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Development of 3D epidermal models: towards the development of a skin model for studies of the autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS)
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The skin is a complex organ mainly responsible for protecting the body from external threats and maintaining homeostasis. It is a complex three-dimensional structure that is composed of two main compartments, the dermis and the epidermis. Due to increasing ethical and legal pressure on animal usage in research, reconstructed 3D human skin models have been gaining popularity. These models mimic human skin architecture in vitro and allow relatively easy manipulation to meet specific needs. Some rare diseases remain poorly studied and could take advantage of this technology. One example is the Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) which is an early-onset neurological disease that was first described in Quebec, Canada, but cases have been reported worldwide. Patients suffer from spasticity and lack of coordination of muscle movements, resulting in an early wheelchair dependence and premature death. ARSACS is caused by loss-of-function mutations in the SACS gene, leading to a defective sacsin protein. Sacsin loss of function has been linked to mitochondrial dysfunction and abnormalities in the organization of intermediate filaments, but the complete picture is still unclear. Evidence of abnormalities in the skin of ARSACS patients has been reported, making this disease an interesting candidate to be studied using in vitro skin models. In this work, two different human keratinocyte cell lines (HaCaT and N/TERT-1) were used to create new human epidermal models using a polycarbonate inert matrix. The localization of different keratins and other markers (keratins 10, 14 and 15, and involucrin) were studied to characterize epidermal differentiation and stratification. Sacsin expression was analyzed in different cell lines and sacsin knockdown was attempted in HaCaT keratinocytes using lentiviral shRNAs. The HaCaT cell line was unable to recreate the normal multi-layer architecture of native skin nor the stratum corneum. This cell line expressed low amounts of the sacsin protein, and no difference was observed between the knockdown and the control by western blot. N/TERT-1 keratinocytes generated a stratified epidermis with all the normal layers present, including the stratum corneum. Complete epidermal differentiation was confirmed by the differential expression of epidermal markers. K14 expression was limited to the basal layer, while K10 was expressed in the upper layers, as expected. Involucrin was mostly expressed in the stratum granulosum and K15 expression was overall very low, indicating a successful differentiation. Sacsin expression was verified in different skin cells (HEKn, HDFn, and N/TERT-1), and N/TERT-1 expressed sacsin in amounts slightly lower than primary human keratinocytes. These findings suggest that the N/TERT-1 cell line has more potential to produce an epidermal skin model with an ARSACS phenotype, which can prove an important tool in future research. Despite the existing knowledge about sacsin structure and function, a lot is still unknown about this protein and how it causes the symptoms underlying ARSACS disease. Advances in this topic could contribute to the development of therapies that could cure or tackle some of ARSACS symptoms to ensure a better quality of life for the patients.
Descrição
Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2021
Palavras-chave
HaCaT N/TERT-1 Modelo de epiderme in vitro ARSACS Sacsin Teses de mestrado - 2021