Role of c-Myc/chloride intracellular channel 4 pathway in lipopolysaccharide-induced neurodegenerative diseases

Abstract

LPS-induced neuronal apoptosis leads to neurodegenerative diseases (NDs). However, the mechanisms underlying NDs pathogenesis remains unclear. The apoptotic response to activation of the c-Myc/chloride intracellular channel (CLIC4) pathway is directed through a mitochondrial pathway. In this study, we aimed to explore the c-Myc/CLIC4 pathway in the progression of NDs induced by lipopolysaccharide (LPS). In an in vivo experiment, the results of HE staining, transmission electron microscopic, immunofluorescence microscopy of cleaved caspase-3 and Bax and the increasing expression of apoptotic pathway related proteins in mitochondria showed that LPS (10 mg/kg) administration damaged mitochondrial and induced hippocampal neuron apoptosis. The Western blot and RT-PCR indicated that LPS induced the activation of c-Myc/CLIC4 pathway. Furthermore, in an in vitro experiment, PC12 cells were exposed to LPS to induce cell injuries to mimic the model of NDs. To further confirm the role of the c-Myc/CLIC4 pathway in LPS-induced neuronal apoptosis, the gene knockout of c-Myc and CLIC4 were performed by CRISPR/Cas9. The results of the flow cytometry assay and Annexin V-FITC/PI showed that knocking out c-Myc and CLIC4 significantly reduced cell apoptosis. The results of Western blot and dual immunofluorescence with Cyt c and TOM20 showed that knocking out c-Myc and CLIC4 significantly reduced the expression of mitochondrial apoptosis-related proteins. Our data confirmed that LPS-induced apoptosis is regulated by the activation of c-Myc/CLIC4 pathway. These results support further research mechanisms underlying neurodegenerative diseases and can provide effective pharmacodynamic targets for the clinical development of therapeutic drugs for neurodegenerative diseases.

Introduction

Neurodegenerative diseases (NDs) are characterized by the loss of neuronal populations in specific regions of the brain (Neal and Richardson, 2018), including Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis and multiple sclerosis (You et al., 2017). With the progress made in the study of NDs, the inflammatory process, oxidative stress, mitochondrial dysfunction and excitotoxicity have been identified as being involved in neuronal degeneration (Jiang et al., 2017; Sery et al., 2013). But the etiologies and pathogenesis of NDs remains poorly understood. Although the etiologies of neurodegenerative diseases may be distinct, different diseases display a similar pathogenesis. Accumulating evidences indicates that apoptosis occurs in and contributes to neurodegenerative disorders’ onset and progression (Gerszon and Rodacka, 2018; Shao et al., 2017). Furthermore, neuronal cell death is the cardinal feature of both acute and chronic NDs (Yuan and Yankner, 2000).

Lipopolysaccharide (LPS), a gram-negative bacterial cell wall component, induces activation of executioner caspases and other signaling cascades that ultimately lead to apoptotic destruction of the cells (Yang et al., 1998). Zhao et al. (2017) reported that LPS is abundant in neocortical and hippocampal extracts from the AD brain. In mice, exposure to LPS also extends to cognitive function (Girard-Joyal and Ismail, 2017). LPS administration is used to induce an in vivo model for neurodegeneration (Noh et al., 2014; Yang et al., 2014). Since the hippocampus has been long recognized as being responsible for learning and memory functions, parasitic invasion of this site may cause neurodegenerative disorders (Chou et al., 2017). Therefore, a large number of studies have used LPS-injured hippocampal neurons as an in vivo model to explore the underlying pathogenesis of neurodegenerative diseases and effective therapeutic targets (Andy et al., 2018; Lee et al., 2018). PC12 cells had general characteristics of neuroendocrine cells, and were easy to subculture compared with primary neurons. Thus, PC12 cells stimulated by LPS were frequently used as the model in vitro for neurodegenerative diseases (Li et al., 2018b). Apoptotic neuronal death is a delayed, multistep process and therefore offers a therapeutic opportunity if one or more of these steps can be interrupted or reversed. So, it is very meaningful to determine the key targets in the regulation of apoptosis in the treatment of NDs.

The chloride intracellular channel (CLIC) family of proteins is ubiquitously expressed, being frequently localized both in the plasma membrane and in intracellular organelles in multiple cell types (Peretti et al., 2015). Among the chloride channel family, the CLIC4 protein was first cloned from the rat hippocampus and functionally studied (Howell et al., 1996). CLIC4, also known as p64H1, RS43, or mtCLIC, is widely expressed in neuronal and non-neuronal tissues (Suginta et al., 2001). CLIC4 is enriched in the outer mitochondrial membrane (Ponnalagu et al., 2016) and it is involved in maintaining the mitochondrial membrane potential (Arnould et al., 2003) to protect the cells from apoptosis. Under the conditions of cytotoxicity and genotoxic stress, CLIC4, as a direct downstream target gene of p53 and c-Myc (Fernandez-Salas et al., 2002), will induce apoptosis through the mitochondrial pathway by decreasing mitochondrial membrane potential, releasing Cytochrome C (Cyt C), and activating caspase-3 (Suh et al., 2004). Yin et al. (2018) observed increased apoptosis along with the upregulation of CLIC4 mRNA after performed LPS and HI. Thus, the CLIC4 levels must be tightly controlled in order to prevent cellular damage in the brain, while maintaining sufficient CLIC4 levels for essential cellular functions. Guo et al. (2018) confirmed that the pathogenesis of neurodegenerative disease is related to the increased protein expression of CLIC4. Although CLIC4 is highly involved in mitochondrial apoptosis pathway, the regulation of CLIC4 in neuronal apoptosis induced by LPS remains to be explored. Whether p53 or c-Myc regulates CLIC4 also needs to be proved.

In the current study, we reported marked upregulation of CLIC4 protein level during neuronal apoptosis induced by LPS. During this process, the activation of c-Myc plays a critical role in neuronal CLIC4 accumulation. c-Myc acts as an upstream effector of CLIC4 to mediate mitochondrial apoptosis pathways in neuronal death models in vivo and in vitro. Our results are beneficial to the understanding of the mechanism of neurodegenerative diseases and indicate that c-Myc/CLIC4 pathway may be a potential mechanism targets for neurological diseases with apoptosis-induced neuronal death.