Abstract
Background:Processing of the amyloid-β protein precursor (AβPP) is neurophysiologically important due to the resulting fragments that regulate synapse biology, as well as potentially harmful due to generation of the 42 amino acid long amyloid β-peptide (Aβ42), which is a key player in Alzheimer’s disease.Objective:Our aim was to clarify the subcellular locations of the fragments involved in the amyloidogenic pathway in primary neurons with a focus on Aβ42 and its immediate substrate AβPP C-terminal fragment (APP-CTF). To overcome the difficulties of resolving these compartments due to their small size, we used super-resolution microscopy.Methods:Mouse primary hippocampal neurons were immunolabelled and imaged by stimulated emission depletion (STED) microscopy, including three-dimensional three-channel imaging, and quantitative image analyses.Results:The first (β-secretase) and second (γ-secretase) cleavages of AβPP were localized to functionally and distally distinct compartments. The β-secretase cleavage was observed in early endosomes in soma, where we were able to show that the liberated N- and C-terminal fragments were sorted into distinct vesicles budding from the early endosomes. Lack of colocalization of Aβ42 and APP-CTF in soma suggested that γ-secretase cleavage occurs in neurites. Indeed, APP-CTF was, in line with Aβ42 in our previous study, enriched in the presynapse but absent from the postsynapse. In contrast, full-length AβPP was not detected in either the pre- or the postsynaptic side of the synapse. Furthermore, we observed that endogenously produced and endocytosed Aβ42 were localized in different compartments.Conclusion:These findings provide critical super-resolved insight into amyloidogenic AβPP processing in primary neurons.
Highlights
Deciphering the molecular landscape of vesicular trafficking and synaptic dysfunction is crucial for understanding the complexity of neurons in physiology and disease
We previously showed that stimulated emission depletion (STED) microscopy, in contrast to confocal microscopy, can be used to resolve different types of Amyloid- peptide (A)42-containing vesicles such as small A42-containing vesicles in the presynapse and larger vesicles with A42 only at the rim in other regions [29]
We have continued our quest to reveal the subcellular details of amyloidogenic amyloid- protein precursor (APP) processing by using state-ofthe-art STED microscopy and pushed the limits to include 3D 3-channel STED microscopy
Summary
Deciphering the molecular landscape of vesicular trafficking and synaptic dysfunction is crucial for understanding the complexity of neurons in physiology and disease. The emerging super-resolution microscopy techniques enable examination of the architecture of synapses and fine structure of organelles at the low nanoscale level. These revolutionizing tools will be critical for understanding the biological systems in the brain and for mapping the pathological processes underlying neurodegenerative disorders, among which Alzheimer’s disease (AD) is the most common one. Processing of the amyloid- protein precursor (APP) is neurophysiologically important due to the resulting fragments that regulate synapse biology, as well as potentially harmful due to generation of the 42 amino acid long amyloid -peptide (A42), which is a key player in Alzheimer’s disease. Conclusion: These findings provide critical super-resolved insight into amyloidogenic APP processing in primary neurons
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