Ozone （O₃） Elicits Neurotoxicity In Spinal Cord Neurons （SCNs） By Inducing ER Ca²⁺ Release And Activating The CaMKII/MAPK Signaling Pathway And XBP1 Prevention Targets

Backround:Environmental ozone (O3) exposure not only causes respiratory disorders, including loss of lung function, exacerbation of asthma, airway damage and lung inflammation, but also affects the functions of the central nervous system (CNS), such as decreased cognitive response, decreased motor activity, headaches, disturbances in the sleep-wake cycle, neuronal dysfunctions, neuronal degeneration and neurochemical alterations. However, as a strong oxidizing agent, O3 remains widely used in the treatment of protrusion of lumbar intervertebral disc (PLID), failed back surgery syndrome (FBSS), soft tissue lesions and arthralgia. Oxygen/ozone treatment of herniated discs is known as an effective and extremely safe procedure because of its analgesic, anti-inflammatory effects and reduction in herniated disk volume. Therefore, whether O3 has neurotoxicity in spinal cord neurons (SCNs) raises concerns in terms of the clinical applications of O3.Previous cellular and biochemical experiments indicate that an inflammatory reaction takes place following O3 exposure in the lung. Although neurobehavioral effects, including memory, motor activity were also altered in these studies, the biochemical changes in the neuronal system after exposure to O3 remain largely unknown. The endoplasmic reticulum (ER) is an essential intracellular organelle, responsible for intracellular calcium (Ca2+) homeostasis and protein folding and processing. The Ca2+ release from the ER is mediated through ER-resident inositol trisphosphate receptors (IP3RS) and the ryanodine receptors (RyRs). Dysfunction of Ca2+ homeostasis in the ER leads to the accumulation of unfolded proteins and activates the ER-stress-induced apoptosis pathway. There are two main pathways through which the ER can mediate apoptosis:the intrinsic pathway activated by impairment of ER function and the mitochondria-dependent pathway, due to Ca2+ overloading from the ER to the mitochondria. The ER Ca2+ release through the RyRs and IP3R is involved in the two types of ER-stress-induced apoptotic pathways. It has been shown that ER and mitochondria are physically and functionally coupled by microdomains that involve RyRs and IP3RS. Moreover, Ca2+ signaling between these organelles can induce an apoptotic crosstalk followed by the mitochondria-specific toxicity as previously mentioned.The ER is a perinuclear, cytoplasmic compartment where proteins and lipids are synthesized. When unfolded and/or misfolded proteins accumulate in the ER, also known as ER stress, the cell invokes responses to remove these defective proteins. This regulatory system is termed the unfolded protein response (UPR). In mammalian cells, several ER-resident transmembrane proteins have been identified as transducers of the ER stress signaling pathway namely:inositol-requiring ER-to-nucleus signal kinase 1 (IRE1); PKR-like ER kinase (PERK) and activating transcription factor 6 (ATF6). In particular, dimerization and phosphorylation of IRE 1, with RNase activity, promotes the splicing of X-box binding protein-1 mRNA (XBP1) in mammalian cells. The activation of RNase activity of IREla by ER stress results in the removal of 26 nucleotides from XBP1 transcripts by unconventional RNA splicing, which converts the inactive form of XBPlmRNA (XBPlu) to an active form (XBPls). The XBPls induces GRP78 expression via a promoter containing the ER response element (ERSE). Accordingly, ER stress results in dimerization of PERK, resulting in the activation of PERK, thereby inducing phosphorylation of eIF2a, the general attenuation of protein translation and increased translation of the transcriptional factor ATF4 protein. Increased translation of ATF4 regulates the expression of downstream genes, such as C/EBP homologous protein (CHOP). Both GRP78 and CHOP, sentinel markers for ER stress under pathologic conditions, bind transiently to newly synthesized proteins translocated into the ER and more permanently to unfolded or misfolded proteins.Based on our preliminary research on low dose ozone influence the calcium channel in vitro of rat spinal neurons and the relation research between calcium overload and apoptosis, the part Ⅰ explores the endoplasmic reticulum calcium release mechanism from the endoplasmic reticulum calcium channel level, and the relation between endoplasmic reticulum calcium release, CaMKII/MAPK signaling pathways and cell apoptosis. In order to study whether XBP1 protect ozone-induced spinal cord neuronal apoptosis, the part Ⅱ detects whether ozone can activate endoplasmic reticulum stress, and discussed whether XBP1 play a protective role and its possible mechanism. The paper investigates whether ozone causes neurons toxicity in spinal cord, the possible mechanisms of toxicity of the ozone toxicity and possible preventive measures from the perspective of the endoplasmic reticulum calcium cascade, which may provide a theoretical basis for the future safer and clinically effective use of medical ozone.Part I Ozone (O3) elicits neurotoxicity in spinal cord neurons (SCNs) by inducing ER Ca2+ release and activating the CaMKII/MAPK signaling pathwayObjective:The present study was designed to explore the cellular injury of primary cultures of SCNs after exposure to a low concentration of O3 and the possible mechanisms.Methods:1. Primary spinal cord neuronal cultures2. Identify of primary spinal cord neuronalObserve the spinal neuron morphology by optical microscope and identify SCNs by MAP-2 and immunofluorescence.3. Assessment of neuronal injuryDetermine the effects of different concentrations of ozone for 60min on cell viability by CCK8 assay.4. Explore whether ozone induced ER Ca2+release by ER Ca2+ imagingAs Ca2+ signaling inhibition ameliorated ozone-induced cell death, the Ca2+ response after ozone exposure in SCN cells was examined. The SCN cells were loaded with Fluo-3/AM and stimulated with O3.5. Determine whether involvement of the IP3/RVR signaling pathway inozone-induced Ca2+release6. Determine whether ozone exposure activated the CaMKII and MAPK signalingpathway through ER Ca2+release by Western blot analysis.7. TUNEL assayApoptosis was detected by the commercially available TUNEL assay kit to detemine Ozone-induced apoptosis in SCNs is dependent on ER Ca2+ release and CaMKII activation.Results:1. Under the inverted phase contrast microscope, the neurons were round in shape, and each surrounded by a clear pale halo. Neuronal bodies were relatively big with abundant cytoplasm. With the prolongation of culturing time, the neuronal bodies kept growing bigger and the processes extended and branched further to form a dense interconnecting reticulation.2. After MAP-2 staining of spinal cord neurons, the purity of cultured neurons was 89.70+4.70%.3. The viability of SCNs was significantly reduced after 1h exposure to O3 in a dose-dependent manner at the range from 20 to 500 μg/mL. The O3 dose at 40μg/mL caused a significantly decrease of cell viability. The Ca2+ chelator prevented O3 exposure-induced cell death. The CaMKII antagonist also inhibited cell death after exposure to O3.4. The SCN cells were loaded with Fluo-3/AM and stimulated with O3. The fluorescence intensity was rapidly increased and recovered to basal levels 5 min later. One-to 1.5-fold increase in fluorescence intensity was observed 90s after O3 stimulation.In the presence of intracellular Ca2+ chelator BAPTA/AM, ozone-induced Ca2+ mobilization was blocked, whereas EGTA had no such effect.5.2APB or dantrolene alone did not affect intracellular Ca2+ levels. However, pretreatment with 2APB or dantrolene significantly inhibited ozone-induced increase of intracellular Ca2+release.6. Exposure to O3 significantly increased p-CaMKII expression in SCN cells. However, the CaMKII inhibitor, KN62, significantly reduced ozone-induced p-CaMKII activation. Exposure to O3 for lh significantly increased the phosphorylation levels of JNK and p38 MAPK, but not that of ERKl/2. The effects of exposure to O3 on the activation of JNK and p38-MAPK could be however abrogated by pretreatment with BAPTA/AM, KN62,2APB or dantrolene.7.24h after treatment, the number of apoptotic cells in the ozone treatment group was ten-fold when compared with the control group. Ozone-induced apoptosis was however blocked by pretreatment with BAPTA/AM or KN62. As the control, BAPTA/AM or KN62 alone did not cause apoptosis in SCNs.Conclusion:The results of this study indicate that exposure to O3 has a potential neurotoxieity in SCN through activating ER Ca2+release and the CaMKII/MAPK signaling pathway. Approaches, such as chelating intracellular Ca2+ and stabilizing neuronal Ca2+ homeostasis could effectively ameliorate the neurotoxieity of exposure to O3. Whilst the current study provides in vitro data regarding the neurotoxicity of exposure to O3 SCN, in vivo evidence supporting these findings, as well as the potential therapeutic implications are still required.Part Ⅱ Protective properties of X box binding protein 1 (XBPl) in ozone (O3)-induced spinal cord neuronal deathObjective:The current study investigated whether exposure to low concentrations of O3 induced increased expression of GRP78 and XBP1 and whether activation of XBP1 is neuroprotective against 03-induced ER stress.Methods:1. Construction and transfection of Adenovirus-mediated XBPls overexpression2. Identification of Adenovirus-mediated XBP1 s overexpressionTo examine in vitro XBP1s expression levels following Ad vector infection, Western blotting and quantitative Real-time-PCR analysis was performed.3. Detect whether O3 activated the ER stress response and the XBP1 splicing Western blotting analysis was performed to detect whether O3 activated the ER stress response and the XBP1 splicing.4. Assessment of neuronal injuryExplore whether adenovirus-mediated XBP1s overexpression prevent ER stress and protects 03-induced cell death in rat primary SCNs.Results:1. Adenovirus mediated gene transfer into the primary SCNsThe primary cultures transduced with adenovirus two days after plating and incubated for 24h showed the best transduction efficiency. With these conditions and an MOI of 100 pfu/cell,80% XBPl positive cells could be seen in the primary SCNs three days after transduction. When the MOI was greater than 300pfu/cell, cytotoxic effects appeared.2. Adenovirus mediated overexpression of XBP1 in primary SCNsThere was an apparent increase in the XBP1 expression after transduction with Adv-XBPl in primary SCNs. The control Ad vectors, Ad-LacZ, did not increase XBPl expression, indicating that transduction with Ad-LacZ alone does not induce any change in the cell systems.3. Ozone exposure induced the ER stress response and XBP1 splicing(1) The ER stress response of SCNs exposed to O3 was evidenced by the increase in GRP78 and CHOP protein levels.(2) Treatment of SCNs with O3 also increased the expression of XBP1 as detected by a specific antibody.4. Adenovirus-mediated XBP1 overexpression prevent ER stress and protects 03-induced cell death in rat primary SCNs(1) After exposed to ozone, however, GRP78 and CHOP expression significantly higher. These differences are significant, suggesting that neuroprotection may be mediated by overexpression XBP1.(2) 03-induced loss of cell viability was significantly attenuated by Adv-XBPl pretreatment.Conclusion:Ozone exposure induced the ER stress response and XBP1 splicing. Moreover, overexpression of XBP1 activation protects against neuronal cell death following O3 exposure and that activation of the XBP1 pathway may provide a preventative approach for the treatment of spinal cord neuronal O3 exposure.