PMC

(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 , 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 , 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).

(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).

(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 [, , ]. The integrity of the purified DCSV are illustrated here by electron 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 [, ].

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).

(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.

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 [, , ]. The DCSV contain cathepsin B that has been identified as a new alternative β-secretase [–], and the well-known β-secretase BACE1 [, ], an aspartyl protease [–]. The γ-secretase complex components are present in these DCSV [], composed of presenilins 1 and 2, nicastrin, Aph-1, and PEN-2 that together function as γ-secretase [, ]. The α-secretase ADAM10 protease is also present in DCSV []. 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.

(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 [], and recombinant non-glycosylated murine is observed at a molecular weight of 37–40 kDa on SDS-PAGE gels []. Glycosylation of endogenous QC is known to modify its apparent molecular weight [].