Publications by authors named "Ulises Ahumada-Castro"

12 Publications

  • Page 1 of 1

transcript variant 2 is a marker of aging and cellular senescence.

Aging (Albany NY) 2021 05 25;13(10):13380-13392. Epub 2021 May 25.

Buck Institute for Research on Aging, Novato, CA 94945, USA.

Cellular senescence is a cell fate response characterized by a permanent cell cycle arrest driven primarily the by cell cycle inhibitor and tumor suppressor proteins p16 and p21. In mice, the p21 encoding locus, , is known to generate two transcripts that produce identical proteins, but one of these transcript variants is poorly characterized. We show that the transcript variant 2, but not the better-studied variant 1, is selectively elevated during natural aging across multiple mouse tissues. Importantly, mouse cells induced to senescence in culture by genotoxic stress (ionizing radiation or doxorubicin) upregulated both transcripts, but with different temporal dynamics: variant 1 responded nearly immediately to genotoxic stress, whereas variant 2 increased much more slowly as cells acquired senescent characteristics. Upon treating mice systemically with doxorubicin, which induces widespread cellular senescence , variant 2 increased to a larger extent than variant 1. Variant 2 levels were also more sensitive to the senolytic drug ABT-263 in naturally aged mice. Thus, variant 2 is a novel and more sensitive marker than variant 1 or total p21 protein for assessing the senescent cell burden and clearance in mice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/aging.203110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8202863PMC
May 2021

In the Right Place at the Right Time: Regulation of Cell Metabolism by IP3R-Mediated Inter-Organelle Ca Fluxes.

Front Cell Dev Biol 2021 2;9:629522. Epub 2021 Mar 2.

Geroscience Center for Brain Health and Metabolism, Center for Integrative Biology, Faculty of Sciences, Universidad Mayor, Santiago, Chile.

In the last few years, metabolism has been shown to be controlled by cross-organelle communication. The relationship between the endoplasmic reticulum and mitochondria/lysosomes is the most studied; here, inositol 1,4,5-triphosphate (IP3) receptor (IP3R)-mediated calcium (Ca) release plays a central role. Recent evidence suggests that IP3R isoforms participate in synthesis and degradation pathways. This minireview will summarize the current findings in this area, emphasizing the critical role of Ca communication on organelle function as well as catabolism and anabolism, particularly in cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fcell.2021.629522DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7960657PMC
March 2021

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).

Autophagy 2021 Jan 8;17(1):1-382. Epub 2021 Feb 8.

University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/15548627.2020.1797280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996087PMC
January 2021

Ca transfer to mitochondria: a spark of life in unexpected conditions.

Mol Cell Oncol 2021 15;8(1):1839341. Epub 2020 Dec 15.

Center for Integrative Biology, Faculty of Sciences, Universidad Mayor, Santiago, Chile.

The inositol 1,4,5-triphosphate receptor (InsP3R)-mediated calcium (Ca) transfer to mitochondria is important to maintain mitochondrial respiration and bioenergetics in normal and cancer cells, even though cancer cells have defective oxidative phosphorylation (OXPHOS). Here, we discuss how tumor mitochondria could become a feasible therapeutic target to treat tumors that depend on reductive carboxylation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/23723556.2020.1839341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7849767PMC
December 2020

Inhibition of InsP3R with Xestospongin B Reduces Mitochondrial Respiration and Induces Selective Cell Death in T Cell Acute Lymphoblastic Leukemia Cells.

Int J Mol Sci 2021 Jan 11;22(2). Epub 2021 Jan 11.

Center for Integrative Biology, Faculty of Sciences, Universidad Mayor, Santiago 8580745, Chile.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy whose chemoresistance and relapse persist as a problem despite significant advances in its chemotherapeutic treatments. Mitochondrial metabolism has emerged as an interesting therapeutic target given its essential role in maintaining bioenergetic and metabolic homeostasis. T-ALL cells are characterized by high levels of mitochondrial respiration, making them suitable for this type of intervention. Mitochondrial function is sustained by a constitutive transfer of calcium from the endoplasmic reticulum to mitochondria through the inositol 1,4,5-trisphosphate receptor (InsP3R), making T-ALL cells vulnerable to its inhibition. Here, we determine the bioenergetic profile of the T-ALL cell lines CCRF-CEM and Jurkat and evaluate their sensitivity to InsP3R inhibition with the specific inhibitor, Xestospongin B (XeB). Our results show that T-ALL cell lines exhibit higher mitochondrial respiration than non-malignant cells, which is blunted by the inhibition of the InsP3R. Prolonged treatment with XeB causes T-ALL cell death without affecting the normal counterpart. Moreover, the combination of XeB and glucocorticoids significantly enhanced cell death in the CCRF-CEM cells. The inhibition of InsP3R with XeB rises as a potential therapeutic alternative for the treatment of T-ALL.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms22020651DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7827595PMC
January 2021

Cancer cells with defective oxidative phosphorylation require endoplasmic reticulum-to-mitochondria Ca transfer for survival.

Sci Signal 2020 07 14;13(640). Epub 2020 Jul 14.

Departments of Physiology and Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

Spontaneous Ca signaling from the InsPR intracellular Ca release channel to mitochondria is essential for optimal oxidative phosphorylation (OXPHOS) and ATP production. In cells with defective OXPHOS, reductive carboxylation replaces oxidative metabolism to maintain amounts of reducing equivalents and metabolic precursors. To investigate the role of mitochondrial Ca uptake in regulating bioenergetics in these cells, we used OXPHOS-competent and OXPHOS-defective cells. Inhibition of InsPR activity or mitochondrial Ca uptake increased α-ketoglutarate (αKG) abundance and the NAD/NADH ratio, indicating that constitutive endoplasmic reticulum (ER)-to-mitochondria Ca transfer promoted optimal αKG dehydrogenase (αKGDH) activity. Reducing mitochondrial Ca inhibited αKGDH activity and increased NAD, which induced SIRT1-dependent autophagy in both OXPHOS-competent and OXPHOS-defective cells. Whereas autophagic flux in OXPHOS-competent cells promoted cell survival, it was impaired in OXPHOS-defective cells because of inhibition of autophagosome-lysosome fusion. Inhibition of αKGDH and impaired autophagic flux in OXPHOS-defective cells resulted in pronounced cell death in response to interruption of constitutive flux of Ca from ER to mitochondria. These results demonstrate that mitochondria play a fundamental role in maintaining bioenergetic homeostasis of both OXPHOS-competent and OXPHOS-defective cells, with Ca regulation of αKGDH activity playing a pivotal role. Inhibition of ER-to-mitochondria Ca transfer may represent a general therapeutic strategy against cancer cells regardless of their OXPHOS status.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/scisignal.aay1212DOI Listing
July 2020

Concerted Action of AMPK and Sirtuin-1 Induces Mitochondrial Fragmentation Upon Inhibition of Ca Transfer to Mitochondria.

Front Cell Dev Biol 2020 25;8:378. Epub 2020 May 25.

Center for Integrative Biology, Faculty of Sciences, Universidad Mayor, Santiago, Chile.

Mitochondria are highly dynamic organelles constantly undergoing fusion and fission. Ca regulates many aspects of mitochondrial physiology by modulating the activity of several mitochondrial proteins. We previously showed that inhibition of constitutive IP3R-mediated Ca transfer to the mitochondria leads to a metabolic cellular stress and eventually cell death. Here, we show that the decline of mitochondrial function generated by a lack of Ca transfer induces a DRP-1 independent mitochondrial fragmentation that at an early time is mediated by an increase in the NAD+/NADH ratio and activation of SIRT1. Subsequently, AMPK predominates and drives the fragmentation. SIRT1 activation leads to the deacetylation of cortactin, favoring actin polymerization, and mitochondrial fragmentation. Knockdown of cortactin or inhibition of actin polymerization prevents fragmentation. These data reveal SIRT1 as a new player in the regulation of mitochondrial fragmentation induced by metabolic/bioenergetic stress through regulating the actin cytoskeleton.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fcell.2020.00378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261923PMC
May 2020

MTOR-independent autophagy induced by interrupted endoplasmic reticulum-mitochondrial Ca communication: a dead end in cancer cells.

Autophagy 2019 02 29;15(2):358-361. Epub 2018 Oct 29.

b Geroscience Center for Brain Health and Metabolism , Santiago , Chile.

The interruption of endoplasmic reticulum (ER)-mitochondrial Ca communication induces a bioenergetic crisis characterized by an increase of MTOR-independent AMPK-dependent macroautophagic/autophagic flux, which is not sufficient to reestablish the metabolic and energetic homeostasis in cancer cells. Here, we propose that upon ER-mitochondrial Ca transfer inhibition, AMPK present at the mitochondria-associated membranes (MAMs) activate localized autophagy via BECN1 (beclin 1). This local response could prevent the proper interorganelle communication that would allow the autophagy-derived metabolites to reach the necessary anabolic pathways to maintain mitochondrial function and cellular homeostasis. Abbreviations: 3MA: 3-methyladenine; ADP: adenosine diphosphate; AMP: adenosine monophosphate; ATG13: autophagy related 13; ATG14: autophagy related 14; ATP: adenosine triphosphate; BECN1: beclin 1; Ca: calcium; DNA: deoxyribonucleic acid; ER: endoplasmic reticulum; GEF: guanine nucleotide exchange factor; ITPR: inositol 1,4,5-trisphosphate receptor; MAMs: mitochondria-associated membranes; MCU: mitochondrial calcium uniporter; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; OCR: oxygen consumption rate; PtdIns3K: class III phosphatidylinositol 3-kinase; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RPTOR: regulatory associated protein of MTOR complex 1; RYRs: ryanodine receptors; STK11/LKB1: serine/threonine kinase 11; TCA: tricarboxylic acid; TSC2: TSC complex subunit 2; ULK1: unc-51 like autophagy activating kinase 1; V-ATPase: vacuolar-type H-ATPase; VDAC: voltage dependent anion channel; XeB: xestospongin B.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/15548627.2018.1537769DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6333456PMC
February 2019

FR58P1a; a new uncoupler of OXPHOS that inhibits migration in triple-negative breast cancer cells via Sirt1/AMPK/β1-integrin pathway.

Sci Rep 2018 09 4;8(1):13190. Epub 2018 Sep 4.

Instituto de Química de Recursos Naturales and Programa de Investigación Asociativa en Cáncer Gástrico, Universidad de Talca, casilla 747, Talca, Chile.

Highly malignant triple-negative breast cancer (TNBC) cells rely mostly on glycolysis to maintain cellular homeostasis; however, mitochondria are still required for migration and metastasis. Taking advantage of the metabolic flexibility of TNBC MDA-MB-231 cells to generate subpopulations with glycolytic or oxidative phenotypes, we screened phenolic compounds containing an ortho-carbonyl group with mitochondrial activity and identified a bromoalkyl-ester of hydroquinone named FR58P1a, as a mitochondrial metabolism-affecting compound that uncouples OXPHOS through a protonophoric mechanism. In contrast to well-known protonophore uncoupler FCCP, FR58P1a does not depolarize the plasma membrane and its effect on the mitochondrial membrane potential and bioenergetics is moderate suggesting a mild uncoupling of OXPHOS. FR58P1a activates AMPK in a Sirt1-dependent fashion. Although the activation of Sirt1/AMPK axis by FR58P1a has a cyto-protective role, selectively inhibits fibronectin-dependent adhesion and migration in TNBC cells but not in non-tumoral MCF10A cells by decreasing β1-integrin at the cell surface. Prolonged exposure to FR58P1a triggers a metabolic reprograming in TNBC cells characterized by down-regulation of OXPHOS-related genes that promote cell survival but comprise their ability to migrate. Taken together, our results show that TNBC cell migration is susceptible to mitochondrial alterations induced by small molecules as FR58P1a, which may have therapeutic implications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-31367-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6123471PMC
September 2018

Mitochondria and Calcium Regulation as Basis of Neurodegeneration Associated With Aging.

Front Neurosci 2018 13;12:470. Epub 2018 Jul 13.

Geroscience Center for Brain Health and Metabolism, Santiago, Chile.

Age is the main risk factor for the onset of neurodegenerative diseases. A decline of mitochondrial function has been observed in several age-dependent neurodegenerative diseases and may be a major contributing factor in their progression. Recent findings have shown that mitochondrial fitness is tightly regulated by Ca signals, which are altered long before the onset of measurable histopathology hallmarks or cognitive deficits in several neurodegenerative diseases including Alzheimer's disease (AD), the most frequent cause of dementia. The transfer of Ca from the endoplasmic reticulum (ER) to the mitochondria, facilitated by the presence of mitochondria-associated membranes (MAMs), is essential for several physiological mitochondrial functions such as respiration. Ca transfer to mitochondria must be finely regulated because excess Ca will disturb oxidative phosphorylation (OXPHOS), thereby increasing the generation of reactive oxygen species (ROS) that leads to cellular damage observed in both aging and neurodegenerative diseases. In addition, excess Ca and ROS trigger the opening of the mitochondrial transition pore mPTP, leading to loss of mitochondrial function and cell death. mPTP opening probably increases with age and its activity has been associated with several neurodegenerative diseases. As Ca seems to be the initiator of the mitochondrial failure that contributes to the synaptic deficit observed during aging and neurodegeneration, in this review, we aim to look at current evidence for mitochondrial dysfunction caused by Ca miscommunication in neuronal models of neurodegenerative disorders related to aging, with special emphasis on AD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fnins.2018.00470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6053519PMC
July 2018

Autophagy protects against neural cell death induced by piperidine alkaloids present in Prosopis juliflora (Mesquite).

An Acad Bras Cienc 2017 Jan-Mar;89(1):247-261

Laboratório de Neuroquímica e Biologia Celular, Avenida Reitor Miguel Calmon, s/n, Universidade Federal da Bahia, Instituto de Ciências da Saúde, Departamento de Bioquímica e Biofísica, 40110-100 Salvador, BA, Brazil.

Prosopis juliflora is a shrub that has been used to feed animals and humans. However, a synergistic action of piperidine alkaloids has been suggested to be responsible for neurotoxic damage observed in animals. We investigated the involvement of programmed cell death (PCD) and autophagy on the mechanism of cell death induced by a total extract (TAE) of alkaloids and fraction (F32) from P. juliflora leaves composed majoritary of juliprosopine in a model of neuron/glial cell co-culture. We saw that TAE (30 µg/mL) and F32 (7.5 µg/mL) induced reduction in ATP levels and changes in mitochondrial membrane potential at 12 h exposure. Moreover, TAE and F32 induced caspase-9 activation, nuclear condensation and neuronal death at 16 h exposure. After 4 h, they induced autophagy characterized by decreases of P62 protein level, increase of LC3II expression and increase in number of GFP-LC3 cells. Interestingly, we demonstrated that inhibition of autophagy by bafilomycin and vinblastine increased the cell death induced by TAE and autophagy induced by serum deprivation and rapamycin reduced cell death induced by F32 at 24 h. These results indicate that the mechanism neural cell death induced by these alkaloids involves PCD via caspase-9 activation and autophagy, which seems to be an important protective mechanism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1590/0001-3765201720160477DOI Listing
March 2018

One-electron reduction of 6-hydroxydopamine quinone is essential in 6-hydroxydopamine neurotoxicity.

Neurotox Res 2013 Jul 6;24(1):94-101. Epub 2013 Feb 6.

Program of Molecular and Clinical Pharmacology, ICBM, Faculty of Medicine, University of Chile, Independencia 1027, Santiago, Chile.

6-Hydroxydamine has widely been used as neurotoxin in preclinical studies related on the neurodegenerative process of dopaminergic neurons in Parkinson's disease based on its ability to be neurotoxic as a consequence of free radical formation during its auto-oxidation to topaminequinone. We report that 50-µM 6-hydroxydopamine is not neurotoxic in RCSN-3 cells derived from substantia nigra incubated during 24 h contrasting with a significant sixfold increase in cell death (16 ± 2 %; P < 0.001) was observed in RCSN-3NQ7 cells expressing a siRNA against DT-diaphorase that silence the enzyme expression. To observe a significant cell death in RCSN-3 cells induced by 6-hydroxydopamine (24 ± 1 %; P < 0.01), we have to increase the concentration to 250 μm while a 45 ± 2 % cell death (P < 0.001) was observed at this concentration in RCSN-3NQ7 cells. The cell death induced by 6-hydroxydopamine in RCSN-3NQ7 cells was accompanied with a (i) significant increase in oxygen consumption (P < 0.01), (ii) depletion of reduced glutathione and (iii) a significant decrease in ATP level (P < 0.05) in comparison with RCSN-3 cells. In conclusion, our results suggest that one-electron reduction of 6-hydroxydopamine quinone seems to be the main reaction responsible for 6-hydroxydopamine neurotoxic effects in dopaminergic neurons and DT-diaphorase seems to play an important neuroprotective role by preventing one-electron reduction of topaminequinone.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12640-013-9382-7DOI Listing
July 2013