Unravelling the role of Hpcal4 as a potential modifier gene for Rett Syndrome

Rett Syndrome (RTT) is a severe neurodevelopmental disorder mainly prevalent among females with 1:10.000 births being affected; it is considered the most frequent cause of profound intellectual disability in females. RTT patients appear to have a normal development up to 6–18 months when the neurological development arrests accompanied by the loss of most early acquired developmental skills, including communication 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). The protein is ubiquitously expressed and is particularly abundant in brain and associated mutations have profound effects on various neural phenotypes such as impairments in synaptogenesis and neuronal maturation. 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. Evidence in literature alongside our RNA-seq data highlighted a downregulation of the Hippocalcin-like 4 (Hpcal4) mRNA in the cerebellum, the hippocampus and the cortex of RTT mouse models lacking Mecp2, in addition to a reduction of the protein levels in the cortex of KO animals. The mRNA encodes for a protein that belongs to the visinin-like protein family (VSNL), which are usually involved in the modulation of voltage-gated Ca2+ channels, kinase modulation and the Ca2+ mediated release of neurotransmitters by vesicles that is relevant to initiate synaptic transmission. However, Hpcal4 functions remain fully undisclosed; indeed, it belongs to the category of Tdark genes, which comprises of protein coding genes with limited or unknown function in literature. Therefore, we aim at clarifying its involvement in neuronal functions in physiological conditions and in RTT and its possible causative link with typically observed RTT neuronal phenotypes. We explored the expression of the protein in the wild type and RTT mouse brain at different time points 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. We further assessed the endogenous expression of the protein in primary hippocampal and cortical neurons and evaluated its colocalization with synaptic markers and determined that the protein is enriched at the level of pre-synaptic compartment. Co-immunoprecipitation of Hpcal4 followed by mass spectrometry analysis in wild type mice cortical lysates revealed several binding interactors that will be validated in future studies, alongside further in vitro approaches that will be applied to understand Hpcal4 mechanism of action and dissect its functional role in neurons. These implemented approaches will be fundamental to determine if Hpcal4 can be flagged as a modifier gene for RTT.