Mechanisms and machinery mediating AMPA receptor anchoring in synaptic plasticity

The brain is made up of nerve cells termed 'neurons', which communicate via specific cell junctions, called synapses. Synapses are small inter-cellular junctions where information between neurons is transmitted and stored (i.e. laid down as memories). Information storage at synapses is believed to be be mediated by changes in synapse efficacy (synaptic plasticity), which involves dynamic changes of protein distribution at the post-synaptic site.

Signaling across synapses is initiated by the release of neurotransmitter chemicals from an 'upstream' neuron at the pre-synaptic site, onto the post-synapse of a downstream recipient cell. A primary neurotransmitter in the mammalian brain is the amino acid glutamate, which activates recipient protein molecules on the post-synapse, called 'receptors'. The glutamate receptor central to the application is the AMPA-receptor (AMPA-R). AMPA-Rs depolarize the post-synaptic membrane in response to binding glutamate and thereby initiate neurotransmission. Depolarization results from a glutamate-triggered influx of positively charged particles (cations), through the AMPA-R, into the post-synaptic neuron resulting in an electrical signal (depolarization). Synaptic potentiation, the substrate for information storage, results from the recruitment of additional AMPA-Rs to the postsynapse resulting in increased cation-influx and more depolarization. Potentiated transmission also requires the concentration or clustering of AMPA-Rs at the synapse and their retention opposite glutamate-release sites.

In this proposal we will investigate mechanisms underlying AMPA-R dynamics at synapses that underlie synaptic plasticity (and thus 'learning'). We will assay the clustering and retention of AMPA-Rs at synapses in response to potentiating stimuli using high-resolution imaging and functional approaches. These will provide a detailed-snapshot of the AMPA-Rs dynamics underlying synaptic plasticity. We have preliminary evidence suggesting that the distal portion of the AMPA-R, the N-terminal domain (or NTD) regulates receptor diffusion/retention and clustering at post-synaptic sites. Hence, the NTD appears to play a currently elusive role in AMPA-R-mediated synapse potentiation and therefore in synaptic learning. AMPA-R retention also stabilizes post-synaptic structures, and our data point to a role for the NTD in this process. Since synapse elimination is a hallmark of
neurodegneration, NTD-mediated receptor retention would have the potential to oppose neurological disorders such as Alzheimer's disease and related dementias. These observations, together with our accumulating evidence for the NTD as novel drug target, highlight a key role for the AMPA-R NTD in synapse function. In summary, our proposed work will provide mechanistic information into the role of AMPARs in synaptic learning and synapse stabilization.

The plastic nature of a synapse, where the flow of information is able to induce rapid and long-lasting changes in synaptic strength, is central to our understanding of learning and memory. The AMPA-type glutamate receptor (AMPAR), is key to this process. Postsynaptically localised AMPARs are activated by presynaptically released glutamate,
depolarising the postsynaptic cell to achieve synaptic transmission. By modulating the extent of AMPAR activation, the strength of transmission can be tuned. Therefore, elucidating the mechanisms controlling the trafficking and localisation of synaptic AMPARs is essential to understanding memory formation.

At hippocampal synapses, the majority of AMPARs consists of GluA1/2 and GluA2/3 heteromers. While the activity-dependent trafficking of GluA1-containing AMPARs to synaptic sites has long been associated with synaptic potentiation, the precise roles and regulation of these different receptors in synaptic transmission and plasticity remain unclear. In this project, we propose to use cutting-edge technology to visualise the dynamics of AMPAR function in synaptic plasticity, in intact neuronal circuits in the most
refined manner to date. CRISPR/Cas9 genome editing coupled with 3D STORM imaging in brain tissue will allow visualisation of the localisation of endogenous AMPAR heteromers within the synapse on the nanoscale. Building on our recent insights into the mechanisms controlling AMPAR synaptic anchoring, we will characterise how interactions
control receptor localisation at synaptic sites and aim to identify proteins that mediate anchoring (using a bioID proximity labeling). We aim to resolve the synaptic receptor rearrangements occurring at baseline conditions and in response to synaptic plasticity. Together, this study aims to reveal the nanoscale rearrangements and mechanisms of synaptic AMPARs during plasticity, which tune synaptic transmission and facilitate information storage in the brain.

Who will benefit from the research?
The main stake holders of this research include other neuroscientists, scholars and students, the pharmaceutical industry
as well as patients suffering from neurological and psychiatric disorders and the general public.

How will they benefit?
Neuroscientists: The molecular organisation of the synapse is a fundamental research question, which is of particular
importance to other neuroscientists interested in synaptic transmission as well as learning and memory mechanisms in the brain.
In addition, pharmacologists, neurologists and psychiatrists would see the importance of understanding the basic receptor
operation at synapses, as this is of importance for understanding drug action, as well as disorders affecting
synaptic function.

Cell biologists: Trafficking and lateral diffusion of receptors is a central problem in cell biology and will be
of interest to the large community studying membrane proteins.

Scholars and students: The research question addressed here is fundamental for understanding brain function, and our
ambition is that the concepts emerging from the data generated in this project will make its way into future textbooks. This
will benefit future generations of students, both in biochemistry, general biology and human and veterinary medicine.

The pharmaceutical industry: The nature of synaptic transmission and how it changes during learning and memory is of
fundamental importance for drug action. Glutamate receptors are a central drug-target, and positive modulators have
therapeutic potential in a number of neurodegenerative diseases and psychiatric disorders, including Alzheimer's disease,
depression and epilepsy. A better understanding of the inner workings of the AMPARs, both at the level of synapses and as drug target, should
permit development of therapies impacting both the health and wealth of the UK. Also of note, currently available drugs
target highly sequence-conserved regions of the receptor (such as perampanel). Their action will therefore be broad-range.
The sequence-diverse NTD has not been extensively studied in AMPARs, and our ongoing and planned work will shed
new light on the inner workings of the NTD in different subtypes of AMPARs.

Patients: The ultimate beneficiary of drug development would be the patient. With the increasing prevalence of dementia in
the population, partly because of an ageing population, there is an urgent need for cognition-enhancing drugs, and the
AMPARs are a promising target.

In addition to patients, the general public is expected to benefit. Understanding how the brain operates is a central scientific
quest, and seen as important by the general public. Memory in particular, and the underlying mechanisms underlying
information storage at synapses, remains one of the central questions in modern neuroscience. We will disseminate our
new understanding in public lectures and workshops, e.g. during the annual 'Cambridge Science Festival'.