Rapamycin treatment increases hippocampal cell viability in an mTOR-independent manner during exposure to hypoxia mimetic, cobalt chloride
Cobalt chloride (CoCl2) induces chemical hypoxia through activation of hypoxia-inducible factor-1 alpha (HIF-1α). Mammalian target of rapamycin (mTOR) is a multifaceted protein capable of regulating cell growth, angiogenesis, metabolism, proliferation, and survival. In this study, we tested the efficacy of a well-known mTOR inhibitor, rapamycin, in reducing oxidative damage and increasing cell viability in the mouse hippocampal cell line, HT22, during a CoCl2-simulated hypoxic insult.
CoCl2 caused cell death in a dose-dependent manner and increased protein levels of cleaved caspase-9 and caspase-3. Rapamycin increased viability of HT22 cells exposed to CoCl2 and reduced activation of caspases-9 and -3. Cells exposed to CoCl2 displayed increased reactive oxygen species (ROS) production and hyperpolarization of the mitochondrial membrane, both of which rapamycin successfully blocked. mTOR protein itself, along with its downstream signaling target, phospho-S6 ribosomal protein (pS6), were significantly inhibited with CoCl2 and rapamycin addition did not significantly lower expression further. Rapamycin promoted protein expression of Beclin-1 and increased conversion of microtubule-associated protein light chain 3 (LC3)-I into LC3-II, suggesting an increase in autophagy. Pro-apoptotic protein, Bcl-2 associated × (Bax), exhibited a slight, but significant decrease with rapamycin treatment, while its anti-apoptotic counterpart, B cell lymphoma-2 (Bcl-2), was to a similar degree upregulated. Finally, the protein expression ratio of phosphorylated mitogen-activated protein kinase (pMAPK) to its unphosphorylated form (MAPK) was dramatically increased in rapamycin and CoCl2 co-treated cells.
Our results indicate that rapamycin confers protection against CoCl2-simulated hypoxic insults to neuronal cells. This occurs, as suggested by our results, independent of mTOR modification, and rather through stabilization of the mitochondrial membrane with concomitant decreases in ROS production. Additionally, inhibition of caspase-9 and -3 activation and stimulation of protective autophagy reduces cell death, while a decrease in the Bax/Bcl-2 ratio and an increase in pMAPK promotes cell survival during CoCl2 exposure. Together these results demonstrate the therapeutic potential of rapamycin against hypoxic injury and highlight potential pathways mediating the protective effects of rapamycin treatment.
KeywordsRapamycin HT22 Cobalt chloride Hypoxia
mitochondrial membrane potential
apoptosis inducing factor
Bcl-2 associated ×
B cell lymphoma
- COX IV
cytochrome C oxidase subunit IV
Dulbecco’s modified eagles medium
hypoxia inducible factor-1alpha
inhibitors of apoptosis
microtuble-associated protein light chain
mitogen-activated protein kinase
mitochondrial outer membrane permeability
mitochondrial permeability transition pore
mammalian target of rapamycin
phosphorylated mitogen-activated protein kinase
phospho-S6 ribosomal protein
reactive oxygen species
traumatic brain injury
tetramethylrhodamine, methyl ester
Cobalt chloride (CoCl2) induces chemical hypoxia through activation of hypoxia inducible factor-1 alpha (HIF-1α), which is a key pathogenic factor in cerebral ischemia in vivo. As such, CoCl2 has been often used as an in vitro hypoxia model, along with oxygen–glucose–deprivation or glutamate exposure, to mimic cerebral ischemia. Cerebral hypoxia, in which the supply of oxygen to the brain is interrupted, is a potent contributor to neurologic dysfunction . Subsequent to an acute lack of oxygen, such as that caused by a stroke or traumatic brain injury (TBI), is the potential for further damage to surviving neuronal tissues due to hyper-activation of inflammatory and various stress or damage-induced cell death pathways, long after the initial insult has been resolved [2, 3, 4].
This secondary response, which occurs with reoxygenation, is multifaceted and may involve increases in lipid peroxidation resulting in release of pro-inflammatory second messenger, arachidonic acid , along with increases in inflammatory cytokines, such as IL-6, IL-1β and TNFα . Calcium overload, increased glutamate-induced excitotoxicity, perturbations in the mitochondrial membrane and increased oxidative stress have all additionally been associated with both hypoxia and reoxygenation responses [1, 7].
The increase in oxidative stress is particularly detrimental to neurons since these cells rely heavily on mitochondrial oxidative phosphorylation for energy production and, at the same time, have relatively low levels of antioxidants compared to other cells . It should be noted that some ROS generation in the cell is normal and ROS itself has important second messenger functions, however, antioxidizing enzymes, such as glutathione peroxidase and superoxide dismutase, must work to prevent over-accumulation of ROS since they are highly reactive and can cause severe damage to cells when left unchecked. ROS-initiated damage to cell membranes, proteins, lipids, and DNA can result in cell death mechanisms being activated [9, 10].
Such a state of oxidative stress is seen, therefore, when an increase in ROS production occurs that outpaces a cell’s endogenous antioxidant mechanisms. This is often the case during reoxygenation where mitochondria may become overwhelmed when first faced with a burst of oxygen or hypoxia-induced cell damage has compromised mitochondrial detoxification functions [7, 11]. Normal detoxification processes are unable to keep up with the increased production of ROS and the system becomes stressed. Given that, the production of ROS occurs in the mitochondria on the electron transport chain, these organelles become particularly susceptible to ROS-induced damage [8, 11, 12]. For example, ROS has been shown to oxidize mitochondrial proteins, lipids, and DNA and is associated with alterations to the mitochondrial membrane potential (Δψm) .
Loss of Δψm, the overall charge difference or electrical gradient across the inner mitochondrial membrane, can severely impact the generation of adenosine triphosphate (ATP) thus affecting a cell’s energetics and increasing susceptibility to death. Disruption of the Δψm has also been noted in cases of hypoxia and during increased ROS production [13, 14, 15]. When oxygen, the final electron accepter in the electron transport chain, is limited during the hypoxic event, electrons flowing through complex IV are hindered and electrons begin to stall in complexes I and III. The lack of electron transfer and concomitant proton pumping across the inner mitochondrial membrane dissipates the Δψm and prevents coupling to ATP synthase . In addition to this loss of ATP production, extreme changes to the Δψm, for example hyperpolarization as reported with stroke and TBI , have been shown to increase ROS production and subsequently trigger formation of the mitochondrial permeability transition pore (mPTP), an opening within the inner mitochondrial membrane that allows for the leakage of ions and other mitochondrial nutrients/proteins. The ensuing changes in concentration gradients along the inner mitochondrial membrane can cause the release of a number of pro-apoptotic components normally sequestered within the intermembrane space.
Such components include the mitochondrial/intrinsic apoptosis pathway initiator proteins, cytochrome C, Smac/Diablo, and apoptosis inducing factor (AIF) [17, 18]. Once released into the cytosol, cytochrome C may combine with pro-caspase-9 and Apaf-1 to form the apoptosome. The apoptosome activates the executioner caspase, caspase-3, a cysteine protease, to induce cell death . Meanwhile, Smac/Diablo works to suppress a group of proteins known as the inhibitors of apoptosis (IAPs) which themselves block caspase activation . AIF migrates to the nucleus where it triggers chromatin condensation and membrane fragmentation . Therefore, damage endured by the mitochondria does not remain confined to these organelles, but rather has important consequences to the overall health of the entire cell.
The initiation of apoptosis can be reduced or enhanced by affecting the level of autophagy that occurs during an insult, such as hypoxia. Autophagy is a catabolic process that breaks down cellular components and nutrients from damaged and dying cells and recycles them potentially to prop up other internal functions or those of neighboring cells. Autophagy has been shown to increase with cerebral hypoxia and other acute brain injuries, but there is still controversy as to whether this increase in autophagy promotes cell survival or instead leads to increased cell death [20, 21]. Its activation can stave off total cell death by replenishing supplies; however, it can also tip the scale towards apoptosis if it becomes over-activated, in which case the cell essentially devours itself. The term mitophagy has been coined to refer to the autophagic process of removing damaged and dysfunctional mitochondria and it has previously been noted that mitophagy is also enhanced with oxidative stress [22, 23].
Rapamycin is a well-documented autophagy activator and is renowned for its ability to decrease mTOR pathway signaling. The mTOR protein can be found in two distinct protein signaling complexes referred to as mTORC1 and mTORC2. In general, mTORC1 responds more directly to environmental stimuli, such as loss of glucose or oxygen, and controls a number of cellular processes, such as protein translation and autophagy, through phosphorylation of downstream kinases, most notably pS6 ribosomal protein, 4E-BP1, and ULK1 [24, 25]. mTORC2 is a recognized effector of insulin signaling and is known to enhance AKT activation . Both complexes respond differently to rapamycin treatment with mTORC1 being more acutely sensitive. mTORC2 typically only becomes disrupted after prolonged, chronic exposure to rapamycin . The interactions between these two pathways, particularly in response to environmental stimuli, is complex and a full description is beyond the scope of this work.
While mTOR activation typically promotes cell survival, in cases of ischemia/reperfusion injury and where oxidative stress is predominant, mTOR signaling may do the opposite as mTOR inhibition has been shown to improve outcomes. For example, rapamycin’s protective effects against hypoxic injury have been documented in the context of cardiac, hepatic, pancreatic, and renal ischemia/reperfusion models [27, 28, 29, 30, 31, 32]. The ability of rapamycin to protect against cerebral hypoxic injury has also been reported recently [33, 34, 35].
In this study, we used a chemical mimetic of hypoxia, CoCl2, to simulate a hypoxic insult in a mouse hippocampal cell line, HT22. CoCl2 has been used for this purpose in a variety of cell lines where its primary effect is to increase HIF-1α . HIF-1α stabilization during hypoxia is a widely accepted phenomenon and is central to the activation and regulation of hypoxia response pathways, thus CoCl2 exposure mimics hypoxia through modulation of these same response pathways. We have previously noted CoCl2’s ability to cause oxidative damage through increased ROS production and hyperpolarization of the mitochondrial membrane in HT22 cells . While not a true measure of hypoxia, we have found it to be a more reliable and consistent model for studying the modifications of hypoxia pathways that are activated similarly during a true hypoxic insult in vivo.
Our objective was to determine if treatment with rapamycin could confer protection against CoCl2-simulated hypoxic injury. Specifically, we looked to determine if rapamycin could mitigate changes in cell viability, mitochondrial membrane potential, and ROS production. Additionally, we sought to identify key pathways that may contribute to rapamycin protection against hypoxia.
Exposure to CoCl2 significantly reduces HT22 cell viability, while treatment with rapamycin increases viability during exposure
We also determined the effects on viability of rapamycin itself using a range of 25 nM to 10 µM. Because rapamycin is dissolved in dimethyl sulfoxide (DMSO) we analyzed our results against DMSO groups containing equal volumes and percent of DMSO. Rapamycin treatment itself had a detrimental effect on HT22 cells averaging a 15% decrease in viability when compared to DMSO-induced viability losses (Fig. 1b). DMSO only became significantly toxic at the two highest doses tested (p ≤ 0.01) and, even then, rapamycin further decreased viability in comparison by 24% (p ≤ 0.001).
Despite these findings, we tested CoCl2-exposed cells together with this same range of rapamycin concentrations. As can be seen in Fig. 1c, starting at 100 nM, rapamycin addition caused significant gains in cell viability culminating in a 21% increase with 500 nM rapamycin treatment (p ≤ 0.01). Even at the highest doses of rapamycin, which from Fig. 1b we knew DMSO would begin to effect viability, we still obtained increases in viability of around 10% (p ≤ 0.001). Since it yielded the greatest increase in viability against CoCl2 without having a DMSO effect and was additionally able to reduce the morphological effects attributed to CoCl2 exposure (Fig. 1d), we choose the 500 nM rapamycin concentration to use in all subsequent experiments.
Rapamycin treatment limits caspase-9 and caspase-3 activation to promote cell survival
Rapamycin treatment restores the Δψm and decreases the production of ROS in CoCl2-exposed cells to limit hypoxia-induced damage
As noted earlier hyperpolarization of the mitochondrial membrane has been linked to increased ROS production. That, and because of the large body of evidence showing hypoxic injury stems from increased ROS production, we examined rapamycin’s ability to modify ROS production in our model. ROS production was measured using another fluorogenic dye, dihydroethidium (DHE), as described in the materials and method. CoCl2 caused a nearly threefold increase in ROS production; an increase of 266% (p ≤ 0.001) (Fig. 3b). Rapamycin by itself had no statistically significant effect on ROS but did cause average ROS production to drop by 15%. Rapamycin added together with CoCl2, significantly reduced ROS production by 66% when compared to CoCl2 alone (p ≤ 0.001) and ROS production remained only slightly elevated above untreated control levels (24%; p ≤ 0.05).
Rapamycin treatment effects during CoCl2 exposure are independent of changes in mTOR, but can interfere with autophagy
Rapamycin treatment enhances autophagy to protect cells during CoCl2 exposure
To further confirm autophagy induction we used Western blot to measure protein expression of LC3-I and LC3-II. Autophagy flux was determined by calculating the ratio of LC3-II/LC3-I in each experimental condition (Fig. 5b). Conversion of LC3-I to LC3-II has been correlated to autophagosome formation that occurs during autophagy and therefore the amount of LC3-II/LC3-I can be compared between samples as an indicator of autophagy induction . We observed that untreated cells had low levels of autophagy induction as the ratio of LC3-II/LC3-I was around 0.5. With CoCl2 exposure LC3-II to LC3-I expression was observed at a 1:1 ratio (p ≤ 0.0.1). Finally, when rapamycin was added during CoCl2 exposure, the LC3-II/LC3-I ratio increased to 2 (p ≤ 0.05).
Rapamycin treatment reduces the Bax/Bcl-2 ratio and increases pMAPK protein expression to promote survival during CoCl2 exposure
At this point, we concluded rapamycin might be exerting its pro-survival influence through mTOR-independent mechanisms. Besides the downstream inhibition of caspase-3 activation and an increase in autophagy, the pathways responsible for mediating the protective effects of rapamycin were unclear. Since mTOR signaling was not significantly altered with treatment, we focused on key survival mediators involved in mitochondrial damage responses that could be linked to ROS production. The Bcl-2 protein family is a key regulator of the mitochondrial apoptosis pathway and contains members responsible for both pro- and anti-apoptotic responses. An imbalance in the ratio of these pro- and anti-apoptotic proteins can determine a cell’s fate by either promoting or hindering mitochondria-initiated apoptosis. In addition to mediating the intrinsic apoptosis pathway, which is of particular interest to our study, these members have also been shown to be involved in oxidative stress-induced neuronal injury .
Another member of the Bcl-2 protein family, with strong ties to the intrinsic cell death pathway, is the apoptosis-promoter, Bax. Bax is often noted to form a complex with Bcl-2 and the Bax/Bcl-2 ratio often acts as a rheostat for cell survival versus apoptosis induction . A low Bax/Bcl-2 ratio is typically seen as favoring cell survival while a high Bax/Bcl-2 ratio will often tip cells towards cytochrome C release and mitochondria-initiated apoptosis. After treatment with CoCl2, we noted that Bax protein expression was increased 6%, but the difference was not statistically significant. However when compared to CoCl2 alone, rapamycin addition significantly decreased Bax expression by 16% (p ≤ 0.05). The changes in Bax expression were nearly equal and opposite to those of Bcl-2. During CoCl2 exposure, the overall Bax/Bcl-2 ratio was significantly decreased (29%; p ≤ 0.05) with rapamycin treatment (Fig. 6a).
After observing the amplification of Bcl-2 and decrease in Bax expression, we measured changes in both unphosphorylated and phosphorylated 44/42 MAPK (pMAPK) proteins. This intracellular signaling molecule is a well-documented promoter of cell survival, particularly in neurons, and has pronounced effects on apoptosis, cell proliferation, growth and differentiation [44, 45]. Furthermore, the activation of MAPK through phosphorylation has also been related to detoxification of ROS [46, 47, 48] and we hypothesized it could play a role in our CoCl2-simulated hypoxia model. As seen in Fig. 6b both MAPK and pMAPK protein expression were significantly reduced by 20 and 85% respectively (p ≤ 0.01 and p ≤ 0.05) in CoCl2-exposed cells. Rapamycin addition increased expression of MAPK by 26% (p ≤ 0.05) and pMAPK by 740% (p ≤ 0.001) when compared to CoCl2 exposed cells. After normalizing both MAPK and pMAPK to β-actin expression, we calculated the ratio of pMAPK to MAPK expression. As seen in Fig. 6b, CoCl2 treatment alone significantly reduced the pMAPK/MAPK expression ratio by 41% (p ≤ 0.01). Rapamycin during CoCl2 exposure not only restored the expression ratio of pMAPK/MAPK, but dramatically upregulated its expression nearly sixfold (p ≤ 0.001). pMAPK/MAPK expression was increased 309% from control levels and 592% from CoCl2 exposure alone.
Using CoCl2 to mimic a hypoxic insult to hippocampal cells, we tested the therapeutic efficacy of rapamycin, a natural compound produced by bacteria and known to have antibiotic, antifungal, and immunosuppressant functions. With the addition of rapamycin during CoCl2 exposure, we observed increased cell viabilities, reduced caspase-9 and caspase-3 activation, stabilization of the Δψm, and significant reduction in ROS production. We believe protection with rapamycin treatment may be conferred independent of changes in mTOR signaling itself, but rather through increased autophagy, a decrease in the Bax/Bcl-2 ratio and increased MAPK activation.
Previous studies examining rapamycin’s ability to limit hypoxia-induced damage, the bulk of which have occurred in the context of cardiac ischemia and reperfusion, have noted overall increases in cell survival [27, 28, 29, 30, 31, 32]. The protective mechanisms most attributed to these increases in cell survival were changes in autophagy and mTOR signaling. However, within these studies, mTOR has been found to have both cardioprotective and cardiotoxic effects complicating the precise role of mTOR in mediating ischemic damage [49, 50]. While we too found rapamycin promoted cell survival in hypoxic neurons, our results indicate a less prominent role for the mTOR signaling pathway. However, it should be noted that CoCl2 exposure, by itself, down-regulated the mTOR signaling pathway as evidenced by significant decreases in mTOR, p-mTOR, and pS6 protein expression (Fig. 4).
This is not surprising, as mTOR has been reported to decrease in the brain during stressful situations, such as glucose deprivation, DNA damage, and hypoxia . However, as with mTOR in cardiac ischemia, there is still uncertainty as to the role of mTOR in mediating cerebral stroke damage in vivo. In our previous studies, we have utilized a transient global cerebral ischemia model in rats to examine the correlations between mTOR signaling and neuronal cell death during fixed reperfusion intervals. At these intervals, we noted marked increases in mTOR pathway components that corresponded to greater cell death. Rapamycin was shown in these cases to reduce mTOR singaling as well as prevent cell death [33, 34, 35].
In our present study, we did not examine samples from a reperfusion or reoxygenation stage, but instead collected neuronal cells while they were still in a hypoxia-simulated environment. A limitation of using a cell line for these experiments is a loss of any whole-animal in vivo effects. The use of HT22 cells, however, allowed us to conduct molecular-based experiments that would otherwise be cumbersome in an animal model. In our analysis we found that, in addition to decreased mTOR expression, cell death was also more pronounced under the CoCl2 exposure condition, with a nearly 80% decrease in cell viability (Fig. 1) and a more than 140-fold increase in cleaved caspase-3 protein expression (Fig. 2). Despite the contrast in mTOR expression levels seen in these two models, rapamycin still conferred protection in both cases. In this regard, the protective actions of rapamycin against CoCl2-simulated hypoxia may be different in that they do not stem from inhibiting an increase in mTOR expression as occurs during reperfusion in an in vivo stroke model. Nor does the protective benefit arise, as our results in Fig. 4 indicate, from any significant reduction in mTOR expression with rapamycin treatment. Beyond the scope of our study was ascertaining the degree to which this depression in mTOR signaling was directly responsible for reducing cell viability during CoCl2 exposure. What we did find, was that rapamycin treatment did not further reduce mTOR expression from the already low levels induced by CoCl2 exposure, nor did treatment in any way reverse this depression. This leads us to conclude that rapamycin protection against CoCl2 is independent of mTOR signaling.
Based on our results, we believe the induction of autophagy, which is often a result of inhibited protein translation/amino acid deprivation following mTOR inhibition, plays a more central role in improving viability during CoCl2 exposure. We know that cells exposed to CoCl2 undergo a great deal of stress. There is a considerable increase in toxic ROS production (Fig. 3b) and the mitochondrial membrane potential is substantially altered (Fig. 3a). Autophagy may serve in this context to remove damaged/dying mitochondria and provide recycled components to help prop up remaining healthy mitochondria in an effort to alleviate oxidative stress. This would explain how rapamycin, an activator of autophagy, was able to decrease ROS production and stabilize the mitochondrial membrane. Evidence of autophagy, specifically autophagy upregulated by rapamycin treatment, conferring protection against cardiac and neurological insults exists in the literature [52, 53, 54]. We saw too in our results that CoCl2-induced reduction of mTOR expression also yielded a significant increase in the LC3-II/LC3-I ratio (Fig. 5a) however this did not produce a survival benefit as CoCl2 exposure increased caspase-3 activation 140-fold and cytochrome c release tenfold (Fig. 2a, b). CoCl2 exposure also, in contrast to the increased LC3-II/LC3-I ratio, reduced the early autophagy mediator, Beclin-1 (Fig. 5b). It was only when rapamycin treatment was added that Beclin-1 expression was restored and the LC3-II/LC3-II ratio again doubled. With this rapamycin-induced increase in autophagy activation, we noted a roughly 20% increase in cell viability (Fig. 1c).
Autophagy may be just one mechanism for rapamycin-induced protection. In addition to the increase in autophagy, we noted an increase in anti-apoptotic Bcl-2 and a simultaneous decrease in pro-apoptotic Bax with rapamycin treatment. Both of these proteins can mediate the mitochondrial intrinsic death pathway by facilitating mitochondrial outer membrane permeability (MOMP) and the release of cytochrome c. Bax is able to insert itself into the outer mitochondrial membrane to trigger MOMP while binding with Bcl-2, also located on the outer mitochondrial membrane, serves to deactivate Bax .
Bcl-2 has long been shown to confer protection against a variety of neurological insults including oxidative stress, excitotoxicity, and cerebral ischemia . Mice deficient in Bcl-2 also experience enhanced oxidative stress and alterations in antioxidants in the brain . It is possible the increase in Bcl-2 expression we saw during rapamycin treatment also contributed to the observed decrease in ROS production with rapamycin addition (Fig. 3b) although we did not measure this connection directly. In opposition to Bcl-2, which confers protection, increased Bax has previously been associated with oxidative damage in neurological models . Bax and Bcl-2 proteins often serve as counterbalances to one another and, with rapamycin addition, the ratio of Bax to Bcl-2 during CoCl2 exposure significantly dropped (Fig. 6a) favoring cell survival. Even though we did not detect significant prevention of cytochrome c release with rapamycin addition, caspase-9 and caspase-3 activation were significantly reduced (Fig. 2a, b) suggesting CoCl2-induced caspase-3 cleavage is independent of cytochrome C release.
Caspases are known to play a pivotal role in ischemia-induced apoptosis. While necrotic cell death predominates in the primary injury location, the surrounding areas often experience apoptosis as a delayed response . This delay allows a therapeutic window in which interventions can be made to prevent the induction of apoptosis in these adjoining cells in hopes of minimizing the overall damage to the patient. Most of this apoptosis during the secondary response is triggered by an increase in ROS production . Interestingly, activation of MAPKs has been related to detoxification of ROS and protection against oxidative stress . Upregulation of MAPK has also been linked to increased Bcl-2 expression on human neuron-like cells . Both of these findings are supported by this present work. We found that both MAPK and pMAPK were significantly reduced during CoCl2 exposure (Fig. 6b). With rapamycin treatment, in which we see protection, pMAPK is significantly upregulated, as well as the total pMAPK/MAPK ratio. Activation of MAPK may also explain why we witnessed a decrease in cleaved caspase-9 and caspase-3 expression, but not cytochrome C release (Fig. 2a, b). MAPK has previously been shown to function downstream of cytochrome c release  and has been shown to interfere specifically with caspase-9 activation . This would explain a reduction in cleaved caspase-9 and caspase-3 expression with rapamycin treatment. MAPK signaling is certainly diverse, and its functions can differ depending on the cell type and context , however, this pro-survival function is in line with the vast majority of studies describing pMAPK signaling in neurons . It is also in line with increased MAPK signaling acting as a protective mechanism against hypoxic insults [57, 62, 63].
In summary, our research indicates that rapamycin confers protection during CoCl2-simulated hypoxia by several mechanisms. At the forefront of these mechanisms are an increase in autophagy and concomitant decrease in apoptosis. While the role of rapamycin acting on autophagy is widely recognized, the action on caspases is more novel and delineation of this response requires further study. Rapamycin-treated cells also experience a reduction in ROS production and restoration of the mitochondrial membrane, two findings highly relevant towards achieving protection against hypoxia. The molecular mechanisms contributing to these gross responses are a decrease in the Bax/Bcl-2 ratio and an increase in pMAPK. Taken together, our results highlight potential pathways outside of mTOR responsible for rapamycin’s protective effects and give credence to rapamycin as a promising agent against neuronal hypoxia.
HT22 is a mouse hippocampal cell line kindly provided by Dr. Jun Panee at the University of Hawaii. HT22 cells were cultured in Dulbecco’s Modified Eagles Medium–High Glucose (GE Healthcare Life Sciences, Marlborough, MA) supplemented with 10% Fetal Bovine Serum, l-glutamine (2 mM), and antibiotics penicillin G and streptomycin (200 units/mL) (Thermo Fisher Scientific, Waltham, MA). Cells were maintained in a % 5 CO2 incubator at 37 °C, and 90–95% humidity. Cells were harvested using a 0.05% trypsin solution (Lonza Bioscience, Walkersville, MD).
Measurement of cell viability via resazurin assay
Cell viability was measured using a water-soluble, indicator dye, resazurin (7-Hydroxy-3H-phenoxazin-3-one 10-oxide), essentially as previously described . Resazurin sodium salt (Acros Organics, Morris Plains, NJ) was dissolved in media at a final concentration of 0.1 mg/mL and 10% volume. This cell-permeable dye is internalized by cultured cells. Actively growing cells metabolize resazurin into a fluorescent form, resorufin. Resultant fluorescence was measured using a PHERAstar Microplate Reader (BMG Labtech, Cary, NC) with a 540-20/590-20 filter. The viability of control cells was arbitrarily set to 100% and the relative fluorescence intensities of experimental groups were converted to relative percentages using the formula: (Relative Fluorescence Intensity of Experimental/Average Relative Fluorescence Intensity of Control) × 100 = % of viable cells.
Cells for western blot analysis were lysed to obtain cytosolic and mitochondrial protein fractions. To obtain these fractions, cells were first lysed in cytosol extraction buffer (250 mM sucrose, 70 mM KCl, 137 mM NaCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4 pH 7.2, and 200 µg/mL digitonin) plus phosphatase and proteinase inhibitors (Pierce and Thermo Fisher Scientific, Waltham, MA) on ice for 5 min. Cells were centrifuged at 1000×g for 5 min at 4 °C, reserving the supernatant as the cytosolic fraction. The cytosolic fraction was further cleared of debris by centrifugation at 20,000×g for 10 min at 4 °C. Meanwhile, the mitochondrial fractions were obtained by incubating the pellet from the first, low-speed centrifugation in two volumes of mitochondrial lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.2% (v/v) Triton X-100, and 0.3% NP-40) plus the above inhibitors.
Where indicated, total cell protein lysates were used for Western blots. To obtain these lysates, cells were incubated on ice for 30 min in RIPA Buffer Solution (Teknova, Hollister, CA) supplemented with the same inhibitors used for cytosolic and mitochondrial fractions. Cells were centrifuged at high speed for 20 min and protein concentrations were measured from the resulting supernatants using standard Bradford Assays (Bio-Rad Laboratories, Hercules, CA).
Protein lysates (20 µg per well) were separated using 4–12% Bis–Tris NuPAGE gels except in the cases of mTOR/phosho-mTOR detection where 3–8% Tris–Acetate NuPAGE gels were used according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). The Bio-Rad Mini Trans-Blot system was used to transfer the separated proteins to PVDF membranes. After transfer, membranes were blocked in a 1:1 solution of Li-COR Odyssey Blocking buffer (Li-COR, Inc., Lincoln, NE) and PBS. Membranes were then probed using the indicated primary antibodies, all obtained from Cell Signaling Technology (Danvers, MA), at 1:1000 dilutions, except in the case of cytosolic loading control β-actin which was diluted 1:5000. IRDye 680LT goat anti-mouse and IRDye 800CW goat anti-rabbit secondary antibodies from Li-COR, Inc (Lincoln, NE) were used at 1:10,000 dilutions for visualization using the Li-COR Odyssey Classic Imaging System scanner. Images obtained using this scanner were analyzed with the Li-COR Image Studio Software version 5.2.5. Fluorescent signals were normalized to loading controls β-actin, or cytochrome C oxidase subunit IV (COX IV) for cytosolic and mitochondrial protein fractions, respectively. Average relative protein expressions of experimental treatment groups were determined by comparison to average expression of the control.
Assay for measurement of reactive oxygen species production
HT22 cells were either untreated or treated for 24 h with 250 µM CoCl2, with and without rapamycin (500 nM), in 96 well plates with cells at around 70% confluence. 5 μM Dihydroethidium (DHE) (Invitrogen, Carlsbad, CA) in DMEM was added during the last 30 min of treatment time with incubation continuing at 37 °C. DHE is a cell permeable dye that becomes oxidized into a fluorescent compound, 2-hydroxyethidium, when the ROS indicator, superoxide, is produced in cells. Increased fluorescence, therefore, corresponds to increased ROS production. At the end of the 24 h treatment time, media was removed and cells were washed twice with PBS. A final volume of 100 µl PBS was added to each well prior to measuring fluorescence using a PHERAstar Microplate Reader with a 590-50/675-50 filter. Background fluorescence was subtracted using additional treatment sets without DHE. To compensate for fluorescence signal changes caused by cell death, resazurin cell viability assays, as described above, were performed in parallel using the same samples used to measure ROS production. Fluorescence measurements were normalized against cell viability to calculate the relative fluorescence values of control versus rapamycin-treated cells in which an increase in fluorescence is indicative of an increase in ROS production.
Assay for measurement of the mitochondrial membrane potential (Δψm)
Measurement of Δψm was performed essentially as previously described . HT22 cells were cultured in 96 well plates to around 70% confluence. Cells were then either untreated or treated with 250 μM CoCl2, with and without rapamycin (500 nM) for 24 h at 37 °C with 500 nM tetramethylrhodamine, methyl ester (TMRM) (Thermo Fisher Scientific, Waltham, MA) being added during the final 30 min of treatment time. During the final 30 min of treatment time, 500 nM tetramethylrhodamine, methyl ester (TMRM) in DMEM medium was added to each well. At the end of treatment time, TMRM-containing media was removed and cells were washed twice with PBS. A final volume of 100 µL PBS was added to each well prior to reading fluorescence with a PHERAstar Microplate Reader (BMG Labtech, Durham, NC) using a 590-50/675-50 filter. Background fluorescence was subtracted using additional treatment sets without TMRM. To compensate for fluorescence changes caused by cell death, resazurin cell viability assays, as described above, were performed in parallel using the same samples used to measure the Δψm. Relative TMRM fluorescence values were calculated by normalizing TMRM fluorescent measurements against cell viability measurements.
Each experiment was repeated at least three times with each individual experiment using triplicate samples. Data are presented as either mean values ± standard deviation (SD), or as a percentage of the control. Statistical analysis was carried out using two-sided t tests, with p-values ≤ 0.05 considered statistically significant.
MZ conceived of the project, performed and analyzed experiments, interpreted results, and wrote the manuscript; CB performed and analyzed experiments, and interpreted results; PL conceived of the project and interpreted results. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.
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Research at BRITE is partially funded by the Golden LEAF Foundation. This funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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