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Optical strategies to investigate NMDA receptor functional diversity and its therapeutic potential

Periodic Reporting for period 1 - OptoNMDA (Optical strategies to investigate NMDA receptor functional diversity and its therapeutic potential)

Reporting period: 2016-04-01 to 2018-03-31

The function of the human brain and its capacity for experience-dependent changes hinge on the dynamics of chemical synapses. At these specialized neuronal sites, chemical transmitters released from presynaptic terminals diffuse across the synaptic cleft and activate receptors localized primarily on the postsynaptic cells, thereby transmitting the flow of information from one neuron to another. Among these receptors, NMDA receptors (NMDARs) form a class activated by glutamate, the major neurotransmitter in the mammalian brain. These receptors play fundamental roles in synaptic transmission and plasticity (the cellular basis of memory). They are also involved in numerous pathologies including stroke, mental retardation or schizophrenia. NMDARs are thus targets of strong therapeutic interest. NMDARs are tetramers usually composed of two GluN1 and two GluN2 subunits encoded by four different genes (GluN2A-D), resulting in a large number of receptor subtypes having distinct anatomical, biophysical, pharmacological and signalling properties. Understanding the functional role of these individual subtypes in the brain is of great importance to develop new strategies to counteract the deleterious effects of NMDAR dysregulation. However, tools allowing targeting of a specific population of NMDARs in a given neuronal circuit are currently lacking. The OptoNMDA research programme proposes the development of an innovative optical approach to selectively enhance the activity of NMDARs containing the GluN2B subunit (GluN2B-NMDARs). This approach consists in developing photo-convertible polyamine (PP) derivatives that can reversibly isomerize between an inactive and active configuration with light to act on the GluN2B-NMDAR polyamine allosteric modulating site in a precise spatio-temporal manner (opto-allostery). By tethering these PPs in the polyamine putative binding site, we managed to create photo-enhanced GluN2B-NMDARs. This project should thus provide critical novel information on NMDARs, and more generally, on the physiology and pathology of excitatory synapses.
To selectively photo-potentiate GluN2B-NMDARs, we envisioned two strategies: one using soluble photoswitchable polyamines (SPPs), which can act on endogenous receptors but can have off-target effects (WP1); and one using tethered photoswitchable polyamines (TPPs), which have almost no off-target effect but require genetic engineering of the receptor (WP2).

Work Package 1: Selectively enhancing GluN2B-NMDARs with SPPs
In collaboration with the Baigl Lab (Ecole Normale Supérieure, Chemistry Department), we had previously designed a photoswitchable spermine, called AzoSp, that combined (i) a spermine moiety that acts as the potentiating ligand; and (ii) an azobenzene moiety that acts as the photoswitch. Azobenzenes can indeed switch from an extended, trans configuration to a bent, cis configuration following illumination with UV light, and back to trans following illumination with visible light. Like its parent compound spermine, AzoSp selectively potentiated GluN2B-containing NMDARs at low concentration, but also induced a very strong inhibition of all NMDAR subtypes by binding to the ion channel pore. The concentrations at which AzoSp induced potentiation and inhibition were not different enough to selectively potentiate GluN2B-NMDARs while avoiding inhibition of the other NMDAR subtypes. We therefore decided to stop working with SPPs and rather focus on Work Package 2.

Work Package 2: Selectively photo-enhancing GluN2B-NMDARs TPPs.
We designed a cysteine-reactive, photoswitchable spermine, called MASp, by attaching to AzoSp a cysteine-reactive maleimide. To covalently attach MASp to the GluN2B subunit, we substituted residues of the spermine putative binding site into cysteines and labeled the cysteine-mutated receptors with MASp. Out of the 23 substitution positions tested on the GluN2B subunit, 5 showed a >30 % photomodulation of the NMDAR current. One position in particular yielded up to 5-fold UV-induced potentiation after MASp labeling. Photomodulation at this position was reversible, reproducible, and allosteric, meaning that it occurred only when the receptor was activated by the agonists. This latter aspect is important, since it allows respecting the temporal pattern of receptor activation in vivo. We chose this mutant as the photo-switchable receptor to introduce in native preparations (WP3). Importantly also, labeling of GluN1/GluN2A receptors, the other major class of NMDARs in the brain, with MASp did not yield any photomodulation. We have therefore reached our goal, which was to selectively and reversibly photo-enhance GluN2B-NMDARs.
This work was presented as a poster at the Young Researcher In Life Sciences conference (Paris, May 2017).

Work Package 3: towards photo-enhancement of GluN2B-NMDAR activity in native tissues
We are currently in the process of characterizing the photomodulation of our photoswitchable receptors in HEK cells and neurons to test for the volatility of the approach and off-target effects. Our next step will be to introduce the mutated receptor in hippocampal slices and investigate the effect of potentiating GluN2B-NMDARs on several physiological processes like synaptic transmission, synaptic plasticity and excitotoxicity.
During this project, we have designed for the first time photo-enhanced NMDA receptors. We are therefore in capacity to provide the neuroscience community with a tool to selectively increase the activity of GluN2B-NMDARs with high spatial and temporal resolution. This will allow deciphering the relative contributions of this specific subpopulation in different processes, such as synaptic plasticity and excitotoxicity.
Contrary to most studies in the field that target the agonist binding-site to photo-activate or -deactivate proteins, our approach targets an allosteric site, meaning that NMDARs need first to be activated by endogenous glutamate, to be able to be photo-enhanced. This approach has the advantage of not interfering with the natural activation pattern of the receptors. This is of considerable importance for NMDARs, since direct activation of these receptors through the agonist binding-sites can lead to their overactivation and subsequent neuronal death. This approach is one the first examples of optical allosteric modulation, or “opto-allostery”, of a membrane receptor and will be able to be generalized to other proteins in the future.
Furthermore, our project is expected to have significant socio-economic impact. It will allow to investigate the relevance of GluN2B-NMDAR potentiation in pathological cases of NMDAR hypofunction, such as age-related mental retardation, cognitive decline or schizophrenia. Aging and mental disorders are currently major national and international socio-economic challenges. The OptoNMDA project may thus have a strong socio-economic impact in the future by leading to new therapeutic strategies to overcome NMDAR-related neurological diseases.
Finally, during the course of the OptoNMDA project, the financed researcher obtained a permanent researcher position at INSERM, in France. Therefore, this grant fully fulfilled its role as a springboard for the researcher to reintegrate a long-term research position in Europe.