The ATM kinase inhibitor KU-55933 provides neuroprotection against hydrogen peroxide-induced cell damage via a γH2AX/p-p53/caspase-3-independent mechanism: Inhibition of calpain and cathepsin D

Highlights

• KU-55933 provided neuroprotection against H₂O₂- and doxorubicin (Dox)- but not staurosporine (St)-induced cell damage.

• KU-55933 mediated greater protection in neuronallly differentiated SH-SY5Y cells.

• KU-55933 blocked phosphorylation of ATM in H₂O₂ and Dox models of cell damage.

• KU-55933 prevented the toxin induced increases in γH2AX, p-p53 and caspase-3 only in the Dox model.

• KU-55933 attenuated the H₂O₂-induced increases in calpain and cathepsin D activity.

Abstract

The role of the kinase ataxia-telangiectasia mutated (ATM), a well-known protein engaged in DNA damage repair, in the regulation of neuronal responses to oxidative stress remains unexplored. Thus, the neuroprotective efficacy of KU-55933, a potent inhibitor of ATM, against cell damage evoked by oxidative stress (hydrogen peroxide, H₂O₂) has been studied in human neuroblastoma SH-SY5Y cells and compared with the efficacy of this agent in models of doxorubicin (Dox)- and staurosporine (St)-evoked cell death. KU-55933 inhibited the cell death induced by H₂O₂ or Dox but not by St in undifferentiated (UN-) and retinoic acid-differentiated (RA)-SH-SY5Y cells, with a more pronounced effect in the latter cell phenotype. Furthermore, this ATM inhibitor attenuated the Dox- but not H₂O₂-induced caspase-3 activity in both UN- and RA-SH-SY5Y cells. Although KU-55933 inhibited the H₂O₂- and Dox-induced activation of ATM, it attenuated the toxin-induced phosphorylation of the proteins H2AX and p53 only in the latter model of cell damage. Moreover, the ATM inhibitor prevented the H₂O₂-evoked increases in calpain and cathepsin D activity and attenuated cell damage to a similar degree as inhibitors of calpain (MDL28170) and cathepsin D (pepstatin A). Finally, we confirmed the neuroprotective potential of KU-55933 against the H₂O₂- and Dox-evoked cell damage in primary mouse cerebellar granule cells and in the mouse hippocampal HT-22 cell line. Altogether, our results extend the neuroprotective portfolio of KU-55933 to a model of oxidative stress, with this effect not involving inhibition of the γH2AX/p-p53/caspase-3 pathway and instead associated with the attenuation of calpain and cathepsin D activity.

Introduction

The ataxia telangiectasia mutated (ATM, EC 2.7.11.1) kinase, as a member of the family of PI3-Ks (phosphatidylinositol 3-kinases), regulates a wide variety of processes, including control of gene expression and the cell cycle, chromatin organization, stress response, cell metabolism and intracellular organization (Shiloh and Ziv, 2013). Loss-of-function mutation in ATM is a cause of ataxia telangiectasia syndrome (A-T) manifested inter alia by impaired coordination, increased risk of cancer, enlarged blood vessels in the skin and eyeballs, immunodeficiency and neurodegeneration (Lee and McKinnon, 2007, McKinnon, 2012). The first identified role of ATM was participation in the response to double strand breakage (DSB), which is thought to be the most detrimental form of DNA damage in mammalian cells (Valerie and Povirk, 2003). When cell homeostasis is preserved, ATM exists in the form of an inactive dimer/tetramer, but in the presence of DSB, ATM undergoes autophosphorylation at Ser1981 (pATM), forms a monomer/dimer and migrates to the nucleus. Histone H2AX is one of the first ATM substrates, and H2AX phosphorylated at Ser139 (γH2AX) is a molecular marker of DSB. Through NBS1 (Nijmegen breakage syndrome 1) protein, ATM kinase is linked with the MRN (MRE11-RAD50-NBS1) complex at the sites of DNA strand breaks, where it phosphorylates proteins, such as the serine/threonine-protein kinase Chk2 (at Thr68) and p53 (at Ser15). This leads to cell cycle arrest and repair of damage, or to apoptosis if the damage is too severe (Bakkenist and Kastan, 2003, Martin, 2008, Stracker et al., 2013). The mechanisms and mutual interconnections between DNA damage and repair, cell cycle regulation, and induction of apoptosis in proliferating cells are quite well known (Iijima et al., 2008a, Iijima et al., 2008b, Matt and Hofmann, 2016, Shiloh and Ziv, 2013); however, these processes are less clear in post-mitotic neurons (Kruman et al., 2004, Lee and McKinnon, 2007, Martin and Wong, 2016). Nevertheless, there are clinical and experimental data demonstrating that the DNA damage-evoked cell-cycle re-entry and apoptosis are important factors contributing to the neuronal cell loss observed in various neurodegenerative disorders (e.g., Alzheimer’s disease, Parkinson’s disease, Huntington's disease and amyotrophic lateral sclerosis) (Camins et al., 2010, Martin, 2008, Mullaart et al., 1990, Pelegrí et al., 2008, Ranganathan and Bowser, 2003, Silva et al., 2014, Smith et al., 2004). Moreover, it has been shown that not only various genotoxic stimuli (e.g., γ-irradiation, camptothecin, etoposide, doxorubicin, methotrexate, homocysteine) but also factors involved in pathomechanisms of neurodegeneration (e.g., β-amyloid, N-methyl-d-aspartate) and some neurotoxins (e.g., 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenylpyridinium iodide (MPP(+)), N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4)) could induce neuronal cell death via DNA damage-induced cell-cycle re-entry and/or apoptosis (Alvira et al., 2007, Bernstein et al., 2011, Camins et al., 2010, Jung et al., 2011; Macleod et al., 2003, Wang et al., 2014, Wang et al., 2015a). Additionally, there are data linking neuroprotective effects of some drugs with the inhibition of DNA damage, as shown for some antidepressant drugs in the DSP4- or camptothecin-induced models of SH-SY5Y cell damage (Wang et al., 2015b).

Although various experimental studies have demonstrated neuroprotective effects of the inhibition of cell-cycle re-entry (e.g., by cyclin-dependent kinase 5 (CDK5) inhibitors) (Cicenas et al., 2015, Neve and McPhie, 2006, Pizarro et al., 2011), the role of the functional modulation of ATM kinase in neuroprotection remains less recognized. Until 2004, when the selective ATM kinase inhibitor KU-55933 was designed (Hickson et al., 2004), non-specific inhibitors such as caffeine or wortmannin were used to inhibit the activity of ATM (Adams et al., 2010, Burma et al., 2001, Guo et al., 2010, Kruman et al., 2004). Among the limited studies on the neuroprotection mediated by ATM inhibition, there are data demonstrating beneficial effects of KU-55933 and/or caffeine in the models of neuronal cell damage induced by MPP(+), β-amyloid or DNA-damaging drugs (etoposide and homocysteine) with mechanisms involving DSB (γH2AX, p-p53), apoptosis and/or cell cycle regulatory proteins (Alvira et al., 2007, Camins et al., 2010, Jung et al., 2011). On the other hand, there are reports suggesting a protective role for ATM based on findings from A-T fibroblasts, which were more sensitive to hydrogen peroxide (H₂O₂)-induced apoptosis (Yu et al., 2015), as well as the observations from Atm-deficient mice of increased oxidative damage in brain tissues (particularly cerebellar Purkinje cells) and loss of dopamine neurons in the nigrostriatal pathway (Barlow et al., 1999, Kirshner et al., 2012). According to current knowledge, the oxidation-evoked ATM activation appear to involve different mechanisms than this one induced by DSB. For example, human fibroblasts treated with H₂O₂ formed active, linked by disulfide bonds ATM dimers which did not phosphorylate H2AX (Guo et al., 2010).

Since the elevated level of reactive oxygen species (ROS) is considered an important factor contributing to neuronal cell loss under various neurodegenerative conditions (Bredesen et al., 2006, Trippier et al., 2013), an investigation of the still-unknown role of ATM kinase in the neuronal response to oxidative stress is of high scientific and clinical importance. Thus, in the present study, we tested the effect of the specific inhibitor of ATM kinase, KU-55933, against H₂O₂-induced neuronal cell damage. Moreover, we compared the obtained results with the efficacy of this agent in the models of neurotoxicity induced by doxorubicin (Dox), a DNA damaging agent and/or inducer of the extracellular apoptotic pathway (Jantas et al., 2008, Jantas and Lason, 2009a, Lopes et al., 2008), and staurosporine (St), an inducer of the mitochondrial apoptotic pathway (Jantas et al., 2009, Koh et al., 1995). Our study, particularly the mechanistic components, was performed in human neuroblastoma SH-SY5Y cells, a commonly used cell line in neurotoxicity/neuroprotection research (Cheung et al., 2009; Lopes et al., 2010, Presgraves et al., 2004). Since there is still ongoing discussion regarding whether SH-SY5Y cells should be differentiated into neurons for neuroprotection studies (Cheung et al., 2009, Jantas et al., 2013, Lopes et al., 2010, Luchtman and Song, 2010, Presgraves et al., 2004, Wenker et al., 2010, Yong-Kee et al., 2011), we used undifferentiated (UN-) and retinoic acid-differentiated (RA)-SH-SY5Y cells. Finally, we confirmed data obtained in human SH-SY5Y cells in mouse primary cerebellar granule cells (CGCs) and the cell lines HT-22 (immortalized mouse hippocampal cells) and C6 (rat glioma).