Age-Dependent Effects of Acute Alcohol Administration in the

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Age-Dependent Effects of Acute Alcohol Administration in the Hippocampal Phosphoproteome Ana Contreras,†,§ Lidia Morales,†,§ Ali Tebourbi,† Miguel Miguéns,‡ Nuria del Olmo,*,† and Carmen Pérez-García† †

Laboratorio de Farmacología, Departamento de Ciencias Farmacéuticas y de la Salud, Facultad de Farmacia. Universidad CEU−San Pablo, 28668 Madrid, Spain ‡ Departamento de Psicología Básica I, Universidad Nacional de Educación a Distancia (UNED), 28040 Madrid, Spain S Supporting Information *

ABSTRACT: Alcohol consumption during adolescence is deleterious to the developing brain and leads to persistent deficits in adulthood. Several results provide strong evidence for ethanol-associated alterations in glutamatergic signaling and impaired synaptic plasticity in the hippocampus. Protein phosphorylation is a wellknown and well-documented mechanism in memory processes, but information on phosphoprotein alterations in hippocampus after ethanol exposure is limited. This study focuses on age-related changes in the hippocampal phosphoproteome after acute alcohol administration. We have compared the phosphoprotein expression in the hippocampus of adult and adolescent Wistar rats treated with a single dose of ethanol (5 g/kg i.p.), using a proteomic approach including phosphoprotein enrichment by immobilized metal affinity chromatography (IMAC). Our proteomic analysis revealed that 13 proteins were differentially affected by age, ethanol administration, or both. Most of these proteins are involved in neuroprotection and are expressed less in young rats treated with ethanol. We conclude that acute alcohol induces important changes in the expression of phosphoproteins in the hippocampus that could increase the risk of neurodegenerative disorders, especially when the alcohol exposure begins in adolescence. alterations in learning-related synaptic plasticity.11 An interesting feature of ethanol is its ability to cause acute memory “blackouts”12,13 that could be related to the inhibitory actions on N-methyl-D-aspartate receptors (NMDA) that play an important role in synaptic plasticity mechanisms in different brain areas including hippocampus.14,15 Exposure to drugs of abuse induce synaptic connectivity in neuronal circuits involved in reward such as the ventral tegmental area (VTA), and the hippocampal-VTA loop may play a critical role in the entry of information into long-term memory 16 to activate the mesocorticolimbic dopaminergic reward system. Moreover, it has been reported that acute and chronic ethanol exposure affects hippocampus both in humans and animals.5,7,17 Acute administration of alcohol produces age-dependent effects showing that adolescent rats are less sensitive to the hypnotic and motor-impairing effects of ethanol, while they are more sensitive to other effects such as the hypothermic effects of the drug.18 Regarding the effects on the hippocampus, experimental results are consistent with the view that this area is more sensitive to the acute effects of alcohol than chronic effects, and it is even more susceptible to the neurotoxic effects of ethanol during adolescence,15 although acute and chronic ethanol exposure does have a marked effect on the hippo-

1. INTRODUCTION Alcohol use disorder is a chronic relapsing brain disease characterized by the loss of the ability to control ethanol intake despite knowledge of detrimental health or personal consequences. Alcohol consumption during adolescence is widespread and young people tend to drink intensively. According to the 2013 National Survey on Drug Use and Health, approximately 5.4 million (about 14.2%) of young people aged 12−20 engaged in heavy episodic (or “binge”) drinking in the United States. Alcohol consumption during adolescence is deleterious to the developing brain and could lead to persistent deficits in adulthood, including alterations in long-term memory and learning skills.1 It is also well-known that ethanol directly alters brain synaptic plasticity2,3 and learning and memory mechanisms.4 Synaptic plasticity measured by long-term potentiation,5,6 structural changes, and alterations in hippocampal function have been observed after ethanol administration.7 Studies in rodents provide clear evidence that chronic ethanol has adverse effects on synaptic function and synaptic plasticity in the hippocampus and cerebral cortex, among other encephalic areas.8,9 Nevertheless, the effects of acute alcohol on synaptic plasticity are currently poorly understood, although impaired plasticity correlates with defects in learning and memory, particularly spatial learning.10 In this regard, some authors showed that acute and chronic ethanol administration produces © 2017 American Chemical Society

Received: September 20, 2017 Published: October 24, 2017 2165

DOI: 10.1021/acs.chemrestox.7b00260 Chem. Res. Toxicol. 2017, 30, 2165−2173

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Chemical Research in Toxicology campus both in humans and animals.5,7,19−22 However, contradictory results were found regarding adolescent sensitivity in hippocampal spatial-dependent memory.23 Ethanol presents a low molecular weight and solubility in both water and lipid and is able to perturb cell membranes with secondary effects on cellular proteins.24 Post-translational modifications are key modulators of protein structure and function,25 and such modifications play a crucial role in controlling various cellular functions in living cells. In this regard, phosphorylation often occurs at multiple residues allowing the protein to adapt to several different functions, including neuronal plasticity.26 Accordingly, phosphorylation has been implicated in memory processes27 and is found to be altered in neurological disorders such as Alzheimer’s disease.28 Proteomics employs high-throughput technologies to study the structure and function of proteins in complex biological samples.29 Indeed, proteomic studies have shown differential protein expression during hippocampal synaptic plasticity.30,31 Studies that previously employed proteomic techniques to study the ethanol-mediated alterations in the brain have been described in detail in an excellent review by Gorini et al. (2014).32 Regarding ethanol-induced age-related hippocampal proteome alterations, Hargreaves et al. (2009)33 suggested that the adolescent hippocampus is more vulnerable to proteomic changes after chronic alcohol exposure and proteins related to glycolysis, glutamate metabolism, neurodegeneration, synaptic function, and cytoskeletal structure were the most affected. Moreover, Swartzwelder et al. (2016)34 found alterations in the GluN2B proteome that persisted into adulthood in adolescent rats that were intermittently exposed to ethanol. However, as far as we know, the age-dependent effects of ethanol on hippocampal phosphoproteome after an acute exposure to the drug have not yet been investigated. In the present study, we sought the identification of phosphoproteins differentially regulated in the rat hippocampus by acute ethanol administration in adolescent and adult rats. In a similar manner to previous studies performed by our group,35,36 we have employed a proteomic approach in which we have combined immobilized metal affinity chromatography (IMAC) for phosphoprotein enrichment, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for protein separation, and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for protein identification.

intraperitoneally in 10 mL/kg of volume. Animals were killed 1 h after the alcohol administration to collect hippocampi for protein identification. The dose of ethanol was selected considering other authors,35 and our previous experiments in which the intake of Wistar adolescent rats ranged from 8−17 g/kg by oral self-administration in overnight sessions. 2.3. Proteomic Analysis. After the rats were decapitated, their brains were rapidly removed and hippocampi were dissected and individually preserved at −80 °C until the proteomic study. We used hippocampi from three animals per group. Since we were interested in the identification of differentially expressed phosphorylated proteins, we used a proteomic approach, previously employed in our laboratory36,38 in which we combined phosphoprotein enrichment, by IMAC, with 2D-PAGE and MALDI-TOF mass spectrometry. 3.1. Extraction and Enrichment in Phosphorylated Proteins. For the extraction and enrichment in phosphorylated proteins, we used the Pierce Phosphoprotein Enrichment Kit (Thermo Scientific, USA), which is based on IMAC and results in highly efficient purification of phosphoprotein containing phosphotyrosine, phosphoserine, and phosphothreonine residues. Tissue samples (n = 3) of each experimental group were processed individually according to the kit manufacturer’s protocol with slight modifications routinely used in our laboratory.36 3.2. 2D-PAGE. For protein separation, we employed the 2D gel electrophoresis protocol routinely used in our laboratory.36,38 Briefly, 300 μL of each sample obtained in the previous step was taken for the rehydration and simultaneous loading of the proteins on an IPG strip (17 cm, 3−10 NL, Bio-Rad, USA) at 50 V, 20 °C, for 12 h in a PROTEAN IEF cell (Bio-Rad, USA). Then, the voltage was increased to 10,000 V and focused for a total of 60,000 Vh. Prior to SDS−PAGE, the strips were equilibrated in a solution containing 0.375 M Tris-HCl (pH 8.8), 6 M urea, 2% SDS, and 20% glycerol (Bio-Rad, USA). The equilibration was carried out in two steps. DTT (2%, Bio-Rad, USA) was added in the first step and iodoacetamide (2.5%, Bio-Rad, USA) in the second one to the equilibration solution. The SDS−PAGE was run in polyacrylamide gels (180 × 200 × 1 mm, 12%) for 6 h at 200 V, 20 °C, in a PROTEAN Plus Dodeca cell (Bio-Rad, USA). The gels were stained with the “Silver Stain” kit (Bio-Rad, USA), according to manufacturer’s protocol. Silver staining was performed in a Dodeca Stainer (Bio-Rad, USA). Three gels from each experimental group were scanned using the densitometer GS-800 (Bio-Rad, USA). Spots were detected, quantified, and matched automatically with the PDQuest v8 software (Bio-Rad, USA) and manually checked. Normalization of the optical density of each spot and statistical analysis were conducted as described in a previous study.36 Spots that showed statistical differences in optical density (P < 0.05) between two groups, with a change in expression of 1.5-fold or more, and that could be clearly visualized in the gels were cut out for mass spectrometry identification. 3.3. Mass Spectrometry Analysis of Protein Spots. Proteins selected for analysis were in-gel reduced, alkylated, and digested with trypsin (Roche, Germany) according to Sechi and Chait (1998).39 1 μL of the supernatant was thin-layer spotted onto a MALDI sample plate. MALDI-time-of-flight (TOF) mass spectrometry analyses were performed at the Proteomics Unit of the UCM in Madrid, a member of the ProteoRed-ISCIII network. A 4800 Plus Analyzer MALDITOF/TOF mass spectrometer (Applied Biosystems, MDS Sciex, Canada) was used and operated in positive reflector mode with an accelerating voltage of 20,000 V. Peptides from the autodigestion of trypsin were used to internally calibrate all mass spectra. The analysis by MALDI-TOF/TOF mass spectrometry produces peptide mass fingerprints and the peptides observed with a signal to noise >10 can be collated as a list of monoisotopic molecular weights. From the mass spectrometry spectra, suitable precursors were selected for tandem mass spectrometry analyses by collision-induced dissociation (CID), using 1 Kv ion reflector mode with a precursor mass window of ±4 Da. The plate model and default calibration were optimized for the tandem mass spectrometry spectra processing. For protein identification, the nonredundant Uniprot/Swiss-Prot (545388 sequences; 193948795 residues) database was searched using Licensed MASCOT

2. EXPERIMENTAL PROCEDURES 2.1. Animals. Male Wistar rats in development phase (4 weeks, body weight 80−120 g) and puberty male Wistar rats (8 weeks, body weight 220−260 g) were used in this study. Throughout the manuscript, “adolescent” refers to 4-week-old animals, and “adult” refers to the 8-week-old group. It is important to consider that, although rodents become sexually mature when they are 6 weeks old on average, social maturity could be reached later, and it should also be noted that sexual maturity does not mark the beginning of adulthood, but rather denotes the beginning of adolescence.37 Animals were group-housed in an automatically controlled environment (23 ± 3 °C, 50% ± 10% relative humidity) with a 12-h light/dark cycle at the beginning of the experiments. They had free access to standard food (Panlab, Barcelona, Spain) and water. All of the animals used in this study were maintained in accordance with European Union Laboratory Animal Care Rules (86/609/ECC directive), and the protocols were approved by the Animal Research Committee of the USP-CEU. 2.2. Alcohol Treatment. A single high dose of ethanol (5 g/kg, n = 3) or saline (control animals, n = 3) was administered 2166


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Chemical Research in Toxicology 2.3 (www.matrixscience.com) through the Global Protein Server v3.6 from ABSCIEX. Search parameters were: oxidized methionine as variable and carbamidomethyl cysteine as fixed modifications; peptide mass tolerance of 50 ppm for peptide mass fingerprints searched, and 80−100 ppm for tandem mass spectrometry searched; a tandem mass spectrometry fragment tolerance of 0.3 Da and up to 1 missed trypsin cleavage site was allowed. We accepted positive protein identification when the Protein Score was greater than the score fixed by MASCOT as significant with p < 0.05. Similarities between theoretical MW (molecular weight) and Ip (isoelectric point) values and the experimental ones were checked. 2.4. Western Blot Analysis. To prove the reproducibility of the proteomic results, some of the proteins identified with the proteomic techniques were also measured by Western blot. We used separate groups of animals with the same ages and following the same treatments as previously described. Hippocampal tissue samples (n = 4 per group) were homogenized in ice-cold lysis buffer. Equal amounts of protein (30 μg) were mixed with Laemmli buffer, then loaded on an SDS-PAGE gel and subjected to electrophoresis. Proteins were transferred to nitrocellulose membranes (GE Healthcare, Barcelona, Spain) by using a transblot apparatus (Bio-Rad, Hercules, CA). The membranes were blocked with 5% dried skimmed milk powder in Tween-PBS for 1 h. Primary antibodiesagainst phosphor-DPYSL2 T514, phosphor-DPYSL2 S522, DPYSL2, phosphor-PSA3 S250, and PSA3 (Abcam, Cambridge, UK)were applied at the appropriate dilution overnight at 4 °C. After washing, appropriate secondary antibodies (antirabbit IgG-peroxidase conjugated) were added for 1 h at a dilution of 1/5000. Blots were washed, incubated in enhanced chemoluminescence reagent (ECL Prime; GE Healthcare), and developed by exposure to autoradiographic films. In order to check the equal loading of samples, blots were re-incubated with β-actin antibody (Affinity Bioreagents, Golden, CO). Blots were detected using the ChemiDoc XRS+ Imaging System (BioRad, Spain). 2.5. Statistical Analysis. In the proteomic experiments, statistical analysis was conducted as described in a previous study.35 Briefly, spots from the gels were detected, quantified, normalized, and matched automatically with PDQuest v8 software (from Bio-Rad, USA); then animal groups were compared in pairs by using the Student’s t test included in the PDQuest v8 software. P < 0.05 was considered as statistically significant. Data from Western blot experiments were analyzed using Student’s t test included in Graphpad Prism 4 program (San Diego, CA, USA). P < 0.05 was considered as statistically significant.

Figure 1. Representative silver-stained 2D gel of the phosphoproteome from the rat hippocampus. Spots labeled with numbers showed significant differences in normalized optical density after comparing the four groups of animals and were identified by mass spectrometry. The complete list of the identified proteins is shown in Table 1.

Table 1) and four in the ethanol-treated rats (abbreviated as ATPB, HSP60, ENOA, PDIA3; aE vs AE, complete name referred in Table 1); interestingly, for all of these proteins, the expression was higher in adult than in adolescent animals. In the comparisons made with animals of the same age but different treatments, we could identify three proteins in the case of adolescent animals (abbreviated as PSA3, GLNA, SODM; aE vs aS, complete names referred in Table 1) and in two proteins in the adult animals (abbreviated as PDIA3, G6PD, AE vs AS, complete names referred in Table 1). For all the proteins identified in this study, the relationship with neuroprotection or neurodegeneration, as well as with ethanol or other drugs of abuse exposure, are indicated in Table 1. We selected some of the most interesting proteins (DPYSL2 and PSA3), identified with the proteomic techniques, to be validated by western-blot assays. We analyzed the total protein content and phosphorylation of DPYSL2 and PSA3. We found a decreased DPYSL2 phosphorylation at Ser522 in young saline (aS) compared to adult saline (AS), which is consistent with the proteomic results. No difference was found in the DPYSL2 phosphorylation at Thr514. In the case of PSA3, we did not find any difference when comparing adult saline (AS) and young saline (aS) in the phosphorylated form at Ser250 (Figure 2).

3. RESULTS The comparison of the 2D patterns between different animal groups resulted in 18 spots of the gel showing significant differences in optical density (Figure 1); proteins were identified by mass spectrometry in only 15 of these spots. One spot (3301) resulted in the identification of a mixture of two proteins (mitogen-activated protein kinase and fructosebisphosphate aldolase C); however, since it is not possible to determine the concentration of each protein in this spot, we cannot take these proteins into consideration. We also had three pairs of spots in which the same protein was identified (1807 and 1808; 2725 and 2807; 7102 and 8101). In all of these pairs, both spots exhibited the same pattern in terms of the change of protein levels and were located close to each other in the gel with limited changes in their horizontal position (Figure 1), suggesting differences in their Ip. These changes in the Ip are usually due to differences in the number of phosphorylated residues or in the type of residues that are phosphorylated.38 Thus, only 11 proteins will be considered for further discussion. When comparing adolescent vs adult rats, four proteins were different between saline treated animals (abbreviated as G6PD, PDXK, DPYSL2, NDKB; aS vs AS, complete name referred in

4. DISCUSSION Our study demonstrates that a single exposure to ethanol is enough to produce differences in hippocampal phosphoproteome and that these differences are dependent on age. These results are in concordance with other authors, who have previously identified age-dependent and long lasting proteomic variations in the hippocampus after repeated doses of ethanol.33,34 The present study also highlighted some interesting proteomic differences between adolescent and adult rats treated with saline. The phosphorylation in different 2167


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Chemical Research in Toxicology

Table 1. Rat Hippocampal Proteins Showing Significant Differences in Normalized Optical Density When Comparing Adolescent and Adult Rats Treated Either with Ethanol (5 g/kg, i.p.) or Salinea ratio of normalized optical densityc spot no.

protein name

UniProt KB IDb

abbreviation

aS/ AS

AE/ AS

aE/ AE

aE/ aS

association with neuroprotection/neurodegeneration (NP/ND) or ethanol (EE) or other drugs of abuse (ODE) exposured

0602

ATP synthase subunit beta

P10719

ATPB

n.s.

n.s.

0.1

n.s.

1102

proteasome subunit alpha type-3 60 kDa heat shock protein, mitochondrial 60 kDa heat shock protein, mitochondrial alpha-enolase

P18422

PSA3

n.s.

n.s.

n.s.

3.5

NP/ND74 EE73/ODE71 ODE (Morphine)62

P63039

HSP60

n.s.

n.s.

0.2

n.s.

NP/ND74

P63039

HSP60

n.s.

n.s.

0.1

n.s.

EE, ODE83

P04764

ENOA

n.s.

n.s.

0.1

n.s.

protein disulfideisomerase A3 protein disulfideisomerase A3 glucose-6-phosphate 1dehydrogenase

P11598

PDIA3

n.s.

n.s.

0.4

n.s.

EE33 NP/ND75 NP/ND70

P11598

PDIA3

n.s.

2.1

n.s.

n.s.

EE69/ODE70

P05370

G6PD

0.4

0.4

n.s.

n.s.

3202

pyridoxal kinase

O35331

PDXK

0.5

n.s.

n.s.

n.s.

3816

dihydropyrimidinaserelated protein 2

P47942

DPYSL2

0.4

n.s.

n.s.

n.s.

4001

nucleoside diphosphate kinase B glutamine synthetase

P19804

NDKB

0.4

n.s.

n.s.

n.s.

NP/ND40,41 EE67 NP/ND42 EE43 NP/ND48 EE49 NP/ND45,46

P09606

GLNA

n.s.

n.s.

n.s.

0.4

superoxide dismutase [Mn], mitochondrial superoxide dismutase [Mn], mitochondrial

P07895

SODM

n.s.

n.s.

n.s.

0.6

NP/ND54 EE52 NP/ND51

P07895

SODM

n.s.

n.s.

n.s.

0.3

EE58

1807 1808 2607 2725 2807 2823

4502 7102 8101 a

AE: Adults rats treated with ethanol; AS: Adults rats treated with saline; aE: adolescent rats treated with ethanol; aS: adolescent rats treated with saline. bProtein code number according to UniProt. The UniProt Knowledgebase (UniProtKB) is the central hub for the collection of functional information on proteins. cNumbers show the ratio of normalized optical density after comparing the corresponding 2 groups of animals when comparisons were significant (p < 0.05); ns (comparison showing not significant differences). dSee text for explanation.

detect any variation in phosphoDPYL2 expression after ethanol exposure, it could also be interesting to further study the possible role of DPYSL2 phosphorylation in age-dependent ethanol effects, since it has been reported that ethanol intake blocks glycogen synthase kinase-3β phosphorylation of DPYSL2. Moreover, the systemic administration of the DPYSL2 inhibitor lacosamide, or knockdown of DPYSL2 in the nucleus accumbens, decreases excessive alcohol intake in mice.49 In Western blotting validation, we have found a decreased DPYSL2 phosphorylation at Ser522 in adolescent saline (aS) compared to adult saline (AS), which is consistent with the present proteomic results (Figure 2). Nevertheless, no difference was found in the DPYSL2 phosphorylation at Thr514. Considering all of these properties together, we can assume that a possible neuronal alteration might be driven by a change in G6PD, PDXK, NDKB, and/or DPYSL2. Since the brain keeps on developing in utero and youth until it becomes mature at a certain age in life,50,51 it may be that we have found here further evidence of the frailty of adolescent brains compared to adult brains. The most intriguing result regarding these four proteins is that alcohol treatment erased the differences between adolescent and adult rats; no statistical differences were found in G6PD, PDXK, DPYSL2, and/or NDKB when we compared the ethanol treated groups (see comparisons aE/aS or aE/AE in Table 1).

residues of all of these proteins has been previously demonstrated according to PhosphositePlus.41 4.1. Age-Related Protein Changes. We found significant, age-related differences in hippocampal expression of four identified proteins: glucose-6-phosphate-1-dehydrogenase (G6PD), pyridoxal kinase (PDXK), nucleoside diphosphate kinase B (NDKB), and dihydropyrimidinase-related protein 2 (DPYSL2). Interestingly, for all of these proteins, the expression was higher in the adult than in the adolescent animals when they were saline administered. G6PD and PDXK are directly related to neuroprotection/neurodegeneration. G6PD protects against endogenous reactive oxygen speciesmediated neurodegeneration in aged mice and against aluminum-induced neurotoxicity in adult rats in several brain areas, including hippocampus,42,43 and PDXK alterations in substancia nigra are associated with Parkinson’s disease.44 It is worth noting that PDXK activity, but not expression, decreases in ethanol-fed animals.45 NDKB is involved in neuronal differentiation and cell proliferation,45,46 and it has been demonstrated that this kinase is regulated in the hippocampus by endocannabinoids.47 Finally, the identification of DPYSL2 seems very interesting, since it has been described that phosphorylation of DPYSL2 by Cdk5 is important for dendritic spine development in cortical neurons in the mouse hippocampus,48 a process that is crucial for learning and memory. Moreover, although we did not 2168


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Figure 2. Western blot analysis of the phosphorylation of DPYSL2 and PSA3. (A) Increased DPYSL2 phosphorylation at Ser522 in adult saline (AS) compared to young saline (aS). Quantitative analysis for the ratio of the pDPYSL2 Ser522 to total DPYSL2. (B) No difference was found in the DPYSL2 phosphorylation at Thr514 comparing adult saline (AS) and young saline (aS). Quantitative analysis for the ratio of the pDPYSL2 Thr514 to total DPYSL2. (C) No difference was found in the PSA3 phosphorylation at Ser250 comparing young saline (aS) and young ethanol (aE). Quantitative analysis for the ratio of the pPSA3 Ser250 to total PSA3. The densitometric ratio was normalized against the YS group. The immunoblots of β-actin were used as a loading control. The results were expressed as the means ± SEM (n = 4). *P < 0.05.

response.60 It has been proposed that alcohol induces the reduction in SODM in endothelium resulting in blood−brain barrier damage.59 In fact, it has been demonstrated that quetiapine, an atypical antipsychotic employed to treat alcoholic patients, diminished ethanol effects in hippocampal areas blocking ethanol-induced oxidative stress in this region in young rats.61 By contrast, as mentioned before, the levels of PSA3, one of the components of proteasome core structure, were higher in adolescent ethanol-treated rats. PSA3 is also altered in the hippocampus and other brain regions after exposure to other drugs such as morphine.63 Moreover, proteasome activity is involved in synaptic plasticity and memory65 and neurodegenerative diseases such as Alzheimer.64 Regarding ethanol exposure, the role of proteasome in alcohol-induced brain damage has also been reported.67 In fact, a different modulation of proteasome activity in the brain that depends on the pattern of alcohol exposure is postulated.66 In this way, chronic ethanol reduces proteasome activity, while acute ethanol increases it, with the latter being consistent with our results. However, in the Western blot analysis we could not find any difference when comparing adult saline (AS) and adolescent saline (aS) in either the total or the phosphorylated form at Ser250 of PSA3.

4.2. Protein Changes in Ethanol-Exposed Adolescent Rats. Three proteins were found to be expressed differently when comparing adolescent rats under acute ethanol exposure with saline-treated adolescent rats: proteasome subunit alpha type 3 (PSA3), glutamine synthetase (GLNA), and mitochondrial superoxide dismutase (SODM). Ethanol intake decreased SODM and GLNA, whereas it increased PSA3 (Table 1). These three elements have a protective role in the brain as they have a detoxifying activity and are cell cycle regulators.52−55 GLNA is an astrocytic enzyme and prevents glutamatedependent excitotoxicity and detoxifies nitrogen.56 In this sense, GLNA is downregulated in the hippocampus of alcoholics.57,58 Thus, we could hypothesize that the acute consumption of ethanol in adolescent animals would induce brain damage probably due to astrocytic pathology affecting glutamate transmission, as has been proposed by other authors.54 With respect to SODM, we observed a reduction in the levels of this protein in adolescent ethanol-treated rats that could be revealing the impairment of antioxidant systems in adolescent animals. Accordingly, some authors have reported that the oxidative damage produced by ethanol exposure in adolescent rats could be the consequence of an insufficient antioxidant 2169


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neuroprotective mechanisms, which could support our previously stated hypothesis for increased vulnerability of adolescent rats to ethanol-induced neurotoxicity in hippocampus. In conclusion, our data show that a single dose of ethanol is sufficient to produce detectable changes in phosphoprotein expression in the rat hippocampus and that these changes are age-related. Further studies would be necessary to determine the longevity of these protein changes and establish how chronic alcohol use could affect their expression. The importance of phosphorylation in terms of protein function and how it affects hippocampal physiology and behavior should also be further studied. Nevertheless, since most of these proteins are related to neuroprotection or neurodegeneration and are downregulated in adolescent rats, it could be suggested that ethanol exposure at a young age might increase the risk of hippocampal damage and the consequent alterations in memory and spatial abilities4,84 which are typical of neurodegenerative diseases.

This would suggest that the differences identified in our proteomic study for phosphoPSA3 could be caused by differences in the phosphorylation levels of other residues. 4.3. Protein Changes in Ethanol-Exposed Adult Rats. Protein changes in adult rats treated with saline or with ethanol have been highlighted in this study. For this comparison, two proteins where clearly identified: protein disulfide-isomerase A3 (PDIA3) and G6PD. As mentioned above, G6PD is a neuroprotective enzyme41 for which we observed a lower expression in adolescent animals vs adults in basal conditions. In adult rats, ethanol also reduces the expression of G6PD, which is consistent with previous studies reporting a decreased activity of this enzyme and increased oxidative stress and subsequent apoptosis in the brain of ethanol-treated rats.68 Nevertheless, other authors have shown that postnatal expression of G6PD in hippocampus remains almost constant during development.69 In the case of PDIA3, increased expression of this protein has been reported in rat embryos exposed to ethanol,70 and this increase was also observed in monkey hippocampus after methamphetamine treatment, which has been proved to be a neuroprotective mechanism.71 In our study, ethanol treatment increased PDIA3 levels in adult animals but not in adolescent animals, which might suggest a higher susceptibility to alcoholinduced brain damage in adolescent rats. 4.4. Age-Related Protein Changes in Ethanol-Exposed Rats. In this proteomic study, the most interesting finding is the variation of the phosphoprotein expression between adolescent and adult rats that are given an acute dose of ethanol. Four proteins stood out and, for all of them, the expression was lower in the hippocampus of adolescent rats: ATP synthase subunit beta (ATPB), alpha enolase (ENOA), 60 kDa heat shock protein (HSP60), and PDIA3. ATPB, which is involved in ATP metabolism,72 is affected by ethanol-induced dysfunctions in a range of tissues, including the brain.73 Within the hippocampus, alterations in ATPB have been reported after chronic nicotine treatment71 and in animal models of Alzheimer’s disease.72 ENOA is considered a multifunctional protein, and its differential expression has been related to several pathologies, such as cancer and Alzheimer’s disease.75,76 ENOA expression in the hippocampus increases with age;77 moreover, a decrease in its expression has been described in the hippocampus of adolescent rats chronically treated with ethanol.33 Accordingly, our results show that a single dose of ethanol also decreases phosphoENOA expression in adolescent rats, and it has been shown that ENOA hyper-phosphorylation is accompanied by decreased enzymatic activity.78 Other authors have shown the up-regulation of protein phosphorylation of hepatic alpha enolase by prenatal alcohol exposure in both 7 day-old and 3 month-old rats.79 HSP60 is a constitutive mitochondrial protein with specific functions in response to oxidative stress.80 In the brain, HSP60 actively participates in many functions, both in normal and pathological conditions. Regarding the hippocampus, HSP60 alterations have been described in animal models of epilepsy81 and Alzheimer’s disease72 as well as after cocaine exposure, with the latter being affected by ethanol cotreatment.82 Finally, we also observed a decrease in PDIA3, a protein that, as mentioned above, has proved to have neuroprotective effects.71,81 In summary, the decrease in ATPB, ENOA, HSP60, and PDIA3 observed in adolescent animals could negatively affect the production of energy, the response to stress, and the

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.7b00260. Additional experimental details and data (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel. 34-913.724.700. ORCID

Nuria del Olmo: 0000-0001-5611-4152 Author Contributions §

These authors contributed equally.

Funding

This work was supported by the Ministerio de Sanidad, Asuntos Sociales e Igualdad (Plan Nacional sobre Drogas P2014-PI029) and the Fundación Universitaria San Pablo-CEU. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank J.M. Garrido, I. Bordallo, and J. Bravo for their assistance with animal care. ABBREVIATIONS 2D-PAGE, two-dimensional polyacrylamide gel electrophoresis; aE, adolescent rats treated with ethanol; AE, adult rats treated with ethanol; ALDOC, fructose-bisphosphate aldolase C; aS, adolescent rats treated with saline; AS, adult rats treated with saline; ATPB, ATP synthase subunit beta; DPYL2, dihydropyrimidinase-related protein 2; ENOA, alpha-enolase; G6PD, glucose-6-phosphate 1-dehydrogenase; GLNA, glutamine synthetase; HSP60, 60 kDa heat shock protein, mitochondrial; IMAC, immobilized metal affinity chromatography; MALDITOF, matrix-assisted laser desorption/ionization time-of-flight; MK01, mitogen-activated protein kinase; NDKB, Nucleoside diphosphate kinase B; PDIA3, protein disulfide-isomerase A3; PDXK, pyridoxal kinase; PSA3, proteasome subunit alpha type3; SODM, superoxide dismutase [Mn], mitochondrial 2170


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