Biology subjects

Malfait, F.

Publications and source records attributed to Malfait, F..

5 recordsLinked to original sources

bioRxiv2024

The chemical chaperone 4-phenylbutyric acid rescues molecular cell defects of COL3A1 mutations that cause vascular Ehlers Danlos Syndrome

PurposeVascular Ehlers Danlos Syndrome (vEDS) is a connective tissue disorder caused by COL3A1 mutations for which there are no treatments due to a limited understanding of underlying mechanisms. We aimed to address this critical knowledge gap, focusing on collagen folding, to establish if targeting protein folding represents a potential therapeutic approach. MethodsWe performed a mechanistic analysis of two novel COL3A1 glycine mutations, G189S and G906R, using primary patient fibroblast cultures, and performed pre-clinical proof-of-concept treatments using FDA-approved chemical chaperones targeting protein folding and/or degradation. ResultsCOL3A1 mutations caused secretion of misfolded collagen III and intracellular collagen retention, leading to matrix defects and endoplasmic reticulum (ER) stress, with increased severity for the more C-terminal mutation. Promoting ER protein folding capacity through the chemical chaperone 4-phenylbutyric acid rescued the ER stress, thermostability of secreted collagen, matrix defects and apoptosis. Optimising treatment duration and dosage helped overcome allele-dependent treatment efficacy. In contrast, protein degradation alone or combined with targeting protein folding did not increase efficacy. ConclusionER stress is a molecular mechanism in vEDS that can be influenced by the position of COL3A1 mutation, and promoting protein folding is a putative mechanism-based therapeutic approach that can rescue intra- and extracellular defects.

genetics↗

bioRxiv2024

Reduced Capsaicin-Induced Mechanical Allodynia and Neuronal Responses in the DRG in the Presence of Shp1 Overexpression

Transient Receptor Potential Vanilloid 1 (TRPV1) is a nonselective cation channel expressed by pain-sensing neurons and has been an attractive target for the development of drugs to treat pain. Recently, Src homology region 2 domain-containing phosphatase-1 (SHP-1) was shown to dephosphorylate TRPV1 in dorsal root ganglia (DRG) neurons, which was linked with alleviating different pain phenotypes. These previous studies were performed in male rodents only and did not directly investigate the role of SHP-1 in TRPV-1 mediated sensitization. Therefore, our goal was to determine the impact of Shp1 overexpression on TRPV1-mediated neuronal responses and capsaicin-induced pain behavior in mice of both sexes. Twelve-week-old male and female mice overexpressing Shp1 (Shp1-Tg) and their wild type (WT) littermates were used. Shp1 overexpression was confirmed in the DRG of Shp1-Tg mice by RNA in situ hybridization and RT-qPCR. Trpv1 and Shp1 were found to be co-expressed in DRG sensory neurons in both genotypes. Functionally, this overexpression resulted in lower magnitude intracellular calcium responses to 200 nM capsaicin stimulation in DRG cultures from Shp1-Tg mice compared to WTs. In vivo, we tested the effects of Shp1 overexpression on capsaicin-induced pain through a model of capsaicin footpad injection. While capsaicin injection evoked nocifensive behavior (paw licking) and paw swelling in both genotypes and sexes, only WT mice developed mechanical allodynia after capsaicin injection. We observed similar level of TRPV1 protein expression in the DRG of both genotypes, however, a higher amount of tyrosine phosphorylated TRPV1 was detected in WT DRG. These experiments suggest that, while SHP-1 does not mediate the acute swelling and nocifensive behavior induced by capsaicin, it does mediate a protective effect against capsaicin-induced mechanical allodynia in both sexes. The protective effect of SHP-1 might be mediated by TRPV1 dephosphorylation in capsaicin-sensitive sensory neurons of the DRG.

neuroscience↗

bioRxiv2022

The matrisome of the murine and human dorsal root ganglion: a transcriptomal approach

The extracellular matrix (ECM) is a dynamic structure composed of a large number of molecules that can be divided into six different categories and are collectively called the matrisome. The ECM plays pivotal roles in physiological and pathological processes in many tissues, including the nervous system. Intriguingly, alterations in ECM molecules/pathways are often associated with painful human conditions and murine experimental pain models. Nevertheless, mechanistic insight into the interplay of normal or defective ECM and pain is largely lacking. To expand the knowledge on ECM composition and synthesis in the peripheral nervous system, we used a transcriptomal approach to investigate the expression and cellular origin of matrisome genes in murine and human dorsal root ganglia (DRG), containing the cell bodies of sensory neurons. Bulk RNA sequencing data showed that over 60% of all matrisome genes were expressed in both murine and human DRG, with proportionally more core matrisome genes (glycoproteins, collagens, and proteoglycans) expressed compared to matrisome-associated genes (ECM-affiliated genes, ECM regulators and secreted factors). Examination of the cellular origin of matrisome expression by single cell RNA sequencing on murine DRG revealed that core matrisome genes, especially collagens, were expressed by vascular leptomeningeal-like (fibroblast) cell types whereas matrisome-associated genes were mainly expressed by neuronal cell types. We analyzed cell-cell communication networks with the CellChat R package and predicted an important role for the Collagen signaling pathway in connecting vascular cell types and nociceptors in murine tissue, which we confirmed by analysis of spatial transcriptomic data from human DRG. RNAscope in situ hybridization and immunohistochemistry confirmed expression of collagens in fibroblasts surrounding nociceptors in human DRG. This study supports the idea that the DRG matrisome may contribute to neuronal signaling in both mouse and human. The identification of the cellular distribution of murine and human matrisome genes provides a framework to study the role of the ECM in peripheral nervous tissue and its effects on pain signaling. HighlightsO_LITranscriptomal analyses of mouse and human dorsal root ganglia (DRG) revealed that over 60% of matrisome genes are expressed by murine and human dorsal root ganglia (DRG), with over 85% of the genes with orthologues overlapping between both species. C_LIO_LIMatrisome-associated genes had the highest expression in both species and included conserved expression of annexins, S100 calcium binding proteins and cathepsins. C_LIO_LICollagens and collagen receptors are expressed by distinct cell types in murine and human DRG, suggesting that the collagen signaling pathway could be involved in cell-cell signaling. C_LI

neuroscience↗

bioRxiv2020

b3galt6 knock-out zebrafish recapitulate β3GalT6-deficiency disorders in human and reveal a trisaccharide proteoglycan linkage region

Proteoglycans are structurally and functionally diverse biomacromolecules found abundantly on cell membranes and in the extracellular matrix. They consist of a core protein linked to glycosaminoglycan chains via a tetrasaccharide linkage region. Here, we show that CRISPR/Cas9-mediated b3galt6 knock-out zebrafish, lacking galactosyltransferase II, which adds the third sugar in the linkage region, largely recapitulate the phenotypic abnormalities seen in human {beta}3GalT6-deficiency disorders. These comprise craniofacial dysmorphism, generalized skeletal dysplasia, skin involvement and indications for muscle hypotonia. In-depth TEM analysis revealed disturbed collagen fibril organization as the most consistent ultrastructural characteristic throughout different affected tissues. Strikingly, despite a strong reduction in glycosaminoglycan content, as demonstrated by anion-exchange HPLC, subsequent LC-MS/MS analysis revealed a small amount of proteoglycans containing a unique linkage region consisting of only three sugars. This implies that formation of glycosaminoglycans with an immature linkage region is possible in a pathogenic context. Our study therefore unveils a novel rescue mechanism for proteoglycan production in the absence of galactosyltransferase II, hereby opening new avenues for therapeutic intervention.

genetics↗

bioRxiv2018

Zebrafish type I collagen mutants faithfully recapitulate human type I collagenopathies

The type I collagenopathies are a group of heterogeneous connective tissue disorders, that are caused by mutations in the genes encoding type I collagen and include specific forms of Osteogenesis Imperfecta (OI) and the Ehlers-Danlos syndrome (EDS). These disorders present with a broad disease spectrum and large clinical variability of which the underlying genetic basis is still poorly understood. In this study, we systematically analyzed skeletal phenotypes in a large set of zebrafish, with diverse mutations in the genes encoding type I collagen, representing different genetic forms of human OI, and the first zebrafish model of human EDS, which harbors characteristic defects in the soft connective tissues. Furthermore, we provide insight into how zebrafish and human type I collagen are compositionally and functionally related, which is relevant in the interpretation of human type I collagen related disease models. Our studies reveal a high degree of inter-genotype variability in phenotypic expressivity that closely correlates with associated OI severity. Further, we demonstrate the potential for select mutations to give rise to variable phenotypic penetrance, mirroring the clinical variability associated with human disease pathology. Therefore, our work suggests the potential for zebrafish to aid in identifying unknown genetic modifiers and mechanisms underlying the phenotypic variability in OI and related disorders. This will improve diagnostic strategies and enable the discovery of new targetable pathways for pharmacological intervention\n\nSIGNIFICANCE STATEMENTType I collagenopathies are a heterogenous group of connective tissue disorders, caused by genetic defects in type I collagen. Inherent to these disorders is a large clinical variability, of which the underlying molecular basis remains undefined. By systematically analyzing skeletal phenotypes in a large set of type I collagen zebrafish mutants we show that zebrafish models are able to both genocopy and phenocopy different forms of human type I collagenopathies, arguing for a similar pathogenetic basis. This study illustrates the potential of zebrafish as a tool to further dissect the molecular basis of phenotypic variability in human type I collagenopathies to improve diagnostic strategies as well as promote the discovery of new targetable pathways for pharmacological intervention of these disorders.

genetics↗