mRNP granule proteins Fmrp and Dcp1a differentially regulate mRNP complexes to contribute to control of muscle stem cell quiescence and activation.

BACKGROUND: During skeletal muscle regeneration, satellite stem cells use distinct pathways to repair damaged myofibers or to self-renew by returning to quiescence. Cellular/mitotic quiescence employs mechanisms that promote a poised or primed state, including altered RNA turnover and translational repression. Here, we investigate the role of mRNP granule proteins Fragile X Mental Retardation Protein (Fmrp) and Decapping protein 1a (Dcp1a) in muscle stem cell quiescence and differentiation. METHODS: Using isolated single muscle fibers from adult mice, we established differential enrichment of mRNP granule proteins including Fmrp and Dcp1a in muscle stem cells vs. myofibers. We investigated muscle tissue homeostasis in adult Fmr1-/- mice, analyzing myofiber cross-sectional area in vivo and satellite cell proliferation ex vivo. We explored the molecular mechanisms of Dcp1a and Fmrp function in quiescence, proliferation and differentiation in a C2C12 culture model. Here, we used polysome profiling, imaging and RNA/protein expression analysis to establish the abundance and assembly status of mRNP granule proteins in different cellular states, and the phenotype of knockdown cells. RESULTS: Quiescent muscle satellite cells are enriched for puncta containing the translational repressor Fmrp, but not the mRNA decay factor Dcp1a. MuSC isolated from Fmr1-/- mice exhibit defective proliferation, and mature myofibers show reduced cross-sectional area, suggesting a role for Fmrp in muscle homeostasis. Expression and organization of Fmrp and Dcp1a varies during primary MuSC activation on myofibers, with Fmrp puncta prominent in quiescence, but Dcp1a puncta appearing during activation/proliferation. This reciprocal expression of Fmrp and Dcp1a puncta is recapitulated in a C2C12 culture model of quiescence and activation: consistent with its role as a translational repressor, Fmrp is enriched in non-translating mRNP complexes abundant in quiescent myoblasts; Dcp1a puncta are lost in quiescence, suggesting stabilized and repressed transcripts. The function of each protein differs during proliferation; whereas Fmrp knockdown led to decreased proliferation and lower cyclin expression, Dcp1a knockdown led to increased cell proliferation and higher cyclin expression. However, knockdown of either Fmrp or Dcp1a led to compromised differentiation. We also observed cross-regulation of decay versus storage mRNP granules; knockdown of Fmrp enhances accumulation of Dcp1a puncta, whereas knockdown of Dcp1a leads to increased Fmrp in puncta. CONCLUSIONS: Taken together, our results provide evidence that the balance of mRNA turnover versus utilization is specific for distinct cellular states.

Small molecule modulator of aggrephagy regulates neuroinflammation to curb pathogenesis of neurodegeneration.

BACKGROUND: Plethora of efforts fails to yield a single drug to reverse the pathogenesis of Parkinson's disease (PD) and related α-synucleopathies. METHODS: Using chemical biology, we identified a small molecule inhibitor of c-abl kinase, PD180970 that could potentially clear the toxic protein aggregates. Genetic, molecular, cell biological and immunological assays were performed to understand the mechanism of action. In vivo preclinical disease model of PD was used to assess its neuroprotection efficacy. FINDINGS: In this report, we show the ability of a small molecule inhibitor of tyrosine kinases, PD180970, to induce autophagy (cell lines and mice midbrain) in an mTOR-independent manner and ameliorate the α-synuclein mediated toxicity. PD180970 also exerts anti-neuroinflammatory potential by inhibiting the release of proinflammatory cytokines such as IL-6 (interleukin-6) and MCP-1 (monocyte chemoattractant protein-1) through reduction of TLR-4 (toll like receptor-4) mediated NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation. In vivo studies show that PD180970 is neuroprotective by degrading the toxic protein oligomers through induction of autophagy and subsiding the microglial activation. INTERPRETATION: These protective mechanisms ensure the negation of Parkinson's disease related motor impairments. FUND: This work was supported by Wellcome Trust/DBT India Alliance Intermediate Fellowship (500159-Z-09-Z), DST-SERB grant (EMR/2015/001946), DBT (BT/INF/22/SP27679/2018) and JNCASR intramural funds to RM, and SERB, DST (SR/SO/HS/0121/2012) to PAA, and DST-SERB (SB/YS/LS-215/2013) to JPC and BIRAC funding to ETA C-CAMP.

Induction of quiescence (G0) in bone marrow stromal stem cells enhances their stem cell characteristics.

Several studies have suggested that bone marrow stromal steam cells (BMSC) exist in a quiescent state (G0) within the in vivo niche; however, an explicit analysis of the biology of G0 state-BMSC has not been reported. We hypothesized that induction of G0 in BMSC might enhance their stem cell properties. Thus, we induced quiescence in BMSC in vitro by (a) suspension culture in a viscous medium or (b) culture on soft polyacrylamide substrate; and examined their molecular and functional phenotype. Induction of G0 was confirmed by bromo-deoxyuridine (BrdU) labelling and analysis of cell cycle gene expression. Upon reactivation and re-entry into cell cycle, G0 state-BMSC exhibited enhanced clonogenic self-renewal, preferential differentiation into osteoblastic rather than adipocytic cells and increased ectopic bone formation when implanted subcutaneously in vivo in immune-deficient mice, compared to asynchronous proliferating (pre-G0) BMSC. Global gene expression profiling revealed reprogramming of the transcriptome during G0 state including significant alterations in relevant pathways and expression of secreted factors, suggesting altered autocrine and paracrine signaling by G0 state-BMSC and a possible mechanism for enhanced bone formation. G0 state-BMSC might provide a clinically relevant model for understanding the in vivo biology of BMSC.

Modulation of Autophagy by a Small Molecule Inverse Agonist of ERRα Is Neuroprotective.

Mechanistic insights into aggrephagy, a selective basal autophagy process to clear misfolded protein aggregates, are lacking. Here, we report and describe the role of Estrogen Related Receptor α (ERRα, HUGO Gene Nomenclature ESRRA), new molecular player of aggrephagy, in keeping autophagy flux in check by inhibiting autophagosome formation. A screen for small molecule modulators for aggrephagy identified ERRα inverse agonist XCT 790, that cleared α-synuclein aggregates in an autophagy dependent, but mammalian target of rapamycin (MTOR) independent manner. XCT 790 modulates autophagosome formation in an ERRα dependent manner as validated by siRNA mediated knockdown and over expression approaches. We show that, in a basal state, ERRα is localized on to the autophagosomes and upon autophagy induction by XCT 790, this localization is lost and is accompanied with an increase in autophagosome biogenesis. In a preclinical mouse model of Parkinson's disease (PD), XCT 790 exerted neuroprotective effects in the dopaminergic neurons of nigra by inducing autophagy to clear toxic protein aggregates and, in addition, ameliorated motor co-ordination deficits. Using a chemical biology approach, we unrevealed the role of ERRα in regulating autophagy and can be therapeutic target for neurodegeneration.

A fine balance: epigenetic control of cellular quiescence by the tumor suppressor PRDM2/RIZ at a bivalent domain in the cyclin a gene.

Adult stem cell quiescence is critical to ensure regeneration while minimizing tumorigenesis. Epigenetic regulation contributes to cell cycle control and differentiation, but few regulators of the chromatin state in quiescent cells are known. Here we report that the tumor suppressor PRDM2/RIZ, an H3K9 methyltransferase, is enriched in quiescent muscle stem cells in vivo and controls reversible quiescence in cultured myoblasts. We find that PRDM2 associates with >4400 promoters in G0 myoblasts, 55% of which are also marked with H3K9me2 and enriched for myogenic, cell cycle and developmental regulators. Knockdown of PRDM2 alters histone methylation at key promoters such as Myogenin and CyclinA2 (CCNA2), and subverts the quiescence program via global de-repression of myogenesis, and hyper-repression of the cell cycle. Further, PRDM2 acts upstream of the repressive PRC2 complex in G0. We identify a novel G0-specific bivalent chromatin domain in the CCNA2 locus. PRDM2 protein interacts with the PRC2 protein EZH2 and regulates its association with the bivalent domain in the CCNA2 gene. Our results suggest that induction of PRDM2 in G0 ensures that two antagonistic programs-myogenesis and the cell cycle-while stalled, are poised for reactivation. Together, these results indicate that epigenetic regulation by PRDM2 preserves key functions of the quiescent state, with implications for stem cell self-renewal.

p38 MAPK regulates G1-S transition in hypoxic cardiac fibroblasts.

Cardiac fibroblast hyperplasia associated with augmented matrix production is central to wound healing following myocardial injury. Regulation of the cardiac fibroblast cell cycle by factors in the diseased myocardium that can potentially modify the hyperplastic response of cardiac fibroblasts has, however, not been investigated. We examined the regulation of the cardiac fibroblast cell cycle by hypoxia, a major constituent of myocardial ischemia. Significant reductions in DNA synthesis and cell number, and flow cytometry indicated decreased G1/S progression in hypoxic adult rat cardiac fibroblasts. Western blot analysis showed reduced levels of cyclin D and cyclin A, induction of p27 and hypophosphorylation of Rb under hypoxia. Skp2, which targets p27 for degradation, was significantly lower and inversely related to p27 protein levels in hypoxic cells. Marked p38 MAPK activation was observed under hypoxia and its inhibition using SB203580 reversed the effects of hypoxia on DNA synthesis, cell cycle phase distribution, p27, and cyclin D1 but not cyclin A. Interestingly, a 2-fold increase in p27 mRNA in hypoxic cells, demonstrated by real-time PCR, was unaffected by SB203580, which, however, reversed the hypoxic inhibition of Skp2. In summary, p38 MAPK is an important determinant of hypoxia-induced G0/G1 block in cardiac fibroblasts. p27 induction in hypoxic cardiac fibroblasts may involve direct transcriptional regulation, independent of p38 MAPK, and post-translational regulation via p38 MAPK-dependent suppression of its degradation by Skp2. The study identifies Skp2 as a potential downstream target of p38 MAPK, suggesting a novel mechanism of G1-S regulation in cardiac fibroblasts exposed to stress conditions.

NF-κB inhibition compromises cardiac fibroblast viability under hypoxia.

Cardiac fibroblasts are reported to be relatively resistant to stress stimuli compared to cardiac myocytes and fibroblasts of non-cardiac origin. However, the mechanisms that facilitate their survival under conditions of stress remain unclear. We explored the possibility that NF-κB protects cardiac fibroblasts from hypoxia-induced cell death. Further, we examined the expression of the antiapoptotic cIAP-2 and Bcl-2 in hypoxic cardiac fibroblasts, and their possible regulation by NF-κB. Phase contrast microscopy and propidium iodide staining revealed that cardiac fibroblasts are more resistant than pulmonary fibroblasts to hypoxia. Electrophoretic Mobility Shift Assay showed that hypoxia activates NF-κB in cardiac fibroblasts. Supershift assay indicated that the active NF-κB complex is a p65/p50 heterodimer. An I-κB-super-repressor was constructed that prevented NF-κB activation and compromised cell viability under hypoxic but not normoxic conditions. Similar results were obtained with Bay 11-7085, an inhibitor of NF-κB. Western blot analysis showed constitutive levels of Bcl-2 and hypoxic induction of cIAP-2 in these cells. NF-κB inhibition reduced cIAP-2 but not Bcl-2 levels in hypoxic cardiac fibroblasts. The results show for the first time that NF-κB is an important effector of survival in cardiac fibroblasts under hypoxic stress and that regulation of cIAP-2 expression may contribute to its pro-survival role.

Genistein abolishes nucleoside uptake by cardiac fibroblasts.

Genistein, a soy isoflavone, is reported to exert significant beneficial action in several human disorders, which has generated immense interest in the mechanisms underlying its effects on diverse cellular processes. It has anti-proliferative action on many cell types, an effect generally attributed to tyrosine kinase inhibition. In this study, genistein was found to cause total inhibition of [3H]-thymidine incorporation into DNA and a modest reduction in [3H]-proline incorporation into protein in primary cultures of cardiac fibroblasts. The decrease in [3H]-thymidine incorporation was not associated with a decrease in cell proliferation but correlated exactly with low intracellular levels of [3H]-thymidine. Genistein dramatically reduced [3H]-thymidine but not [3H]-proline uptake by these cells in which the equilibrative nucleoside transporter may be the major route of nucleoside uptake. The effect was irreversible and was demonstrable in pulmonary fibroblasts as well. The findings suggest that nucleoside uptake mechanisms may be a novel target of genistein action in cardiac fibroblasts and point to serious limitations in using genistein to assess the role of tyrosine kinase in cell proliferation by the standard technique of [3H]-thymidine incorporation.