Molecular Aspects and Mechanisms Underlying Darier’s Disease: The Role of NOTCH1 and SERCA2 Protein Interaction

Darier's disease (DD, OMIM #124200) is a rare genodermatosis with autosomal dominant inheritance affecting approximately 1 in 50,000 individuals. Clinically, it manifests as skin lesions in the form of hyperkeratotic papules which evolve into crusty plaques, mainly located in areas exposed to UV radiation, heat or friction. These lesions can get worse with bacteria, fungi, and yeasts, and typically appear between the ages of 6 and 20 years. Histopathologically, DD is characterized by acantholysis (loss of intercellular adhesion), dyskeratosis (premature and abnormal keratinisation) and rounded keratinocytes (corps ronds). The disease is caused by pathogenic variants in the ATP2A2 gene (located in 12q23-24.1), which encodes the SERCA2 protein (Sarco/Endoplasmic Reticulum Calcium ATPase 2), an ATP-dependent pump involved in the active transport of calcium from the cytosol to the lumen of the endoplasmic reticulum (ER). Missense or nonsense variants, deletions, insertions or splicing alterations in the ATP2A2 gene impair SERCA2 function, thereby altering intracellular calcium homeostasis. This may trigger an ER stress response and cell apoptosis, contributing to the pathogenesis of the disease. However, the molecular mechanisms underlying the pathological process and the epidermal aberrant clinical phenotype are not yet fully understood. Our research hypothesis is based on previous studies conducted on other disease models such as T-cell leukemia, in which SERCA2 directly modulates the activity of the NOTCH1 receptor signaling pathway. Moreover, the NOTCH1-activated signaling pathway plays a key role in physiological keratinocyte differentiation by inhibiting the proliferation of basal precursors after its activation with terminal cell differentiation. Based on these observations, our study, for the first time, investigates potential alterations in the pathway activity mediated by NOTCH1 receptor in patients affected by DD. Our study involved the recruitment of 19 patients affected by DD and 20 healthy controls. Blood samples were collected to detect genetic variants in ATP2A2 by gene sequencing in order to create a plasmid library for the in vitro over-expression of the identified patient-specific ATP2A2 variants. In vitro assays, including Real-time PCR, Western blot, Immunofluorescence, and Luciferase assays, were conducted by overexpressing plasmid library on HACAT (Human Aneuploid Keratinocyte) and HEK293T (Human Embryonic Kidney 293T) cell lines. Moreover, from the same patients a punch skin biopsy has been collected for generation of primary keratinocytes and fibroblasts cell cultures to evaluate the activity of the NOTCH1-mediated signaling pathway. Bioptic ex-vivo analysis were performed, such as RNA-sequencing, Immunofluorescence, Immunohistochemistry and Spatial Transcriptomics assays. The results indicate a significant dysregulation of NOTCH1 activity in DD samples compared to wild-type controls. A genotype-phenotype correlation was observed: different pathogenic variants of ATP2A2 result in varying levels of gene dysregulation in NOTCH1 targets, which are associated with differences in disease severity. Moreover, transcriptome analysis revealed a specific “transcriptional signature” of DD, highlighting not only the alteration of NOTCH1-related genes but also those involved in metabolic pathways, ribosomal structure, mitochondrial activity, and immune and inflammatory responses. These findings expand the understanding of disease pathogenesis and may suggest new potential therapeutic targets.