Central nervous system (CNS) is one of the complex systems in the body that consists of brain and spinal cord. Any disease or traumatic assault may lead to the degeneration of CNS including loss of homeostasis. CNS injuries constitute a major cause of morbidity and mortality includes the life threatening injuries such as traumatic brain injury (TBI) and spinal cord injury (SCI). TBI and SCI are caused by both primary and secondary injuries influencing the cascades of cellular and molecular events, which will cause further damage in the system, and loss of body functions. The consequences of the secondary injury include mitochondrial dysfunction, neurotransmitter accumulation, blood-brain barrier (BBB) and blood spinal cord barrier disruption, apoptosis, excitotoxic damage, initiation of inflammatory, and immune processes which is followed by initial primary mechanical trauma. Secondary injury involves the production of highly reactive species, reactive oxygen species (ROS), reactive nitrogen species (RNS), or free radicals which will cause damage to protein structure, DNA, and cell membrane and leads to oxidative stress which plays a major role in the pathophysiology of CNS injury. The progression of the damage starts from the primary impact on brain or spinal cord and will continue for hours, days, and weeks after the initial mechanical insult which will result in tissue damage (Samantaray et al., 2009; Khalatbary et al., 2010; Bains and Hall, 2012b; Bhalala et al., 2013; Naseem and Parvez, 2014). Plus the stress response, autophagy is a highly essential cellular response to damage and influences the improvement and progression of post-traumatic disease (Wang et al., 2015). The term autophagy, from Greek “self-eating” refers to a range of processes, including chaperone-mediated autophagy, microautophagy and macroautophagy, which regulated process of degradation and recycling of cellular constituents, participated in organelle turnover and the bioenergetic management of starvation of spinal cord injury. The mammalian target of rapamycin (mTOR), a conserved serine/threonine kinase, is the catalytic subunit of two fundamentally distinct complexes: complexes-mTOR complex 1 (mTORC1) and complexes-mTOR complex 2 (mTORC2) that individually plays an essential role in the control of cell proliferation. Both complexes localized to distinctive subcellular sections, thus affecting their initiation and role (Wullschleger et al., 2006; Betz and Hall, 2013). The mTORC1 stimulate protein synthesis by mRNA translation and cell development by entering the G1 phase of the cell cycle, however mTORC2, firstly identified as a regulator of the actin cytoskeleton, has been indicated to phosphorylate members of the AGC kinase family, including Akt, which is linked to several pathological conditions (Menon and Manning, 2008; Foster and Fingar, 2010; Sparks and Guertin, 2010). They have distinctive downstream targets, different biological functions and importantly, different sensitivity to the drug rapamycin. mTORC1 is pharmacologically inhibited by short-term rapamycin management, whereas mTORC2 is resistant to short-term rapamycin treatment, although long-term treatment can prevent mTORC2 complex assembly (Phung et al., 2006; Sarbassov et al., 2006). One of the most important mTOR inhibitor studied until today was Rapamycin. Rapamycin, an inhibitor of the mTOR pathway, can extend lifespan and improve age-related functional decline in mice, thereby providing the first proof of principal that a pharmaceutical agent can slow the aging process in mammals. These outcomes have proven robust in repeated studies; however, their potential translational relevance towards a means to slow aging or prevent age-related disease in otherwise healthy humans remains unclear. Part of the challenge in addressing the potential of rapamycin (or its analogs) as a pro-longevity therapeutic lies in its known clinical risks for adverse side effects. Primary amongst these are metabolic defects that include hyperglycemia, hyperlipidemia, insulin resistance and increased incidence of new-onset type 2 diabetes. In healthy rodents, treatment with rapamycin also causes a relatively rapid, dose-dependent impairment of markers of glucose homeostasis. The natures of the metabolic effects/defects caused by rapamycin remain ambiguous regarding their role in longevity and healthy aging. Fang et al. suggested the effects of rapamycin on metabolism depend on the length of treatment with a detrimental effect on glucose metabolism in the short-term whereas mice treated chronically with rapamycin actually became insulin-sensitive. On the other hand, Blagosklonny has proposed that the presumed metabolic impairments caused by rapamycin may simply be a consequence of its action as a “starvation-mimetic” and, further, may be fundamentally required for its pro-longevity effect (Blagosklonny, 2011; Wilkinson et al., 2012; Fang et al., 2013; Miller et al., 2014). Considering a lot of Rapamycin-induced side effect, in these years, a number of inhibitors of the PI3K/AKT/mTOR pathway has been identified such as Temsirolimus and KU0063794. Temsirolimus was the first mTORC1 inhibitor investigated in clinical trials in the late 1990s in patients with cancer. Is an ester derivative of rapamycin and it is a specific inhibitor of mTORC1 that interferes with the synthesis of proteins that regulate proliferation, growth, and survival of tumor cells. Treatment with temsirolimus leads to cell cycle arrest in the G1 phase and stops tumor angiogenesis by reducing synthesis of VEGF (Duran et al., 2006). KU0063794 is a second-generation mTOR inhibitor targeting mTORC1 and mTORC2, including p70S6K, 4E-BP1 and Akt. Specifically, inhibits the phosphorylation of S6K1 and 4E-BP1, which are downstream substrates of mTORC1, and it inhibits Akt phosphorylation on Ser473, which is the target of mTORC2 (Garcia-Martinez et al., 2009; Zhang et al., 2013). In a recent study it has been demonstrated that KU0063794 decreasing the viability and growth of renal cell carcinoma cell lines, Caki-1 and 786-O, and showed anti-fibrotic activity in Keloid disease (Syed et al., 2013; Zhang et al., 2013). Previously it has been showed that mTOR plays a key role in modulation of macrophage/microglia activation, reduction of IL-1β and TNFα production, expression of nitric oxide synthase, prevention of apoptosis neuronal loss and demyelination both in the first and second phases of the damage after injury (Kanno et al., 2012). Moreover, it has been demonstrated that rapamycin treatment significantly improved the neurological recovery from SCI and increased the number of surviving neurons at the lesion epicenter (Chen et al., 2013b). However, the mechanism of autophagy related inflammation after SCI and TBI is still unclear. So, in this regard in this thesis we have evaluated the effect of Ku0063794, as potential treatments for inflammation in SCI and TBI models.

The role of mTOR signaling pathway in Brain and Spinal Cord Injury

CORDARO, MARIKA
2017-01-23

Abstract

Central nervous system (CNS) is one of the complex systems in the body that consists of brain and spinal cord. Any disease or traumatic assault may lead to the degeneration of CNS including loss of homeostasis. CNS injuries constitute a major cause of morbidity and mortality includes the life threatening injuries such as traumatic brain injury (TBI) and spinal cord injury (SCI). TBI and SCI are caused by both primary and secondary injuries influencing the cascades of cellular and molecular events, which will cause further damage in the system, and loss of body functions. The consequences of the secondary injury include mitochondrial dysfunction, neurotransmitter accumulation, blood-brain barrier (BBB) and blood spinal cord barrier disruption, apoptosis, excitotoxic damage, initiation of inflammatory, and immune processes which is followed by initial primary mechanical trauma. Secondary injury involves the production of highly reactive species, reactive oxygen species (ROS), reactive nitrogen species (RNS), or free radicals which will cause damage to protein structure, DNA, and cell membrane and leads to oxidative stress which plays a major role in the pathophysiology of CNS injury. The progression of the damage starts from the primary impact on brain or spinal cord and will continue for hours, days, and weeks after the initial mechanical insult which will result in tissue damage (Samantaray et al., 2009; Khalatbary et al., 2010; Bains and Hall, 2012b; Bhalala et al., 2013; Naseem and Parvez, 2014). Plus the stress response, autophagy is a highly essential cellular response to damage and influences the improvement and progression of post-traumatic disease (Wang et al., 2015). The term autophagy, from Greek “self-eating” refers to a range of processes, including chaperone-mediated autophagy, microautophagy and macroautophagy, which regulated process of degradation and recycling of cellular constituents, participated in organelle turnover and the bioenergetic management of starvation of spinal cord injury. The mammalian target of rapamycin (mTOR), a conserved serine/threonine kinase, is the catalytic subunit of two fundamentally distinct complexes: complexes-mTOR complex 1 (mTORC1) and complexes-mTOR complex 2 (mTORC2) that individually plays an essential role in the control of cell proliferation. Both complexes localized to distinctive subcellular sections, thus affecting their initiation and role (Wullschleger et al., 2006; Betz and Hall, 2013). The mTORC1 stimulate protein synthesis by mRNA translation and cell development by entering the G1 phase of the cell cycle, however mTORC2, firstly identified as a regulator of the actin cytoskeleton, has been indicated to phosphorylate members of the AGC kinase family, including Akt, which is linked to several pathological conditions (Menon and Manning, 2008; Foster and Fingar, 2010; Sparks and Guertin, 2010). They have distinctive downstream targets, different biological functions and importantly, different sensitivity to the drug rapamycin. mTORC1 is pharmacologically inhibited by short-term rapamycin management, whereas mTORC2 is resistant to short-term rapamycin treatment, although long-term treatment can prevent mTORC2 complex assembly (Phung et al., 2006; Sarbassov et al., 2006). One of the most important mTOR inhibitor studied until today was Rapamycin. Rapamycin, an inhibitor of the mTOR pathway, can extend lifespan and improve age-related functional decline in mice, thereby providing the first proof of principal that a pharmaceutical agent can slow the aging process in mammals. These outcomes have proven robust in repeated studies; however, their potential translational relevance towards a means to slow aging or prevent age-related disease in otherwise healthy humans remains unclear. Part of the challenge in addressing the potential of rapamycin (or its analogs) as a pro-longevity therapeutic lies in its known clinical risks for adverse side effects. Primary amongst these are metabolic defects that include hyperglycemia, hyperlipidemia, insulin resistance and increased incidence of new-onset type 2 diabetes. In healthy rodents, treatment with rapamycin also causes a relatively rapid, dose-dependent impairment of markers of glucose homeostasis. The natures of the metabolic effects/defects caused by rapamycin remain ambiguous regarding their role in longevity and healthy aging. Fang et al. suggested the effects of rapamycin on metabolism depend on the length of treatment with a detrimental effect on glucose metabolism in the short-term whereas mice treated chronically with rapamycin actually became insulin-sensitive. On the other hand, Blagosklonny has proposed that the presumed metabolic impairments caused by rapamycin may simply be a consequence of its action as a “starvation-mimetic” and, further, may be fundamentally required for its pro-longevity effect (Blagosklonny, 2011; Wilkinson et al., 2012; Fang et al., 2013; Miller et al., 2014). Considering a lot of Rapamycin-induced side effect, in these years, a number of inhibitors of the PI3K/AKT/mTOR pathway has been identified such as Temsirolimus and KU0063794. Temsirolimus was the first mTORC1 inhibitor investigated in clinical trials in the late 1990s in patients with cancer. Is an ester derivative of rapamycin and it is a specific inhibitor of mTORC1 that interferes with the synthesis of proteins that regulate proliferation, growth, and survival of tumor cells. Treatment with temsirolimus leads to cell cycle arrest in the G1 phase and stops tumor angiogenesis by reducing synthesis of VEGF (Duran et al., 2006). KU0063794 is a second-generation mTOR inhibitor targeting mTORC1 and mTORC2, including p70S6K, 4E-BP1 and Akt. Specifically, inhibits the phosphorylation of S6K1 and 4E-BP1, which are downstream substrates of mTORC1, and it inhibits Akt phosphorylation on Ser473, which is the target of mTORC2 (Garcia-Martinez et al., 2009; Zhang et al., 2013). In a recent study it has been demonstrated that KU0063794 decreasing the viability and growth of renal cell carcinoma cell lines, Caki-1 and 786-O, and showed anti-fibrotic activity in Keloid disease (Syed et al., 2013; Zhang et al., 2013). Previously it has been showed that mTOR plays a key role in modulation of macrophage/microglia activation, reduction of IL-1β and TNFα production, expression of nitric oxide synthase, prevention of apoptosis neuronal loss and demyelination both in the first and second phases of the damage after injury (Kanno et al., 2012). Moreover, it has been demonstrated that rapamycin treatment significantly improved the neurological recovery from SCI and increased the number of surviving neurons at the lesion epicenter (Chen et al., 2013b). However, the mechanism of autophagy related inflammation after SCI and TBI is still unclear. So, in this regard in this thesis we have evaluated the effect of Ku0063794, as potential treatments for inflammation in SCI and TBI models.
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