Elsevier

Neuroscience Letters

Volume 566, 30 April 2014, Pages 125-130
Neuroscience Letters

Activity-independent release of the amyloid β-peptide from rat brain nerve terminals

https://doi.org/10.1016/j.neulet.2014.02.050Get rights and content

Highlights

  • A small amount of production was detected in pure synaptic vesicle fractions.

  • Large amounts of Aβ are released from synaptosomes without stimulation.

  • Stimulation of synaptosomes with KCl or 4-AP did not alter the release of Aβ.

Abstract

Synaptic degeneration is one of the earliest hallmarks of Alzheimer disease. The molecular mechanism underlying this degeneration is not fully elucidated but one key player appears to be the synaptotoxic amyloid β-peptide (Aβ). The exact localization of the production of Aβ and the mechanisms whereby Aβ is released remain elusive. We have earlier shown that Aβ can be produced in crude synaptic vesicle fractions and it has been reported that increased synaptic activity results in increased secreted but decreased intracellular Aβ levels. Therefore, we considered whether Aβ could be produced in synaptic vesicles and/or released through the same mechanisms as neurotransmitters in synaptic vesicle exocytosis. Small amounts of Aβ were found to be produced in pure synaptic vesicle preparations. We also studied the release of glutamate and Aβ from rat cortical nerve terminals (synaptosomes). We found that large amounts of Aβ were secreted from non-stimulated synaptosomes, from which glutamate was not released. On the contrary, we could not detect any differences in Aβ release between non-stimulated synaptosomes and synaptosomes stimulated with KCl or 4-aminopyridine, whereas glutamate release was readily inducible in this system. To conclude, our results indicate that the major release mechanism of Aβ from isolated nerve terminals differs from the synaptic release of glutamate and that the activity-dependent increase of secreted Aβ, reported by several groups using intact cells, is likely dependent on post-synaptic events, trafficking and/or protein synthesis mechanisms.

Introduction

Synaptic degeneration is one of the hallmarks that correlates best with cognitive decline in Alzheimer disease (AD) [1] and results in loss of communication between the neurons. However, the molecular mechanisms for this degeneration remain largely elusive. A likely causative agent is the amyloid β-peptide (Aβ) that has been proven to be synaptotoxic in vitro and in vivo [2], [3], [4]. Aβ is produced by the sequential cleavage of the Aβ precursor protein (APP) by β-secretase, to yield APP-CTFβ and soluble APPβ (sAPPβ), followed by γ-secretase of APP-CTFβ to yield Aβ. Whereas β-secretase cleavage is conducted by a single protein, BACE1, γ-secretase is a large transmembrane protein complex consisting of at least four subunits; presenilin (PS1 or PS2), nicastrin, Aph1 (Aph1a or Aph1b) and Pen2 [5]. The γ-secretase cleavage site is not fixed to a single position and thus several different peptides are produced, whereof Aβ40 is the most abundant while longer variants such as Aβ42 and Aβ43 are more toxic and more prone to aggregate [6], [7], [8]. In an alternative pathway, APP is cleaved by α-secretase instead of β-secretase, yielding sAPPα and APP-CTFα, which upon γ-secretase cleavage yields a harmless p3 product instead of Aβ. β- and γ-secretase processing most probably occur in intracellular compartments, since endocytosis of APP is required for the majority of the Aβ production [9]. A recent study [10] showed that large amounts of Aβ were constitutively released from neurons. In addition, several studies suggest that intracellular Aβ might be more detrimental to the brain than secreted Aβ [11], [12] and that the intracellular Aβ concentrations are elevated in neurons at risk in AD brains [13]. It is therefore possible that defective Aβ secretion at least partially is responsible for the synaptic degeneration in AD. In order to counteract this potential defect, it is important to elucidate the mechanisms by which this secretion occurs. However, despite that almost 30 years have passed since the discovery of Aβ, these mechanisms still remains elusive. Exosomes, formed from late endosomes, have been proposed as executors of Aβ secretion but, at least in neuroblastoma cells, only about 1% of the secreted Aβ was found to be associated with exosomes [14]. Hence, the major pathway for Aβ secretion is still unknown. We and others have earlier shown that Aβ can be produced in crude synaptic vesicle preparations and that γ- and β-secretase components as well as APP are present in ultra-pure synaptic vesicles [15], [16]. Interestingly, a number of studies have shown that induction of synaptic activity results in increased secreted and decreased intracellular Aβ levels [17], [18], [19], [20]. The combined results from these studies made us speculate that Aβ might be produced in synaptic vesicles and released through normal synaptic vesicle exocytosis in the same manner as neurotransmitters. To test this hypothesis, we analyzed Aβ production in pure synaptic vesicle preparations and found that a fraction of Aβ indeed is produced in these preparations. However, we also analyzed glutamate and Aβ release from rat brain nerve terminals (synaptosomes) and found that large amounts of Aβ were released from non-stimulated synaptosomes, from which no glutamate was released. These results suggest that the major release mechanism of Aβ from isolated nerve terminals is activity-independent and distinct from synaptic vesicle exocytosis.

Section snippets

Animals

Male Wistar rats (Charles River) were sacrificed by carbon dioxide. The animals used in this study were treated according to Swedish or German (TierSchG) national guidelines as well as guidelines from Karolinska Institutet and the Max Planck Institute. The study was approved by the Animal research ethical committee of southern Stockholm (S73-11). No experiments were performed on live animals.

Preparation of pure synaptic vesicles

Synaptic vesicles were prepared as previously described [21]. Briefly, synaptic vesicles were released

small amount of Aβ is produced in pure synaptic vesicle preparations

To follow up our and other's previous results showing enriched Aβ production in crude synaptic vesicle fractions and the presence of γ- and β-secretase components as well as APP in highly pure (control pored glass chromatography (CPG) purified) synaptic vesicle preparations [15], [16], we investigated whether Aβ could be produced also in these CPG-purified synaptic vesicle fractions. Electron microscopy confirmed that the vast majority of this preparation consisted of small vesicles of the size

Discussion

In this study, we tested the hypothesis that Aβ is produced in synaptic vesicles and released through the same mechanism as neurotransmitters. We indeed detected a small but significant production of Aβ40 in pure synaptic vesicles preparations even though the production was not enriched compared to homogenates. Given the large amounts of synaptic vesicles in the brain and the fast recycling of these vesicles, the small amounts of Aβ produced by synaptic vesicles could still be of importance. We

Conclusions

We conclude that although small amounts of Aβ can be produced in synaptic vesicle preparations, the major release mechanism of Aβ from isolated nerve terminals is activity-independent and distinct from synaptic vesicle exocytosis.

Acknowledgements

We are very grateful to Julia Preobraschenski and Janina Boyken (Max Planck Institute, Göttingen) for synaptic vesicle preparation and expert help in establishing the synaptosomal preparations, respectively, Lennart Brodin, Karolinska Institutet for valuable advice and Roger Strömberg, Karolinska Institutet for providing the fluorometer. This study was supported by grants from the Alzheimer's Association (NIRG-12-237941), Swedish Brain Power, Alzheimerfonden, Demensfonden, Knut och Alice

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