Disruption of the Homer1 coiled-coiled domain by a novel de novo human HOMER1 variant impairs protein scaffolding, calcium signalling, and synaptogenesis

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 ²⁺ signalling, dendritic spine morphogenesis and multiple forms of synaptic plasticity. Here we report a novel substitution mutation in the human HOMER1 gene, HOMER1 ^R297W , and demonstrate that Homer1b/c ^R297W expression dominant-negative like effect on Homer1-dependent functions. In dorsal root ganglion (DRG) sensory neurons, Homer1b/c1 ^R297W impairs axonal growth cone turning to gradients of brain-derived neurotrophic factor (BDNF), a process that requires functional store-operated Ca ²⁺ entry (SOCE). Accordingly, we found that SOCE was significantly blunted in both Homer1b/c ^R297W DRG growth cones and hippocampal neuron soma. In hippocampal neurons, Homer1b/c ^R297W lowered dendritic spine density and reduced endoplasmic reticulum infiltration into spines. Homer1b/c ^R297W 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 ^R297W 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 ^R297W 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.