Ullas Kolthur-Seetharam

Control of AIF-mediated cell death by the functional interplay of SIRT1 and PARP-1 in response to DNA damage

Cell survival after genotoxic stress is determined by a counterbalance of pro- andanti-death factors. Sirtuins (SIRTs) are deacetylases that promote cell survival whereaspoly(ADP-ribose) polymerases (PARPs) can act both as survival and death inducingfactor and the two protein families are strictly dependent on NAD+ for their activities.Here we report that SIRT1 modulates PARP-1 activity upon DNA damage. Activation ofSIRT1 by resveratrol leads to reduced PARP-1 activity and there is a drastic increase inPAR synthesis in sirt1-null cells. The unbalanced regulation of PARP-1 in the absence ofSIRT1 results in AIF (apoptosis inducing factor)-mediated cell death. Our findingsestablish a functional link between the two NAD+-dependent enzyme systems andprovide a physiological interpretation for the mechanism of death in cells lacking SIRT1.

Sirt1 and mir‐9 expression is regulated during glucose‐stimulated insulin secretion in pancreatic β‐islets

MicroRNA mir‐9 is speculated to be involved in insulin secretion because of its ability to regulate exocytosis. Sirt1 is an NAD‐dependent protein deacetylase and a critical factor in the modulation of cellular responses to altered metabolic flux. It has also been shown recently to control insulin secretion from pancreatic β‐islets. However, little is known about the regulation of Sirt1 and mir‐9 levels in pancreatic β‐cells, particularly during glucose‐dependent insulin secretion. In this article, we report that mir‐9 and Sirt1 protein levels are actively regulated in vivo in β‐islets during glucose‐dependent insulin secretion. Our data also demonstrates that mir‐9 targets and regulates Sirt1 expression in insulin‐secreting cells. This targeting is relevant in pancreatic β‐islets, where we show a reduction in Sirt1 protein levels when mir‐9 expression is high during glucose‐dependent insulin secretion. This functional interplay between insulin secretion, mir‐9 and Sirt1 expression could be relevant in diabetes. It also highlights the crosstalk between an NAD‐dependent protein deacetylase and microRNA in pancreatic β‐cells.

dSir2 in the Adult Fat Body, but Not in Muscles, Regulates Life Span in a Diet-Dependent Manner

Sir2, an evolutionarily conserved NAD(+)-dependent deacetylase, has been implicated as a key factor in mediating organismal life span. However, recent contradictory findings have brought into question the role of Sir2 and its orthologs in regulating organismal longevity. In this study, we report that Drosophila Sir2 (dSir2) in the adult fat body regulates longevity in a diet-dependent manner. We used inducible Gal4 drivers to knock down and overexpress dSir2 in a tissue-specific manner. A diet-dependent life span phenotype of dSir2 perturbations (both knockdown and overexpression) in the fat body, but not muscles, negates the effects of background genetic mutations. In addition to providing clarity to the field, our study contrasts the ability of dSir2 in two metabolic tissues to affect longevity. We also show that dSir2 knockdown abrogates fat-body dFOXO-dependent life span extension. This report highlights the importance of the interplay between genetic factors and dietary inputs in determining organismal life spans.

The Histone Deacetylase SIRT1 Controls Male Fertility in Mice Through Regulation of Hypothalamic-Pituitary Gonadotropin Signaling

Abstract Sirtuins (SIRTs) are class-III NAD-dependent histone deacetylases (HDACs) that regulate various physiological processes. Inactivation of SIRT1 in the mouse leads to male sterility, but the molecular mechanisms responsible for this phenotype have not been determined. Here we show that fetal testis development appears normal in Sirt1−/− mice. In contrast, the first round of spermatogenesis arrests before the completion of meiosis with abundant apoptosis of pachytene spermatocytes, abnormal Leydig and Sertoli cell maturation, and strongly reduced intratesticular testosterone levels. We show that this phenotype is the consequence of diminished hypothalamic gonadotropin-releasing hormone expression and strongly reduced luteinizing hormone levels. Rather than having an intrinsic effect on male germ cells per se, our results show that SIRT1 regulates spermatogenesis at postnatal stages by controlling hypothalamus-pituitary gonadotropin (HPG) signaling. In addition to its well studied role in control of metabolism and energy homeostasis, our results thus reveal a novel and critical function of SIRT1 in controlling HPG signaling. This phenotype is more severe than those previously described using mice bred on different genetic backgrounds, and highlights the fact that SIRT1 function is strongly modified by other genetic loci.

NAD: a master regulator of transcription.

Cellular processes such as proliferation, differentiation and death are intrinsically dependent upon the redox status of a cell. Among other indicators of redox flux, cellular NAD(H) levels play a predominant role in transcriptional reprogramming. In addition to this, normal physiological functions of a cell are regulated in response to perturbations in NAD(H) levels (for example, due to alterations in diet/metabolism) to maintain homeostatic conditions. Cells achieve this homeostasis by reprogramming various components that include changes in chromatin structure and function (transcription). The interdependence of changes in gene expression and NAD(H) is evolutionarily conserved and is considered crucial for the survival of a species (by affecting reproductive capacity and longevity). Proteins that bind and/or use NAD(H) as a co-substrate (such as, CtBP and PARPs/Sirtuins respectively) are known to induce changes in chromatin structure and transcriptional profiles. In fact, their ability to sense perturbations in NAD(H) levels has been implicated in their roles in development, stress responses, metabolic homeostasis, reproduction and aging or age-related diseases. It is also becoming increasingly clear that both the levels/activities of these proteins and the availability of NAD(H) are equally important. Here we discuss the pivotal role of NAD(H) in controlling the functions of some of these proteins, the functional interplay between them and physiological implications during calorie restriction, energy homeostasis, circadian rhythm and aging.

Mitochondrial alterations and oxidative stress in an acute transient mouse model of muscle degeneration: Implications for muscular dystrophy and related muscle pathologies

Background: Human muscular dystrophies and inflammatory myopathies share common pathological events. Results: The cardiotoxin (CTX) model displayed acute and transient muscle degeneration and all the cellular events usually implicated in human muscle pathology. Conclusion: Mitochondrial alterations and oxidative stress significantly contribute to muscle pathogenesis. Significance: The CTX model is valuable in understanding the mechanistic and therapeutic paradigms of muscle pathology. Muscular dystrophies (MDs) and inflammatory myopathies (IMs) are debilitating skeletal muscle disorders characterized by common pathological events including myodegeneration and inflammation. However, an experimental model representing both muscle pathologies and displaying most of the distinctive markers has not been characterized. We investigated the cardiotoxin (CTX)-mediated transient acute mouse model of muscle degeneration and compared the cardinal features with human MDs and IMs. The CTX model displayed degeneration, apoptosis, inflammation, loss of sarcolemmal complexes, sarcolemmal disruption, and ultrastructural changes characteristic of human MDs and IMs. Cell death caused by CTX involved calcium influx and mitochondrial damage both in murine C2C12 muscle cells and in mice. Mitochondrial proteomic analysis at the initial phase of degeneration in the model detected lowered expression of 80 mitochondrial proteins including subunits of respiratory complexes, ATP machinery, fatty acid metabolism, and Krebs cycle, which further decreased in expression during the peak degenerative phase. The mass spectrometry (MS) data were supported by enzyme assays, Western blot, and histochemistry. The CTX model also displayed markers of oxidative stress and a lowered glutathione reduced/oxidized ratio (GSH/GSSG) similar to MDs, human myopathies, and neurogenic atrophies. MS analysis identified 6 unique oxidized proteins from Duchenne muscular dystrophy samples (n = 6) (versus controls; n = 6), including two mitochondrial proteins. Interestingly, these mitochondrial proteins were down-regulated in the CTX model thereby linking oxidative stress and mitochondrial dysfunction. We conclude that mitochondrial alterations and oxidative damage significantly contribute to CTX-mediated muscle pathology with implications for human muscle diseases.

Chromosome territories reposition during DNA damage-repair response

BackgroundLocal higher-order chromatin structure, dynamics and composition of the DNA are known to determine double-strand break frequencies and the efficiency of repair. However, how DNA damage response affects the spatial organization of chromosome territories is still unexplored.ResultsOur report investigates the effect of DNA damage on the spatial organization of chromosome territories within interphase nuclei of human cells. We show that DNA damage induces a large-scale spatial repositioning of chromosome territories that are relatively gene dense. This response is dose dependent, and involves territories moving from the nuclear interior to the periphery and vice versa. Furthermore, we have found that chromosome territory repositioning is contingent upon double-strand break recognition and damage sensing. Importantly, our results suggest that this is a reversible process where, following repair, chromosome territories re-occupy positions similar to those in undamaged control cells.ConclusionsThus, our report for the first time highlights DNA damage-dependent spatial reorganization of whole chromosomes, which might be an integral aspect of cellular damage response.

Specialization of the general transcriptional machinery in male germ cells

In adult animals, spermatogenesis involves a continuous differentiation of the spermatogonial stem and progenitor cell population into mature sperm. A unique aspect of this developmental process is the germ cell-specific expression and function of paralogues of components of the general transcription machinery, notably subunits of TFIID. Genetic and biochemical studies show that these paralogues play critical, but mechanistically distinct roles in Drosophila and mouse spermatogenesis.

Fat Body dSir2 Regulates Muscle Mitochondrial Physiology and Energy Homeostasis Nonautonomously and Mimics the Autonomous Functions of dSir2 in Muscles

ABSTRACT Sir2 is an evolutionarily conserved NAD+-dependent deacetylase which has been shown to play a critical role in glucose and fat metabolism. In this study, we have perturbed Drosophila Sir2 (dSir2) expression, bidirectionally, in muscles and the fat body. We report that dSir2 plays a critical role in insulin signaling, glucose homeostasis, and mitochondrial functions. Importantly, we establish the nonautonomous functions of fat body dSir2 in regulating mitochondrial physiology and insulin signaling in muscles. We have identified a novel interplay between dSir2 and dFOXO at an organismal level, which involves Drosophila insulin-like peptide (dILP)-dependent insulin signaling. By genetic perturbations and metabolic rescue, we provide evidence to illustrate that fat body dSir2 mediates its effects on the muscles via free fatty acids (FFA) and dILPs (from the insulin-producing cells [IPCs]). In summary, we show that fat body dSir2 is a master regulator of organismal energy homeostasis and is required for maintaining the metabolic regulatory network across tissues.

14-3-3γ-mediated transport of plakoglobin to the cell border is required for the initiation of desmosome assembly in vitro and in vivo

ABSTRACT The regulation of cell–cell adhesion is important for the processes of tissue formation and morphogenesis. Here, we report that loss of 14-3-3&ggr; leads to a decrease in cell–cell adhesion and a defect in the transport of plakoglobin and other desmosomal proteins to the cell border in HCT116 cells and cells of the mouse testis. 14-3-3&ggr; binds to plakoglobin in a PKC&mgr;-dependent fashion, resulting in microtubule-dependent transport of plakoglobin to cell borders. Transport of plakoglobin to the border is dependent on the KIF5B–KLC1 complex. Knockdown of KIF5B in HCT116 cells, or in the mouse testis, results in a phenotype similar to that observed upon 14-3-3&ggr; knockdown. Our results suggest that loss of 14-3-3&ggr; leads to decreased desmosome formation and a decrease in cell–cell adhesion in vitro, and in the mouse testis in vivo, leading to defects in testis organization and spermatogenesis.