Protein Signature of Human Skin Fibroblasts Allows the Study of the Molecular Etiology of Rare Neurological Diseases
Abstract Background: The elucidation of pathomechanisms leading to the manifestation of rare (genetically caused) neurological diseases including neuromuscular diseases (NMD) represents an important step toward the understanding of the genesis of the respective disease and might help to define starting points for (new) therapeutic intervention concepts. However, these “discovery studies” are often limited by the availability of human biomaterial. Moreover, given that results of next-generation-sequencing approaches frequently result in the identification of ambiguous variants, testing of their pathogenicity is crucial but also depending on patient-derived material. Results: To systematically address the question if human skin fibroblasts might serve as valuable biomaterial for (molecular) studies of NMD, using proteomic profiling, we generated a protein library by decreasing protein complexity via pH8-based sample fractionation: cataloguing of 8280 proteins revealed the expression of a variety of such linked to genetic forms of motoneuron diseases, congenital myasthenic syndromes, neuropathies and muscle disorders. In silico-based pathway analyses revealed expression of a variety of proteins involved in muscle contraction and such decisive for neuronal function and maintenance suggesting the suitability of human skin fibroblasts to study the etiology of NMD. Based on these findings, next we aimed to further demonstrate the suitability of this in vitro model to study NMD by a use case: utilizing a data independent acquisition approach, the proteomic signature of whole protein extracts of fibroblasts derived from an Allgrove-patient was studied. Paradigmatic dysregulated proteins were confirmed in muscle biopsy of the patient and protein-functions could be linked to neurological symptoms known for this disease. Moreover, protoemic investigation of nuclear protein composition allowed the identification of protein-dysregulations according with structural perturbations observed in the muscle biopsy. As proteomic data suggested a perturbed lipid homeostasis, BODIPY-staining was performed on fibroblasts and coherent anti-stokes Raman scattering microscopy on muscle biopsy. Results of both investigations suggest altered lipid storage as part of the underlying disease-etiology. Conclusions: our combined data reveal that human fibroblasts may serve as an in vitro system to study the molecular etiology of rare neurological diseases exemplified on Allgrove syndrome in an unbiased fashion.