Inhibition of cytoplasmic p53 differentially modulates Ca 2+ signaling and cellular viability in young and aged striata Rodrigo Portes Ureshino a, ⁎, Yi-Te Hsu b , Lúcia Garcez do Carmo a , César Henrique Yokomizo c , Iseli Lourenço Nantes c , Soraya Soubhi Smaili a a Department of Pharmacology, Federal University of São Paulo, São Paulo, SP 04044-020, Brazil b Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA c Human and Natural Sciences Center, Federal University of ABC, Santo André, SP 09210-170, Brazil abstract article info Article history: Received 11 April 2014 Received in revised form 12 July 2014 Accepted 24 July 2014 Available online 30 July 2014 Section Editor: Werner Zwerschke Keywords: Aging p53 Striatum Glutamate Calcium Oxygen consumption The p53 protein, a transcription factor with many gene targets, can also trigger apoptosis in the cytoplasm. The disruption of cell homeostasis, such as Ca 2+ signaling and mitochondrial respiration, contributes to the loss of vi- ability and ultimately leads to cell death. However, the link between Ca 2+ signaling and p53 signaling remains unclear. During aging, there are alterations in cell physiology that are commonly associated with a reduced adap- tive stress response, thus increasing cell vulnerability. In this work, we examined the effects of a cytoplasmic p53 inhibitor (pifithrin μ) in the striatum of young and aged rats by evaluating Ca 2+ signaling, mitochondrial respi- ration, apoptotic protein expression, and tissue viability. Our results showed that pifithrin μ differentially modu- lated cytoplasmic and mitochondrial Ca 2+ in young and aged rats. Cytoplasmic p53 inhibition appeared to reduce the mitochondrial respiration rate in both groups. In addition, p53 phosphorylation and Bax protein levels were elevated upon cytoplasmic p53 inhibition and could contribute to the reduction of tissue viability. Following glu- tamate challenge, pifithrin μ improved cell viability in aged tissue, reduced reactive oxygen species (ROS) gener- ation, and reduced mitochondrial membrane potential (ΔΨm). Taken together, these results indicate that cytoplasmic p53 may have a special role in cell viability by influencing cellular Ca 2+ homeostasis and respiration and may produce differential effects in the striatum of young and aged rats. © 2014 Elsevier Inc. All rights reserved. 1. Introduction DNA damage is related to a variety of cellular responses and ulti- mately leads to cell death. This remarkable event is associated with neu- rodegenerative diseases, such as Alzheimer's disease (de la Monte et al., 1997) and Parkinson's disease (Biswas et al., 2005). During aging, accu- mulating DNA mutations can cause toxic effects to biological systems (Kirkwood, 1989; Vijg et al., 2005). p53 is known as the “supra-guard- ian” of DNA (Lane, 1992), as it has a central role in the detection of DNA damage, the repair of DNA, and the induction of apoptosis. p53 induces apoptosis via gene transcription or by the transactivation of cytoplasmic proteins. It has been reported that p53 co-localizes with mitochondria after cell stress (Dumont et al., 2003; Marchenko et al., 2000; Sansome et al., 2001) to promote apoptosis (Mihara et al., 2003). p53 can modulate Bax expression (Miyashita and Reed, 1995) and transactivation (Chipuk et al., 2004; Moll et al., 2005), leading to Bax oligomerization on mitochondrial outer membranes (Mikhailova et al., 2003) and promotes the release of proapoptotic factors, such as cyto- chrome c (Manon et al., 1997). The DNA-binding domain of p53 regulates its transcriptional activities and is also important for interactions with Bcl- 2 in the cytoplasm (Moll et al., 2005). PUMA, another member of the Bcl-2 family induced by p53, has been suggested to release p53 from the p53- Bcl-2 complex, allowing it to activate Bax (Chipuk et al., 2005). Pifithrin μ (PFTμ) is an inhibitor of cytoplasmic p53 that prevents the formation of p53–Bcl-2 and p53–Bcl-xL complexes, which may reduce Bax and Bak oligomerization in the mitochondrial outer membrane and protect cell against apoptosis (Strom et al., 2006). Furthermore, PFTμ also in- hibits heat shock 70 kDa protein (HSP 70) and disrupts interactions with targets such as p53 (Leu et al., 2009). The prevention of p53 trans- port to the mitochondria by HSP 70 disrupts its translocation and subse- quent effects. Increased oxidative DNA damage during aging may lead to altered gene expression, cause abnormal cell proliferation or apoptosis, and contribute to the cell senescence (Mullart et al., 1990). Mitochondria are the main sites of reactive oxygen species (ROS) generation. ROS Experimental Gerontology 58 (2014) 120–127 Abbreviations: PFTμ, pifithrin μ; ROS, reactive oxygen species; ΔΨm, mitochondri- al membrane potential; ETC, electron transport chain; aCSF, arti ficial, sucrose- supplemented cerebral–spinal fluid; Ca 2+ m, mitochondrial Ca 2+ ; Ca 2+ c, cytoplas- mic Ca 2+ ; TBHP, tert-butyl hydroperoxide; G6PD, glucose-6-phosphate dehydroge- nase; GSH, glutathione; HSP 70, heat shock 70 kDa protein; PUMA, p53 upregulated modulator of apoptosis. ⁎ Corresponding author at: Rua Três de Maio, n. 100, São Paulo, SP 04044-020, Brazil. E-mail address: rodrigo_ureshino@yahoo.com.br (R.P. Ureshino). http://dx.doi.org/10.1016/j.exger.2014.07.014 0531-5565/© 2014 Elsevier Inc. All rights reserved. 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