Protein signature of human skin fibroblasts allows the study of the molecular etiology of rare neurological diseases

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.

Human skin fibroblasts were used to generate a spectral library using pH8-fractionation of followed by nano LC-MS/MS. Afterwards, Allgrove-patient derived fibroblasts were subjected to a data independent acquisition approach. In addition, proteomic signature of an enriched nuclear protein fraction was studied. Proteomic findings were confirmed by immunofluorescence in a muscle biopsy derived from the same patient and cellular lipid homeostasis in the cause of Allgrove syndrome was analysed by fluorescence (BODIPY-staining) and coherent anti-Stokes Raman scattering (CARS) microscopy.

To systematically address the question if human skin fibroblasts might serve as valuable biomaterial for (molecular) studies of NMD, we generated a protein library cataloguing 8280 proteins including 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 diversity 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: the proteomic signature of fibroblasts derived from an Allgrove-patient was studied. Dysregulation of paradigmatic proteins could be confirmed in muscle biopsy of the patient and protein-functions could be linked to neurological symptoms known for this disease. Moreover, proteomic investigation of nuclear protein composition allowed the identification of protein-dysregulations according with structural perturbations observed in the muscle biopsy. BODIPY-staining on fibroblasts and CARS microscopy on muscle biopsy suggest altered lipid storage as part of the underlying disease etiology.

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.