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.