Mild acidosis protects neurons during oxygen-glucose deprivation by

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Mild acidosis protects neurons during oxygen-glucose deprivation by reducing loss of mitochondrial respiration Mingyue Zhu, Dongliang Zhang, Chen Zhou, and Zhen Chai ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00737 • Publication Date (Web): 05 Mar 2019 Downloaded from http://pubs.acs.org on March 6, 2019

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ACS Chemical Neuroscience

Mild acidosis protects neurons during oxygen-glucose deprivation by reducing loss of mitochondrial respiration

Ming-Yue Zhu, Dong-Liang Zhang, Chen Zhou* and Zhen Chai* State Key Laboratory of Membrane Biology, School of Life Sciences, Peking University, Beijing 100871, China

*Corresponding authors: Zhen Chai, State Key Laboratory of Membrane Biology, College of Life Sciences, Peking University, Beijing 100871, China. Tel: 86-10-62757830; Fax: 86-10-62751526; Email: [email protected]

Chen Zhou, State Key Laboratory of Membrane Biology, College of Life Sciences, Peking University, Beijing 100871, China. Tel: 86-10-62765106; Fax: 86-10-62765106; Email: [email protected]

Running title Mild acidosis protects mitochondrial respiration during neuronal ischemia

Keywords mild acidosis, ischemia, ATP, respiration, mitochondria, neuron

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Abstract Brain ischemia is often accompanied by brain acidosis and this acidosis can affect ischemic neuronal injury. Ischemic neuronal injury is initiated by a decrease in ATP production which mainly relies on mitochondrial oxidative phosphorylation. Ischemia often causes mitochondrial dysfunction, and acidosis has been found to affect mitochondrial function, suggesting that acidosis accompanying ischemia may influence neurons by targeting mitochondrial metabolism. However, the effects of acidosis on mitochondrial energy metabolism during ischemia lacks thorough investigation. Here, we found that mild acidosis significantly reduced neuronal death possibly by slowing the process of ATP deprivation during oxygen-glucose deprivation (OGD), an in-vitro ischemic model. The maintaining of neuronal ATP depended on protecting mitochondrial ATP production. Further investigation of mitochondrial function revealed that mild acidosis alleviated OGD-induced collapse of mitochondrial membrane potentials as well as damage to respiratory function, at least in part by reducing impacts on complex I and II activities. Inhibition of complex I activity aggravated neuronal death, which suggests that the contribution of mild acidosis to maintaining complex I activity promoted neuronal survival during OGD. Our findings reveal maintaining mitochondrial respiration as a new possible protective mechanism of mild acidosis during ischemia, on neurons.

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Introduction Ischemic stroke is currently one of the leading causes of human death, but routine clinical treatments are still limited to restoring blood supply using recombinant tissue plasminogen activator or removing blood clots by mechanical thrombectomy1. Neuroprotective therapies have not yet become standard clinical practice2, possibly due to a limited understanding of the processes and mechanisms involved in ischemic injury. Brain acidosis is an important satellite phenomenon during ischemia. Ischemic brain tissue can reach pH 6.5 and can even drop as low as pH 6.0 depending on ischemic severity and blood glucose level3–5. However, the influence of acidosis on ischemic neuronal injury remains controversial and its mechanisms require further investigation. Severe acidosis can cause detrimental effects by activating acid-sensing ion channels, decreasing antioxidant activity, and stressing the endoplasmic reticulum6–9. However, mild acidosis often plays a protective role against ischemia by inhibiting NMDA receptors10,11. One study which partially explained this contradiction found that acidosis caused acidotoxicity but eliminated excitotoxicity during ischemia12. In addition to inhibiting NMDA receptors, mild acidosis also protects neurons by targeting NADPH oxidase and mitochondria13–15.

Ischemia damages mitochondria, resulting in deficient energy metabolism and neuronal death16,17. Ischemia decreases mitochondrial membrane potentials, damages electron transport complexes and mtDNA, alters mitochondrial membrane permeability, and fragments mitochondria, all of which affect mitochondrial respiratory function18–23. Protecting or restoring the supply of ATP and processes protecting mitochondria such as mitophagy, which decreases the proportion of damaged mitochondria, and mitochondrial biogenesis, which produces healthy mitochondria can reduce neuronal death from ischemia24–28. Previous studies have highlighted the possibility that acidosis can attenuate ischemic injury by targeting mitochondria14,15,18,29. Transient acidic postconditioning, applied by inhalation of 20% CO2, was found to alleviate ischemia/reperfusion-induced 3



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collapse of mitochondrial membrane potentials, to inhibit opening of mitochondrial permeability transition pores (mPTP), and to facilitate mitophagy14,29. Another study demonstrated that mild acidosis can promote mitochondrial elongation during hypoxia to protect neurons15. Acid-sensing ion channel 1a are also located in mitochondria and are suspected to be an important mPTP regulator30.

Despite the important role of mitochondrial function in ischemic injury and the observed effects of acidosis on mitochondria, there is limited research on the protective effects of mild acidosis during ischemia on mitochondrial metabolism. Studies have only shown that mild acidosis can promote the elongation of neuronal mitochondria and reprogram mitochondrial metabolic pathways during hypoxia15. However, the effects of mild acidosis on mitochondrial metabolism during severe ischemia remain unclear. To investigate this question, we used oxygen-glucose deprivation (OGD), a classic in-vitro model of ischemia, to study mitochondrial ATP generation and respiration under neutral and mildly acidic conditions.

Results and Discussion

Mild acidosis attenuates OGD-induced reduction of mitochondrial ATP production Brain acidosis accompanies ischemia and can affect not only neurons31,32 but also other cell types in the brain33,34. In order to study the influence of acidosis on neurons and exclude the indirect impacts from other cells, we used primary cultured rat cortical neurons in our experiments. During severe ischemic injury, such as global ischemia, brain oxygen and glucose levels decrease to near-zero35; this condition is well-modeled by OGD. OGD is the most commonly used in-vitro ischemic model and has provided useful and reliable insight into the mechanisms underlying ischemia36. Previous research has shown that mild acidosis can alleviate OGD-induced neuronal injury10,11. We confirmed this protective effect of mild acidosis (pH 6.5) with an LDH assay used to evaluate neuronal cell death. Mild acidosis significantly reduced LDH release during OGD and the reperfusion period (Fig. 1A). 4


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Acidosis alone did not increase LDH release (Fig. 1A), possibly due to the limited severity and longevity of acidosis acting on primary cultured neurons. To rule out the possibility that the solutions were acidized during OGD, the pH of the treating solutions after 3 h OGD treatment were measured. The pH values were 7.33±0.01 and 6.52±0.01 in pH 7.4 group and pH 6.5 group respectively. The results reveal that HEPES in the solutions could be sufficient to maintain the pH 7.4 or 6.5. Insufficient energy supply initiates ischemic injury16. To investigate whether the protective effect of mild acidosis is related to energy metabolism, we measured neuronal ATP levels after different durations of OGD treatment. Results showed that neuronal ATP levels decreased soon after OGD initiation, but mild acidosis significantly slowed the process of ATP deprivation (Fig. 1B). We found that there were not significant differences of the amount of protein between pH 7.4 and 6.5 groups during OGD (Fig. S1). This data suggested that the changes of ATP level were resulted from the changes of ATP content in neuron rather than cell number. Neuronal ATP level depends on ATP consumption and generation. Protein synthesis and neuronal excitability are two important contributors to neuronal ATP consumption37,38. We used 100 µg/ml cycloheximide (CHX) to inhibit protein synthesis15,39, and 1 µM tetrodotoxin (TTX) to inhibit action potential firing38,40. Results showed that inhibiting protein synthesis elevated neuronal ATP level after 1 h OGD, while the elevation of neuronal ATP by mild acidosis was similar in the presence or absence of CHX (Fig. S2). Inhibiting action potential firing did not significantly affect neuronal ATP levels in either neutral or acidic conditions (Fig. S3). These results suggest that mild acidosis during OGD does not increase neuronal ATP by reducing ATP consumption through the processes of inhibiting protein synthesis or neuronal excitability. Under normal circumstances, mitochondrial respiration supplies most neuronal ATP, while during ischemia the proportion provided by glycolysis increases41. Further investigation was required to determine whether mild acidosis protects neuronal ATP production and the possible mechanisms underlying such a function.

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Fig 1. Mild acidosis reduced the impacts of OGD on neuronal ATP level. (A) Mild acidosis during OGD reduced neuronal death. The figure shows relative LDH release during 3 h OGD and 3 h reperfusion. One-way ANOVA, *P < 0.05 vs pH 6.5; **P < 0.01 vs pH 7.4; ##P < 0.01 vs OGD/Reperfusion pH 7.4. n = 3 independent experiments. (B) Mild acidosis slowed OGDinduced neuronal ATP decrease. The figure shows neuronal ATP levels measured after the indicated treatments. Data are normalized to the “1 h pH 7.4” treatment. Two-way ANOVA, **P < 0.01 pH 7.4 vs OGD pH 7.4 or pH 6.5 vs OGD pH 6.5; ##P < 0.01 OGD pH 7.4 vs OGD pH 6.5. n = 3 independent experiments.

First, we used mitochondrial respiration inhibitors to test whether ATP generation in our OGD model depends on mitochondria and what proportion of ATP the mitochondria produce. We use three different compounds to inhibit mitochondrial ATP production. FCCP is a common mitochondrial uncoupling agent used to decrease mitochondrial inner membrane potential. Oligomycin is a complex V (ATP synthase) inhibitor used to inhibit ATP synthesis. Antimycin A is a complex III inhibitor used to inhibit electron transport. We measured neuronal ATP level after 1 h or 2 h OGD at pH 7.4 or pH 6.5. The results show that all three compounds decreased neuronal ATP levels to nearly zero at both pH 7.4 and pH 6.5 (Fig. 2A). These results show that neuronal ATP production still mainly depends on mitochondrial respiration even during OGD under both neutral and acidic conditions. To further test whether mild acidosis increases mitochondrial ATP generation during OGD, we measured ATP in mitochondria. Since we found that neither mild acidosis nor OGD changed mitochondrial mass, based on the protein level of mitochondrial outer membrane protein TOM20 (Fig. 2B), we normalized the ATP levels by mitochondrial protein concentration to exclude different mitochondrial isolation efficiencies in the different groups. The results show that 1 h or 2 h OGD caused obvious decreases in 6


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mitochondrial ATP at pH 7.4, while mild acidosis (pH 6.5) significantly improved this phenomenon (Fig. 2C). Mild acidosis restored mitochondrial ATP production during OGD injury. To investigate the relationship between the effects of mild acidosis on mitochondrial ATP production and neuronal cell survival, we used FCCP to decrease the efficiencies of mitochondrial ATP production under acidic conditions. 5 nM FCCP did not reduce neuronal ATP production at pH 6.5 under normoxic conditions, but it did reduce neuronal ATP production at pH 6.5 during OGD to a level similar to that at pH 7.4 (Fig. S4A). Consistent with the results for neuronal ATP level, 5 nM FCCP did not cause neuronal death at pH 6.5 under normoxic conditions, but did increase neuronal death at pH 6.5 during OGD and reperfusion injury to a level similar to that at pH 7.4 (Fig. S4B). These results suggest that mild acidosis protects neurons during ischemia by maintaining mitochondrial ATP production.

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Fig 2. Mild acidosis limited reductions in mitochondrial ATP generation during OGD. (A) ATP generation mainly depended on mitochondrial respiration during OGD. The figure shows neuronal ATP level measured after 1 h (top) or 2 h (bottom) OGD treatment. Data were normalized with “pH 7.4 (no OGD)” treatment. FCCP, mitochondrial uncoupling agent, 1 μM. Oligo (Oligomycin), complex V inhibitor, 2.5 μM. AA (Antimycin A), complex III inhibitor, 2.5 μM. One-way ANOVA, **P < 0.01 vs pH 7.4; ##P < 0.01 vs pH 6.5. n = 3 independent experiments. (B) Mild acidosis did not affect mitochondrial mass. The figure shows western blot analyses of TOM20 and β-actin protein level after 1 h (top) or 2 h (bottom) treatment. Data were normalized with pH 7.4. One-way ANOVA, P > 0.05. n = 4 independent experiments. (C) Mild acidosis reduced the impacts of OGD on mitochondrial ATP level. The figure shows mitochondrial ATP levels normalized respectively by isolated mitochondrial protein concentrations after 1 h (left) or 2 h (right) treatment. Data were normalized with pH 7.4. One-

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way ANOVA, **P < 0.01 pH 7.4 vs OGD pH 7.4 or pH 6.5 vs OGD pH 6.5; #P < 0.05 vs OGD pH 7.4. n = 4 independent experiments.

Previous studies have shown that mild acidosis increases mitochondrial ATP production during hypoxia by reprogramming mitochondrial metabolism15, which is consistent with our results. However, these studies also showed that a significant neuronal ATP decrease required more than 6 h hypoxia treatment at neutral pH, while our OGD model showed a significant ATP decrease within 1 h OGD treatment. This difference suggests that OGD and hypoxia produce very different energy metabolism conditions for neurons. Other studies support our hypothesis, revealing severe mitochondrial dysfunction caused by OGD but not hypoxia42. In consideration of this obvious difference between OGD and hypoxia, we further investigated how mild acidosis influences mitochondrial function during OGD.

Mild acidosis attenuates OGD-induced mitochondrial dysfunction Mitochondrial membrane potential is an important indicator of mitochondrial function. We measured mitochondrial membrane potential using TMRE and found that mild acidosis significantly attenuated OGD-induced decreases in mitochondrial membrane potential (Fig. 3A). Mitochondrial membrane potential mainly depends on the proton gradient across the mitochondrial inner membrane, which is generated by mitochondrial respiration43. To directly assess respiratory function, we measured neuronal oxygen-consumption rate (OCR). First, we measured basal respiration. The basal respiration rate decreased after OGD at pH 7.4, while mild acidosis restored respiration to a near-normal level (Fig. 3B). We also measured mitochondrial respiratory capacity by adding FCCP, which pushes mitochondria toward their maximal respiration rate. The maximal rate decreased after OGD at both pH 7.4 and pH 6.5, but mild acidosis greatly attenuated this decrease (Fig. 3B). Rotenone and antimycin A, the inhibitors of complex I and complex III respectively, were added to completely inhibit mitochondrial respiration so that non-mitochondrial respiration could be measured. The results show that non-mitochondrial OCR accounted for only a 9



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very small portion of neuronal OCR (Fig. 3B). It is rational to use neuronal OCR to evaluate mitochondrial respiratory function.

Fig 3. Mild acidosis attenuated OGD-induced mitochondrial dysfunction. (A) Mitochondrial membrane potential. The figure shows TMRE fluorescence imaging after indicated treatments (left) and the quantification of TMRE fluorescence (right). Two-way ANOVA, **P < 0.01 pH 7.4 vs OGD pH 7.4 or pH 6.5 vs OGD pH 6.5; ##P < 0.01 OGD pH 7.4 vs OGD pH 6.5. n = 16 imaging fields from 4 independent experiments. (B) Neuronal OCR measurements. FCCP, 2 μM. AA, 0.5 μM. Rot (rotenone), 0.5 μM. Basal respiration is the average of the first through the forth data points. Maximal respiration is the average of the fifth through the seventh data points. One-way ANOVA, *P < 0.05 vs pH 6.5; **P < 0.01 vs pH 7.4; #P < 0.05 vs pH 7.4; ##P < 0.01 vs OGD pH 7.4. n = 40 samples from 4 independent experiments.

Collapse of mitochondrial membrane potential and damage to respiratory function are common consequences of ischemic injury17 and both were reproduced in our OGD model. Other research has shown that mild acidosis can promote mitochondrial energy metabolism15,44, but it has not been investigated whether mild acidosis can attenuate 10


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ischemia-induced mitochondrial respiratory damage. Our results clearly show that mild acidosis treatment for 2 h did not alter mitochondrial respiration under normoxic conditions, but it did attenuate mitochondrial respiratory damage caused by OGD injury. Increasing mitophagy to eliminate damaged mitochondria26,45 or promoting mitochondrial biogenesis to produce healthy mitochondria27,28 can play protective roles in ischemia. Mitophagy should be accompanied by a decrease in mitochondrial mass whereas biogenesis should be accompanied by an increase. Acidic postconditioning after ischemia has been shown to protect against ischemic injury by promoting mitophagy. However, in our experiments, we did not find differences in mitochondrial mass between the different treatments (Fig. 2B). Acid-sensing ion channels (ASICs) are important receptors responding to acidification. ASIC1a is not only a calcium-permeable ion channel on cytomembrane6 but also an important mPTP regulator located on inner mitochondrial membrane30. However, inhibiting ASIC1a using 100 ng/ml Psalmotoxin (PcTx1)6 did not significantly change neuronal viability at pH 6.5 in our OGD model (65.9%±8.5% without PcTx1 and 63.4%±12.0% with PcTx1), which suggested that ASIC1a was not involved in the effect of mild acidosis in our experiments. Mild acidosis attenuates OGD-induced decreases in mitochondrial complex I and II activities Next, we investigated how mild acidosis attenuates mitochondrial respiratory damage during OGD. Electrons move from mitochondrial complex I or II to complex III, and eventually to complex IV, where electrons are donated to oxygen causing oxygen consumption43. Since mild acidosis was found to attenuate OGD-induced OCR decreases in basal and maximal respiration, we further examined the influence of mild acidosis on the activities of complexes in the mitochondrial electron transport chain. The activities of the mitochondrial complexes were detected by measuring neuronal OCR. This was carried out using saponin to permeabilize neuronal membranes so that the specific substrates for each complex could directly reach the mitochondria (Fig. S5). The activities of complexes 11



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I, II, and III decreased after 2 h OGD treatment at neutral pH, while the activity of complex IV remained steady (Fig. 4). This may be due to the sensitivity of complexes I, II, and III, which all contain an Fe-S center, to the massive quantities of reactive oxygen species (ROS) that are generated during OGD46. Mild acidosis can alleviate OGD-induced damage to complexes I and II (Fig. 4A, B), which are complexes that influence the entry of electrons into the electron transport chain. We did not observe changes in complex I protein levels (Fig. S6) between the neutral and mild acidosis conditions, which suggests that mild acidosis improves complex I activity by a mechanism other than increasing protein level.

Fig 4. Mild acidosis attenuated OGD-induced decreases in mitochondrial complex I and II activities. (A) complex I activity. n = 19 samples from 4 independent experiments. (B) complex II activity. n = 16-18 samples from 4 independent experiments. (C) complex III activity. n = 1315 samples from 3 independent experiments. (D) complex IV activity. n = 14-15 samples from 3 independent experiments. One-way ANOVA. *P < 0.05 pH 7.4 vs OGD pH 7.4 or pH 6.5 vs OGD pH 6.5. **P < 0.01 vs pH 7.4. #P < 0.05; ##P < 0.01 vs OGD pH 7.4.

Inhibiting complex I or complex II causes neuronal death47,48. To evaluate the relationship between the protective effects of mild acidosis and mitochondrial complex activity, we used rotenone to inhibit complex I activity. Rotenone 25 nM causes partial inhibition of complex I to a level less than 50%49. This concentration of rotenone alone did not cause 12


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neuronal death, but it significantly increased neuronal death in combination with OGD treatment (Fig. 5). Completely inhibiting complex I activity with 1 μM rotenone induced obvious neuronal death (Fig. 5). 1 μM rotenone along with OGD treatment at pH 7.4 did not further increase neuronal death compared with the 25 nM rotenone concentration (Fig. 5). One possible explanation for this phenomenon is that OGD treatment and 25 nM rotenone together caused near complete inhibition of complex I activity, while 1 μM rotenone cannot further enhance this inhibitory effect. However, 1 μM rotenone added to the OGD pH 6.5 condition further increased neuronal death compared with 25 nM rotenone (Fig. 5). This may be because OGD together with 25 nM rotenone only partially inhibited mitochondrial complex I activity at pH 6.5, while 1 μM rotenone further increased the inhibitory effect. We observed no difference in neuronal death between pH 7.4 and pH 6.5 when complex I was completely inhibited with 1 μM rotenone during OGD treatment (Fig. 5). These results suggest that the protection effect of mild acidosis on complex I activity contributed to alleviation of neuronal death.

Fig 5. Effect of complex I inhibitor on neuronal death. Relative LDH release during 3 h OGD treatment and 3 h reperfusion. One-way ANOVA, **P < 0.01; ns P > 0.05. n = 4 independent experiments.

Ischemic injury can cause the activities of mitochondrial complexes to decrease19,20,42. Previous studies have shown that acidosis can increase the protein levels of several complexes during hypoxia15 or increase the activity of complex IV50 during OGD. These 13



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studies and our results, which show that mild acidosis attenuates OGD-induced reductions in the activities of mitochondrial complexes I and II, demonstrate that acidosis protected mitochondrial function during OGD by targeting electron transport chain complexes. Impaired mitochondrial energy metabolism has contributed to neuronal death in neurodegenerative diseases51. Maintaining the integrity of the electron transport chain can inhibit mitochondrial apoptosis52, while inhibiting complexes I or II can result in neuronal death47,48. And inhibiting complex I activity can attenuate the protective effect of mild acidosis on neuronal survival. Therefore, we conclude that mild acidosis protects neurons from ischemic injury by attenuating damage to mitochondrial complexes.

Conclusions Mild acidosis slowed OGD-induced neuronal ATP deprivation, mainly by protecting mitochondrial ATP production. OGD-induced damage to mitochondrial membrane potential and respiratory function were also alleviated by mild acidosis. Our findings suggest that this occurs because mild acidosis protects the activities of mitochondrial complexes I and II during OGD, thereby promoting neuronal survival. Despite the importance of mitochondrial function during neuronal OGD injury, the effects of mild acidosis accompanying ischemia on mitochondrial function has lacked investigation. The closest study to ours investigated hypoxic conditions (4 to 30 hours), but not OGD conditions, in their injury model15. That study found that the primary attenuating mechanism of mild acidosis was its promotion of mitochondrial elongation to increase neuronal ATP generation, which protects neurons from hypoxic injury. By integrating these results with our own, we can speculate that mild acidosis protects neuronal mitochondrial function from various directions and is based on the length and severity of an ischemic attack. Still, future work is needed to focus on the mechnisms underlying how mild acidosis attenuates decreases in the activities of mitochondrial complexes during ischemia. 14


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Materials and Methods

Cortical neuron culture All experimental procedures with animals were approved by the Peking University Animal Care and Use Committee. We used both male and female Sprague-Dawley rats at embryonic day 18 to culture primary cortical neurons. Cortexes were first dissected and then digested by 0.125% trypsin (0.25% trypsin from Gibco plus an equal volume of Hanks’ solution) for 2 min at 37 °C. Next, cortexes were placed into a trypsin inhibitor solution (Sigma, 1 mg/ml) for 10 min. After washing the cortexes four times, we dispersed the cortexes into individual cells and then collected cells by centrifugation at 1000 rpm for 7 min. The cells were resuspended in neuronal culture medium (neurobasal medium supplemented with 2% B27, 1% GlutMax, and 0.5% penicillin-streptomycin, all from Gibco) and diluted to a density of 5×105 cells/ml. Cells were plated onto 10 cm dishes, 6well plates, 24-well plates, and 24-well seahorse plates containing 21 ml, 2.4 ml, 0.5 ml, and 125 µl of cell resuspension solution, respectively. All plates were coated with 50 µg/mL poly-D-lysine (Sigma). Cells were maintained in neuronal culture medium for 9-14 days before use. 10 µM arabinosylcytosine C (Sigma) was added on Day 3 to inhibit glia proliferation. Media was fully replaced on Day 4 and half replaced on Day 8.

Oxygen-glucose deprivation (OGD) The artificial cerebral spinal fluid (ACSF) used in this study for the OGD treatment contained 141 mM NaCl, 2.5 mM KCl, 2.4 mM CaCl2, 1.3 mM MgCl2, 1.25 mM Na2HPO4, and 10 mM HEPES and was adjusted to a pH of 7.4 or 6.5 using NaOH or HCl. ACSF for the control group contained 10 mM glucose. Solutions were prewarmed to 37 °C before use. ACSF for the OGD group was pre-balanced with pure N2 for at least 1 h before use. Neurons were then treated in the solutions for the indicated time period, during which the pH of the solutions stay almost constant. For reperfusion, the culture was refreshed with 15



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neuronal culture medium at pH 7.4.

Cellular ATP measurement Cellular ATP concentrations were measured using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). In brief, the neurons used in this experiment were cultured in 24-well plates. We first made working solutions according to the assay kit instructions. Then we added 125 µl neurobasal medium (Gibco) and 125 µl working solution to each well. The solutions were mixed with a shaker at 150 rpm for 2 min and then incubated at room temperature for 30 min. Solutions were transferred to white 96-well plates and luminescences were read using a BioTek Cytation5. Each experimental condition had multiple replicate wells.

Mitochondrial ATP measurement After the neurons, cultured in 10 cm dishes, were exposed to each indicated treatment, neuronal mitochondria were extracted. Neurons were first harvested in 2 ml mitochondrial isolation buffer (MIB), which contained 210 mM D-mannitol, 70 mM sucrose, 10 mM HEPES, 2 mM EGTA, and 0.1% fatty acid free BSA (Sigma), with KOH used to adjust the pH to 7.4. The neurons plus solution were homogenized using a 5-ml glass/Teflon tissue grinder with 15 slow up-downs and 10 fast up-downs at 300rpm on ice. The homogenate was then concentrated using 1050 g at 4 °C for 10 min. The supernatant was collected and the deposit was resuspended in MIB, followed by reconcentration using 1050 g at 4 °C for 5 min. The supernatant was again collected and combined with the supernatant collected in the previous step. This combination was concentrated using 8600 g at 4 °C for 10 min. The supernatant was discarded and the deposit was resuspended in MIB without EGTA and BSA. The mitochondrial suspension was then concentrated using 14600 g at 4 °C for 10 min. The supernatant was discarded and the deposit was resuspend in MIB without EGTA and BSA to get the final mitochondrial suspension. 16


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Mitochondrial ATP and protein concentration were determined for the mitochondrial suspensions. Mitochondrial ATP was measured using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). In brief, equal volumes of mitochondrial suspension and working solution (prepared from the assay kit) were mixed in a white 96-well plate using a shaker at 150 rpm for 2 min. The plates were then incubated at room temperature for 30 min and luminescences were read using a BioTek Cytation5.

The protein concentrations of the mitochondrial suspensions were measured using the PierceTM BCA Protein Assay Kit (Thermo Scientific). The working solution for this assay was prepared using reagents A and B from the kit at a ratio of 50:1. 200 μl of the working solution was added to 25 μl of mitochondrial suspension in a 96-well plate. The plate was mixed gently and incubated at 37 °C for 30 min. Absorption was read at 570 nm (A570) and 630 nm (A630). We used the value of A570 minus A630 to evaluate protein concentration. A series of known concentrations of BSA solution were used to make a standard curve which was used to determine the exact protein concentration of each sample. The mitochondrial ATP level was normalized with the corresponding mitochondrial protein concentration to exclude the impact of mitochondrial isolation efficiency.

Western blot analysis Neurons were cultured in 6-well plates. After experimental treatments, neurons were lysed with strong RIPA lysis buffer supplemented with 1 mM PMSF (Beyotime). The cell pellets were disrupted using ultrasonication and concentrated using 12000 rpm at 4 °C for 10 min. Supernatants were collected and protein concentrations were measured as described in the “Mitochondrial ATP measurement” section using the PierceTM BCA Protein Assay Kit. Protein samples were denatured in loading buffer at 100 °C for 5 min and then immediately placed on ice. The prepared protein samples were separated on 15% SDS-PAGE gels and 17



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transferred to PVDF membranes. The membranes were blocked with 5% milk for 1 h and incubated with anti-TOM20 (1:100, Santa Cruz) overnight. The membranes were washed and incubated with HRP-conjugated anti-rabbit (1:1000) and HRP-conjugated anti-betaactin (1:5000, EASYBIO). Bands were imaged and densities were analyzed.

Mitochondrial membrane potential measurement Neurons were cultured in 24-well plates. After treatment, neurons were incubated in 150 nM TMRE at 37 °C for 15 min. Cells were washed with ACSF (with glucose) and TMRE fluorescence at EX 510 nm and EM 590 nm was imaged using an UltraVIEW VoX from PerkinElmer.

Neuron oxygen consumption rate (OCR) measurement Neurons were cultured in 24-well XF Cell Culture Microplates. Neuronal OCR was detected using a Seahorse XFe24 Analyzer. The detailed procedure followed the instructions from the Agilent company. In brief, we prepared XF assay medium using XF base medium (Agilent), 10 mM glucose, 1 mM sodium pyruvate, and 2 mM L-glutamine and adjusted the pH to 7.4 using NaOH. After treatment, neurons were washed twice with XF assay medium and then placed in fresh assay medium for OCR measurement. Four basal OCR measurements were taken first. Then FCCP (2 µm) was added and three measurements for maximal OCR were taken. Finally, antimycin A (0.5 µM) and rotenone (0.5 µM) were added and three measurements for non-mitochondrial OCR were taken. Each measurement included 3 min mixing, 3 min waiting, and 3 min measuring.

Mitochondrial complex activity measurement We followed the experimental procedures given in a paper published in Nature Protocols 53.

In brief, neurons were cultured in 24-well XF Cell Culture Microplates. A mannitol and

sucrose buffer (MAS) for OCR detection was prepared with 70 mM sucrose, 220 mM 18


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mannitol, 10 mM KH2PO4, 5 mM MgCl2, 2 mM HEPES, and 1 mM EGTA and adjusted to pH 7.2 using KOH. Before use, 0.4% fatty acid-free BSA was added to the MAS to make an MASB buffer. After treatment, neurons were washed twice with MASB and placed into an MASB solution with 50 µg/ml saponin for OCR measurement. Saponin was used to permeabilize neuronal plasma membranes to allow direct contact between the substrates and mitochondria. Two measurements were made for basal OCR. Then substrates for a specific mitochondrial complex combined with 1 mM ADP were added and two measurements were taken for measurements of complex activity. The specific substrates were as follows: 5 mM pyruvate and 2.5 mM malate for complex I; 10 mM succinate and 1 µM rotenone for complex II; 0.5 mM duroquinol for complex III; 0.5 mM N,N,N',N'Tetramethyl-p-phenylene-diamine (TMPD) and 2 mM ascorbate for complex IV. Next, 1 µg/ml oligomycin was added to inhibit complex V (ATP synthesis) activity and two measurements were made to evaluate the non-ATP synthesis OCR. Finally, inhibitors for specific complexes were also added and two measurements for OCR not related to the specific complex were collected. Inhibitors for different complexes were as follows: rotenone 1 µM for complex I; Malonate 40 µM for complex II; Antimycin A 2 µM for complex III; potassium azide 20 mM for complex IV. Each measurement included 2 min mixing, 2 min waiting, and 2 min measuring. During data processing, the average value of the last two measurements was subtracted from every measurements to exclude OCR unrelated to the specific complex.

Lactate Dehydrogenase (LDH) assay Lactate Dehydrogenase (LDH) release was detected using the CytoTox96® Nonradioactive Cytotoxicity Assay (Promega). In brief, equal volumes of working solution and sample were mixed, pipetted into a 96-well plate, and incubated at room temperature for 30 min. Absorption was then read at 490 nm. Data were normalized with the maximum LDH release measured after adding lysis buffer to get the relative LDH release. 19



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Supporting Information Supplementary materials and methods and supplementary figures mentioned in the article.

Author Information Corresponding authors *Tel: 86-10-62757830; Fax: 86-10-62751526; Email: [email protected] *Tel: 86-10-62765106; Fax: 86-10-62765106; Email: chenzhou@ pku.edu.cn

Author Contributions Ming-Yue Zhu designed and carried out the experiments, collected and analyzed the data, and wrote the manuscript. Dong-Liang Zhang carried out the experiments and collected and analyzed the data. Zhen Chai and Chen Zhou designed the project, guided the research, and revised the manuscript.

Funding This research was supported by the National Basic Research Program of China (grant no. 2017YFA0105202) and the National Natural Science Foundation of China (grant no. 31671111).

Note The authors declare no conflicts of interest.

Acknowledgements The authors thank Professor Shiqiang Wang for helpful research discussions; Professor Xinxiang Zhang and Dr. Xiaohui Zhang for their help with looking for the target; Dr. Xuexin Fan for her advice on mitochondrial extraction and blue native PAGE; Dr. Guilan Li for her help with the microplate reader and seahorse analyzer; Dr Hui Li for her help 20


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with seahorse analyzer; and Dr. Hongxia Lv for her help with UltraVIEW VoX. We also thank the State Key Laboratory of Membrane Biology for its support.

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27



90x46mm (600 x 600 DPI)


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Fig 1. Mild acidosis reduced the impacts of OGD on neuronal ATP level. (A) Mild acidosis during OGD reduced neuronal death. The figure shows relative LDH release during 3 h OGD and 3 h reperfusion. One-way ANOVA, *P < 0.05 vs pH 6.5; **P < 0.01 vs pH 7.4; ##P < 0.01 vs OGD/Reperfusion pH 7.4. n = 3 independent experiments. (B) Mild acidosis slowed OGD-induced neuronal ATP decrease. The figure shows neuronal ATP levels measured after the indicated treatments. Data are normalized to the “1 h pH 7.4” treatment. Two-way ANOVA, **P < 0.01 pH 7.4 vs OGD pH 7.4 or pH 6.5 vs OGD pH 6.5; ##P < 0.01 OGD pH 7.4 vs OGD pH 6.5. n = 3 independent experiments. 134x53mm (600 x 600 DPI)



Fig 2. Mild acidosis limited reductions in mitochondrial ATP generation during OGD. (A) ATP generation mainly depended on mitochondrial respiration during OGD. The figure shows neuronal ATP level measured after 1 h (top) or 2 h (bottom) OGD treatment. Data were normalized with “pH 7.4 (no OGD)” treatment. FCCP, mitochondrial uncoupling agent, 1 μM. Oligo (Oligomycin), complex V inhibitor, 2.5 μM. AA (Antimycin A), complex III inhibitor, 2.5 μM. One-way ANOVA, **P < 0.01 vs pH 7.4; ##P < 0.01 vs pH 6.5. n = 3 independent experiments. (B) Mild acidosis did not affect mitochondrial mass. The figure shows western blot analyses of TOM20 and β-actin protein level after 1 h (top) or 2 h (bottom) treatment. Data were normalized with pH 7.4. One-way ANOVA, P > 0.05. n = 4 independent experiments. (C) Mild acidosis reduced the impacts of OGD on mitochondrial ATP level. The figure shows mitochondrial ATP levels normalized respectively by isolated mitochondrial protein concentrations after 1 h (left) or 2 h (right) treatment. Data were normalized with pH 7.4. One-way ANOVA, **P < 0.01 pH 7.4 vs OGD pH 7.4 or pH 6.5 vs OGD pH 6.5; #P < 0.05 vs OGD pH 7.4. n = 4 independent experiments. 168x177mm (300 x 300 DPI)


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Fig 3. Mild acidosis attenuated OGD-induced mitochondrial dysfunction. (A) Mitochondrial membrane potential. The figure shows TMRE fluorescence imaging after indicated treatments (left) and the quantification of TMRE fluorescence (right). Two-way ANOVA, **P < 0.01 pH 7.4 vs OGD pH 7.4 or pH 6.5 vs OGD pH 6.5; ##P < 0.01 OGD pH 7.4 vs OGD pH 6.5. n = 16 imaging fields from 4 independent experiments. (B) Neuronal OCR measurements. FCCP, 2 μM. AA, 0.5 μM. Rot (rotenone), 0.5 μM. Basal respiration is the average of the first through the forth data points. Maximal respiration is the average of the fifth through the seventh data points. One-way ANOVA, *P < 0.05 vs pH 6.5; **P < 0.01 vs pH 7.4; #P < 0.05 vs pH 7.4; ##P < 0.01 vs OGD pH 7.4. n = 40 samples from 4 independent experiments. 174x130mm (300 x 300 DPI)



Fig 4. Mild acidosis attenuated OGD-induced decreases in mitochondrial complex I and II activities. (A) complex I activity. n = 19 samples from 4 independent experiments. (B) complex II activity. n = 16-18 samples from 4 independent experiments. (C) complex III activity. n = 13-15 samples from 3 independent experiments. (D) complex IV activity. n = 14-15 samples from 3 independent experiments. One-way ANOVA. *P < 0.05 pH 7.4 vs OGD pH 7.4 or pH 6.5 vs OGD pH 6.5. **P < 0.01 vs pH 7.4. #P < 0.05; ##P < 0.01 vs OGD pH 7.4. 107x102mm (600 x 600 DPI)


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Fig 5. Effect of complex I inhibitor on neuronal death. Relative LDH release during 3 h OGD treatment and 3 h reperfusion. One-way ANOVA, **P < 0.01; ns P > 0.05. n = 4 independent experiments. 120x58mm (600 x 600 DPI)


Mild acidosis protects neurons during oxygen-glucose deprivation by

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