GLUD1 MUTATIONS ASSOCIATED WITH INTELLECTUAL DISABILITY ALTER SYNAPTIC CLUSTERING AND RECEPTOR MEMBRANE DYNAMICS, AS WELL AS GROUP I MGLU RECEPTOR–DEPENDENT REGULATION

The ionotropic glutamate delta receptor GluD1 is widely expressed in the nervous system and plays key roles in synapse formation, function, and plasticity, yet the underlying molecular and cellular mechanisms remain poorly understood. Although classified as an ionotropic receptor, GluD1 does not bind glutamate; instead, it interacts with cerebellins, D-serine, and GABA to form trans-synaptic bridges and trigger transmembrane signaling. We recently identified two GluD1 mutations, R161H and T752M, associated with intellectual disability, which impair group I metabotropic glutamate receptor (mGlu1/5) signaling and reduce excitatory synapse density. Here, we investigated how these mutations alter GluD1 synaptic nanoscale organization and its functional coupling to mGlu1/5.
Primary rat hippocampal neurons were transfected with HA-tagged GluD1 wild-type, R161H, or T752M constructs. We combined super-resolution microscopy, single-particle tracking, and immunostaining to analyze receptor clustering and mobility, together with calcium imaging to assess neuronal responses. We found that GluD1 forms organized clusters at excitatory synapses whose spatial organization is governed by a diffusion-capture mechanism. Notably, the R161H and T752M mutations differentially altered both the size of GluD1 clusters and its diffusion properties. Functionally, both mutants strongly reduced the intracellular calcium rise triggered by mGlu1/5 activation with DHPG. Moreover, mGlu1/5 activation regulated GluD1 synaptic dynamics, and this regulation was disrupted by both mutations.
Altogether, our results show that disease-associated GluD1 mutations perturb receptor nanoscale organization and membrane dynamics, as well as synaptic signaling, thereby providing a mechanistic link between altered receptor organization and synaptic dysfunction in neurodevelopmental disorders.