Investigation of the pathobiology of myofibrillar myopathies in zebrafish

Myofibrillar myopathies (MFMs) are a group of muscle diseases exhibiting progressive muscle weakness, and are characterised at the cellular level by structural failure of the muscle, and the formation of cytoplasmic protein aggregates. There is an immense amount of variation in the clinical features presented including the age of onset, which ranges from infancy to late adulthood; the selective involvement of cardiac and respiratory muscle groups; and severity covering the full spectrum from mild muscle weakness to premature lethality. Mutations in eight genes have been implicated in myofibrillar myopathy (MFM), all of which encode for proteins localised to the Z-disk of the sarcomere. The remarkable consistency in pathology between all MFMs suggests a common mechanism of disease, which is currently unknown. Therefore, to identify this common mechanism I generated zebrafish models of filamin C (FLNC) and BCL2-related athanogene 3 (BAG3)-related MFM. I overexpressed fluorescently tagged, full-length, MFM causing mutant FLNC and BAG3, and showed that the presence of either mutant protein results in protein aggregation, which is the first hallmark feature of MFM. Loss of either of the two proteins on the other hand, leads to contraction dependent fibre failure at the Z-disk. Remarkably, the MFM causing mutant proteins were capable of rescuing the loss of function fiber failure phenotype dismissing a role of haploinsufficiency in MFM. I demonstrate instead that aggregates include not only the mutant protein but also result in the sequestration of wildtype protein, and other proteins required for muscle integrity. The sequestration eventually leads to insufficient protein being available in the muscle therefore resulting in fibre failure and muscle weakness. My studies therefore describe how dominant mutations in FLNC and BAG3 result in muscle failure and, provide an explanation for the delayed onset and progressive nature of the disease. A key revelation from my studies is the role of toxic aggregates in triggering muscle weakness. I demonstrate that stimulation of the autophagic protein degradation pathway can reduce the number of cells affected by aggregates. I have therefore not only determined the mechanism of disease in MFM but also provided a promising avenue for the development of therapies, and suitable animal models in which to evaluate them

Investigation of the pathobiology of myofibrillar myopathies in zebrafish

Myofibrillar myopathies (MFMs) are a group of muscle diseases exhibiting progressive muscle weakness, and are characterised at the cellular level by structural failure of the muscle, and the formation of cytoplasmic protein aggregates. There is an immense amount of variation in the clinical features presented including the age of onset, which ranges from infancy to late adulthood; the selective involvement of cardiac and respiratory muscle groups; and severity covering the full spectrum from mild muscle weakness to premature lethality. Mutations in eight genes have been implicated in myofibrillar myopathy (MFM), all of which encode for proteins localised to the Z-disk of the sarcomere. The remarkable consistency in pathology between all MFMs suggests a common mechanism of disease, which is currently unknown. Therefore, to identify this common mechanism I generated zebrafish models of filamin C (FLNC) and BCL2-related athanogene 3 (BAG3)-related MFM. I overexpressed fluorescently tagged, full-length, MFM causing mutant FLNC and BAG3, and showed that the presence of either mutant protein results in protein aggregation, which is the first hallmark feature of MFM. Loss of either of the two proteins on the other hand, leads to contraction dependent fibre failure at the Z-disk. Remarkably, the MFM causing mutant proteins were capable of rescuing the loss of function fiber failure phenotype dismissing a role of haploinsufficiency in MFM. I demonstrate instead that aggregates include not only the mutant protein but also result in the sequestration of wildtype protein, and other proteins required for muscle integrity. The sequestration eventually leads to insufficient protein being available in the muscle therefore resulting in fibre failure and muscle weakness. My studies therefore describe how dominant mutations in FLNC and BAG3 result in muscle failure and, provide an explanation for the delayed onset and progressive nature of the disease. A key revelation from my studies is the role of toxic aggregates in triggering muscle weakness. I demonstrate that stimulation of the autophagic protein degradation pathway can reduce the number of cells affected by aggregates. I have therefore not only determined the mechanism of disease in MFM but also provided a promising avenue for the development of therapies, and suitable animal models in which to evaluate them

KBTBD13 is an actin-binding protein that modulates muscle kinetics

International audienceThe mechanisms that modulate the kinetics of muscle relaxation are critically important for muscle function. A prime example of the impact of impaired relaxation kinetics is nemaline myopathy caused by mutations in KBTBD13 (NEM6). In addition to weakness, NEM6 patients have slow muscle relaxation, compromising contractility and daily life activities. The role of KBTBD13 in muscle is unknown, and the pathomechanism underlying NEM6 is undetermined. A combination of transcranial magnetic stimulation-induced muscle relaxation, muscle fiber- and sarcomere-contractility assays, low-angle x-ray diffraction, and superresolution microscopy revealed that the impaired muscle-relaxation kinetics in NEM6 patients are caused by structural changes in the thin filament, a sarcomeric microstructure. Using homology modeling and binding and contractility assays with recombinant KBTBD13, Kbtbd13-knockout and Kbtbd13R408C-knockin mouse models, and a GFPlabeled Kbtbd13-transgenic zebrafish model, we discovered that KBTBD13 binds to actin - a major constituent of the thin filament - and that mutations in KBTBD13 cause structural changes impairing muscle-relaxation kinetics. We propose that this actin-based impaired relaxation is central to NEM6 pathology

Erratum to: Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) (Autophagy, 12, 1, 1-222, 10.1080/15548627.2015.1100356

non present

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field