Periodic Reporting for period 1 - DynamiK (Exploring the role of NMDA receptors in K+ channel nanoscale organisation, surface dynamics and function)
Período documentado: 2023-10-01 hasta 2025-09-30
N-methyl-D-aspartate receptors (NMDARs) are a class of neurotransmitter receptors that bind glutamate, the major excitatory neurotransmitter in the brain. NMDARs support essential brain functions, including neuronal communication and development, learning, and memory. These receptors form a pore that allows the passage of essential ions like calcium and sodium across the neuron membrane to modulate neuronal activity. Indeed, through calcium-driven signalling, NMDARs are best known for regulating the strength of synapses (the communication points between neurons) in a process termed synaptic plasticity, which is thought to underly learning and memory. However, the advancement of imaging techniques in past years has further revealed that the role of NMDAR goes beyond their calcium permeability. The way NMDARs move around the cell surface and how they are organized at a nanoscale level in specialized domains can also significantly shape synaptic plasticity.
About 15 years ago, researchers found circulating antibodies targeting NMDARs (NMDAR-Abs) in patients with autoimmune encephalitis, schizophrenia and psychosis, and which cause severe psychiatric, cognitive and neurological symptoms. This remarkable discovery reshaped how several neurological and psychiatric disorders are diagnosed and further substantiated the idea that NMDAR function is more than being an ion channel. At the cellular level, the antibodies disturb the nanoscale organization and surface diffusion of NMDARs, causing the receptors to be internalized. This reduces NMDAR function and weakens synaptic plasticity, even though calcium flow through the receptors remains unchanged, once again corroborating an ionotropic-independent role of NMDARs. A puzzling clinical feature of NMDAR-Abs is that many patients also experience seizures, associated with neuronal hyperexcitation, despite the antibody-induced reduction of NMDAR excitatory function. Indeed, NMDAR-Abs make cells more excitable (meaning that they are more easily activated) and cause excessive excitation in hippocampal networks, explaining the seizures. However, the mechanisms behind this particular effect of the NMDAR-Abs are still not understood.
Considering the relevance of NMDARs in the regulation of multiple neuronal dimensions and their implication in several neurological/psychiatric disorders, it is essential that we learn the full landscape of NMDAR physiology to understand their associated pathology. One key piece of knowledge that is missing so far is the characterization of the surface environment (where NMDAR-Abs act, for instance) surrounding NMDARs, including knowing who their neighbors are and how they behave at the cell surface as well. In this project, we aimed to do precisely this. By employing advanced biochemical and super-resolution microscopy approaches, we aimed at creating a snapshot of the surface interactome of NMDARs and investigate how it might be remodeled in the presence of NMDAR-Abs.
Identifying important interactors of NMDARs (which could potentially also be corrupted by NMDAR-Abs) will not only provide further insight into the physiological role of NMDARs but also shed light on the full pathogenic mechanisms elicited by the antibodies.
About 15 years ago, researchers found circulating antibodies targeting NMDARs (NMDAR-Abs) in patients with autoimmune encephalitis, schizophrenia and psychosis, and which cause severe psychiatric, cognitive and neurological symptoms. This remarkable discovery reshaped how several neurological and psychiatric disorders are diagnosed and further substantiated the idea that NMDAR function is more than being an ion channel. At the cellular level, the antibodies disturb the nanoscale organization and surface diffusion of NMDARs, causing the receptors to be internalized. This reduces NMDAR function and weakens synaptic plasticity, even though calcium flow through the receptors remains unchanged, once again corroborating an ionotropic-independent role of NMDARs. A puzzling clinical feature of NMDAR-Abs is that many patients also experience seizures, associated with neuronal hyperexcitation, despite the antibody-induced reduction of NMDAR excitatory function. Indeed, NMDAR-Abs make cells more excitable (meaning that they are more easily activated) and cause excessive excitation in hippocampal networks, explaining the seizures. However, the mechanisms behind this particular effect of the NMDAR-Abs are still not understood.
Considering the relevance of NMDARs in the regulation of multiple neuronal dimensions and their implication in several neurological/psychiatric disorders, it is essential that we learn the full landscape of NMDAR physiology to understand their associated pathology. One key piece of knowledge that is missing so far is the characterization of the surface environment (where NMDAR-Abs act, for instance) surrounding NMDARs, including knowing who their neighbors are and how they behave at the cell surface as well. In this project, we aimed to do precisely this. By employing advanced biochemical and super-resolution microscopy approaches, we aimed at creating a snapshot of the surface interactome of NMDARs and investigate how it might be remodeled in the presence of NMDAR-Abs.
Identifying important interactors of NMDARs (which could potentially also be corrupted by NMDAR-Abs) will not only provide further insight into the physiological role of NMDARs but also shed light on the full pathogenic mechanisms elicited by the antibodies.
To capture a snapshot of the surface environment around NMDARs, we first employed a staining approach with a fluorescent dye (NHS-ester) that labels all proteins at the neuronal cell surface and then used dSTORM microscopy, a super-resolution imaging technique that maps individual molecules with much higher detail than conventional microscopy, allowing for their nanoscale reconstruction. We found that protein organization and density are similar across the neuron surface, including at synapses, which are naturally very protein-dense. Remarkably, NMDAR-Abs rapidly remodel surface proteins in non-synaptic areas, while synaptic proteins are affected only after several hours, revealing a widespread reorganization of the neuronal surface.
We next aimed to identify the surface interactome of NMDARs. To do this, we developed a biochemical approach that labels all surface proteins within a short distance of the receptor. We engineered a plasmid in which the main NMDAR subunit, GluN1, is fused to a horseradish peroxidase (HRP) enzyme. When hydrogen peroxide is added, HRP converts a non-permeant biotin compound into reactive molecules that attach to proteins within roughly 20 nm, allowing us to tag all surface proteins surrounding NMDARs. These biotin-labelled proteins can then be isolated and purified using streptavidin-coated beads and identified by liquid chromatography–tandem mass spectrometry, a quantitative proteomics technique that can detect and sequence proteins in small samples. Using this approach, we identified hundreds of proteins enriched around NMDARs, spanning multiple protein families such as adhesion molecules, receptors, ion channels, and extracellular matrix components. Importantly, the abundance of many of these proteins around NMDARs was changed in the presence of NMDAR-Abs, confirming that the antibodies cause extensive reshaping of the surface protein environment surrounding NMDARs.
Finally, we examined how NMDAR-Abs affect one of the potential interactors of NMDARs. Potassium (K⁺) channels are of particular interest because they not only interact functionally with NMDARs but also play a central role in controlling neuronal excitability, which we know is disrupted by NMDAR-Abs. We found that the antibodies alter both the synaptic localization and the nanoscale organization of a specific subfamily of K⁺ channels. Ongoing experiments are now testing whether the function of this channel is also impaired, and critically, whether genetic or pharmacological inhibition of this channel can counteract the hyperexcitability caused by NMDAR-Abs.
We next aimed to identify the surface interactome of NMDARs. To do this, we developed a biochemical approach that labels all surface proteins within a short distance of the receptor. We engineered a plasmid in which the main NMDAR subunit, GluN1, is fused to a horseradish peroxidase (HRP) enzyme. When hydrogen peroxide is added, HRP converts a non-permeant biotin compound into reactive molecules that attach to proteins within roughly 20 nm, allowing us to tag all surface proteins surrounding NMDARs. These biotin-labelled proteins can then be isolated and purified using streptavidin-coated beads and identified by liquid chromatography–tandem mass spectrometry, a quantitative proteomics technique that can detect and sequence proteins in small samples. Using this approach, we identified hundreds of proteins enriched around NMDARs, spanning multiple protein families such as adhesion molecules, receptors, ion channels, and extracellular matrix components. Importantly, the abundance of many of these proteins around NMDARs was changed in the presence of NMDAR-Abs, confirming that the antibodies cause extensive reshaping of the surface protein environment surrounding NMDARs.
Finally, we examined how NMDAR-Abs affect one of the potential interactors of NMDARs. Potassium (K⁺) channels are of particular interest because they not only interact functionally with NMDARs but also play a central role in controlling neuronal excitability, which we know is disrupted by NMDAR-Abs. We found that the antibodies alter both the synaptic localization and the nanoscale organization of a specific subfamily of K⁺ channels. Ongoing experiments are now testing whether the function of this channel is also impaired, and critically, whether genetic or pharmacological inhibition of this channel can counteract the hyperexcitability caused by NMDAR-Abs.
This study delivers the first comprehensive map of the NMDAR surface interactome, providing crucial insight into the physiological surface environment of the receptor. Remarkably, the project unveils a profound remodeling of the neuronal surface induced by NMDAR-Abs, globally reorganizing the surface proteome and altering the abundance of proteins specifically surrounding NMDARs. Potassium channels emerge as potential contributors to these effects, but their role in driving antibody-induced neuronal dysfunction remains to be established.