Cells

Bdelloid rotifers are known to survive desiccation and high doses of ionizing radiation. This extreme resistance is notably due to their capacity to cope with numerous DNA double-strand breaks (DSBs). Genes encoding key components of the non-homologous end joining (NHEJ) DNA repair pathway are strongly upregulated in the bdelloid rotifer Adineta vaga following exposure to ionizing radiation. Considering the notably high doses tolerated by these organisms, their capacity to efficiently restore genome integrity is particularly striking. Although NHEJ is generally regarded as less accurate than homologous recombination (HR), the absence of major genomic rearrangements in the descendants of irradiated rotifers suggests that DNA repair occurs with high fidelity. Terwagne et al. recently reported a delayed repair in germline nuclei, occurring during oocyte development when homologous chromosomes pair, thereby enabling template-based repair through HR. In this study, we established an in situ hybridization approach on A. vaga cryosections to investigate the spatial and temporal expression of key actors involved in NHEJ, HR, and Base excision repair (BER) pathways in somatic and germline tissues. We show that NHEJ (KU80) and BER-related genes (PARPs) as well as A. vaga Ligase E (putatively involved in DNA repair) are expressed early after radiation exposure in the somatic syncytium. In contrast, HR-related genes (Rad51: two paralogs, Rad54), as well as PCNA (involved in DNA replication, NER, BER, HR) are expressed later in maturing oocytes, indicating the activation of a delayed homologous recombination repair pathway in germline nuclei. Nurse cells, which express genes associated with both HR and NHEJ pathways, may rely on both mechanisms for their own DNA repair while also supplying mRNAs to the maturing oocyte. Our results provide new evidence for a differential regulation of DNA DSB repair pathways between soma and germline in bdelloids, with NHEJ predominating in somatic tissues and HR in the germline of A. vaga. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/722046v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@3b1f3borg.highwire.dtl.DTLVardef@17f5eb5org.highwire.dtl.DTLVardef@122ef14org.highwire.dtl.DTLVardef@7e4413_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOAbstract Figure:C_FLOATNO Summary of in situ hybridization results: genes coding for actors of NHEJ are expressed in the somatic nuclei and in the nurse nuclei of Adineta vaga individuals 2.5 hours post X-rays radiation, while genes coding for HR actors and PCNA (involved in multiple pathways including DNA replication and DNA repair: NER, BER, MR, HR) are expressed in the nurse nuclei 2.5 hours post radiation, and later in the maturing oocyte during oogenesis and in the laid eggs. Genes coding for actors highly expressed post-radiation, involved in the BER pathway appear to be only expressed in the somatic syncytium 2.5 hours post radiation, as well as the gene coding for the Ligase E, likely involved in DNA repair. C_FIG

Parkinsons disease (PD) is the second most common neurodegenerative disease, resulting from accumulation of -synuclein (-syn) in midbrain dopaminergic neurons and progressive neuronal loss. The most relevant species of -syn, oligomers, may exert neurotoxicity in a variety of mechanisms. Accumulation of misfolded -syn in the endoplasmic reticulum (ER) lumen induces ER stress conditions that leads to activation of the Unfolded Protein Response (UPR) and its main sensor PKR-like ER kinase (PERK). PERK is critical for cell fate determination - under prolonged ER stress, it may direct cell towards pro-apoptotic pathways. Targeting of -syn aggregation or UPR by genetic and pharmacological approaches proved effective in preclinical models of PD by previous research. Thus, in the present study, we aimed to determine the potential effect of combination of small-molecule inhibitors of -syn aggregation and ER stress-mediated PERK signaling (namely anle138b and AMG44) in a novel, 3D in vitro model of PD. We demonstrate that combination of both anti-aggregation and ER stress-targeting approaches amplifies neuroprotection against PD in organoid model in terms of increased neuronal metabolic activity, decreased -syn phosphorylation and aggregation, reduced dopaminergic cell death, and restoration of proteostasis.

Common sex chromosome aneuploidies (SCAs) often present with cognitive and cardiovascular dysfunction in humans. To address SCA effects on gene expression and DNA methylation in relevant cell types, we differentiated neural precursor cells (NPCs) and cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) with different numbers of sex chromosomes, including isogenic and independent lines. As expected, the expression of genes that escape X inactivation (escapees) mostly increases with the number of inactive X chromosomes (Xi). However, allelic analysis shows dampening of escapees specifically on the Xi in XXY compared to XX in both NPCs and CMs, revealing a novel type of dosage compensation in SCA. In contrast, Y-linked gene expression is higher in XXY versus XY NPCs, but the opposite is observed in CMs. This may explain the greater number of differentially expressed autosomal genes in NPCs versus CMs with an added Y chromosome, while effects of added X chromosomes are similar between cell types. Concordantly, changes in autosomal DNA methylation are mainly driven by the presence of a Y chromosome. These findings highlight the cell-type specificity of sex-linked and autosomal gene regulation in SCA conditions. HighlightsO_LISex chromosome aneuploidy induces cell-type specific changes in gene expression C_LIO_LIDampening of the inactive X chromosome in XXY alleviate X overexpression C_LIO_LIHigh Y-linked gene expression in XXY neuronal precursor cells but not cardiomyocytes C_LIO_LISex chromosome aneuploidy disrupts sex biases in autosomal gene expression C_LI

Although calmodulin is best known as an intracellular calcium sensor, it also possesses calcium-independent functions in unicellular organisms. This is exemplified by the budding yeast S. cerevisiae calmodulin, which binds its essential targets, the pericentrin-like protein Spc110 and type I and V myosins, without needing calcium. Whether such calcium-independent cellular functions are conserved in other yeasts and vertebrates nevertheless remains an open question. Here, we examined the calcium-independent functions of the fission yeast S. pombe calmodulin Cam1 by measuring its intracellular distribution. Using quantitative fluorescence microscopy, we assessed the intracellular localization of two cam1 mutants, where binding of Ca2+ had been compromised by mutations in their EF hands, compared to the wild type protein. Both Cam1-2V and -3V reduced their localization by 90% to the yeast microtubule-organizing center spindle pole bodies (SPB). In contrast, these two mutants did not affect the myosin-dependent localization to the equatorial division plane and to the cell tips. Replacing the endogenous cam1 with cam1-2V decreased the SPB localization of pericentrin Pcp1 by 69%, without changing the localization of either type V or I myosins. Over-expression of Pcp1 rescued the mitotic defects of cam1-2V cells at the restrictive temperature. Surprisingly, the cytokinesis of this cam1 mutant was largely normal. We concluded that fission yeast calmodulin Cam1 depends on Ca2+to be a component of SPBs, suggesting that calcium plays a critical role in the assembly of SPBs.

In our society, ageing, longevity, and neurodegenerative diseases are major concerns of public health. Recently, in Drosophila, we have identified a new cluster of three snoRNAs, including jouvence, and showed that each of them affect longevity and neurodegeneration. As these snoRNAs are required in the epithelium of the gut, these results point-out a causal relationship between the epithelium of the gut and the neurodegenerative lesions through the metabolic parameters, indicating a gut-brain axis. Here, we demonstrate that each snoRNA pseudouridylates a specific site on ribosomal-RNA, which consequently affects the amount of ribosomes as well as the translational efficacy. Moreover, using TRAP experiment assay, we also show that these lacks of pseudouridylations modify the translation of specific genes involved in lipid metabolism. Consequently, these lead to a chronic deregulation of trigycerides and sterols levels, whose correlate to an increase of neurogenerative lesions in old flies, as well as to a modification of longevity.

IP3R-Grp75-VDAC1 protein complex at the mitochondria-ER contact sites (MERCS) is involved in response to nutrients and control of glucose and energy metabolism, however, early alterations of the complex and MERCS in response to increased fat intake remain inconclusive. We investigated early effects of high-fat diet (HFD) on IP3R-Grp75-VDAC1 protein expression in correlation with ER-mitochondrial interaction in the liver of mice. Five-week-old mice were fed an HFD or a standard diet (SD) for 2 weeks (2W) or 8 weeks (8W). MERCS fractionation by a gradient ultracentrifugation, Western blot, transmission electron microscopy (TEM), Oroboros high-resolution respirometry were used to analyse liver tissues, while real-time PCR was used to profile genes responsive to HFD. No macroscopic morphological or functional alterations were observed in mice at 2W, while, expectedly, at 8W of HFD mice gained weight and glucose intolerance. Total IP3R protein was reduced at both 2W and 8W points by a post-transcriptional mechanism, while in MERCS, IP3R, VDAC1 and Grp75 were reduced at 8W time-point. TEM analysis revealed a significant reduction of mitochondrial coverage by MERCS, mitochondrial fragmentation and shortening of ER-mitochondria distance already at 2W time-point. Mitochondrial function and metabolism were largely spared. Markers of altered protein homeostasis such as Lmp2, Mecl-1 and Lmp7 showed an early upregulation. In conclusion, HFD induces early alterations in liver MERCS that precede gain of weight and glucose intolerance, suggesting their primary role in obesity and metabolic diseases and as potential therapeutic target.

Lymphocyte-extracellular matrix (ECM) interactions occur intermittently throughout the lymphocytes life cycle. Alterations in blood insulin levels following feeding modulates naive lymphocyte trafficking and adhesion to fibronectin via a pathway involving insulin-like growth factor-1 receptor (IGF-1R), phospholipase C gamma 1 (PLC-{gamma}1) and {beta}2 integrin activation. Lymphocytes exert traction forces, on the ECM during the process of extravasation. While these forces are essential for several homeostatic processes, the role of insulin in modulating lymphocyte-derived traction forces upon ECM adhesion is unknown. The aim of the current study was to investigate the effect of insulin on the traction generated by lymphocytes when adhered onto a fibronectin-coated substrate. Jurkat T-cells were placed on a fibronectin layer (50{micro}g/ml, 100{micro}m thickness) coated on polyacrylamide gels of stiffness 400Pa with red fluorescence beads as fiduciary markers. The cellular force generated by Jurkat T-cells was mapped using traction force microscopy. To elucidate the role of PLC-{gamma}1 in cellular force generation, the traction of Jurkat T-cells lacking PLC-{gamma}1, as well as those of a knockout cell where PLC-{gamma}1 was restored were quantified and compared with wild-type Jurkat T-cells. Lack of PLC-{gamma}1 attenuated adhesion when compared to wild-type Jurkat T-cells. Additionally, the traction force generated by each cell type decreased with increasing concentration of extracellular calcium. Treatment of adherent Jurkat T-cells with insulin increased traction in lower extracellular calcium condition while a dip was observed when a high extracellular calcium was present, in comparison to the untreated cells. However, the effect of insulin treatment was lost in the case of Jurkat T-cells lacking PLC-{gamma}1. Together these results indicate that insulin regulates traction force generated by adherent Jurkat T-cells via a process involving PLC-{gamma}1, in a calcium dependent manner.

Astroglia can counteract the harmful effects of -synuclein (-SYN) protofibrils and reverse premature cellular senescence by promoting tunneling nanotubes (TNTs). However, the mechanism behind this recovery is unknown. This study is the first to examine TNT-mediated mechanical stability in senescent astroglial recovery. We demonstrate that disruption of Lamin A/C in -SYN-protofibrils-treated senescent cells reduces actin-cytoskeleton stress, as measured by nucleus flatness index and isometric scale factor from quantitative microscopy. ROCK (Rho-associated kinase) inhibition, which is crucial for reducing actin-cytoskeleton tension, promotes TNTs. Small molecules like Cytochalasin-D, Nocodazole, and Jasplakinolide, which inhibit TNTs by altering actin tension other than ROCK pathway, cannot reverse senescence. RNA-sequence heatmaps reveal changes in senescence-, integrin-, and ROCK-pathway genes; STRING links these to the Hippo pathway. Experimental results show that cytosolic YAP translocation, a key regulator of Hippo pathway, is vital for TNT formation and actin-based stability in U87-MG astrocytoma and primary astrocytes. Interestingly, TNTs form between two cells with different actin tensions: one exhibits low actin tension with Hippo signaling on, while the other has higher actin tension with Hippo signaling off. The most notable observation is the high abundance of YAP inside the TNTs, along with actin. The study shows that TNTs maintain mechanical stability through Lamin A/C integrity and actin tension in -SYN-induced senescent astroglia, thereby protecting the cells, reversing senescence. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=163 SRC="FIGDIR/small/711517v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@192307dorg.highwire.dtl.DTLVardef@ad8b75org.highwire.dtl.DTLVardef@19ece78org.highwire.dtl.DTLVardef@1056395_HPS_FORMAT_FIGEXP M_FIG C_FIG

Chaperonins complex into double-ringed octamers to aid peptide folding. Recent evidence implicates dysfunctional chaperonin subunits in cancer and neurodegenerative diseases because their deregulation exacerbates cellular injury. Nevertheless, a gap of knowledge exists regarding the expression and localization of chaperonin subunits in relation to amyloidogenic processes in Alzheimers disease (AD). Here, we show that reduced levels of chaperonin-containing TCP-1 subunits 2 (CCT2) and 3 (CCT3) stratify AD, with the subcellular distribution of their residua being mutually exclusive with both {beta}-amyloid and hyperphosphorylated tau in neurons. We find CCT3 localized to a subset of glial fibrillary acidic protein-positive astrocytes in AD. Increased oxidative stress in vitro upregulated CCT3 expression in astrocyte-like U251 cells. Conversely, CCT3, but not CCT2, loss-of-function in neuron-like SH-SY5Y cells increased intracellular {beta}-amyloid load. These data suggest that CCT2/CCT3 are faithful disease-state indicators and implicate CCT3 in oxidative stress-dependent cellular damage pathways.

We present an automated image-analysis methodology for quantitative assessment of primary cilia in cultured human primary fibroblasts. The proposed approach implements a modular processing pipeline combining deep-learning based nuclear segmentation with intensity-driven segmentation of the ciliary axoneme and basal body. These partial segmentations are integrated using a distance-based association strategy, enabling automated reconstruction of individual cilia and subsequent extraction of geometrical features. Performance was evaluated against manually segmented ground truth. While manual annotations showed a systematic underestimation of cilia length, both automated and manual analyses produced consistent population-level trends and identical statistical discrimination across sample groups. Application of the pipeline revealed a progressive reduction in cilia length and ciliation frequency with increasing passage number, demonstrating the sensitivity of the method to subtle morphological changes. The proposed framework enables scalable, reproducible, and objective quantification of primary cilia morphology and provides a computational tool applicable to high-throughput studies of cellular ageing and related phenotypes. All the code related to this work is available through GitHub: https://github.com/reyesaldasoro/Cilia.

The most severe phenotype of alpha-1-antitrypsin deficiency (AATD) is caused by the Z-mutation within the SERPINA1 gene. The Glu342Lys substitution causes misfolding and polymerisation of the alpha-1-antitrypsin (AAT) protein, its accumulation in the ER and increases the susceptibility of hepatocytes towards ER-stress. Here, we present an induced pluripotent stem cell (iPSC)-based hepatic model to study AATD. We demonstrate that iPSCs from AATD patients differentiate equally well to hepatocyte-like cells (HLCs) as control iPSCs. We detected ZAAT polymers in patient-derived HLCs which could be reduced by SAHA or CBZ treatment. Transcriptome analyses revealed major differences in metabolism and signalling between control and AATD HLCs and indicated increased stress levels affecting intracellular organelles. Importantly, the transcriptomes of control and patient-derived cells separated into distinct clusters with respect to the expression of Heat-shock protein (HSP) encoding genes. SAHA treatment increased expression of various HSPs which might contribute towards reduced ZAAT polymers.

At the Wisconsin National Primate Research Center, we have identified a family of rhesus carrying the microtubule-associated protein tau (MAPT) R406W mutation linked to frontotemporal dementia (FTD). Rhesus induced pluripotent stem cells (RhiPSCs) derived from these monkeys present a unique opportunity for in vitro modeling and comparison with cells derived from MAPT R406W human carriers. Here, we report the development of a reproducible method to generate RhiPSCs compliant with the standards of the International Society for Stem Cell Research (ISSCR) to support in vitro modeling of FTD-MAPT R406W. Our stepwise approach identified efficient methods for fibroblast derivation, fibroblast reprogramming to RhiPSC, and RhiPSC maintenance over continued culture. To derive fibroblasts from MAPT wild type (WT) and R406W monkeys, a combination of manual processing and overnight enzymatic digestion was required to maximize the number of low passage fibroblasts available for reprogramming. Fibroblast reprogramming to RhiPSC using Sendai viral vectors versus oriP/EBNA1 episomal plasmids revealed the latter as most efficient. Electroporation conditions for oriP/EBNA1 reprogramming were optimized to maximize plasmid uptake and cell survival. Ultimately, eight RhiPSC lines were derived from 4 donor rhesus monkeys (n=2 WT, n=2 R406W; two clonal lines per donor) and fully characterized according to ISSCR standards. RhiPSC stemness and genetic stability was best maintained on mouse embryonic fibroblast feeders in Universal Primate Pluripotency Stem Cell medium, as opposed to Essential 12 medium supplemented with IWR1, which produced cytogenetic abnormalities. Rhesus neural progenitor cells were generated using a monolayer protocol and expressed PAX6 and NESTIN after 21 days of differentiation. Our reliable method will be useful to labs seeking to derive RhiPSCs for preclinical studies. Overall, the RhiPSCs generated from MAPT R406W carriers will be a critical resource for evaluating the molecular underpinnings of tau-related neurodegeneration across primate species.

Cystatin-related epididymal spermatogenic (CRES) is a member of a reproductive subgroup of family 2 cystatins and part of a functional amyloid-containing extracellular matrix (ECM) with host defense functions in the epididymal lumen. CRES and the endogenous epididymal amyloid matrix exhibit high plasticity, forming distinct amyloid structures, with antimicrobial functions tailored to specific bacterial strains. We recently demonstrated that CRES/CRES amyloid is also present in the mammalian brain ECM. In this study, we utilized a CRES knockout (KO) mouse model to determine if the loss of CRES altered the structure of the hippocampal and cortical ECM and impacted brain function. We report that CRES KO male and female mice exhibited sex-specific structural changes in both the loose/interstitial and perineuronal net (PNN) ECM. Additionally, young CRES KO males--but not females--displayed deficits in learning, memory and executive functioning when compared to WT controls. In contrast, older CRES KO males did not exhibit behavioral deficits, a finding that correlated with the observation of a similar PNN ECM structure when compared to WT mice. Our data suggest that CRES functions as a regulatory or structural component of the brain ECM, contributing to its sex-specific structural organization and plasticity, affecting sex-specific behaviors of learning and memory.

Colorectal cancer (CRC) cachexia induces skeletal muscle dysfunction, impeding quality of life and worsening cancer prognosis. Multiple preclinical models, including the widely used mouse model of subcutaneous inoculation with the C26 colorectal carcinoma cell line, have been developed to study the biological mechanisms of CRC cachexia and elucidate potential new treatments. It has been proposed that a distinct cell line of the same origin, namely CT26, is relatively non-cachexic. However, studies evaluating the relative potential of C26 and CT26 cells to induce cancer cachexia in parallel have been limited. The differences in the biological mechanisms by which C26 and CT26 impact skeletal muscle mass and function have also not been fully elucidated. In the current study, we investigated the differential capacity of C26 and CT26 to induce cancer cachexia using both an in vitro cancer-muscle cell co-culture and an in vivo syngeneic mouse model. Our results show that both C26 and CT26 cells induced significant atrophy of murine C2C12 skeletal myotubes. In the mouse model, while C26 and CT26 both reduced skeletal muscle mass and fat mass, only C26 tumors led to loss of body weight and impaired skeletal muscle force output. We further show that C26 tumor-bearing mice exhibit greater muscle inflammation than CT26 tumor-bearing mice. In addition, mice bearing C26 and CT26 tumors showed differential regulation of the innate immune responses and muscle protein turnover. Overall, our data suggests that although both C26 and CT26 cells do exhibit cachexic effects, C26 cells induce greater loss in body weight, fat mass, skeletal muscle mass, and physical function via promoting chronic inflammation and deregulating protein balance of skeletal muscle.

Anthracyclines, including doxorubicin, are widely used chemotherapeutic agents, but dose-dependent cardiotoxicity limits their clinical utility and increases the risk of heart failure in cancer survivors. In paediatric patients, female sex is a significant risk factor for anthracycline-associated cardiotoxicity, yet pre-clinical studies rarely investigate sex differences in immature hearts. Here, we provide a proteomic dataset from primary cardiomyocytes, isolated from postnatal day 2 rat hearts and treated with a clinically relevant concentration of doxorubicin. Analysis of proteins present in all samples identified candidates previously shown to be regulated by doxorubicin in adult hearts, as well as candidates that may be specifically regulated in young hearts. This dataset provides a resource for generating hypotheses on molecular mechanisms contributing to sex differences in juvenile doxorubicin-induced cardiotoxicity.

Pathogenic RYR1 variants are associated with a set of rare neuromuscular disorders termed RYR1-related disorders (RYR1-RD). Clinical manifestations of RYR1-RD include proximal/axial muscle weakness, delayed motor milestones, impaired mobility, muscle pain, and fatigue. Muscle-specific microRNAs (miRNAs) are mostly expressed in muscle tissue and can be detected peripherally in plasma. Using a digital detection system, here we identified and quantified differential amounts of miRNAs in six adult (four monoallelic and two biallelic) RYR1-RD patient plasma samples compared to controls. Overall, 51 differentially expressed miRNAs were identified and hsa-miR-4454+hsa-miR-7975, in particular, was significantly overexpressed relative to controls (+ 39-fold, P=0.00285). Exploration of these differentially expressed miRNAs warrant further investigation as potential biomarkers of RYR1-RD.

Leucine-Rich Repeat Kinase 2 (LRRK2) is a signaling molecule involved in Parkinsons disease pathomechanisms. In disease, the LRRK2 protein displays both a toxic gain of kinase function and a loss of phosphorylation at heterophosphosites found in an extended loop of the LRR domain. RAB GTPases, such as RAB29, have been identified as upstream activators of LRRK2. Indeed, co-expression of LRRK2 with RAB29 induces a hyperactivation of LRRK2 kinase activity, however the role of the LRRK2 heterologous phosphorylation status in its activation remains unknown. Here, our aim was to determine the role of LRRK2 heterologous phosphorylation on its activation by RAB29. Using single and compound phosphodead or phosphomimetic mutants of LRRK2 we show differential sensitivity of LRRK2 phosphomutants to activation by RAB29, with phosphodead mutants being more susceptible to be activated than phosphomimetic mutants. Interestingly, we find that the single phosphodead S910A LRRK2 mutant displays an activation of LRRK2 kinase activity similar to that observed for the compound phosphodead 6xS>A LRRK2 mutant (S860A/S910A/S935A/S955A/S973A/S976A). Time-course analysis revealed that phosphodead mutants displayed higher but also faster activation by RAB29. In addition, both physical interaction between LRRK2 and RAB29 as well as RAB29-induced recruitment of LRRK2 to the trans-Golgi network (TGN) was enhanced by phosphodead compared to phosphomimetic mutants. To confirm effects on native LRRK2, we tested a panel of ten nanobodies targeting LRRK2 that stabilized LRRK2 phosphorylation at varying levels. Nanobodies stabilizing LRRK2 at low S935 phosphorylation levels showed enhanced RAB29-induced activation compared to nanobodies not affecting pS935 LRRK2. Finally, we tested whether LRRK2 heterologous phosphorylation could affect centrosome cohesion deficits, a phenotype that has been linked to LRRK2 hyperactivation, and found that both the phosphodead LRRK2 as well as a nanobody stabilizing dephosphorylated LRRK2 enhanced the centrosome cohesion deficit. Our findings indicate that hyperactivability of LRRK2 is directly related to its heterologous phosphorylation status, with dephosphorylation leading to strong hyperactivation of LRRK2 by upstream activating RABs, and phosphorylated LRRK2 showing the opposite. This implies that strategies favoring LRRK2 phosphorylation will have therapeutic benefit.

A hallmark of damaged skeletal muscle fibers is displaced myonuclei that are no longer peripherally positioned. Displaced myonuclei are dogmatically thought to be derived exclusively from muscle stem cell (satellite cell) fusion. Using a surgical resection muscle injury model and in vivo recombination-independent resident myonuclear labeling, we detail the prevalence, time course, and origin of displaced myonuclei in response to a non-chemically-mediated muscle trauma. We found that: 1) non-satellite cell-derived (resident) displaced myonuclei emerge seven days after surgical injury in similar proportion to exogenous (satellite cell-derived) displaced myonuclei in intact muscle fibers, with a biased prevalence in myosin heavy chain IIB muscle fibers, 2) muscle fibers with multiple ([≥]2) displaced resident myonuclei was an unexpected but noteworthy feature of muscle fibers 7 days after injury, 3) embryonic myosin-expressing fibers at seven days post-surgery expectedly contain predominantly satellite-cell derived displaced myonuclei, but a subset have displaced resident myonuclei, and 4) satellite cell numbers in intact muscle do not increase until 7 days post-surgery. These data may help inform whether to target satellite cell-initiated processes, myonuclear-initiated processes, or both to facilitate muscle fiber injury repair. This information could lead to more effective therapeutic strategies for treating muscle trauma.

This report identifies a bidirectional signaling axis connecting lipid metabolism to nuclear mechanotransduction, with the potential to control fatty acid/triglyceride metabolism. The sterol regulatory element-binding (SREBP) family of transcription factors control fatty acid, triglyceride and cholesterol synthesis and metabolism. The family consists of three members: SREBP1a, SREBP1c, and SREBP2, that are regulated by intracellular cholesterol levels and insulin signaling. The SREBP2-dependent control of the LDL receptor gene is a well-established target for cholesterol-lowering therapeutics and the activity of SREBP1c is an attractive target in metabolic disease. In the current report, we identify SYNE4 (nesprin-4), a component of the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, as a direct target of the SREBP family of transcription factors, and show that nesprin-4 in turn supports SREBP1c function. We identify functional SREBP binding sites in the human SYNE4 promoter and demonstrate that these are required for the sterol- and SREBP-dependent regulation of the promoter. Furthermore, we show that the endogenous SYNE4 gene is also regulated by SREBP1/2 and intracellular sterol levels. Interestingly, SREBP2 is responsible for the sterol regulation of the SYNE4 gene in HepG2 cells, while SREBP1 is the major regulator in MCF7 cells, demonstrating that diberent cell types use diberent SREBP paralogs to regulate the same promoter/gene. Importantly, we find that nesprin-4 is a positive regulator of SREBP1c expression and function in HepG2 cells and during the diberentiation of human adipose-derived stem cells. In summary, the current report identifies a novel regulatory interaction between lipid metabolism and the LINC complex. Importantly, we demonstrate that this signaling axis is bidirectional, forming a closed loop that has the potential to control SREBP1c activity and thereby fatty acid and triglyceride synthesis/metabolism. Based on our data, we propose that the nesprin-4-dependent regulation of SREBP1c could represent a novel therapeutic target in metabolic disease.

Cartilage is characterized by a highly specialized extracellular matrix (ECM) secreted by chondrocytes and limited self-regenerative capacity. In vivo investigations of chondrogenesis are limited by difficult and traumatic access, especially in humans. While it is known for decades that disturbances of chondrocyte differentiation and changed cartilage ECM composition cause severe skeletal phenotypes in vertebrates, a detailed molecular understanding of chondrogenesis and cartilage ECM formation is still missing, especially in the context of human genetic skeletal diseases. ATDC5 cells, derived from AT805 mouse teratocarcinoma cells, have been used in the past to model chondrogenic differentiation, however, most studies have investigated few major cellular differentiation markers only so that the composition of the secreted ECM as well as effects on the ATDC5 transcriptome upon differentiation are still unclear. Here, we performed time-resolved transcriptomic and ECM proteomic analyses of differentiating ATDC5 cells. Both datasets confirmed the formation of a cartilage-like matrix with increasing expression of key chondrocyte genes over the course of differentiation. ECM proteomics further revealed a number of ECM components not previously reported in ATDC5 cells or the secreted ECM, encompassing collagens, proteoglycans, glycoproteins and other secreted factors. Overall, our findings provide a more detailed molecular characterization of ATDC5 chondrogenesis and highlight the potential of this model system for ECM-focused studies.