GluA3 as a key regulator of dendritic structure and synaptic plasticity

AMPA-type glutamate receptors (AMPARs), composed of GluA1–GluA4 subunits, mediate fast excitatory synaptic transmission in the brain. Among AMPARs’ subunits, the physiopathological role of GluA3 remains poorly understood. To address this, we downregulated GluA3 using an shRNA-based approach in both in vitro and in vivo models. Primary rat hippocampal neurons were transduced with either shRNA-GRIA3 or scrambled RNA viral vectors at DIV10, and analyses were performed at DIV20. GluA3 silencing significantly reduced dendritic arbor complexity and increased the proportion of thin spines, indicating impaired structural maturation. At the molecular level, GluA3 downregulation selectively decreased surface expression of GluA1-containing AMPARs, as well as GluN2A- and GluN2B-containing NMDARs. Consistently, live calcium imaging revealed a marked reduction in spontaneous NMDAR-mediated calcium transients, while glutamate release was not affected. Moreover, repeated glutamate uncaging-induced single-spine stimulation resulted in defective spine enlargement and abnormal calcium accumulation, likely due to impaired recruitment of GluA1-containing AMPARs to the postsynaptic membrane, as indicated by SEP-GluA1 fluorescence. RNA-seq analysis further identified dysregulation of key activity-dependent genes, including CAMK2A and MAPK1 (encoding ERK1), as well as pathways involved in protein trafficking and neuronal development. Rescue experiments demonstrated that re-expression of ERK1 was sufficient to restore dendritic arborization, thereby identifying ERK1 signaling as downstream effector of GluA3. Notably, GluA3 silencing exhibited a clear time-dependent effect: early silencing (DIV3) caused more severe deficits, whereas late silencing (DIV14) had minimal impact. Considering the temporal specificity and the role of GluA3 in primary hippocampal neurons, we downregulated its expression in the CA1 region of the hippocampus in both adolescent and adult male mice. We detected alterations in dendritic complexity comparable to in vitro observations, further supporting a role for GluA3 as an upstream regulator of neuronal architecture. Ongoing behavioral studies are currently being conducted to determine whether GluA3 loss ultimately leads to cognitive deficits.