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

Frykman S, Hur JY, Frånberg J, Aoki M, Winblad B, Nahalkova J, Behbahani H, Tjernberg LO - PLoS ONE (2010)

Bottom Line: In cell lines, active gamma-secretase has been found to localize primarily to the Golgi apparatus, endosomes and plasma membranes.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Care Sciences and Society (NVS), Karolinska Institutet Dainippon Sumitomo Pharma Alzheimer Center, Novum, Huddinge, Sweden. susanne.frykman@ki.se

ABSTRACT

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.

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Related in: MedlinePlus

γ-Secretase activity in synaptic fractions.A) Aβ40 production from endogenous substrate was assessed by incubating the fractions at 37°C for 16 h with or without the γ-secretase inhibitor L-685,458. The Aβ40 levels were analyzed by ELISA and the levels in the samples with L-685,458 were subtracted from the levels without L-685,458. B) Aβ40 production after the addition of 20 ng of an exogenous substrate, C99-FLAG, was obtained as above with the difference that 20 ng of C99-FLAG was added prior to incubation. C) AICD production was assessed as above and the samples without L-685,458 were analyzed with immunoblotting. D) Degradation of AICD was investigated by adding synthetic AICD to the sample and incubating at 37°C for 16 h in the presence of L-685,458. H, homogenate; SM, synaptic membranes; SV, synaptic vesicles; P3, 100 000×g pellet; C = control (buffer and AICD only). Data are presented as mean values +/− SD (n = 4). *, p<0.05; **, p<0.01.
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pone-0008948-g003: γ-Secretase activity in synaptic fractions.A) Aβ40 production from endogenous substrate was assessed by incubating the fractions at 37°C for 16 h with or without the γ-secretase inhibitor L-685,458. The Aβ40 levels were analyzed by ELISA and the levels in the samples with L-685,458 were subtracted from the levels without L-685,458. B) Aβ40 production after the addition of 20 ng of an exogenous substrate, C99-FLAG, was obtained as above with the difference that 20 ng of C99-FLAG was added prior to incubation. C) AICD production was assessed as above and the samples without L-685,458 were analyzed with immunoblotting. D) Degradation of AICD was investigated by adding synthetic AICD to the sample and incubating at 37°C for 16 h in the presence of L-685,458. H, homogenate; SM, synaptic membranes; SV, synaptic vesicles; P3, 100 000×g pellet; C = control (buffer and AICD only). Data are presented as mean values +/− SD (n = 4). *, p<0.05; **, p<0.01.

Mentions: We found high Aβ40 production, assessed as above, from endogenous APP-derived substrates (Figure 3A) as well as from added C99-FLAG (Figure 3B) in both synaptic membranes and synaptic vesicles. The activity was clearly enriched as compared to the homogenate and the endogenous production was higher than in P3 (100 000×g pellet). P3 showed low Aβ40 production in this experiment but the AICD production was increased compared to homogenate (Figure 3C). Intriguingly, when we investigated the AICD production, we could not detect any AICD in synaptic vesicles whereas the AICD production in synaptic membranes was high (Figure 3C). There were no detectable AICD levels in the samples treated with L-685,458 (data not shown). To investigate whether the absence of AICD was due to AICD degradation, we incubated synthetic AICD with synaptic membranes, synaptic vesicles or the P3 pellet in presence of L-685,458. Whereas there was no degradation of AICD in synaptic membranes or in P3, the levels of AICD in synaptic vesicles was indeed dramatically decreased (Figure 3D). Regarding the intermediate purification steps, the production of both Aβ and AICD was low in the P2 and synaptosome fraction and similar to synaptic membranes in the LP1 fraction (data not shown).


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

Frykman S, Hur JY, Frånberg J, Aoki M, Winblad B, Nahalkova J, Behbahani H, Tjernberg LO - PLoS ONE (2010)

γ-Secretase activity in synaptic fractions.A) Aβ40 production from endogenous substrate was assessed by incubating the fractions at 37°C for 16 h with or without the γ-secretase inhibitor L-685,458. The Aβ40 levels were analyzed by ELISA and the levels in the samples with L-685,458 were subtracted from the levels without L-685,458. B) Aβ40 production after the addition of 20 ng of an exogenous substrate, C99-FLAG, was obtained as above with the difference that 20 ng of C99-FLAG was added prior to incubation. C) AICD production was assessed as above and the samples without L-685,458 were analyzed with immunoblotting. D) Degradation of AICD was investigated by adding synthetic AICD to the sample and incubating at 37°C for 16 h in the presence of L-685,458. H, homogenate; SM, synaptic membranes; SV, synaptic vesicles; P3, 100 000×g pellet; C = control (buffer and AICD only). Data are presented as mean values +/− SD (n = 4). *, p<0.05; **, p<0.01.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2812513&req=5

pone-0008948-g003: γ-Secretase activity in synaptic fractions.A) Aβ40 production from endogenous substrate was assessed by incubating the fractions at 37°C for 16 h with or without the γ-secretase inhibitor L-685,458. The Aβ40 levels were analyzed by ELISA and the levels in the samples with L-685,458 were subtracted from the levels without L-685,458. B) Aβ40 production after the addition of 20 ng of an exogenous substrate, C99-FLAG, was obtained as above with the difference that 20 ng of C99-FLAG was added prior to incubation. C) AICD production was assessed as above and the samples without L-685,458 were analyzed with immunoblotting. D) Degradation of AICD was investigated by adding synthetic AICD to the sample and incubating at 37°C for 16 h in the presence of L-685,458. H, homogenate; SM, synaptic membranes; SV, synaptic vesicles; P3, 100 000×g pellet; C = control (buffer and AICD only). Data are presented as mean values +/− SD (n = 4). *, p<0.05; **, p<0.01.
Mentions: We found high Aβ40 production, assessed as above, from endogenous APP-derived substrates (Figure 3A) as well as from added C99-FLAG (Figure 3B) in both synaptic membranes and synaptic vesicles. The activity was clearly enriched as compared to the homogenate and the endogenous production was higher than in P3 (100 000×g pellet). P3 showed low Aβ40 production in this experiment but the AICD production was increased compared to homogenate (Figure 3C). Intriguingly, when we investigated the AICD production, we could not detect any AICD in synaptic vesicles whereas the AICD production in synaptic membranes was high (Figure 3C). There were no detectable AICD levels in the samples treated with L-685,458 (data not shown). To investigate whether the absence of AICD was due to AICD degradation, we incubated synthetic AICD with synaptic membranes, synaptic vesicles or the P3 pellet in presence of L-685,458. Whereas there was no degradation of AICD in synaptic membranes or in P3, the levels of AICD in synaptic vesicles was indeed dramatically decreased (Figure 3D). Regarding the intermediate purification steps, the production of both Aβ and AICD was low in the P2 and synaptosome fraction and similar to synaptic membranes in the LP1 fraction (data not shown).

Bottom Line: In cell lines, active gamma-secretase has been found to localize primarily to the Golgi apparatus, endosomes and plasma membranes.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Care Sciences and Society (NVS), Karolinska Institutet Dainippon Sumitomo Pharma Alzheimer Center, Novum, Huddinge, Sweden. susanne.frykman@ki.se

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus