Farewell to Professor David Yaffe - A pillar of the myogenesis field.

It is with great sadness that we have learned about the passing of Professor David Yaffe (1929-2020, Israel). Yehi Zichro Baruch - May his memory be a blessing. David was a man of family, science and nature. A native of Israel, David grew up in the historic years that preceded the birth of the State of Israel. He was a member of the group that established Kibbutz Revivim in the Negev desert, and in 1948 participated in Israel's War of Independence. David and Ruth eventually joined Kibbutz Givat Brenner by Rehovot, permitting David to be both a kibbutz member and a life-long researcher at the Weizmann Institute of Science, where David received his PhD in 1959. David returned to the Institute after his postdoc at Stanford. Here, after several years of researching a number of tissues as models for studying the process of differentiation, David entered the myogenesis field and stayed with it to his last day. With his dedication to the field of myogenesis and his commitment to furthering the understanding of the People and the Land of Israel throughout the international scientific community, David organized the first ever myogenesis meeting that took place in Shoresh, Israel in 1975. This was followed by the 1980 myogenesis meeting at the same place and many more outstanding meetings, all of which brought together myogenesis, nature and scenery. Herein, through the preparation and publication of this current manuscript, we are meeting once again at a "David Yaffe myogenesis meeting". Some of us have been members of the Yaffe lab, some of us have known David as his national and international colleagues in the myology field. One of our contributors has also known (and communicates here) about David Yaffe's earlier years as a kibbutznick in the Negev. Our collective reflections are a tribute to Professor David Yaffe. We are fortunate that the European Journal of Translational Myology has provided us with tremendous input and a platform for holding this 2020 distance meeting "Farwell to Professor David Yaffe - A Pillar of the Myogenesis Field".

Altered presynaptic ultrastructure in excitatory hippocampal synapses of mice lacking dystrophins Dp427 or Dp71.

Mental retardation is a feature of X-linked Duchenne muscular dystrophy (DMD) which likely results from the loss of the brain full-length (Dp427) and short C-terminal products of the dystrophin gene, such as Dp71. The loss of Dp427 or Dp71 is known to alter hippocampal glutamate-dependent synaptic transmission and plasticity in mice. Although dystrophins have a selective postsynaptic expression in brain, a putative role in retrograde regulation of transmitter release was suggested by studies in Drosophila. Here we used electron microscopy to analyze the distribution of synaptic vesicles in CA1 hippocampal axospinous non perforated-excitatory synapses of mice lacking Dp427 or Dp71 compared to control littermates. We found that the density of morphologically-docked vesicles is increased and the vesicle size is reduced in mice lacking Dp427, while in Dp71-null mice there is a decrease in the density of vesicles located in the vicinity of the active zone and an increase in the vesicle size and in the width of synaptic clefts. This is the first indication that the loss of mammalian brain dystrophins impacts on the presynaptic ultrastructural organization of central glutamatergic synapses, which may explain some of the alterations of synapse function and plasticity that contribute to intellectual disability in DMD.

Cloned myogenic cells can transdifferentiate in vivo into neuron-like cells.

BACKGROUND: The question of whether intact somatic cells committed to a specific differentiation fate, can be reprogrammed in vivo by exposing them to a different host microenvironment is a matter of controversy. Many reports on transdifferentiation could be explained by fusion with host cells or reflect intrinsic heterogeneity of the donor cell population. METHODOLOGY/PRINCIPAL FINDINGS: We have tested the capacity of cloned populations of mouse and human muscle progenitor cells, committed to the myogenic pathway, to transdifferentiate to neurons, following their inoculation into the developing brain of newborn mice. Both cell types migrated into various brain regions, and a fraction of them gained a neuronal morphology and expressed neuronal or glial markers. Likewise, inoculated cloned human myogenic cells expressed a human specific neurofilament protein. Brain injected donor cells that expressed a YFP transgene controlled by a neuronal specific promoter, were isolated by FACS. The isolated cells had a wild-type diploid DNA content. CONCLUSIONS: These and other results indicate a genuine transdifferentiation phenomenon induced by the host brain microenvironment and not by fusion with host cells. The results may potentially be relevant to the prospect of autologous cell therapy approach for CNS diseases.

Dystrophin-dependent and -independent AQP4 pools are expressed in the mouse brain.

In a recent study, we demonstrated that in the plasma membrane AQP4 is organized into several distinct large multisubunit complexes. In this study, we analysed whether these pools are similarly affected in dystrophin-deficient mice and immunolocalized the sites of dystrophin-dependent and -independent AQP4 pools. Western blot performed on two-dimensional Blue Native/SDS-PAGE membranes indicated that, among the AQP4 pools, it was mainly a large multisubunit complex that was specifically affected in dystrophin-deficient mice (DP71 and mdx3cv mice). This dystrophin-dependent AQP4 pool was immunolocalized in perivascular astrocytes, since it was found to be significantly altered in both types of dystrophin-deficient mice. Dystrophin-independent pools were immunolocalized in the granular cell layer of the cerebellum and in the subpial endfoot layer and ependymal cells in the brain. These data provide a better understanding on the association between AQP4 and the dystrophin-glycoprotein complex in the central nervous system.

Kir4.1 and AQP4 associate with Dp71- and utrophin-DAPs complexes in specific and defined microdomains of Müller retinal glial cell membrane.

The dystrophin-associated proteins (DAPs) complex consisting of dystroglycan, syntrophin, dystrobrevin, and sarcoglycans in muscle cells is associated either with dystrophin or its homolog utrophin. In rat retina, a similar complex was found associated with dystrophin-Dp71 that serves as an anchor for the inwardly rectifying potassium channel Kir4.1 and the aqueous pore, aquaporin-4 (AQP4). Here, using immunofluorescence imaging of isolated retinal Müller glial cells and co-immunoprecipitation experiments performed on an enriched Müller glial cells end-feet fraction, we investigated the effect of Dp71 deletion on the composition, anchoring, and membrane localization of the DAPs-Kir4.1 and/or -AQP4 complex. Two distinct complexes were identified in the end-feet fraction associated either with Dp71 or with utrophin. Upon Dp71 deletion, the corresponding DAPs complex was disrupted and a compensating utrophin upregulation was observed, accompanied by diffuse overall staining of Kir4.1 along the Müller glial cells and redistribution of the K(+) conductance. Dp71 deficiency was also associated with a marked reduction of AQP4 and beta-dystroglycan expression. Furthermore, it was observed that the Dp71-DAPs dependent complex could be, at least partially, associated with a specific membrane fraction. These results demonstrate that Dp71 has a central role in the molecular scaffold responsible for anchoring AQP4 and Kir4.1 in Müller cell end-feet membranes. They also show that despite its close relationship to the dystrophin proteins and its correlated upregulation, utrophin is only partially compensating for the absence of Dp71 in Müller glial cells.

Dystrophin deficiency in Drosophila reduces lifespan and causes a dilated cardiomyopathy phenotype.

A number of studies have been conducted recently on the model organism Drosophila to determine the function of genes involved in human disease, including those implicated in neurological disorders, cancer and metabolic and cardiovascular diseases. The simple structure and physiology of the Drosophila heart tube together with the available genetics provide a suitable in vivo assay system for studying cardiac gene functions. In our study, we focus on analysis of the role of dystrophin (Dys) in heart physiology. As in humans, the Drosophila dys gene encodes multiple isoforms, of which the large isoforms (DLPs) and a truncated form (Dp117) are expressed in the adult heart. Here, we show that the loss of dys function in the heart leads to an age-dependent disruption of the myofibrillar organization within the myocardium as well as to alterations in cardiac performance. dys RNAi-mediated knockdown in the mesoderm also shortens lifespan. Knockdown of all or deletion of the large isoforms increases the heart rate by shortening the diastolic intervals (relaxation phase) of the cardiac cycle. Morphologically, loss of the large DLPs isoforms causes a widening of the cardiac tube and a lower fractional shortening, a phenotype reminiscent of dilated cardiomyopathy. The dilated dys mutant phenotype was reversed by expressing a truncated mammalian form of dys (Dp116). Our results illustrate the utility of Drosophila as a model system to study dilated cardiomyopathy and other muscular-dystrophy-associated phenotypes.

Role of mental retardation-associated dystrophin-gene product Dp71 in excitatory synapse organization, synaptic plasticity and behavioral functions.

BACKGROUND: Duchenne muscular dystrophy (DMD) is caused by deficient expression of the cytoskeletal protein, dystrophin. One third of DMD patients also have mental retardation (MR), likely due to mutations preventing expression of dystrophin and other brain products of the DMD gene expressed from distinct internal promoters. Loss of Dp71, the major DMD-gene product in brain, is thought to contribute to the severity of MR; however, the specific function of Dp71 is poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: Complementary approaches were used to explore the role of Dp71 in neuronal function and identify mechanisms by which Dp71 loss may impair neuronal and cognitive functions. Besides the normal expression of Dp71 in a subpopulation of astrocytes, we found that a pool of Dp71 colocalizes with synaptic proteins in cultured neurons and is expressed in synaptic subcellular fractions in adult brains. We report that Dp71-associated protein complexes interact with specialized modular scaffolds of proteins that cluster glutamate receptors and organize signaling in postsynaptic densities. We then undertook the first functional examination of the brain and cognitive alterations in the Dp71-null mice. We found that these mice display abnormal synapse organization and maturation in vitro, altered synapse density in the adult brain, enhanced glutamatergic transmission and reduced synaptic plasticity in CA1 hippocampus. Dp71-null mice show selective behavioral disturbances characterized by reduced exploratory and novelty-seeking behavior, mild retention deficits in inhibitory avoidance, and impairments in spatial learning and memory. CONCLUSIONS/SIGNIFICANCE: Results suggest that Dp71 expression in neurons play a regulatory role in glutamatergic synapse organization and function, which provides a new mechanism by which inactivation of Dp71 in association with that of other DMD-gene products may lead to increased severity of MR.

Dissecting muscle and neuronal disorders in a Drosophila model of muscular dystrophy.

Perturbation in the Dystroglycan (Dg)-Dystrophin (Dys) complex results in muscular dystrophies and brain abnormalities in human. Here we report that Drosophila is an excellent genetically tractable model to study muscular dystrophies and neuronal abnormalities caused by defects in this complex. Using a fluorescence polarization assay, we show a high conservation in Dg-Dys interaction between human and Drosophila. Genetic and RNAi-induced perturbations of Dg and Dys in Drosophila cause cell polarity and muscular dystrophy phenotypes: decreased mobility, age-dependent muscle degeneration and defective photoreceptor path-finding. Dg and Dys are required in targeting glial cells and neurons for correct neuronal migration. Importantly, we now report that Dg interacts with insulin receptor and Nck/Dock SH2/SH3-adaptor molecule in photoreceptor path-finding. This is the first demonstration of a genetic interaction between Dg and InR.

Regeneration and transdifferentiation potential of muscle-derived stem cells propagated as myospheres.

We have isolated from mouse skeletal muscle a subpopulation of slow adherent myogenic cells that can proliferate for at least several months as suspended clusters of cells (myospheres). In the appropriate conditions, the myospheres adhere to the plate, spread out, and form a monolayer of MyoD(+) cells. Unlike previously described myogenic cell lines, most of the myosphere cells differentiate, without cell fusion, into thin mononucleated contractile fibers, which express myogenin and skeletal muscle myosin heavy chain. The presence of Pax-7 in a significant proportion of these cells suggests that they originate from satellite cells. The addition of leukemia inhibitory factor to the growth medium of the myospheres enhances proliferation and dramatically increases the proportion of cells expressing Sca-1, which is expressed by several types of stem cells. The capacity of myosphere cells to transdifferentiate to other mesodermal cell lineages was examined. Exposure of cloned myosphere cells to bone morphogenetic protein resulted in suppression of myogenic differentiation and induction of osteogenic markers such as alkaline phosphatase and osteocalcin. These cells also sporadically differentiated to adipocytes. Myosphere cells could not, so far, be induced to transdifferentiate to hematopoietic cells. When inoculated into injured muscle, myosphere-derived cells participated in regeneration, forming multinucleated cross-striated mature fibers. This suggests a potential medical application.

The Drosophila homologue of the dystrophin gene - introns containing promoters are the major contributors to the large size of the gene.

We show that the drosophila gene encoding the dystrophin-like protein (DLP) is as complex as the mammalian dystrophin gene. Three 5' promoters and three internal promoters regulate the expression of three full-length and three truncated products, respectively. The existence of this complex gene structure in such evolutionary remote organisms suggests that both types of products have diverse important functions. The promoters of both the DLP gene and the mammalian dystrophin gene are located in very large introns. These introns contribute significantly to the large size of the genes. The possible relevance of the conservation of the large size of introns containing promoters to the regulation of promoter activity is discussed.

Dystrophin Dp71 expression is down-regulated during myogenesis: role of Sp1 and Sp3 on the Dp71 promoter activity.

Dp71 expression is present in myoblasts but declines during myogenesis to avoid interfering with the function of dystrophin, the predominant Duchenne muscular dystrophy gene product in differentiated muscle fibers. To elucidate the transcriptional regulatory mechanisms operating on the developmentally regulated expression of Dp71, we analyzed the Dp71 expression and promoter activity during myogenesis of the C2C12 cells. We demonstrated that the cellular content of Dp71 transcript and protein decrease in myotubes as a consequence of the negative regulation that the differentiation stimulus exerts on the Dp71 promoter. Promoter deletion analysis showed that the 224-bp 5'-flanking region, which contains several Sp-binding sites (Sp-A to Sp-D), is responsible for the Dp71 promoter basal activity in myoblasts as well as for down-regulation of the promoter in differentiated cells. Electrophoretic mobility shift and chromatin immunoprecipitation assays indicated that Sp1 and Sp3 transcription factors specifically bind to the Sp-binding sites in the minimal Dp71 promoter region. Site-directed mutagenesis assay revealed that Sp-A is the most important binding site for the proximal Dp71 promoter activity. Additionally, cotransfection of the promoter construct with Sp1- and Sp3-expressing vectors into Drosophila SL2 cells, which lack endogenous Sp family, confirmed that these proteins activate specifically the minimal Dp71 promoter. Endogenous Sp1 and Sp3 proteins were detected only in myoblasts and not in myotubes, which indicates that the lack of these factors causes down-regulation of the Dp71 promoter activity in differentiated cells. In corroboration, efficient promoter activity was restored in differentiated muscle cells by exogenous expression of Sp1 and Sp3.

Acute pathophysiological effects of muscle-expressed Dp71 transgene on normal and dystrophic mouse muscle.

products of the dystrophin gene range from the 427-kDa full-length dystrophin to the 70.8-kDa Dp71. Dp427 is expressed in skeletal muscle, where it links the actin cytoskeleton with the extracellular matrix via a complex of dystrophin-associated proteins (DAPs). Dystrophin deficiency disrupts the DAP complex and causes muscular dystrophy in humans and the mdx mouse. Dp71, the major nonmuscle product, consists of the COOH-terminal part of dystrophin, including the binding site for the DAP complex but lacks binding sites for microfilaments. Dp71 transgene (Dp71tg) expressed in mdx muscle restores the DAP complex but does not prevent muscle degeneration. In wild-type (WT) mouse muscle, Dp71tg causes a mild muscular dystrophy. In this study, we tested, using isolated extensor digitorum longus muscles, whether Dp71tg exerts acute influences on force generation and sarcolemmal stress resistance. In WT muscles, there was no effect on isometric twitch and tetanic force generation, but with a cytomegalovirus promotor-driven transgene, contraction with stretch led to sarcolemmal ruptures and irreversible loss of tension. In MDX muscle, Dp71tg reduced twitch and tetanic tension but did not aggravate sarcolemmal fragility. The adverse effects of Dp71 in muscle are probably due to its competition with dystrophin and utrophin (in MDX muscle) for binding to the DAP complex.

Targeted inactivation of dystrophin gene product Dp71: phenotypic impact in mouse retina.

The abnormal retinal neurotransmission observed in Duchenne muscular dystrophy (DMD) patients and in some genotypes of mice lacking dystrophin has been attributed to altered expression of short products of the dystrophin gene. We have investigated the potential role of Dp71, the most abundant C-terminal dystrophin gene product, in retinal electrophysiology. Comparison of the scotopic electroretinograms (ERG) between Dp71-null mice and wild-type (wt) littermates revealed a normal ERG in Dp71-null mice with no significant changes of the b-wave amplitude and kinetics. Analysis of DMD gene products, utrophin and dystrophin-associated proteins (DAPs), showed that Dp71 and utrophin were localized around the blood vessels, in the ganglion cell layer (GCL), and the inner limiting membrane (ILM). Dp71 deficiency was accompanied by an increased level of utrophin and decreased level of beta-dystroglycan localized in the ILM, without any apparent effect on the other DAPs. Dp71 deficiency was also associated with an impaired clustering of two Müller glial cell proteins-the inwardly rectifying potassium channel Kir4.1 and the water pore aquaporin 4 (AQP4). Immunostaining of both proteins decreased around blood vessels and in the ILM of Dp71-null mice, suggesting that Dp71 plays a role in the clustering and/or stabilization of the two proteins. AQP4 and Kir4.1 may also be involved in the regulation of the ischemic process. We found that a transient ischemia resulted in a greater damage in the GCL of mice lacking Dp71 than in wt mice. This finding points at a crucial role played by Dp71 in retinal function.

Exogenous Dp71 is a dominant negative competitor of dystrophin in skeletal muscle.

Dystrophin, the protein which is absent or non-functional in Duchenne muscular dystrophy, consists of four main domains: an N-terminal actin binding domain, a rod shaped domain of spectrin-like repeats, a cysteine-rich domain and a unique C-terminal domain. In muscle, dystrophin forms a linkage between the cytoskeletal actin and a group of membrane proteins (dystrophin associated proteins). The N-terminal domain binds to the cytoskeletal actin and the association with the dystrophin associated proteins is mediated mainly by the cysteine-rich and C-terminal domains of dystrophin. The dystrophin gene also encodes two isoforms of non-muscle dystrophins and a number of smaller products consisting of the two C-terminal domains with different extensions into the spectrin-like repeat domain. Dp71, which consist of the C-terminal and the cysteine-rich domains of dystrophin, is the major product of the gene in all non-muscle tissues tested so far, but it is absent in differentiated skeletal muscle. In an attempt to understand the functions of Dp71, we produced transgenic mice over-expressing this protein in several tissues. The highest levels of exogeneous Dp71 were detected in skeletal muscle, in association with the sarcolemma. This resulted in muscle damage similar to that found in mice which lack dystrophin. The data indicates that Dp71 competes with dystrophin for the binding to the dystrophin associated proteins. Since Dp71 lacks the actin binding domain, it cannot form the essential linkage between the dystrophin associated proteins complex and the cytoskeleton.