The transcriptional co-activator PGC-1␣ and the NAD ؉ -dependent deacetylase SIRT1 are considered important inducers of mitochondrial biogenesis because in the nucleus they regulate transcription of nucleus-encoded mitochondrial genes. We demonstrate that PGC-1␣ and SIRT1 are also present inside mitochondria and are in close proximity to mtDNA. The mitochondrial proteome includes ϳ1500 proteins among which only 13 are expressed by the mitochondrial genome (1). Accordingly, abundance, morphology, and functional properties of mitochondria are finely controlled at the nuclear genome, where an interconnected network of transcription factors regulates the expression of mitochondrial proteins, including those that control replication and transcription of the mitochondrial genome. The same transcriptional network senses alterations of energetic homeostasis of the cells and is able to adapt mitochondrial function and biogenesis in response to nutrient availability and energy demand (2, 3).In the last few years, the NAD ϩ -dependent protein deacetylase sirtuin 1 (SIRT1) is emerging as a crucial regulator of mitochondrial biogenesis (4). Mammalian SIRT1 is homologous to the yeast silent information regulator 2 (Sir2) and belongs to class III histone deacetylates (HDACIII) that include other sirtuins (SIRT2-7) with specific subcellular localization and protein substrates. The dependence of SIRT1 on NAD ϩ links its enzymatic activity directly to the energy status of the cell, thus being activated in conditions of nutrient deprivation such as fasting and caloric restriction (5). Besides its role in modification of chromatin and silencing of transcription, by heterochromatin formation through histones modification, SIRT1 targets a wide range of transcriptional factors, including protein 53 (p53) and forkhead box O (FoxO) (6, 7). However, by regulating peroxisome proliferator-activated receptor ␥ co-activator 1␣ (PGC-1␣), through a functional protein-protein interaction, SIRT1 influences the activity of one of the most versatile metabolic transcriptional co-activators of genes involved in energy metabolism (5,8,9). In particular, PGC-1␣ represents an upstream inducer of genes of mitochondrial metabolism by positively affecting the activity of some hormone nuclear receptors (peroxisome proliferator-activated receptor ␥ and estrogen-related receptor ␣) and nuclear transcription factors (NRF-1,2) (10). NRF-1 is a downstream effector of SIRT1/PGC-1␣ and activates the expression of OxPhos components, mitochondrial transporters, and ribosomal proteins. Additionally, NRF-1 regulates the activation of the Tfam, Tfb1m, and Tfb2m promoters and indirectly affects the expression of Cox genes, Glut4 and PGC-1␣ itself (1). Importantly, the coordination of the two genomes seems to be exclusively achieved by the nucleus-encoded proteins TFAM, 2 TFB1M, and TFB2M, among which the mitochondrial transcription factor A seems to play a central role being essential for transcription, replication, and maintenance of mtDNA (11,12). mtDNA is packaged i...

Impairment of redox homeostasis has been extensively associated with obesity, as a consequence of the chronic inflammatory state present in overweight subjects. Deregulation of glutathione (GSH), the most important non-enzymatic intracellular anti-oxidant, induces insulin resistance in mature adipocytes, but data are lacking about its effects on adipogenesis. In this report we demonstrate that during adipogenesis of 3T3-L1 cells the GSH/GSSG ratio decreases, shifting redox status towards oxidizing conditions. Moreover, we demonstrate that inhibition of GSH synthesis, obtained by treatment with L-buthionine-sulfoximine (BSO), enhances C/EBPβ LAP/LIP ratio and PPARγ expression during mitotic clonal expansion (MCE) stimulating adipogenesis. On the contrary, GSH ethyl ester (GSHest) supplementation completely abrogates this process also in the presence of BSO. GSH decrement during the first 24 h of adipogenesis is sufficient to induce higher triglyceride accumulation in differentiated adipocytes with respect to control, whereas GSHest treatment inhibits lipid droplets formation. We further demonstrate that Resveratrol (RV) could exert anti-adipogenic properties also by increasing GSH content through γ-glutamyl-cysteine ligase (GCL) induction. Overall data indicate that in pre-adipocytes the decrease of GSH accelerates adipogenesis, suggesting that the use of agents able to maintain GSH redox status in adipose tissue, such as RV, could be promising in stopping the lipogenic loop of obesity.

Superoxide dismutases (SODs) represent the first line of defense against oxidative stress, which is considered an essential factor in several neurodegenerative diseases and aging. We investigated the role of the copper,zinc superoxide dismutase (SOD1) in the maintenance of intracellular redox homeostasis by analyzing the early effects of SOD1 down-regulation in SH-SY5Y neuroblastoma cells. Through the use of small interference RNA, SOD1 was efficiently down-regulated at 48 h after transfection without any significant effect on cell viability. The steady-state concentration of superoxide was significantly increased after 12 h, when SOD1 was only slightly decreased, and progressively returned to values close to those observed in control cells. The superoxide increase was buffered by the enhanced levels of antioxidant glutathione (GSH); however, GSH increase was not sufficient to avoid damage to proteins in terms of carbonyls. GSH-depleting agents, such as BSO or diamide, further increased protein damage and committed SOD1 deficient cells to death, confirming the pivotal role played by this antioxidant. Although SOD1 declined mostly in the cytosolic compartment, mitochondria were significantly affected with impairment of the mitochondrial transmembrane potential and a decrease in ATP production. Together with these effects carbonylation of mitochondrial proteins was detected and in particular a consistent carbonylation and decrease of the antiapoptotic protein Bcl-2. These conditions induced a high susceptibility of SOD1-depleted cells to treatment with the mitochondrial reactive oxygen species producing agent rotenone. Overall, the results demonstrate that loss of SOD1 leads to severe damage of mitochondria, suggesting an important biological role for this enzyme in the preservation of mitochondrial homeostasis.

Garlic organosulphur compounds have been successfully used as redox anti-proliferative agents. In this work, we dissect the effects of diallyl disulphide (DADS) focusing on the events upstream of cell cycle arrest and apoptosis induced in neuroblastoma SH-SY5Y cells. We demonstrate that DADS is able to cause early morphological changes, cytoskeleton oxidation, microfilaments reduction and depolymerization of microtubules. These events are attenuated in cells stably overexpressing the antioxidant enzyme SOD1, suggesting that superoxide plays a crucial role in destabilizing cytoskeleton. Moreover, we evidence that the main microtubules-associated protein Tau undergoes PP1-mediated dephosphorylation as demonstrated by treatment with okadaic acid as well as by immunoreaction with anti-Tau-1 antibody, which specifically recognizes its dephosphorylated forms. Tau dephosphorylation is inhibited by the two-electron reductants NAC and GSH ester but not by SOD1. The inability of DADS to induce apoptosis in neuroblastoma-differentiated cells gives emphasis to the anti-proliferative activity of DADS, which can be regarded as a promising potent anti-neuroblastoma drug by virtue of its widespread cytoskeleton disrupting action on proliferating cells.

We have studied the effects of superoxide production after Cu,Zn superoxide dismutase (SOD1) down-regulation by RNA interference. We demonstrated that SOD1 depletion induced, only in neuroblastoma cells, a decrease in actin and beta-tubulin content and accumulation of neurofilament light chain and Tau proteins. Alterations of cell morphology and the microfilament network were also observed, together with the up-regulation of the Cdk5/p35 pathway, which is involved in the regulation of actin polymerization. The decrease of filamentous actin was transient and was recovered through the activation of p38/Hsp27 MAPK pathway, as well as after treatment with N-acetyl-L-cysteine. The importance of p38 in the recovery of cytoskeleton was confirmed by experiments carried out in the presence of its inhibitor SB203580, which induced cell death. Our data demonstrate that SOD1 is essential for the preservation of cytoskeleton integrity, by maintaining physiological concentration of reactive oxygen species and inhibiting the activation of the neuronal specific Cdk5/p35 pathway.