The impact of canonical Wnt transcriptional repressors TLE3 and TLE4 on postsynaptic transcription at the neuromuscular junction.

Here, we investigated the role of the canonical Wnt signaling pathway transcriptional regulators at the neuromuscular junction. Upon applying a denervation paradigm, the transcription levels of Ctnnb1, Tcf7l1, Tle1, Tle2, Tle3, and Tle4 were significantly downregulated. A significant decrease in canonical Wnt signaling activity was observed using the denervation paradigm in Axin2-lacZ reporter mice. Alterations in the transcriptional profile of the myogenic lineage in response to agrin (AGRN) suggested that TLE3 and TLE4, family members of groucho transducin-like enhancer of split 3 (TLE3), transcriptional repressors known to antagonize T cell factor/lymphoid enhancer factor (TCF)-mediated target gene activation, could be important regulators of canonical Wnt signaling activity at the postsynapse. Knockouts of these genes using CRISPR/Cas9 gene editing in primary skeletal muscle stem cells, called satellite cells, led to decreased AGRN-dependent acetylcholine receptor (CHRN) clustering and reduced synaptic gene transcription upon differentiation of these cells. Overall, our findings demonstrate that TLE3 and TLE4 participate in diminishing canonical Wnt signaling activity, supporting transcription of synaptic genes and CHRN clustering at the neuromuscular junction.

The YAP1/TAZ-TEAD transcriptional network regulates gene expression at neuromuscular junctions in skeletal muscle fibers.

We examined YAP1/TAZ-TEAD signaling pathway activity at neuromuscular junctions (NMJs) of skeletal muscle fibers in adult mice. Our investigations revealed that muscle-specific knockouts of Yap1 or Taz, or both, demonstrate that these transcriptional coactivators regulate synaptic gene expression, the number and morphology of NMJs, and synaptic nuclei. Yap1 or Taz single knockout mice display reduced grip strength, fragmentation of NMJs, and accumulation of synaptic nuclei. Yap1/Taz muscle-specific double knockout mice do not survive beyond birth and possess almost no NMJs, the few detectable show severely impaired morphology and are organized in widened endplate bands; and with motor nerve endings being mostly absent. Myogenic gene expression is significantly impaired in the denervated muscles of knockout mice. We found that Tead1 and Tead4 transcription rates were increased upon incubation of control primary myotubes with AGRN-conditioned medium. Reduced AGRN-dependent acetylcholine receptor clustering and synaptic gene transcription were observed in differentiated primary Tead1 and Tead4 knockout myotubes. In silico analysis of previously reported genomic occupancy sites of TEAD1/4 revealed evolutionary conserved regions of potential TEAD binding motifs in key synaptic genes, the relevance of which was functionally confirmed by reporter assays. Collectively, our data suggest a role for YAP1/TAZ-TEAD1/TEAD4 signaling, particularly through TAZ-TEAD4, in regulating synaptic gene expression and acetylcholine receptor clustering at NMJs.

The desmin mutation R349P increases contractility and fragility of stem cell-generated muscle micro-tissues.

AIMS: Desminopathies comprise hereditary myopathies and cardiomyopathies caused by mutations in the intermediate filament protein desmin that lead to severe and often lethal degeneration of striated muscle tissue. Animal and single cell studies hinted that this degeneration process is associated with massive ultrastructural defects correlating with increased susceptibility of the muscle to acute mechanical stress. The underlying mechanism of mechanical susceptibility, and how muscle degeneration develops over time, however, has remained elusive. METHODS: Here, we investigated the effect of a desmin mutation on the formation, differentiation, and contractile function of in vitro-engineered three-dimensional micro-tissues grown from muscle stem cells (satellite cells) isolated from heterozygous R349P desmin knock-in mice. RESULTS: Micro-tissues grown from desmin-mutated cells exhibited spontaneous unsynchronised contractions, higher contractile forces in response to electrical stimulation, and faster force recovery compared with tissues grown from wild-type cells. Within 1 week of culture, the majority of R349P desmin-mutated tissues disintegrated, whereas wild-type tissues remained intact over at least three weeks. Moreover, under tetanic stimulation lasting less than 5 s, desmin-mutated tissues partially or completely ruptured, whereas wild-type tissues did not display signs of damage. CONCLUSIONS: Our results demonstrate that the progressive degeneration of desmin-mutated micro-tissues is closely linked to extracellular matrix fibre breakage associated with increased contractile forces and unevenly distributed tensile stress. This suggests that the age-related degeneration of skeletal and cardiac muscle in patients suffering from desminopathies may be similarly exacerbated by mechanical damage from high-intensity muscle contractions. We conclude that micro-tissues may provide a valuable tool for studying the organization of myocytes and the pathogenic mechanisms of myopathies.

Alkali ion influence on structure and stability of fibrillar amyloid-ß oligomers.

Alzheimer's disease is characterized by the aggregation of Amyloid-ß (Aß) peptide into oligomers, fibrils and plaques. Many factors influencing this process as well as the stability of the various Aß aggregates are known to date, and include the concentration and type of metal ions. Most experimental and theoretical studies have concentrated on heavy metal ions, like Fe2+, Zn2+, or Cu2+, while the smaller alkali ions Li+, Na+, and K+ have not gained much attention notwithstanding their role and ubiquity in physiological environments. In this work, we applied atomistic molecular dynamics simulations to investigate the potential role of these alkali ions in stabilizing fibrillar Aß oligomers of different size and topology, i.e., single and double filament systems comprising 3-24 peptide chains per filament. We find a pronounced difference on the molecular level in the interaction behavior with free carboxylate groups of the Aß oligomer: Li+ forms stable bridged interactions, whereas K+ interacts more transiently and lacks bridging. The behavior of Na+ is in between, so that this ion-protein interaction obeys the renowned Hofmeister series. These differences are also reflected in the ability of the alkali ions to stabilize the oligomer secondary structure. The stabilizing effect is most pronounced for the smaller fibrillar oligomers, suggesting that the type of alkali ion critically affects the initial stages of fibril formation. Our findings thus offer a molecular explanation for the observation that the polymorphisms of Aß fibril structures are caused by differences in the surrounding ionic environment. Graphical abstract Influence of alkali ions on the structure and stability of fibrillar amyloid-ß oligomers.

In mammalian skeletal muscle, phosphorylation of TOMM22 by protein kinase CSNK2/CK2 controls mitophagy.

In yeast, Tom22, the central component of the TOMM (translocase of outer mitochondrial membrane) receptor complex, is responsible for the recognition and translocation of synthesized mitochondrial precursor proteins, and its protein kinase CK2-dependent phosphorylation is mandatory for TOMM complex biogenesis and proper mitochondrial protein import. In mammals, the biological function of protein kinase CSNK2/CK2 remains vastly elusive and it is unknown whether CSNK2-dependent phosphorylation of TOMM protein subunits has a similar role as that in yeast. To address this issue, we used a skeletal muscle-specific Csnk2b/Ck2ß-conditional knockout (cKO) mouse model. Phenotypically, these skeletal muscle Csnk2b cKO mice showed reduced muscle strength and abnormal metabolic activity of mainly oxidative muscle fibers, which point towards mitochondrial dysfunction. Enzymatically, active muscle lysates from skeletal muscle Csnk2b cKO mice phosphorylate murine TOMM22, the mammalian ortholog of yeast Tom22, to a lower extent than lysates prepared from controls. Mechanistically, CSNK2-mediated phosphorylation of TOMM22 changes its binding affinity for mitochondrial precursor proteins. However, in contrast to yeast, mitochondrial protein import seems not to be affected in vitro using mitochondria isolated from muscles of skeletal muscle Csnk2b cKO mice. PINK1, a mitochondrial health sensor that undergoes constitutive import under physiological conditions, accumulates within skeletal muscle Csnk2b cKO fibers and labels abnormal mitochondria for removal by mitophagy as demonstrated by the appearance of mitochondria-containing autophagosomes through electron microscopy. Mitophagy can be normalized by either introduction of a phosphomimetic TOMM22 mutant in cultured myotubes, or by in vivo electroporation of phosphomimetic Tomm22 into muscles of mice. Importantly, transfection of the phosphomimetic Tomm22 mutant in muscle cells with ablated Csnk2b restored their oxygen consumption rate comparable to wild-type levels. In sum, our data show that mammalian CSNK2-dependent phosphorylation of TOMM22 is a critical switch for mitophagy and reveal CSNK2-dependent physiological implications on metabolism, muscle integrity and behavior.

Wnt/ß-catenin signaling via Axin2 is required for myogenesis and, together with YAP/Taz and Tead1, active in IIa/IIx muscle fibers.

Canonical Wnt/ß-catenin signaling plays an important role in myogenic differentiation, but its physiological role in muscle fibers remains elusive. Here, we studied activation of Wnt/ß-catenin signaling in adult muscle fibers and muscle stem cells in an Axin2 reporter mouse. Axin2 is a negative regulator and a target of Wnt/ß-catenin signaling. In adult muscle fibers, Wnt/ß-catenin signaling is only detectable in a subset of fast fibers that have a significantly smaller diameter than other fast fibers. In the same fibers, immunofluorescence staining for YAP/Taz and Tead1 was detected. Wnt/ß-catenin signaling was absent in quiescent and activated satellite cells. Upon injury, Wnt/ß-catenin signaling was detected in muscle fibers with centrally located nuclei. During differentiation of myoblasts expression of Axin2, but not of Axin1, increased together with Tead1 target gene expression. Furthermore, absence of Axin1 and Axin2 interfered with myoblast proliferation and myotube formation, respectively. Treatment with the canonical Wnt3a ligand also inhibited myotube formation. Wnt3a activated TOPflash and Tead1 reporter activity, whereas neither reporter was activated in the presence of Dkk1, an inhibitor of canonical Wnt signaling. We propose that Axin2-dependent Wnt/ß-catenin signaling is involved in myotube formation and, together with YAP/Taz/Tead1, associated with reduced muscle fiber diameter of a subset of fast fibers.

LAP proteins are localized at the post-synaptic membrane of neuromuscular junctions and appear to modulate synaptic morphology and transmission.

Erbin, Lano, Scribble, and Densin-180 belong to LAP (leucine-rich repeats and PDZ domain) adaptor proteins involved in cell signaling pathways. Previously, we identified Erbin, Lano, and Scribble, but not Densin-180, in muscle cells, where they are involved in regulating the aggregation of nicotinic acetylcholine receptors in vitro. Here, we analyzed their cellular localization at the neuromuscular junction (NMJ) in skeletal muscles of mice. Erbin, Lano, and Scribble were significantly accumulated at NMJs and localized in different synaptic cells. Moreover, we used mouse mutants to analyze the role of Erbin at the NMJ. We used two Erbin mutant mouse strains that either completely lack Erbin protein (Erbinnull/null ) or express a truncated Erbin mutant where the carboxy-terminal PDZ domain is replaced by ß-galactosidase (ErbinΔC/ΔC ) thereby abolishing its interaction with ErbB receptor tyrosine kinases. Neither the lack of the PDZ domain of Erbin, nor its complete absence interfered with the general localization of LAP proteins at NMJs, but Lano and Scribble transcript levels were up-regulated in homozygous Erbin-null muscles. Furthermore, grip strength was reduced and neural transmission impaired in homozygous aged Erbin-null but not Erbin-ΔC mice. Erbin-null skeletal muscles did not reveal any conspicuous impairment of the muscle fiber. Localization of other NMJ marker proteins was not affected either. Quantitative 3D morphometry showed that NMJs of Erbin-null muscles were significantly smaller and fragmented in the soleus. We speculate that Erbin, Lano, and Scribble act at the post-synaptic membrane of NMJs in a concerted fashion to regulate nicotinic acetylcholine receptors cluster morphology and neural transmission. Cover Image for this issue: doi: 10.1111/jnc.13340.