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Pyroglutamate-amyloid-β and glutaminyl cyclase are colocalized with amyloid-β in secretory vesicles and undergo activity-dependent, regulated secretion - PubMed

Pyroglutamate-amyloid-β and glutaminyl cyclase are colocalized with amyloid-β in secretory vesicles and undergo activity-dependent, regulated secretion

Holger Cynis et al. Neurodegener Dis. 2014.

Abstract

Background and aims: N-truncated pyroglutamate (pGlu)-amyloid-β [Aβ(3-40/42)] peptides are key components that promote Aβ peptide accumulation, leading to neurodegeneration and memory loss in Alzheimer's disease. Because Aβ deposition in the brain occurs in an activity-dependent manner, it is important to define the subcellular organelle for pGlu-Aβ(3-40/42) production by glutaminyl cyclase (QC) and their colocalization with full-length Aβ(1-40/42) peptides for activity-dependent, regulated secretion. Therefore, the objective of this study was to investigate the hypothesis that pGlu-Aβ and QC are colocalized with Aβ in dense-core secretory vesicles (DCSV) for activity-dependent secretion with neurotransmitters.

Methods: Purified DCSV were assessed for pGlu-Aβ(3-40/42), Aβ(1-40/42), QC, and neurotransmitter secretion. Neuron-like chromaffin cells were analyzed for cosecretion of pGlu-Aβ, QC, Aβ, and neuropeptides. The cells were treated with a QC inhibitor, and pGlu-Aβ production was measured. Human neuroblastoma cells were also examined for pGlu-Aβ and QC secretion.

Results: Isolated DCSV contain pGlu-Aβ(3-40/42), QC, and Aβ(1-40/42) with neuropeptide and catecholamine neurotransmitters. Cellular pGlu-Aβ and QC undergo activity-dependent cosecretion with Aβ and enkephalin and galanin neurotransmitters. The QC inhibitor decreased the level of secreted pGlu-Aβ. The human neuroblastoma cells displayed regulated secretion of pGlu-Aβ that was colocalized with QC.

Conclusions: pGlu-Aβ and QC are present with Aβ in DCSV and undergo activity-dependent, regulated cosecretion with neurotransmitters.

© 2014 S. Karger AG, Basel.

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Figures

Figure 1
Figure 1. Secretory vesicles provide activity-dependent of neurotransmitters
(a) Neuronal secretion of neurotransmitters

. Secretory vesicles of neurons provide synthesis, storage, and regulated secretion of neuropeptides, catecholamines, and bioactive neurotransmitter molecules that are critical for cell-cell communication in the nervous system.

(b) Dense core secretory vesicles (DCSV) observed by electron microscopy

. Secretory vesicles of the dense core secretory vesicle type were isolated from chromaffin cells of bovine adrenal medulla tissue by differential sucrose gradient centrifugation, which results in a highly purified preparation of homogeneous DCSV [25, 28, 29]. The integrity of the purified DCSV are illustrated here by electron microscopy.

Figure 2
Figure 2. Glutaminyl cyclase (QC) activity and QC protein in dense core secretory vesicles
(a) QC activity in DCSV

. QC activity (black symbols) in purified dense core secretory vesicles (DCSV) of chromaffin cells (also known as chromaffin granules) was measured in time-course assays. Heat-inactivated secretory vesicles (red symbols) and omission of the auxiliary enzyme of the test (green symbols) showed no activity.

(b) QC enzyme protein in DCSV

. Purified DCSV were subjected to anti-QC western blots. Endogenous QC immunoreactive bands of ~60 kDa and 48 kDa were observed. Selectivity of anti-QC to detect these immunoreactive bands was demonstrated by conducting the western blot with only the secondary anti-rabbit serum (omitting the primary anti-QC serum), which resulted in the absence of immunoreactivity (unpublished data). It is noted that endogenous bovine pituitary QC has been observed with an apparent molecular weight on SDS-PAGE of ~40–45 kDa [59], and recombinant non-glycosylated murine is observed at a molecular weight of 37–40 kDa on SDS-PAGE gels [31]. Glycosylation of endogenous QC is known to modify its apparent molecular weight [102].

Figure 3
Figure 3. Activity-dependent co-secretion of pGluAβ and glutaminyl cyclase (QC) from neuronal-like chromaffin cells
(a) Regulated secretion of pGlu-Aβ

. Regulated secretion was induced by KCl depolarization and by nicotine treatment of neuronal-like chromaffin cells (90 min. incubation time), with inclusion of unstimulated controls. The secretion media was collected for measurement of pGlu-Aβ(3-40).

(b) Regulated secretion of QC activity

. The secretion media from the experiments of figure 3a were collected and measured for QC activity. *Statistically significant for comparison of stimulated cells (by KCl or nicotine) compared to control unstimulated cells (p < 0.05, student’s t-test).

Figure 4
Figure 4. Activity-dependent, regulated secretion of Aβ and peptide neurotransmitters
(a and b) Regulated secretion of Aβ(1-40) and Aβ(1-42).

Regulated secretion from chromaffin cells was stimulated by KCl depolarization and by nicotine, as conducted in fig. 3, and Aβ(1-40) and Aβ(1-42) in the secretion media were measured.

(c and d) Regulated secretion of (Met)enkephalin and galanin peptide neurotransmitters

. Regulated secretion from chromaffin cells was stimulated by KCl depolarization and by nicotine, as conducted in fig. 3, and the neuropeptides (Met)enkephalin and galanin in the secretion media were measured. +Statistically significant for comparison of stimulated cells (by KCl or nicotine) compared to control unstimulated cells (p < 0.05, student’s t-test).

Figure 5
Figure 5. Inhibitor of QC reduces pGluAβ released from the regulated secretory pathway

Chromaffin cells were incubated with or without the QC inhibitor PQ529 for 18 hrs. Cells were then subjected to regulated secretion induced by KCl depolarization (for 90 min.), and controls consisted of unstimulated cells. The secretion media was collected and measured for concentrations of pGlu-Aβ(3-40). Data show that the inhibitor substantially reduced the amount of pGlu-Aβ(3-40) released from the regulated secretory pathway, representing activity-dependent secretion. **Statistically significant comparison of PQ529 and control (without PQ529) treated cells undergoing KCl depolarization induced secretion of pGlu-Aβ(3-40) (p < 0.05, student’s t-test). *Statistically significant comparison of KCl and control cells (without inhibitor treatment) with respect to secretion of pGlu-Aβ(3-40) (p < 0.05, student’s t-test).

Figure 6
Figure 6. Cellular pGluAβ and glutaminyl cyclase (QC) are co-localized with the enkephalin neurotransmitter present in secretory vesicles, analyzed by immunofluorescence microscopy
(a) pGlu-Aβ and enkephalin localization

. The localization of pGlu-Aβ (green fluorescence) and (Met)enkephalin (red fluorescence) were assessed by immunofluorescent deconvolution microscopy. The merged images display areas of co-localization (yellow fluorescence, as shown by the arrows).

(b) QC and enkephalin localization

. The subcellular localization of QC (green fluorescence) with enkephalin (red fluorescence) present in secretory vesicles is illustrated by immunofluorescence microscopy. The merged images (yellow fluorescence) illustrate co-localization of QC and enkephalin (examples of co-localization are shown by arrows).

(c) QC and 6E10 APP/Aβ localization

. The localization of QC (green fluorescence) and 6E10 immunoreactivity (red fluorescence), representing Aβ- and APP-related forms, in chromaffin cells was assessed by immunofluorescence microscopy. The merged images illustrate areas of co-localization (yellow fluorescence). Controls that omitted the primary antisera and used only the secondary fluorescence-labelled antisera resulted in absence of immunofluorescence signals. The 6E10 antibody, generated to antigen Aβ(1-16), recognizes various Aβ and APP-related forms [103, 104].

Figure 7
Figure 7. IMR32 neuroblastoma cells: activity-dependent co-secretion of pGluAβ with Aβ peptides
(a) pGlu-Aβ(3-40) secretion stimulated by KCl depolarization

. Human IMR32 neuroblastoma cells were subjected to secretion induced by KCl depolarization (90 min.), and pGlu-Aβ(3-40) in the secretion media was measured by ELISA. *Statistically significant for KCl compared to control (unstimulated) (p < 0.05, student’s t-test).

(b) Aβ(1-40) secretion induced by KCl

. Aβ(1-40) was measured in secretion media of cells subjected to KCl depolarization, as described in part 8a. *Statistically significant for KCl compared to control (unstimulated) (p < 0.05, student’s t-test).

(c) Aβ(1-42) secretion induced by KCl

. Aβ(1-42) was measured in secretion media of cells subjected to KCl depolarization, as described in part 8a. *Statistically significant for KCl compared to control (unstimulated) (p < 0.05, student’s t-test).

(d) Colocalization of pGlu-Aβ and QC

. The subcellular localization of pGluAβ (red fluorescence) and QC (green fluorescence) in the human IMR32 neuroblastoma cells was assessed by immunofluorescence microscopy. Merged images illustrate co-localization of pGlu-Aβ and QC. Controls that omitted the primary antisera and used only the secondary fluorescence-labelled antisera resulted in absence of immunofluorescence signals for pGlu-Aβ and QC.

Figure 8
Figure 8. pGlu-Aβ and Aβ peptides with QC and the APP processing machinery in secretory vesicles containing neurotransmitters

The secretory vesicles (DCSV type) isolated from model neuronal-like chromaffin cells were demonstrated in this study to contain pGlu-Aβ(3-40/42) and QC, combined with Aβ(1-40/42). Prior studies indicate the presence of β-secretases, γ-secretase complex, and α-secretase in DCSV [58, 59, 62]. The DCSV contain cathepsin B that has been identified as a new alternative β-secretase [–61], and the well-known β-secretase BACE1 [58, 62], an aspartyl protease [–57]. The γ-secretase complex components are present in these DCSV [58], composed of presenilins 1 and 2, nicastrin, Aph-1, and PEN-2 that together function as γ-secretase [63, 64]. The α-secretase ADAM10 protease is also present in DCSV [58]. These findings illustrate the presence of the APP processing machinery for production of pGlu-Aβ and Aβ peptides in neurotransmitter secretory vesicles containing neuropeptides and catecholamines.

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