Abstract
<jats:title>Abstract</jats:title> <jats:p> Rare de novo variants in synaptic scaffolding proteins are increasingly recognized for their roles in driving abnormal neuronal connectivity underlying conditions such as epilepsy and autism spectrum disorder (ASD). Homer1b/c, a synaptic scaffolding protein, regulates a wide suite of synaptic functions including Ca <jats:sup>2+</jats:sup> signalling, dendritic spine morphogenesis and multiple forms of synaptic plasticity. Here we report a novel substitution mutation in the human <jats:italic>HOMER1</jats:italic> gene, <jats:italic> HOMER1 <jats:sup>R297W</jats:sup> </jats:italic> , and demonstrate that Homer1b/c <jats:sup>R297W</jats:sup> expression dominant-negative like effect on Homer1-dependent functions. In dorsal root ganglion (DRG) sensory neurons, Homer1b/c1 <jats:sup>R297W</jats:sup> impairs axonal growth cone turning to gradients of brain-derived neurotrophic factor (BDNF), a process that requires functional store-operated Ca <jats:sup>2+</jats:sup> entry (SOCE). Accordingly, we found that SOCE was significantly blunted in both Homer1b/c <jats:sup>R297W</jats:sup> DRG growth cones and hippocampal neuron soma. In hippocampal neurons, Homer1b/c <jats:sup>R297W</jats:sup> lowered dendritic spine density and reduced endoplasmic reticulum infiltration into spines. Homer1b/c <jats:sup>R297W</jats:sup> hippocampal neurons also exhibited decreased synaptic metabotropic glutamate receptor 5 (mGluR5) expression and blunted dendritic Ca²⁺ increases following group-I mGluR activation. Super resolution imaging using direct stochastic optical reconstruction microscopy (dSTORM) further demonstrated that Homer1b/c <jats:sup>R297W</jats:sup> diminishes receptor clustering, uncoupling it from crucial binding partners including IP3R, mGluR5 and STIM1/2. Taken together, these findings highlight the importance of Homer1b/c’s tetrameric scaffolding in shaping axon guidance, dendritic spine dynamics and synaptic Ca²⁺ signalling. Disruption of these processes by Homer1b/c <jats:sup>R297W</jats:sup> offers valuable mechanistic insights into how rare de novo variants and altered protein scaffolding can contribute to the connectivity deficits implicated in neurodevelopmental and neurological disorders. </jats:p>