ABSTRACT
Mesenchymal stem cells (MSCs) are promising cells for regenerative medicine therapies because they can differentiate towards multiple cell lineages. However, the occurrence of cellular senescence and the acquiring of the senescence-associated secretory phenotype (SASP) limit their clinical use. Since the transcription factor TWIST1 influences expansion of MSCs, its role in regulating cellular senescence was investigated. The present study demonstrated that silencing of TWIST1 in MSCs increased the occurrence of senescence, characterised by a SASP profile different from irradiation-induced senescent MSCs. Knowing that senescence alters cellular metabolism, cellular bioenergetics was monitored by using the Seahorse XF apparatus. Both TWIST1-silencing-induced and irradiation-induced senescent MSCs had a higher oxygen consumption rate compared to control MSCs, while TWIST1-silencing-induced senescent MSCs had a low extracellular acidification rate compared to irradiation-induced senescent MSCs. Overall, data indicated how TWIST1 regulation influenced senescence in MSCs and that TWIST1 silencing-induced senescence was characterised by a specific SASP profile and metabolic state.
Subject(s)
Mesenchymal Stem Cells , Senescence-Associated Secretory Phenotype , Cellular Senescence , Energy Metabolism , Gene Expression RegulationABSTRACT
Macroautophagy selectively degrades dysfunctional mitochondria by a process known as mitophagy. Here we demonstrate the involvement of transglutaminase 2 (TG2) in the turnover and degradation of damaged mitochondria. In TG2-ablated cells we observed the presence of a large number of fragmented mitochondria that display decreased membrane potential, downregulation of IF1 along with increased Drp1 and PINK1 levels, two key proteins regulating the mitochondrial fission. Of note, we demonstrate that in healthy mitochondria, TG2 interacts with the dynamic proteins Drp1 and Fis1; interestingly, their interaction is largely reduced upon induction of the fission process by carbonyl cyanide m-chlorophenyl hydrazine (CCCP). In keeping with these findings, mitochondria lacking TG2 are more susceptible to CCCP treatment. As a consequence of accumulation of damaged mitochondria, cells lacking TG2 increased their aerobic glycolysis and became sensitive to the glycolytic inhibitor 2-deoxy-D-glucose (2-DG). In contrast, TG2-proficient cells are more resistant to 2-DG-induced apoptosis as the caspase 3 is inactivated through the enzyme's crosslinking activity. The data presented in this study show that TG2 plays a key role in cellular dynamics and consequently influences the energetic metabolism.
Subject(s)
Autophagy/physiology , GTP-Binding Proteins/metabolism , Mitochondria/metabolism , Transglutaminases/metabolism , Aerobiosis , Animals , Energy Metabolism , GTP-Binding Proteins/deficiency , Glycolysis , HEK293 Cells , Humans , Mice , Mice, Knockout , Mitochondria/enzymology , Mitochondria/pathology , Protein Glutamine gamma Glutamyltransferase 2 , Transglutaminases/deficiencyABSTRACT
DNA damage and ageing share expression changes involving alterations in many aspects of metabolism, suppression of growth and upregulation of defence and genome maintenance systems. "Omics" technologies have permitted large-scale parallel measurements covering global cellular constituents and aided the identification of specific response pathways that change during ageing and after DNA damage. We have set out to identify genes with highly conserved response patterns through meta-analysis of mRNA expression datasets collected during natural ageing and accelerated ageing caused by a Transcription-Coupled Nucleotide Excision Repair (TC-NER) defect in a diverse set of organs and tissues in mice, and from in vitro UV-induced DNA damage in a variety of murine cells. The identified set of genes that show similar expression patterns in response to organ ageing (accelerated and normal), and endogenously and exogenously induced DNA damage, consists of genes involved in anti-oxidant systems and includes the transcription factor Bach2 as one of the most consistent markers. BACH2 was originally identified as a partner of the small Maf proteins and antagonist of the NRF2 anti-oxidant defence pathway and has been implicated in B-cell differentiation and immune system homeostasis. Although BACH2 has never before been associated with UV-induced damage or ageing, it shows a strong downregulation in both conditions. We have characterized the dynamics of Bach2 expression in response to DNA damage and show that it is a highly sensitive responder to transcription-blocking DNA lesions. Gene expression profiling using Affymetrix microarray analysis after siRNA-mediated silencing of Bach2 identified cell cycle and transcription regulation as the most significantly altered processes consistent with a function as transcription factor affecting proliferation.
Subject(s)
Aging/genetics , Basic-Leucine Zipper Transcription Factors/metabolism , DNA Damage/genetics , Animals , Basic-Leucine Zipper Transcription Factors/genetics , Biomarkers , Cell Survival/genetics , Gene Expression Regulation , HEK293 Cells , Humans , Mice , Models, Animal , NIH 3T3 Cells , Oligonucleotide Array Sequence Analysis , Radiation, Ionizing , Ultraviolet RaysABSTRACT
Huntington's disease (HD) is a dominant genetic neurodegenerative disorder. The pathology affects principally neurons in the basal ganglia circuits and terminates invariably in death. There is compelling necessity for safe and effective therapeutic strategies to arrest, or even retard the progression of the pathogenesis. Recent findings indicate the autophagy-lysosome systems as appealing targets for pharmacological intervention. Autophagy exerts a critical role in controlling neuronal protein homeostasis, which is perturbed in HD, and is compromised in the pathogenesis of several neurodegenerative diseases. Type 2 transglutaminase (TG2) plays an important role both in apoptosis and autophagy regulation, and accumulates at high levels in cells under stressful conditions. TG2 inhibition, achieved either via drug treatments or genetic approaches, has been shown to be beneficial for the treatment of HD in animal models. In this review we will discuss the relevance of TG2 to the pathogenesis of HD, in an effort to define novel therapeutic avenues.
Subject(s)
GTP-Binding Proteins/physiology , Huntington Disease/enzymology , Transglutaminases/physiology , Animals , Autophagy/physiology , Cell Death/physiology , GTP-Binding Proteins/antagonists & inhibitors , Humans , Huntington Disease/drug therapy , Mice , Mitochondria/enzymology , Mitochondria/physiology , Models, Animal , Protein Glutamine gamma Glutamyltransferase 2 , Transglutaminases/antagonists & inhibitorsSubject(s)
Adenosine Triphosphate/biosynthesis , GTP-Binding Proteins/metabolism , Myocardial Reperfusion Injury/prevention & control , Myocytes, Cardiac/metabolism , Transglutaminases/metabolism , Animals , GTP-Binding Proteins/deficiency , GTP-Binding Proteins/genetics , In Vitro Techniques , Male , Membrane Potentials , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitochondria, Heart/metabolism , Myocardial Reperfusion Injury/metabolism , Myocytes, Cardiac/cytology , Protein Glutamine gamma Glutamyltransferase 2 , Receptors, Adrenergic, alpha-1/deficiency , Receptors, Adrenergic, alpha-1/genetics , Receptors, Adrenergic, alpha-1/metabolism , Signal Transduction , Transglutaminases/deficiency , Transglutaminases/geneticsSubject(s)
Apoptosis , Autophagy , Protein Precursors/physiology , Thymosin/analogs & derivatives , Thymosin/physiology , Animals , Caspases/metabolism , Cell Death , Humans , Huntington Disease/metabolism , Huntington Disease/pathology , Mice , Mitochondria/metabolism , Models, Biological , Neurons/metabolism , Neurons/pathology , Signal TransductionABSTRACT
By crossing Huntington's disease (HD) R6/1 transgenic mice with 'tissue' transglutaminase (TG2) knock-out mice, we have demonstrated that this multifunctional enzyme plays an important role in the neuronal death characterising this disorder in vivo. In fact, a large reduction in cell death is observed in R6/1, TG2(-/-) compared with R6/1 transgenic mice. In addition, we have shown that the formation of neuronal intranuclear inclusions (NII) is potentiated in absence of the 'tissue' transglutaminase. These phenomena are paralleled by a significant improvement both in motor performances and survival of R6/1, TG2(-/-) versus R6/1 mice. Taken together these findings suggest an important role for tissue transglutaminase in the regulation of neuronal cell death occurring in Huntington's disease.
