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. Author manuscript; available in PMC: 2020 Sep 1.
Published in final edited form as: Alcohol. 2018 Dec 7;79:25–35. doi: 10.1016/j.alcohol.2018.12.002

Minocycline Attenuates Ethanol-induced Cell Death and Microglial Activation in the Developing Spinal Cord

Zhenhua Ren 1,2, Xin Wang 2, Mei Xu 2, Jacqueline A Frank 2, Jia Luo 2,3,#
PMCID: PMC6555701  NIHMSID: NIHMS1516173  PMID: 30529756

Abstract

Developmental exposure to ethanol may cause fetal alcohol spectrum disorders (FASD), and immature central nervous system (CNS) is particularly vulnerable to ethanol. In addition to the developing brain, we previously showed that ethanol also caused neuroapoptosis, microglial activation, and neuroinflammation in the spinal cord. Minocycline is an antibiotic that inhibits microglial activation and alleviates neuroinflammation. We sought to determine whether minocycline could protect spinal cord neurons against ethanol-induced damage. In this study, we showed that minocycline significantly inhibited ethanol-induced caspase-3 activation, microglial activation, and the expression of pro-inflammatory cytokines in the developing spinal cord. Moreover, minocycline blocked ethanol-induced activation of glycogen synthase kinase 3 beta (GSK3β) a key regulator of microglial activation. Meanwhile, minocycline significantly restored ethanol-induced inhibition of protein kinase B (AKT), mammalian target of the rapamycin (mTOR), and ERK1/2 signaling pathways, which were important pro-survival signaling pathways for neurons. Together, minocycline may attenuate ethanol-induced damage to the developing spinal cord by inhibiting microglial activation/neuroinflammation and restoring the pro-survival signaling.

Keywords: Alcohol, glycogen synthase kinase 3, fetal alcohol spectrum disorders, inflammation, neuroprotection

Introduction

Fetal alcohol spectrum disorders (FASD) are caused by alcohol exposure during the development, and characterized by a spectrum of structural anomalies along with neurocognitive and behavioral disabilities (Riley, Infante, & Warren, 2011). According to the National Institute on Alcohol Abuse and Alcoholism (NIAAA), 10% of pregnant women have drunk alcohol in the past month, and 20-30% drank at some point during the pregnancy in the United States. In North American, approximately 4.7% of women who are pregnant are alcoholics (McHugh, Wigderson, & Greenfield, 2014). Alcohol crosses the blood brain barrier (BBB) and the developing central nervous system (CNS) is particularly vulnerable to alcohol exposure (Crews, Lawrimore, Walter, & Coleman, 2017; Kane & Drew, 2016). Fetal alcohol exposure is the leading cause of mental retardation (Adebiyi, Mukumbang, Okop, & Beytell, 2018; May & Gossage, 2001; Nash, Sheard, Rovet, & Koren, 2008). FASD causes drastic medical and social burdens (de Sanctis, Memo, Pichini, Tarani, & Vagnarelli, 2011; Joya et al., 2012; Memo, Gnoato, Caminiti, Pichini, & Tarani, 2013). Despite attempts to increase public awareness of the risks involved, the number of women drinking during pregnancy has not declined in the USA (Alshaarawy, Breslau, & Anthony, 2016; Ebrahim, Diekman, Floyd, & Decoufle, 1999). Ethanol adversely impacts the developing CNS and causes neuroimmune responses (Crews et al., 2017; Kane & Drew, 2016; Komada et al., 2017; Pascual et al., 2017). Ethanol-induced neuron death and neuroinflammation may underlie some neuropathological and cognitive defects observed in FASD (Chastain & Sarkar, 2014; Drew & Kane, 2014; Kane & Drew, 2016; X. Wang et al., 2018). Therefore, it is important to develop effective strategies to ameliorate alcohol-induced CNS damages.

Although ethanol-induced damage to the developing brain has been well-established, the effects of ethanol on the developing spinal cord, however, receive little attention. Using a well-established third trimester equivalent mouse model of ethanol exposure, we have recently demonstrated that ethanol exposure during the development caused permanent loss of neurons in the spinal cord which was accompanied by microglial activation and neuroinflammation (Ren et al., 2017). Minocycline is a second generation broad-spectrum antibiotic, and the most lipid-soluble of the tetracycline-class antibiotics that is capable of crossing the BBB (X. Wang et al., 2018). In various models of neurodegenerative disease, such as Alzheimer’s disease (AD), Huntington’s disease (HD), and Parkinson’s disease (PD), minocycline has demonstrated neurorestorative as well as neuroprotective properties (Budni et al., 2016; M. Chen et al., 2000; Seidl & Potashkin, 2011). Minocycline has a potent anti-microglial activation and anti-inflammatory property (Moller et al., 2016; Tikka & Koistinaho, 2001), which is a possible mechanism for its neuroprotection in neurodegenerative disorders (Budni et al., 2016; S. D. Chen, Yin, Hwang, Tang, & Yang, 2012; Cox, Varma, & Banik, 2015; Dheen, Kaur, & Ling, 2007; Seidl & Potashkin, 2011).

The current study sough to determine whether minocycline’s neuroprotective and anti-inflammatory property can alleviate ethanol-induced damages to the developing spinal cord. Using the third trimester equivalent mouse model of ethanol exposure, we demonstrated here that minocycline significantly ameliorated ethanol-induced neuroapoptosis in dorsal horn of spinal cord, and inhibited ethanol-stimulated microglial activation/neuroinflammation. We also investigated the cell signaling pathways that may be involved in minocycline neuroprotection.

Material and Methods

Reagents

The detailed information of primary antibodies used for immunohistochemistry (IHC) is shown in Table 1, and for immunoblotting (IB) in Table 2. Anti-β-actin antibody and DAB staining kit were obtained from Sigma-Aldrich (St. Louis, MO). HRP-conjugated anti-rabbit, anti-mouse, anti-goat and anti-rat secondary antibodies were purchased from GE Healthcare Life Sciences (Piscataway, NJ). Ketamine/xylazine was obtained from Butler Schein Animal Health (Dublin, OH). Minocycline was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Reagents used in this project were obtained from Sigma Chemical Co. (St. Louis, MO, USA) unless stated otherwise.

Table 1.

Primary antibodies used for Immunohistochemistry staining

Name of Antibody Target Antigen Manufacturer Catalog Number Species Raised Dilution Used Related publications
cleaved
caspase-3		 Asp175 Cell Signaling CS9661S Rabbit, Polyclonal 1:3000 PMID: 22632376
Iba-1 C-terminus Wako Chemicals 019-19741 Rabbit, Polyclonal 1:1000 PMID: 22956822
CD68 FA-11 AbD Serotec MCA1957 Rat, monoclonal 1:500 PMID: 12480978
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Table 2.

Primary antibodies used for Immunoblotting

Name of Antibody Target Antigen Manufacturer Catalog Number Species Raised Dilution Used Related publications
cleaved caspase-3 Asp175 Cell Signaling CS9661 Rabbit, Polyclonal 1:1000 PMID: 23319806
Cleaved PARP Asp214 Cell Signaling CS9548 Mouse, monoclonal 1:1000 PMID: 24516177
caspase 8 1C12 Cell Signaling CS9746 Mouse, monoclonal 1:1000 PMID: 28439094
Caspase 12 Q6UXS9 AbCam ab18766 Rabbit, Polyclonal 1:1000 PMID: 23209735
Il-6 AbCam ab6672 Rabbit, Polyclonal 1:1000 PMID: 28450925
MCP-1 PMP35 AbD Serotec AAM43 Rabbit, Polyclonal 1:3000 PMID: 9884336
MCPIP P-12 Santa Cruz sc-136750 Goat, Polyclonal 1:1000 PMID: 22036805
CD68 FA-11 AbD Serotec MCA1957 Rat, monoclonal 1:1000 PMID: 27829437
P2X7 H-265 Santa Cruz sc-25698 Rabbit, Polyclonal 1:1000 PMID: 24286344
Iba-1 C-terminus Wako Chemicals 019-19741 Rabbit, Polyclonal 1:1000 PMID: 22504638
p-GSK3β (Ser9) 5B3 Cell Signaling CS9323 Rabbit, monoclonal 1:1000 PMID: 27901475
GSK3β 27C10 Cell Signaling CS9315 Rabbit, monoclonal 1:1000 PMID: 22796213
Active β catenin Ser33/37/Thr41 Cell Signaling CS8814 Rabbit, monoclonal 1:1000 PMID: 28049723
β catenin Ser37 Cell Signaling CS9562 Rabbit, Polyclonal 1:1000 PMID: 28237967
p-CREM Ser133 Cell Signaling CS9198 Rabbit, monoclonal 1:1000 PMID: 28587388
CREM 48H2 Cell Signaling CS9197 Rabbit, monoclonal 1:1000 PMID: 28587388
p-c-Jun Ser63 Cell Signaling CS9261 Rabbit, monoclonal 1:1000 PMID: 29285221
p-Akt Ser473 Cell Signaling CS9271 Rabbit, Polyclonal 1:1000 PMID: 25942043
Akt Cell Signaling CS9272 Rabbit, Polyclonal 1:1000 PMID: 28250437
p-PI3K Tyr199 Cell Signaling CS4228 Rabbit, Polyclonal 1:1000 PMID: 15592455
p-PDK1 Ser241 Cell Signaling CS3061 Rabbit, Polyclonal 1:1000 PMID: 10801415
p-mTOR Ser2481 Cell Signaling CS2974 Rabbit, Polyclonal 1:1000 PMID: 12901945
p-ERK1/2 Thr202/Tyr204 Cell Signaling CS9101 Rabbit, Polyclonal 1:1000 PMID: 28646232
ERK1/2 Cell Signaling CS 9102 Rabbit, Polyclonal 1:1000 PMID: 17496916
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Animals and ethanol exposure

C57BL/6J mice were obtained from Jackson Laboratories (Bar Harbor, Maine) and maintained in the Division of Laboratory Animal Resources of the University of Kentucky Medical Center. All procedures were performed in accordance with the guidelines set by the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Kentucky. Animals were maintained in a 12/12 hours light/dark cycle with a temperature of 22±1°C and relative humidity of 60±5%, and received standard chow and water ad libitum. A well-established mouse model of postnatal ethanol exposure was used (Carloni, Mazzoni, & Balduini, 2004; Ikonomidou et al., 2001; Olney et al., 2002). Ethanol and minocycline were administrated on postnatal day 5 (PD5) pups. Pups were weighed and toe-clipped. Animals of similar weight were randomly assigned to four experimental groups: control (Con), minocycline (Min), ethanol (EtOH group), and ethanol plus minocycline (EtOH + Min). To preclude potential litter effects, 4-5 litters were used for each experiment and only one pup from each litter was assigned to an experimental group. Pups of mixed sex with roughly equal number of male and female pups were used in this study. Minocycline was administered by two subcutaneous (SC) injections at a concentration of 30 mg/kg each at 12 and 2 hours prior to ethanol exposure (Guasti et al., 2009). Pups received a total of 5 g/kg ethanol in two SC injections; each was of 2.5 g/kg ethanol and 2 hours apart (Alimov et al., 2013). We have previously determined that the blood alcohol centration (BAC) was 338 mg/dl 8 hours after the first injection (Ke et al., 2011). Control animals received SC injections of the same volume of sterile saline. Eight hours after the first injection, mice were euthanized, and the spinal cords were dissected and processed for further analysis.

Tissue preparation and immunobiotting

Animals were anesthetized with intraperitoneal injection of ketamine/xylazine (100 mg/kg/10 mg/kg), and the entire spinal cord was dissected and immediately frozen in dry ice and then stored at −80°C. The protein was extracted and subjected to immunoblotting analysis as previously described (X. Wang et al., 2018). Briefly, tissues were homogenized in an ice cold lysis buffer containing 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM EGTA, 0.5% NP-40, 0.25% SDS, 1 mM PMSF, 5 μg/ml leupeptin, and 5 μg/ml aprotinin. Homogenates were centrifuged at 20,000 g for 30 min at 4°C and the supernatant fraction was collected. After determining protein concentration, aliquots of the protein samples (30 μg) were separated on a SDS-polyacrylamide gel by electrophoresis. The separated proteins were transferred to nitrocellulose membranes. The membranes were blocked with 5% BSA in 0.01 M PBS (pH 7.4) and 0.05% Tween-20 (TPBS) at room temperature for 1 hour. Subsequently, the membranes were probed with primary antibodies overnight at 4°C. The final dilutions for primary antibodies were showed in the Table 2. After three washes (5 min each) in TPBS, the membranes were incubated with a secondary antibody conjugated to horseradish peroxidase. The immune complexes were detected by the enhanced chemiluminescence substrate (GE Healthcare, Chalfont, Buckinghamshire, UK). The density of immunoblotting was quantified with the software of Image lab 5.2 (Bio-Rad Laboratories, Hercules, CA).

Immunohistochemistry

The procedure for immunohistochemistry (IHC) has been previously described with some modifications (X. Wang et al., 2018). Briefly, animals were deeply anesthetized with intraperitoneal injection of ketamine/xylazine and then intracardially perfused with PBS followed by 4% paraformaldehyde in PBS (pH7.4). The spinal cord tissues from lumbar and thoracic sections were removed, post fixed in 4% paraformaldehyde for an additional 24 hours and then transferred to sucrose solution (concentration from 10% to 30%) until the tissues sunk to the bottom. The tissues were frozen in OCT compound and sectioned at 10 μm in a sagittal plane using a Cryostat Microtone (Thermo Scientific). The sections were incubated in 0.3% H2O2/30% methanol in PBS for 10 min. After washing with PBS, sections were mounted on slides and dried. The slides were then blocked with 5% goat serum and 0.5% Triton X-100 in PBS for 1 h at room temperature. After blocking, the slides were treated with primary antibodies overnight at 4°C. The details of primary antibodies used for immunohistochemistry were showed in the Table 1. After washing with PBS, slides were incubated with biotin-conjugated goat anti-rabbit or goat anti-rat secondary antibody (1: 800) for 1 hour at room temperature and then washed with PBS. Avidin–biotin–peroxidase complex was prepared according to the manufacturer’s instructions. The slides were developed in 0.05% 3,3-diaminobenzidine (DAB) (Sigma-Aldrich, Inc.) containing 0.003% H2O2 in PBS. The sections were then dehydrated through graded alcohol, and cleared with xylene and mounted with synthetic resin. The images were recorded using an Olympus BX51 microscope. Negative controls were performed by omitting the primary antibody. For Iba-1 staining, the positive cells were counted in twenty randomly selected fields using the software of Image lab 5.2 (Bio-Rad Laboratories, Hercules, CA). Ten sections from five animals were analyzed for each group.

Statistical analysis

The data were expressed as mean ± SEM. Statistical significance was determined by a one-way ANOVA followed by Scheffe’s post-doc test at a significance level of 5%. The analyses were performed using SPSS software (SPSS, Chicago, IL, USA).

Results

Minocycline alleviates ethanol-induced apoptosis in the developing spinal cord

Our previous study showed that developmental ethanol exposure causes permanent loss of spinal cord neurons (Ren et al., 2017). We sought to determine whether minocycline offer protection against ethanol-induced neuronal death in the developing spinal cord. As shown in Fig. 1, the alterations in cleaved caspase-3 were analyzed by one-way ANOVA with the treatments as a variable. A significant alteration in cleaved caspase-3 was observed, [F(3,24)=84.37; p<0.0001]. Ethanol exposure caused a drastic increase in the expression of cleaved caspase-3 in the spinal cord of PD5 mice (p<0.0001) (Fig. 1A and 1B). Consistent with our previous findings (Ren et al., 2017), the cleaved-capase-3 positive cells were mainly located in the lamina II-MI of the dorsal horn of spinal cord. Minocycline significantly diminished ethanol-induced caspase-3 activation (p<0.0001) (Fig. 1A and 1B). In addition, ethanol increased the expression of cleaved PARP, [F(3,24)=24.43; p<0.001]; caspase 8 [F(3,24)=28.68; p<0.001]; and caspase 12, [F(3,24)=31.90; p<0.0001]; whereas minocycline significantly inhibited ethanol-induced increase of these apoptotic markers (p<0.05) (Fig. 1B and 1C).

Figure 1. Effect of minocycline on ethanol-induced neuroapoptosis in the developing spinal cord.

Figure 1.

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A: C57BL6 mice of postnatal (PD) 5 were exposed to ethanol as described in the Materials and Methods. Minocycline was administered by two subcutaneous injections at a dose of 30 mg/kg each on 12 h and 2 h before the ethanol exposure. Eight hours after ethanol exposure, mice were sacrificed and the spinal cord was processed for IHC of cleaved caspase-3. Upper panel, Bar = 200 μm; Lower panel, Bar = 100 μm. B: The expression of cleaved caspase-3 and PARP in the spinal cord were determined with immunoblotting. C: The expression of cleaved caspase-8 and caspase-12 in the spinal cord were determined with immunoblotting. For immunoblotting, the expression of these proteins was quantified and normalized to the expression of β-actin. Each data point was the mean ± SEM of three independent experiments. *p< 0.05 and **p < 0.01, statistically significant difference from control group; #p < 0.05 and ##p < 0.01, statistically significant difference from ethanol group. Four-five animals were analyzed for each group.

Minocycline inhibits ethanol-induced increase of cytokines and chemokines in the developing spinal cord

We examined the effect of ethanol minocycline on the expression of pro-inflammatory cytokines/chemokines in the developing spinal cord (Fig. 2). Inerleukin-6 (IL-6) is a pro-inflammatory cytokine that is involved CNS infections and injuries (Vallieres & Rivest, 1999; W. Y. Wang, Tan, Yu, & Tan, 2015). Monocyte chemoattractant protein-1 (MCP1, also known as CCL2) is a pro-inflammatory chemokine and up-regulated in many CNS disorders (Dimitrijevic, Stamatovic, Keep, & Andjelkovic, 2007; Hinojosa, Garcia-Bueno, Leza, & Madrigal, 2011; Yao & Tsirka, 2014). Monocyte chemotactic protein-1-induced protein-1 (MCPIP1), a transcriptional activator, is downstream of CCR2 and regulates the expression of MCP-1, IL-1 β and TNFα (Jura, Skalniak, & Koj, 2012). Ethanol increased the expression of IL-6, [F(3,24)=18.42; p<0.05]; MCP-1, [F(3,24)=20.12; p<0.05]; and MCPIP, [F(3,24)=60.63; p<0.05]; whereas minocycline significantly inhibited ethanol-induced increase of these pro-inflammatory mediators (p<0.05) (Fig. 2).

Figure 2. Effect of minocycline on ethanol-induced cytokines and chemokines.

Figure 2.

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The treatment of minocycline and ethanol was same as described in Fig. 1. The spinal cord tissues were processed for immunoblotting analysis (A). The expression of IL-6 (B), MCP-1 (C), and MCPIP (D) was quantified and normalized to the expression of β-actin. Each data point was the mean ± SEM of three independent experiments. *p< 0.05 and **p < 0.01, statistically significant difference from control group; #p < 0.05 and ##p < 0.01, statistically significant difference from ethanol group.

Minocycline inhibits ethanol-induced microglial activation in the developing spinal cord

Microglial activation plays a key role in the neuroinflammation and is associated with many CNS disorders (Dheen et al., 2007). Since minocycline is a “microglia inhibitor”, we sought to determine whether minocycline was able to inhibit ethanol-induced microglial activation. Iba-1 is specifically expressed microglia and upregulated following CNS injury (Ito et al., 1998), ischemia (Ito, Tanaka, Suzuki, Dembo, & Fukuuchi, 2001), and a number of neurological disorders (Imai & Kohsaka, 2002). The resting microglia cell is characterized by a small cell body and much elaborated thin processes, while the activated microglia have fewer and thicker processes with larger cell body (Z. Chen et al., 2012). Microglial activation was assessed morphologically by IHC of Iba-1. Ethanol significantly increased the number of active microglia (Fig. 3A and 3C), and upregulated the expression level of Iba-1 in the developing spinal cord, [F(3,24)=106.3; p<0.0001] (Fig. 3B). CD68, a cell surface marker of M1-poalrized microglia, is inflammatory markers in the brain and indicative of microglial activation (Jeong, Ji, Min, & Joe, 2013; Kobayashi et al., 2013). P2X7 is a regulator of inflammatory responses, and expressed by most immune cells, including the monocyte-derived cell lineages (Adinolfi et al., 2018). The activation of P2X7 is associated with microglial activation and cytotoxicity (Volonte, Apolloni, Skaper, & Burnstock, 2012). Ethanol increased the expression of CD68, [F(3,24)=106.3; p<0.0001]; and P2X7, [F(3,24)=62.77; p<0.001] protein levels (Fig. 3E). The increased CD68 expreesion was further confirmed by the IHC study (Fig. 3D).

Figure 3. Effect of minocycline on ethanol-induced microglia activation.

Figure 3.

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The treatment of minocycline and ethanol was same as described in Fig. 1. A: Microglia was identified by IHC of Iba-1 in the spinal cord. Upper panel, Bar = 200 μm; Lower panel, Bar = 100 μm. B: The expression of Iba-1 in the spinal cord were determined with immunoblotting. C: The number of Iba-1-positive cells in the dorsal horn of spinal cord was determined as described in the Materials and Methods. D: The infiltration of macrophages in the spinal cord was determined by CD68 IHC. Bar = 100 μm. E: The expression of CD68 and P2X7 in the spinal cord were determined with immunoblotting. For immunoblotting, the expression of these proteins was quantified and normalized to the expression of β-actin. Each data point was the mean ± SEM of three independent experiments. *p< 0.05 and **p < 0.01, statistically significant difference from control group; #p < 0.05 and ##p < 0.01, statistically significant difference from ethanol group. Four-five animals were analyzed for each group.

Minocycline blocked ethanol-induced increase of Iba-1 expression and the number of active microglia (p<0.01) (Fig. 3A-C). It also inhibited ethanol-induced increase of CD68 (p<0.01) and P2X7 (p<0.05) (Fig. 3D and 3E). It is interesting to note that minocycline alone also increased CD68 expression (p<0.01); however, when ethanol and minocycline were applied together, minocycline significantly inhibited ethanol-induced increase of CD68 (Fig. 3E).

Minocycline inhibits ethanol-induced GSK3β activation in the developing spinal cord

Glycogen synthase kinase 3 beta (GSK3β) dysregulation is involved in the pathogenesis of numerous neurological disorders, affecting the CNS encompassing both neuroinflammation and neurodegenerative diseases (Golpich et al., 2015; Maixner & Weng, 2013; Sandberg, Patil, D’Angelo, Weber, & Mallard, 2014; Verdile et al., 2015). GSK3β and its cellular signaling cascades play an important role in the regulation of neuroinflammation (Jope, Yuskaitis, & Beurel, 2007; Maixner & Weng, 2013). The activity of GSK3β is mainly regulated by the phosphorylation at serine 9 which results in the inhibition of GSK3β (Golpich et al., 2015; Luo, 2009; Maixner & Weng, 2013). Ethanol significantly decreased the expression of phosphorylated GSK3β [p-GSK3β (Ser9)] in the developing spinal cord, [F(3,24)=21.04; p<0.05] (Fig. 4A and 4B), indicating an activation of GSK3β. Minocycline treatment blocked ethanol-induced activation of GSK3β (p<0.05) (Fig. 4A and 4B).

Figure 4. Effect of minocycline on ethanol-induced GSK3β activation.

Figure 4.

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The treatment of minocycline and ethanol was same as described in Fig. 1. The spinal cord tissues were processed for immunoblotting analysis (A). The expression of phospho-GSK3β (Ser9) (B), active β-catenin (C), phospho-CREB (Ser133) (D), and phospho-c-Jun (Ser63) (E) was quantified and normalized to the expression of β-actin. Each data point was the mean ± SEM of three independent experiments. *p< 0.05 and **p < 0.01, statistically significant difference from control group; #p < 0.05 and ##p < 0.01, statistically significant difference from ethanol group.

We then examined the effect of ethanol on the substrates of GSK3β, including β-catenin, cAMP response element-binding protein (CREB), and c-Jun (Golpich et al., 2015; Gotschel et al., 2008; Zhou et al., 2004). β-catenin is a substrate of GSK3β; activation of GSK3β destabilizes β-catenin while inactivation of GSK3β increases the stabilized (non-phosphorylated) form of β-catenin, resulting in higher levels of β-catenin (Zhu, Kang, Huang, He, & Xie, 2009). Ethanol decreased the active form of β-catenin, [F(3,24)=54.99; p<0.001], and minocycline blocked ethanol inhibition of active β-catenin (p<0.01) (Fig. 4A and 4C). Interestingly, minocycline treatment alone increased the expression of active β-catenin (p<0.01) (Fig. 4A and 4C). Phosphorylated GSK3β enters the nucleus and acts upon and binds to a series of transcription factors, including CREB (Hur & Zhou, 2010). CREB is a critical component of the neuroprotective transcriptional network. CREB dysregulation contributes to an array of neuropathological conditions (Qiao et al., 2018; Sakamoto, Karelina, & Obrietan, 2011). Ethanol dramatically inhibited CREB phosphorylation, [F(3,24)=42.40; p<0.01]; whereas minocycline offset ethanol’s effect (p<0.01) (Fig. 4A and 4D). Phosphorylated c-Jun contributes to the formation of the activator protein 1 (AP-1) transcription factor complex which is involved in the expression of many pro-inflammatory genes and cytokines, such as IL-6 and TNF-α (J. Zhao et al., 2016). p-c-Jun is also one of the GSK3β target proteins (Luo, 2009). Minocycline blocked ethanol-induced inhibition of phosphorylated c-Jun at serine 63 in the developing spinal cord, [F(3,24)=56.38; p<0.01] (Fig. 4A and 4E).

Minocycline blocks ethanol-induced inhibition of pro-survival signaling in the developing spinal cord

The phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) axis is involved in cell growth, proliferation, differentiation, motility, and survival (King, Yeomanson, & Bryant, 2015; Porta, Paglino, & Mosca, 2014). PI3K/AKT signaling inhibits pro-inflammatory pathways, and enhances neuron survival (Crowder & Freeman, 1998; Han & Holtzman, 2000; Xu et al., 2017). In addition, AKT is upstream of GSK3β and the activation of AKT inhibits GSK3β activity through inducing its phosphorylation at serine 9 (F. Zhang, Phiel, Spece, Gurvich, & Klein, 2003). Ethanol inhibited AKT phosphorylation at serine 473, [F(3,24)=43.33; p<0.01], while minocycline restored the phosphorylation of AKT (p<0.01), offsetting ethanol’s effect (Fig. 5A and 5B). Interestingly, ethanol and minocycline induced a modest increase in phosphorylation of PI3K, [F(3,24)=34.98; p<0.05] (Fig. 5A and 5C). Phosphoinositide-dependent protein kinase 1 (PDK1) is another upstream regulator of AKT; it phosphorylates and activates AKT (Liu, Cheng, Roberts, & Zhao, 2009). Ethanol decreased the phosphorylation of PDK1 at serine 241, [F(3,24)=61.86; p<0.001]; whereas minocycline restored the expression of p-PDK1 (p<0.01) (Fig. 5A and 5D).

Figure 5. Minocycline reverses ethanol-induced inhibition of pro-survival signaling.

Figure 5.

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The treatment of minocycline and ethanol was same as described in Fig. 1. The spinal cord tissues were processed for immunoblotting analysis (A). The expression of phospho-AKT (Ser473) (B), phospho-PI3K (Tyr199) (C), phospho-PDK1 (Ser241) (D), phospho-mTOR (Ser2481) (E), and phospho-ERK1/2 (Thr202 and Tyr204) (F) was quantified and normalized to the expression of β-actin. Each data point was the mean ± SEM of three independent experiments. *p< 0.05 and **p < 0.01, statistically significant difference from control group; #p < 0.05 and ##p < 0.01, statistically significant difference from ethanol group.

Mammalian target of the rapamycin (mTOR) is a serine/threonine protein kinase, and regulate various cellular processes including cell survival (Hay & Sonenberg, 2004; Lipton & Sahin, 2014). Ethanol inhibited mTOR phosphorylation at serine 2481, [F(3,24)=26.59; p<0.05], and minocycline reversed ethanol-induced inhibition (p<0.05) (Fig. 5A and 5E). ERK1/2 is important pro-survival regulators for neurons (Gudasheva, Logvinov, Antipova, & Seredenin, 2013; Ortuno-Sahagun et al., 2014). Ethanol inhibited ERK1/2 phosphorylation, [F(3,24)=15.15; p<0.05]; whereas minocycline reversed ethanol-induced inhibition (p<0.05) (Fig. 5A and 5F).

Discussion

Using a well-established third trimester equivalent mouse model of ethanol exposure (Alimov et al., 2013; Olney et al., 2002; X. Wang et al., 2018), we demonstrated that minocycline effectively inhibited apoptosis in dorsal horn of spinal cord, microglial activation, and the expression of pro-inflammatory factors. Minocycline blocked ethanol-induced activation of GSK3β, an important regulator of pro-inflammatory response. Ethanol inhibited pro-survival signals for neurons, such as AKT, mTOR, and ERK1/2; whereas minocycline restored these signals. Together, our results suggest a therapeutic potential of minocycline for FASD.

Ethanol exposure during the development including prenatal and early postnatal periods in rodents caused glial activation and neuroinflammation in the CNS (Ahlers, Karacay, Fuller, Bonthius, & Dailey, 2015; Bodnar, Hill, & Weinberg, 2016; Chastain & Sarkar, 2014; Kane, Smith, Miranda, & Kable, 2012; Raineki et al., 2017; Ren et al., 2017; Saito, Chakraborty, Hui, Masiello, & Saito, 2016). Ethanol-induced increases of pro-inflammatory cytokines and chemokines, such as IL-6, IL-1β, TNF-α, and MCP-1 are believed to mediate many detrimental effects on the developing brain (Drew, Johnson, Douglas, Phelan, & Kane, 2015; Kane & Drew, 2016; Pascual et al., 2017; Q. Zhao et al., 2016). Our previous studies showed that that ethanol-induced neuroapoptosis was accompanied with microglial activation and increase in pro-inflammatory cytokines/chemokines in the both developing brain and spinal cord (X. Wang et al., 2018). Minocycline is postulated to be a “microglia inhibitor” and has potent anti-inflammatory property (Moller et al., 2016). It is proposed that minocycline may protect cells against apoptotic death through downregulating pro-inflammatory cytokine output (Giuliani, Hader, & Yong, 2005). We recently showed that minocycline attenuated ethanol-induced neuroapoptosis in the brain of early postnatal mice by inhibiting microglial activation (X. Wang et al., 2018). The current study extended this finding to the developing spinal cord and confirmed that minocycline was effective in protecting the developing spinal cord against ethanol-induced damage. These findings indicate that minocycline’s neuroprotection may be general for the developing CNS rather than region/structure-specific. It was noted that minocycline alone increased the expression of cytokines and some apoptosis markers. Minocycline is a broad-spectrum tetracycline antibiotic and able to cross the BBB. Minocycline was reported to cause some autoimmune responses (Mongey & Hess, 2008; Ochsendorf, 2010). Although the exact mechanisms are unknown, minocycline-induced changes may be related to its effect on immune responses. Recent studies demonstrated that tigecycline, a tetracycline derivative was effective reducing ethanol intake and withdrawal in adult mice (Bergeson, Nipper, Jensen, Helms, & Finn, 2016; Martinez et al., 2016). It would be interesting to determine whether minocycline could protect mature brain against ethanol’s insult, since microglial activation/ neuroinflammation has been observed in adult brain following ethanol exposure (Coleman, Zou, & Crews, 2017; Vetreno, Patel, Patel, Walter, & Crews, 2017; Yang et al., 2014).

Minocycline-mediated inhibition of microglial activation was evident by decreasing the number of Iba-1- and CD68-positive cells (Fig. 3). The ATP-gated P2X7 ion channel is an abundant microglial protein in the CNS that plays an important pathological role in executing ATP-driven danger signal transduction. Recent studies indicate that the activation of P2X7 is associated with microglial activation and cytotoxicity (Bhattacharya & Biber, 2016; Volonte et al., 2012). The activation of microglial P2X7 has been shown to cause neuroinflammation, which in turn induces neurodegeneration by releasing pro-inflammatory cytokines (Bhattacharya & Biber, 2016; Ren et al., 2017; Skaper, Debetto, & Giusti, 2010; Volonte et al., 2012). Emerging findings have generated great interest and excitement around targeting the P2X7 as a potential drug target for CNS disorders. We showed that minocycline effectively blocked ethanol-induced upregulation of P2X7 (Fig. 3E), suggesting that minocycline’s neuroprotection may be mediated by its action on P2X7.

GSK3β dysregulation has been implied in the pathogenesis of numerous neuroinflammatory and neurodegenerative diseases (Golpich et al., 2015; Reddy, 2013; Ren et al., 2017). GSK3β-mediated cellular signaling regulates microglial activation and neuroinflammation (Golpich et al., 2015; Maixner & Weng, 2013). GSK3β plays an important role in ethanol-induced neurodegeneration and inhibition of differentiation in the developing brain and cultured neuronal cells (G. Chen et al., 2009; Y. Liu et al., 2009; Luo, 2009; Ren et al., 2017; X. Wang et al., 2018). The current study indicated that minocycline inhibited ethanol-induced activation of GSK3β in the developing spinal cord by increasing its phosphorylation in Ser9. This finding is consistent with the previous observation that ethanol caused Ser9 dephosphorylation in the developing brain (G. Chen et al., 2009; Y. Liu et al., 2009; X. Wang et al., 2018). A series of transcription factors, such as β-catenin, CREB, and c-Jun are substrates of GSK3β (Gotschel et al., 2008; Zhou et al., 2004). β-catenin participates in both pro- and anti-inflammatory responses (Bonhaus, Burge, & McNamara, 1987; Duan et al., 2007; Li et al., 2014). Minocycline blocked ethanol-induced increase of active β-catenin in the developing spinal cord. CREB is a critical component of the neuroprotective transcriptional network, and its dysregulation contributes to an array of neuropathological conditions (Qiao et al., 2018; Sakamoto et al., 2011). Minocycline reversed ethanol-induced inhibition of CREB phosphorylation. Phosphorylated c-Jun contributes to the formation of the activator protein 1 (AP-1) transcription factor complex which is involved in the expression of many pro-inflammatory genes and cytokines, such as IL-6 and TNF-α (J. Zhao et al., 2016). Therefore, minocycline may block ethanol-induced GSK3β activation and subsequent cell signaling that mediate microglial activation and neuroinflammation.

In addition to microglial activation/neuroinflammation, ethanol may directly target neurons and cause neuroapoptosis. We therefore examined several important cell signaling pathways that regulate neuronal survival, namely PI3K/AKT, mTOR, and ERK1/2. We showed that minocycline effectively attenuated ethanol inhibition of these signaling pathways, suggesting that minocycline’s protection may also result from its promotion of pro-survival signaling in neurons. There is considerable cross talk between cell survival and inflammation signaling. For example, AKT is upstream of GSK3β and its activation inhibits GSK3β activity; AKT is an important pro-survival protein and may also modulate inflammatory responses through GSK3β pathway (H. Zhang et al., 2014). Our results demonstrated that ethanol inhibited AKT phosphorylation at serine 473, and minocycline reversed ethanol inhibition of AKT phosphorylation. It appeared that effect of ethanol on AKT phosphorylation was mediated by PDK1 rather than PI3K because ethanol did not inhibit PI3K but reduce PDK1 phosphorylation. mTOR is upstream of GSK3β as well, and the phosphorylation of mTOR complex-1 (mTORC1) results in the inhibition of GSK3β (H. H. Zhang, Lipovsky, Dibble, Sahin, & Manning, 2006). mTOR pathway regulates both cell survival and inflammation (Ghosh et al., 2016; Smith et al., 2014; Srivastava, Shperdheja, Baybis, Ferguson, & Crino, 2016). ERK1/2 is also an important regulator of neuron survival and involved in microglial activation/neuroinflammation in the brain (Badshah et al., 2016; Dheen et al., 2007; Sun & Nan, 2017; J. Zhao et al., 2016). Minocycline blocked ethanol-induced inhibition of PDK1, AKT, mTOR, and ERK1/2 (Fig. 5), suggesting that it may target both neuron survival and inflammatory signaling in the developing spinal cord.

We showed in this study that ethanol activated pro-inflammatory signaling, such as GSK3β, but inhibited pro-survival signaling, such as AKT, ERK1/2. However, in animal studies it is difficult to draw a conclusion regarding the effects of ethanol on the sequence of cell signaling cascades and the interaction among these signal pathways. Depending on the experimental models, alcohol exposure paradigm or cell types being investigated, the effects of ethanol on the signaling pathways may vary. For example, Blanco et al. (2005) (Blanco, Valles, Pascual, & Guerri, 2005) showed that ethanol stimulated TLR4-dependent ERK1/2 and JNK in cultured astrocytes. On the other hand, acute ethanol exposure did not affect the p38 MAPK, JNK or ERK phosphorylation in the brain of adult mice and cultured hippocampal cells (Wu et al., 2012).

Potential future studies

Although the current study suggests that minocycline may have neuroprotective property against ethanol-induced spinal cord damage, more investigation of the dosage and timeline of minocycline’s action is necessary. For example, administration of ethanol after ethanol exposure needs to be investigated because it is more relevant to clinical interventions. Our previously study demonstrated that ethanol during the development caused a permanent loss of spinal cord neurons in the adulthood (Ren et al., 2017). Dysfunctions of dorsal horn interneurons may contribute to symptoms of chronic pain such as the increased sensitivity to noxious stimuli (hyperalgesia) (Zeilhofer, Benke, & Yevenes, 2012). Future study needs to determine whether ethanol causes physiological or behavioral alterations associated with the functions of these neurons. It is also important to determine whether minocycline’s protection is long lasting and maintained in the adulthood.

  • Minocycline protected the developing spinal cord against ethanol-induced damage

  • Minocycline inhibited ethanol-induced microglia activation

  • Minocycline blocked ethanol-induced activation of GSK3β

  • Minocycline reversed ethanol inhibition of pro-survival signaling

Acknowledgement

This research is supported by grants from the National Institutes of Health (NIH) (AA017226 and AA015407). It is also supported in part by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development [Biomedical Laboratory Research and Development: Merit Review (BX001721)], and the National Natural Science Foundation of China (81372693).

Abbreviation

AKT

protein kinase B

AD

Alzheimer’s disease

BBB

blood brain barrier

CNS

central nervous system

CCR2

chemokine (C–C motif) receptor 2

cAMP

response element-binding protein

FASD

fetal alcohol spectrum disorder

GSK3β

Glycogen synthase kinase 3 beta

HD

Huntington’s disease

Iba-1

ionized calcium binding adaptor molecule 1

IL

interleukin

MCP-1

monocyte chemoattractant protein-1

MCPIP1

Monocyte chemotactic protein-1-induced protein-1

mTOR

mammalian target of the rapamycin

PD

Parkinson’s disease

PARP

Poly (ADP-ribose) polymerase

PD

Postnatal day

PDK1

phosphoinositide-dependent protein kinase 1

PI3K

phosphatidylinositol 3 kinase

TNFα

tumor necrosis factor-α

Footnotes

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