CD82 Aggravates Sevoflurane - Induced Neurotoxicity by Regulating TRPM7 in Developing Neurons

1. Introduction

Sevoflurane, isoflurane, ketamine and other drugs are the most common anesthetics in surgical operations [1]. However, mounting studies have found that anesthetics could induce long-term neurotoxicity and cause cognitive impairment in infant brains [1, 2]. Sevoflurane was reported to induce neuronal apoptosis and abnormal protein deposition, resulting in disruption of cerebral homeostasis and cognitive impairment [3]. Moreover, sevoflurane could also alter metabolomic profiles in neonatal rats and induce neurotoxicity and neurobehavioral dysfunction [4]. Therefore, therapeutic strategies should be developed to protect against sevoflurane anesthesia-induced neurotoxicity in the developing brains.

CD82, a member of tetraspanins, is expressed in most eukaryotes and regulate cell adhesion, cell signaling and membrane trafficking [5]. CD82 was found to associate with cis with$\alpha$4 integrin to mediate cell adhesion in hematopoietic stem/progenitor cell [5]. CD82 was also found to be involved in tumor cell migration and invasion [6]. Recently, CD82 has been found to negatively regulate the oligodendrocyte development and myelination [7]. In addition, CD82 was reported to be a potential biomarker for patients with central nervous system involvement [8]. Since CD82 could promote secretion of beta-amyloid peptide, thereby mediating age-related cognitive impairment [9], the effect of CD82 on sevoflurane-induced neurotoxicity was then evaluated in this study.

Transient receptor potential melastatin (TRPM7) is widely distributed in periphery and central nervous system and could regulate cell proliferation, death, differentiation and migration [10]. In the nervous system, TRPM7 is involved in a key neurobiological process from synaptic transmission to cognitive function under physiological conditions [11], thereby mediating neurotoxicity, nerve injury and neuronal death [11]. For example, TRPM7 functions as a zinc influx channel, and potentiates the neurotoxicity induced by Zn${}^{\mathrm{2+}}$ [12]. Inhibition of TRPM7 could demonstrate neuroprotective effect against hypoxic-ischemic injury in neonatal neurons [13]. Moreover, previous study has shown that CD82 could activate caspase-3 to cleave TRPM7, thereby promoting the phosphorylation of Numb to participate in beta-amyloid peptide secretion [9]. Therefore, this study hypothesized that CD82 might promote cleavage of TRPM7 to mediate sevoflurane-induced neurotoxicity.

This study was performed to examine the protein expression of CD82 and TRPM7 in hippocampal neurons after sevoflurane treatment. Additionally, the role of CD82/TRPM7 on sevoflurane-induced neurotoxicity in hippocampal neurons was also determined.

2. Methods

3. Results

3.1 Expression of CD82 and TRPM7 in sevoflurane-induced neurons

Hippocampal neurons were isolated from neonatal rats (postnatal day 1 or 2), and then exposed to 1.8 % sevoflurane for 6, 12, 24 or 48 hours. Cell viability of neurons was decreased time dependently by sevoflurane exposure (Fig. 1A). Protein expressions of CD82 and TRPM7 were increased by sevoflurane exposure (Fig. 1B) in a time-dependent manner, suggesting the potential regulatory ability of CD82/TRPM7 on sevoflurane-induced neurotoxicity.

Fig. 1. Expression of CD82 and TRPM7 in sevoflurane-induced neurons.
(A) Cell viability of hippocampal neurons was decreased time dependently by sevoflurane exposure.
(B) Protein expression of CD82 and TRPM7 were increased time dependently by sevoflurane exposure. *p $<$ 0.05, **p $<$ 0.01.

3.2 Knockdown of CD82 attenuated sevoflurane-induced neurotoxicity

Sevoflurane decreased the cell viability in neurons (Fig. 2A), while pre-transfection with siCD82 attenuated the inhibitory effect of sevoflurane on neuronal viability (Fig. 2A). Moreover, cell apoptosis of neurons was promoted followed sevoflurane treatment (Fig. 2B). However, knockdown of CD82 reversed sevoflurane-induced promoting of cell apoptosis (Fig. 2B), indicating that knockdown of CD82 attenuated sevoflurane-induced neurotoxicity.

Fig. 2.Knockdown of CD82 attenuated sevoflurane-induced neurotoxicity.
(A) Pre-transfection with siCD82 attenuated sevoflurane-induced decrease in neuronal viability.
(B) Pre-transfection with siCD82 attenuated sevoflurane-induced increase in neuronal apoptosis. **, ## p $<$ 0.01.

3.3 Sevoflurane promoted cleavage of TRPM7 through enhance of CD82

Western blot analysis demonstrated that sevoflurane treatment increased the expression of cleaved TRPM7 and cleaved caspase 3 (Fig. 3). However, the increase in cleaved TRPM7 and cleaved caspase 3 were attenuated in neurons pre-transfected with siCD82 (Fig. 3), indicating that sevoflurane promoted cleavage of TRPM7 through enhance of CD82.

Fig. 3.Sevoflurane promoted cleavage of TRPM7 through enhance of CD82.
Pre-transfection with siCD82 attenuated sevoflurane-induced increase oin CD82, cleaved TRPM7 and cleaved caspase 3 expression. **, ## p $<$ 0.01.

3.4 CD82 aggravated sevoflurane-induced neurotoxicity through cleavage of TRPM7

To evaluate the role of CD82/TRPM7 in sevoflurane-induced neurotoxicity in the developing neurons, neurons were co-transfected with pcDNA-CD82 and siTRPM7, and then exposed to sevoflurane. Over-expression of CD82 enhanced the expression of CD82, cleaved TRPM7 and cleaved caspase 3 in sevoflurane-induced neurons (Fig. 4A). However, the increased cleaved TRPM7 and cleaved caspase 3 in sevoflurane-induced neurons transfected with pcDNA-CD82 were reversed by additional transfection with siTRPM7 (Fig. 4A), confirming that sevoflurane promoted cleavage of TRPM7 through the enhanced expression of CD82. Moreover, forced CD82 expression aggravated sevoflurane-induced neurotoxicity with decreased cell viability (Fig. 4B) and increased cell apoptosis (Fig. 4C). However, knockdown of TRPM7 counteracted the promoting effect of CD82 on sevoflurane-induced neurotoxicity through the increase in cell viability (Fig. 4B) and decrease in cell apoptosis (Fig. 4C), suggesting that CD82 aggravated sevoflurane-induced neurotoxicity through cleavage of TRPM7 in the developing neurons.

Fig. 4.CD82 aggravated sevoflurane-induced neurotoxicity through cleavage of TRPM7.
(A) Over-expression of CD82 enhanced expression of CD82, cleaved TRPM7 and cleaved caspase 3 in sevoflurane-induced neurons. Knockdown of TRPM7 attenuated CD82 over-expression-induced increase of cleaved TRPM7 and cleaved caspase 3.
(B) Forced CD82 expression aggravated sevoflurane-induced neurotoxicity with decreased cell viability, while knockdown of TRPM7 counteracted with the promotive effect of CD82 on sevoflurane-induced neurotoxicity through increase of cell viability.
(C) Forced CD82 expression aggravated sevoflurane-induced neurotoxicity with increased cell apoptosis, while knockdown of TRPM7 counteracted with the promotive effect of CD82 on sevoflurane-induced neurotoxicity through decrease of cell apoptosis. #, & p $<$ 0.05, **, ##, && p $<$ 0.01.

4. Discussion

Ions, including Ca ${}^{\mathrm{2+}}$ and Mg ${}^{\mathrm{2+}}$, are important for brain development [14], and overload of the ions via ion channels could contribute anesthesia-induced neurotoxicity [14]. TRPM7 traces metal ions to maintain cell metabolism [10], and blockers or RNA interference for TRPM7 have been shown to attenuate Zn ${}^{\mathrm{2+}}$-induced neurotoxicity [12] and protect hippocampal neurons against ischemia-induced cell death [15]. Since TRPM7 was cleaved by CD82 during age-related cognitive impairment [9], the role of CD82/TRPM7 axis in sevoflurane-induced neurotoxicity in the developing neurons was then validated in this study.

Preclinical study has shown that children exposed to sevoflurane demonstrated higher apoptosis status of neural stem cells [16]. Moreover, anesthetic sevoflurane could also induce neurotoxicity in neuronal cells with decreased cell viability and increased cell apoptosis [17]. The data from this study showed that exposure of the isolated neurons from neonatal rats to sevoflurane induced severely neurotoxicity with decreased cell viability and increased cell apoptosis. Furthermore, CD82 and TRPM7 were increased in neurons post sevoflurane exposure, suggesting the regulatory role of CD82/TRPM7 on sevoflurane-induced neurotoxicity in the developing neurons.

Functional assays indicated that forced CD82 aggravated sevoflurane-induced neurotoxicity with increased cell viability and decreased cell apoptosis of neurons, while knockdown of CD82 decreased the cell viability and increased the cell apoptosis, therebt alleviating sevoflurane-induced neurotoxicity. In addition, TRPM7 silencing attenuated CD82 over-expression-induced neurotoxicity in sevoflurane-induced neurons, demonstrating that CD82 might aggravate sevoflurane-induced neurotoxicity through regulation of TRPM7. Antioxidant defense was increased in children exposed to sevoflurane [16], and levels of reactive oxygen species were also accumulated in neonatal rats post sevoflurane exposure [4]. The reduction of reactive oxygen species was shown to be helpful for the amelioration of sevoflurane-induced neurotoxicity [17]. TRPM7 could release Zn${}^{\mathrm{2+}}$ through sensation of oxidative stress [18]. Therefore, whether CD82/TRPM7 could regulate oxidative stress in neurons to aggravate sevoflurane-induced neurotoxicity should be further investigated. Moreover, exposure of sevoflurane was reported to induce autophagy of hippocampal neurons [19]. CD82 was required for the interaction with Toll-like receptor 9 to dictate autophagy [20] and TRPM7-mediated Ca${}^{\mathrm{2+}}$ influx could regulate basal autophagy [21]. Therefore, CD82/TRPM7 might also regulate autophagy in sevoflurane-induced neurons to aggravate the neurotoxicity.

Sevoflurane exposure has been shown to elevate caspase-3 activation [4], and caspase 3 could promote cleavage of TRPM7 for disassociation of the $\alpha$ -kinase domain, thus participating in cell apoptosis [22]. Over-expression of CD82 promoted TRPM7 $\alpha$-kinase cleavage through activation of caspase 3 [9]. Western blot analysis in this study demonstrated that forced CD82 expression in neurons increased the protein expression of cleaved TRPM7 and cleaved caspase 3, while knockdown of CD82 decreased the protein expression. Moreover, knockdown of TRPM7 attenuated CD82 over-expression-induced increase in cleaved TRPM7 and cleaved caspase 3, suggesting that CD82 might aggravate sevoflurane-induced neurotoxicity through TRPM7 cleavage. Additionally, since suppression of TRPM7 could induce cell death of gastric cancer [23], the effects of TRPM7 on cell viability and apoptosis of sevoflurane-induced neurons should be investigated in the further study. Administration of RNA interference of CD82 and TRPM7 in neonatal rats might be performed to provide in vivo evidence for the regulatory role of CD82/TRPM7 on sevoflurane-induced neurotoxicity.

5. Conclusions

This study demonstrated that enhanced CD82 and TRPM7 were related to sevoflurane-induced neurotoxicity. CD82 promoted the cleavage of TRPM7, resulting in up-regulation of CD82 and cleaved TRPM7 in hippocampus of developing brains. In addition, pretreatment with siCD82 decreased cleaved TRPM7 and cleaved caspase 3 in neurons and mitigated sevoflurane-induced decrease in cell viability and increase in cell apoptosis, thereby ameliorating sevoflurane-induced neurotoxicity. These findings proposed that sevoflurane-induced neurotoxicity in the developing brains could be attenuated by knockdown of CD82.

Acknowledgements

Thanks to all the peer reviewers and editors for their opinions and suggestions.

Conflict of interest

The authors state that there are no conflicts of interest to disclose.

Funding

This work was supported by the Fundamental Research Funds for the Central Universities (Grant No. xzy012019121).

Ethics approval

All animal experiments were approved by the Biomedical Ethics Committee of Ninth Hospital of Xi’an (Approval no.2020-046) for the use of animals and conducted in accordance with the National Institutes of Health Laboratory Animal Care and Use Guidelines.

Statement of Informed Consent

Not applicable.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors’ contributions

Qing Shu designed the study, supervised the data collection, Xiaoyan Zhao analyzed the data, interpreted the data, Xin Geng and Xiaoye Wang prepare the manuscript for publication and reviewed the draft of the manuscript. All authors have read and approved the manuscript.