Activity-independent release of the amyloid β-peptide from rat brain nerve terminals
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
References (27)
- et al.
Evidence that production and release of amyloid beta-protein involves the endocytic pathway
J. Biol. Chem.
(1994) - et al.
Large quantities of Abeta peptide are constitutively released during amyloid precursor protein metabolism in vivo and in vitro
J. Biol. Chem.
(2011) - et al.
Abeta secretion and plaque formation depend on autophagy
Cell Rep.
(2013) - et al.
APP processing and synaptic function
Neuron
(2003) - et al.
Synaptic activity regulates interstitial fluid amyloid-beta levels in vivo
Neuron
(2005) - et al.
Molecular anatomy of a trafficking organelle
Cell
(2006) Substance P release from K+ -depolarized rat brain synaptosomes at one-second resolution
Brain Res.
(1983)- et al.
Activity-induced convergence of APP and BACE-1 in acidic microdomains via an endocytosis-dependent pathway
Neuron
(2013) - et al.
Physical basis of cognitive alterations in Alzheimer's disease: synapse loss is the major correlate of cognitive impairment
Ann. Neurol.
(1991) - et al.
Diffusible, nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins
PNAS
(1998)
Amyloid-beta protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory
Nat. Med.
Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo
Nature
Proteolytic processing of Alzheimer's beta-amyloid precursor protein
J. Neurochem.
Cited by (13)
Synaptic dysfunction in early phases of Alzheimer's Disease
2022, Handbook of Clinical NeurologyCitation Excerpt :This indicates that these vesicles differ from the synaptic vesicles responsible for neurotransmitter release (Yu et al., 2018). In agreement with this observation, the major release mechanism of Aβ from isolated nerve terminals differs from the synaptic release of glutamate (Lundgren et al., 2014). The machinery required for Aβ generation is present in the presynaptic terminals and can affect synaptic transmission modulating Aβ release or through other pathways (Barthet and Mulle, 2020).
Synaptosome as a tool in Alzheimer's disease research
2020, Brain ResearchCitation Excerpt :Indeed, studies have confirmed the presence of APP and γ-secretase-mediated production of the amyloid fragments in isolated synaptic vesicles of the presynaptic compartments (Frykman et al., 2010; Groemer et al., 2011). Further, to understand whether extracellular secretion of Aβ from the synaptic vesicles is induced by neuronal activity, Lundgren and co-workers employed rat cortical synaptosomes and stimulated them with KCl or 4-aminopyridine, and observed increased glutamate, but not Aβ, secretion in comparison to that of non-stimulated synaptosomes (Lundgren et al., 2014). These findings indicate that the major mechanism of Aβ secretion from synaptosomes is activity-independent and differs from the activity-induced release mechanisms for glutamate.
Maturation and processing of the amyloid precursor protein is regulated by the potassium/sodium hyperpolarization-activated cyclic nucleotide-gated ion channel 2 (HCN2)
2017, Biochemical and Biophysical Research CommunicationsCitation Excerpt :This could possibly be due to different kinetics in production and degradation of these different pools of Aβ or that the cells assure constant levels of intracellular Aβ by regulating its secretion. Similarly, we have previously found that when incubating isolated nerve terminals at 37 °C, presumably inducing Aβ production, only the secreted and not the intracellular levels were increased over time [15]. Apart from an effect on secreted Aβ levels, sAPP and APP-CTF levels were also reduced by HCN2 siRNA treatment.
Endogenous APP accumulates in synapses after BACE1 inhibition
2016, Neuroscience ResearchCitation Excerpt :Whether the presynaptic compartment contributes to activity-dependent Aβ production from endogenous APP is not clear. For instance, Aβ release from synaptosomes was found not to depend on activity (Lundgren et al., 2014). Further work will be required to elucidate the role of the presynaptic compartment in APP processing.
Opposing functions for retromer and rab11 in extracellular vesicle traffic at presynaptic terminals
2021, Journal of Cell Biology