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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 components and activity in a discontinuous iodixanol gradient.Postmitochondrial supernatant from rat brain was layered on a 2.5–30% iodixanol gradient and fractions were collected from the bottom of the tube. A) Western blots showing the γ-secretase components, the substrate APP and the subcellular markers N-cadherin (plasma membrane), syntaxin 13 (early endosomes), γ-adaptin (trans-Golgi network), GM130 (cis-Golgi), ERGIC53 (ER-Golgi intermediate compartment) and KDEL (endoplasmatic reticulum). Each marker was analyzed in 3–4 gradients. B) Protein concentration in the fractions. Note that the protein concentration does not peak in the same fraction as the γ-secretase components or activity. The line is added just to guide the eye. C) Aβ40 production from endogenous substrate. The fractions were incubated over night at 37°C with or without L-685,458 and the Aβ40 concentration was measured by ELISA. Production was calculated as concentration without inhibitor minus concentration with inhibitor. D) Aβ40 production with added exogenous substrate (C99-FLAG). The fractions were incubated as above but with the addition of C99-FLAG. Due to slight discrepancies in the peak fraction between experiments which resulted in large standard deviations for each fraction, we have chosen to show a representative experiment rather than the mean value. The peak fraction was, however, always fraction 5, 6 or 7 and the enrichment in the peak fraction was always at least three-fold. Each experiment was repeated 5 times. E) Quantification of the subcellular markers in the different fractions. The fractions with highest γ-secretase activity are indicated by the dotted box. Mean values (% of optical density (OD) in the fraction with the highest density for each marker) are plotted (n = 3–4). Again, the standard deviations were high due to shifts in the gradient and have been removed to avoid a too disordered picture. The shift of fractions with the highest density of different markers also results in that the mean values don't reach 100% in most cases. The lines were added just to guide the eye.
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pone-0008948-g001: γ-Secretase components and activity in a discontinuous iodixanol gradient.Postmitochondrial supernatant from rat brain was layered on a 2.5–30% iodixanol gradient and fractions were collected from the bottom of the tube. A) Western blots showing the γ-secretase components, the substrate APP and the subcellular markers N-cadherin (plasma membrane), syntaxin 13 (early endosomes), γ-adaptin (trans-Golgi network), GM130 (cis-Golgi), ERGIC53 (ER-Golgi intermediate compartment) and KDEL (endoplasmatic reticulum). Each marker was analyzed in 3–4 gradients. B) Protein concentration in the fractions. Note that the protein concentration does not peak in the same fraction as the γ-secretase components or activity. The line is added just to guide the eye. C) Aβ40 production from endogenous substrate. The fractions were incubated over night at 37°C with or without L-685,458 and the Aβ40 concentration was measured by ELISA. Production was calculated as concentration without inhibitor minus concentration with inhibitor. D) Aβ40 production with added exogenous substrate (C99-FLAG). The fractions were incubated as above but with the addition of C99-FLAG. Due to slight discrepancies in the peak fraction between experiments which resulted in large standard deviations for each fraction, we have chosen to show a representative experiment rather than the mean value. The peak fraction was, however, always fraction 5, 6 or 7 and the enrichment in the peak fraction was always at least three-fold. Each experiment was repeated 5 times. E) Quantification of the subcellular markers in the different fractions. The fractions with highest γ-secretase activity are indicated by the dotted box. Mean values (% of optical density (OD) in the fraction with the highest density for each marker) are plotted (n = 3–4). Again, the standard deviations were high due to shifts in the gradient and have been removed to avoid a too disordered picture. The shift of fractions with the highest density of different markers also results in that the mean values don't reach 100% in most cases. The lines were added just to guide the eye.

Mentions: We prepared a 10 000×g supernatant from rat brain and loaded this fraction on a discontinuous 2.5 to 30% iodixanol gradient. The γ-sectretase components nicastrin, PS1-CTF, PS2-CTF, Aph-1aL and Pen-2 were enriched in fraction 5–7 of this gradient, corresponding to an iodixanol concentration of 7.5–15% (Figure 1A), while the highest protein concentration was found in lighter fractions (Figure 1B). The direct substrate for γ-secretase cleavage, the APP-CTFs co-fractionated with the γ-secretase components whereas the full-length APP was more widely distributed (Figure 1A). To measure γ-secretase activity, the fractions were incubated at 37°C with or without the γ-secretase inhibitor L-685,458 and assayed for endogenous Aβ40 production by ELISA (Figure 1C). Since it is possible that the substrate concentration is a limiting factor, we also assayed for total γ-secretase activity by the addition of the exogenous substrate C99-FLAG which corresponds to β-secretase cleaved APP (Figure 1D). To calculate the γ-secretase dependent Aβ40 production, the Aβ40 levels found in the presence of the γ-secretase inhibitor L-685,458 were subtracted from the levels found in the absence of the L-685,458. Unfortunately, we were not able to detect Aβ42 in this experimental setup. Both the endogenous Aβ40 production and the total γ-secretase activity were enriched in fractions 5–7, although the peak activity fraction varied slightly between experiments. We quantified the levels of the subcellular markers by western blotting in the different fractions and found that the marker that showed the best correlation with γ-secretase activity was the early endosomal marker syntaxin 13 (Figure 1E). In addition, the plasma membrane marker N-cadherin correlated well with γ-secretase activity. The ER-Golgi intermediate compartment marker, ERGIC-53, and the trans-Golgi network marker γ-adaptin had two peaks of which one correlated with γ-secretase activity and the other one was found in lighter fractions. The ER marker KDEL was found in heavier fractions whereas the cis-Golgi marker GM130 was found in slightly lighter fractions although some overlap with γ-secretase occurred also for these markers (Figure 1E). Thus, active γ-secretase co-fractionates with an endosomal/plasma membrane enriched fraction in an iodixanol gradient prepared from rat brain.


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 components and activity in a discontinuous iodixanol gradient.Postmitochondrial supernatant from rat brain was layered on a 2.5–30% iodixanol gradient and fractions were collected from the bottom of the tube. A) Western blots showing the γ-secretase components, the substrate APP and the subcellular markers N-cadherin (plasma membrane), syntaxin 13 (early endosomes), γ-adaptin (trans-Golgi network), GM130 (cis-Golgi), ERGIC53 (ER-Golgi intermediate compartment) and KDEL (endoplasmatic reticulum). Each marker was analyzed in 3–4 gradients. B) Protein concentration in the fractions. Note that the protein concentration does not peak in the same fraction as the γ-secretase components or activity. The line is added just to guide the eye. C) Aβ40 production from endogenous substrate. The fractions were incubated over night at 37°C with or without L-685,458 and the Aβ40 concentration was measured by ELISA. Production was calculated as concentration without inhibitor minus concentration with inhibitor. D) Aβ40 production with added exogenous substrate (C99-FLAG). The fractions were incubated as above but with the addition of C99-FLAG. Due to slight discrepancies in the peak fraction between experiments which resulted in large standard deviations for each fraction, we have chosen to show a representative experiment rather than the mean value. The peak fraction was, however, always fraction 5, 6 or 7 and the enrichment in the peak fraction was always at least three-fold. Each experiment was repeated 5 times. E) Quantification of the subcellular markers in the different fractions. The fractions with highest γ-secretase activity are indicated by the dotted box. Mean values (% of optical density (OD) in the fraction with the highest density for each marker) are plotted (n = 3–4). Again, the standard deviations were high due to shifts in the gradient and have been removed to avoid a too disordered picture. The shift of fractions with the highest density of different markers also results in that the mean values don't reach 100% in most cases. The lines were added just to guide the eye.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2812513&req=5

pone-0008948-g001: γ-Secretase components and activity in a discontinuous iodixanol gradient.Postmitochondrial supernatant from rat brain was layered on a 2.5–30% iodixanol gradient and fractions were collected from the bottom of the tube. A) Western blots showing the γ-secretase components, the substrate APP and the subcellular markers N-cadherin (plasma membrane), syntaxin 13 (early endosomes), γ-adaptin (trans-Golgi network), GM130 (cis-Golgi), ERGIC53 (ER-Golgi intermediate compartment) and KDEL (endoplasmatic reticulum). Each marker was analyzed in 3–4 gradients. B) Protein concentration in the fractions. Note that the protein concentration does not peak in the same fraction as the γ-secretase components or activity. The line is added just to guide the eye. C) Aβ40 production from endogenous substrate. The fractions were incubated over night at 37°C with or without L-685,458 and the Aβ40 concentration was measured by ELISA. Production was calculated as concentration without inhibitor minus concentration with inhibitor. D) Aβ40 production with added exogenous substrate (C99-FLAG). The fractions were incubated as above but with the addition of C99-FLAG. Due to slight discrepancies in the peak fraction between experiments which resulted in large standard deviations for each fraction, we have chosen to show a representative experiment rather than the mean value. The peak fraction was, however, always fraction 5, 6 or 7 and the enrichment in the peak fraction was always at least three-fold. Each experiment was repeated 5 times. E) Quantification of the subcellular markers in the different fractions. The fractions with highest γ-secretase activity are indicated by the dotted box. Mean values (% of optical density (OD) in the fraction with the highest density for each marker) are plotted (n = 3–4). Again, the standard deviations were high due to shifts in the gradient and have been removed to avoid a too disordered picture. The shift of fractions with the highest density of different markers also results in that the mean values don't reach 100% in most cases. The lines were added just to guide the eye.
Mentions: We prepared a 10 000×g supernatant from rat brain and loaded this fraction on a discontinuous 2.5 to 30% iodixanol gradient. The γ-sectretase components nicastrin, PS1-CTF, PS2-CTF, Aph-1aL and Pen-2 were enriched in fraction 5–7 of this gradient, corresponding to an iodixanol concentration of 7.5–15% (Figure 1A), while the highest protein concentration was found in lighter fractions (Figure 1B). The direct substrate for γ-secretase cleavage, the APP-CTFs co-fractionated with the γ-secretase components whereas the full-length APP was more widely distributed (Figure 1A). To measure γ-secretase activity, the fractions were incubated at 37°C with or without the γ-secretase inhibitor L-685,458 and assayed for endogenous Aβ40 production by ELISA (Figure 1C). Since it is possible that the substrate concentration is a limiting factor, we also assayed for total γ-secretase activity by the addition of the exogenous substrate C99-FLAG which corresponds to β-secretase cleaved APP (Figure 1D). To calculate the γ-secretase dependent Aβ40 production, the Aβ40 levels found in the presence of the γ-secretase inhibitor L-685,458 were subtracted from the levels found in the absence of the L-685,458. Unfortunately, we were not able to detect Aβ42 in this experimental setup. Both the endogenous Aβ40 production and the total γ-secretase activity were enriched in fractions 5–7, although the peak activity fraction varied slightly between experiments. We quantified the levels of the subcellular markers by western blotting in the different fractions and found that the marker that showed the best correlation with γ-secretase activity was the early endosomal marker syntaxin 13 (Figure 1E). In addition, the plasma membrane marker N-cadherin correlated well with γ-secretase activity. The ER-Golgi intermediate compartment marker, ERGIC-53, and the trans-Golgi network marker γ-adaptin had two peaks of which one correlated with γ-secretase activity and the other one was found in lighter fractions. The ER marker KDEL was found in heavier fractions whereas the cis-Golgi marker GM130 was found in slightly lighter fractions although some overlap with γ-secretase occurred also for these markers (Figure 1E). Thus, active γ-secretase co-fractionates with an endosomal/plasma membrane enriched fraction in an iodixanol gradient prepared from rat brain.

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