GINS complex subunit 2 (GINS2) plays a protective role in alcohol-induced brain injury

Abstract Acute alcohol intoxication is a central nervous system disease that accounts for a large number of hospital admissions. In the present study, we have explored the role of GINS complex subunit 2 (GINS2) in acute alcohol intoxication and alcohol-induced brain injury. We began by determining that GINS2 mRNA expression was significantly increased in the serum of patients with alcohol abuse. We then found that GINS2 is increased in mouse brains after alcohol consumption. To explore the role of GINS2 in alcohol-induced microglia function, we knocked down GINS2 in mouse microglia and then treated the cells with alcohol. Knockdown of GINS2 significantly increased alcohol-induced ROS production and the oxidative stress marker malondialdehyde. To explore if GINS2 is involved in alcohol-induced microglia apoptosis, we examined cell viability in GINS2 knockdown cells by TUNEL staining and caspase activity assays. Consistently, results showed that alcohol-induced cell apoptosis was promoted by knockdown of GINS2. Finally, we assessed expression levels of inflammatory factors in GINS2 knockdown microglial cells as well as the effects of GINS2 knockdown on NF-κB signalling. Inflammatory factors were stimulated by alcohol and further promoted by GINS2 knockdown, and GINS2 knockdown promoted alcohol-induced NF-κB activity in microglia.


Introduction
Alcohol consumption is deeply entrenched in societies throughout the world as an accepted cultural practice as well as one of the greatest causes of morbidity and mortality [1,2]. Its negative effects are wide ranging, impacting communities and the individual's health both chronically and acutely [3]. In the United States, excessive alcohol consumption accounts for 1 in 10 deaths among working-age adults and is a leading cause of premature mortality [4]. In the present study, we have explored the role of GINS complex subunit 2 (GINS2) in acute alcohol intoxication and alcoholinduced brain injury. Ultimately, we have determined that GINS2 plays a crucial protective role in microglia against alcohol-induced brain injury and NF-jB-mediated inflammation. The central nervous system (CNS) largely mediates both chronic and acute alcohol intoxication [5]. Chronic alcohol intoxication causes altered excitatory and inhibitory signalling cascades as well as neuroinflammatory responses that ultimately results in lasting structural brain abnormalities [6,7]. Acutely, alcohol intoxication causes dramatic impairments in attention, visuomotor control, short-term memory, mood, and cognitive performance [8]. While the short-and longterm effects of alcohol consumption on cognition and behaviour have been extensively studied, more research at the genetic and molecular levels are necessary for clinically assessing and mediating the harmful outcomes. There are currently no clinically reliable objective biomarkers of alcohol abuse or alcoholism, but investigating the molecular and genetic underpinnings of alcohol pathologyas we have in this studypromises to improve diagnosis and treatment of alcohol abuse [9]. Some areas of the brain are more susceptible to the harmful effects of alcohol than others. For example, the hippocampus, white matter, cerebellum, prefrontal cortex and glial cells are especially susceptible to damage [10]. Alcohol's effects also vary based on age, with greatly noticeable differences between the developing and developed brain [11,12]. However, consistent across both adolescent and adult brains is the significant effect alcohol has on neuroinflammation and subsequent neurotoxicity [13,14]. Acute alcohol intoxication induced neuroimmune gene expression which increases oxidative stress and subsequent neurodegeneration [15]. This alcohol-induced, aberrant molecular activity is part of a complex proinflammatory pathway that includes enhanced microglia and astrocyte activation as well as increased NF-jB signalling, which upregulates transcription of cytokines, oxidases, and proteases [16,17].
Activation of proinflammatory monocytes is a hallmark of alcohol-induced neurodegenerative processes [18,19]. Microglia, which are the brain's resident macrophage that develops from monocytes, can function as pro-or anti-inflammatory mediators in response to CNS immune insults [20,21]. While microglia can help establish cell homeostasis following alcohol exposure [22], they also exacerbate oxidative stress and neuroinflammation [23,24]. Acute alcohol intoxication activates microglia almost immediately, resulting in NF-jB signalling and increases in several cytokines, including interleukin-1b (IL-1b) [25] and tumour necrosis factor (TNF)-a [26]. In this study, we therefore focused on the effects of acute alcohol intoxication on microglia, specifically how their functions are impacted by knockdown of GINS complex subunit 2 (GINS2), which we determined was upregulated by alcohol in rodent brain tissues in a 2015 study [27]. Ultimately, we established that GINS2 knockdown increases alcohol-induced brain inflammation, oxidative pressure, microglia endoplasmic reticulum (ER) stress, microglia apoptosis, and NF-jB activity.

Human samples
The study was approved by the Ethics Committee of the Fourth Affiliated Hospital of Harbin Medical University. This study includes 10 men with alcohol abuse who were hospitalized in the Neurology Department of the Fourth Affiliated Hospital of Harbin Medical University. Written informed consent was received from all patients. Fresh serum samples were collected from all participants and frozen in liquid nitrogen and then stored at À80 C.

Animals
This study was approved by the Institutional Animal Care and Use Committee (IACUC) of Harbin Medical University. 36 C57BL/6 mice (5-6 weeks old) were randomly divided into four groups. Alcohol consumption followed a two-bottle choice-drinking in the dark paradigm [28,29]. For 3 weeks, the animals received 0% or 15% alcohol from separate bottles during a 3 h period starting 3 h into the dark cycle to avoid side preference. The groups were pair-fed diets with equal amounts of water. Study mice were sacrificed under anaesthesia and brain tissues were excised for analysis.

Culture of primary microglial cells
Mouse primary microglial cell cultures were prepared as previously described [27,30].

Western blot
We performed western blot as previously described [31]. Antibodies against GINS2, ATF4, CHOP, GRP78, p65, Histone H1 and b-actin were purchased from Santa Cruz Biotechnologies (Santa Cruz). Protein levels of Histone H1 and b-actin were used as loading controls.

ROS measurement
Intracellular ROS production was used to measure oxidative pressure. The microglial cells (1 Â 10 5 ) were seeded in sixwell plates and resuspended in 10 lM DCF-DA for 30 min at 37 C after treatment. Flow cytometry was then used to analyse ROS levels in the stained cells.

Malondialdehyde and superoxide dismutase measurement
The kits for Malondialdehyde (MDA) levels and superoxide dismutase (SOD) activity measurement (Beyotime, Beijing) were used according to the manufacturer's instructions.

Caspase activity assays
Caspase activity assays were performed as previously described [31].

Statistical analysis
Result analysis were showed as mean ± SD from at least three separate experiments. One-way ANOVA was used to analyse differences between groups. p < .05 was considered statistically significant.

Treatment with alcohol induces GINS2 production in microglial cells and brain tissues
From our previous microarray data, we found that GINS2 was upregulated by alcohol in rodent brain tissues [27]. Therefore, in the present study we examined GINS2 mRNA expression in the serum of patients with alcohol abuse and found that GINrS2 expression was significantly increased (Figure 1(A)). To investigate whether GINS2 plays a role in alcohol-induced brain injury, we analysed GINS2 expression in the brains of mice fed with alcohol. We found that GINS2 mRNA and protein level is increased in mouse brains after alcohol consumption (Figure 1(B) and 1(C)). In addition, there was no difference in weight gain between the control and alcohol-feed groups (Figure 1(D)). To assess the effects of alcohol on microglia, we treated primary microglial cells with different concentrations of alcohol and then analysed GINS2 expression 12 h post-treatment. GINS2 expression was significantly higher after alcohol treatment in a dose-dependent manner (Figure 1(E) and 1(F)). Together, these results indicate that alcohol increases GINS2 expression in brain tissues and primary microglia cells.

GINS2 knockdown enhances oxidative pressure induced by alcohol
To explore the role of GINS2 in alcohol-induced microglial cell function, we knocked down GINS2 gene in mouse microglial cells and then exposed the cells to 75 mM alcohol. Knockdown efficiency was confirmed by western blotting (Figure 2(A)). Because prior research has established a close relationship between oxidant stress and alcohol-induced brain injury [32], we used flow cytometry to assess the effect of alcohol treatment on intracellular ROS levels. As shown in Figure 2(B), GINS2 knockdown significantly increased alcoholinduced ROS production. To further assess oxidative pressure, we assessed levels of MDA, a degradation product of membrane lipid oxidation. GINS2 knockdown significantly enhanced alcohol-induced MDA production (Figure 2(C)). We then examined levels of the endogenous anti-oxidative enzyme SOD. GINS2 knockdown significantly decreased the amount of free SOD in alcohol treated microglial cells (Figure 2(D)). Overall, these results indicate that reduced GINS2 expression increases alcohol-induced oxidative pressure in microglial cells.

GINS2 knockdown increases ER stress in microglial cells treated with alcohol
Prior research has determined that endoplasmic reticulum (ER) stress is highly associated with alcohol-induced brain cell injury [33,34]. We therefore examined three biomarkers of ER stress (ATF4, CHOP and GRP78) in microglial cells. As shown in Figure 3, treatment with alcohol promoted expression of ATF4, CHOP and GRP78, and GINS2 knockdown further increased the levels of these markers. Taken together, the above findings strongly suggest that GINS2 knockdown enhances alcohol-induced ER stress in microglial cells.

GINS2 knockdown increases alcohol-induced apoptosis in microglial cells
To explore if GINS2 is involved in alcohol-induced apoptosis in microglial cells, we examined cell viability in control and GINS2 knockdown cells. In control cells, cell viability decreased by 50% with alcohol treatment, whereas in GINS2 knockdown cells, cell viability decreased by almost 60% with alcohol treatment (Figure 4(A)). TUNEL staining, caspase-3 and caspase-9 activity assays consistently showed that alcohol-induced cell apoptosis was promoted by knockdown of GINS2 (Figure 4(B)-(D)). Overall, these results suggest that the presence of GINS2 protects against alcohol-induced apoptosis in microglial cells.

GINS2 regulates NF-jB signalling
To investigate whether GINS2 regulates alcohol-induced rodent brain inflammation, we evaluated expression of    inflammatory factors in GINS2 knockdown microglial cells. IL-1b, IL-6, IL-10, TNF-a and IFN-c expression was stimulated by alcohol and further promoted by GINS2 knockdown ( Figure 5). To determine whether NF-jB signalling is affected by alcohol-induced, GINS2-mediated inflammation, we treated GINS2 knockdown mouse microglial cells with alcohol and extracted nuclear proteins for p65 expression. Alcohol promoted p65 nuclear translocation, which was enhanced by GINS2 knockdown (Figure 6(A)). NF-jB luciferase activity and cell fluorescence staining assays confirmed that GINS2 knockdown in microglial cells further promoted alcohol-induced NF-jB activity (Figures 6(B) and 4(C)). These results indicate that GINS2 knockdown increases alcohol-induced brain inflammation and alcohol-induced NF-jB activity.

Discussion
Despite its presence in almost all societies, alcohol consumption continues to contribute greatly to the global burden of disease, accounting for around 3.8% of global deaths and 4.6% of global disability-adjusted-life-years [35]. Acute alcohol intoxication occurs following ingestion of significant amounts of alcohol over a short period of time. Symptoms of acute alcohol intoxication vary with severity and can include impaired judgement, memory deficits, inappropriate aggressive behaviour, coma, and other harmful cognitive-behavioural conditions [36]. While alcohol has been extensively studied, especially for its social and economic consequences, the genetic and molecular mechanisms of alcohol's effects on the CNS have not been fully elucidated. In the present study, using patient serum, an experimental mouse model of acute alcohol intoxication, and primary mouse microglia, we carried out extensive analyses of the effects of GINS2 knockdown on alcohol-induced neuroinflammation and neurotoxicity. Ultimately, our study has established a crucial, protective role for GINS2 in acute alcohol intoxication, thus providing the basis for further research into clinical biomarkers of acute alcohol intoxication and effective clinical treatments.
We focused on the activity of GINS2 because our prior research using microarray data revealed that alcohol upregulates GINS2 expression in rodent brain tissues [27]. GINS was originally identified in 2000 by Lai et al. as an evolutionarily conserved protein product in Caenorhabditis elegans [37], but the first study to focus entirely on the GINS multiprotein complex was conducted by Takayama et al. [38]. In this study, it was determined that GINS forms part of a protein complex required for the initiation of chromosomal DNA replication in yeast. Since then, GINS has been shown to associate with Cdc45 and Mcm2-7 in a helicase complex for leading strand DNA synthesis in human cells [39]. Not only is the GINS complex crucial for normal human physiology in the domain of cell proliferation but it has also been shown to play roles in a variety of pathologies, largely cancers [40]. However, no studies have focused on its function in the CNS or alcohol-induced CNS changes. The present study therefore contributes substantially to the growing body of work concerning GINS2 function in pathology and opens the door for more research into its role in the CNS. Furthermore, this study establishes a previously little explored relationship between GINS2 and oxidative pressure, ER stress, and NF-jB signalling.
Microglia are the resident macrophages of the CNS, and they are highly active in the healthy and pathological CNS, responding to disturbances in blood vessels, neurons, and the blood brain barrier, among many other fundamental CNS components [41]. As the primary immune effectors of the CNS derived from circulating monocytes, microglia act as antigen presenting cells and travel to sites of tissue damage to secrete cytokines and other proinflammatory constituents [42]. They serve crucial, sometimes seemingly contradictory, functions in neurodegenerative diseases [43]. In response to neurological insults, microglia adopt variable phenotypes, which can be anti-or proinflammatory depending on the specific CNS challenge [20]. Additionally, microglia can sometimes be induced to produce ROS that causes neurodegeneration, such as in the case of the organochlorinated pesticide dieldrin [44], or they can scavenge oxygen-derived free radicals, such as during excitotoxin-induced neurodegeneration [45]. In the present study, we have determined that GINS2 knockdown in microglia has a wide range of negative consequences for microglia when treated with alcohol, including increased oxidative pressure, ER stress, and apoptosis. These results strongly suggest that GINS2 serves a protective role in microglia when its expression is induced by alcohol consumption.
Even though it is generally believed that alcohol consumption induces an inflammatory state systemically [46] and in the CNS [19], the effects of acute alcohol intoxication on immune regulation are not entirely understood. Alcohol consumption has been shown to stimulate toll-like receptor 2 (TLR2) and TLR4 expression in microglia, thus increasing ROS, neuronal apoptosis, and proinflammatory mediators [47]. Furthermore, in a rodent model of binge drinking, alcohol exposure increased hippocampal microglia activation, thus inducing a heightened state of neuroinflammation [48]. NF-jB signalling is integral to alcohol-induced neuroinflammation, as alcohol can directly increase NF-jB transcription of proinflammatory genes, such as through alcohol's release of the TLR4 agonist High Mobility Group Box 1 [49]. This alcohol-induced proinflammatory signalling can be attenuated, for example, with the anti-inflammatory drug xanthohumol, which inhibits microglial inflammatory responses by reducing NF-jB signalling [50]. By demonstrating that GINS2 knockdown increases alcohol-induced NF-jB signalling, this study indicates that GINS2 expression may induce microglia to serve a more anti-inflammatory role to reduce alcoholinduced neuroinflammation.
Overall, the present study has achieved compelling results regarding the role of GINS2 in acute alcohol intoxication, specifically in microglia integrity and neuroinflammation. The results suggest that GINS2 expression can potentially serve as a biomarker of alcohol-induced neuroinflammation and can help identify microglia that act in an inflammatory capacity. Perhaps most importantly, the results here open new avenues for understanding the complex nature of alcoholinduced neurotoxicity and neuroinflammation.

Author contributions
CL and RW performed the experiments and collected data. CL and YZ analysed the data and wrote the manuscript. YZ designed the work.

Disclosure statement
No potential conflict of interest was reported by the authors.