Amide Alkaloids as Privileged Sources of Senomodulators for Therapeutic Purposes in Age-Related Diseases.

Nature is an important source of bioactive compounds and has continuously made a large contribution to the discovery of new drug leads. Particularly, plant-derived compounds have long been identified as highly interesting in the field of aging research and senescence. Many plants contain bioactive compounds that have the potential to influence cellular processes and provide health benefits. Among them, Piper alkaloids have emerged as interesting candidates in the context of age-related diseases and particularly senescence. These compounds have been shown to display a variety of features, including antioxidant, anti-inflammatory, neuroprotective, and other bioactive properties that may help counteracting the effects of cellular aging processes. In the review, we will put the emphasis on piperlongumine and other related derivatives, which belong to the Piper alkaloids, and whose senomodulating potential has emerged during the last several years. We will also provide a survey on their potential in therapeutic perspectives of age-related diseases.

Critical contribution of mitochondria in the development of cardiomyopathy linked to desmin mutation.

BACKGROUND: Beyond the observed alterations in cellular structure and mitochondria, the mechanisms linking rare genetic mutations to the development of heart failure in patients affected by desmin mutations remain unclear due in part, to the lack of relevant human cardiomyocyte models. METHODS: To shed light on the role of mitochondria in these mechanisms, we investigated cardiomyocytes derived from human induced pluripotent stem cells carrying the heterozygous DESE439K mutation that were either isolated from a patient or generated by gene editing. To increase physiological relevance, cardiomyocytes were either cultured on an anisotropic micropatterned surface to obtain elongated and aligned cardiomyocytes, or as a cardiac spheroid to create a micro-tissue. Moreover, when applicable, results from cardiomyocytes were confirmed with heart biopsies of suddenly died patient of the same family harboring DESE439K mutation, and post-mortem heart samples from five control healthy donors. RESULTS: The heterozygous DESE439K mutation leads to dramatic changes in the overall cytoarchitecture of cardiomyocytes, including cell size and morphology. Most importantly, mutant cardiomyocytes display altered mitochondrial architecture, mitochondrial respiratory capacity and metabolic activity reminiscent of defects observed in patient's heart tissue. Finally, to challenge the pathological mechanism, we transferred normal mitochondria inside the mutant cardiomyocytes and demonstrated that this treatment was able to restore mitochondrial and contractile functions of cardiomyocytes. CONCLUSIONS: This work highlights the deleterious effects of DESE439K mutation, demonstrates the crucial role of mitochondrial abnormalities in the pathophysiology of desmin-related cardiomyopathy, and opens up new potential therapeutic perspectives for this disease.

Behavior, Neural Structure, and Metabolism in Adult Male Mice Exposed to Environmentally Relevant Doses of Di(2-ethylhexyl) Phthalate Alone or in a Phthalate Mixture.

BACKGROUND: We have previously shown that chronic exposure of adult male mice to low doses of di(2-ethylhexyl) phthalate (DEHP) altered male sexual behavior and induced down-regulation of the androgen receptor (AR) in the neural circuitry controlling this behavior. OBJECTIVES: The cellular mechanisms induced by chronic exposure of adult male mice to low doses of DEHP alone or in an environmental phthalate mixture were studied. METHODS: Two-month-old C57BL/6J males were exposed orally for 8 wk to DEHP alone (0, 5, or 50µg/kg/d) or to DEHP (50µg/kg/d) in a phthalate mixture. Behavior, dendritic density per 50-µm length, pre-/postsynaptic markers, synapse ultrastructure, and bioenergetic activity were analyzed. RESULTS: Mice exposed to DEHP either alone or in a phthalate mixture differed in mating, emission of ultrasonic vocalizations, and the ability to attract receptive females in urinary preference tests from control mice. Analyses in the medial preoptic area, the key hypothalamic region involved in male sexual behavior, showed lower dendritic spine density and protein levels of glutamate receptors and differences in other postsynaptic components and presynaptic markers between the treated groups. Ultrastructural observation of dendritic synapses by electron microscopy showed comparable morphology between the treated groups. Metabolic analyses highlighted differences in hypothalamic metabolites of males exposed to DEHP alone or in a phthalate mixture compared to control mice. These differences included lower tryptophan and higher NAD+ levels, respectively, a precursor and end product of the kynurenine pathway of tryptophan metabolism. The protein amounts of the xenobiotic aryl hydrocarbon receptor, one of the targets of this metabolic pathway and known negative regulator of the AR, were higher in the medial preoptic area of exposed male mice. DISCUSSION: Differences in behavior of male mice exposed to environmental doses of phthalates were associated with differences in neural structure and metabolism, with possibly a key role of the kynurenine pathway of tryptophan metabolism in the effects mediated by these substances. https://doi.org/10.1289/EHP11514.

The C-Terminal Acidic Tail Modulates the Anticancer Properties of HMGB1.

Energy metabolism reprogramming was recently listed as a hallmark of cancer. In this process, the switch from pyruvate kinase isoenzyme type M1 to pyruvate kinase isoenzyme type M2 (PKM2) is believed to play a crucial role. Interestingly, the activity of the active form of PKM2 can efficiently be inhibited by the high-mobility group box 1 (HMGB1) protein, leading to a rapid blockage of glucose-dependent aerobic respiration and cancer cell death. HMGB1 is a member of the HMG protein family. It contains two DNA-binding HMG-box domains and an acidic C-terminal tail capable of positively or negatively modulating its biological properties. In this work, we report that the deletion of the C-terminal tail of HMGB1 increases its activity towards a large panel of cancer cells without affecting the viability of normal immortalized fibroblasts. Moreover, in silico analysis suggests that the truncated form of HMGB1 retains the capacity of the full-length protein to interact with PKM2. However, based on the capacity of the cells to circumvent oxidative phosphorylation inhibition, we were able to identify either a cytotoxic or cytostatic effect of the proteins. Together, our study provides new insights in the characterization of the anticancer activity of HMGB1.

Glyoxal Induces Senescence in Human Keratinocytes through Oxidative Stress and Activation of the Protein Kinase B/FOXO3a/p27KIP1 Pathway.

Senescence is a well-characterized cellular state associated with specific markers such as permanent cell proliferation arrest and the secretion of messenger molecules by cells expressing the senescence-associated secretory phenotype. The senescence-associated secretory phenotype composition depends on many factors such as the cell type or the nature of the stress that induces senescence. Because the skin constitutes a barrier with the external environment, it is particularly subjected to different types of stresses and consequently prone to premature cellular aging. The dicarbonyl compounds glyoxal (GO) and methylglyoxal are precursors of advanced glycation end products, whose presence marks normal and pathological aging. In this study, we show that GO treatment provokes oxidative stress by increasing ROS and advanced glycation end-products levels and induces senescence in human keratinocytes. Furthermore, GO-induced senescence bears a unique molecular progression profile: an early-stage senescence when protein kinase B‒FOXO3a-p27KIP1 pathway mediates cell cycle arrest and a late-stage senescence maintained by the p16INK4/pRb pathway. Moreover, we characterized the resulting secretory phenotype during early-stage senescence by mass spectrometry. Our study provides evidence that GO can affect keratinocyte functions and act as a driver of human skin aging. Hence, senotherapeutics aimed at modulating GO-associated senescence phenotype hold promising potential.

Egr1 loss-of-function promotes beige adipocyte differentiation and activation specifically in inguinal subcutaneous white adipose tissue.

In mice, exercise, cold exposure and fasting lead to the differentiation of inducible-brown adipocytes, called beige adipocytes, within white adipose tissue and have beneficial effects on fat burning and metabolism, through heat production. This browning process is associated with an increased expression of the key thermogenic mitochondrial uncoupling protein 1, Ucp1. Egr1 transcription factor has been described as a regulator of white and beige differentiation programs, and Egr1 depletion is associated with a spontaneous increase of subcutaneous white adipose tissue browning, in absence of external stimulation. Here, we demonstrate that Egr1 mutant mice exhibit a restrained Ucp1 expression specifically increased in subcutaneous fat, resulting in a metabolic shift to a more brown-like, oxidative metabolism, which was not observed in other fat depots. In addition, Egr1 is necessary and sufficient to promote white and alter beige adipocyte differentiation of mouse stem cells. These results suggest that modulation of Egr1 expression could represent a promising therapeutic strategy to increase energy expenditure and to restrain obesity-associated metabolic disorders.

Mitochondrial Lon protease - depleted HeLa cells exhibit proteome modifications related to protein quality control, stress response and energy metabolism.

The ATP-dependent Lon protease is located in the mitochondrial matrix and oxidized proteins are among its primary targets for their degradation. Impairment of mitochondrial morphology and function together with apoptosis were observed in lung fibroblasts depleted for Lon expression while accumulation of carbonylated mitochondrial proteins has been reported for yeast and HeLa Lon deficient cells. In addition, age-related mitochondrial dysfunction has been associated with an impairment of Lon expression. Using a HeLa cell line stably transfected with an inducible shRNA directed against Lon, we have previously observed that Lon depletion results in a mild phenotype characterized by an increase of both production of reactive oxygen species and level of oxidized proteins (Bayot et al., 2014, Biochimie, 100: 38-47). In this study using the same cell line, we now show that Lon knockdown leads to modifications of the expression of a number of specific proteins involved in protein quality control, stress response and energy metabolism, as evidenced using a 2D gel-based proteomic approach, and to alteration of the mitochondrial network morphology. We also show that these effects are associated with decreased proliferation and can be modulated by culture conditions in galactose versus glucose containing medium.

The role of Pitx2 and Pitx3 in muscle stem cells gives new insights into P38α MAP kinase and redox regulation of muscle regeneration.

Skeletal muscle regeneration depends on satellite cells. After injury these muscle stem cells exit quiescence, proliferate and differentiate to regenerate damaged fibres. We show that this progression is accompanied by metabolic changes leading to increased production of reactive oxygen species (ROS). Using Pitx2/3 single and double mutant mice that provide genetic models of deregulated redox states, we demonstrate that moderate overproduction of ROS results in premature differentiation of satellite cells while high levels lead to their senescence and regenerative failure. Using the ROS scavenger, N-Acetyl-Cysteine (NAC), in primary cultures we show that a physiological increase in ROS is required for satellite cells to exit the cell cycle and initiate differentiation through the redox activation of p38α MAP kinase. Subjecting cultured satellite cells to transient inhibition of P38α MAP kinase in conjunction with NAC treatment leads to their rapid expansion, with striking improvement of their regenerative potential in grafting experiments.

Redox regulation by Pitx2 and Pitx3 is critical for fetal myogenesis.

During development, major metabolic changes occur as cells become more specialized within a lineage. In the case of skeletal muscle, differentiation is accompanied by a switch from a glycolytic proliferative progenitor state to an oxidative postmitotic differentiated state. Such changes require extensive mitochondrial biogenesis leading to increased reactive oxygen species (ROS) production that needs to be balanced by an antioxidant system. Our analysis of double conditional Pitx2/3 mouse mutants, both in vivo during fetal myogenesis and ex vivo in primary muscle cell cultures, reveals excessive upregulation of ROS levels leading to DNA damage and apoptosis of differentiating cells. This is a consequence of downregulation of Nrf1 and genes for antioxidant enzymes, direct targets of Pitx2/3, leading to decreased expression of antioxidant enzymes, as well as impairment of mitochondrial function. Our analysis identifies Pitx2 and Pitx3 as key regulators of the intracellular redox state preventing DNA damage as cells undergo differentiation.

Pitx2 and Pitx3 transcription factors: two key regulators of the redox state in adult skeletal muscle stem cells and muscle regeneration.

Adult tissue homeostasis and regeneration rely on tissue stem cell populations that generate committed precursors and differentiated cells while maintaining a pool of stem cells. In adult skeletal muscle, such cells, called satellite cells, remain quiescent at the periphery of muscle fibers. Upon injury they undergo activation, proliferation and differentiation to replace damaged fibers and also self-renew to reconstitute the muscle stem cell pool. During regeneration, the transition from a quiescent muscle stem cell to a differentiated fiber is accompanied by major metabolic changes. Such changes, and notably the switch from a glycolytic proliferative progenitor state to an oxidative post-mitotic differentiated state, require extensive mitochondrial biogenesis that takes place at the onset of differentiation and leads to increased ROS production. However, it is unclear whether this enhanced ROS production/mitochondrial content reflects an adaptation to the rising energy demand or whether it constitutes an essential regulation element of the differentiation program.To investigate the potential role of this metabolic switch and more specifically of reactive oxygen species during muscle regeneration, we took advantage of mouse mutants for Pitx2 and Pitx3 genes. Both genes are involved in foetal myogenesis where they have been identified as key regulators of the redox state preventing excessive ROS levels and DNA damage as cells undergo differentiation. We have now analyzed adult single and double Pitx2:Pitx3 conditional mutant mouse lines targeted to the muscle stem cell compartment. Double mutant satellite cells undergo senescence with impaired regeneration after injury, whereas in single Pitx3 mutants, premature differentiation occurs. We show that these effects are directly linked to dose-dependent changes in ROS levels and can be reversed by lowering ROS with the N-acetylcystein, supporting the notion that a controlled increase in ROS is required for differentiation of muscle stem cells.

Lying low but ready for action: the quiescent muscle satellite cell.

The muscle satellite cell is essential for skeletal muscle regeneration. It is located on the muscle fibre, under the basal lamina as a quiescent cell, which becomes activated after injury, when it leaves the fibre, proliferates, and either undergoes myogenesis to form new fibres or reconstitutes the satellite cell pool. In this review, we discuss the cellular environment of the quiescent cell, including the extracellular matrix, which constitutes its niche. Cell adhesion molecules and some signalling pathways reinforce its quiescent state, whereas other signals lead to activation. We discuss how the satellite cell is ready to respond with the appropriate receptors, but protects its quiescence by mechanisms that include immobilization of ligands by extracellular matrix components and synthesis of inhibitors for intracellular signalling pathways and for metalloproteinases that break down the matrix and promote ligand processing and receptor activation. The quiescent satellite cell is also well protected against toxins and oxidative stress. It has a low metabolic rate, as shown by few active mitochondria and anaerobic glycolysis. Different subpopulations of quiescent satellite cells can be distinguished on the basis of cell surface markers and stem cell-like properties. We discuss the latter in the context of the small proportion of satellite cells that express high levels of Pax7, or that are derived from cells that have never activated the Myf5 myogenic determination gene. However, many quiescent satellite cells transcribe Myf5, but do not enter myogenesis because of post-transcriptional regulation, which prevents Myf5 protein accumulation. Post-transcriptional regulation, through microRNA repression of a potential cell cycle activator, further illustrates how these cells are ready for action.

The selector gene Pax7 dictates alternate pituitary cell fates through its pioneer action on chromatin remodeling.

The anterior and intermediate lobes of the pituitary gland derive from the surface ectoderm. They provide a simple system to assess mechanisms of developmental identity established by tissue determinants. Each lobe contains a lineage expressing the hormone precursor pro-opiomelanocortin (POMC): the corticotropes and melanotropes. The T-box transcription factor Tpit controls terminal differentiation of both lineages. We now report on the unique role of Pax7 as a selector of intermediate lobe and melanotrope identity. Inactivation of the Pax7 gene results in loss of melanotrope gene expression and derepression of corticotrope genes. Pax7 acts by remodeling chromatin and allowing Tpit binding to a new subset of enhancers for activation of melanotrope-specific genes. Thus, the selector function of Pax7 is exerted through pioneer transcription factor activity. Genome-wide, the Pax7 pioneer activity is preferentially associated with composite binding sites that include paired and homeodomain motifs. Pax7 expression is conserved in human and dog melanotropes and defines two subtypes of pituitary adenomas causing Cushing's disease. In summary, expression of Pax7 provides a unique tissue identity to the pituitary intermediate lobe that alters Tpit-driven differentiation through pioneer and classical transcription factor activities.

Divergent transcriptional activities determine limb identity.

Limbs develop using a common genetic programme despite widely differing morphologies. This programme is modulated by limb-restricted regulators such as hindlimb (HL) transcription factors Pitx1 and Tbx4 and the forelimb (FL) Tbx5. Both Tbx factors have been implicated in limb patterning and growth, but their relative activities and underlying mechanisms remain unclear. In this paper, we show that Tbx4 and Tbx5 harbour conserved and divergent transcriptional regulatory domains that account for their roles in limb development. In particular, both factors share an activator domain and the ability to stimulate limb growth. However, we find that Tbx4 is the primary effector of HL identity for both skeletal and muscle development; this activity relies on a repressor domain that is inactivated by a human TBX4 small-patella syndrome mutation. We propose that limb identity is largely achieved by default in FL, whereas a specific repressor activity unique to Tbx4 determines HL identity.

Pitx2 defines alternate pathways acting through MyoD during limb and somitic myogenesis.

The MyoD gene is part of the core regulatory network that governs skeletal myogenesis and acts as an essential determinant of the myogenic cell fate. Although generic regulatory networks converging on this gene have been described, the specific mechanisms leading to MyoD expression in muscles of different ontology remain misunderstood. We now show that the homeobox gene Pitx2 is required for initial activation of the MyoD gene in limb muscle precursors through direct binding of Pitx2 to the MyoD core enhancer. Whereas Myf5 and Mrf4 are dispensable for limb muscle progenitor fate, inactivation of Myf5 and Mrf4 in Pitx2 mutants results in a drastic decrease of limb MyoD expression. Thus, Pitx2 and Myf5 define parallel genetic pathways for limb myogenesis. We show a similar dependence on Pitx2 and Myf5(Mrf4) in myotome, where MyoD expression is initially activated by Myf5 and Mrf4. In their absence, MyoD expression is eventually rescued by a Pax3-dependent mechanism. We now provide evidence that Pitx2 contributes to the rescue of MyoD expression and that it acts downstream of Pax3. We thus propose that myogenic differentiation of somite-derived muscle cells relies on two parallel genetic pathways, with the Pitx2 pathway being of primary importance for limb myogenesis but the Myf5 and Mrf4 pathway predominating in myotome. Muscle-specific wiring of regulatory networks composed of similar transcription factors thus underlies development of distinct skeletal muscles.

A muscle-specific promoter directs Pitx3 gene expression in skeletal muscle cells.

The Pitx homeobox transcription factor genes have been implicated in different developmental processes, including determination of hind limb identity for Pitx1, left-right asymmetry for Pitx2, and eye development and survival of midbrain dopaminergic neurons for Pitx3. Pitx1 and Pitx2 have partly redundant activities in craniofacial development, including in pituitary organogenesis, as indicated by their names. These genes also exhibit redundant activities in the control of hind limb bud growth. Recent studies have shown expression of the three Pitx genes in muscle, with Pitx3 being the most widely expressed in all skeletal muscles. We now report the identification of a muscle-specific promoter within the Pitx3 gene that is situated between the first exon for eye and brain expression and exon 2 that contains the initiator ATG codon. Sequences proximal to this muscle-specific exon 1 are essential and sufficient to confer muscle-specific expression in transgenic mice, they are responsive to myogenic basic helix-loop-helix regulatory factors, and they recruit these factors in vivo. In agreement with exclusive use of the muscle-specific promoter in aphakia mice that are deleted of the brain promoter, the trimethyl-lysine 4 histone H3 promoter signature shifts to this promoter in embryonic day 13 ak limb bud muscle cells. Myogenic basic helix-loop-helix regulatory factor activation of Pitx3 transcription may be part of a positive feedback loop contributing to establishment of the myogenic program.

Sequential expression and redundancy of Pitx2 and Pitx3 genes during muscle development.

The myogenic program is controlled by different groups of transcription factors acting during muscle development, including bHLH muscle regulatory factors (MRFs), the paired factors Pax3 and Pax7 and the homeobox factors Six1 and Six4. This program is critically dependent on MRFs that target downstream muscle-specific genes. We now report the expression of Pitx2 and Pitx3 transcription factors throughout muscle development. Pitx2 is first expressed in muscle progenitor cells of the dermomyotome and myotome. The onset of myoblast differentiation is concomitant with expression of Pitx3; its expression is maintained in all skeletal muscles while Pitx2 expression decreases thereafter. We have generated Pitx3 mutant mice and this deficiency does not significantly perturb muscle development but it is completely compensated by the maintenance of Pitx2 expression in all skeletal muscles. These experiments suggest that Pitx genes are important for myogenesis and that Pitx2 and Pitx3 may have partly redundant roles.

Identification of a new hybrid serum response factor and myocyte enhancer factor 2-binding element in MyoD enhancer required for MyoD expression during myogenesis.

MyoD is a critical myogenic factor induced rapidly upon activation of quiescent satellite cells, and required for their differentiation during muscle regeneration. One of the two enhancers of MyoD, the distal regulatory region, is essential for MyoD expression in postnatal muscle. This enhancer contains a functional divergent serum response factor (SRF)-binding CArG element required for MyoD expression during myoblast growth and muscle regeneration in vivo. Electrophoretic mobility shift assay, chromatin immunoprecipitation, and microinjection analyses show this element is a hybrid SRF- and MEF2 Binding (SMB) sequence where myocyte enhancer factor 2 (MEF2) complexes can compete out binding of SRF at the onset of differentiation. As cells differentiate into postmitotic myotubes, MyoD expression no longer requires SRF but instead MEF2 binding to this dual-specificity element. As such, the MyoD enhancer SMB element is the site for a molecular relay where MyoD expression is first initiated in activated satellite cells in an SRF-dependent manner and then increased and maintained by MEF2 binding in differentiated myotubes. Therefore, SMB is a DNA element with dual and stage-specific binding activity, which modulates the effects of regulatory proteins critical in controlling the balance between proliferation and differentiation.

Binding of serum response factor to cystic fibrosis transmembrane conductance regulator CArG-like elements, as a new potential CFTR transcriptional regulation pathway.

CFTR expression is tightly controlled by a complex network of ubiquitous and tissue-specific cis-elements and trans-factors. To better understand mechanisms that regulate transcription of CFTR, we examined transcription factors that specifically bind a CFTR CArG-like motif we have previously shown to modulate CFTR expression. Gel mobility shift assays and chromatin immunoprecipitation analyses demonstrated the CFTR CArG-like motif binds serum response factor both in vitro and in vivo. Transient co-transfections with various SRF expression vector, including dominant-negative forms and small interfering RNA, demonstrated that SRF significantly increases CFTR transcriptional activity in bronchial epithelial cells. Mutagenesis studies suggested that in addition to SRF other co-factors, such as Yin Yang 1 (YY1) previously shown to bind the CFTR promoter, are potentially involved in the CFTR regulation. Here, we show that functional interplay between SRF and YY1 might provide interesting perspectives to further characterize the underlying molecular mechanism of the basal CFTR transcriptional activity. Furthermore, the identification of multiple CArG binding sites in highly conserved CFTR untranslated regions, which form specific SRF complexes, provides direct evidence for a considerable role of SRF in the CFTR transcriptional regulation into specialized epithelial lung cells.