Biology subjects

Lopez-Marquez, A.

Publications and source records attributed to Lopez-Marquez, A..

4 recordsLinked to original sources

bioRxiv2025

Generation and Characterization of Col6a1 knock-in mice: A Promising Pre-Clinical Model for Collagen VI-Related Dystrophies

Collagen VI Related Dystrophies (COL6-RD) are congenital muscle diseases, typically inherited as an autosomal dominant trait. A frequent type of mutation involves glycine substitutions in the triple helical domain of collagen VI alpha chains, exerting a dominant-negative effect on the unaltered protein. Despite this, no prior animal model captured this mutation type. Using CRISPR/Cas9, we generated transgenic mice with the equivalent of the human COL6A1 c.877 G>A; p. Gly293Arg mutation. We characterized their skeletal muscle phenotype over time, utilizing computer-aided tools applied to standardized parameters of muscle pathology and function. Knock-in mice exhibited early-onset reduced muscle weight, myopathic histology, increased fibrosis, reduced collagen VI expression, muscle weakness, and impaired respiratory function. These features provide adequate outcome measures to assess therapeutic interventions. The different automated image analysis methods deployed here analyze thousands of features simultaneously, enhancing accuracy in describing muscle disease models. Overall, the Col6a1 Ki Gly292Arg mouse model offers a robust platform to deepen our understanding of COL6-RD and advance its therapeutic landscape. Summary StatementWe generated and characterized over time the first mouse model representing dominant negative glycine substitutions in the alpha chains of collagen VI that are a frequent cause of Collagen VI-Related Dystrophies.

neuroscience↗

bioRxiv2024

Collablots: Quantification of collagen VI levels and its structural disorganisation in cell cultures from patients with collagen VI-related dystrophies

AimsThis study aims to develop a quantitative method for assessing collagen VI expression in cell cultures, which is crucial for the diagnosis and treatment of collagen VI-related dystrophies. MethodsWe developed a combined in-cell western (ICW) and on-cell western (OCW) assay, that we have called collablot to quantify collagen VI and its organisation in the extracellular matrix of cell cultures from patients and healthy controls. To optimise it, we optimised cell density and the protocols to induce collagen expression in cultures, as well as the cell fixation and permeabilisation methods. This was completed with a thorough selection of collagen antibodies and a collagen hybridising peptide (CHP). We then used collablots to compare cultures from patients and controls and evaluate therapeutic interventions in the cultures. ResultsCollablots enabled the quantification of collagen VI expression in both control and patient cells, aligning with immunocytochemistry findings and detecting variations in collagen VI expression following treatment of the cultures. Additionally, CHP analysis revealed a marked increase in collagen network disruption in patients compared to the controls. ConclusionsThe collablot assay represents an optimal method for quantifying collagen VI expression and its organisation in culture and assessing the effect of therapies. Key Points- Evaluating therapies for collagen VI-related dystrophies (COL6-RD) requires the quantification of collagen VI levels. - Collablot assays are a novel method for quantifying collagen VI expression and its structural organisation in cell culture. - Due to the significant role of phenotype heterogeneity in this complex disease, quantifying collagen alone might not be adequate for diagnosing COL6-RD, but the addition of a peptide to quantify collagen disorganisation could help in the characterisation of patient cultures.

neuroscience↗

bioRxiv2024

Cell-derived matrices mechanics as a functional read-out in Collagen VI-related Congenital Muscular Dystrophies

Atomic force microscopy-force spectroscopy (AFM-FS) applied to biological samples can provide information on the micro- and nanoscale mechanical properties of tissues, which are a determinant of cellular behavior. Mechanical properties are largely determined by the structure and composition of the extracellular matrix (ECM), for which several in vitro models have been developed, including cell-derived matrices (CDMs). CDMs are decellularized natural ECMs assembled by cells, that closely mimic the in vivo stromal fiber organization and molecular content. Here we applied AFM-FS to evaluate the nanomechanical properties of CDMs obtained from primary skin fibroblast cultures of patients affected by collagen VI-related congenital muscular dystrophies (COL6-RDs). COL6-RDs are a set of neuromuscular conditions caused by pathogenic variants in any of the three major COL6 genes, which result in deficiency or dysfunction of the COL6 incorporated into the ECM of connective tissues. Current diagnosis includes the genetic confirmation of the disease and categorization of the phenotype based on maximum motor ability, as no direct correlation exists between genotype and phenotype of COL6-RDs. The development of new tools able to identify phenotype traits can significantly contribute to the diagnosis and prognosis of COL6-RDs. We describe differences in the elastic modulus (E) among CDMs deriving from patients with different clinical phenotypes, as well as the restoration of E in CDMs obtained from genetically edited cells. Results anticipate the potential of the nanomechanical analysis of CDMs as a complementary clinical tool, providing phenotypic information about COL6-RDs and their response to gene therapies.

biophysics↗

bioRxiv2023

PAH DEFICIENT PATHOLOGY IN HUMANIZED c.1066-11G>A PHENYLKETONURIA MICE

We have generated using CRISPR/Cas9 technology a partially humanized mouse model of the neurometabolic disease phenylketonuria (PKU), carrying the highly prevalent PAH variant c.1066-11G>A. This variant creates an alternative 3 splice site, leading to the inclusion of 9 nucleotides coding for 3 extra amino acids between Q355 and Y356 of the protein. Homozygous Pah c.1066-11A mice, with a partially humanized intron 10 sequence with the variant, accurately recapitulate the splicing defect and present almost undetectable hepatic PAH activity. They exhibit fur hypopigmentation, lower brain and body weight and reduced survival. Blood and brain phenylalanine levels are elevated, along with decreased tyrosine, tryptophan and monoamine neurotransmitter levels. They present behavioral deficits, mainly hypoactivity and diminished social interaction, locomotor deficiencies and an abnormal hind-limb clasping reflex. Changes in the morphology of glial cells, increased GFAP and Iba1 staining signals and decreased myelinization are observed. Hepatic tissue exhibits nearly absent PAH protein, reduced levels of chaperones DNAJC12 and HSP70 and increased autophagy markers LAMP1 and LC3BII, suggesting possible coaggregation of mutant PAH with chaperones and subsequent autophagy processing. This PKU mouse model with a prevalent human variant represents a useful tool for pathophysiology research and for novel therapies development.

pathology↗