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Lamin A is a nuclear lamina constituent expressed in differentiated cells. Mutations in the LMNA gene cause several diseases, including muscular dystrophy and cardiomyopathy. Among the nuclear envelope partners of lamin A are Sad1 and UNC84 domain-containing protein 1 (SUN1) and Sad1 and UNC84 domain-containing protein 2 (SUN2), which mediate nucleo-cytoskeleton interactions critical to the anchorage of nuclei. In this study, we show that differentiating human myoblasts accumulate farnesylated prelamin A, which elicits upregulation and recruitment of SUN1 to the nuclear envelope and favors SUN2 enrichment at the nuclear poles. Indeed, impairment of prelamin A farnesylation alters SUN1 recruitment and SUN2 localization. Moreover, nuclear positioning in myotubes is severely affected in the absence of farnesylated prelamin A. Importantly, reduced prelamin A and SUN1 levels are observed in Emery–Dreifuss muscular dystrophy (EDMD) myoblasts, concomitant with altered myonuclear positioning. These results demonstrate that the interplay between SUN1 and farnesylated prelamin A contributes to nuclear positioning in human myofibers and may be implicated in pathogenetic mechanisms.

Farnesylated prelamin A is a processing intermediate produced in the lamin A maturation pathway. Accumulation of a truncated farnesylated prelamin A form, called progerin, is a hallmark of the severe premature ageing syndrome, Hutchinson-Gilford progeria. Progerin elicits toxic effects in cells, leading to chromatin damage and cellular senescence and ultimately causes skin and endothelial defects, bone resorption, lipodystrophy and accelerated ageing. Knowledge of the mechanism underlying prelamin A turnover is critical for the development of clinically effective protein inhibitors that can avoid accumulation to toxic levels without impairing lamin A/C expression, which is essential for normal biological functions. Little is known about specific molecules that may target farnesylated prelamin A to elicit protein degradation. Here, we report the discovery of rapamycin as a novel inhibitor of progerin, which dramatically and selectively decreases protein levels through a mechanism involving autophagic degradation. Rapamycin treatment of progeria cells lowers progerin, as well as wild-type prelamin A levels, and rescues the chromatin phenotype of cultured fibroblasts, including histone methylation status and BAF and LAP2alpha distribution patterns. Importantly, rapamycin treatment does not affect lamin C protein levels, but increases the relative expression of the prelamin A endoprotease ZMPSTE24. Thus, rapamycin, an antibiotic belonging to the class of macrolides, previously found to increase longevity in mouse models, can serve as a therapeutic tool, to eliminate progerin, avoid farnesylated prelamin A accumulation, and restore chromatin dynamics in progeroid laminopathies.

Adaptive responses of skeletal muscle regulate the nuclear shuttling of the sarcomeric protein Ankrd2 that can transduce different stimuli into specific adaptations by interacting with both structural and regulatory proteins. In a genome-wide expression study on Ankrd2-knockout or -overexpressing primary proliferating or differentiating myoblasts, we found an inverse correlation between Ankrd2 levels and the expression of proinflammatory genes and identified Ankrd2 as a potent repressor of inflammatory responses through direct interaction with the NF- κ B repressor subunit p50. In particular, we identified Gsk3 β as a novel direct target of the p50/Ankrd2 repressosome dimer and found that the recruitment of p50 by Ankrd2 is dependent on Akt2-mediated phosphorylation of Ankrd2 upon oxidative stress during myogenic differentiation. Surprisingly, the absence of Ankrd2 in slow muscle negatively affected the expression of cytokines and key calcineurin-dependent genes associated with the slow-twitch muscle program. Thus, our findings support a model in which alterations in Ankrd2 protein and phosphorylation levels modulate the balance between physiological and pathological inflammatory responses in muscle.

Serine/threonine kinase Akt/PKB intracellular distribution undergoes rapid changes in response to agonists such as Platelet-derived growth factor (PDGF) or Insulin-like growth factor (IGF). The concept has recently emerged that Akt subcellular movements are facilitated by interaction with nonsubstrate ligands. Here we show that Akt is bound to the actin skeleton in in situ cytoskeletal matrix preparations from PDGF-treated Saos2 cells, suggesting an interaction between the two proteins. Indeed, by immunoprecipitation and subcellular fractioning, we demonstrate that endogenous Akt and actin physically interact. Using recombinant proteins in in vitro binding and overlay assays, we further demonstrate that Akt interacts with actin directly. Expression of Akt mutants strongly indicates that the N-terminal PH domain of Akt mediates this interaction. More important, we show that the partition between actin bound and unbound Akt is not constant, but is modulated by growth factor stimulation. In fact, PDGF treatment of serum-starved cells triggers an increase in the amount of Akt associated with the actin skeleton, concomitant with an increase in Akt phosphorylation. Conversely, expression of an Akt mutant in which both Ser473 and Thr308 have been mutated to alanine completely abrogates PDGF-induced binding. The small GTPases Rac1 and Cdc42 seem to facilitate actin binding, possibly increasing Akt phosphorylation.

Background: Skeletal muscle disorders associated with mutations of lamin A/C gene include autosomal Emery–Dreifuss muscular dystrophy and limb girdle muscular dystrophy 1B. The pathogenic mechanism underlying these diseases is unknown. Recent data suggest an impairment of signalling mechanisms as a possible cause of muscle malfunction. A molecular complex in muscle cells formed by lamin A/C, emerin, and nuclear actin has been identified. The stability of this protein complex appears to be related to phosphorylation mechanisms. Objective: To analyse lamin A/C phosphorylation in control and laminopathic muscle cells. Methods: Lamin A/C N-terminal phosphorylation was determined in cultured mouse myoblasts using a specific antibody. Insulin treatment of serum starved myoblast cultures was carried out to evaluate involvement of insulin signalling in the phosphorylation pathway. Screening of four Emery–Dreifuss and one limb girdle muscular dystrophy 1B cases was undertaken to investigate lamin A/C phosphorylation in both cultured myoblasts and mature muscle fibres. Results: Phosphorylation of lamin A was observed during myoblast differentiation or proliferation, along with reduced lamin A/C phosphorylation in quiescent myoblasts. Lamin A N-terminus phosphorylation was induced by an insulin stimulus, which conversely did not affect lamin C phosphorylation. Lamin A/C was also hyperphosphorylated in mature muscle, mostly in regenerating fibres. Lamin A/C phosphorylation was strikingly reduced in laminopathic myoblasts and muscle fibres, while it was preserved in interstitial fibroblasts. Conclusions: Altered lamin A/C interplay with a muscle specific phosphorylation partner might be involved in the pathogenic mechanism of Emery–Dreifuss muscular dystrophy and limb girdle muscular dystrophy 1B.

We refer to our article by Vittoria Cenni et al. published in the European Journal of Histochemistry.

Adaptive responses of skeletal muscle regulate the nuclear shuttling of the sarcomeric protein Ankrd2 that can transduce different stimuli into specific adaptations by interacting with both structural and regulatory proteins. In a genome-wide expression study on Ankrd2-knockout or -overexpressing primary proliferating or differentiating myoblasts, we found an inverse correlation between Ankrd2 levels and the expression of proinflammatory genes and identified Ankrd2 as a potent repressor of inflammatory responses through direct interaction with the NF-κB repressor subunit p50. In particular, we identified Gsk3β as a novel direct target of the p50/Ankrd2 repressosome dimer and found that the recruitment of p50 by Ankrd2 is dependent on Akt2-mediated phosphorylation of Ankrd2 upon oxidative stress during myogenic differentiation. Surprisingly, the absence of Ankrd2 in slow muscle negatively affected the expression of cytokines and key calcineurin-dependent genes associated with the slow-twitch muscle program. Thus, our findings support a model in which alterations in Ankrd2 protein and phosphorylation levels modulate the balance between physiological and pathological inflammatory responses in muscle.

Surveillance of Echinococcus multilocularis at the edge of its range is hindered by fragmented distributional patterns and low prevalence in definitive hosts. Thus, tests with adequate levels of sensitivity are especially important for discriminating between infected and non-infected areas. In this study we reassessed the prevalence of E . multilocularis at the southern border of its distribution in Province of Bolzano (Alto Adige, northeastern Alps, Italy), to improve surveillance in wildlife and provide more accurate estimates of exposure risk. We compared the diagnostic test currently implemented for surveillance based on coproscopy and multiplex PCR (CMPCR) to a real-time quantitative PCR (qPCR) in 235 fox faeces collected in 2019 and 2020. The performances of the two tests were estimated using a scraping technique (SFCT) applied to the small intestines of a subsample (n = 123) of the same foxes as the reference standard. True prevalence was calculated and the sample size required by each faecal test for the detection of the parasite was then estimated. True prevalence of E . multilocularis in foxes (14.3%) was markedly higher than reported in the last decade, which was never more than 5% from 2012 to 2018 in the same area. In addition, qPCR showed a much higher sensitivity (83%) compared to CMPCR (21%) and agreement with the reference standard was far higher for qPCR (0.816) than CMPCR (0.298) meaning that for the latter protocol, a smaller sample size would be required to detect the disease. Alto Adige should be considered a highly endemic area. Routine surveillance on definitive hosts at the edges of the E . multilocularis distribution should be applied to smaller geographic areas, and rapid, sensitive diagnostic tools using directly host faeces, such as qPCR, should be adopted.

BackgroundRemibrutinib is a selective, covalent and potent inhibitor of Bruton’s tyrosine kinase (BTK) in development for several autoimmune and auto-allergic indications showing promising clinical safety and efficacy (Kaul et al 2021, Maurer et al 2022). Sjögren’s (SjS) is a chronic autoimmune disorder characterized by dryness in the eyes and mouth, fatigue, joint pain, and reduced quality of life (Mariette et al 2018). Remibrutinib may be particularly promising for the treatment of SjS due to its ability to target underlying B cell abnormalities and showed promising efficacy in a Ph2 SjS trial (Dörner et al 2022).ObjectivesThe aim of this exploratory biomarker study within the LOUiSSe trial was to identify protein and transcript signatures in the blood of patients with active SjS that are associated with remibrutinib treatment.MethodsWe conducted the LOUiSSe Ph2 clinical trial (NCT04035668) to evaluate the safety and efficacy of remibrutinib in patients with moderate to severe SjS. The study design was a randomized, double-blind, placebo-controlled, multi-center trial. Blood samples were collected at baseline and 24 weeks after treatment began. Protein profiles were generated using a Multiplex 7K aptamer profiling, and transcriptomics analysis was performed on RNA extracted from whole blood. By comparing the treatment and the placebo groups over time using multivariate linear regression models, we identified proteins and genes that were differentially modulated by remibrutinib. The gene signature identified by Yao et al (2009) was used as a reference for the interferon signature.ResultsWe observed a consistent downregulation of 35 proteins associated with B cell activity, including CD23, FCRL1, FAIM3, and FCRL4, in all treatment groups receiving remibrutinib compared to the placebo group after 24 weeks of treatment. These modulated proteins are involved in multiple pathways related to B-cell activation and function, including B-cell receptor signaling, Fc receptor signaling, and platelet activation. Whole blood bulk mRNA sequencing revealed that the remibrutinib group showed a significant downregulation of a 34-gene signature, most of which are known to be important for B-cell activation and immunoglobulin production. The top two most significantly downregulated genes, FCRL5 and SOX5, were previously identified as being highly enriched in a circulating tissue-like memory B-cell subset. This observation is also corroborated by the proteomics profiling analysis, where FCRL4 known to co-localize in the same memory B-cell subset (Verstappen et al 2020, Götz et al 2008), was heavily decreased by the treatment. Despite the specific and strong effects on B-cells, estimation of cell type proportions using RNA-seq revealed no significant changes, suggesting that remibrutinib does not affect the overall B-cell numbers and the relative proportions of its major subsets, like plasma cells, naïve and memory B-cells. Results from both proteomics and transcriptomics profiling suggest that remibrutinib treatment does not modulate the interferon signature.ConclusionOur analysis of serum proteins and whole blood transcripts showed that remibrutinib treatment was associated with a significant downregulation of proteins and genes enriched in FcRL4+ B cells, a subset of tissue-resident memory B cells that are expanded in inflamed tissues and found in the salivary glands of SjS patients. These findings suggest that remibrutinib may have a particularly strong inhibitory effect on this subset. Additionally, many genes and proteins involved in B-cell activation, differentiation, and maturation were also downregulated, indicating that remibrutinib has a potent effect on multiple B-cell pathways.References[1]Kaul et al, Clin Transl Sci. 2021[2]Mariette X, et al. N Engl J Med. 2018;[3]Maurer, M. et al. 2022[4]Dörner T, et al, Arthritis Rheumatol. 2022[5]Verstappen GM, et al. J of Autoimmunity 2020[6]Götz R.A. Ehrhardt, et al. J Exp Med. 2008[7]Yihong Yao et al, Americ Coll Reum 2018Acknowledgements:NIL.Disclosure of InterestsAndrea Grioni Shareholder of: Novartis, Employee of: Novartis, Aida Santos da Costa Shareholder of: Novartis, Employee of: Novartis, Enrico Ferrero Shareholder of: Novartis, Employee of: Novartis, Valeria De Luca Shareholder of: Novartis, Employee of: Novartis, Rainer Hillenbrand Shareholder of: Novartis, Employee of: Novartis, Andre da Costa Shareholder of: Novartis, Employee of: Novartis, Bruno Cenni Shareholder of: Novartis, Employee of: Novartis, Martin Kaul Shareholder of: Novartis, Employee of: Novartis, Thomas Dörner Consultant of: study support and honoraria for scientific advice: Novartis, Eli Lilly, EMD, BMS; Janssen; AbbVie, Roche/GNE, Richard Siegel Shareholder of: Novartis, Employee of: Novartis.

Patterns of fibre elongation and orientation for the cruciate and collateral ligaments of the human knee joint and for the patellar tendon have not yet been established in three-dimensions. These patterns are essential for understanding thoroughly the contribution of these soft tissues to joint function and of value in surgical treatments for a more conscious assessment of the knee status. Measurements from 10 normal cadaver knees are here reported using an accurate surgical navigation system and consistent anatomical references, over a large flexion arc, and according to current recommended conventions. The contours of relevant sub-bundles were digitised over the corresponding origins and insertions on the bones. Representative fibres were calculated as the straight line segments joining the centroids of these attachment areas. The most isometric fibre was also taken as that whose attachment points were at the minimum change in length over the flexion arc. Changes in length and orientation of these fibres were reported versus the flexion angle. A good general repeatability of intra- and inter-specimens was found. Isometric fibres were found in the locations reported in the literature. During knee flexion, ligament sub-bundles slacken in the anterior cruciate ligament, and in the medial and lateral collateral ligaments, whereas they tighten in the posterior cruciate ligament. In each cruciate ligament the two compounding sub-bundles have different extents for the change in fibre length, and also bend differently from each other on both tibial planes. In the collateral ligaments and patellar tendon all fibres bend posteriorly. Patellar tendon underwent complex changes in length and orientation, on both the tibial sagittal and frontal planes. For the first time thorough and consistent patterns of geometrical changes are provided for the main knee ligaments and tendons after careful fibre mapping.