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Beta-amyloid peptides undergo regulated co-secretion with neuropeptide and catecholamine neurotransmitters - PubMed

Beta-amyloid peptides undergo regulated co-secretion with neuropeptide and catecholamine neurotransmitters

Thomas Toneff et al. Peptides. 2013 Aug.

Abstract

Beta-amyloid (Aβ) peptides are secreted from neurons, resulting in extracellular accumulation of Aβ and neurodegeneration of Alzheimer's disease. Because neuronal secretion is fundamental for the release of neurotransmitters, this study assessed the hypothesis that Aβ undergoes co-release with neurotransmitters. Model neuronal-like chromaffin cells were investigated, and results illustrate regulated, co-secretion of Aβ(1-40) and Aβ(1-42) with peptide neurotransmitters (galanin, enkephalin, and NPY) and catecholamine neurotransmitters (dopamine, norepinephrine, and epinephrine). Regulated secretion from chromaffin cells was stimulated by KCl depolarization and nicotine. Forskolin, stimulating cAMP, also induced co-secretion of Aβ peptides with peptide and catecholamine neurotransmitters. These data suggested the co-localization of Aβ with neurotransmitters in dense core secretory vesicles (DCSV) that store and secrete such chemical messengers. Indeed, Aβ was demonstrated to be present in DCSV with neuropeptide and catecholamine transmitters. Furthermore, the DCSV organelle contains APP and its processing proteases, β- and γ-secretases, that are necessary for production of Aβ. Thus, Aβ can be generated in neurotransmitter-containing DCSV. Human IMR32 neuroblastoma cells also displayed regulated secretion of Aβ(1-40) and Aβ(1-42) with the galanin neurotransmitter. These findings illustrate that Aβ peptides are present in neurotransmitter-containing DCSV, and undergo co-secretion with neuropeptide and catecholamine neurotransmitters that regulate brain functions.

Keywords: APP; Aβ; BSA; Catecholamines; DCSV; Dense core secretory vesicles; NPY; Neuropeptides; Neurotransmitters; PBS; Regulated secretion; amyloid precursor protein; bovine serum albumin; dense core secretory vesicles; neuropeptide Y; phosphate-buffered saline; β-Amyloid; β-amyloid.

Copyright © 2013 Elsevier Inc. All rights reserved.

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Figures

Fig. 1
Fig. 1

Regulated secretion of Aβ peptides from neuronal chromaffin cells stimulated by KCl depolarization and nicotine. Regulated secretion from neuronal-like chromaffin cells in primary culture was stimulated by KCl depolarization (50 mM) and nicotine (10 μM) (15 min incubation). At the end of this treatment period, secretion media was collected and analyzed for concentrations of the Aβ(1–40) and Aβ(1–42) peptides (panels (a) and (b), respectively). *p < 0.05 (Student's t-test).

Fig. 2
Fig. 2

Regulated co-secretion of peptide and catecholamine neurotransmitters from neuronal chromaffin cells with Aβ peptides. Regulated secretion from chromaffin cells in primary culture was stimulated by KCl depolarization (50 mM) and nicotine (10 μM) (15 min incubation), as described in Fig. 1. The secretion media was measured for Aβ peptides (illustrated in Fig. 1). Data in this Fig. 2 show co-secretion of the peptide neurotransmitters galanin, NPY, and (Met)enkephalin (panels (a)–(c), respectively), and co-secretion with the catecholamine neurotransmitters dopamine, norepinephrine, and epinephrine (panels (d)–(f), respectively). *p < 0.05 (Student's t-test).

Fig. 3
Fig. 3

Forskolin stimulates secretion of Aβ peptides from chromaffin cells. Chromaffin cells were treated with forskolin (2 h) to induce regulated secretion. The secretion media was collected and levels of Aβ peptides were measured. Forskolin induced the regulated, co-secretion of Aβ(1–40) and Aβ(1–42) (panels (a) and (b)). *p < 0.05 (Student's t-test).

Fig. 4
Fig. 4

Forskolin stimulates the co-secretion of peptide and catecholamine neurotransmitters with Aβ peptides from chromaffin cells. As described in Fig. 3, chromaffin cells were treated with forskolin (2 h) to stimulate regulated secretion. The secretion media was collected after the forskolin treatment, and levels of peptide and catecholamine neurotransmitters were measured, to evaluate their secretion with Aβ peptides (data shown in Fig. 3). Results of this Fig. 4 show that the peptide neurotransmitters galanin, NPY, and (Met)enkephalin (panels (a)–(c)) and the catecholamine neurotransmitters dopamine, norepinephrine, and epinephrine (panels (d)–(f)) are co-secreted with Aβ peptides (Fig. 3) from chromaffin cells,. Data show that forskolin stimulates the regulated co-secretion of Aβ peptides with neurotransmitters. *p < 0.05 (Student's t-test).

Fig. 5
Fig. 5

Cellular localization of Aβ/APP immunoreactivity with galanin and NPY neurotransmitters. (a) Aβ/APP localization with galanin. Localization of Aβ and APP precursor forms (detected by 6E10 immunoreactivity, red fluorescence) with the galanin peptide neurotransmitter (green fluorescence) was assessed by immunofluorescence deconvolution microscopy. The co-localization of Aβ/APP with galanin was assessed by their merged images, with areas of co-localization illustrated by yellow fluorescence (shown by arrows). The 6E10 antibody detects Aβ peptides and its APP precursor protein. (b) Aβ/APP localization with NPY in chromaffin cells. Localization of Aβ and APP precursor forms (assessed by 6E10 immunoreactivity, red fluorescence) with the NPY peptide neurotransmitter (green fluorescence) was evaluated by immunofluorescence deconvolution microscopy. The co-localization of Aβ/APP with NPY was assessed by their merged images, with areas of co-localization shown by yellow fluorescence (see arrows). Controls conducted with only secondary antibody (labeled with Alexa Fluor) showed no fluorescence, thus indicating that the immunofluorescence was due to the primary antisera to Aβ/APP, galanin, and NPY. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 6
Fig. 6

Neurotransmitter dense core secretory vesicles (DCSV) contain Aβ and APP. (a) Electron microscopy of dense core secretory vesicles (DCSV) isolated from chromaffin cells. The homogeneity and integrity of DCSV purified from adrenal medullary chromaffin cells was illustrated by electron microscopy, conducted as described in the methods. (b) APP and Aβ in DCSV: antibody epitopes. Endogenous full-length APP (amyloid precursor protein) is the precursor of Aβ, present in DCSV [45,48]. Antisera to different epitopes of APP are illustrated for antibodies 1656, 22C11, R7, 1659, and R1, with peptide antigen sequences described in the methods and procedures. The presence of Aβ with APP in DCSV indicates the presence of β- and γ-secretase components in the DCSV organelle. (c) Aβ in DCSV. Western blot with antisera 1656 directed to Aβ(17–28) detected a band of ∼4.5 kDa, consistent with the presence of monomeric Aβ. Replicate lanes (two lanes) are shown. (d) APP in DCSV. APP was observed in DCSV by western blots with antisera 1656, 22C11, R7, and R1 directed to epitope regions of APP (shown in Fig. 6b). APP of ∼100–110 kDa on SDS-PAGE gels of western blots was observed with these antisera. These antisera also detected bands of ∼ 87–90 kDa that are consistent in apparent molecular weight to sAPPα and sAPPβ.

Fig. 7
Fig. 7

Amyloidogenic β- and γ-secretase components are present in neurotransmitter-containing dense core secretory vesicles (DCSV). Western blots assessed the presence of β-secretase and γ-secretase components in dense core sescretory vesicles (DCSV) purified from chromaffin cells. These DCSV contain the β-secretase components of the recently identified cathepsin B (∼28 kDa mature form, and ∼35 kDa proenzyme) [15] and the well known BACE1 (∼ 60–70 kDa) [21,59], illustrated by western blots (panels (a) and (b), respectively). Western blots illustrated that γ-secretase complex components are present, consisting of presenilin 1 (panel (c))of ∼50 kDa and ∼28 kDa representing full-length and cleaved forms [9,56,58], presenilin 2 (panel d) of ∼50 kDa and ∼25 kDa bands representing full-length and cleaved forms [24], nicastrin (panel (e)) of ∼150 kDa and ∼100 kDa bands [23], Aph-1 (panel (f)) of ∼25 kDa [29], and PEN-2 (panel (g)) of ∼10–12 kDa band [23,32]. Arrows indicate the bands observed as secretase components, with their apparent molecular weights observed.

Fig. 8
Fig. 8

ADAM10, representing α-secretase, is present in neurotransmitter secretory vesicles of the DCSV type. The presence of ADAM10 in DCSV isolated from neuronal chromaffin cells was assessed by anti-ADAM10 western blots. The western blots illustrate the presence of ∼95 kDa and ∼60 kDa forms of ADAM10, representing proform and mature forms of ADAM10, respectively [27,42].

Fig. 9
Fig. 9

Human IMR32 neuroblastoma cells display regulated co-secretion of Aβ peptides with the galanin neurotransmitter. Regulated secretion was stimulated by KCl depolarization (50 mM in media, for 90 min.) of human IMR32 neuroblastoma cells. Controls include cells that were not treated with KCl. The secretion media was collected for measurements of Aβ(1–40), Aβ(1–42), and the galanin neurotransmitter (panels (a), (b), and (c), respectively). *p < 0.05 (Student's t-test).

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