Ji-Yeun Hur

Active γ‐secretase is localized to detergent‐resistant membranes in human brain

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

Synaptic and Endosomal Localization of Active γ-Secretase in Rat Brain

Background A key player in the development of Alzheimers disease (AD) is the γ-secretase complex consisting of at least four components: presenilin, nicastrin, Aph-1 and Pen-2. γ-Secretase is crucial for the generation of the neurotoxic amyloid β-peptide (Aβ) but also takes part in the processing of many other substrates. In cell lines, active γ-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 γ-secretase in the affected organ of AD, namely the brain. Principal Findings We show by subcellular fractionation of rat brain that high γ-secretase activity, as assessed by production of Aβ40, 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 Aβ40 production and contained high amounts of the γ-secretase components. Further purification of the synaptic vesicles verified the presence of the γ-secretase components in these compartments. The localization of an active γ-secretase in synapses and endosomes was confirmed in rat brain sections and neuronal cultures by using a biotinylated γ-secretase inhibitor together with confocal microscopy. Significance The information about the subcellular localization of γ-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 γ-secretase.

Identification of Novel γ-Secretase-associated Proteins in Detergent-resistant Membranes from Brain

Background: In AD, APP can be processed in lipid rafts, and γ-secretase-associated proteins (GSAPs) can affect Aβ production. Results: We identify novel GSAPs in detergent-resistant membranes (DRMs). Conclusion: VDAC1 and CNTNAP1 associate with γ-secretase in DRMs and affect APP processing with less effect on Notch processing. Significance: Novel GSAPs that regulate Aβ production can be used as AD therapeutic targets. In Alzheimer disease, oligomeric amyloid β-peptide (Aβ) species lead to synapse loss and neuronal death. γ-Secretase, the transmembrane protease complex that mediates the final catalytic step that liberates Aβ from its precursor protein (APP), has a multitude of substrates, and therapeutics aimed at reducing Aβ production should ideally be specific for APP cleavage. It has been shown that APP can be processed in lipid rafts, and γ-secretase-associated proteins can affect Aβ production. Here, we use a biotinylated inhibitor for affinity purification of γ-secretase and associated proteins and mass spectrometry for identification of the purified proteins, and we identify novel γ-secretase-associated proteins in detergent-resistant membranes from brain. Furthermore, we show by small interfering RNA-mediated knockdown of gene expression that a subset of the γ-secretase-associated proteins, in particular voltage-dependent anion channel 1 (VDAC1) and contactin-associated protein 1 (CNTNAP1), reduced Aβ production (Aβ40 and Aβ42) by around 70%, whereas knockdown of presenilin 1, one of the essential γ-secretase complex components, reduced Aβ production by 50%. Importantly, these proteins had a less pronounced effect on Notch processing. We conclude that VDAC1 and CNTNAP1 associate with γ-secretase in detergent-resistant membranes and affect APP processing and suggest that molecules that interfere with this interaction could be of therapeutic use for Alzheimer disease.

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.

Identification of two novel synaptic γ-secretase associated proteins that affect amyloid β-peptide levels without altering Notch processing

Synaptic degeneration is one of the earliest hallmarks of Alzheimer disease (AD) and results in loss of cognitive function. One of the causative agents for the synaptic degeneration is the amyloid β-peptide (Aβ), which is formed from its precursor protein by two sequential cleavages mediated by β- and γ-secretase. We have earlier shown that γ-secretase activity is enriched in synaptic compartments, suggesting that the synaptotoxic Aβ is produced locally. Proteins that interact with γ-secretase at the synapse and regulate the production of Aβ can therefore be potential therapeutic targets. We used a recently developed affinity purification approach to identify γ-secretase associated proteins (GSAPs) in synaptic membranes and synaptic vesicles prepared from rat brain. Liquid chromatography-tandem mass spectrometry analysis of the affinity purified samples revealed the known γ-secretase components presenilin-1, nicastrin and Aph-1b along with a number of novel potential GSAPs. To investigate the effect of these GSAPs on APP processing, we performed siRNA experiments to knock down the expression of the GSAPs and measured the Aβ levels. Silencing of NADH dehydrogenase [ubiquinone] iron-sulfur protein 7 (NDUFS7) resulted in a decrease in Aβ levels whereas silencing of tubulin polymerization promoting protein (TPPP) resulted in an increase in Aβ levels. Treatment with γ-secretase inhibitors often results in Notch-related side effects and therefore we also studied the effect of the siRNAs on Notch processing. Interestingly, silencing of TPPP or NDUFS7 did not affect cleavage of Notch. We also studied the expression of TPPP and NDUFS7 in control and AD brain and found NDUFS7 to be highly expressed in vulnerable neurons such as pyramidal neurons in the hippocampus, whereas TPPP was found to accumulate in intraneuronal granules and fibrous structures in hippocampus from AD cases. In summary, we here report on two proteins, TPPP and NDUFS7, which interact with γ-secretase and alter the Aβ levels without affecting Notch cleavage.

Erlin-2 is associated with active γ-secretase in brain and affects amyloid β-peptide production

The transmembrane protease complex γ-secretase is responsible for the generation of the neurotoxic amyloid β-peptide (Aβ) from its precursor (APP). Aβ has a causative role in Alzheimer disease, and thus, γ-secretase is a therapeutic target. However, since there are more than 70 γ-secretase substrates besides APP, selective inhibition of APP processing is required. Recent data indicates the existence of several γ-secretase associated proteins (GSAPs) that affect the selection and processing of substrates. Here, we use a γ-secretase inhibitor for affinity purification of γ-secretase and associated proteins from microsomes and detergent resistant membranes (DRMs) prepared from rat or human brain. By tandem mass spectrometry we identified a novel brain GSAP; erlin-2. This protein was recently reported to reside in DRMs in the ER. A proximity ligation assay, as well as co-immunoprecipitation, confirmed the association of erlin-2 with γ-secretase. We found that a higher proportion of erlin-2 was associated with γ-secretase in DRMs than in soluble membranes. siRNA experiments indicated that reduced levels of erlin-2 resulted in a decreased Aβ production, whereas the effect on Notch processing was limited. In summary, we have found a novel brain GSAP, erlin-2, that resides in DRMs and affects Aβ production.

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

P2-279 AFFINITY PULLDOWN OF GAMMA-SECRETASE AND ASSOCIATED PROTEINS FROM HUMAN AND RAT BRAIN Yasuhiro Teranishi, Ji-Yeun Hur, Hedvig Welander, Jenny Frånberg, Mikio Aoki, Bengt Winblad, Susanne Frykman, Lars O. Tjernberg, Pharmacology Research Laboratories Research Division, Dainippon Sumitomo Pharma Co., Ltd., Suita, Japan; Karolinska Institutet and Dainippon Sumitomo Pharma Alzheimer Center (KASPAC), Karolinska Institutet, Huddinge, Sweden; Genomic Science Laboratories, Functional Genomics Group, Dainippon Sumitomo Pharma Co., Ltd., Osaka, Japan. Contact e-mail: yasuhiro-teranishi@ds-pharma.co.jp

Human brain proteins showing neuron‐specific interactions with γ‐secretase

The transmembrane protease complex γ‐secretase is a key enzyme in Alzheimer disease pathogenesis as it liberates the neurotoxic amyloid β‐peptide (Aβ); however, the mechanism of regulation of its activity in various cell types and subcellular compartments is largely unknown. Several γ‐secretase inhibitors have been developed, but none have been released due to side‐effects that appear to arise from reduced processing of Notch, one of many γ‐secretase substrates. Hence, it is desirable to specifically inhibit Aβ production. In our previous studies, we have identified several γ‐secretase‐associated proteins (GSAPs) from brain, which affect Aβ production without having any major effects on Notch processing. In the present study using detergent‐resistant membranes prepared from brain, we have identified four GSAPs that affect Aβ production to a greater extent than Notch processing. We evaluated the interaction between GSAPs and γ‐secretase in various cell types and their mRNA expression in various human organs. Using an in situ proximity ligation assay, we demonstrated that many GSAPs showed considerably greater interaction with γ‐secretase in neurons than in human embryonic kidney cells stably over‐expressing APP, and showed that several GSAPs are highly expressed in human brain. This study underscores the importance of studying protein‐protein interactions in relevant cell types, and suggests that reducing Aβ production by interfering with brain‐ or neuron‐specific γ‐secretase/GSAP interactions may reduce the risk of unwanted side‐effects associated with treatment of Alzheimer disease.

P4-200: Active γ-secretase is enriched in synaptic membranes prepared from rat brain

Background: Synaptic degeneration is the first pathological hallmark in Alzheimer’s disease and the one that correlates best with disease progression. The mechanism for synaptic degeneration is unknown but a strong candidate as a causative agent is amyloid -peptide (A ) oligomers that may cause Ca dysregulation and oxidative stress. A is produced by the processing of the amyloid precursor protein (APP) by two sequential proteolytic events. The last cleavage is performed by a large membrane-bound protein complex called -secretase consisting of at least for components. In addition to APP, -secretase cleaves a large number of substrates which complicates its potential use as a theurapeutical target. In order to design specific -secretase inhibitors/ regulators it is important to increase the knowledge about this enzyme. Several studies have determined the subcellular localisation of -secretase to the late secretory-endosomal pathways using different cell-lines. However, the localization in brain has not been reported. Since the synapses seem to be particularly vulnerable for A toxicity we wanted to examine if -secretase is localized to the synapses. Methods: We prepared synaptic membranes and synaptic vesicles using sucrose gradients and synaptosomal lysis, and assayed these fractions for subcellular markers, -secretase components and -secretase activity as measured by AICD and A production. Results: The synaptic membrane fraction was enriched in PSD-95, a post-synaptic membrane marker, but also contained endosomal markers. The -secretase activity was dramatically enriched in this fraction, as assayed by endogenous AICD and A production. It is possible that the -secretase in synaptic membranes could be differently regulated compared to other parts of the brain and we speculate that synaptic membrane preparations could be useful for finding -secretase inhibitors/ regulators that specifically interacts with the synaptic A production. Conclusions: Gamma-secretase activity is enriched in synaptic membranes and high local production of A might contribute to the synaptic degeneration seen in Alzheimer’s disease.