Rapamycin Confers Neuroprotection against Colistin-Induced

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Rapamycin confers neuroprotection against colistin-induced oxidative stress, mitochondria dysfunction and apoptosis through the activation of autophagy and mTOR/Akt/CREB signaling pathways Chongshan Dai, Giuseppe D Ciccotosto, Roberto Cappai, Yang Wang, Shusheng Tang, Daniel Hoyer, Elena K. Schneider, Tony Velkov, and Xilong Xiao ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00323 • Publication Date (Web): 19 Dec 2017 Downloaded from http://pubs.acs.org on December 20, 2017

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ACS Chemical Neuroscience

Rapamycin confers neuroprotection against colistin-induced oxidative stress, mitochondria dysfunction and apoptosis through the activation of autophagy and mTOR/Akt/CREB signaling pathways

Chongshan Dai1, Giuseppe D. Ciccotosto2, Roberto Cappai2, Yang Wang1, Shusheng Tang1, Daniel Hoyer3,4,5, Elena K. Schneider6, Tony Velkov6*, Xilong Xiao1,* 1

Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, China Agricultural University, No.2 Yuanmingyuan West Road, Beijing 100193, P. R. China. 2Department of Pathology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Vic., Australia. 3 Department of Pharmacology & Therapeutics, School of Biomedical Sciences, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, Parkville, VIC, 3010, Australia. 4The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, 30 Royal Parade, Parkville, VIC, 3052, Australia. 5 Department of Molecular Medicine, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA. 6Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia.

Running title: Rapamycin attenuates colistin neurotoxicity *Joint corresponding authors: Xilong Xiao. Telephone: +86 10 6273 3857; Fax: +86 10 6273 1032. E-mail: [email protected]. Tony Velkov. Telephone: +61 3 9903 9539. Fax: +61 3 9903 9583. E-mail: [email protected]

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Abstract Our previous studies showed colistin-induced neurotoxicity involves apoptosis and oxidative damage. The present study demonstrates a neuroprotective effect of rapamycin against colistin-induced neurotoxicity in vitro and in vivo. In a mouse model, colistin treatment (18 mg/kg/d; 14 days) produced marked neuronal mitochondria damage in the cerebral cortex and increased activation of caspase-9 and -3. Rapamycin co-treatment (2.5 mg/kg/d) effectively reduced this neurotoxic effect. In an in vitro mouse neuroblastoma-2a (N2a) cell culture model, rapamycin pre-treatment (500 nM) reduced colistin (200 µM) induced cell death from ~50% to 72%. Moreover, rapamycin showed a marked neuroprotective effect in the N2a cells by decreasing intracellular reactive oxygen species (ROS) production and by up-regulating the activities of the anti-ROS enzymes superoxide dismutase and catalase, and recovering GSH levels to normal. Moreover, rapamycin pre-treatment protected against

colistin-induced

mitochondrial

dysfunction,

caspase

activation

and

subsequent apoptosis by up-regulating autophagy and activating the Akt/CREB, NGF and Nrf2 pathways, while inhibiting p53 signaling. Taken together, this is the first study to demonstrate that rapamycin protects against colistin-induced neurotoxicity by activating autophagy, inhibiting oxidative stress, mitochondria dysfunction and apoptosis. Our data highlight that regulating autophagy to rescue neurons from apoptosis may become a new targeted therapy to relieve the adverse neurotoxic effects associated with colistin therapy.

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Keywords:

Rapamycin;

Colistin;

Neurotoxicity;

Apoptosis;

Oxidative

stress;

Mitochondrial dysfunction.

Introduction The increasing emergence of multidrug resistant (MDR) Gram-negative pathogens in particular Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa and the scarcity of new antibiotics in the pipeline is a worldwide health crisis.1, 2 Polymyxins (colistin and polymyxin B) are lipopeptide antibiotics used as a last-line treatment for MDR infections caused by these problematic Gram-negative pathogens.3, 4 A shortcoming of polymyxin therapy is dose-limiting nephrotoxicity and a notable incidence of neurotoxicity.4-6 Although, there has been considerable progress towards understanding the molecular pathology of polymyxin-induced nephrotoxicity,7-9 the molecular mechanisms underlying the associated neurotoxicity remains largely uncharacterized. We have previously shown colistin-induced neuronal cell death involves reactive oxygen species (ROS)-mediated oxidative stress, the activation of the p53, death receptor pathways and mitochondrial dysfunction, followed by apoptosis.10-12 Rapamycin (also known as sirolimus), is a lipophilic macrocyclic lactone antibiotic produced by the bacterium Streptomyces hygroscopicus originally found in the soil of Easter Island.13,

14

Rapamycin has been approved by the US Food and Drug

Administration as an immunosuppressant drug to prevent rejection following organ transplantation, to treat lymphangioleiomyomatosis and is also used to coat coronary 3



stents.13, 15, 16 Rapamycin has been shown to display some notable pharmacological activities, due to its potent inhibition of the serine/threonine kinase termed the mechanistic target of rapamycin (mTOR) also known as FK506-binding protein.17 Feeding mice with rapamycin reduced ROS release in brain mitochondria.18 In another study, rapamycin was shown to reduce neuronal apoptotic cell death in models of Parkinson's Disease by inducing autophagy and activation of the Akt pathway, a pro-survival signal.19 Singh and colleagues demonstrated that rapamycin treatment could attenuate oxidative stress and apoptosis (though the induction of autophagy) in a rat model of amyloid-β-Induced-induced neurodegeneration.20 In our previous study, we showed that colistin treatment of mouse neuroblastoma-2a (N2a) cells produced an increased expression of Beclin1 and LC3II/I ratio, indicating that the activation of autophagy ensues as a result of polymyxin-induced neurotoxicity.10 In the present study, we provide demonstrable evidence that rapamycin effects a neuroprotective activity against colistin-induced neurotoxicity in vitro and in vivo, in neuronal N2A cell culture and in a C57BL/6 mouse model. We show that the neuroprotective mechanism of rapamycin involves suppression of oxidative stress, amelioration of mitochondrial dysfunction and rescue from apoptosis via the activation of autophagy and mTOR/Akt/CREB signaling pathways.

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Results and Discussion Colistin treatment induces apoptosis and autophagy the cerebral cortex of mice and in N2a cells Colistin treatment for 2 weeks (accumulated dose 252 mg/kg) significantly up-regulated

the

expression

of

apoptosis

markers,

Bcl-2,

Bax,

CytC,

cleaved-capase-9 and cleaved-casapse-3 proteins, autophagy markers, LC3II, p62 and Beclin1 proteins, and Nrf2 pathway proteins, Nrf2 and HO-1, in the cerebral cortex (Fig. S1). In N2a cells, treatment with 100 µM colistin for 24 h induced a marked increase production of GFP-LC3 dot formation compared to the untreated cells (Fig. S2).

Rapamycin improves the histopathology and mitochondrial survival in cerebral cortex of mice treated with colistin Histopathological examination of the cerebral cortex of mice treated with colistin for 2 weeks (cumulated dose 252 mg/kg) revealed slight changes including deeply stained nuclei and neuronal cell damage that were not evident in the untreated control and rapamycin + colistin groups (Fig. 1A), the histopathological scores decreased to 0.67 (Fig. 1B). We performed TEM imaging to assess ultrastructural changes in the cerebral cortex of mice treated with colistin. The results revealed marked mitochondrial injury in the colistin per se treatment group, which was evident from the decreased number of and swollen, ruptured and vacuolated mitochondria, with 5



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deteriorated cristae (Fig. 1C). Rapamycin treatment at 2.5 mg/kg for 14 days markedly decreased the colistin-induced mitochondrial damage; moreover, only slight mitochondrial cristae deletion was evident in the colistin + rapamycin group. There were no abnormal changes of mitochondrial morphology or ultrastructure in cerebral cortex tissue in the rapamycin per se treatment group.

Rapamycin ameliorates mitochondrial dysfunction in the cerebral cortex of mice treated with colistin Compared to the untreated control group, colistin treatment significantly increased the level of the Ca2+-induced MPT to 128% (p

Rapamycin Confers Neuroprotection against Colistin-Induced

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