THE INTERPLAY BETWEEN HPCAL4 AND MECP2: IDENTIFICATION AND CHARACTERIZATION OF A NOVEL PUTATIVE TARGET FOR RETT SYNDROME THERAPY

Rett Syndrome (RTT) is a severe neurodevelopmental disorder and the leading cause of intellectual disability in females with 1 in 10,000 births being affected. The symptoms start to manifest in between 6 and 18 months of age and these include intellectual disability, epilepsy and impairments of social and motor skills. Most cases (90-95%) arise from sporadic mutations within the X-linked gene coding for the methyl-CpG binding protein 2 (MeCP2). It is an important epigenetic regulator that is ubiquitously expressed and particularly abundant in brain and important for proper neuronal maturation and function. In fact, MeCP2 deficiency in neurons is responsible for impairment of fundamental aspects of neuronal development and function including defects in dendritic arborization and spine density. Besides Mecp2, several other genes have been associated with RTT or RTT-like phenotypes and the number of disease-candidate genes has grown over the years. Therefore, identifying novel molecular players may provide new insights into RTT pathophysiology and uncover potential modifier genes that influence disease progression and severity. In this work, we investigated the neuronal calcium sensor hippocalcin-like 4 (Hpcal4), which encodes for a protein belonging to the visinin-like (VSNL) subfamily of EF-hand Ca2+-sensing proteins. Evidence in literature alongside previous transcriptomic data from our laboratory, consistently reported a downregulation of this gene in RTT patients and mouse models. However, HPCAL4 functions remain fully undisclosed; indeed, it belongs to the category of T-dark genes, which comprises of protein coding genes with limited or unknown function in literature. Therefore, we aimed in this project to clarify Hpcal4’s involvement in neuronal functions in physiological conditions and determine if it can be flagged as a modifier gene for RTT by contributing to the observed RTT phenotypes. We explored the expression of the protein in the wild type and RTT mouse brain and found that it is regionally modulated along brain development and deficiently expressed in the symptomatic RTT brain. We additionally analyzed the protein levels in primary neuronal cultures where they were found to be increased during neuronal maturation and reduced in Mecp2 KO neurons. By assessing its subcellular level, we found that HPCAL4 is present in the soma and dendrites, enriched in the pre-synaptic compartment, and localizes to the plasma membrane in an activity-dependent manner. Proteomic analysis revealed that HPCAL4 interacts with a set of proteins with well established synaptic functions, including synaptic vesicles recycling and neurotransmitter release. Finally, we characterized the phenotypes resulting from its downregulation in neurons and found similar defects that are present in RTT neurons, including reduction in dendritic arborization accompanied by impairments in neuronal maturation. In conclusion, our study highlights a putative novel function for HPCAL4 in neuronal maturation and synaptic processes. Its decreased levels in RTT models, together with the phenotypes observed upon its downregulation, suggest that it may contribute to RTT pathophysiology as a modifier gene.