Protection of melatonin against acidosis‐induced neuronal injuries

Abstract Acidosis, a common feature of cerebral ischaemia and hypoxia, plays a key role in these pathological processes by aggravating the ischaemic and hypoxic injuries. To explore the mechanisms, in this research, we cultured primary neurons in an acidic environment (potential of hydrogen [pH]6.2, 24 hours) to mimic the acidosis. By proteomic analysis, 69 differentially expressed proteins in the acidic neurons were found, mainly related to stress and cell death, synaptic plasticity and gene transcription. And, the acidotic neurons developed obvious alterations including increased neuronal death, reduced dendritic length and complexity, reduced synaptic proteins, tau hyperphosphorylation, endoplasmic reticulum (ER) stress activation, abnormal lysosome‐related signals, imbalanced oxidative stress/anti‐oxidative stress and decreased Golgi matrix proteins. Then, melatonin (1 × 10−4 mol/L) was used to pre‐treat the cultured primary neurons before acidic treatment (pH6.2). The results showed that melatonin partially reversed the acidosis‐induced neuronal death, abnormal dendritic complexity, reductions of synaptic proteins, tau hyperphosphorylation and imbalance of kinase/phosphatase. In addition, acidosis related the activations of glycogen synthase kinase‐3β and nuclear factor‐κB signals, ER stress and Golgi stress, and the abnormal autophagy‐lysosome signals were completely reversed by melatonin. These data indicate that melatonin is beneficial for neurons against acidosis‐induced injuries.


| INTRODUC TI ON
The brain is a highly energy-consuming organ, expending more energy than any other organ in proportion to its size. When the energy delivery, production, utilization and storage of brain are insufficient or impaired, the acid-base disturbance, especially acidosis, will occur. Clinically, brain acidosis often results either from an increase in tissue partial pressure of carbon dioxide (PCO 2 ) during hypercapnia, or from the accumulation of the by-products of anaerobic metabolism, such as lactate and protons, during hypoxia. 1 The extracellular potential of hydrogen (pH) will drop to 6.5-6.0 during ischaemia in normoglycaemic conditions and even below 6.0 in severe ischaemia or hyperglycaemic conditions. 1,2 In brain, the acid-base equilibrium is essential for neurotransmission and neuronal excitability. Gamma-aminobutyric acid A (GABAA) receptors, various voltage-gated ion channels and transient receptor potential vanilloid 1 are modulated by an acidic pH. 3 Almost all in vivo studies have shown that acidosis is a common feature of ischaemia and further aggravates the damage, resulting in disturbance in ion homeostasis, excitotoxicity and a number of cell death-mediating pathways, which are responsive to inflammatory and oxidative stress mediators ultimately neuronal death. 4,5 It was previously shown that lactic acidosis in astrocytes under ischaemic conditions accelerated hypoxia and neuronal damage, leading to more severe infarctions. 6 A reduced pH has also been implicated in seizures, 7 Down's syndrome, 8 Pick's disease 8 and neurodegenerative diseases including Alzheimer's disease (AD) and Huntington's disease. 8 Thus, to elucidate the acidosis-induced neuronal injuries is critical for brain protection from acidosis.
Melatonin (N-acetyl-5-methoxytryptamine), a tryptophan metabolite synthesized mainly in the pineal gland produced primarily by the pineal gland at night, has multiple biological activities, including antioxidants, anti-inflammatory, neuroprotection, anti-depression, antinociception and antianxiety. 9 In addition, it is also important in stabilizing the structures of synapses and enhancing the functions of synapses, and further benefit to cognition. 10 Although the mechanism is still unclear, studies have shown that melatonin has protective effects on the hypercapnia and hypoxia sensitivity in rats. 11,12 It has been found that acute foetal hypoxia induced cerebral oxidative stress and inflammation in the late gestation brain, while melatonin abolished the primary and secondary increases in brain hydroxyl radical formation and reduced cerebral lipid peroxidation, thereby reducing brain damage. 13,14 In addition, systemic or transdermal neonatal melatonin administration significantly reduced the neuropathology and encephalopathy signs associated with perinatal asphyxia. 15 Other studies have shown that melatonin reduced oxidative stress and cell damage in the foetal sheep brain in response to severe hypoxia 16 and protected against hippocampal cell loss following cerebral ischaemia and reperfusion in foetal and neonatal rats by improving mitochondrial injury. 17 In this study, we studied the effect of melatonin pre-treatment on the acidosis-induced neuronal injuries in vitro.
The primary neurons were cultured in an acidic environment (pH6.2) for 24 hours to mimic the acidosis after growing in the normal medium for 14 days. By proteomic analysis, 69 differentially expressed proteins were found, which were mainly related to stress and cell death, synaptic plasticity and gene transcription. Then, before being cultured in the acidic medium, the primary neurons were exposed in melatonin (1 × 10 −4 mol/L, Mel neurons) or dimethyl sulphoxide (DMSO) (0.01%, Veh neurons) for 24 hours. It was observed that melatonin pre-treatment increased the neuronal cell viability, and the total length and the complexity of dendrites, and reversed the abnormalities of synaptic proteins and tau hyperphosphorylation caused by acidosis. Compared with the neurons cultured in the normal medium (Con neurons), the increased levels of nuclear factor-κB (NF-κB) p65, glucose-regulated protein 78 (GRP78/Bip), activating transcription factor6 (ATF6) and the inhibition of nuclear factor erythroid 2-related factor 2 (Nrf2) were found in Veh neurons, but not in Mel neurons. Thus, all these in vitro data of this study illustrated that melatonin is beneficial for neurons against acidosis-induced injuries.
The primary antibodies used for immunofluorescence staining and Western blotting are listed in Table 1.

| Cell counting kit-8 assay
The cell suspension was added to a 96-well plate at a suitable density and maintained at 37°C in a humidified incubator with 5% CO 2 .
The cells were then exposed to different experimental conditions.

| TdT-mediated dUTP nick end labelling staining
The cultured neurons were fixed in 4% (wt/vol) paraformaldehyde

| Enzyme-linked immunosorbent assay (ELISA)
The neuronal superoxide dismutase 1 (SOD1) levels were assayed by ELISA. The neurons were washed with PBS for 5 minutes, the neurons were digested by trypsin, and the cells were lysed and centrifuged at 5000 × g for 10 minutes at 4°C to obtain the supernatant.

| Proteomic analysis
The integrated approach used to quantify dynamic changes in the primary cultured rat neurons included isobaric tags for relative and absolute quantification (iTRAQ), high-performance liquid chromatography (HPLC) fractionation and mass spectrometry-based quantitative proteomics. The cellular proteins were digested by trypsin, and the obtained peptides from the denatured protein were labelled by iTRAQ

| Western blotting
Protein concentrations were measured using a bicinchoninic acid kit (Thermo Fisher Scientific) after extracted protein from neuron. Imaging System (Li-Cor Bioscience).

| Immunofluorescence staining
The cultured neurons were washed in PBS for 5 minutes, fixed in 4% PFA for 30 minutes and then washed with PBS containing 0.1% Triton X-100 for 15 minutes. After being ruptured membrane in PBS supplemented with 0.5% Triton X-100 for 10 minutes, the cells were incubated in 3% BSA to block non-specific sites for 30 minutes at 25°C and then washed with PBS for 30 minutes.
The cells were incubated with the primary antibodies at 4°C for 24 hours. The cells were then washed with PBS for 30 minutes and were subsequently incubated with secondary antibodies for 1 hour at 37°C. The cells were incubated with DAPI for 10 minutes and sealed with 50% glycerine after being washed with PBS. The images were observed by a laser confocal microscope (LSM780; Zeiss, Heidelberg, Germany), and the fluorescence images were analysed by the software affiliated.

| Simple neurite tracer analysis and Sholl analysis
For assessment of neuron morphology and neuronal complexity, neurons were immunofluorescence staining with microtubule-as-

| Statistical analysis
Data were presented as means ± SEM. Statistical analysis was performed using SPSS 12.0 (SPSS Inc, Chicago, IL, USA). Statistical analysis was performed by using the unpaired Student's t test or one-way ANOVA followed by the Tukey post hoc test. Null hypotheses were rejected at P < 0.05.

| Decreases in extracellular pH value induced neuronal injury
After growing in the normal medium (pH = 7.5) for 14 DIV, the pri-
In Mel neurons, the levels of NF-κB p65 and p-Nrf2 returned to the levels as Con neurons had ( Figure 3D,E).
By immunofluorescence staining of MAP2, the morphological changes of the neurons were analysed. Con neurons had a total dendritic length of 1690.0 ± 63.64 μm, while the Veh neurons had a significantly decreased total dendritic length (691.6 ± 77.25 μm) ( Figure 3F,G).
By Sholl analysis, we studied the dendritic complexities of neurons. In the concentric circle analysis, the Veh neurons had obviously decreased maximum number of dendritic intersections (5.3 ± 0.56, Figure 3H,I) and fewer total dendritic interactions (71.3 ± 8.31, Figure 3H,J) compared to Con neurons (181.0 ± 20.01 and 11.3 ± 1.67, respectively).

| Melatonin attenuates acidosis-induced synaptic abnormality and tau hyperphosphorylation
In the differentially expressed proteins, several synaptic-associated proteins were included, for example solute carrier family 17 (sodiumdependent inorganic phosphate cotransporter), member 6 (SLC17A6), Ly6h, which was found in most hippocampal pyramidal neurons, is involved in the regulation of α7 nicotinic acetylcholine receptors transport and nicotine-induced glutamate signalling enhancement. 35 eEF2K, also known as calcium/calmodulin-dependent protein kinase III (CaMKIII), is involved in changes in synaptic plasticity and learning and memory. 36,37 Knockdown of eEF2K by RNA interference reduced the dendritic spine stability and inhibited the expression of dendritic brain-derived neurotrophic factor (BDNF). 38 LRP6 is a coreceptor for Wnt signalling, and its deficiency was reported contributes to synaptic abnormalities. 39 Figure 4D).
It is well established that hyperphosphorylated tau, a MAP, induces morphological and functional abnormalities of neurons. 48,49 By Western blotting, we found the Veh neurons had higher levels of phosphorylated tau at Thr231 (pT231, 152.6%), Ser396 (pS396, 181.6%), Ser214 (pS214, 289.6%) and Ser404 (pS404, 134.9%), and a lower level of non-phosphorylated tau at Ser198/199/202 (recognized by Tau1) than Con neurons, while Mel neurons only had the higher level of phosphorylated tau at Ser214 (Figure 4E,F). In addition, no evident changes were found in the levels of total tau probed by Tau5.

| Melatonin regulates organelle stress responses induced by acidosis
Neuron has various organelles such as the endoplasmic reticulum Similar to the ER, the GA can sense and transduce death signals through its own unique molecular machinery in cell death pathways. 56 It has also been reported that during apoptotic cell death, there is a crosstalk between ER, mitochondria and GA. 57  Additionally, lysosome also acts as a signalling organelle that senses nutrient availability and generates an adaptive response, which is important for cellular homeostasis. It was reported lysosomal calcium release activates the master autophagy regulator transcription factor EB (TFEB). 58 Here, levels of autophagy activation marker including beclin1 (autophagosome nucleation) and LC3II/I (autophagosome formation) showed no difference after acidic treatment.
Macroautophagy (autophagy) is a highly conserved intracellular

| D ISCUSS I ON
In this research, we treated the cultured primary neurons with an acidic environment (pH6.2, 24 hours) to mimic the acidosis. By Acidosis is commonly associated with increased levels of inflammation, oxidative stress 59,60 and activating NF-κB signalling. 61 In cerebral ischaemia, NF-κB signalling pathway plays an exceptionally important role due to its pleiotropic effects, unique regulatory mechanisms, and a large number of activating signalling pathways and genes it controls. 62 Activating NF-κB was shown to induce some cytotoxic factors to exacerbate inflammation and oxidative stress and promote apoptosis. 63 Some organelles of neuron, such as the ER, mitochondria and lysosome, 64-66 are susceptible to the reduction F I G U R E 5 Melatonin regulates organelle stress responses induced by acidosis. A, The differentially expressed proteins related to organelle stress are listed with a ratio (pH6.2/Con), including ATP synthase F (0) complex subunit C2, mitochondrial (ATP5G2), carbamoyl phosphate synthetase 1 (CPS1), phosphatidylserine synthase 1 (PSS1) and midkine (MDK). Red colour indicates the increased proteins (P < 0.05), and green indicates the decreased ones (P < 0.05). Levels of Beclin1, microtubule-associated protein light chain 3 (LC3), ras-related protein rab-7a (Rab7), lysosomal-associated membrane protein 1 (LAMP1), LAMP2, glucose-regulated protein 78 (GRP78/Bip) and activating transcription factor 6 (ATF6) were measured by Western blotting (B) and quantitatively analysed (C). Levels of Golgi matrix proteins including trans-Golgi network protein 46 (TGN46), Golgi reassembly stacking protein 55 (GRASP55), GRASP65 and Golgi matrix proteins golgin-84 (Gol-84) were measured by Western blotting (D) and quantitatively analysed (E). Data were presented as means ± SEM (n = 4/group). *P < 0.05, **P < 0.01, *** P < 0.001 vs Con, # P < 0.05, ## P < 0.01 vs Veh of pH and involved in the inflammation and oxidative stress-induced neuronal damage. Here, in the acidotic neurons with higher NF-κB and lower phosphorylated Nrf2, we detected the ER stress activation, abnormal lysosome-related signals, imbalanced oxidation/anti-oxidation and decreased Golgi matrix proteins.
Dendrites are closely related to the morphology and number of synapses, and affect the induction of synaptic plasticity. 67  also remained unchanged. Tau, a MAP, is important in stabilizing microtubules, and its hyperphosphorylation leads to the destabilization and dysfunctions of the microtubule network, and dysregulation in synaptic plasticity. 49 Here, we found the neurons cultured in pH6.2 medium had much higher phosphorylation levels of tau at Thr231 (pT231), Ser396 (pS396), Ser214 (pS214) and Ser404 (pS404) sites.
Abnormal hyperphosphorylation of tau is the result of imbalance of tau kinases and phosphatases. GSK3β is one major kinase, and PP2A is thought to be the major phosphatase for tau dephosphorylation in AD. 50 In this research, inhibition of PP2A, AKT and ERK1/2 and activation of GSK3β were observed in acidotic neurons.
GSK3β was shown activated with the elevation of GRP78 during ER stress-induced tau hyperphosphorylation with spatial memory deficits in rats. 55,69 In this study, GRP78 and ATF6 were increased in the acidotic neurons, indicting the activation of ER stress.
Furthermore, we observed decreased GRASP65, Gol-84, LAMP1, Rab7 and LC3II/I in the acidotic neurons. Double knockout of GRASP65 and GRASP55 dispersed the Golgi stack into single cisternae and tubulovesicular structures. 70 Pharmacological intervention or overexpression of the C-terminal fragment of GRASP65 inhibited fragmentation and decreased or delayed neuronal cell death. 57 In addition, our previous study has shown that down-regulation of Gol-84 induces Golgi fragmentation with hyperphosphorylation of tau. 71 Decreased Sesn3, LC3II/I, LAMP1 and Rab7 indicated the dysregulation of autophagy-lysosome signals. NF-κB has an essential role in inflammation and is an important player in the pathophysiology of neuronal impairments, with roles in cell death and synaptic dysfunction. 72,73 It was found that there is a crosstalking between the NF-κB pathway and GSK-3β signalling pathways. GSK-3β, which was initially identified as a key regulator of insulin-dependent glycogen synthesis, is known to be a mediator of a number of major signalling pathways including the PI3K pathway, the Wnt pathway and Notch pathway. It was shown that GSK-3β activation is required for NF-κB activation, 74 and GSK-3β inhibition caused a dramatic decrease in NF-κB activity. 75 However, NF-κB activation by tumour necrosis factor requires AKT activation. 76 The exact molecular mechanism of GSK-3β mediated NF-κB modulation is still elusive and requires further clarification.
In summary, acidosis-induced rat primary neurons underwent significant apoptosis, synaptic abnormality and tau hyperphosphorylation. Melatonin partially reversed acidosis-induced neuronal death, abnormal dendritic complexity, reduced synaptic proteins, tau hyperphosphorylation and imbalance of kinase/phosphatase probably via rescuing GSK-3β and NF-κB activation. Additionally, acidosis-induced ER stress, Golgi stress and abnormal autophagy-lysosome signals were completely reversed by melatonin. In conclusion, we verified that melatonin has a protective effect on acidosis-induced neuronal dysfunctions and ultimately decreases neuronal death from acidosis.

ACK N OWLED G EM ENTS
This work was supported by grants from the Natural Science

Foundation of China (91539112) and Integrated Innovative Team for
Major Human Diseases Program of Tongji Medical College.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

AUTH O R CO NTR I B UTI O N S
QT and X-WZ initiated, designed and supervised the study.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data available on request from the authors.