The interplay between HPCAL4 and MeCP2: identification and characterization of a novel putative target for Rett syndrome therap

To dissect the possible timing of HPCAL4 contribution to neurodevelopment and Rett Syndrome, we developed a three-sided spatio-temporal approach aimed at revealing: 1) Hpcal4 protein expression pattern in WT and RTT mice and neurons; 2) endogenous Hpcal4 subcellular localization and 3) Hpcal4-downregulation effects on calcium homeostasis in resting and depolarized neurons. We found that Hpcal4 protein level increases during cortical and hippocampal maturation, while it decreases in cerebellum and brainstem of WT mice. In P20 Mecp2 KO male brains, Hpcal4 protein level was unaffected compared to WT, despite a general downregulation at P40 and P60, being more prominent in the cerebellum at P40. Consistently with this, Hpcal4 protein expression increased in maturing WT cortical neurons and decreased starting from DIV19 KO ones. Concerning Hpcal4 localization, we found it diffused in the neuronal soma, dendrites and spines in resting condition, while upon depolarization, it appears to be more associated with the plasma membrane. Colocalization analysis with presynaptic markers revealed no preferential localization neither in the inhibitory, nor in the excitatory presynaptic compartment, but the portion of the excitatory presynaptic markers colocalizing with Hpcal4 increased with neuronal maturation. To test the involvement of Hpcal4 in the functional activity of the cortical network, shRNA mimicked the downregulation observed in RTT samples. In Hpcal4-downregulated neurons an increase in the global MFR was observed, despite no alterations in neuronal excitability, the basal calcium level was reduced. Increased glutamatergic and a slight reduction of GABAergic transmission resulted in higher E/I ratio. These data indicate that Hpcal4 regulates global network activity by setting the basal Ca2+ level in neurons, which influences the E/I synaptic ratio. Lastly, a proteomic approach identified three Hpcal4 targets, confirming its function at the synaptic level.