The purpose of this study is to screen for and test the effectiveness of dantrolene (Dl) as a novel drug to improve Darier disease (DD).
By using various in vitro models which recapitulate the abnormal ER-Ca2+ homeostasis seen in DD cells, we tested whether the novel drug Dl (which inhibits the intracellular influx of Ca2+) is able to impact mechanistic factors associated with the pathological basis of DD. These include ER-Ca2+ homeostasis, cell-to-cell adhesion, and ER stress. Firstly, in normal human epidermal keratinocyte (HEKA) cells treated with thapsigargin (Tg) (a non-competitive inhibitor of SERCA2) as well as in HEKA cells with ATP2A2 genetically knocked down (siATP2A2), we demonstrated using confocal imaging that ER-Ca2+ loss is retained with the addition of Dl. In addition, through western blot analysis from both models we demonstrated that Dl is able to rescue the downregulation of SERCA2.
Next, using both a presto blue cell viability assay, as well as a caspase 3/7 apoptosis assay, we demonstrated in both previously mentioned in vitro models that Dl promoted cell viability.
Additionally, when investigating the impact Dl has on ER stress, we found through qPCR analysis that several ER stress-related genes were significantly lower in dantrolene treated HEKA models. Importantly, Dl was also found to significantly reduce the genetic expression of ER stress genes in fibroblasts derived from DD patient biopsies. We also demonstrated that Dl promoted the transcription of several cell adhesion genes in the HEKA cell models, as well as in DD fibroblasts. Next, using a dispase mechanical assay, we found that in both in vitro models, again, Dl promoted cell adhesion. Finally, we also performed confocal microscopy using markers for cell adhesion (β-catenin, OCLN and DSG-1) in both in vitro models and found that Dl promoted the expression of them all.
So far, through investigations of pathological mechanisms underpinning abnormal ER-Ca2+ homeostasis seen in DD, we have demonstrated that Dl may have therapeutic potential in the treatment of DD.

LB006 - Correcting endoplasmic reticulum calcium dyshomeostasis as a novel treatment for Darier disease

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