PyroGlu-Aβ and Glutaminyl Cyclase are Co-Localized with Aβ in Secretory Vesicles and Undergo Activity-Dependent Secretion

Abstract

Background and Aims

N-truncated pGlu-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 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, and their localization with full-length Aβ(1-40/42) peptides for regulated secretion. Therefore, the objective of this study was to investigate the hypothesis that pGlu-Aβ and glutaminyl cyclase (QC) are co-localized with Aβ in secretory vesicles (DCSV) for activity-dependent secretion with neurotransmitters.

Methods

Purified DCSV was assessed for pGlu-Aβ(3-40/42), Aβ(1-40/42), QC, and neurotransmitters. Neuronal-like chromaffin cells were analyzed for co-secretion of pGlu-Aβ, QC, Aβ, and neuropeptides. Cells were treated with a QC inhibitor and pGlu-Aβ was measured. Human neuroblastoma cells were also examined for pGlu-Aβ and QC.

Results

Isolated DCSV contain pGlu-Aβ(3-40/42), QC, and Aβ(1-40/42) with neuropeptide and catecholamine neurotransmitters. Cellular pGlu-Aβ(3-40/42) and QC undergo activity-dependent co-secretion with Aβ(1-40/42) and enkephalin and galanin neurotransmitters. A QC inhibitor decreased levels of pGlu-Aβ. Human neuroblastoma cells displayed regulated secretion of pGlu-Aβ that is co-localized with QC.

Conclusions

PyroGlu-Aβ and QC are present with Aβ in DCSV, and undergo activity-dependent, regulated co-secretion with neurotransmitters.

Introduction

Accumulation of neurotoxic β-amyloid peptides (Aβ) in brain represents a key factor in the development of memory deficits in Alzheimer’s disease (AD) [1–4]. Aβ peptides of multiple forms are present in AD brains. Notably, affected AD brains contain the N-terminally truncated pyroglutamate (pGlu-) forms of Aβ(3-40/42) (pGlu-Aβ) as a major portion of total Aβ peptides, which includes the known full-length Aβ(1-40/42) peptides [5–7]. The accumulation of pGlu-Aβ(3-40/42) in brain occurs before that of the Aβ(1-40/42) peptides [5]. Significantly, pGlu-Aβ facilitates the seeding of Aβ peptides into neurotoxic oligomers [8] that are thought to participate in development of memory deficits in AD [9]. Increasing brain levels of pGluAβ(3-42) results in aggravated memory deficits and amyloid plaque accumulation in the 5XFAD mouse model of AD [10], illustrating the key role of N-truncated pGlu-modified forms of Aβ in AD.

The pGlu-Aβ(3-40/42) peptides start with N-terminal glutamate, the third amino-terminal residue of Aβ(1-40/42). Notably, this residue is converted to pGlu by the enzyme glutaminyl cyclase (QC) [8, 11, 12]. Glutaminyl cyclase is elevated in AD brains [5–7]. Moreover, inhibition of QC [13, 14] results in decreased brain pGlu-Aβ, and QC gene knockout results in decreased brain pGlu-Aβ with improved behavioral deficits in 5XFAD mice [12]. These findings indicate that glutaminyl cyclase is involved in development of AD via formation of pGlu-Aβ.

Aβ peptides have been demonstrated to be released from brain neurons in an electrical activity-dependent manner [15–19]. Neural activity modulates the formation and secretion of Aβ [20, 21]. Recent evidence indicates that endogenous neuronal activity regulates the regional brain vulnerability to Aβ deposition [22]. Significantly, neuronal activity is fundamental for regulated secretion of neurotransmitters [23, 24]. These features of Aβ peptides raise the question of whether pGlu-Aβ and its biosynthetic enzyme QC are co-secreted with Aβ and neurotransmitters in a regulated, activity-dependent manner. Therefore, this study investigated the hypothesis that co-secretion of truncated pGlu-Aβ and full-length Aβ occurs with neurotransmitters stored and released from secretory vesicles, resulting in extracellular Aβ peptide forms. These studies utilized neuronal chromaffin cells that display activity-dependent secretion of neurotransitters and also produce Aβ [17, 19], as well as human IMR32 neuroblastoma cells.

Results show that secretory vesicles of the dense core secretory vesicle (DCSV) type contain pGlu-Aβ with QC and Aβ, combined with catecholamine and peptide neurotransmitters. Cellular pGlu-Aβ and QC undergo co-secretion from the regulated secretory pathway of neuronal-like chromaffin cells stimulated by nicotine or KCl depolarization. pGlu-Aβ also undergoes regulated co-secretion with Aβ and the peptide neurotransmitters (Met)enkephalin and galanin peptide. Treatment of cells with a QC inhibitor resulted in decreased levels of pGlu-Aβ released from the regulated secretory pathway. Furthermore, human neuroblastoma cells displayed regulated co-secretion of pGlu-Aβ(3-40) with Aβ(1-40/42), combined with co-localization of pGlu-Aβ and QC. These findings support the hypothesis that pGlu-Aβ and QC undergo co-secretion with Aβ and peptide neurotransmitters from the regulated secretory pathway.

Experimental Procedures

Content of pGlu-Aβ, QC, Aβ peptides, and neurotransmitters in dense core secretory vesicles (DCSV)

Secretory vesicles were purified from fresh bovine adrenal medulla to evaluate the content of QC, pGlu-Aβ, Aβ peptides, (Met)enkephalin and galanin peptide neurotransmitters, and the catecholamine neurotransmitters dopamine, norepinephrine, and epinephrine. These dense core secretory vesicles (DCSV), also known as chromaffin granules (CG), were isolated by differential sucrose density gradient centrifugation as previously described [25–29]. The density gradient isolation procedure has been established to yield secretory vesicles of high purity based on assessment of organelle markers and electron microscopy [25–29]. The procedure results in purified secretory vesicles that lack biochemical markers for other subcellular organelles of lysosomes (acid phosphatase marker), cytoplasm (lactate dehydrogenase marker), mitochondria (fumarase and glutamate dehydrogenase markers), and endoplasmic reticulum (glucose-6-phosphatase marker). Enzyme markers in the purified secretory vesicle preparation represent less than 1% of total homogenate markers. Furthermore, electron microscopy and enzyme markers have confirmed the integrity and purity of the isolated DCSV [25–29].

The presence of QC in the isolated DCSV was assessed by monitoring of QC enzymatic activity as we have described [30–32]. QC in chromaffin granules was subjected to immunoprecipitation with anti-QC, conducted as we have described [33], followed by western blot with anti-QC sera (rabbit) performed by western blot procedures as we have described previously [25, 33–35].

For measurement of Aβ peptides and neurotransmitters, purified secretory vesicles were lysed by freeze-thawing in buffer (50 mM Na-acetate, 50 mM NaCl, 1 mM EDTA) containing a cocktail of protease inhibitors (pepstatin A, leupeptin, chymostatin, and E64c at 10 μM each, and PMSF at 500 μM). An acid extract (0.1 N acetic acid) was prepared from the lysed secretory vesicles (as described previously [25, 26, 34, 35]) for measurement of pGlu-Aβ(3-40), pGlu-Aβ(3-42), Aβ(1-40), Aβ(1-42), (Met)enkephalin, and galanin, and the catecholamines dopamine, norepinephrine, and epinephrine. DCSV samples were subjected to ELISA measurements of pGlu-Aβ(3-40) (#27418, IBL International, Toronto, Canada), pGlu-Aβ(3-42) (#27716, IBL), Aβ(1-40) (#27718, IBL), Aβ(1-42) (#27712, IBL), with RIA assays for (Met)enkephalin and galanin (RIAs were conducted as previously reported [28, 33, 35]. The well-established ELISA kits have been characterized to demonstrate their specificities for the particular peptides (by the manufacturer, IBL). The ELISA assays for Aβ(1-40) and Aβ(1-42) do not crossreact with each other nor to the pGlu-Aβ peptides, the ELISA assay for pGlu-Aβ(3-40) does not detect pGlu-Aβ(3-42) or Aβ(1-40/42), and the ELISA assay for pGlu-Aβ(3-42) specifically detects this N-truncated peptide rather than pGlu-Aβ(3-40) and does not detect Aβ(1-40/42). Glutaminyl cyclase activity was measured in the media, as we have described [30–32], using H-Gln-βNA as substrate in a fluorometric assay. Dopamine, norepinephrine, and epinephrine catecholamines were measured by radioenzymatic assays as we have previously described [36, 37]. Protein content of the purified DCSV was measured by the Bio-Rad DC protein assay (Lowry) kit (Biorad, Hercules, CA). Contents of Aβ peptides and neurotransmitters were expressed as pg per μg protein.

Neuronal-like chromaffin cells in primary culture

Neuronal-like chromaffin cells in primary culture are obtained from the adrenal medulla of the sympathetic nervous system. Chromaffin cells were prepared from fresh adrenal medulla tissue (bovine) as we have previously described [25, 35, 38]. Briefly, chromaffin cells were dissected from fresh adrenal glands, dissociated in a collagenase/DNase solution at 37°C, filtered, and centrifuged. Cells were plated onto fibronectin coated dishes (EMD Chemicals, Gibbston, NJ, USA) in media containing Dulbecco’s Modified Eagle Medium (DMEM) (Cellgro, Manassas, VA, USA), 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA) and penicillin/streptomycin. Cells were maintained at 37°C and 6% CO₂.

Regulated secretion of pGlu-Aβ, QC, Aβ, and peptide neurotransmitters from chromaffin cells

After six days in culture, chromaffin cells were subjected to KCl depolarization or treatment with nicotine to stimulate regulated, activity-dependent secretion. Cells were incubated in standard release medium (SRM, as described [28] with 0.25 μg/ml BSA (bovine serum albumin) for 90 minutes at 37° C, followed by removal of media, which represents basal secretion. Cells were then stimulated in SRM media containing KCl (50 mM) or nicotine (10 μM) for 90 minutes (37° C), followed by collection of media representing regulated secretion. Secretion media is collected with addition of a cocktail of protease inhibitors (1 mM EDTA, 500 μM AEBSF, 10 μM each of E64c, leupeptin, pepstatin A, and chymostatin). The secretion media was concentrated by ultrafiltration through a 2 kDa cut-off membrane (Vivaspin 2 2K MWCO Hydrosart, Sartorius, Goettingen, Germany) which retains Aβ. The concentrated sample was subjected to measurements of pGlu-Aβ(3-40), pGlu-Aβ(3-42), Aβ(1-40), Aβ(1-42), (Met)enkephalin, and galanin as described above for DCSV. Glutaminyl cyclase activity was measured in the media, as we have described previously [29, 30], using H-Gln-βNA as substrate in a fluorometric assay.

Inhibition of endogenous QC in chromaffin cells and levels of secreted pGlu-Aβ(3-40)

Chromaffin cells were incubated with the QC inhibitor PQ529 (Probiodrug AG, Halle, Germany) at 50 μM for 18 hours. Cells were then subjected to regulated secretion induced by treating cells with KCl (50 mM) for 90 minutes. Control incubation without KCl was also included. Cell culture media was then collected for measurement of pGlu-Aβ(3-40) by ELISA (as described above).

Cellular immunofluorescence localization of pGlu-Aβ and QC with (Met)enkephalin neurotransmitter present in secretory vesicles

Immunofluorescence microscopy of chromaffin cells was conducted to assess the sub-cellular localization of pGlu-Aβ and QC, compared to (Met)enkephalin present in dense core secretory vesicles (DCSV) that undergoe regulated secretion. Chromaffin cells in culture were fixed for immunofluorescence deconvolution microscopy, conducted as previously described [33–35]. The primary antibodies used for immunocytochemistry were rabbit anti-QC 1301 (1:250, Probiodrug AG, Halle, Germany), mouse anti-(Met)enkephalin (1:100, Abcam #23503, Cambridge, MA), mouse anti-pGlu-Aβ (1:50, Probiodrug, Halle, Germany) detecting the N-terminus of both pGlu-Aβ(3-40) and pGlu-Aβ(3-42), rabbit anti-Met-enkephalin (1:50, Millipore, Billerica, MA), and mouse anti-APP clone 6E10 (1:100, Covance, Princetown, NJ). The primary antibodies were detected with the secondary antibodies anti-rabbit IgG-Alexa Fluor 594 (goat) (1:200 dilution, red fluorescence, Molecular Probes, Eugene, Oregon) and with anti-mouse IgG Alexa Fluor 488 (goat) (1:200 dilution, green fluorescence, Molecular Probes, Eugene, Oregon). Immunofluorescent images were analyzed by the Delta Vision Spectris Image Deconvolution Systems on an Olympus IX70 epi-fluorescence microscope using the software Softworx Explorer from Applied Precision, as we have described [33, 39]. As control, immunostaining with only secondary antibody was performed; these controls demonstrated a lack of immunofluorescence, indicating the specificity of the primary immunoreactivity observed.

Human IMR neuroblastoma cells and regulated secretion of pGlu-Aβ with Aβ peptides

Human IMR32 neuroblastoma cells were obtained from ATCC (American Type Tissue Culture, Crystal City, VA) and cultured according to the manufacturer’s instructions. Activity-dependent secretion was achieved by stimulating secretion by incubation with KCl (50 mM) in the media for 90 minutes. Control secretion for 90 minutes without KCl was included. The secretion media was collected, concentrated, and subjected to measurements of pGlu-Abeta(3-40), Aβ(1-40), and Aβ(1-42) (ELISA kits from IBL). Cellular localization of pGlu-Aβ and QC was conducted by immunofluorescence confocal microscopy, as described above for chromaffin cells.

Statistical analyses of data

Secretion of Aβ peptides and neurotransmitters were conducted in triplicate in each experiment, and experiments were repeated three times. Data were assessed for statistical signficance at p < 0.05 by the student’s t-test.

Results

Secretory vesicles contain pGlu-Aβ and QC, with Aβ with neurotransmitters

Several studies in the field demonstrate that Aβ undergoes activity-dependent, regulated secretion from neurons [15–18], implicating release from secretory vesicles that store and secrete neurotransmitters (Fig. 1a). Therefore, studies here investigated the hypothesis that pGlu-Aβ and its biosynthetic enzyme glutaminyl cyclase may be present with Aβ and neurotransmitters in secretory vesicles for extracellular release.

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.

Dense core secretory vesicles (DCSV) of neuronal-like chromaffin cells store neurotransmitter contents for activity-dependent, regulated secretion, and were isolated for this study. DCSV were purified by sucrose density gradient centrifugation; the integrity of the isolated DCSV was illustrated by electron microscopy (Fig. 1b). Previous reports have documented the high purity of these isolated DCSV conducted by assessing enzyme markers for subcellular organelles [25, 28, 29].

The isolated DCSV contain pGlu-Aβ(3-40), pGlu-Aβ(3-42), Aβ(1-40), and Aβ(1-42), quantitated by ELISA assays (Table 1). The concentrations of pGlu-Aβ(3-40) and pGlu-Aβ(3-42) (at approximately 0.022 pg/mg and 0.070 pg/mg protein) were observed at 10% and 82% of the concentrations of Aβ(1-40) and Aβ(1-42), respectively. Moreover, the DCSV contain enkephalin and galanin peptide neurotransmitters, as well as the catecholamines dopamine, norepinephrine, and epinephrine (Table 1). These data show that the DCSV contain pGlu-Aβ and Aβ peptides with peptide and catecholamine neurotransmitters.

Table 1.

Aβ Peptides and Neurotransmitters in Purified Dense Core Secretory Vesicles

Secretory Vesicle Component	Concentration
Beta-amyloid peptides:	Content (pg/mg protein)
pGluAβ(3-40)	0.022 ± 0.004
pGluAβ(3-42)	0.070 ± 0.009
Aβ(1-40)	0.220 ± 0.010
Aβ(1-42)	0.085 ± 0.004
Peptide Neurotransmitters:	Content (pg/mg protein)
(Met)enkephalin	215,000 ± 14.4
Galanin	408 ± 0.015
Catecholamine Neurotransmitters:	Content (pg/mg protein)
Dopamine	1,150 ± 0.13
Norepinephrine	4,560 ± 0.34
Epinephrine	6,490 ± 1.02

The concentrations of Aβ peptides and neurotransmitters in purified chromaffin dense core secretory vesicles were measured by assays as described in the methods.

The presence of QC activity in the isolated DCSV was demonstrated by an activity assay using Gln-βNA as substrate. The time-dependent formation of the QC product is illustrated (Fig. 2a). Heat inactivation as well as omitting the auxiliary enzyme of the assay underlined the specificity of the test. Furthermore, QC enzyme protein is present in DCSV, observed by anti-QC western blots (Fig. 2b).

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].

pGlu-Aβ and QC undergo activity-dependent, regulated secretion with Aβ and peptide neurotransmitters from neuronal-like chromaffin cells

Activity-dependent secretion of neurotransmitters from neuronal-like chromaffin cells was stimulated by KCl depolarization which represents regulated secretion and by nicotine which activates the nicotinic cholinergic receptor [38, 40–42]. KCl depolarization and nicotine activation of the cholinergic receptor of neuronal-like chromaffin cells are utilized in the field to assess activity-dependent, regulated secretion [35, 40, 43–50]. KCl depolarization and nicotine stimulated the secretion of pGlu-Aβ(3-40) (Fig. 3a). Stimulated secretion (with KCl) was several-fold greater than basal secretion (no KCl). High KCl and nicotine stimulated the release of ~3–4% of total cellular Aβ(3-40) (data not shown), but KCl and nicotine stimulated the predominate portion of secreted Aβ(3-40) compared to the condition of basal constitutive secretion. Notably, QC, the biosynthetic enzyme for pGlu-Aβ, undergoes activity-dependent co-secretion with pGlu-Aβ(3-40) (Fig. 3b).

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).

Furthermore, KCl and nicotine stimulated the secretion of Aβ(1-40) and Aβ(1-42) (Fig. 4). Because regulated, activity-dependent secretion is fundamental for release of neurotransmitters, the peptide neurotransmitters (Met)enkephalin and galanin were evaluated in these experiments. (Met)enkephalin and galanin were secreted in an activity-dependent manner, stimulated by KCl depolarization or nicotine (Fig. 4). These data illustrate the co-secretion of pGlu-Aβ(3-40) and QC activity with Aβ(1-40/42) that are co-secreted with the neurotransmitters (Met)enkephalin and galanin.

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).

Inhibition of endogenous QC reduces pGlu-Aβ in the regulated secretory pathway

The role of QC in the production of pGlu-Aβ was evaluated with the QC inhibitor PQ529 [51]. Chromaffin cells were treated with PQ529 (50 μM) for 18 hours, and then subjected to regulated secretion stimulated by KCl depolarization. Results demonstrated that PQ529 significantly reduced the amounts of pGlu-Aβ(3-40) released via the regulated secretory pathway (Fig. 5). Basal, constitutive levels of secreted pGlu-Aβ(3-40) were not reduced by PQ529, although a modest, insignificant decrease with PQ529 was observed. These findings support the role of QC for producing pGlu-Aβ in DCSV via the regulated secretory pathway that provides activity-dependent secretion.

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).

Cellular localization of pGlu-Aβ and QC with enkephalin in secretory vesicles

Neurotransmitter and neurosecretory components that undergo regulated secretion are stored in secretory vesicles. Therefore, the localization of pGlu-Aβ and QC, with the (Met)enkephalin neurotransmitter was demonstrated by immunofluorescence confocal microscopy (Fig. 6). Data show the cellular co-localization of pGlu-Aβ with (Met)enkephalin in chromaffin cells (Fig. 6a). The punctate pattern of pGlu-Aβ subcellular localization coincides with that of (Met)enkephalin that is present in secretory vesicles, shown by their merged yellow immunofluoresence (Fig. 6a). Furthermore, QC is also co-localized with enkephalin (Fig. 6b), indicating the presence of of both QC and pGlu-Aβ in enkephalin neurotransmitter-containing secretory vesicles. These data are consistent with the presence of pGlu-Aβ and QC in isolated secretory vesicles that contain neurotransmitters (Table 1 and Fig. 2).

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].

The localization of QC with Aβ- and APP-related (amyloid precursor protein) forms was assessed by the 6E10 antibody which detects Aβ- and APP-related immunoreactivites. Data show punctate localization of QC and 6E10 immunoreactivity showing co-localization (Fig. 6c). These results support the hypothesis for the presence of pGlu-Aβ and QC with APP- and Aβ-related peptide forms in secretory vesicles.

Human neuroblastoma cells display activity-dependent, regulated secretion of pGluAβ with Aβ peptides

Evaluation in a human neuroblastoma cell line (IMR32) was conducted to assess regulated secretion of pGlu-Aβ in human neuronal model. Secretion of pGlu-Aβ(3-40) was stimulated by KCl, compared to basal control cells (no KCl) (Fig. 7a). In addition, regulated co-secretion of Aβ(1-40) and Aβ(1-42) was stimulated by KCl (Fig. 7b, c). Immunofluorescence microscopy illustrated the co-localization of pGlu-Aβ with QC (Fig. 7d). PyroGlu-Aβ and QC are each observed in discrete, punctate patterns of subcellular distribution. Merging of their images displaed their co-localization (shown by the yellow immunofluorescence). These data demonstrate the regulated co-secretion of pGlu-Aβ with Aβ peptides, and co-localization of pGlu-Aβ with QC in human neuroblastoma.

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.

Discussion

This study demonstrates activity-dependent, regulated co-secretion of pGlu-Aβ and QC with Aβ and peptide neurotransmitters that are released from secretory vesicles. The isolated secretory vesicles (dense core secretory vesicle, DCSV, type) contain pGlu-Aβ with QC, full-length Aβ peptides, combined with peptide and catecholamine neurotransmitters. Data show that release of DCSV contents results in co-secretion of pGlu-Aβ with QC, Aβ, and peptide neurotransmitters from the regulated secretory pathway of neuronal-like chromaffin cells. Cellular immunofluorescence microscopy also illustrated co-localization of pGlu-Aβ with QC and enkephalin, and of QC with APP- and Aβ-related immunoreactivity. Treatment of cells with a QC inhibitor resulted in reduced levels of pGlu-Aβ released from the regulated secretory pathway, indicating the role of QC for production of pGlu-Aβ. Furthermore, human neuroblastoma cells display regulated secretion of pGlu-Aβ and Aβ, and pGlu-Aβ is co-localized with QC. These data demonstrate that pGlu-Aβ and QC undergo regulated co-secretion with Aβ from neurotransmitter secretory vesicles to provide extracellular pGlu-Aβ with Aβ that accumulate in AD brains.

The presence of pGlu-Aβ and QC with Aβ in neurotransmitter secretory vesicles raises the question of how are Aβ and pGlu-Aβ produced in this organelle? The reason for this question is that it may be hypothesized that APP and its processing secretases may be present in these secretory vesicles for production of Aβ, and for QC production of pGlu-Aβ (illustrated in Fig. 8). Our previous studies show that these secretory vesicles contain the amyloid precursor protein (APP) [17, 52, 53] and Aβ peptides, indicating the presence of secretases that convert APP to Aβ. Indeed, the β- and γ-secretases, are present in the secretory vesicles with APP. The β-secretase BACE1 [54–57] is present in DCSV [19, 58]. A wild-type β-secretase was recently identified as cathepsin B [59–61] and is present in DCSV [58, 59]. The presenilin 1 γ-secretase component is present in secretory vesicles [58, 62], as well as the nicastrin, Aph-1, and Pen-2 components [58] of the γ-secretase complex [63, 64]. Subsequent to the secretases, N-terminal truncation of full-length Aβ is predicted to occur to generate Aβ(3-40/42) that serves as substrate for QC to produce pGlu-Aβ(3-40/42) by QC [65–68]. It will be of interest in future studies to determine the relative levels of N-truncated Aβ peptides (Aβ(2-40/42) and Aβ(3-40/42) compared to pGlu-Aβ(3-40/42), utilizing mass spectrometry to distinguish these peptide species. Thus, the secretory vesicle contains the APP processing proteases that produce Aβ peptides, combined with QC for production of pGlu-Aβ(3-40/42) (Fig. 8). The range of pH conditions for activities of these secretases [69–74] coincide with the internal pH conditions of these secretory vesicles [75–77]. The secretory vesicle organelle, thus, contains the APP processing machinery for producing Aβ and pGlu-Aβ peptide forms.

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 [59–61], and the well-known β-secretase BACE1 [58, 62], an aspartyl protease [54–57]. 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.

These data support the hypothesis that pGlu-Aβ is produced by glutaminyl cyclase (QC) in secretory vesicles that release neurotransmitter components in a regulated, activity-dependent manner. QC converts the N-terminal glutamate of Aβ(3-40/42) peptides to pyroglutamate of pGluAβ(3-40/42). The QC substrates Aβ(3-40/42) are presumably produced by N-terminal truncation of Aβ(1-40/42). The presence of full-length Aβ(1-40/42) in secretory vesicles [19, 58, 59] is compatible with biosynthesis of pGlu-Aβ from Aβ in these secretory vesicles.

The dense core secretory vesicle is a key organelle for regulated, activity-dependent release of peptide and catecholamine neurotransmitters. Indeed, regulated secretion of pGluAβ and Aβ occurs with the peptide neurotransmitters (Met)enkephalin and galanin. Evidence for co-secretion of Aβ peptides with neurotransmitters indicates involvement of activity-dependent deposition of Aβ peptides occuring with secretion of neurotransmitters in AD brains, as demonstrated in vivo [21, 22]. It will be of interest in future studies to examine the variety of secretagogues known to induce neurotransmitter secretion [78–83] for effects on stimulating the regulated secretion pGlu-Aβ peptides to gain further understanding of factors controlling pGlu-Aβ release. It is noted that the DCSV isolated from the peripheral sympathetic nervous system (from adrenal medulla) contain pGlu-Aβ, whereas it is not detectable in normal aged human brains [84]. Thus, the DCSV utilized in this study provides a model to understand the neurobiology of pGlu-Aβ.

Further, it is noted that it is not yet known whether pGlu-Aβ is located in other subcellular organelles that contain full-length Aβ(1-40/42). Full-length Aβ is present in endosomes [85–89], lysosomes [90–92], autophagosomes [93–96], exosomes [97, 98], and other related subcellular organelles. It will be important in future studies to compare the organelle locations of pGlu-Aβ and Aβ peptides that will provide understanding of the neuronal trafficking of pGlu-Aβ compared to Aβ.

The neurobiology of regulated, activity-dependent secretion of pGlu-Aβ and Aβ is significant with respect to the functional role of pGlu-Aβ in neurodegeneration and memory loss in AD. pGlu-Aβ comprises a major portion of Aβ species in AD, demonstrated to compose the majority of total Aβ peptides compared to Aβ(1-40) and Aβ(1-42) [5–7]. Quantification indicates the presence of pGlu-Aβ peptides at similar and greater levels than Aβ(1-40) and Aβ(1-42) in AD brains. Also, pGlu-Aβ is present as oligomeric complexes in AD brains [99]. In vitro studies show that pGlu-Aβ displays a higher aggregation propensity, as well as stronger tendency to seed aggregation of other Aβ species [8, 9, 100]. Moreover, pGlu-Aβ is present in AD brains but not in normal aged brains [84].

In vivo studies illustrate the neurotoxicity of pGlu-Aβ for memory deficits. Over-expression of pGlu-Aβ in mice correlates with Aβ and behavioral deficits [10, 12] as well as neuronal loss and impaired long-term potentiation [101]. These studies also showed that pGlu-Aβ formation is dependent on QC since knockout of QC in transgenic mice resulted in reduced levels of brain pGlu-Aβ. These findings support a role of pGlu-Aβ for amyloid plaque formation and memory loss in AD.

Reduction of pGlu-Aβ formation may provide a therapeutic strategy for AD since inhibition of QC results in decreased brain pGlu-Aβ [13, 14] and QC gene knockout decreases brain pGlu-Aβ with improved behavioral deficits [12]. These findings indicate that glutaminyl cyclase is involved in development of AD via formation of pGlu-Aβ that contributes to the disease condition.

Based on results of this study, inhibition of QC reduces the amount of pGlu-Aβ released from the regulated secretory pathway. It will be of interest in future studies to assess the effects of QC inhibition on regulated, activity-dependent deposition of amyloid in AD animal model brains. Production of pGlu-Aβ by QC in secretory vesicles that produce and secrete Aβ peptides and neurotansmitters in an activity-dependent manner suggests a close association of pGlu-Aβ and Aβ neurotoxicities with neurotransmitter functions.