Losing connections in Alzheimer disease : the amyloid precursor protein processing machinery at the synapse

Synaptic degeneration is one of the earliest characteristics of Alzheimer disease (AD). The amyloid β-peptide (Aβ) plays a critical role in the pathology of AD and therefore thorough understanding of its production and functions is of outmost importance. Aβ is generated by sequential cleavage of the amyloid precursor protein (APP) by the β-secretase BACE1 and by the γ-secretase. In an alternative, non-amyloidogenic pathway, APP is cleaved by the α-secretase ADAM10 instead of BACE1, precluding Aβ formation. Increased synaptic activity has been associated with increased secretion of Aβ and since our lab had previously shown that Aβ can be produced at the synapse, we hypothesised that Aβ is produced inside synaptic vesicles and released through normal synaptic vesicle exocytosis. We found that small amounts of Aβ can be produced in synaptic vesicles, although these vesicles do not appear to be the main site of Aβ production.

To study the secretion, synaptosomes (functional, pinched off, nerve endings) were isolated from rat brain and we could demonstrate that Aβ is continuously secreted from synapses in an activity-independent manner through a mechanism that is distinct from normal neurotransmitter release. While further investigating the highly pure synaptic vesicles, both ADAM10 and BACE1, as well as their cleavage products, APP C-terminal fragments (CTFs), were found to be greatly enriched in these vesicles compared to total brain homogenate. Yet, presenilin was the only enriched component of the γ-secretase complex. In addition, these Western blotting findings were confirmed by in situ proximity ligation assay (PLA) showing close proximity of both ADAM10 and BACE1 to the synaptic vesicle marker synaptophysin in intact mouse primary hippocampal neurons. Active γ-secretase, on the other hand, only gave rise to few PLA-signals, indicating that the first cleavage step in Aβ production takes place in synaptic vesicles while γ-secretase cleavage takes place elsewhere. Subsequently the synaptic location of the secretases was confirmed also in adult rat and human brain. Again using PLA, we could demonstrate that both ADAM10 and BACE1 were in close proximity to both synaptophysin and the postsynaptic density marker PSD-95 as well as to their substrate APP in both human and rat adult brain hippocampus and cortex. Also APP was in close proximity to both synaptophysin and PSD-95.

In addition to the known synaptotoxicity of Aβ, a number of studies have implied important and toxic roles for other APP-derived fragments, such as CTF-β or the synaptotoxic Aα-η. Alternative cleavage of APP by η-secretase gives rise to CTF-η, which is further cleaved to Aα-η by ADAM10. However, which of these fragments that is most abundant in AD brain had, to our knowledge, not been elucidated. When performing SDS-PAGE and Western blotting we found that a 25 kDa CTF (likely corresponding to CTF-η) was abundant in human brain but present at much lower levels in rat and mouse brain. The 25 kDa CTF was also present in macaque and guinea pig brain but the levels of this fragment was not increased in the brain of a mouse model overexpressing the human APP gene with a Swedish/London mutation. This implies that it is the environment in the human brain, rather than the human APP gene itself, that determines whether the 25 kDa CTF is formed or not. Furthermore, we investigated whether AD patients have altered levels of the 25 kDa CTF in their brains but could not detect any significant differences between AD and control brain homogenate. Altogether, this thesis has contributed with new knowledge about synaptic release of Aβ and the synaptic localisation of the APP processing enzymes. It has thus highlighted the complexity and species differences of APP processing and its regulation. Implementation of this knowledge may facilitate future development of more specific and efficient treatment strategies for AD.

List of scientific papers

I. Lundgren J. L., Ahmed S., Winblad B., Gouras G. K., Tjernberg L. O. and Frykman S. (2014). Activity-independent release of the amyloid-β peptide from rat brain nerve terminals. Neuroscience Letters. 566:125-130.
https://doi.org/10.1016/j.neulet.2014.02.050

II. Lundgren J. L., Ahmed S., Schedin Weiss S., Gouras G. K., Winblad B., Tjernberg L. O. and Frykman S. (2015). ADAM10 and BACE1 are localized to synaptic vesicles. Journal of Neurochemistry. 135:606-615.
https://doi.org/10.1111/jnc.13287

III. Lundgren J. L., Vandermeulen L., Sandebring-Matton A., Ahmed S., Winblad B., Di Luca M., Tjernberg L. O., Marcello E. and Frykman S. Similar pre- and postsynaptic distribution of ADAM10 and BACE1 in rat and human adult brain. [Manuscript]

IV. Haytural H., Lundgren J. L., Jorda T., Seed Ahmed M., Winblad B., Årsland D., Graff C., Barthet G., Tjernberg L. O. and Frykman S. A 25 kilodalton amyloid precursor protein C-terminal fragment is abundant in human but not in mouse or rat brain. [Manuscript]