Myomir dysregulation and reactive oxygen species in aged human satellite cells

AbstractSatellite cells that reside on the myofibre surface are crucial for the muscle homeostasis and regeneration. Aging goes along with a less effective regeneration of skeletal muscle tissue mainly due to the decreased myogenic capability of satellite cells. This phenomenon impedes proper maintenance and contributes to the age-associated decline in muscle mass, known as sarcopenia. The myogenic potential impairment does not depend on a reduced myogenic cell number, but mainly on their difficulty to complete a differentiation program. The unbalanced production of reactive oxygen species in elderly people could be responsible for skeletal muscle impairments. microRNAs are conserved post-transcriptional regulators implicated in numerous biological processes including adult myogenesis. Here, we measure the ROS level and analyze myomiR (miR-1, miR-133b and miR-206) expression in human myogenic precursors obtained from Vastus lateralis of elderly and young subjects to provide the molecular signature responsible for the differentiation impairment of elderly activated satellite cells

Constitutive IP₃ signaling underlies the sensitivity of B-cell cancers to the Bcl-2/IP₃ receptor disruptor BIRD-2

Anti-apoptotic Bcl-2 proteins are upregulated in different cancers, including diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL), enabling survival by inhibiting pro-apoptotic Bcl-2-family members and inositol 1,4,5-trisphosphate (IP3) receptor (IP3R)-mediated Ca2+-signaling. A peptide tool (Bcl-2/IP3R Disruptor-2; BIRD-2) was developed to abrogate the interaction of Bcl-2 with IP3Rs by targeting Bcl-2′s BH4 domain. BIRD-2 triggers cell death in primary CLL cells and in DLBCL cell lines. Particularly, DLBCL cells with high levels of IP3R2 were sensitive to BIRD-2. Here, we report that BIRD-2-induced cell death in DLBCL cells does not only depend on high IP3R2-expression levels, but also on constitutive IP3 signaling, downstream of the tonically active B-cell receptor. The basal Ca2+ level in SU-DHL-4 DLBCL cells was significantly elevated due to the constitutive IP3 production. This constitutive IP3 signaling fulfilled a pro-survival role, since inhibition of phospholipase C (PLC) using U73122 (2.5 µM) caused cell death in SU-DHL-4 cells. Milder inhibition of IP3 signaling using a lower U73122 concentration (1 µM) or expression of an IP3 sponge suppressed both BIRD-2-induced Ca2+ elevation and apoptosis in SU-DHL-4 cells. Basal PLC/IP3 signaling also fulfilled a pro-survival role in other DLBCL cell lines, including Karpas 422, RI-1 and SU-DHL-6 cells, whereas PLC inhibition protected these cells against BIRD-2-evoked apoptosis. Finally, U73122 treatment also suppressed BIRD-2-induced cell death in primary CLL, both in unsupported systems and in co-cultures with CD40L-expressing fibroblasts. Thus, constitutive IP3 signaling in lymphoma and leukemia cells is not only important for cancer cell survival, but also represents a vulnerability, rendering cancer cells dependent on Bcl-2 to limit IP3R activity. BIRD-2 seems to switch constitutive IP3 signaling from pro-survival into pro-death, presenting a plausible therapeutic strategy

Extracellular GTP is a Potent Water- Transport Regulator via Aquaporin 5 Plasma-Membrane Insertion in M1-CCD Epithelial Cortical Collecting Duct Cells

Background/Aims: Extracellular GTP is able to modulate some specific functions in neuron, glia and muscle cell models as it has been demonstrated over the last two decades. In fact, extracellular GTP binds its specific plasma membrane binding sites and induces signal transduction via [Ca(2+)]i increase. We demonstrate, for the first time, that extracellular GTP is able to modulate cell swelling in M1-CCD cortical collecting duct epithelial cells via upregulation of aquaporin 5 (AQP5) expression. Methods: We used videoimaging, immunocitochemistry, flow cytometry, confocal techniques, Western blotting and RT-PCR for protein and gene expression analysis, respectively. Results: We demonstrate that AQP5 mRNA is up-regulated 7 h after the GTP exposure in the cell culture medium, and its protein level is increased after 12-24 h. We show that AQP5 is targeted to the plasma membrane of M1-CCD cells, where it facilitates cell swelling, and that the GTP-dependent AQP5 up-regulation occurs via [Ca(2+)]i increase. Indeed, GTP induces both oscillating and transient [Ca(2+)]i increase, and specifically the oscillating kinetic appears to be responsible for blocking cell cycle in the S-phase while the [Ca(2+)]i influx, with whatever kinetic, seems to be responsible for inducing AQP5 expression. Conclusion: The role of GTP as a regulator of AQP5-mediated water transport in renal cells is of great importance in the physiology of renal epithelia, due to its possible physiopathological implications. GTP-dependent AQP5 expression could act as osmosensor. In addition, the data presented here suggest that GTP might play the same role in other tissues where rapid water transport is required for cell volume regulation and maintenance of the homeostasis. © 2014 S. Karger AG, Basel. ispartof: Cellular Physiology and Biochemistry vol:33 issue:3 pages:731-46 ispartof: location:Germany status: publishe

Long-term culture of patient-derived cardiac organoids recapitulated Duchenne muscular dystrophy cardiomyopathy and disease progression

Duchenne Muscular Dystrophy (DMD) is an X-linked neuromuscular disease which to date is incurable. The major cause of death is dilated cardiomyopathy however, its pathogenesis is unclear as existing cellular and animal models do not fully recapitulate the human disease phenotypes. In this study, we generated cardiac organoids from patient-derived induced pluripotent stem cells (DMD-COs) and isogenic-corrected controls (DMD-Iso-COs) and studied if DMD-related cardiomyopathy and disease progression occur in the organoids upon long-term culture (up to 93 days). Histological analysis showed that DMD-COs lack initial proliferative capacity, displayed a progressive loss of sarcoglycan localization and high stress in endoplasmic reticulum. Additionally, cardiomyocyte deterioration, fibrosis and aberrant adipogenesis were observed in DMD-COs over time. RNA sequencing analysis confirmed a distinct transcriptomic profile in DMD-COs which was associated with functional enrichment in hypertrophy/dilated cardiomyopathy, arrhythmia, adipogenesis and fibrosis pathways. Moreover, five miRNAs were identified to be crucial in this dysregulated gene network. In conclusion, we generated patient-derived cardiac organoid model that displayed DMD-related cardiomyopathy and disease progression phenotypes in long-term culture. We envision the feasibility to develop a more complex, realistic and reliable in vitro 3D human cardiac-mimics to study DMD-related cardiomyopathies

Bcl-xL acts as an inhibitor of IP3R channels, thereby antagonizing Ca2+-driven apoptosis

Anti-apoptotic Bcl-2-family members not only act at mitochondria but also at the endoplasmic reticulum, where they impact Ca dynamics by controlling IP receptor (IPR) function. Current models propose distinct roles for Bcl-2 vs. Bcl-xL, with Bcl-2 inhibiting IPRs and preventing pro-apoptotic Ca release and Bcl-xL sensitizing IPRs to low [IP] and promoting pro-survival Ca oscillations. We here demonstrate that Bcl-xL too inhibits IPR-mediated Ca release by interacting with the same IPR regions as Bcl-2. Via in silico superposition, we previously found that the residue K87 of Bcl-xL spatially resembled K17 of Bcl-2, a residue critical for Bcl-2’s IPR-inhibitory properties. Mutagenesis of K87 in Bcl-xL impaired its binding to IPR and abrogated Bcl-xL’s inhibitory effect on IPRs. Single-channel recordings demonstrate that purified Bcl-xL, but not Bcl-xL, suppressed IPR single-channel openings stimulated by sub-maximal and threshold [IP]. Moreover, we demonstrate that Bcl-xL-mediated inhibition of IPRs contributes to its anti-apoptotic properties against Ca-driven apoptosis. Staurosporine (STS) elicits long-lasting Ca elevations in wild-type but not in IPR-knockout HeLa cells, sensitizing the former to STS treatment. Overexpression of Bcl-xL in wild-type HeLa cells suppressed STS-induced Ca signals and cell death, while Bcl-xL was much less effective in doing so. In the absence of IPRs, Bcl-xL and Bcl-xL were equally effective in suppressing STS-induced cell death. Finally, we demonstrate that endogenous Bcl-xL also suppress IPR activity in MDA-MB-231 breast cancer cells, whereby Bcl-xL knockdown augmented IPR-mediated Ca release and increased the sensitivity towards STS, without altering the ER Ca content. Hence, this study challenges the current paradigm of divergent functions for Bcl-2 and Bcl-xL in Ca-signaling modulation and reveals that, similarly to Bcl-2, Bcl-xL inhibits IPR-mediated Ca release and IPR-driven cell death. Our work further underpins that IPR inhibition is an integral part of Bcl-xL’s anti-apoptotic function.The work was supported by Grants from the Research Foundation—Flanders (FWO) (G.0901.18N), by the Research Council of the KU Leuven (OT14/101, C14/19/099, C14/19/101, and AKUL/19/34), the Interuniversity Attraction Poles Program (Belgian Science Policy; IAP-P7/13), the Central European Leuven Strategic Alliance (CELSA/18/040), and the Canadian Institutes Health Research (FDN143312). NR and HI are recipient of postdoctoral fellowships of the FWO; HI obtained a travel grant from the FWO to perform work in DIY’s laboratory. GB, JBP and DIY are part of the FWO Scientific Research Network CaSign (W0.019.17N). Work in DIY’s lab is supported by NIH (NIDCR) grant DE014756. DWA holds the Tier 1 Canada Research Chair in Membrane Biogenesis. The Switch laboratory was supported by the Flanders institute for Biotechnology (VIB), the University of Leuven, the Fund for Scientific Research Flanders (Hercules Foundation/FWO AKUL/15/34—G0H1716N). NL is funded by the Stichting Alzheimer Onderzoek (SAO-FRA 2020/0013) and is recipient of FWO postdoctoral fellowships (12P0919N and 12P0922N to NL)

LRRK2 phosphorylation status and kinase activity regulate (macro)autophagy in a Rab8a/Rab10-dependent manner

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common genetic cause of Parkinson’s disease (PD), with growing importance also for Crohn’s disease and cancer. LRRK2 is a large and complex protein possessing both GTPase and kinase activity. Moreover, LRRK2 activity and function can be influenced by its phosphorylation status. In this regard, many LRRK2 PD-associated mutants display decreased phosphorylation of the constitutive phosphorylation cluster S910/S935/S955/S973, but the role of these changes in phosphorylation status with respect to LRRK2 physiological functions remains unknown. Here, we propose that the S910/S935/S955/S973 phosphorylation sites act as key regulators of LRRK2-mediated autophagy under both basal and starvation conditions. We show that quadruple LRRK2 phosphomutant cells (4xSA; S910A/S935A/S955A/S973A) have impaired lysosomal functionality and fail to induce and proceed with autophagy during starvation. In contrast, treatment with the specific LRRK2 kinase inhibitors MLi-2 (100 nM) or PF-06447475 (150 nM), which also led to decreased LRRK2 phosphorylation of S910/S935/S955/S973, did not affect autophagy. In explanation, we demonstrate that the autophagy impairment due to the 4xSA LRRK2 phospho-dead mutant is driven by its enhanced LRRK2 kinase activity. We show mechanistically that this involves increased phosphorylation of LRRK2 downstream targets Rab8a and Rab10, as the autophagy impairment in 4xSA LRRK2 cells is counteracted by expression of phosphorylation-deficient mutants T72A Rab8a and T73A Rab10. Similarly, reduced autophagy and decreased LRRK2 phosphorylation at the constitutive sites were observed in cells expressing the pathological R1441C LRRK2 PD mutant, which also displays increased kinase activity. These data underscore the relation between LRRK2 phosphorylation at its constitutive sites and the importance of increased LRRK2 kinase activity in autophagy regulation and PD pathology

APOL1 C-Terminal Variants May Trigger Kidney Disease through Interference with APOL3 Control of Actomyosin

The C-terminal variants G1 and G2 of apolipoprotein L1 (APOL1) confer human resistance to the sleeping sickness parasite Trypanosoma rhodesiense, but they also increase the risk of kidney disease. APOL1 and APOL3 are death-promoting proteins that are partially associated with the endoplasmic reticulum and Golgi membranes. We report that in podocytes, either APOL1 C-terminal helix truncation (APOL1Δ) or APOL3 deletion (APOL3KO) induces similar actomyosin reorganization linked to the inhibition of phosphatidylinositol-4-phosphate [PI(4)P] synthesis by the Golgi PI(4)-kinase IIIB (PI4KB). Both APOL1 and APOL3 can form K+ channels, but only APOL3 exhibits Ca2+-dependent binding of high affinity to neuronal calcium sensor-1 (NCS-1), promoting NCS-1-PI4KB interaction and stimulating PI4KB activity. Alteration of the APOL1 C-terminal helix triggers APOL1 unfolding and increased binding to APOL3, affecting APOL3-NCS-1 interaction. Since the podocytes of G1 and G2 patients exhibit an APOL1Δ or APOL3KO-like phenotype, APOL1 C-terminal variants may induce kidney disease by preventing APOL3 from activating PI4KB, with consecutive actomyosin reorganization of podocytes.info:eu-repo/semantics/publishe

An explainable model of host genetic interactions linked to COVID-19 severity

We employed a multifaceted computational strategy to identify the genetic factors contributing to increased risk of severe COVID-19 infection from a Whole Exome Sequencing (WES) dataset of a cohort of 2000 Italian patients. We coupled a stratified k-fold screening, to rank variants more associated with severity, with the training of multiple supervised classifiers, to predict severity based on screened features. Feature importance analysis from tree-based models allowed us to identify 16 variants with the highest support which, together with age and gender covariates, were found to be most predictive of COVID-19 severity. When tested on a follow-up cohort, our ensemble of models predicted severity with high accuracy (ACC = 81.88%; AUCROC = 96%; MCC = 61.55%). Our model recapitulated a vast literature of emerging molecular mechanisms and genetic factors linked to COVID-19 response and extends previous landmark Genome-Wide Association Studies (GWAS). It revealed a network of interplaying genetic signatures converging on established immune system and inflammatory processes linked to viral infection response. It also identified additional processes cross-talking with immune pathways, such as GPCR signaling, which might offer additional opportunities for therapeutic intervention and patient stratification. Publicly available PheWAS datasets revealed that several variants were significantly associated with phenotypic traits such as "Respiratory or thoracic disease", supporting their link with COVID-19 severity outcome.A multifaceted computational strategy identifies 16 genetic variants contributing to increased risk of severe COVID-19 infection from a Whole Exome Sequencing dataset of a cohort of Italian patients

Carriers of ADAMTS13 Rare Variants Are at High Risk of Life-Threatening COVID-19

Thrombosis of small and large vessels is reported as a key player in COVID-19 severity. However, host genetic determinants of this susceptibility are still unclear. Congenital Thrombotic Thrombocytopenic Purpura is a severe autosomal recessive disorder characterized by uncleaved ultra-large vWF and thrombotic microangiopathy, frequently triggered by infections. Carriers are reported to be asymptomatic. Exome analysis of about 3000 SARS-CoV-2 infected subjects of different severities, belonging to the GEN-COVID cohort, revealed the specific role of vWF cleaving enzyme ADAMTS13 (A disintegrin-like and metalloprotease with thrombospondin type 1 motif, 13). We report here that ultra-rare variants in a heterozygous state lead to a rare form of COVID-19 characterized by hyper-inflammation signs, which segregates in families as an autosomal dominant disorder conditioned by SARS-CoV-2 infection, sex, and age. This has clinical relevance due to the availability of drugs such as Caplacizumab, which inhibits vWF-platelet interaction, and Crizanlizumab, which, by inhibiting P-selectin binding to its ligands, prevents leukocyte recruitment and platelet aggregation at the site of vascular damage

Gain- and Loss-of-Function CFTR Alleles Are Associated with COVID-19 Clinical Outcomes

Carriers of single pathogenic variants of the CFTR (cystic fibrosis transmembrane conductance regulator) gene have a higher risk of severe COVID-19 and 14-day death. The machine learning post-Mendelian model pinpointed CFTR as a bidirectional modulator of COVID-19 outcomes. Here, we demonstrate that the rare complex allele [G576V;R668C] is associated with a milder disease via a gain-of-function mechanism. Conversely, CFTR ultra-rare alleles with reduced function are associated with disease severity either alone (dominant disorder) or with another hypomorphic allele in the second chromosome (recessive disorder) with a global residual CFTR activity between 50 to 91%. Furthermore, we characterized novel CFTR complex alleles, including [A238V;F508del], [R74W;D1270N;V201M], [I1027T;F508del], [I506V;D1168G], and simple alleles, including R347C, F1052V, Y625N, I328V, K68E, A309D, A252T, G542*, V562I, R1066H, I506V, I807M, which lead to a reduced CFTR function and thus, to more severe COVID-19. In conclusion, CFTR genetic analysis is an important tool in identifying patients at risk of severe COVID-19