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. Methods 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. Results 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. 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.

Homozygous WASHC4 variant in two sisters causes a syndromic phenotype defined by dysmorphisms, intellectual disability, profound developmental disorder, and skeletal muscle involvement

“…Indeed, our proteomic study indicated altered expression of numerous proteins decisive for proper function of the neuromuscular axis (supplementary material, Table S3). This molecular finding not only provides first insights into the biochemical origin of the broad neurological involvement of WASHC4 deficiency but also confirms the suitability of fibroblasts for identifying pathophysiological processes of neuronal relevance [15]. Along this line, these data suggest that a molecular interplay of dysregulated proteins might contribute to the clinical manifestation of symptoms.…”

“…To identify proteins, which are affected by loss of functional WASHC4, proteomic profiling was carried out on skin fibroblasts derived from a WASHC4 patient. This approach was prompted by the molecular observation that a variety of proteins of neurological relevance are expressed in these cells [15]. Indeed, our proteomic study indicated altered expression of numerous proteins decisive for proper function of the neuromuscular axis (supplementary material, Table S3).…”

Intracellular Lipid Accumulation and Mitochondrial Dysfunction Accompanies Endoplasmic Reticulum Stress Caused by Loss of the Co-chaperone DNAJC3

“…To obtain insights into the mechanism underlying the pathophysiology of mutations in DNAJC3, unbiased proteomic studies utilizing patient-derived fibroblasts were carried out (Figure 1). While fibroblasts are not the primary site of clinical manifestation, these cell models have been previously shown to be suitable to study the etiology of rare neurological diseases (Hentschel et al, 2021) and show very similar levels of DNAJC3 expression to peripheral nerve (Supplementary Figure 1). Results of this profiling suggest that lipid metabolism is affected by DNAJC3 mut mutations (Figure 1) as Sterol-O-acyltransferase 1 (SOAT1), an enzyme responsible for the esterification of fatty acids, especially cholesterol (Das et al, 2008), was found to be decreased.…”

“…Studies on DNAJC3-silenced rat and human β cells did not affect insulin content and secretion but sensitized cells to ER stress, triggering mitochondrial apoptosis (Lytrivi et al, 2021). To further unravel this pathophysiological route here, we focused on elucidating the pathomechanism of DNAJC3-related pathology by performing proteomics in patient-derived primary fibroblasts, a suitable model to study the etiology of rare neurological diseases (Hentschel et al, 2021), and further investigation of the suggested cellular phenotype.…”

Proteomic Analysis of Marinesco–Sjogren Syndrome Fibroblasts Indicates Pro-Survival Metabolic Adaptation to SIL1 Loss

“…A recent study indicated that the protein signature of human skin fibroblasts may help understand the pathogenic mechanisms of neuromuscular diseases [18]. We decided to study the proteome of MSS patient fibroblasts (HF-MSS) with a view to understanding the molecular changes involved in the pathogenesis of this syndrome.…”