Post by Shireen Parimoo
What's the science?
Amyloid precursor protein (APP), as its name suggests, is a precursor to amyloid-β, which is implicated in Alzheimer’s disease. Although the build up of amyloid-β plaques are a hallmark of Alzheimer’s disease pathology, the physiological role of amyloid-β in the human brain is not well understood. Studies have found that APP undergoes ectodomain shedding, which leads to the extracellular release of a part of the protein: secreted APP (sAPP). sAPPa, is thought to influence synaptic transmission and neuronal plasticity. However, the physiological mechanism by which sAPPa carries out that role in the brain – such as which cell-membrane receptor(s) it binds to – is currently unknown. This week in Science, Rice and colleagues used in vivo and in vitro methods to examine the interaction between sAPPa and cell-membrane receptors, and its effect on synaptic processes.
How did they do it?
The authors extracted hippocampal synaptosomes (synapses; the connections between neurons) from wild type (i.e. normal) rat brains and then used (i) biochemical fractionation to separate the components of the synaptosome, and (ii) structured illumination imaging (a high-resolution microscopy technique) to detect APP. Using sAPPa as bait, the authors used affinity purification mass spectrometry to isolate and subsequently identify cell-membrane proteins that bound to sAPPa. They then used biolayer interferometry to examine the interaction between sAPPa and a certain type of receptor - the GABA receptor. This technique involved incubating biosensors with specific receptor domains and placing them into an sAPPa solution to facilitate molecular binding. The biosensors measure the change in light waves as sAPPa binds to and unbinds from the receptor domains. Isothermal titration calorimetry was used to measure how strongly sAPP, as well as the Acidic (AcD) and Extension (ExD) domains of the APP695 protein (an APP isoform), bind to a certain GABA receptor domain, called sushi 1.
To investigate the effect of sAPPa on GABA receptor activity, the authors measured mini excitatory (mEPSCs) and inhibitory post-synaptic currents (mIPSCs) in mouse hippocampal neuronal cultures following the application of sAPPa, AcD, ExD, or AcD-ExD. They also applied low-voltage and burst stimulation to CA3 neurons and recorded field EPSCs. Finally, they isolated the peptides that form the ExD domain of sAPPa to identify the smallest unit that could bind to the sushi 1 domain and influence synaptic transmission. This was done both in vitro using the methods outlined above and in vivo using 2-photon calcium imaging in the hippocampal neurons of anesthetized mice.
What did they find?
The authors found an abundance of a certain GABA receptor subunit (GABABR1a) in hippocampal synapses, and that sAPPa bound to the sushi 1 peptide domain of the receptor with high affinity. The AcD-ExD region of APP695 also had high affinity for the sushi 1 domain, but when the two functional domains were examined separately, only ExD bound to sushi 1, but not AcD. This means that APP695 strongly binds to the GABABR1a subunit because of the interaction between their sAPPa-ExD and sushi 1 functional domains, respectively.
The interaction between sAPPa and the GABA receptor also affected neuronal transmission in mouse hippocampal neurons. Low frequency stimulation of hippocampal (CA3) neurons after sAPPa application decreased the amplitude of field EPSCs, indicating a reduction in synaptic transmission. Burst stimulation increased short-term facilitation in the presence of sAPPa, suggesting decreased neurotransmitter release. However, these effects were not observed when the ExD domain was deleted. Furthermore, both sAPPa and AcD-ExD reduced the frequency of mEPSCs and mIPSCs, whereas AcD alone had no effect. Finally, the authors identified a 17- amino acid sequence (APP 17-mer) within the ExD domain that was sufficient to bind to the sushi 1 domain and modulate neuronal activity. In fact, in vivo imaging revealed that, like sAPPa, APP 17-mer suppressed neuronal activity in mouse hippocampal neurons. This means that the ExD functional domain of APP – specifically a 17-amino acid sequence within ExD – is necessary and sufficient to suppress synaptic activity through interactions with GABABR1 receptors.
What's the impact?
This is the first study to uncover the mechanism underlying the effect of APP on synaptic activity. The authors identified GABABR1 as the primary receptor that sAPP binds to, and systematically determined that their interaction suppressed neuronal transmission and neurotransmitter release. Prior to this study, the specific role of APP in the synapse was not understood. These findings have important implications for developing therapeutics to target illnesses mediated by GABAB receptors.
Rice et al. Secreted amyloid-b precursor protein functions as a GABABR1a ligand to modulate synaptic transmission. Science (2019). Access the original scientific publication here.