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NMDA receptor diversity: from molecular dynamics to synaptic physiopathology

Periodic Reporting for period 4 - NMDADYN (NMDA receptor diversity: from molecular dynamics to synaptic physiopathology)

Reporting period: 2021-05-01 to 2022-10-31

NMDA receptors (NMDARs) are a class of neurotransmitter receptors that play critical role in brain development and plasticity. Recent studies have revealed that these glutamate-gated ion channels are more complex than initially thought, undergoing tight subunit-specific regulation by an array of endogenous modulators and existing as multiple subtypes, each with its own anatomical, functional and signaling properties. Such complexity raises key questions regarding the conformational changes that this multi-domain receptor undergoes, the physiological relevance of its subunit plurality and the microenvironment’s impact on receptor and circuit function.

The objective of this multi-scale project is to create and implement the spatially and temporally sensitive tools required to break the barriers to our understanding of NMDAR diversity and modulation. To address these challenges, we are using and developing innovative strategies at the crossroads of protein engineering, biological chemistry and neuroscience to achieve a molecular level control of NMDARs that is subunit-specific, reversible and usable both in vitro and in vivo. Using a bottom-up approach, the project contains four aims covering molecular, cellular and behavioral levels. The first two investigate NMDAR structural mechanisms and exploit this knowledge to develop new optochemical receptor tools. The next two address physiological questions using these tools as well as novel mouse lines.

Aim 1: Characterize NMDAR conformational dynamics and allosteric transitions
Aim 2: Engineer a family of light-controlled NMDARs (‘Opto-allostery’)
Aim 3: Understand the role of specific NMDAR populations in neuronal functions
Aim 4: Explore the receptor’s synaptic microenvironment in normal and disease states

Overall, we expect to provide fundamental insights into the intricate workings of an essential class of brain receptors and further our comprehension of neuronal excitatory transmission in normal and pathological conditions.
Key advances have been made on the following topics:

- NMDAR structural dynamics and allosteric mechanisms
NMDARs form large heterotetrameric molecular complexes. A distinguishing feature of NMDARs is their high conformational mobility which underlies their remarkable allosteric potential. Understanding the functional coupling of the various domains remains a major challenge. Using a combination of experimental approaches and in silico analysis, we identified a rotation motion at the interface between the two constitute dimers as a key structural mechanism in GluN2B NMDAR allostery. Surprisingly, we found that in GluN2A receptors, long-range allosteric coupling proceeds through a distinct interface, within local LBD dimers. These results provide the first integrated views of long-distance domains coupling in full-length NMDARs, and highlight the potential of subunit-subunit interfaces as novel sites for pharmacological manipulation of glutamate receptors.

- Molecular optogenetics and NMDAR functional diversity
Reprogramming receptor to artificially respond to light has strong potential for molecular studies and interrogation of biological functions. In the last years, we have designed a set of light controllable NMDARs using either photosensitive amino acids or ligands. In a first work, we provided the first demonstration of real-time detection of molecular rearrangements due to reversible light-switching of single amino acid side-chains inserted into a neuronal receptor. In a second and still ongoing work, we developed a photoswitchable allosteric potentiator for GluN2B-receptors exploiting the GluN2B-selective polyamine modulatory site. We have now obtained full experimental validation of this receptor tool both in vitro, ex vivo and in vivo. Ongoing experiments are currently performed to decipher the specific roles of GluN2B receptors in synapse function and behavior.

- A new family of glycine excitatory NMDA receptors
Based on the discovery of a pharmacological agent (CGP-78608) that uniquely and massively potentiates GluN1/GluN3 receptors (by preventing their desensitization), we discovered that GluN1/GluN3A receptors form glutamate-insensitive NMDARs expressed at high levels in specific brain regions, where they constitute a new type of neuronal receptors gated by glycine. These results have profound implication on our understanding of NMDAR signaling diversity and open new vistas on the roles of glycine in neurotransmission.

- The microenvironment at excitatory synapses
A striking feature of NMDARs is that they can be regulated by their microenvironment in a localized and subunit-specific manner. Tight receptor regulation is critical for normal brain physiology. We focused on NMDAR modulation by two atypical ligands, D-serine and zinc. Using an original ZnT3-Cre mouse line, we are currently mapping the distribution of zincergic neurons and pathways throughout the brain. We also discovered that vesicular zinc is not limited to excitatory synapses but is also accumulated by a specific subpopulation of interneurons in the cortex and hippocampus. Using an original inducible knock-in mouse line that harbor glycine-impaired NMDARs, we unveiled striking effects on synaptic plasticity pointing to major differences in the availability and role of glycine and D-serine as NMDAR co-agonists and synapse regulators.
1. We have set-up a dual strategy based on the combination of experimental and computational approaches to decipher the molecular mechanism and structural dynamics of NMDARs. Using this multi-level approach, we have provided the first integrated views of how these large multi-domain multi-subunit receptor complexes operate at the molecular and structural level. These studies have allowed the identification of novel regions important for important for receptor regulation with important implications regarding NMDAR pharmacology and in vivo regulation.

2. Using innovative optopharmacological approaches, we have designed a set of precise light-controllable NMDAR subunits allowing manipulation and interrogation of receptor function with unprecedented resolution (molecular, spatial and temporal). We are now implementing these receptor tools into native settings (awake behaving animals) to decode the role of specific NMDAR populations in brain signaling and behavior. Essential novel information regarding NMDAR signaling diversity and molecular physiology are expected.

3. We have discovered a new family of glutamate-insensitive neuronal NMDA receptors activated by glycine only and composed of the GluN3A subunit (glycine excitatory GluN1/GluN3A receptors). This discovery has broad ranging implications for the study and exploration of GluN3A receptors in brain development and function. It also profoundly reshapes our understanding of NMDAR diversity as well as glycinergic neurotransmission. Key results on the mode of action and roles of these newly discovered receptors are expected in the near future.

4. We have generated original mouse lines (ZnT3-Cre, condition GluN1 glycine/D-serine knock-in) that have started to provide essential novel insights on the composition, plasticity and functional importance of the synaptic microenvironment. These new animal models provide unmatched means to explore the regulation of NMDA receptors and its dynamics during normal and pathological brain function.