Synaptotagmin-3 is Involved in the Mechanism of Forgetting

Post by: Amanda McFarlan

What's the science?

Synaptotagmins are a group of calcium-sensitive membrane-trafficking proteins that are known to be involved in regulating activity-dependent endo- and exocytosis at the synapse (the space between neurons). Recent findings have revealed that contrary to Synaptotagmin-1 and Synaptotagmin-2 (localized to synaptic vesicle membranes), Synaptotagmin-3 is localized to the plasma membrane. This suggests that Synaptotagmin-3 may have an important role at the synaptic membrane, including the internalization of receptors (i.e. endocytosis) that is necessary for synaptic depression. Synaptic depression is a critical process in forgetting a memory. This week in Science, Awasthi and colleagues investigated the role of Synaptotagmin-3 in mediating activity-induced internalization of receptors at the synapse, long term depression (LTD) and forgetting.

How did they do it?

The authors used several experimental techniques to determine the role of Synaptotagmin-3 in mediating LTD and forgetting. First, they used immunohistochemistry to localize the expression of Synaptotagmin-3 in the mouse brain. Then, they investigated whether Synaptotagmin-3 was involved in activity-dependent endocytosis at the synapse. To do this, they used immunohistochemistry to identify surface Synaptotagmin-3 (at the postsynaptic membrane) and internalized Synaptotagmin-3 (inside the postsynaptic neuron) in Synaptotagmin-3 knockout neurons (neurons without Synaptotagmin-3) before and after stimulation with AMPA or NMDA. Additionally, the authors explored whether Synaptotagmin-3 endocytosed receptors. They used pull-down assays to identify the protein binding partners of Synaptotagmin-3 and western blots to determine the effect of inhibiting the 3Y tail of the GluA2 subunit of the AMPA receptor (a subunit important for receptor endocytosis) on Synaptotagmin-3 binding to GluA2. Next, the authors used whole-cell recordings in wild-type (i.e. control mice) and Synaptotagmin-3 knockout mice to investigate whether the absence of Synaptotagmin-3 disrupted LTD. Finally, the authors used a water maze spatial memory task to determine the role of Synaptotagmin-3 in forgetting. In this task, wild-type and Synaptotagmin-3 knockout mice were placed in a pool of opaque water and learned to find a hidden platform using visual cues over several days of training. On the test day, the platform was removed, and the mice were allowed to search for the platform for 60 seconds while their movements were tracked.

What did they find?

The authors determined that Synaptotagmin-3 expression was most abundant in dendrites (i.e. the branches of a neuron located on the postsynaptic side of a synapse) compared to axons, and could be found in the hippocampus, cortex, thalamus, and striatum. Following the stimulation with AMPA or NMDA in Synaptotagmin-3 knockout neurons, the authors found a significant increase in internalized Synaptotagmin-3 compared to controls which suggests that Synaptotagmin-3 is involved in activity-dependent endocytosis at the synapse. Next, pull-down assays showed that Synaptotagmin-3 interacts with two proteins that are important for receptor endocytosis, AP-2 and BRAG2, as well as the GluA2 subunit of the AMPA receptor. Further investigation using Western blots revealed that Synaptotagmin-3 interacts with GluA2 at its 3Y tail, since competitive inhibition of the 3Y tail disrupted the Synaptotagmin-3-GluA2 binding. Whole-cell patch-clamp recordings in Synaptotagmin-3 knockout mice revealed that LTD was abolished in the absence of Synaptotagmin-3 compared to wild-type mice (i.e. control mice). Together, these findings suggest that endocytosis of AMPA receptors, which is necessary for LTD, requires Synaptotagmin-3. Finally, the water maze spatial memory task revealed that Synaptotagmin-3 knockout mice were able to learn normally, however they had deficits in forgetting compared to wild-type mice. During the test trial, wild-type mice searched for the platform the most in the first 10-20 seconds, and then switched to a dispersed search pattern. Synaptotagmin-3 knockout mice, however, continued searching for the platform throughout the entire trial, suggesting that the mouse’s ability to forget the location of the platform was impaired in the absence of Synaptotagmin-3.

What's the impact?

This is the first study to provide evidence of a molecular ‘forgetting mechanism’ that requires Synaptotagmin-3. The authors showed that Synaptotagmin-3 is required for the internalization of AMPA receptors, resulting in synaptic depression and forgetting. Altogether, this research is essential for understanding an important aspect of memory, forgetting.

Awasthi et al. Synaptotagmin-3 drives AMPA receptor endocytosis, depression of synapse strength, and forgetting. Science (2019).Access to the original scientific publication here.