenilin 1 (PS1), the catalytic subunit of gamma-secretase, for gamma-secretase complex formation and activity on Notch and APP processing. Deletion of exon 10 resulted in impaired PS1 endoproteolysis, gamma-secretase complex formation, and had a differential effect on Abeta-peptide production. Although the production of Abeta38, Abeta39, and Abeta40 was severely impaired, the effect on Abeta42 was affected to a lesser extent, implying that the production of the AD-related Abeta42 peptide is separate from the production of the Abeta38, Abeta39, and Abeta40 peptides. Interestingly, formation of the intracellular domains of both APP and Notch was intact, implying a differential cleavage activity between the epsilon/S3 and gamma sites. The most C-terminal amino acids of the hydrophilic loop were important for regulating APP processing. In summary, the large hydrophilic loop of PS1 appears to differentially regulate the relative production of different Abeta peptides without affecting Notch processing, two parameters of significance when considering gamma-secretase as a target for pharmaceutical intervention in AD.

Minor contribution of presenilin 2 for γ-secretase activity in mouse embryonic fibroblasts and adult mouse brain.

γ-Secretase plays an important function in the development of Alzheimer disease, since it participates in the production of the toxic amyloid ß-peptide (Aß) from the amyloid precursor protein (APP). Besides APP, γ-secretase cleaves many other substrates resulting in adverse side effects when γ-secretase inhibitors are used in clinical trials. γ-Secretase is a membrane bound protein complex consisting of at least four subunits, presenilin (PS), nicastrin, Aph-1 and Pen-2. PS and Aph-1 exist as different homologs (PS1/PS2 and Aph-1a/Aph-1b, respectively), which generates a variation in complex composition. PS1 and PS2 appears to have distinct roles since PS1 is essential during embryonic development whereas PS2 deficient mice are viable with a mild phenotype. The molecular mechanism behind this diversity is, however, largely unknown. In order to investigate whether PS1 and PS2 show different substrate specificity, we used PS1 or PS2 deficient mouse embryonic fibroblasts to study the processing on the γ-secretase substrates APP, Notch, N-cadherin, and ephrinB. We found that whereas depletion of PS1 severely affected the cleavage of all substrates, the effect of PS2 depletion was minor. In addition, less PS2 was found in active γ-secretase complexes. We also studied the effect of PS2 depletion in adult mouse brain and, in concordance with the results from the mouse embryonic fibroblasts, PS2 deficiency did not alter the cleavage of the two most important substrates, APP and Notch. In summary, this study shows that the contribution of PS2 on γ-secretase activity is of less importance, explaining the mild phenotype of PS2-deficient mice.

Affinity pulldown of γ-secretase and associated proteins from human and rat brain.

γ-Secretase is a transmembrane protease complex responsible for the processing of a multitude of type 1 transmembrane proteins, including amyloid precursor protein (APP) and Notch. A functional complex is dependent on the assembly of four proteins: presenilin (PS), nicastrin, Aph-1 and Pen-2. Little is known about how the substrates are selected by γ-secretase, but it has been suggested that γ-secretase associated proteins (GSAPs) could be of importance. For instance, it was recently reported from studies in cell lines that TMP21, a transmembrane protein involved in trafficking, binds to γ-secretase and regulates the processing of APP-derived substrates without affecting Notch cleavage. Here, we present an efficient and selective method for purification and analysis of γ-secretase and GSAPs. Microsomal membranes were prepared from rat or human brain and incubated with a γ-secretase inhibitor coupled to biotin via a long linker and a S-S bridge. After pulldown using streptavidin beads, bound proteins were eluted under reducing conditions and digested by trypsin. The tryptic peptides were subjected to LC-MS/MS analysis, and proteins were identified by sequence data from MS/MS spectra. All of the known γ-secretase components were identified. Interestingly, TMP21 and the PS associated protein syntaxin1 were associated to γ-secretase in rat brain. We suggest that the present method can be used for further studies on the composition of the γ-secretase complex.

Abeta43 is more frequent than Abeta40 in amyloid plaque cores from Alzheimer disease brains.

One hallmark of Alzheimer disease (AD) is the extracellular deposition of the amyloid beta-peptide (Abeta) in senile plaques. Two major forms of Abeta are produced, 40 (Abeta40) and 42 (Abeta42) residues long. The most abundant form of Abeta is Abeta40, while Abeta42 is more hydrophobic and more prone to form toxic oligomers and the species of particular importance in early plaque formation. Thus, the length of the hydrophobic C-terminal seems to be very important for the oligomerization and neurotoxicity of the Abeta peptide. Here we investigated which Abeta species are deposited in AD brain. We analyzed plaque cores, prepared from occipital and frontal cortex, from sporadic and familial AD cases and performed a quantitative study using Abeta standard peptides. Cyanogen bromide was used to generate C-terminal Abeta fragments, which were analyzed by HPLC coupled to an electrospray ionisation ion trap mass spectrometer. We found a longer peptide, Abeta43, to be more frequent than Abeta40. No variants longer than Abeta43 could be observed in any of the brains. Immunohistochemistry was performed and was found to be in line with our findings. Abeta1-43 polymerizes rapidly and we suggest that this variant may be of importance for AD.

Active gamma-secretase is localized to detergent-resistant membranes in human brain.

Several lines of evidence suggest that polymerization of the amyloid beta-peptide (Abeta) into amyloid plaques is a pathogenic event in Alzheimer's disease (AD). Abeta is produced from the amyloid precursor protein as the result of sequential proteolytic cleavages by beta-secretase and gamma-secretase, and it has been suggested that these enzymes could be targets for treatment of AD. gamma-Secretase is an aspartyl protease complex, containing at least four transmembrane proteins. Studies in cell lines have shown that gamma-secretase is partially localized to lipid rafts, which are detergent-resistant membrane microdomains enriched in cholesterol and sphingolipids. Here, we studied gamma-secretase in detergent-resistant membranes (DRMs) prepared from human brain. DRMs prepared in the mild detergent CHAPSO and isolated by sucrose gradient centrifugation were enriched in gamma-secretase components and activity. The DRM fraction was subjected to size-exclusion chromatography in CHAPSO, and all of the gamma-secretase components and a lipid raft marker were found in the void volume (> 2000 kDa). Co-immunoprecipitation studies further supported the notion that the gamma-secretase components are associated even at high concentrations of CHAPSO. Preparations from rat brain gave similar results and showed a postmortem time-dependent decline in gamma-secretase activity, suggesting that DRMs from fresh rat brain may be useful for gamma-secretase activity studies. Finally, confocal microscopy showed co-localization of gamma-secretase components and a lipid raft marker in thin sections of human brain. We conclude that the active gamma-secretase complex is localized to lipid rafts in human brain.

gamma-Secretase dependent production of intracellular domains is reduced in adult compared to embryonic rat brain membranes.

BACKGROUND: gamma-Secretase is an intramembrane aspartyl protease whose cleavage of the amyloid precursor protein (APP) generates the amyloid beta-peptide (Abeta) and the APP intracellular domain. Abeta is widely believed to have a causative role in Alzheimer's disease pathogenesis, and therefore modulation of gamma-secretase activity has become a therapeutic goal. Besides APP, more than 50 substrates of gamma-secretase with different cellular functions during embryogenesis as well as adulthood have been revealed. Prior to gamma-secretase cleavage, substrates are ectodomain shedded, producing membrane bound C-terminal fragments (CTFs). PRINCIPAL FINDINGS: Here, we investigated gamma-secretase cleavage of five substrates; APP, Notch1, N-cadherin, ephrinB and p75 neurotrophin receptor (p75-NTR) in membranes isolated from embryonic, young or old adult rat brain by analyzing the release of the corresponding intracellular domains (ICDs) or Abeta40 by western blot analysis and ELISA respectively. The highest levels of all ICDs and Abeta were produced by embryonic membranes. In adult rat brain only cleavage of APP and Notch1 could be detected and the Abeta40 and ICD production from these substrates was similar in young and old adult rat brain. The CTF levels of Notch1, N-cadherin, ephrinB and p75-NTR were also clearly decreased in the adult brain compared to embryonic brain, whereas the APP CTF levels were only slightly decreased. CONCLUSIONS: In summary our data suggests that gamma-secretase dependent ICD production is down-regulated in the adult brain compared to embryonic brain. In addition, the present approach may be useful for evaluating the specificity of gamma-secretase inhibitors.

Synaptic and endosomal localization of active gamma-secretase in rat brain.

BACKGROUND: A key player in the development of Alzheimer's disease (AD) is the gamma-secretase complex consisting of at least four components: presenilin, nicastrin, Aph-1 and Pen-2. gamma-Secretase is crucial for the generation of the neurotoxic amyloid beta-peptide (Abeta) but also takes part in the processing of many other substrates. In cell lines, active gamma-secretase has been found to localize primarily to the Golgi apparatus, endosomes and plasma membranes. However, no thorough studies have been performed to show the subcellular localization of the active gamma-secretase in the affected organ of AD, namely the brain. PRINCIPAL FINDINGS: We show by subcellular fractionation of rat brain that high gamma-secretase activity, as assessed by production of Abeta40, is present in an endosome- and plasma membrane-enriched fraction of an iodixanol gradient. We also prepared crude synaptic vesicles as well as synaptic membranes and both fractions showed high Abeta40 production and contained high amounts of the gamma-secretase components. Further purification of the synaptic vesicles verified the presence of the gamma-secretase components in these compartments. The localization of an active gamma-secretase in synapses and endosomes was confirmed in rat brain sections and neuronal cultures by using a biotinylated gamma-secretase inhibitor together with confocal microscopy. SIGNIFICANCE: The information about the subcellular localization of gamma-secretase in brain is important for the understanding of the molecular mechanisms of AD. Furthermore, the identified fractions can be used as sources for highly active gamma-secretase.

Rat brain gamma-secretase activity is highly influenced by detergents.

Gamma-secretase is important for the development of Alzheimer's disease, since it is a crucial enzyme for the generation of the pathogenic amyloid beta-peptide (Abeta). Most data on gamma-secretase is derived from studies in cell lines overexpressing gamma-secretase components or amyloid precursor protein (APP), and since gamma-secretase is a transmembrane protein complex, detergents have been frequently used to facilitate the studies. However, no extensive comparison of the influence of different detergents at different concentrations on gamma-secretase activity in preparations from brain has been made. Here, we establish the optimal conditions for gamma-secretase activity in rat brain, using an activity assay detecting endogenous production of the APP intracellular domain, which is generated when gamma-secretase cleaves the APP C-terminal fragments. We performed a subcellular fractionation and noted the highest gamma-secretase activity in the 100000g pellet and that the optimal pH was around 7. We found that gamma-secretase was active for at least 16 h at 37 degrees C and that the endogenous substrate levels were sufficient for activity measurements. The highest activity was obtained in 0.4% CHAPSO, which is slightly below the critical micelle concentration (0.5%) for this detergent, but the complex was not solubilized efficiently at this concentration. On the other hand, 1% CHAPSO solubilized a substantial amount of the gamma-secretase components, but the activity was low. The activity was fully restored by diluting the sample to 0.4% CHAPSO. Therefore, using 1% CHAPSO for solubilization and subsequently diluting the sample to 0.4% is an appropriate procedure for obtaining a soluble, highly active gamma-secretase from rat brain.