on International journal of molecular sciences
by Caiazza C, D'Agostino M, Passaro F, Faicchia D, Mallardo M, Paladino S, Pierantoni GM, Tramontano D
Acute administration of a high level of extracellular citrate displays an anti-proliferative effect on both in vitro and in vivo models. However, the long-term effect of citrate treatment has not been investigated yet. Here, we address this question in PC3 cells, a prostate-cancer-derived cell line. Acute administration of high levels of extracellular citrate impaired cell adhesion and inhibited the proliferation of PC3 cells, but surviving cells adapted to grow in the chronic presence of 20 mM citrate. Citrate-resistant PC3 cells are significantly less glycolytic than control cells. Moreover, they overexpress short-form, citrate-insensitive phosphofructokinase 1 (PFK1) together with full-length PFK1. In addition, they show traits of mesenchymal-epithelial transition: an increase in E-cadherin and a decrease in vimentin. In comparison with PC3 cells, citrate-resistant cells display morphological changes that involve both microtubule and microfilament organization. This was accompanied by changes in homeostasis and the organization of intracellular organelles. Thus, the mitochondrial network appears fragmented, the Golgi complex is scattered, and the lysosomal compartment is enlarged. Interestingly, citrate-resistant cells produce less total ROS but accumulate more mitochondrial ROS than control cells. Consistently, in citrate-resistant cells, the autophagic pathway is upregulated, possibly sustaining their survival. In conclusion, chronic administration of citrate might select resistant cells, which could jeopardize the benefits of citrate anticancer treatment.
on Redox biology
by Sagliocchi S, Cicatiello AG, Di Cicco E, Ambrosio R, Miro C, Di Girolamo D, Nappi A, Mancino G, De Stefano MA, Luongo C, Raia M, Ogawa-Wong AN, Zavacki AM, Paladino S, Salvatore D, Dentice M
Thyroid hormone (TH) is a key metabolic regulator that acts by coordinating short- and long-term energy needs. Accordingly, significant metabolic changes are observed depending on thyroid status. Although it is established that hyperthyroidism augments basal energy consumption, thus resulting in an enhanced metabolic state, the net effects on cellular respiration and generation of reactive oxygen species (ROS) remain unclear. To elucidate the effects of augmented TH signal in muscle cells, we generated a doxycycline-inducible cell line in which the expression of the TH-activating enzyme, type 2 deiodinase (D2), is reversibly turned on by the "Tet-ON" system. Interestingly, increased intracellular TH caused a net shift from oxidative phosphorylation to glycolysis and a consequent increase in the extracellular acidification rate. As a result, mitochondrial ROS production, and both the basal and doxorubicin-induced production of cellular ROS were reduced. Importantly, the expression of a set of antioxidant genes was up-regulated, and, among them, the mitochondrial scavenger Sod2 was specifically induced at transcriptional level by D2-mediated TH activation. Finally, we observed that attenuation of oxidative stress and increased levels of SOD2 are key elements of the differentiating cascade triggered by TH and D2, thereby establishing that D2 is essential in coordinating metabolic reprogramming of myocytes during myogenic differentiation. In conclusion, our findings indicate that TH plays a key role in oxidative stress dynamics by regulating ROS generation. Our novel finding that TH and its intracellular metabolism act as mitochondrial detoxifying agents sheds new light on metabolic processes relevant to muscle physiology.
on The Journal of cell biology
by Venditti R, Rega LR, Masone MC, Santoro M, Polishchuk E, Sarnataro D, Paladino S, D'Auria S, Varriale A, Olkkonen VM, Di Tullio G, Polishchuk R, De Matteis MA
ER-TGN contact sites (ERTGoCS) have been visualized by electron microscopy, but their location in the crowded perinuclear area has hampered their analysis via optical microscopy as well as their mechanistic study. To overcome these limits we developed a FRET-based approach and screened several candidates to search for molecular determinants of the ERTGoCS. These included the ER membrane proteins VAPA and VAPB and lipid transfer proteins possessing dual (ER and TGN) targeting motifs that have been hypothesized to contribute to the maintenance of ERTGoCS, such as the ceramide transfer protein CERT and several members of the oxysterol binding proteins. We found that VAP proteins, OSBP1, ORP9, and ORP10 are required, with OSBP1 playing a redundant role with ORP9, which does not involve its lipid transfer activity, and ORP10 being required due to its ability to transfer phosphatidylserine to the TGN. Our results indicate that both structural tethers and a proper lipid composition are needed for ERTGoCS integrity.
on Journal of cellular physiology
by Di Cristo F, Finicelli M, Digilio FA, Paladino S, Valentino A, Scialò F, D'Apolito M, Saturnino C, Galderisi U, Giordano A, Melone MAB, Peluso G
Mitochondrial dysfunction seems to play a fundamental role in the pathogenesis of neurodegeneration in Huntington's disease (HD). We assessed possible neuroprotective actions of meldonium, a small molecule affecting mitochondrial fuel metabolism, in in vitro and in vivo HD models. We found that meldonium was able to prevent cytotoxicity induced by serum deprivation, to reduce the accumulation of mutated huntingtin (mHtt) aggregates, and to upregulate the expression of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) in mHTT-expressing cells. The PGC-1α increase was accompanied by the increment of mitochondrial mass and by the rebalancing of mitochondrial dynamics with a promotion of the mitochondrial fusion. Meldonium-induced PGC-1α significantly alleviated motor dysfunction and prolonged the survival of a transgenic HD Drosophila model in which mHtt expression in the nervous system led to progressive motor performance deficits. Our study strongly suggests that PGC-1α, as a master coregulator of mitochondrial biogenesis, energy homeostasis, and antioxidant defense, is a potential therapeutic target in HD.
on Critical reviews in biochemistry and molecular biology
by Lebreton S, Zurzolo C, Paladino S
Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are a class of proteins attached to the extracellular leaflet of the plasma membrane via a post-translational modification, the glycolipid anchor. The presence of both glycolipid anchor and protein portion confers them unique features. GPI-APs are expressed in all eukaryotes, from fungi to plants and animals. They display very diverse functions ranging from enzymatic activity, signaling, cell adhesion, cell wall metabolism, neuritogenesis, and immune response. Likewise other plasma membrane proteins, the spatio-temporal organization of GPI-APs is critical for their biological activities in physiological conditions. In this review, we will summarize the latest findings on plasma membrane organization of GPI-APs and the mechanism of its regulation in different cell types. We will also examine the involvement of specific GPI-APs namely the prion protein PrP, the Folate Receptor alpha and the urokinase plasminogen activator receptor in human diseases focusing on neurodegenerative diseases and cancer.
on Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology
by Paladino S, Conte A, Caggiano R, Pierantoni GM, Faraonio R
A general hallmark of neurological diseases is the loss of redox homeostasis that triggers oxidative damages to biomolecules compromising neuronal function. Under physiological conditions the steady-state concentrations of reactive oxygen species (ROS) and reactive nitrogen species (RNS) are finely regulated for proper cellular functions. Reduced surveillance of endogenous antioxidant defenses and/or increased ROS/RNS production leads to oxidative stress with consequent alteration of physiological processes. Neuronal cells are particularly susceptible to ROS/RNS due to their biochemical composition. Overwhelming evidences indicate that nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-linked pathways are involved in protective mechanisms against oxidative stress by regulating antioxidant and phase II detoxifying genes. As such, Nrf2 deregulation has been linked to both aging and pathogenesis of many human chronic diseases, including neurodegenerative ones such as Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis. Nrf2 activity is tightly regulated by a fine balance between positive and negative modulators. A better understanding of the regulatory mechanisms underlying Nrf2 activity could help to develop novel therapeutic interventions to prevent, slow down or possibly reverse various pathological states. To this end, microRNAs (miRs) are attractive candidates because they are linked to intracellular redox status being regulated and, post-transcriptionally, regulating key components of ROS/RNS pathways, including Nrf2.
on Molecular therapy. Methods & clinical development
by De Pasquale V, Sarogni P, Pistorio V, Cerulo G, Paladino S, Pavone LM
Mucopolysaccharidoses (MPSs) are inherited metabolic diseases caused by the deficiency of lysosomal enzymes needed to catabolize glycosaminoglycans (GAGs). Four therapeutic options are currently considered: enzyme replacement therapy, substrate reduction therapy, gene therapy, and hematopoietic stem cell transplantation. However, while some of them exhibit limited clinical efficacy and require high costs, others are still in development. Therefore, alternative treatments for MPSs need to be explored. Here we describe an innovative therapeutic approach based on the use of a recombinant protein that is able to bind the excess of extracellular accumulated heparan sulfate (HS). We demonstrate that this protein is able to reduce lysosomal defects in primary fibroblasts from MPS I and MPS IIIB patients. We also show that, by masking the excess of extracellular accumulated HS in MPS fibroblasts, fibroblast growth factor (FGF) signal transduction can be positively modulated. We, therefore, suggest the use of a competitive binding molecule for HS in MPSs as an alternative strategy to prevent the detrimental extracellular substrate storage.
on Molecular medicine (Cambridge, Mass.)
by Izzo A, Mollo N, Nitti M, Paladino S, Calì G, Genesio R, Bonfiglio F, Cicatiello R, Barbato M, Sarnataro V, Conti A, Nitsch L
Trisomy of chromosome 21 (TS21) is the most common autosomal aneuploidy compatible with postnatal survival with a prevalence of 1 in 700 newborns. Its phenotype is highly complex with constant features, such as mental retardation, dysmorphic traits and hypotonia, and variable features including heart defects, susceptibility to Alzheimer's disease (AD), type 2 diabetes, obesity and immune disorders. Overexpression of genes on chromosome-21 (Hsa21) is responsible for the pathogenesis of Down syndrome (DS) phenotypic features either in a direct or in an indirect manner since many Hsa21 genes can affect the expression of other genes mapping to different chromosomes. Many of these genes are involved in mitochondrial function and energy conversion, and play a central role in the mitochondrial dysfunction and chronic oxidative stress, consistently observed in DS subjects.Recent studies highlight the deep interconnections between mitochondrial dysfunction and DS phenotype. In this short review we first provide a basic overview of mitochondrial phenotype in DS cells and tissues. We then discuss how specific Hsa21 genes may be involved in determining the disruption of mitochondrial DS phenotype and biogenesis. Finally we briefly focus on drugs that affect mitochondrial function and mitochondrial network suggesting possible therapeutic approaches to improve and/or prevent some aspects of the DS phenotype.
on Cell death & disease
by Fasano D, Parisi S, Pierantoni GM, De Rosa A, Picillo M, Amodio G, Pellecchia MT, Barone P, Moltedo O, Bonifati V, De Michele G, Nitsch L, Remondelli P, Criscuolo C, Paladino S
Recently, a new form of autosomal recessive early-onset parkinsonism (PARK20), due to mutations in the gene encoding the phosphoinositide phosphatase, Synaptojanin 1 (Synj1), has been reported. Several genes responsible for hereditary forms of Parkinson's disease are implicated in distinct steps of the endolysosomal pathway. However, the nature and the degree of endocytic membrane trafficking impairment in early-onset parkinsonism remains elusive. Here, we show that depletion of Synj1 causes drastic alterations of early endosomes, which become enlarged and more numerous, while it does not affect the morphology of late endosomes both in non-neuronal and neuronal cells. Moreover, Synj1 loss impairs the recycling of transferrin, while it does not alter the trafficking of the epidermal growth factor receptor. The ectopic expression of Synj1 restores the functions of early endosomes, and rescues these trafficking defects in depleted cells. Importantly, the same alterations of early endosomal compartments and trafficking defects occur in fibroblasts of PARK20 patients. Our data indicate that Synj1 plays a crucial role in regulating the homeostasis and functions of early endosomal compartments in different cell types, and highlight defective cellular pathways in PARK20. In addition, they strengthen the link between endosomal trafficking and Parkinson's disease.
on Pharmaceutical research
by Melone MAB, Dato C, Paladino S, Coppola C, Trebini C, Giordana MT, Perrone L
Oxidative stress is a hallmark of Alzheimer's Disease (AD) and promotes tau phosphorylation. Since Thioredoxin Interacting protein (TXNIP), the inhibitor of the anti-oxidant system of Thioredoxin, is up regulated in the hippocampus of AD patients, we investigated whether TXNIP plays a role in promoting tau phosphorylation and whether Verapamil, an inhibitor of TXNIP expression, prevents TXNIP downstream effects.