Research paperSimvastatin preconditioning confers neuroprotection against hypoxia-ischemia induced brain damage in neonatal rats via autophagy and silent information regulator 1 (SIRT1) activation
Introduction
Preconditioning (PC) is a phenomenon in which brief episodes of a sublethal insult induce protection against subsequent damaging injuries. This phenomenon, also known as hormesis, represents an evolutionary-based adaptive response that allows organisms to survive and thrive in challenging environments (Arumugam et al., 2006; Mattson, 2014). In the brain, PC obtained with a brief and nonlethal episode of ischemia confers protection against a subsequent ischemia-reperfusion (I/R) injury through up-regulation of endogenous protective mechanisms (Liu et al., 2009). One of the key pathways mediating the PC protective effect is autophagy, a catabolic process used for the degradation of protein aggregates and dysfunctional organelles, including mitochondria, the endoplasmic reticulum, and peroxisomes (Sheng et al., 2010; Yan et al., 2011). Autophagy is essential for reprogramming the metabolism and balancing sources of energy for cell survival in the challenging environment caused by ischemia. This adaptive response is orchestrated through multifaceted cellular programs involving the concerted action of several pathways that regulate nutrient uptake, metabolism, cell cycle and growth control, and survival/death programs (Kroemer et al., 2010). The mechanistic target of rapamycin (mTOR) and the NAD-dependent protein deacetylase SIRT1 are currently considered essential players in these adaptive responses and are both critical modulators of the autophagy machinery (Jung et al., 2010; Lee et al., 2008). mTOR is considered the central regulator of autophagy and responds to different stimuli promoting protein translation and synthesis, cell growth, and survival (Jung et al., 2010). mTOR is a serine-threonine kinase that interacts with several proteins to form two distinct complexes, mTORC1 and mTORC2, which show different subunit compositions, sensitivities to rapamycin, and functions (Laplante and Sabatini, 2012). SIRT1, on the other hand, regulates the autophagy machinery by deacetylating the autophagy-related proteins ATG5, ATG7 and LC3 and, indirectly, via the activation of AMPK and the inhibition of mTOR (Ghosh et al., 2010).
Autophagy increases after acute ischemic episodes, and pharmacological treatments known to protect against ischemia, as well as ischemic PC, have been shown to potentiate autophagy after injury (Xie et al., 2018). Pharmacological inhibition of autophagy, on the other hand, blocks neuroprotection (Buckley et al., 2014; Carloni et al., 2010), confirming the critical role played by autophagy in neuroprotection.
In addition to ischemia, several drugs have been shown to provide PC-like effects (Koronowski et al., 2015; Matejovska et al., 2008), including statins (Hassan et al., 2019; Sun et al., 2019). Statins are 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors widely prescribed to lower cholesterol in hyperlipidemic patients at risk of cardiovascular disease. Statins have shown neuroprotective effects in adult and neonatal models of ischemic brain injury (Carloni et al., 2009; Saeedi Saravi et al., 2017). The beneficial effects of statins are due to both their lipid and non-lipid lowering properties, the latter of which are defined as “pleiotropic” effects. In the cardiovascular system, these pleiotropic properties include anti-inflammatory actions, plaque stabilization, improved endothelial function, and inhibition of vascular smooth muscle cell proliferation (Oesterle et al., 2017); hence, this class of drugs confers proven beneficial prophylactic effects. However, whether statin therapy might precondition the brain, improving stroke outcome in patients, is still under debate. We previously reported the efficacy of simvastatin (Sim) administration in a neonatal model of hypoxia-ischemia (HI)-induced brain injury. Neuroprotection was shown when the drug was administered repeatedly or as a single dose before, but not after HI (Balduini et al., 2003), pointing to a preconditioning-like effect of the drug (Balduini et al., 2009). The aim of the present study was to gain more insight into the preconditioning-like effect of Sim in a neonatal model of HI by examining in greater detail the activation of autophagy induced by Sim and the role of mTOR and SIRT1 in its neuroprotective effect. The effect of Sim preconditioning was studied both in the initial phase of brain damage, when the complex mechanisms underlying the neurodegenerative process are in an early stage, and during the late phase of the neurodegenerative process.
Section snippets
Cerebral hypoxia-ischemia (HI)
All surgical and experimental procedures were carried out following the Italian regulations for the care and use of laboratory animals (according to the EU Directive 86/609/EEC) and were approved by the Animal Care Committee of the University of Urbino Carlo Bo. On postnatal day 7 (PN7), Sprague-Dawley pup rats (Charles River) were anesthetized with 5% isoflurane in N2O/O2 (70/30%) mixture, and subjected to permanent ligation of the right common carotid artery followed by 2.5 h hypoxia (92%
Autophagy increased in simvastatin-treated hypoxic-ischemic rats and its inhibition blocked neuroprotection
To assess autophagy activation after Sim preconditioning, we administered MDC, which labels acidic endosomes, lysosomes, and late-stage autophagosomes (Perry et al., 2009). We then analyzed labeling at 2 h and 24 h after the ischemic insult. MDC-positive autophagosome-like structures are upregulated in vivo under conditions that increase autophagy (Carloni et al., 2010; Klionsky et al., 2016). As shown in Fig. 1, MDC labeling was not observed in vehicle-treated or Sim-treated control rats, nor
Discussion
Autophagy is an adaptive strategy that allows cells to survive bioenergetic stress or other pathological conditions. Although excessive autophagy can cause cell death, enhanced and controlled autophagy is protective, providing essential substrates during starvation and removing damaged proteins and organelles. We report here that Sim preconditioning potentiated the increased autophagic response induced by HI in neonatal rats. The neuroprotective effect of the statin appeared to be strictly
Funding
This research was supported by grants from the Department of Biomolecular Sciences of the University of Urbino Carlo Bo.
Declaration of authorship
S.C. conceptualized and designed the study, performed the data acquisition and drafted the initial manuscript. W.B. critically reviewed and revised the manuscript. Both of the authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work.
Declaration of Competing Interest
The authors declare that there is no conflict of interest.
Acknowledgments
We thank Claudia Scopa for excellent technical assistance.
References (60)
- et al.
Hormesis/preconditioning mechanisms, the nervous system and aging
Ageing Res. Rev.
(2006) - et al.
Simvastatin reduces caspase-3 activation and inflammatory markers induced by hypoxia-ischemia in the newborn rat
Neurobiol. Dis.
(2006) - et al.
Protective role of autophagy in neonatal hypoxia-ischemia induced brain injury
Neurobiol. Dis.
(2008) - et al.
Simvastatin acutely reduces ischemic brain damage in the immature rat via Akt and CREB activation
Exp. Neurol.
(2009) - et al.
Increased autophagy reduces endoplasmic reticulum stress after neonatal hypoxia-ischemia: role of protein synthesis and autophagic pathways
Exp. Neurol.
(2014) - et al.
Factors involved in rosuvastatin induction of insulin sensitization in rats fed a high fat diet
Nutr. Metab. Cardiovasc. Dis.
(2013) - et al.
Multiple site acetylation of Rictor stimulates mammalian target of rapamycin complex 2 (mTORC2)-dependent phosphorylation of Akt protein
J. Biol. Chem.
(2012) - et al.
Long-term atorvastatin improves age-related endothelial dysfunction by ameliorating oxidative stress and normalizing eNOS/iNOS imbalance in rat aorta
Exp. Gerontol.
(2014) p70S6 kinase phosphorylation for pharmacodynamic monitoring
Clin. Chim. Acta
(2012)- et al.
SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity
Cell
(2006)