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Mechanical stress induced by contractions constantly threatens the integrity of muscle Z-disc, a crucial force-bearing structure in striated muscle. The PDZ-LIM proteins have been proposed to function as adaptors in transducing mechanical signals to preserve the Z-disc structure, however the underlying mechanisms remain poorly understood. Here, we show that LDB3, a well-characterized striated muscle PDZ-LIM protein, modulates mechanical stress signaling through interactions with the mechanosensing domain in filamin C, its chaperone HSPA8, and PKCα in the Z-disc of skeletal muscle. Studies of Ldb3Ala165Val/+ mice indicate that the myopathy-associated LDB3 p.Ala165Val mutation triggers early aggregation of filamin C and its chaperones at muscle Z-disc before aggregation of the mutant protein. The mutation causes protein aggregation and eventually Z-disc myofibrillar disruption by impairing PKCα and TSC2-mTOR, two important signaling pathways regulating protein stability and disposal of damaged cytoskeletal components at a major mechanosensor hub in the Z-disc of skeletal muscle., Pathak et al. identify LDB3, a striated muscle PDZ-LIM protein, as a signaling adaptor in a major mechanosensor assembly through interactions with filamin C, HSPA8, and PKCα. When LDB3 is mutated, PKCα and TSC2-mTOR mediated homeostasis is impaired, leading to protein aggregation myopathy.

Mutations in GBA1 encountered in Gaucher disease are a leading risk factor for Parkinson disease and associated Lewy body disorders. Many GBA1 mutation carriers, especially those with severe or null GBA1 alleles, have earlier and more progressive parkinsonism. To model the effect of partial glucocerebrosidase deficiency on neurological progression in vivo, mice with a human A53T α-synuclein (SNCAA53T) transgene were crossed with heterozygous null gba mice (gba+/−). Survival analysis of 84 mice showed that in gba+/−//SNCAA53T hemizygotes and homozygotes, the symptom onset was significantly earlier than in gba+/+//SNCAA53T mice (p-values 0.023–0.0030), with exacerbated disease progression (p-value < 0.0001). Over-expression of SNCAA53T had no effect on glucocerebrosidase levels or activity. Immunoblotting demonstrated that gba haploinsufficiency did not lead to increased levels of either monomeric SNCA or insoluble high molecular weight SNCA in this model. Immunohistochemical analyses demonstrated that the abundance and distribution of SNCA pathology was also unaltered by gba haploinsufficiency. Thus, while the underlying mechanism is not clear, this model shows that gba deficiency impacts the age of onset and disease duration in aged SNCAA53T mice, providing a valuable resource to identify modifiers, pathways and possible moonlighting roles of glucocerebrosidase in Parkinson pathogenesis.

BACKGROUND CUG-BP, Elav-like family member 1 (CELF1) is a multifunctional RNA binding protein found in a variety of adult and embryonic tissues. In the heart, CELF1 is found exclusively in the myocardium. However, the roles of CELF1 during cardiac development have not been completely elucidated. RESULTS Myofibrillar organization is disrupted and proliferation is reduced following knockdown of CELF1 in cultured chicken primary embryonic cardiomyocytes. In vivo knockdown of Celf1 in developing Xenopus laevis embryos resulted in myofibrillar disorganization and a trend toward reduced proliferation in heart muscle, indicating conserved roles for CELF1 orthologs in embryonic cardiomyocytes. Loss of Celf1 also resulted in morphogenetic abnormalities in the developing heart and gut. Using optical coherence tomography, we showed that cardiac contraction was impaired following depletion of Celf1, while heart rhythm remained unperturbed. In contrast to cardiac muscle, loss of Celf1 did not disrupt myofibril organization in skeletal muscle cells, although it did lead to fragmentation of skeletal muscle bundles. CONCLUSIONS CELF1 is required for normal myofibril organization, proliferation, morphogenesis, and contractile performance in the developing myocardium. Developmental Dynamics 245:854-873, 2016. © 2016 Wiley Periodicals, Inc.

Z-disc-associated, alternatively spliced, PDZ motif-containing protein (ZASP) is a principal component of the sarcomere. The three prevalent isoforms of ZASP in skeletal muscle are generated by alternative splicing of exons 9 and 10. The long isoforms, either having (ZASP-L) or lacking exon 10 (ZASP-LΔex10), include an N-terminal PDZ domain, an actin-binding region (ABR) with a conserved motif (ZM), and three C-terminal LIM domains. The short isoform (ZASP-S) lacks the LIM domains. Mutations, A147T and A165V, within the ZM of ZASP-LΔex10 cause myofibrillar myopathy, but the mechanism is unknown. We have prepared these proteins, their ABR, and the respective mutant variants in recombinant form, characterized them biophysically, and analyzed their actin-binding properties by surface plasmon resonance and electron microscopy. All the proteins were physically homogeneous and monomeric and had circular dichroic spectra consistent with partially folded conformations. Comparison of the NMR HSQC spectra of ZASP-S and the PDZ domain showed that the ABR is unstructured. ZASP-S and its mutant variants and ZASP-LΔex10 all bound to immobilized G-actin with high affinity (K(d) ≈ 10(−8) to 10(−9) M). Constructs of the isolated actin-binding region missing exon 10 (ABRΔ10) bound with lower affinity (K(d) ≈ 10(−7) M), but those retaining exon 10 (ABR+10) did so only weakly (K(d) ≈ 10(−5) M). ZASP-S, and the ABRΔ10, also induced F-actin and array formation, even in conditions of low ionic strength and in the absence of KCl and Mg(2+) ions. Interestingly, the ZM mutations A147T and A165V did not affect any of the results described above.

CUG-BP, Elav-like family member 1 (CELF1) is a highly conserved RNA binding protein that regulates pre-mRNA alternative splicing, polyadenylation, mRNA stability, and translation. In the heart, CELF1 is expressed in the myocardium, where its levels are tightly regulated during development. CELF1 levels peak in the heart during embryogenesis, and aberrant up-regulation of CELF1 in the adult heart has been implicated in cardiac pathogenesis in myotonic dystrophy type 1, as well as in diabetic cardiomyopathy. Either inhibition of CELF activity or over-expression of CELF1 in heart muscle causes cardiomyopathy in transgenic mice. Nonetheless, many of the cardiac targets of CELF1 regulation remain unknown. In this study, to identify cardiac targets of CELF1 we performed cross-linking immunoprecipitation (CLIP) for CELF1 from embryonic day 8 chicken hearts. We identified a previously unannotated exon in MYH7B as a novel target of CELF1-mediated regulation. We demonstrated that knockdown of CELF1 in primary chicken embryonic cardiomyocytes leads to increased inclusion of this exon and decreased MYH7B levels. We also investigated global changes in the transcriptome of primary embryonic cardiomyocytes following CELF1 knockdown in a published RNA-seq dataset. Pathway and network analyses identified strong associations between CELF1 and regulation of cell cycle and translation. Important regulatory proteins, including both RNA binding proteins and a cardiac transcription factor, were affected by loss of CELF1. Together, these data suggest that CELF1 is a key regulator of cardiomyocyte gene expression.