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Oxygen matters: tissue culture oxygen levels affect mitochondrial function and structure as well as responses to HIV viroproteins

Oxygen matters: tissue culture oxygen levels affect mitochondrial function and structure as well... Citation: Cell Death and Disease (2011) 2, e246; doi:10.1038/cddis.2011.128 & 2011 Macmillan Publishers Limited All rights reserved 2041-4889/11 www.nature.com/cddis Oxygen matters: tissue culture oxygen levels affect mitochondrial function and structure as well as responses to HIV viroproteins This article has been corrected since Online Publication and a corrigendum has also been published 1,2 1 1 ,1,2 LM Tiede , EA Cook , B Morsey and HS Fox* Mitochondrial dysfunction is implicated in a majority of neurodegenerative disorders and much study of neurodegenerative disease is done on cultured neurons. In traditional tissue culture, the oxygen level that cells experience is dramatically higher (21%) than in vivo conditions (1–11%). These differences can alter experimental results, especially, pertaining to mitochondria and oxidative metabolism. Our results show that primary neurons cultured at physiological oxygen levels found in the brain showed higher polarization, lower rates of ROS production, larger mitochondrial networks, greater cytoplasmic fractions of mitochondria and larger mitochondrial perimeters than those cultured at higher oxygen levels. Although neurons cultured in either physiological oxygen or atmospheric oxygen exhibit significant increases in mitochondrial reactive oxygen species (ROS) production when treated with the human immunodeficiency virus (HIV) virotoxin trans-activator of transcription, mitochondria of neurons cultured at physiological oxygen underwent depolarization with dramatically increased cell death, whereas those cultured at atmospheric oxygen became hyperpolarized with no increase in cell death. Studies with a second HIV virotoxin, negative regulation factor (Nef), revealed that Nef treatment also increased mitochondrial ROS production for both the oxygen conditions, but resulted in mitochondrial depolarization and increased death only in neurons cultured in physiological oxygen. These results indicate a role for oxidative metabolism in a mechanism of neurotoxicity during HIV infection and demonstrate the importance of choosing the correct, physiological, culture oxygen in mitochondrial studies performed in neurons. Cell Death and Disease (2011) 2, e246; doi:10.1038/cddis.2011.128; published online 22 December 2011 Subject Category: Neuroscience Mitochondria are implicated in a variety of neurodegenerative generation. Though HIV-associated dementia and encepha- conditions including aging, Parkinson’s disease, Huntington’s litis have decreased with successful treatment of HIV by disease, multiple sclerosis and Alzheimer’s disease. A prime combination antiretroviral therapy, cognitive deficits still occur 8,9 function of mitochondria is to reduce oxygen to water during within otherwise healthy HIV-infected individuals have been oxidative phosphorylation to produce adenosine tri-phosphate revealed. Prime candidates for inducing neuronal dysfunction (ATP). Altered levels of oxygen are detected by the mitochon- are viral proteins, as production of early viral products can drion and communicated to the cell as a whole via reactive persist even in the presence of therapy and absence of 1,2 oxygen species (ROS). The vast majority of tissue culture productive viral replication. is currently conducted in incubators with an oxygen tension One such protein, the trans-activator of transcription (Tat), matching that of atmospheric oxygen levels (21%). However, activates transcription of the viral genome with other well- this is much greater than the oxygen levels experienced in vivo. documented functions, and has been shown to be neuro- 10–18 Although exposure to lower physiological oxygen conditions toxic and cause both mitochondrial hyperpolarization 19–23 (2–5%) has been documented to increase survival, proliferation and depolarization. Interestingly, examination in immune 3–5 and dopaminergic differentiation of cultured neurons, most cells studied under various tissue culture oxygen levels experiments continue to be done predominantly at atmospheric revealed that Tat was toxic to lymphocytes cultured in oxygen levels. This transcends the nervous system as it has atmospheric oxygen, but stimulated cells cultured at physio- been found that immune cells, when cultured at physiological logically relevant oxygen. 6,7 oxygen, exhibit markedly altered responses to stimuli. Another viral protein, the ‘negative regulation factor’ (Nef), 25–28 One disconcerting phenomenon associated with persistent can be transferred to uninfected cells and released into 29,30 human immunodeficiency virus (HIV) infection is neurode- the extracellular environment. Nef has also been shown 1 2 Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, USA and Nebraska Center for Virology, University of Nebraska Lincoln, Lincoln, NE, USA *Corresponding author: HS Fox, Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, 985800 Nebraska Medical Center, Omaha, NE 68198-5800, USA. Tel: þ 402 559 4821; Fax: þ 402 559 7495; E-mail: [email protected] Keywords: mitochondria; culture oxygen; neurodegeneration; neurons; HIV Abbreviations: ROS, Reactive oxygen species; HIV, human immunodeficiency virus; Tat, trans-activator of transcription; Nef, negative regulation factor; Dhr123, dyhydrorhodamine 123; ATP, adenosine tri-phosphate; ADP, adenosine di-phosphate; RSNs, rat striatal neurons; Mg, magnesium Received 16.5.11; revised 14.11.11; accepted 14.11.11; Edited by A Finazzi-Agro ´ Tissue culture oxygen levels LM Tiede et al 31–33 to be toxic to a variety of cell types in brain, and found to neurons to 3.8 for neurons cultured in 2% oxygen. These increase ROS production, depolarize mitochondria, and results can be seen in Figure 2a. As such the optimal oxygen increase activated caspase production in astrocytes cultured level was 2% as determined by the maximum mitochondrial under hypoglycemic conditions. membrane potential because high mitochondrial potential Here, we investigated the influence of tissue culture oxygen indicates a greater capacity for ATP production. These levels in neurotoxicity of HIV viroproteins in primary rat striatal results were easily repeatable in three biological replicates. neurons (RSNs). Although levels of ROS production induced by Tat were similar regardless of oxygen levels, effects of Tat on mitochondrial polarization and neuron survival depended Mitochondrial ROS production rates vary with tissue critically on oxygen levels, perhaps contributing to divergent culture oxygen levels. Dihydrorhodamine 123 is a results reported by others and with important ramifications for fluorophore that is localized to mitochondria and fluoresces the mechanism of toxicity. Similarly, Nef treatment increased only upon oxidation and as such can be used to make ROS production regardless of oxygen level, but resulted in comparative measures of the rate of ROS production. The mitochondrial depolarization and increased cell death only in ROS production rate was greatest for the neurons cultured in neurons cultured under physiological oxygen conditions. 5% oxygen (0.18 ADU/min). Lower values were obtained in the atmospheric condition with an ROS production rate deter- mined to be 0.07 ADU/min, and the lowest (0.002 ADU/min) Results in the neurons cultured at 2% oxygen as shown in Figure 2b. As high levels of ROS are damaging to cells, these results Mitochondrial polarization changes with tissue culture indicate that in terms of minimizing ROS production culturing oxygen levels. Mitochondrial polarization is a critical RSNs at 2% oxygen is the most favorable. measure of mitochondrial function and was determined using ratiometric JC-1 imaging. In the presence of high membrane potential, JC-1 forms dimers and the fluorescence shifts from green to red. By assessing the ratio of the red to green fluorescence, mitochondrial potential can be compared between samples with the higher ratio corresponding to a higher mitochondrial membrane potential. The images in Figure 1 clearly indicate that the highest mitochondrial potential in primary RSNs occurred in the 2% oxygen culture condition. The mitochondrial polarization values ranged from 1.33 under uncoupling conditions Uncoupled Atmospheric Oxygen a b 2% Oxygen c d 10µm Figure 1 JC-1 polarization ratio images for uncoupled neurons (a) neurons cultured in atmospheric oxygen (b), 5% oxygen (c) or 2% oxygen (d) Images were obtained by dividing the red JC-1 fluorescence image by the green JC-1 Figure 2 Quantification of effects of culture oxygen on mitochondrial fluorescence image and using a 16-color lookup table. The black indicates a ratio polarization (a) as given by the JC-1 polarization ratio and reactive oxygen species value of 0 (background) while the white represents the maximum polarization. Scale production (b) indicated by the rate of Dhr123 fluorescence increase. *Po0.05, bar indicates 10 mm **Po0.01 and ***Po0.001 for N¼ 3 biological replicates Cell Death and Disease Tissue culture oxygen levels LM Tiede et al Figure 3 Images of neurons labeled with Mitotracker Red that have been cultured in atmospheric (a) 5% oxygen (b) and 2% oxygen (c) as well as quantification of mitochondrial morphology including network size (d), area fraction (e) and perimeter (f) determined from all Mitotracker Red images. *Po0.05 and **Po0.01 for N¼ 3 biological replicates Mitochondrial morphology and abundance differ according shown in Figure 3e, the area fraction constituted 30% in to tissue culture oxygen levels. Mitotracker Red CM-H XRos neurons cultured at 2% oxygen but dropped to 22% cultured in was used to label functional mitochondria in neurons to atmospheric oxygen. For the perimeter (Figure 3f), neurons investigate changes in the mitochondrial distribution and cultured in 2% oxygen were maximal at 290 pixels, whereas morphology (Figure 3). Although many parameters of shape this dropped to 172 pixels at atmospheric oxygen. These and size could be analyzed, we have chosen to focus on results agree with those seen in transmission electron network size, denoting the average size of the extensive microscopy images (Figure 4) where at 2% oxygen the mitochondrial networks in the neurons, perimeter, representing mitochondria were seen as having the greatest length and the outline of the networks, and fraction, which indicates the elongated shapes, whereas those cultured in atmospheric proportion of the area of the image occupied by the oxygen tended to be shorter in length with a higher proportion mitochondria. of rounded mitochondria. The network size is maximal (1066 pixels) in neurons cultured at 2% oxygen, and decreases to 630 pixels in RSNs Mitochondrial polarization changes with Tat treatment. cultured at 5% oxygen and again to 321 pixels in neurons As the neurotoxic effects of Tat are well studied, but cultured in atmospheric oxygen (Figure 3d). The area fraction mechanistic explanations are quite diverse and sometimes and perimeter both follow the same decreasing trend. As contradictory, we first examined the effects of Tat on mito- Cell Death and Disease Tissue culture oxygen levels LM Tiede et al Figure 5 Quantification of effects of Tat on mitochondrial polarization (a)as given by the JC-1 polarization ratio and reactive oxygen species production (b) indicated by the rate of Dhr123 fluorescence increase. *Po0.05 and **Po0.01 for N¼ 3 biological replicates chondria from primary neurons cultured under physiological (2%) and atmospheric (21%) oxygen. As above, the baseline polarization ratio of the controls for the untreated 2% oxygen cultures is significantly higher than that of the atmospheric conditions (Figure 5a). When treated with Tat (100 ng/ml), there was a significant (Po0.01) hyperpolarization of mitochondria in neurons cultured in atmospheric oxygen more than doubling the ± ± polarization ratio from 1.6 0.2 in controls to 3.8 0.5 with Tat treatment (Figure 5a). In contrast, neurons cultured in 2% oxygen level underwent significant (Po0.05) mitochondrial depolarization with Tat treatment, here more than halving the polarization ratio from controls with a 4.3 0.2 ratio to 1.6±0.1 with Tat treatment (Figure 5a). ROS production increases following Tat treatment. In Figure 5b an increase of ROS is clearly indicated by the heightened rate of fluorescence increase in neuronal cultures treated with 100 ng/ml Tat for both 2% oxygen and atmospheric oxygen conditions. The increase is significantly (Po0.01) greater for the case of 2% oxygen Figure 4 Transmission electron microscopy images of mitochondria in neurons than for the cultures maintained in atmospheric oxygen. cultured in atmospheric, 5% and 2% oxygen. Scale bars indicate 100 nm However, this result appears to be primarily the result of the lower ROS production rate observed in the 2% oxygen culture untreated condition compared with the untreated Cell Death and Disease Tissue culture oxygen levels LM Tiede et al atmospheric oxygen cultures, as there is not a statistically Heat-inactivated Nef Nef 10µm significant difference in the rates of ROS production for the Tat-treated cultures. Thus, our work reveals disparate findings regarding the effects of Tat on neurons. Using parallel cultures under different oxygen conditions, non-physiological atmospheric conditions led to hyperpolarization in response to Tat, whereas with physio- logical oxygen depolarization occurred. In both the oxygen conditions ROS production increased, however, the magnitude of the changes is different for the two cases examined here. Mitochondrial polarization changes with Nef treatment under different oxygen conditions. Having demonstrated our ability to find different cellular responses to the virotoxin Tat depending on physiological or non-physiological oxygen conditions, we then turned our attention to Nef. Although Nef is less well studied for its neurotoxic properties, the effects of Nef on infected and uninfected cells has gained significant importance with the recent demonstration of the mechanisms of efficient Nef secretion from infected cells and uptake by uninfected cells and its intracellular transfer from infected to uninfected cells. These both lead to striking in vitro and Figure 6 JC-1 polarization ratio images for neurons cultured in atmospheric in vivo effects on a number of cell types and physiological 26–30 oxygen (a and b) or 2% oxygen (c and d) following treatment with Nef (b and d)or systems. heat-inactivated Nef (a and c). Images were obtained by dividing the red JC-1 We first treated cultures with Nef as well as heat-inactivated fluorescence image by the green JC-1 fluorescence image and using a 16-color Nef as a control. In neuronal cultures maintained in atmo- lookup table. The black indicates a ratio value of 0 (background) while the white spheric oxygen cultures treatment with 100 ng/ml Nef (Figures represents the maximum polarization. Scale bar indicates 10 mm 6a, b and 7a) did not change polarization ratios. In contrast, in cultures maintained at physiological 2% oxygen, the mito- chondrial polarization ratio was significantly (Po0.0001) decreased for the Nef-treated neurons (Figure 6c) compared with the heat-inactivated Nef-treated neurons (Figure 6d) as can be seen in Figure 7a. The control polarization ratio was ± ± 3.8 0.6, dropping to 0.99 0.06 with active Nef. Mitochondrial ROS production increases with Nef treatment. Similar to the results seen with Tat treatment, the rate of mitochondrial ROS production increased upon treatment with Nef for neurons cultured in both 2% oxygen and atmospheric oxygen (Figure 7b; Supplementary Figure 1) when compared with their heat-inactivated Nef controls. The ROS production rates did not vary significantly between the Nef-treated cultures of either oxygen condition, though the basal rates were an order of magnitude lower (0.0004 0.0002 arb/min) for the 2% oxygen controls than for the atmospheric (0.007 0.002 arb/min), indicating a greater increase in ROS production for cells cultured at 2% oxygen when treated with Nef. Both Tat and Nef decrease ATP levels in neurons cultured at physiological oxygen concentrations. As we found difference with both Tat and Nef effects on mitochondria due to oxygen conditions, we next assessed their effects on the primary function of mitochondria, production of ATP. Magnesium (Mg) Green fluoresces 2þ 2þ when bound to Mg . As ATP binds Mg more strongly than adenosine di-phosphate (ADP), Mg Green imaging can Figure 7 Quantification of effects of Nef on mitochondrial polarization (a)as be used to infer changes in the ATP/ADP ratio over time. given by the JC1 polarization ratio and reactive oxygen species production (b) As shown in, the normalized ratio of Mg Green fluorescent indicated by the rate of Dhr123 fluorescence increase. *Po0.05 and **Po0.01 and rates was vastly greater in the 2% oxygen cultures following ***Po0.001 for N¼ 3 biological replicates Cell Death and Disease 2% Oxygen Atmospheric Oxygen Tissue culture oxygen levels LM Tiede et al Tat or Nef treatment than in the atmospheric cultures Increased cell death observed in neurons treated with (Figure 8a). As ATP binds Mg more efficiently than ADP, Tat and Nef cultured at physiological oxygen. Calcein/ the increased free Mg suggests the possibility of more propidium iodide (PI) staining is a valuable tool used to dramatically decreased ATP production in the neurons determine the viability of cultured cells. Calcein is taken up by cultured in 2% oxygen condition upon exposure to Tat or Nef. the cells but is fluorescent only when acted on by esterases Using the luciferase assay as a direct measure of the ATP and indicates enzyme activity considered to be a marker of concentration, we determined that there was a significant healthy cells. PI is a membrane-impermeable dye that labels (Po0.01) increase in the amount of ATP from 191 3 nmol/ DNA, and as such cells exhibiting PI fluorescence have lost 10 000 cells to 253 16 nmol/10 000 cells following 8 h of membrane integrity and are considered to be dead or dying. treatment with 100 ng/ml Nef in atmospheric oxygen Neurons cultured in atmospheric oxygen and treated with Nef (Figure 8b) and no significant change upon treatment with exhibited a significant (Po0.01) increase in cell death only Tat. For the neurons cultured in 2% oxygen, however, the when treated with the highest level (100 ng/ml) of Nef concentration of ATP significantly (Po0.05) decreased from compared with heat-inactivated controls (Figure 9a), and ± ± 200 6 nM/10 000 cells to 184 2 nM/10 000 cells with an 8-h none at all upon treatment with any concentration of Tat. treatment with 100 ng/ml Nef. Strikingly, there was a dramatic However, neurons cultured in physiological 2% oxygen change found upon treatment of neurons cultured in 2% exhibited a dose-dependent response to Tat and Nef even oxygen with 100 ng/ml Tat where ATP concentration dropped in cultures treated with as little as 25 ng/ml of Tat or Nef to 168 6 nmol/10 000 cells following treatment with Tat. (Figure 9b). Figure 8 Changes in ATP following Tat or Nef treatment were assessed in two different ways: using magnesium green to measure the changes in free magnesium Figure 9 Comparison of the percent of cell death indicated by Calcein and PI concentration (a) that can indicate changes in the ADP/ATP ratio, and the luciferase staining of neurons cultured with atmospheric oxygen (a) or 2% oxygen (b) when assay (b) that directly measures the amount of ATP present in a sample. *Po0.05 treated with Tat, Nef or heat-inactivated Nef. *Po0.05 and **Po0.01 and and **Po0.01 for N¼ 3 biological replicates ***Po0.0001 for a minimum of N¼ 3 biological replicates Cell Death and Disease Tissue culture oxygen levels LM Tiede et al Discussion of neurons to Tat remains the same regardless of the oxygen conditions, which has not been proven at this point. It is clear from our results that the unchallenged state of the Owing to the recent burst of finding on the effects of Nef on mitochondria in primary neurons differs depending on the 18,29 uninfected cells, the demonstration of the physiological oxygen conditions during cell culture. This is not unreasonable 29,30 secretion of Nef from infected cells, and the relative given the delicate balance that must be maintained in order to paucity of studies on Nef and neurons, we then focused our maximize energy production and minimize the damage from studies on Nef. For Nef there was no effect of the protein on ROS in mitochondria. Our results indicate that there is a state the polarization of mitochondria in neurons cultured in in which maximal mitochondrial function is achieved with atmospheric oxygen, but the decrease of potential in the 2% minimal ROS production, maximal energy capacity and oxygen neuron cultures was equal to that of neurons treated increased mitochondrial network size and the fraction of the with 10 mM carbonyl cyanide m-chlorophenylhydrazone cell occupied by mitochondria. As the result is in the range of (Figure 2) to cause uncoupling of oxidative phosphorylation. oxygen levels expected in the striatum of the rat brain, it This level of mitochondrial uncoupling usually results in seems reasonable to conclude that the optimal mitochondrial gradual cell death if maintained for prolonged periods. It is conditions correspond most closely to the native state of the possible that the observed cell death can be attributed directly mitochondria in vivo. to the loss of ATP production that occurred with the dramatic The use of less than atmospheric oxygen in culture can loss of mitochondrial membrane potential experienced by Nef- raise the question of a hypoxic threshold. It is reasonable to treated cultures maintained in 2% oxygen levels. Although the expect that there exist different optimal oxygen levels for the mechanism of this cell death was not directly studied, during cells derived from different sources corresponding to their calcein/PI imaging, condensation of chromatin and evidence natural oxygen level in the organism. In our case 2% oxygen is of apoptotic bodies were observed (data not shown), within the range of oxygen levels expected in the striatum of indicating that apoptosis is the likely manner of neuron death the rat brain. Although decreased polarization of mitochondria resulting from Nef treatment. and increased ROS production are typically noted in studies of As we observed little or no cell death in the atmospheric 36,37 hypoxia, the opposite was observed in the RSNs cultured oxygen cultures treated with Nef and Tat compared with the in 2% oxygen. Taken together with our other results make it 2% oxygen cultures while ROS production increased in both highly unlikely that the 2% oxygen culture condition repre- culture conditions, it is reasonable to conclude that ROS sents a hypoxic case for these particular primary cells. production is not solely responsible for the Nef-induced For Tat, determining the mechanism of toxicity of this HIV- toxicity observed in the 2% oxygen cultures. Rather it is more associated protein has generated a large amount of data that likely that the increased ROS is more detrimental because of has been contradictory at times. Tat has been shown to both the decreased ATP levels resulting from Nef treatment that is 19–23 hyperpolarize and depolarize mitochondria, and we have critical in repair and replacement of damaged lipids and clearly shown that this is indeed the case for RSNs that have proteins. As the neurons cultured in atmospheric oxygen been cultured in differing oxygen conditions. Although the exhibited increased ATP levels, it is likely that these cells had hyperpolarization result has become more accepted in recent the energy capacity necessary to make needed repairs to literature, this result may not have physiological relevance as damaged proteins and organelles preventing cell death. indicated by the depolarization of neurons cultured in 2% Therefore, one proposed mechanism for the Nef-induced oxygen, which is similar to the neuron’s in vivo environment. toxicity is that decreased energy production resulting from It is important to note that culture oxygen is not likely the mitochondrial depolarization by Nef results in a reduced cause of the divergent results reported by others; however, capacity to repair proteins damaged by increased levels of our results clearly indicate that perturbations in the culture ROS and results in eventual apoptosis only in the case of conditions can dramatically alter the response of neuron neurons cultured in 2% oxygen (Figure 10). mitochondria to Tat. One caveat that applies to functional imaging studies is that Although ROS production rates were increased by Tat there are potential issues with the dye used. In the case of treatment in both the cells cultured at 2% oxygen and JC-1 permanent dimerization has sometimes been noted in atmospheric oxygen, the increase was more dramatic in the the literature. Upon uncoupling with FCCP after making initial cells cultured in 2% oxygen. Coupled with the loss of ATP measurements the JC-1 polarization ratios always returned production (see Figure 9) resulting from such dramatic loss of within error to the levels obtained from administering dye to mitochondrial potential, the ROS production could thus be the neurons after uncoupling. Also, experimental results were extremely detrimental under these more physiological condi- duplicated using the dye tetramethylrhodamine, ethyl ester tions. It is also important to note that the increase in ROS (TMRE), though TMRE provided less reproducibility from production may be derived from disruptions of oxidative sample to sample and week to week. In addition, in the case of phosphorylation resulting in increased production of ROS dyhydrorhodamine 123 (Dhr123), the accumulation of dye in rather than an unrelated effect of Tat for both the cases of mitochondria is dependent upon the potential of the mitochon- hyperpolarization and depolarization. Taken together, it dria. Although this can sometimes skew the results, as we appears that neurons are more strongly affected by Tat than obtained the lowest levels of fluorescence with the highest previous studies have led us to believe, and that mitochondrial levels of polarization, this does not seem to be an issue in this dysfunction may further complicate other processes already study. Also as all time courses showed an increase in noted to be involved in Tat-related toxicity in neurons. fluorescence over time, quenching did not appear to be a However, this mechanism requires that the initial response significant contributor to the results. Cell Death and Disease Atmospheric O Tissue culture oxygen levels LM Tiede et al Nef ROS ROS Mitochondria Mitochondria Unchanged Mitochondrial Potential Decreased Mitochondrial Potential ATP Production Likely Unchanged Decreased ATP Production Increased Oxidative Damage Increased Oxidative damage Repair damaged Neuronal Death proteins Figure 10 Diagram of proposed mechanism of Nef toxicity for neurons cultured in atmospheric oxygen as well as physiological (2%) oxygen indicating the pathway to repair the oxidatively damaged proteins or neuronal death, respectively, beginning with alterations in the native mitochondrial structure, function and protein content It is notable that Mg Green fluorescence was increased that could limit the translation of knowledge from tissue culture upon treatment with Tat and Nef in both atmospheric and 2% to in vivo cases. cultures though ATP levels only dropped significantly in the The fact that there are differences in not only the responses case of the 2% cultures. This indicates that there is another of cultured neurons to HIV-associated proteins, but in the source of free Mg resulting from treatment with Tat and Nef natural states of the neurons depending on culture oxygen that is separate from decreased ATP levels and common to levels may not be immediately obvious. However, it is both the culture conditions, correlating with the increased important to remember that oxygen, although necessary to ROS observed in all cultures treated with Tat and Nef. neuron survival, is inherently toxic. Furthermore neurons, like The road from discovery to application can be extraordina- most other cells, have the ability to adapt to their environment rily complicated in the case of disease and treatment. Things for survival in culture. Hence, cells cultured in atmospheric learned in cultured cells have to be confirmed in animal oxygen may well be expected to have increased capacity to models. Treatments of disease must be translated from the reduce ROS via increased protein levels of glutathione, laboratory to the clinic. There are many opportunities along superoxide dismutase and other ROS scavengers. The ratios this road for things to break down. In order to minimize the of the different metabolic proteins and their corresponding disruptions and failures in attempts to bring successful subunits may also differ in order to regulate energy production treatments to the clinic, it is important that the model systems under varying oxygen levels. Further experiments are are able to accurately represent the disease state, so that the necessary to determine whether this does indeed occur and mechanisms of disease discovered in these models are what relevance it might have to the responses of neurons not representative of the natural disease state. Our results show only to HIV-associated proteins but also other neuropathic that use of the appropriate oxygen condition reveals not only processes. small effects noted in the atmospheric condition as in the case of Nef, but also that vastly different results can occur as was Materials and Methods apparent in the treatment of neurons with Tat. The vastly Neuron harvest and tissue culture. Primary neurons were isolated from different mitochondrial response of cultured neurons to Tat rat striatum from E18 embryos (Brain Bits, Springfield, IL, USA) by trituration and could be extremely misleading in the understanding of the centrifugation according to the suggested protocol. Cells were counted and plated process of Tat toxicity, a perfect example of a complication in 35 mm, poly-D-lysine-coated dishes (MatTek, Ashland, MA, USA) with No. 1.5 Cell Death and Disease Physiological O 2 Tissue culture oxygen levels LM Tiede et al coverslip buttoms at 5 10 cells per dish for imaging or in poly-D-lysine-coated Neurobasal medium to obtain a final concentrations of 25, 50, 75 and 100 ng/ml. 6-well plates at 2 10 cells per well for western blots. Cells were cultured in HIV Nef protein (NIH AIDS Research and Reference Reagent Program) solutions Neurobasal MEM supplemented with B-27 and L-glutamate. Our initial studies were prepared in the same manner and diluted in media to final concentrations of examined a range of oxygen conditions that are generally considered physiological. 25, 50, 75 and 100 ng/ml. For imaging studies of mitochondrial potential, structure After evaluating mitochondrial morphology and function in neurons cultured at and ROS production, neurons were incubated in 100 ng/ml Tat or 100 ng/ml Nef for atmospheric, 9, 5 and 2% oxygen, we determined that 9% oxygen was not 1 h before the beginning of imaging. For toxicity curves, neurons were incubated significantly different than the atmospheric condition and all of our data here focuses with Neurobasal media containing Tat or Nef for 72 h. Nef that had been heat on atmospheric, 5 and 2% oxygen conditions. For the atmospheric condition, cells inactivated at 100 1C overnight was used as a negative control in all Nef were cultured in a standard tissue culture incubator containing 5% carbon dioxide, experiments and deoxygenated water alone was used as a control in the whereas for the 2 and 5% oxygen condition neurons were placed in a chamber experiments involving Tat. (Billups-Rothenberg, Del Mar, CA, USA) charged with a gas mixture containing 2% oxygen or 5% oxygen, 5% carbon dioxide and the remainder nitrogen. Oxygen Luciferase assay. Cells were treated for 24 h with Tat, Nef, or the heat- levels in culture were confirmed using an oxygen electrode. Media were inactivated controls. They were lifted, centrifuged at 7000 r.p.m. and flash frozen half-exchanged after 5 days and then every 3–4 days thereafter until neurons had before storing at 801C. After thawing, samples were treated and analyzed been in culture for 14 days. Unless otherwise noted materials were obtained from according to provided protocols for the ATP Determination Kit (Invitrogen). Sigma-Aldrich (St. Louis, MO, USA). Fixation for electron microscopy. Neurons were cultured for 14 days Imaging. Cells were incubated in the appropriate dye and then imaged on a according to the conditions above. Media were removed and coverslips were Nikon swept-field confocal microscope using a  60 oil-immersion (1.45 NA) washed three times with PBS before fixation in 3% paraformaldehyde, 1.5% objective. A live cell imaging chamber (Pathology Devices, Inc., Westminster, MD, glutaraldehyde, 0.1 M sodium cacodylate buffer at a pH of 7.4, and post fixed in 1% USA) was used to maintain appropriate temperature, oxygen and carbon dioxide osmium tetraoxide in 0.10 M sodium cacodylate buffer for 30 min. Afterwards conditions during imaging (media oxygenation confirmed using an oxygen coverslips were dehydrated through an ethanol series and embedded using araldite electrode). For measuring mitochondrial polarization, cells were incubated in and polymerized overnight at 651C before sectioning. Thin sections were stained 2 nM JC-1 (Invitrogen, Carlsbad, CA, USA) for 20 min. Images were acquired using with uranyl acetate and Reynold’s lead citrate before examination on a FEI Tecnai a 488-nm excitation, and 536/40-nm and 593/40-nm emission filters for the green G2 transmission electron microscope operated at 80 kV. monomers and red dimers, respectively. To examine the rate of ROS produced in the mitochondria, neurons were incubated in 10 mM Dhr123 (Invitrogen) for 20 min Conflict of Interest before imaging. The excitation wavelength used was again 488 nm with a 536/40-nm fluorescence emission filter. Images were taken at a rate of 1/min for The authors declare no conflict of interest. 10 min before a new region of interest (ROI) was selected. For mitochondrial morphology experiments cells were incubated in 25 nM Mitotracker Red CM- H XRos (Invitrogen) for 20 min. Images were acquired using 568 nm excitation Acknowledgements. This research was supported by the National Institutes wavelength and a 593/40-nm emission filter was used to collect the resulting of Health grants MH073490 and MH062261, Nebraska Tobacco Settlement fluorescence. Biomedical Research Development Funds, through a fellowship under Ruth L For calcein and PI staining, cells were incubated in 10 mM calcein (Invitrogen) Kirschstein National Research Service Award 5 T32 AI060547 from the National with 10 mM PI (Invitrogen) for 30 min. Cells were imaged on a Zeiss Cell Observer Institute of Allergy and Infectious Diseases (PI Dr. Charles Wood, Nebraska Center microscope (Carl Zeiss, Thornwood, NY, USA) with appropriate filters to determine for Virology, University of Nebraska at Lincoln) and National Center for Research whether cells exhibited the presence or absence of calcein and PI staining. Grant Number P20 RR16469 supporting the NE-INBRE Scholars program (PI Dr. James Turpen, UNMC). This is manuscript #11 from the UNMC Center for Image analysis. To determine membrane polarization, the ratio of red to green Integrative and Translational Neuroscience. We thank Dr. Howard Gendelman for fluorescence of JC-1 images was calculated using NIH Image J. The outline of the use of the swept-field confocal microscope and comments on the manuscript. individual cells was circled using the freehand tool to create ROIs and saved in the We would also like to thank the Core Electron Microscopy Research Facility at the multimeasure tool. The average intensity of the same ROIs was analyzed in both the University of Nebraska Medical Center and Tom Bargar for the use of and expertize red and green channels. Then the ratio was obtained for each region and the with the transmission electron microscopy. The following reagents were obtained average of the ratio was obtained over all ROIs for all images. Polarization images through the NIH AIDS Research and Reference Reagent Program, Division of were obtained by dividing the red image by the green image and by selecting a AIDS, NIAID, NIH: HIV-1 Nef (Catalogue no. 11478) and HIV-1 Tat (Catalogue 16-color lookup table. no. 2222, from Dr. John Brady and DAIDS, NIAID). To calculate the rate of ROS production in neurons cultured under various conditions, Dhr123 time series were analyzed in Image J. As previously described, ROIs were selected and then propagated through the stack using the multimeasure 1. Chandel NS, Maltepe E, Goldwasser E, Mathieu CE, Simon MC, Schumacker PT. tool. Average fluorescence values were normalized to the first image ROI. Then all Mitochondrial reactive oxygen species trigger hypoxia-induced transcription. Proc Natl normalized values were averaged at each time point. These average values were Acad Sci USA 1998; 95: 11715–11720. plotted and then analyzed with a linear fit to determine the slope. The slope was 2. 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Human immunodeficiency virus-1 Tat protein and methamphetamine interact synergistically to published by Nature Publishing Group. This work is impair striatal dopaminergic function. J Neurochem 2002; 83: 955–963. licensed under the Creative Commons Attribution-Noncommercial-Share 24. Sahaf B, Atkuri K, Heydari K, Malipatlolla M, Rappaport J, Regulier E et al. Culturing of Alike 3.0 Unported License. To view a copy of this license, visit http:// human peripheral blood cells reveals unsuspected lymphocyte responses relevant to HIV disease. Proc Natl Acad Sci USA 2008; 105: 5111–5116. creativecommons.org/licenses/by-nc-sa/3.0/ Supplementary Information accompanies the paper on Cell Death and Disease website (http://www.nature.com/cddis) Cell Death and Disease http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cell Death & Disease Springer Journals

Oxygen matters: tissue culture oxygen levels affect mitochondrial function and structure as well as responses to HIV viroproteins

Tiede, L M; Cook, E A; Morsey, B; Fox, H S
Cell Death & Disease , Volume 2 (12) – Dec 22, 2011
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Life Sciences; Life Sciences, general; Biochemistry, general; Cell Biology; Immunology; Cell Culture; Antibodies
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10.1038/cddis.2011.128
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Abstract

Citation: Cell Death and Disease (2011) 2, e246; doi:10.1038/cddis.2011.128 & 2011 Macmillan Publishers Limited All rights reserved 2041-4889/11 www.nature.com/cddis Oxygen matters: tissue culture oxygen levels affect mitochondrial function and structure as well as responses to HIV viroproteins This article has been corrected since Online Publication and a corrigendum has also been published 1,2 1 1 ,1,2 LM Tiede , EA Cook , B Morsey and HS Fox* Mitochondrial dysfunction is implicated in a majority of neurodegenerative disorders and much study of neurodegenerative disease is done on cultured neurons. In traditional tissue culture, the oxygen level that cells experience is dramatically higher (21%) than in vivo conditions (1–11%). These differences can alter experimental results, especially, pertaining to mitochondria and oxidative metabolism. Our results show that primary neurons cultured at physiological oxygen levels found in the brain showed higher polarization, lower rates of ROS production, larger mitochondrial networks, greater cytoplasmic fractions of mitochondria and larger mitochondrial perimeters than those cultured at higher oxygen levels. Although neurons cultured in either physiological oxygen or atmospheric oxygen exhibit significant increases in mitochondrial reactive oxygen species (ROS) production when treated with the human immunodeficiency virus (HIV) virotoxin trans-activator of transcription, mitochondria of neurons cultured at physiological oxygen underwent depolarization with dramatically increased cell death, whereas those cultured at atmospheric oxygen became hyperpolarized with no increase in cell death. Studies with a second HIV virotoxin, negative regulation factor (Nef), revealed that Nef treatment also increased mitochondrial ROS production for both the oxygen conditions, but resulted in mitochondrial depolarization and increased death only in neurons cultured in physiological oxygen. These results indicate a role for oxidative metabolism in a mechanism of neurotoxicity during HIV infection and demonstrate the importance of choosing the correct, physiological, culture oxygen in mitochondrial studies performed in neurons. Cell Death and Disease (2011) 2, e246; doi:10.1038/cddis.2011.128; published online 22 December 2011 Subject Category: Neuroscience Mitochondria are implicated in a variety of neurodegenerative generation. Though HIV-associated dementia and encepha- conditions including aging, Parkinson’s disease, Huntington’s litis have decreased with successful treatment of HIV by disease, multiple sclerosis and Alzheimer’s disease. A prime combination antiretroviral therapy, cognitive deficits still occur 8,9 function of mitochondria is to reduce oxygen to water during within otherwise healthy HIV-infected individuals have been oxidative phosphorylation to produce adenosine tri-phosphate revealed. Prime candidates for inducing neuronal dysfunction (ATP). Altered levels of oxygen are detected by the mitochon- are viral proteins, as production of early viral products can drion and communicated to the cell as a whole via reactive persist even in the presence of therapy and absence of 1,2 oxygen species (ROS). The vast majority of tissue culture productive viral replication. is currently conducted in incubators with an oxygen tension One such protein, the trans-activator of transcription (Tat), matching that of atmospheric oxygen levels (21%). However, activates transcription of the viral genome with other well- this is much greater than the oxygen levels experienced in vivo. documented functions, and has been shown to be neuro- 10–18 Although exposure to lower physiological oxygen conditions toxic and cause both mitochondrial hyperpolarization 19–23 (2–5%) has been documented to increase survival, proliferation and depolarization. Interestingly, examination in immune 3–5 and dopaminergic differentiation of cultured neurons, most cells studied under various tissue culture oxygen levels experiments continue to be done predominantly at atmospheric revealed that Tat was toxic to lymphocytes cultured in oxygen levels. This transcends the nervous system as it has atmospheric oxygen, but stimulated cells cultured at physio- been found that immune cells, when cultured at physiological logically relevant oxygen. 6,7 oxygen, exhibit markedly altered responses to stimuli. Another viral protein, the ‘negative regulation factor’ (Nef), 25–28 One disconcerting phenomenon associated with persistent can be transferred to uninfected cells and released into 29,30 human immunodeficiency virus (HIV) infection is neurode- the extracellular environment. Nef has also been shown 1 2 Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, USA and Nebraska Center for Virology, University of Nebraska Lincoln, Lincoln, NE, USA *Corresponding author: HS Fox, Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, 985800 Nebraska Medical Center, Omaha, NE 68198-5800, USA. Tel: þ 402 559 4821; Fax: þ 402 559 7495; E-mail: [email protected] Keywords: mitochondria; culture oxygen; neurodegeneration; neurons; HIV Abbreviations: ROS, Reactive oxygen species; HIV, human immunodeficiency virus; Tat, trans-activator of transcription; Nef, negative regulation factor; Dhr123, dyhydrorhodamine 123; ATP, adenosine tri-phosphate; ADP, adenosine di-phosphate; RSNs, rat striatal neurons; Mg, magnesium Received 16.5.11; revised 14.11.11; accepted 14.11.11; Edited by A Finazzi-Agro ´ Tissue culture oxygen levels LM Tiede et al 31–33 to be toxic to a variety of cell types in brain, and found to neurons to 3.8 for neurons cultured in 2% oxygen. These increase ROS production, depolarize mitochondria, and results can be seen in Figure 2a. As such the optimal oxygen increase activated caspase production in astrocytes cultured level was 2% as determined by the maximum mitochondrial under hypoglycemic conditions. membrane potential because high mitochondrial potential Here, we investigated the influence of tissue culture oxygen indicates a greater capacity for ATP production. These levels in neurotoxicity of HIV viroproteins in primary rat striatal results were easily repeatable in three biological replicates. neurons (RSNs). Although levels of ROS production induced by Tat were similar regardless of oxygen levels, effects of Tat on mitochondrial polarization and neuron survival depended Mitochondrial ROS production rates vary with tissue critically on oxygen levels, perhaps contributing to divergent culture oxygen levels. Dihydrorhodamine 123 is a results reported by others and with important ramifications for fluorophore that is localized to mitochondria and fluoresces the mechanism of toxicity. Similarly, Nef treatment increased only upon oxidation and as such can be used to make ROS production regardless of oxygen level, but resulted in comparative measures of the rate of ROS production. The mitochondrial depolarization and increased cell death only in ROS production rate was greatest for the neurons cultured in neurons cultured under physiological oxygen conditions. 5% oxygen (0.18 ADU/min). Lower values were obtained in the atmospheric condition with an ROS production rate deter- mined to be 0.07 ADU/min, and the lowest (0.002 ADU/min) Results in the neurons cultured at 2% oxygen as shown in Figure 2b. As high levels of ROS are damaging to cells, these results Mitochondrial polarization changes with tissue culture indicate that in terms of minimizing ROS production culturing oxygen levels. Mitochondrial polarization is a critical RSNs at 2% oxygen is the most favorable. measure of mitochondrial function and was determined using ratiometric JC-1 imaging. In the presence of high membrane potential, JC-1 forms dimers and the fluorescence shifts from green to red. By assessing the ratio of the red to green fluorescence, mitochondrial potential can be compared between samples with the higher ratio corresponding to a higher mitochondrial membrane potential. The images in Figure 1 clearly indicate that the highest mitochondrial potential in primary RSNs occurred in the 2% oxygen culture condition. The mitochondrial polarization values ranged from 1.33 under uncoupling conditions Uncoupled Atmospheric Oxygen a b 2% Oxygen c d 10µm Figure 1 JC-1 polarization ratio images for uncoupled neurons (a) neurons cultured in atmospheric oxygen (b), 5% oxygen (c) or 2% oxygen (d) Images were obtained by dividing the red JC-1 fluorescence image by the green JC-1 Figure 2 Quantification of effects of culture oxygen on mitochondrial fluorescence image and using a 16-color lookup table. The black indicates a ratio polarization (a) as given by the JC-1 polarization ratio and reactive oxygen species value of 0 (background) while the white represents the maximum polarization. Scale production (b) indicated by the rate of Dhr123 fluorescence increase. *Po0.05, bar indicates 10 mm **Po0.01 and ***Po0.001 for N¼ 3 biological replicates Cell Death and Disease Tissue culture oxygen levels LM Tiede et al Figure 3 Images of neurons labeled with Mitotracker Red that have been cultured in atmospheric (a) 5% oxygen (b) and 2% oxygen (c) as well as quantification of mitochondrial morphology including network size (d), area fraction (e) and perimeter (f) determined from all Mitotracker Red images. *Po0.05 and **Po0.01 for N¼ 3 biological replicates Mitochondrial morphology and abundance differ according shown in Figure 3e, the area fraction constituted 30% in to tissue culture oxygen levels. Mitotracker Red CM-H XRos neurons cultured at 2% oxygen but dropped to 22% cultured in was used to label functional mitochondria in neurons to atmospheric oxygen. For the perimeter (Figure 3f), neurons investigate changes in the mitochondrial distribution and cultured in 2% oxygen were maximal at 290 pixels, whereas morphology (Figure 3). Although many parameters of shape this dropped to 172 pixels at atmospheric oxygen. These and size could be analyzed, we have chosen to focus on results agree with those seen in transmission electron network size, denoting the average size of the extensive microscopy images (Figure 4) where at 2% oxygen the mitochondrial networks in the neurons, perimeter, representing mitochondria were seen as having the greatest length and the outline of the networks, and fraction, which indicates the elongated shapes, whereas those cultured in atmospheric proportion of the area of the image occupied by the oxygen tended to be shorter in length with a higher proportion mitochondria. of rounded mitochondria. The network size is maximal (1066 pixels) in neurons cultured at 2% oxygen, and decreases to 630 pixels in RSNs Mitochondrial polarization changes with Tat treatment. cultured at 5% oxygen and again to 321 pixels in neurons As the neurotoxic effects of Tat are well studied, but cultured in atmospheric oxygen (Figure 3d). The area fraction mechanistic explanations are quite diverse and sometimes and perimeter both follow the same decreasing trend. As contradictory, we first examined the effects of Tat on mito- Cell Death and Disease Tissue culture oxygen levels LM Tiede et al Figure 5 Quantification of effects of Tat on mitochondrial polarization (a)as given by the JC-1 polarization ratio and reactive oxygen species production (b) indicated by the rate of Dhr123 fluorescence increase. *Po0.05 and **Po0.01 for N¼ 3 biological replicates chondria from primary neurons cultured under physiological (2%) and atmospheric (21%) oxygen. As above, the baseline polarization ratio of the controls for the untreated 2% oxygen cultures is significantly higher than that of the atmospheric conditions (Figure 5a). When treated with Tat (100 ng/ml), there was a significant (Po0.01) hyperpolarization of mitochondria in neurons cultured in atmospheric oxygen more than doubling the ± ± polarization ratio from 1.6 0.2 in controls to 3.8 0.5 with Tat treatment (Figure 5a). In contrast, neurons cultured in 2% oxygen level underwent significant (Po0.05) mitochondrial depolarization with Tat treatment, here more than halving the polarization ratio from controls with a 4.3 0.2 ratio to 1.6±0.1 with Tat treatment (Figure 5a). ROS production increases following Tat treatment. In Figure 5b an increase of ROS is clearly indicated by the heightened rate of fluorescence increase in neuronal cultures treated with 100 ng/ml Tat for both 2% oxygen and atmospheric oxygen conditions. The increase is significantly (Po0.01) greater for the case of 2% oxygen Figure 4 Transmission electron microscopy images of mitochondria in neurons than for the cultures maintained in atmospheric oxygen. cultured in atmospheric, 5% and 2% oxygen. Scale bars indicate 100 nm However, this result appears to be primarily the result of the lower ROS production rate observed in the 2% oxygen culture untreated condition compared with the untreated Cell Death and Disease Tissue culture oxygen levels LM Tiede et al atmospheric oxygen cultures, as there is not a statistically Heat-inactivated Nef Nef 10µm significant difference in the rates of ROS production for the Tat-treated cultures. Thus, our work reveals disparate findings regarding the effects of Tat on neurons. Using parallel cultures under different oxygen conditions, non-physiological atmospheric conditions led to hyperpolarization in response to Tat, whereas with physio- logical oxygen depolarization occurred. In both the oxygen conditions ROS production increased, however, the magnitude of the changes is different for the two cases examined here. Mitochondrial polarization changes with Nef treatment under different oxygen conditions. Having demonstrated our ability to find different cellular responses to the virotoxin Tat depending on physiological or non-physiological oxygen conditions, we then turned our attention to Nef. Although Nef is less well studied for its neurotoxic properties, the effects of Nef on infected and uninfected cells has gained significant importance with the recent demonstration of the mechanisms of efficient Nef secretion from infected cells and uptake by uninfected cells and its intracellular transfer from infected to uninfected cells. These both lead to striking in vitro and Figure 6 JC-1 polarization ratio images for neurons cultured in atmospheric in vivo effects on a number of cell types and physiological 26–30 oxygen (a and b) or 2% oxygen (c and d) following treatment with Nef (b and d)or systems. heat-inactivated Nef (a and c). Images were obtained by dividing the red JC-1 We first treated cultures with Nef as well as heat-inactivated fluorescence image by the green JC-1 fluorescence image and using a 16-color Nef as a control. In neuronal cultures maintained in atmo- lookup table. The black indicates a ratio value of 0 (background) while the white spheric oxygen cultures treatment with 100 ng/ml Nef (Figures represents the maximum polarization. Scale bar indicates 10 mm 6a, b and 7a) did not change polarization ratios. In contrast, in cultures maintained at physiological 2% oxygen, the mito- chondrial polarization ratio was significantly (Po0.0001) decreased for the Nef-treated neurons (Figure 6c) compared with the heat-inactivated Nef-treated neurons (Figure 6d) as can be seen in Figure 7a. The control polarization ratio was ± ± 3.8 0.6, dropping to 0.99 0.06 with active Nef. Mitochondrial ROS production increases with Nef treatment. Similar to the results seen with Tat treatment, the rate of mitochondrial ROS production increased upon treatment with Nef for neurons cultured in both 2% oxygen and atmospheric oxygen (Figure 7b; Supplementary Figure 1) when compared with their heat-inactivated Nef controls. The ROS production rates did not vary significantly between the Nef-treated cultures of either oxygen condition, though the basal rates were an order of magnitude lower (0.0004 0.0002 arb/min) for the 2% oxygen controls than for the atmospheric (0.007 0.002 arb/min), indicating a greater increase in ROS production for cells cultured at 2% oxygen when treated with Nef. Both Tat and Nef decrease ATP levels in neurons cultured at physiological oxygen concentrations. As we found difference with both Tat and Nef effects on mitochondria due to oxygen conditions, we next assessed their effects on the primary function of mitochondria, production of ATP. Magnesium (Mg) Green fluoresces 2þ 2þ when bound to Mg . As ATP binds Mg more strongly than adenosine di-phosphate (ADP), Mg Green imaging can Figure 7 Quantification of effects of Nef on mitochondrial polarization (a)as be used to infer changes in the ATP/ADP ratio over time. given by the JC1 polarization ratio and reactive oxygen species production (b) As shown in, the normalized ratio of Mg Green fluorescent indicated by the rate of Dhr123 fluorescence increase. *Po0.05 and **Po0.01 and rates was vastly greater in the 2% oxygen cultures following ***Po0.001 for N¼ 3 biological replicates Cell Death and Disease 2% Oxygen Atmospheric Oxygen Tissue culture oxygen levels LM Tiede et al Tat or Nef treatment than in the atmospheric cultures Increased cell death observed in neurons treated with (Figure 8a). As ATP binds Mg more efficiently than ADP, Tat and Nef cultured at physiological oxygen. Calcein/ the increased free Mg suggests the possibility of more propidium iodide (PI) staining is a valuable tool used to dramatically decreased ATP production in the neurons determine the viability of cultured cells. Calcein is taken up by cultured in 2% oxygen condition upon exposure to Tat or Nef. the cells but is fluorescent only when acted on by esterases Using the luciferase assay as a direct measure of the ATP and indicates enzyme activity considered to be a marker of concentration, we determined that there was a significant healthy cells. PI is a membrane-impermeable dye that labels (Po0.01) increase in the amount of ATP from 191 3 nmol/ DNA, and as such cells exhibiting PI fluorescence have lost 10 000 cells to 253 16 nmol/10 000 cells following 8 h of membrane integrity and are considered to be dead or dying. treatment with 100 ng/ml Nef in atmospheric oxygen Neurons cultured in atmospheric oxygen and treated with Nef (Figure 8b) and no significant change upon treatment with exhibited a significant (Po0.01) increase in cell death only Tat. For the neurons cultured in 2% oxygen, however, the when treated with the highest level (100 ng/ml) of Nef concentration of ATP significantly (Po0.05) decreased from compared with heat-inactivated controls (Figure 9a), and ± ± 200 6 nM/10 000 cells to 184 2 nM/10 000 cells with an 8-h none at all upon treatment with any concentration of Tat. treatment with 100 ng/ml Nef. Strikingly, there was a dramatic However, neurons cultured in physiological 2% oxygen change found upon treatment of neurons cultured in 2% exhibited a dose-dependent response to Tat and Nef even oxygen with 100 ng/ml Tat where ATP concentration dropped in cultures treated with as little as 25 ng/ml of Tat or Nef to 168 6 nmol/10 000 cells following treatment with Tat. (Figure 9b). Figure 8 Changes in ATP following Tat or Nef treatment were assessed in two different ways: using magnesium green to measure the changes in free magnesium Figure 9 Comparison of the percent of cell death indicated by Calcein and PI concentration (a) that can indicate changes in the ADP/ATP ratio, and the luciferase staining of neurons cultured with atmospheric oxygen (a) or 2% oxygen (b) when assay (b) that directly measures the amount of ATP present in a sample. *Po0.05 treated with Tat, Nef or heat-inactivated Nef. *Po0.05 and **Po0.01 and and **Po0.01 for N¼ 3 biological replicates ***Po0.0001 for a minimum of N¼ 3 biological replicates Cell Death and Disease Tissue culture oxygen levels LM Tiede et al Discussion of neurons to Tat remains the same regardless of the oxygen conditions, which has not been proven at this point. It is clear from our results that the unchallenged state of the Owing to the recent burst of finding on the effects of Nef on mitochondria in primary neurons differs depending on the 18,29 uninfected cells, the demonstration of the physiological oxygen conditions during cell culture. This is not unreasonable 29,30 secretion of Nef from infected cells, and the relative given the delicate balance that must be maintained in order to paucity of studies on Nef and neurons, we then focused our maximize energy production and minimize the damage from studies on Nef. For Nef there was no effect of the protein on ROS in mitochondria. Our results indicate that there is a state the polarization of mitochondria in neurons cultured in in which maximal mitochondrial function is achieved with atmospheric oxygen, but the decrease of potential in the 2% minimal ROS production, maximal energy capacity and oxygen neuron cultures was equal to that of neurons treated increased mitochondrial network size and the fraction of the with 10 mM carbonyl cyanide m-chlorophenylhydrazone cell occupied by mitochondria. As the result is in the range of (Figure 2) to cause uncoupling of oxidative phosphorylation. oxygen levels expected in the striatum of the rat brain, it This level of mitochondrial uncoupling usually results in seems reasonable to conclude that the optimal mitochondrial gradual cell death if maintained for prolonged periods. It is conditions correspond most closely to the native state of the possible that the observed cell death can be attributed directly mitochondria in vivo. to the loss of ATP production that occurred with the dramatic The use of less than atmospheric oxygen in culture can loss of mitochondrial membrane potential experienced by Nef- raise the question of a hypoxic threshold. It is reasonable to treated cultures maintained in 2% oxygen levels. Although the expect that there exist different optimal oxygen levels for the mechanism of this cell death was not directly studied, during cells derived from different sources corresponding to their calcein/PI imaging, condensation of chromatin and evidence natural oxygen level in the organism. In our case 2% oxygen is of apoptotic bodies were observed (data not shown), within the range of oxygen levels expected in the striatum of indicating that apoptosis is the likely manner of neuron death the rat brain. Although decreased polarization of mitochondria resulting from Nef treatment. and increased ROS production are typically noted in studies of As we observed little or no cell death in the atmospheric 36,37 hypoxia, the opposite was observed in the RSNs cultured oxygen cultures treated with Nef and Tat compared with the in 2% oxygen. Taken together with our other results make it 2% oxygen cultures while ROS production increased in both highly unlikely that the 2% oxygen culture condition repre- culture conditions, it is reasonable to conclude that ROS sents a hypoxic case for these particular primary cells. production is not solely responsible for the Nef-induced For Tat, determining the mechanism of toxicity of this HIV- toxicity observed in the 2% oxygen cultures. Rather it is more associated protein has generated a large amount of data that likely that the increased ROS is more detrimental because of has been contradictory at times. Tat has been shown to both the decreased ATP levels resulting from Nef treatment that is 19–23 hyperpolarize and depolarize mitochondria, and we have critical in repair and replacement of damaged lipids and clearly shown that this is indeed the case for RSNs that have proteins. As the neurons cultured in atmospheric oxygen been cultured in differing oxygen conditions. Although the exhibited increased ATP levels, it is likely that these cells had hyperpolarization result has become more accepted in recent the energy capacity necessary to make needed repairs to literature, this result may not have physiological relevance as damaged proteins and organelles preventing cell death. indicated by the depolarization of neurons cultured in 2% Therefore, one proposed mechanism for the Nef-induced oxygen, which is similar to the neuron’s in vivo environment. toxicity is that decreased energy production resulting from It is important to note that culture oxygen is not likely the mitochondrial depolarization by Nef results in a reduced cause of the divergent results reported by others; however, capacity to repair proteins damaged by increased levels of our results clearly indicate that perturbations in the culture ROS and results in eventual apoptosis only in the case of conditions can dramatically alter the response of neuron neurons cultured in 2% oxygen (Figure 10). mitochondria to Tat. One caveat that applies to functional imaging studies is that Although ROS production rates were increased by Tat there are potential issues with the dye used. In the case of treatment in both the cells cultured at 2% oxygen and JC-1 permanent dimerization has sometimes been noted in atmospheric oxygen, the increase was more dramatic in the the literature. Upon uncoupling with FCCP after making initial cells cultured in 2% oxygen. Coupled with the loss of ATP measurements the JC-1 polarization ratios always returned production (see Figure 9) resulting from such dramatic loss of within error to the levels obtained from administering dye to mitochondrial potential, the ROS production could thus be the neurons after uncoupling. Also, experimental results were extremely detrimental under these more physiological condi- duplicated using the dye tetramethylrhodamine, ethyl ester tions. It is also important to note that the increase in ROS (TMRE), though TMRE provided less reproducibility from production may be derived from disruptions of oxidative sample to sample and week to week. In addition, in the case of phosphorylation resulting in increased production of ROS dyhydrorhodamine 123 (Dhr123), the accumulation of dye in rather than an unrelated effect of Tat for both the cases of mitochondria is dependent upon the potential of the mitochon- hyperpolarization and depolarization. Taken together, it dria. Although this can sometimes skew the results, as we appears that neurons are more strongly affected by Tat than obtained the lowest levels of fluorescence with the highest previous studies have led us to believe, and that mitochondrial levels of polarization, this does not seem to be an issue in this dysfunction may further complicate other processes already study. Also as all time courses showed an increase in noted to be involved in Tat-related toxicity in neurons. fluorescence over time, quenching did not appear to be a However, this mechanism requires that the initial response significant contributor to the results. Cell Death and Disease Atmospheric O Tissue culture oxygen levels LM Tiede et al Nef ROS ROS Mitochondria Mitochondria Unchanged Mitochondrial Potential Decreased Mitochondrial Potential ATP Production Likely Unchanged Decreased ATP Production Increased Oxidative Damage Increased Oxidative damage Repair damaged Neuronal Death proteins Figure 10 Diagram of proposed mechanism of Nef toxicity for neurons cultured in atmospheric oxygen as well as physiological (2%) oxygen indicating the pathway to repair the oxidatively damaged proteins or neuronal death, respectively, beginning with alterations in the native mitochondrial structure, function and protein content It is notable that Mg Green fluorescence was increased that could limit the translation of knowledge from tissue culture upon treatment with Tat and Nef in both atmospheric and 2% to in vivo cases. cultures though ATP levels only dropped significantly in the The fact that there are differences in not only the responses case of the 2% cultures. This indicates that there is another of cultured neurons to HIV-associated proteins, but in the source of free Mg resulting from treatment with Tat and Nef natural states of the neurons depending on culture oxygen that is separate from decreased ATP levels and common to levels may not be immediately obvious. However, it is both the culture conditions, correlating with the increased important to remember that oxygen, although necessary to ROS observed in all cultures treated with Tat and Nef. neuron survival, is inherently toxic. Furthermore neurons, like The road from discovery to application can be extraordina- most other cells, have the ability to adapt to their environment rily complicated in the case of disease and treatment. Things for survival in culture. Hence, cells cultured in atmospheric learned in cultured cells have to be confirmed in animal oxygen may well be expected to have increased capacity to models. Treatments of disease must be translated from the reduce ROS via increased protein levels of glutathione, laboratory to the clinic. There are many opportunities along superoxide dismutase and other ROS scavengers. The ratios this road for things to break down. In order to minimize the of the different metabolic proteins and their corresponding disruptions and failures in attempts to bring successful subunits may also differ in order to regulate energy production treatments to the clinic, it is important that the model systems under varying oxygen levels. Further experiments are are able to accurately represent the disease state, so that the necessary to determine whether this does indeed occur and mechanisms of disease discovered in these models are what relevance it might have to the responses of neurons not representative of the natural disease state. Our results show only to HIV-associated proteins but also other neuropathic that use of the appropriate oxygen condition reveals not only processes. small effects noted in the atmospheric condition as in the case of Nef, but also that vastly different results can occur as was Materials and Methods apparent in the treatment of neurons with Tat. The vastly Neuron harvest and tissue culture. Primary neurons were isolated from different mitochondrial response of cultured neurons to Tat rat striatum from E18 embryos (Brain Bits, Springfield, IL, USA) by trituration and could be extremely misleading in the understanding of the centrifugation according to the suggested protocol. Cells were counted and plated process of Tat toxicity, a perfect example of a complication in 35 mm, poly-D-lysine-coated dishes (MatTek, Ashland, MA, USA) with No. 1.5 Cell Death and Disease Physiological O 2 Tissue culture oxygen levels LM Tiede et al coverslip buttoms at 5 10 cells per dish for imaging or in poly-D-lysine-coated Neurobasal medium to obtain a final concentrations of 25, 50, 75 and 100 ng/ml. 6-well plates at 2 10 cells per well for western blots. Cells were cultured in HIV Nef protein (NIH AIDS Research and Reference Reagent Program) solutions Neurobasal MEM supplemented with B-27 and L-glutamate. Our initial studies were prepared in the same manner and diluted in media to final concentrations of examined a range of oxygen conditions that are generally considered physiological. 25, 50, 75 and 100 ng/ml. For imaging studies of mitochondrial potential, structure After evaluating mitochondrial morphology and function in neurons cultured at and ROS production, neurons were incubated in 100 ng/ml Tat or 100 ng/ml Nef for atmospheric, 9, 5 and 2% oxygen, we determined that 9% oxygen was not 1 h before the beginning of imaging. For toxicity curves, neurons were incubated significantly different than the atmospheric condition and all of our data here focuses with Neurobasal media containing Tat or Nef for 72 h. Nef that had been heat on atmospheric, 5 and 2% oxygen conditions. For the atmospheric condition, cells inactivated at 100 1C overnight was used as a negative control in all Nef were cultured in a standard tissue culture incubator containing 5% carbon dioxide, experiments and deoxygenated water alone was used as a control in the whereas for the 2 and 5% oxygen condition neurons were placed in a chamber experiments involving Tat. (Billups-Rothenberg, Del Mar, CA, USA) charged with a gas mixture containing 2% oxygen or 5% oxygen, 5% carbon dioxide and the remainder nitrogen. Oxygen Luciferase assay. Cells were treated for 24 h with Tat, Nef, or the heat- levels in culture were confirmed using an oxygen electrode. Media were inactivated controls. They were lifted, centrifuged at 7000 r.p.m. and flash frozen half-exchanged after 5 days and then every 3–4 days thereafter until neurons had before storing at 801C. After thawing, samples were treated and analyzed been in culture for 14 days. Unless otherwise noted materials were obtained from according to provided protocols for the ATP Determination Kit (Invitrogen). Sigma-Aldrich (St. Louis, MO, USA). Fixation for electron microscopy. Neurons were cultured for 14 days Imaging. Cells were incubated in the appropriate dye and then imaged on a according to the conditions above. Media were removed and coverslips were Nikon swept-field confocal microscope using a  60 oil-immersion (1.45 NA) washed three times with PBS before fixation in 3% paraformaldehyde, 1.5% objective. A live cell imaging chamber (Pathology Devices, Inc., Westminster, MD, glutaraldehyde, 0.1 M sodium cacodylate buffer at a pH of 7.4, and post fixed in 1% USA) was used to maintain appropriate temperature, oxygen and carbon dioxide osmium tetraoxide in 0.10 M sodium cacodylate buffer for 30 min. Afterwards conditions during imaging (media oxygenation confirmed using an oxygen coverslips were dehydrated through an ethanol series and embedded using araldite electrode). For measuring mitochondrial polarization, cells were incubated in and polymerized overnight at 651C before sectioning. Thin sections were stained 2 nM JC-1 (Invitrogen, Carlsbad, CA, USA) for 20 min. Images were acquired using with uranyl acetate and Reynold’s lead citrate before examination on a FEI Tecnai a 488-nm excitation, and 536/40-nm and 593/40-nm emission filters for the green G2 transmission electron microscope operated at 80 kV. monomers and red dimers, respectively. To examine the rate of ROS produced in the mitochondria, neurons were incubated in 10 mM Dhr123 (Invitrogen) for 20 min Conflict of Interest before imaging. The excitation wavelength used was again 488 nm with a 536/40-nm fluorescence emission filter. Images were taken at a rate of 1/min for The authors declare no conflict of interest. 10 min before a new region of interest (ROI) was selected. For mitochondrial morphology experiments cells were incubated in 25 nM Mitotracker Red CM- H XRos (Invitrogen) for 20 min. Images were acquired using 568 nm excitation Acknowledgements. This research was supported by the National Institutes wavelength and a 593/40-nm emission filter was used to collect the resulting of Health grants MH073490 and MH062261, Nebraska Tobacco Settlement fluorescence. Biomedical Research Development Funds, through a fellowship under Ruth L For calcein and PI staining, cells were incubated in 10 mM calcein (Invitrogen) Kirschstein National Research Service Award 5 T32 AI060547 from the National with 10 mM PI (Invitrogen) for 30 min. Cells were imaged on a Zeiss Cell Observer Institute of Allergy and Infectious Diseases (PI Dr. Charles Wood, Nebraska Center microscope (Carl Zeiss, Thornwood, NY, USA) with appropriate filters to determine for Virology, University of Nebraska at Lincoln) and National Center for Research whether cells exhibited the presence or absence of calcein and PI staining. Grant Number P20 RR16469 supporting the NE-INBRE Scholars program (PI Dr. James Turpen, UNMC). This is manuscript #11 from the UNMC Center for Image analysis. To determine membrane polarization, the ratio of red to green Integrative and Translational Neuroscience. We thank Dr. Howard Gendelman for fluorescence of JC-1 images was calculated using NIH Image J. The outline of the use of the swept-field confocal microscope and comments on the manuscript. individual cells was circled using the freehand tool to create ROIs and saved in the We would also like to thank the Core Electron Microscopy Research Facility at the multimeasure tool. The average intensity of the same ROIs was analyzed in both the University of Nebraska Medical Center and Tom Bargar for the use of and expertize red and green channels. Then the ratio was obtained for each region and the with the transmission electron microscopy. The following reagents were obtained average of the ratio was obtained over all ROIs for all images. 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Human immunodeficiency virus-1 Tat protein and methamphetamine interact synergistically to published by Nature Publishing Group. This work is impair striatal dopaminergic function. J Neurochem 2002; 83: 955–963. licensed under the Creative Commons Attribution-Noncommercial-Share 24. Sahaf B, Atkuri K, Heydari K, Malipatlolla M, Rappaport J, Regulier E et al. Culturing of Alike 3.0 Unported License. To view a copy of this license, visit http:// human peripheral blood cells reveals unsuspected lymphocyte responses relevant to HIV disease. Proc Natl Acad Sci USA 2008; 105: 5111–5116. creativecommons.org/licenses/by-nc-sa/3.0/ Supplementary Information accompanies the paper on Cell Death and Disease website (http://www.nature.com/cddis) Cell Death and Disease

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