Characterization of a novel collagen VI knockout zebrafish model and its application for mechanistic insights and drug repurposing in congenital myopathies

Collagen VI (COL6) is a distinctive extracellular matrix protein expressed in various tissues and playing key roles in a wide range of processes. The most critical role of COL6 is displayed by skeletal muscle, and mutations of genes encoding for COL6 chains are responsible for various inherited muscle diseases in humans, collectively known as “COL6-related myopathies” (COL6-RM). Studies in animal models allowed to unravel some mechanisms underlying COL6-RM. Works in COL6 null (Col6a1-/-) mice revealed that ablation of COL6 results in decreased muscle strength accompanied by organelle alterations, mitochondrial dysfunction and apoptosis of myofibers. However, our current knowledge on how extracellular COL6 influences and regulates such intracellular mechanisms is still partial. In this context, we exploited the zebrafish model as it represents a powerful tool to visualize the spatio-temporal in vivo activation of signaling pathways, thanks to its numerous advantages including embryo transparency and rapid external development. During the first part of my PhD project, I characterized a novel zebrafish COL6 knockout line. The obtained results show that lack of COL6 leads to defective slow muscle organization, which impairs swimming capabilities during both development and adult life. Moreover, COL6 KO fish embryos display altered motor neuron development, together with autophagy and organelle defects. These results proved that COL6 KO zebrafish are a valuable in vivo tool to model COL6-RM, since they recapitulate the main features displayed by patients, also highlighting that they provide a notable in vivo platform for drug screenings and for identifying new potential therapeutic treatments. The second part of my PhD work was focused on an in vivo drug repurposing screen to test the efficacy of compounds from an FDA-approved library in rescuing muscle structure and function in the COL6 KO zebrafish model. Indeed, COL6-RM still need safe and definitive therapies and the development of new drugs for such rare and severe diseases is economically unsustainable. Therefore, I screened more than 300 compounds and identified several drugs able to significantly ameliorate the motility defects of COL6 KO embryos. Interestingly, some of the identified drugs are involved in the modulation of cAMP signaling, a pathway never associated to COL6-RM so far. Taking advantage of a cAMP reporter line, I found that COL6 KO embryos display decreased cAMP response activity in the developing muscle fibers. To further establish whether the cAMP pathway is indeed a beneficial target, I treated embryos with some cAMP-elevating agents, which indeed successfully replicated the outcomes displayed by treatments with candidate drugs. Moreover, the identified drugs were also successful in activating cAMP signaling in slow muscle precursors, promoting their specification during development, and in ameliorating muscle fiber organization, with increased axon elongation of motor neurons. Notably, some of the drugs were also effective in promoting mitochondrial biogenesis, thus pointing at regulation of mitochondrial homeostasis as a further molecular target for COL6-RM. Altogether, the results obtained with this part of the present thesis work not only allowed to identify FDA-approved drugs with beneficial effects in the functional recovery of defects displayed by COL6 KO fish, but also led to the identification of thus far unexplored molecular pathways affected by COL6 deficiency and impacting on muscle health. These data allow to pave the way for future studies aimed at dissecting the molecular mechanism underlying COL6-RM and point at cAMP signaling as a druggable target for the amelioration of the myopathic symptoms caused by COL6 deficiency.