Deciphering the role of the Tdark gene HPCAL4 in neurodevelopment and Rett syndrome

HPCAL4 is a Tdark gene encoding a neuronal calcium sensor with an unexplored role in neurodevelopment and disease. Since MECP2 deficiency in Rett syndrome (RTT) disrupts Ca²⁺-dependent pathways essential for dendritic arborization, synaptic density, and neuronal signaling, we found it intriguing that Hpcal4 is downregulated in both the human RTT cerebellum and the RTT mouse cortex, suggesting its potential involvement in RTT pathogenesis. To investigate this, we characterized Hpcal4 expression across neuronal development in WT and RTT mice, defined its subcellular localization under resting and stimulated conditions and assessed its downregulation impact on calcium homeostasis and synaptic activity. Expression analysis revealed that Hpcal4 is developmentally regulated and significantly reduced in early symptomatic Mecp2 deficient mice and Mecp2 null primary neurons. Through proximity labeling and co-immunoprecipitation, we identified Hpcal4-interacting proteins, highlighting its synaptic role. Imaging further confirmed its widespread distribution across neurons, showing a shift toward the plasma membrane upon depolarization, with accumulation in the presynaptic compartment. Finally, functional analysis through shRNA-mediated HPCAL4 downregulation mimicked RTT-associated changes, such as increased MFR, reduced basal calcium levels and altered E/I balance. These findings highlight HPCAL4 as a key modulator of neuronal activity, calcium homeostasis and synaptic vesicle cycle, implicating it in RTT pathogenesis.