Abstract

   graphic file with name ao2c01008_0009.jpg

   Antiepileptic drugs have been shown to be associated with inducing or
   exacerbating adverse psychotropic reaction, including aggressive
   behavior. Perampanel, the first pharmacological compound approved by
   the FDA in 2012, is an effective antiepileptic drug for intractable
   epilepsy but induces severe aggression. So far, the underlying
   molecular mechanisms of aggression induced by perampanel remain
   incompletely understood. In the present study, a model of aggressive
   behavior based on the clinical use of perampanel was established and
   resident–intruder test and open field test were performed. Changes in
   hippocampal protein profiles were detected by tandem mass tag (TMT)
   proteomics. The behavioral results indicated that long-term use of
   perampanel increased the aggressive behavior of C57BL/6J mice.
   Proteomic analysis revealed that 93 proteins were significantly altered
   in the hippocampus of the perampanel-treated group (corrected p <
   0.05), which were divided into multiple functional groups, mainly
   related to synaptic function, synaptogenesis, postsynaptic density
   protein, neurite outgrowth, AMPA-type glutamate receptor
   immobilization, and others. Bioinformatic analysis showed that
   differentially expressed proteins were involved in synaptic plasticity
   and the Ras signaling pathway. Furthermore, validation results by
   western blot demonstrated that glutamate receptor 1 (GluA1) and
   phosphorylation of mitogen-activated protein kinase (ERK1/2) were
   notably up-regulated, and synaptophysin (Syn) and postsynaptic density
   95 (PSD95) were down-regulated in perampanel-treated mice. Therefore,
   our results provide valuable insight into the molecular mechanisms of
   aggressive behavior induced by perampanel, as well as potential options
   for safety treatment of perampanel in the future.

1. Introduction

   Antiepileptic drugs have been shown to be associated with inducing
   adverse psychotropic reaction, including aggressive behavior.^[42]1
   Perampanel (PER), as the latest generation of antiepileptic drug, is a
   negative allosteric (noncompetitive)
   α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor
   antagonist with a novel pharmacological mechanism of action,^[43]2
   which is licensed as adjunctive treatment of partial-onset seizures in
   patients with epilepsy aged 12 years and older.^[44]3 Pooled data from
   phase III studies showed an increased rate of hostile/aggressive
   adverse events in patients who received PER compared with those who
   received placebo.^[45]4 There is a black box warning by the FDA about
   serious psychiatric and behavioral reactions in the prescribing
   information, including “aggression, hostility, and irritability”.^[46]5
   There is increasing evidence suggesting that irritability and
   aggression are the most serious mental responses that occurred at
   dose-dependent rates.^[47]6 A pharmacokinetic–pharmacological study
   confirmed a significant correlation between the blood concentration of
   PER and the incidence of aggressive behavior.^[48]7 According to a 4
   year clinical extension study, the most common adverse events that led
   to drug discontinuation were irritability (1.2%) and fatigue
   (1.1%).^[49]8 Moreover, aggressive behavior caused by PER not only
   impacts the use of drugs but also brings difficulty with continuing the
   treatment of epilepsy. Consequently, studying the molecular mechanisms
   of aggressive behavior induced by PER is of paramount importance for
   safer application of this promising agent.

   Aggression is a complex phenomenon, involving the interaction among
   genetic, neurobiological, and psychosocial factors.^[50]9 So far, the
   underlying molecular mechanisms of aggression remains incompletely
   understood. Early animal studies had shown brain regions implicated in
   aggressive behavior including the prefrontal cortex, nucleus accumbens,
   hypothalamus, amygdala, and striatum.^[51]10 Indeed, structural
   abnormalities in any of these brain regions could increase the risk of
   aggressive behavior.^[52]11 Additionally, studies have shown that
   neurotransmitters and hormones also affect the occurrence of aggressive
   behavior.^[53]12 However, aggressive behavior is a multifactorial
   disease that involves abnormal molecular alterations.^[54]13 A recent
   study has implicated that aggressive behavior is involved in
   neurodevelopmental processes and synaptic plasticity.^[55]14 It may
   indicate that aggressive behavior does not only involve
   neurotransmitter function but also involve molecules in establishing
   neuronal circuits and brain plasticity.^[56]15 The hippocampus is a key
   anatomical brain region involved in learning and memory, anxiety, and
   depression.^[57]16 It is highly susceptible to drugs and environmental
   toxins.^[58]17 A structural magnetic resonance imaging study found that
   a decreased volume of the hippocampal cortex and structural brain
   abnormalities were strongly associated with aggressive behavior.^[59]18
   In addition, another study revealed that the hippocampal CA2 region to
   the lateral septal circuit disinhibits social aggression.^[60]19
   Although the hippocampus is known to be important for declarative
   memory, how the hippocampus regulates aggression is less well
   understood. Proteomics, as a high-throughput technique, can
   indiscriminately study the protein response and behavior pattern under
   different stress conditions and is widely applied to gain a deeper
   understanding of drug action, which contributes to the exploration of
   the induction mechanism of drug-mediated toxicity and provision of
   potential biomarker candidates.^[61]20−[62]22 Of note, to date, very
   few studies have employed a proteomic approach in revealing protein
   markers of aggressive behavior induced by PER. Therefore, the aim of
   our study was to determine whether administration of perampanel to mice
   would induce alterations in hippocampal protein profiles and to
   elucidate the possible neurobiological mechanisms of aggressive
   behavior.

   In the present study, a model of aggressive behavior was established to
   simulate the clinical use of perampanel for 2 months, and changes in
   hippocampal protein profiles were detected using tandem mass labeling
   (TMT) proteomics. Bioinformatic analysis showed that differentially
   expressed proteins (DEPs) were involved in synaptic plasticity and the
   Ras signaling pathway. In addition, changes in synaptic marker proteins
   and key molecules of the Ras signaling pathway were further verified by
   western blot. These results provide profound implications of the
   molecular mechanisms for PER-induced aggressive behavior, as well as
   the disturbance of proteomic profiles supporting PER-induced aggressive
   behavior.

2. Materials and Methods

2.1. Animals and Treatment

   This experiment was performed in accordance with the Guidelines for The
   Care and Use of Laboratory Animals of the National Institutes of Health
   and approved by the Ethics Committee for the Use of Laboratory Animals
   of Ningxia Medical University (Z2019/023, approved 10 November 2019).
   Sixty 8 week-old male C57BL/6J mice (21 ± 0.5 g) were selected from the
   Experimental Animal Center of Ningxia Medical University. All mice were
   reared under standard feeding conditions, including temperature (22 ± 2
   °C), relative humidity (35–50%), light/dark cycle for 12 h, and free
   food and water. All mice were grouped in transparent plastic cages (24
   × 13.5 × 13 cm) with five mice per cage. Mice were randomly divided
   into two groups: resident mice and intruder mice (30 mice in each
   group). The intruder mice were not given any treatment, only free
   feeding and water. Resident mice were assigned to two experimental
   groups: drug “perampanel” group (PER) and control “vehicle” group (Con)
   (n = 15 each group). The Con group was intragastrically given 0.5%
   carboxymethyl cellulose-sodium (CMC-NA) solution (0.1 mL/g), mice in
   the PER group were intragastrically given PER/CMC-Na suspension (0.015
   mg/kg, PER dissolved in CMC-NA), and the dose of PER was selected
   according to the current clinically published literature.^[63]23 The
   mice were administered intragastrically continuously for 60 days, which
   took place between 3 pm and 5 pm per day. The initial dose of PER was
   0.015 mg/kg at the 1st month, and the dose was increased to 0.03 mg/kg
   at the 2nd month. The resident–intruder test was performed at the 61st
   day.

2.2. Open Field Test

   Mice were placed in a closed template area (50 cm × 50 cm × 40 cm) and
   allowed to explore freely for 10 min, and the mouse movement was
   monitored using Smart 3.0 video tracking software (Panlab, Spain). The
   experiment was carried out in the behavioral laboratory with sound
   insulation, light intensity, temperature, and humidity suitable and
   consistent. At the beginning of the experiment, the experimental
   equipment was cleaned to avoid the residual information of the last
   animal affecting the results. The mice in each group were placed in the
   box in turn, and the experimenter quickly evacuated the video area. The
   system automatically records the moving distance (cm) and time (s) of
   the mice in the central and surrounding areas and analyzes the data.
   The experiment began at the 61st day and 1 h before the beginning of
   the resident–intruder test.

2.3. Aggressive Behavior Test

   Aggressive behavior is assessed using the resident–intruder standard
   procedure.^[64]24 Before the behavior test, the sawdust of the
   residential home cage was not cleaned for 1 week. The entire behavioral
   room was lit at 40 lux, and the mice were acclimated to the environment
   for 1 h. At the beginning of the resident–intruder test, one mouse was
   left in the home cage, while four mice were moved to a new cage. One
   intruder mouse was placed into the home cage containing one resident
   mouse. The intruder mouse weighed about 10% less than the resident
   mouse. Behavior was videotaped with an iPhone mounted directly above
   the cages for 10 min. The video recordings were later analyzed by two
   trained observers who were blinded to the treatment groups. The latency
   to the first attack, the total attack number, and duration were
   measured. Mice with the aggression score of more than 3 points were
   screened and entered the sampling stage. The aggressive intensity was
   assessed based on the aggression scale: 0 points, no aggressive
   behavior; 1 point, attack number ≤ 3, sniffing, wagging their tails,
   chasing; 2 points, attack number ≤ 10, attacking the head, flanking,
   suppressing, squeaking, and violently attacking the opponent; 3 points,
   attack number ≤ 20, in addition to the above performance, there is
   mutual bite, falling or climbing on the intruder; 4 points, attack
   number > 20, bite violently, form a ball, bite each other, and continue
   to attack after yielding.

2.4. Protein Extraction and Digestion

   Three minutes after the end of the last aggressive behavior test, mice
   were anesthetized by intraperitoneal injection of 3% pentobarbital
   sodium solution at a dose of 50 mg/kg. After that, the mice were
   directly killed by neck cutting. The hippocampal tissues were removed
   quickly on ice and frozen in liquid nitrogen before being stored in a
   refrigerator at −80 °C for further analysis. SDT (4% SDS, 100 mM
   Tris–HCl, pH 7.6) buffer was used for sample lysis and protein
   extraction. The amount of protein was quantified with the BCA Protein
   Assay Kit (Bio-Rad, USA). Protein digestion by trypsin was performed
   according to the filter-aided sample preparation (FASP) procedure
   described by Mann et al.^[65]25 The peptide content was estimated by UV
   light spectral density at 280 nm using an extinction coefficient of 1.1
   of 0.1% (g/L) solution that was calculated on the basis of the
   frequency of tryptophan and tyrosine in vertebrate proteins.

2.5. Tandem Mass Tag (TMT) Labeling

   One hundred micrograms of peptide mixture of each sample was labeled
   using TMT reagent according to the manufacturer’s instructions (Thermo
   Scientific). Peptides of the two groups were labeled with different TMT
   labels: three biological repeats of the control group were labeled with
   TMT-126, TMT-127, and TMT-128 and three biological repeats of the PER
   group were labeled with TMT-129, TMT-130, and TMT-131.

2.6. High-pH Reversed-Phase Fractionation

   Labeled peptides were fractionated into 10 different fractions by the
   High pH Reversed-Phase Peptide Fractionation Kit (Thermo Scientific).
   Briefly, the dried peptide mixture was reconstituted and acidified with
   0.1% TFA solution and loaded to an equilibrated, high-pH,
   reversed-phase fractionation spin column. The collected fractions were
   desalted on C18 Cartridges (Empore SPE Cartridges C18 (standard
   density); bed I.D., 7 mm; volume, 3 mL; Sigma) and concentrated by
   vacuum centrifugation.

2.7. LC–MS/MS Analysis and Protein Identification

   Samples were analyzed using a Q Exactive mass spectrometer (Thermo
   Scientific) that was coupled to an Easy nLC1200 (Thermo Fisher
   Scientific). The peptides (1 μg) were loaded onto a reversed-phase trap
   column (Thermo Scientific Acclaim PepMap100, 100 μm × 2 cm, nanoViper
   C18) connected to a C18 reversed-phase analytical column (Thermo
   Scientific Easy Column, 10 cm long, 75 μm inner diameter, 3 μm resin)
   in buffer A (0.1% formic acid) and separated with a linear gradient of
   buffer B (84% acetonitrile and 0.1% formic acid) at a flow rate of 300
   nL/min controlled by Intelli Flow technology. The MS raw data for each
   sample were searched using the MASCOT engine (Matrix Science, London,
   UK; version 2.2) embedded into Proteome Discoverer 1.4 software for
   identification and quantitation analysis. The false discovery rate was
   set as ≤0.01. The protein ratios were calculated as the median of only
   unique peptides of the protein. All peptide ratios were normalized by
   the median protein ratio, and the median protein ratio should be “1”
   after the normalization. Fold changes were based on the 95% confidence
   interval of the variance between the two replicate standard TMT-plex
   reporter ion ratios (n = 6849, 95%, 1.007–1.011). By considering that
   the samples were deviated from 1-fold expression (no change in TMT
   reporter ion intensities vs control intensity values), statistical
   analysis was used with p < 0.05 as a criterion for significance.

2.8. Bioinformatic Analyses

   Cluster 3.0 and Java Treeview software were used to perform the
   hierarchical clustering analysis. Functional annotation of Gene
   Ontology (GO) was derived from the software program Blast2GO
   ([66]https://blast.ncbi.nlm.nih.gov/). Proteins were classified based
   on three categories: biological process, cellular component, and
   molecular function. Following annotation steps, the studied proteins
   were blasted against the online Kyoto Encyclopedia of Genes and Genomes
   (KEGG) database ([67]http://www.genome.jp/kegg/) to retrieve their KEGG
   anthology identifications and were subsequently mapped to pathways in
   KEGG. Enrichment analysis was applied based on Fisher’s exact test.
   Benjamini–Hochberg correction for multiple testing was further applied
   to adjust derived p-values. Also, only functional categories and
   pathways with p-values under a threshold of 0.05 were considered
   significant. The protein–protein interaction (PPI) information of the
   studied proteins was retrieved from the IntAct molecular interaction
   database by their gene symbols and STRING software
   ([68]http://string-db.org). The results were imported into Cytoscape
   software (version 3.6.0) to visualize and further analyze functional
   protein–protein interaction networks.

2.9. Western Blot

   Isolation of hippocampal proteins was described above. The samples (20
   μg for each sample) were separated on 12% polyacrylamide SDS–PAGE gel
   (83 × 73 × 15 mm) and then transferred to polyvinylidene difluoride
   membranes, which were incubated with anti-synaptophysin monoclonal
   antibody (Abcam, cat. #: ab32127, 1:1000), anti-PSD95 monoclonal
   antibody (Abcam, cat. #: ab2723, 1:200), anti-ERK1(phospho T202) +
   ERK2(phospho T185) monoclonal antibody (Abcam, cat. #: ab214036,
   1:1000), anti-glutamate receptor 1 (AMPA subtype) monoclonal antibody
   (Abcam, cat. #: ab109450, 1:2000), or anti-alpha tubulin polyclonal
   antibody (Abcam, cat. #: ab7291, 1:5000) overnight at 4 °C and then
   incubated with horseradish peroxidase (HRP) conjugated anti-rabbit or
   anti-goat secondary antibody (Proteintech, 1:10000) for 3 h at room
   temperature. Protein bands were visualized using a Supersignal West
   Pico Chemiluminescence kit (Pierce).

2.10. Statistics

   For histograms, volcano plots, and clustering analysis, proteomic data
   are log-transformed and normalized when appropriate. Statistical
   analysis was performed using SPSS software version 13.0 (IBM Software,
   New York, USA). Data are expressed as mean ± standard deviation (SD).
   Comparative analysis of the quantitative data was performed using
   Student’s t-test with statistically significant difference at p < 0.05.

3. Results

3.1. Perampanel Exposure-Induced Aggressive Behavior

   Aggressive behavior is considered to be one of clinical phenotypes
   resulting from patients with long-term use of PER. Based on these
   reports, we developed a mice model to study the aggressive behavior
   induced by PER. Before assessing the level of aggression, we measured
   the locomotor activity in an open field test. The total distance
   traveled and central duration of PER-treated mice were not different
   from the Con group (Student’s t-test, p > 0.05,[69]Figure [70]1
   ), indicating that PER did not affect the locomotor function of mice.
   Regarding aggressive behavior, compared with the control group,
   PER-treated mice showed a decrease in latency to the first attack and
   an increase in the number of attacks and total duration of attacks
   (Student’s t-test, p < 0.001, [71]Figure [72]2). These results
   indicated that long-term exposure to PER increased the aggressive
   behavior of C57BL/6J mice.

Figure 1.

   [73]Figure 1
   [74]Open in a new tab

   Locomotor activity of PER and CON groups in the open field test. No
   significant difference in the total distance and center time was found
   between Con and PER groups in mice, respectively, which was shown in a
   computer-generated trace of the animal’s movements over 10 min (n = 6
   mice per group; Student’s t-test). (A–D) Data are presented as mean ±
   SD.

Figure 2.

   Figure 2
   [75]Open in a new tab

   Aggressive behavior test in two groups. (A) Latency to the first attack
   (p < 0.001); (B) number of attacks (p < 0.001); (C) attack duration (p
   < 0.01). Data represent mean ± SD (n = 6 per group). **p < 0.01 and
   ***p < 0.001 indicate significant statistical difference between
   groups.

3.2. Differential Expression of Hippocampal Proteins

   In the current study, PER exposure disrupted hippocampal proteomic
   profiles of mice. A total of 6849 proteins were identified by one or
   more unique peptides from the protein database (false discovery rate <
   1%) by TMT-based quantitative proteomic analysis. Corresponding to 6849
   proteins, 93 proteins were significantly altered in the hippocampus
   tissue of the PER group (corrected p < 0.05); we used GO annotation to
   identify the functional significances of all identified proteins, which
   were divided into multiple functional groups, mainly related to
   synaptic function, synaptogenesis, synaptic plasticity, postsynaptic
   density protein, vesicular transport, neurite outgrowth, AMPA-type
   glutamate receptor immobilization, Ras GTPase, and others. The detailed
   information of the differentially expressed proteins (DEPs) is listed
   in [76]Table [77]1
   . Among the DEPs, 59 proteins were up-regulated and 34 proteins were
   significantly down-regulated in perampanel-treated mice compared with
   the control group. We also conducted DEP clustering using the
   hierarchical cluster analysis and generated heatmap in which the two
   comparable groups can be easily recognized ([78]Figure [79]3A,B),
   highlighting the notable differences between PER and Con groups.

Table 1. List of Quantitatively Modulated Differentially Expressed Proteins
in the Hippocampus Identified by TMT Proteomics Following PER Exposure[80]^a.

   accession description Cov Uniq M/C t-test p-value
   [81]Q80YS6 actin filament-associated protein 1, GN = Afap1 1 1
   2.771190952 0.000318871
   [82]Q8K3K8 optineurin, GN = Optn 2 1 1.392116859 0.009811607
   [83]P07903 DNA excision repair protein ERCC-1, GN = Ercc1 7 1
   1.310416285 0.007013598
   [84]P97328 ketohexokinase, GN = Khk 2 1 1.280888319 0.028568005
   [85]Q9CWQ3 mitochondrial inner membrane protease ATP23 homolog, GN =
   Atp23 4 1 1.218091972 0.026972971
   [86]P29351 tyrosine-protein phosphatase non-receptor type 6, GN = Ptpn6
   3 2 1.211774969 0.002227745
   [87]O08967 cytohesin-3, GN = Cyth3 12 1 1.20656536 0.033370933
   [88]Q99K95 replication termination factor 2, GN = Rtf2 3 1 1.194115843
   0.036390341
   [89]Q91ZE0 trimethyllysine dioxygenase, mitochondrial, GN = Tmlhe 2 1
   1.190253971 0.030854053
   [90]Q8VBT0 thioredoxin-related transmembrane protein 1, GN = Tmx1 23 8
   1.187900082 0.029794138
   [91]Q61333 tumor necrosis factor alpha-induced protein 2, GN = Tnfaip2
   1 1 1.167683579 0.024939168
   [92]Q61249 immunoglobulin-binding protein 1, GN = Igbp1 21 6
   1.165655296 0.008190139
   [93]P70460 vasodilator-stimulated phosphoprotein, GN = Vasp 9 3
   1.157812095 0.022705772
   [94]Q5DQR4 syntaxin-binding protein 5-like, GN = Stxbp5l 21 18
   1.14660469 0.019967117
   [95]Q0VBL3 RNA-binding protein 15, GN = Rbm15 3 3 1.146047039
   0.029562494
   [96]P15208 insulin receptor, GN = Insr 4 5 1.140066392 0.003800028
   [97]Q8BGA3 leucine-rich repeat transmembrane neuronal protein 2, GN =
   Lrrtm2 6 3 1.123499392 0.049398248
   [98]P56393 cytochrome c oxidase subunit 7B, GN = Cox7b 10 2 1.120774032
   0.028593212
   [99]Q8BKI2 trinucleotide repeat-containing gene 6B protein, GN = Tnrc6b
   1 2 1.119896434 0.048432502
   [100]Q8BLU0 leucine-rich repeat transmembrane protein FLRT2, GN = Flrt2
   6 4 1.119183137 0.031456573
   [101]P70372 ELAV-like protein 1, GN = Elavl1 35 10 1.109275956
   0.043608033
   [102]Q99MP8 BRCA1-associated protein, GN = Brap 4 2 1.108830808
   0.022466448
   [103]Q99JF5 diphosphomevalonate decarboxylase, GN = Mvd 13 5
   1.108657253 0.048063462
   [104]F6SEU4 Ras/Rap GTPase-activating protein SynGAP, GN = Syngap1 45
   49 1.108032821 0.015276438
   [105]O35144 telomeric repeat-binding factor 2, GN = Terf2 1 1
   1.102550161 0.017687406
   [106]Q68FF0 uncharacterized protein KIAA1841, GN = Kiaa1841 2 2
   1.100563703 0.040458368
   [107]Q8JZP2 synapsin-3, GN = Syn3 30 11 1.098423376 0.021453436
   [108]Q9CZN4 protein shisa-9, OS = Mus musculus, OX = 10090, GN = Shisa9
   15 6 1.093834958 0.027522023
   [109]Q8BLB7 lethal(3)malignant brain tumor-like protein 3, GN = L3mbtl3
   1 1 1.08984348 0.018365628
   [110]P05622 platelet-derived growth factor receptor beta, GN = Pdgfrb 3
   3 1.089357506 0.002160346
   [111]Q8R2H9 phosphoethanolamine/phosphocholine phosphatase, GN =
   Phospho1 19 6 1.087790485 0.018061833
   [112]Q4ACU6 SH3 and multiple ankyrin repeat domains protein 3, GN =
   Shank3 31 41 1.076513927 0.022212105
   [113]P52189 inward rectifier potassium channel 4, GN = Kcnj4 9 4
   1.07594632 0.002652225
   [114]Q91YQ3 cold shock domain-containing protein C2, GN = Csdc2 25 5
   1.069415813 0.001378977
   [115]Q8VHQ3 protein phosphatase 1 regulatory inhibitor subunit 16B, GN
   = Ppp1r16b 7 4 1.068784484 0.043779232
   [116]Q3TC72 fumarylacetoacetate hydrolase domain-containing protein 2A,
   GN = Fahd2 57 12 1.067599347 0.044817035
   [117]Q78RX3 small integral membrane protein 12, GN = Smim12 27 3
   1.065361562 0.04945411
   [118]Q9D009 putative lipoyltransferase 2, GN = Lipt2 16 4 1.063020604
   0.04778066
   [119]Q9JK45 potassium voltage-gated channel subfamily KQT member 5, GN
   = Kcnq5 1 1 1.061013369 0.049625226
   [120]Q9WTU6 mitogen-activated protein kinase 9, GN = Mapk9 25 5
   1.060252203 0.021940713
   [121]Q3UIU2 NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit
   6, GN = Ndufb6 41 6 1.059345305 0.048990551
   [122]P46735 unconventional myosin-Ib, GN = Myo1b 20 21 1.056683157
   0.049709811
   [123]Q91YX5 Acyl-CoA:lysophosphatidylglycerol acyltransferase 1, GN =
   Lpgat1 21 8 1.048716656 0.040300128
   [124]Q9ER39 torsin-1A, GN = Tor1a 2 1 1.047414076 0.022220112
   [125]Q99J56 derlin-1, GN = Derl1 11 3 1.046398534 0.015974799
   [126]Q69ZZ6 transmembrane and coiled-coil domains protein 1, GN = Tmcc1
   20 9 1.0450555 0.049090563
   [127]Q62108 disks large homolog 4, OS = Mus musculus, OX = 10090, GN =
   Dlg4 54 30 1.041440349 0.015860967
   [128]Q811I0 ATP synthase mitochondrial F1 complex assembly factor 1, GN
   = Atpaf1 34 10 1.041126873 0.011658994
   [129]Q8BFR4 N-acetylglucosamine-6-sulfatase, GN = Gns 11 6 1.038349909
   0.042754672
   [130]Q3UH99 protein shisa-6, OS = Mus musculus, OX = 10090, GN = Shisa6
   19 9 1.036913837 0.023913209
   [131]Q9D0M1 phosphoribosyl pyrophosphate synthase-associated protein 1,
   GN = Prpsap1 47 11 1.034563714 0.00578867
   [132]Q9JM52 misshapen-like kinase 1, OS = Mus musculus, OX = 10090, GN
   = Mink1 27 27 1.033506063 0.003627109
   [133]Q60738 zinc transporter 1, GN = Slc30a1 12 5 1.032821845
   0.042890271
   [134]Q91YM4 FAST kinase domain-containing protein 4, GN = Tbrg4 5 4
   1.032820741 0.014491126
   [135]Q921W4 quinone oxidoreductase-like protein 1, GN = Cryzl1 30 9
   1.032033008 0.034037391
   [136]Q8JZN7 mitochondrial Rho GTPase 2, GN = Rhot2 9 5 1.030800469
   0.029075355
   [137]Q9Z2I8 succinate–CoA ligase [GDP-forming] subunit beta,
   mitochondrial, GN = Suclg2 39 18 1.027400076 0.035306881
   [138]Q99MR0 actin-like protein 6B, GN = Actl6b 21 6 1.024814164
   0.036668562
   [139]Q78PG9 coiled-coil domain-containing protein 25, GN = Ccdc25 19 4
   1.023158245 0.035135783
   [140]E9PUL5 proline-rich transmembrane protein 2, GN = Prrt2 37 8
   0.982084039 0.018897725
   [141]Q63829 COMM domain-containing protein 3, GN = Commd3 23 5
   0.980662277 0.034438143
   [142]Q9CQF4 mitochondrial transcription rescue factor 1, GN = Mtres1 13
   3 0.97898691 0.040461715
   [143]Q9DCT2 NADH dehydrogenase iron–sulfur protein 3, GN = Ndufs3 48 14
   0.968398505 0.022386004
   [144]Q8CH72 E3 ubiquitin-protein ligase TRIM32, GN = Trim32 22 13
   0.965597426 0.03594526
   [145]Q8R4F1 netrin-G2, GN = Ntng2 4 3 0.965217544 0.000994181
   [146]P11103 poly [ADP-ribose] polymerase 1, GN = Parp1 9 8 0.964471006
   0.040301374
   [147]Q91VK4 integral membrane protein 2C, GN = Itm2c 34 6 0.962686811
   0.0424762
   [148]Q91ZH7 phospholipase ABHD3, GN = Abhd3 10 4 0.960783265
   0.008175226
   [149]Q91X97 neurocalcin-delta, GN = Ncald 77 7 0.958409806 0.033373857
   [150]O08644 ephrin type-B receptor 6, GN = Ephb6 6 5 0.94969615
   0.038122544
   [151]P70408 cadherin-10, GN = Cdh10 16 10 0.946552755 0.030902986
   [152]Q9QUN9 dickkopf-related protein 3, GN = Dkk3 18 6 0.945815891
   0.014993276
   [153]Q9WV76 AP-4 complex subunit beta-1, GN = Ap4b1 2 1 0.939870283
   0.026232922
   [154]Q3TQF0 F-box only protein 31, GN = Fbxo31 2 1 0.938756477
   0.023650774
   [155]O55033 cytoplasmic protein NCK2, GN = Nck2 23 8 0.934122038
   0.006039627
   [156]P23953 carboxylesterase 1C, GN = Ces1c 11 6 0.933377651
   0.022455845
   [157]Q3UFY7 7-methylguanosine phosphate-specific 5′-nucleotidase, GN =
   Nt5c3b 15 4 0.932564335 0.042808153
   [158]O08677 kininogen-1, GN = Kng1 11 6 0.931376613 0.046635589
   [159]Q8R2U6 diphosphoinositol polyphosphate phosphohydrolase 2, GN =
   Nudt4 25 1 0.928412788 0.00365918
   [160]P32848 parvalbumin alpha, GN = Pvalb 73 12 0.927436716 0.036932494
   [161]O35089 protein cornichon homolog 2, GN = Cnih2 6 1 0.921967153
   0.030133405
   [162]Q8VE96 solute carrier family 35 member F6, GN = Slc35f6 3 1
   0.920103006 0.011340532
   [163]Q9ESB3 histidine-rich glycoprotein, GN = Hrg 3 2 0.914787183
   0.042555487
   [164]Q9CQ25 mitotic-spindle organizing protein 2, GN = Mzt2 9 1
   0.914477964 0.046210189
   [165]Q9CQM0 nicolin-1, GN = Nicn1 4 1 0.90052069 0.001735753
   [166]O70250 phosphoglycerate mutase 2, GN = Pgam2 34 5 0.899310792
   0.04662173
   [167]Q61838 pregnancy zone protein, GN = Pzp 20 30 0.893129596
   0.008922305
   [168]Q8K013 GTP-binding protein 10, GN = Gtpbp10 2 1 0.87500987
   0.046151603
   [169]P02798 metallothionein-2, GN = Mt2 13 1 0.859901481 0.019934253
   [170]P01864 Ig gamma-2A chain C region secreted form 15 3 0.859231943
   0.026535001
   [171]Q8BGZ7 keratin, type II cytoskeletal 75, GN = Krt75 13 2
   0.843522803 0.03382069
   [172]Q3V016 homeodomain-interacting protein kinase 4, GN = Hipk4 1 1
   0.733170586 0.002472431
   [173]Q2WF71 leucine-rich repeat and fibronectin type III
   domain-containing protein 1, GN = Lrfn1 12 1 0.732267503 0.035409077
   [174]Open in a new tab
   ^a

   Note: Cov, covered by identified peptides; Uniq, unique peptide number;
   M, PER group; C, Con group.

Figure 3.

   [175]Figure 3
   [176]Open in a new tab

   Characteristics of the identified proteins in PER and CON groups. (A)
   Hierarchical cluster analysis of 93 differentially expressed proteins.
   Whole hippocampal samples are represented in the columns, and the IDs
   of differentially expressed proteins are delineated in rows. The color
   bar located below the figure shows that red indicates significant
   up-regulation and blue represents down-regulation. M1, M2, and M3,
   three replicates of perampanel-treated mice; C1, C2, and C3, three
   replicates of control mice. (B) Volcano plot for the DEPs in the
   comparison group according to fold change (M/C) and p value (t-test).
   Red and blue dots indicate significantly up-regulated and
   down-regulated proteins, respectively, and proteins with no difference
   are gray.

3.3. Enrichment Analysis of Differentially Expressed Proteins

   To better understand how the functional role of DEPs was disrupted by
   PER exposure, GO annotation was used to categorize according to
   molecular function (MF), cellular component (CC), and biological
   process (BP). To detect significantly enriched biological function
   types, we performed GO enrichment analysis and ranked the terms by
   enrichment score (−log10(p value)). According to the GO enrichment
   results, the top 20 enriched items were displayed by the enrichment
   factors ([177]Figure [178]4
   ).

Figure 4.

   [179]Figure 4
   [180]Open in a new tab

   GO functional enrichment analysis of the DEPs in three categories,
   biological process, molecular function, and cellular component. The
   p-value represents the enriched degree.

   The biological process of the identified proteins mainly involved
   regulation of neuronal synaptic plasticity, regulation of AMPA receptor
   activity, postsynaptic density organization, regulation of synapse
   structure or activity, vocalization behavior, regulation of
   vesicle-mediated transport, and anatomical structure homeostasis. The
   results for molecular function showed the strong enrichments of SH3
   domain binding, neurexin family protein binding, structural constituent
   of postsynaptic density, nerve growth factor binding, and neuroligin
   family protein binding. Cellular component annotation displayed the
   intrinsic component of postsynaptic density membrane, synaptic
   membrane, dendritic spine membrane, dendrite membrane, anchored
   component of synaptic membrane, and neuron projection membrane. KEGG
   pathway analysis (p < 0.05) revealed that the statistically significant
   enriched terms included the Ras signaling pathway, IL-17 signaling
   pathway, JAK–STAT signaling pathway, Rap1 signaling pathway, focal
   adhesion, axon guidance, glycine, serine, and threonine metabolism,
   cell adhesion molecules, and neuroactive ligand–receptor interaction
   ([181]Figure [182]5
   ). The Ras signaling pathway and axon guidance are the most significant
   changes in up-regulation and down-regulation pathways.

Figure 5.

   [183]Figure 5
   [184]Open in a new tab

   KEGG pathway enrichment analysis of the DEPs. The p-value represents
   the enriched degree. Orange, significant increase; blue, significant
   decrease.

3.4. STRING Analysis of Protein–Protein Interaction Networks

   To find out the correlation between protein interactions and related
   biological processes, a protein–protein interaction (PPI) network of
   the 93 DEPs was constructed by the STRING database ([185]Figure [186]6
   ).

Figure 6.

   [187]Figure 6
   [188]Open in a new tab

   Protein–protein interaction network analysis of the DEPs using STRING.
   The line thickness represents the confidence scores, and thicker
   connection lines indicate the higher confidence of protein–protein
   interaction. Nodes are labeled with gene names.

   Proteins regarding synaptic function accounted for about half of all
   regulated proteins. It was obviously revealed that at least four
   closely coexpressed clusters were displayed in the complex PPI network,
   including Vasp, Nck2, DLg4, and Shisa6. These clusters were mainly
   associated with neural development and dendritic outgrowth, Ras
   pathway, postsynaptic density protein, and AMPA-type glutamate
   receptor. The neural development and dendritic outgrowth-related
   cluster included Vasp, Actl6b, Ptpn6, Insr, and Mapk9. Proteins in the
   Ras pathway-related cluster included Nck2, Insr, Ephb6, Mink1, Pdgfrb,
   and Syngap1. Proteins in the postsynaptic density protein cluster
   included Dlg4, Syngap1, Lrrtm2, Shank3, Pvalb, Lrfn1, Prrt2, and Ntng2.
   In addition, the AMPA-type glutamate receptor-related cluster included
   Shisa6, Shisa9, Cnih2, and Dlg4. Taken together, the bioinformatic
   analysis of our study revealed that synaptic plasticity and the Ras
   signaling pathway may play an important role in the pathogenesis of
   aggressive behavior.

3.5. Validation of Differentially Expressed Proteins

   To confirm the potential bioinformatic analysis significance in
   aggressive behavior induced by PER, further verification of Syn, PSD95,
   p-ERK1/2, and GluA1 was performed using western blot. The following
   reasons were used for choosing the four proteins: (a) most significant
   proteins for functional enrichment and KEGG analysis; (b) signal
   proteins of synaptic plasticity and the Ras signaling pathway; (c)
   relevance to synaptic function and commercially available antibodies;
   (d) perampanel is a relatively safe drug, and the only drug target is
   the AMPA receptor. The results of western blot showed that expression
   of Syn and PSD95 was down-regulated and that of p-ERK and GluA1
   proteins was up-regulated in the PER-treated mice compared to the
   control groups (p < 0.05 and p < 0.001; [189]Figure [190]7
   ).

Figure 7.

   [191]Figure 7
   [192]Open in a new tab

   Western blot validations of Syn, PSD95, p-ERK1/2, and GluA1 proteins in
   the hippocampus. (A) Western blot images. (B) The protein expression
   levels were quantitatively analyzed with the tubulin level. Data are
   presented as mean ± SD. Student’s t-test, *p < 0.05, ***p < 0.001
   versus the control group. n = 6 for each group.

4. Discussion

   There is increasing amount of clinical research revealing that the
   long-term use of perampanel for refractory partial-onset epilepsy has
   caused an abundance of concern about the adverse events and health
   risks of aggressive behavior.^[193]26−[194]28 However, the underlying
   pathogenesis and neurobiological significance remain poorly understood.
   In our current study, we have carried out quantitative proteomic
   methods to identify changes in protein profile alternations induced by
   PER in the hippocampus of an aggressive behavior mice model. Half of
   DEPs were mainly directed to neurite growth, postsynaptic density, AMPA
   receptor, and Ras signaling pathway. Furthermore, western blot assay
   further demonstrated the alteration of Syn, PSD95, GluA1, and p-ERK in
   PER-treated mice. Gene function enrichment and pathway visualization
   analysis objectively displayed that dysregulation of synaptic
   plasticity and the Ras signaling pathway might mainly mediate the
   pathological mechanism.

   Our study successfully established aggressive behavior in mice after
   oral administration of PER at a dose of 2 mg/kg per day for 2 months.
   Resident–intruder test results showed that aggressive behavior was
   significantly enhanced in the PER group compared to the control group.
   To our knowledge, no previous animal studies have formalized the
   representation of PER-induced aggressive behavior in male mice.
   Nonetheless, these results are comparable with the already published
   controlled randomized clinical studies. Irritability and aggressive
   behavior were listed in perampanel’s instructions since it was first
   marketed.^[195]29 There was evidence shown that around 25% patients
   with severe drug-resistant focal epilepsy revealed PER-related
   aggressiveness.^[196]23 In a recent prospective study, Goji and
   Kanemoto indicated that PER increases assessment scores indicative of
   aggression.^[197]30 Collectively, these observations seen in
   experimental animal models are qualitatively consistent with previous
   cases of aggressive behavior.

   Proteomics has recently been exploited to analyze protein expression
   profiling and contributes to revealing the signal pathways of external
   environmental stimuli and pathological processes of drug action as well
   as the biological regulatory mechanisms associated with disease
   pathology.^[198]31,[199]32 However, there are no reports on studies of
   PER-induced aggression using proteomic methods. Our results showed that
   PER-induced changes in hippocampal proteins were manifested as a
   disturbance of synaptic plasticity and the Ras signaling pathway.

   We found that PER treatment induced the decrease in Syn and PSD95
   proteins in the hippocampus. Synaptic plasticity-related proteins
   mainly include synaptophysin and postsynaptic density 95,^[200]33 which
   have received increasing attention as a means of modifying innate and
   adaptive behavioral responses.^[201]34 Changes in synaptic
   plasticity-related proteins reflect their roles in axonal growth,
   synaptic reconstruction, and synaptic connection.^[202]35 The most
   significantly enriched pathway associated with aggressive behavior in
   humans is axon guidance.^[203]36 Previous work has shown that perinatal
   fluoxetine exposure resulted in significantly decreased rates of
   hippocampal synaptophysin density, which was correlated with increased
   social aggression.^[204]37 In another study, maternal separation mice
   showed reduced synaptic function at the hippocampal synapses, which was
   significantly associated with more aggressive behavior.^[205]38 In
   additional animal work, Stxbp1+/– mice that exhibited impaired synaptic
   function and excitatory neurons showed a pronounced greater number and
   prolonged duration of aggression than the wild-type mice.^[206]39 In
   our opinion, synaptic protein and neurite growth seem to play an
   important regulatory role in aggressive behavior.

   Postsynaptic density 95 (PSD95) is highly gathered at the synapses and
   accounted for the assembly of the abundant protein complexes
   indispensable for synaptic plasticity and neural development.^[207]40
   PSD95 plays a crucial role in capturing and immobilizing surface AMPA
   receptors,^[208]41 which interacted with the stargazin auxiliary
   subunit to anchor and increase the number of AMPA receptors at the
   synapse.^[209]42 Synaptic morphological plasticity and constructional
   plasticity are the material foundation of functional
   plasticity.^[210]43 PSD95 has been extensively related to the
   modulation of pro-social and emotional behavior.^[211]44 A previous
   study showed that male PSD95^+/– mice demonstrated higher levels of
   aggression.^[212]45 In addition, Dlgap2^–/– mice exerted a shorter and
   thinner PSD with respect to wild-type mice in cortical synapses; thus,
   Dlgap2^–/– mice displayed heightened aggressive behavior.^[213]46 The
   DLG5 gene related to neuritogenesis was associated with aggressive
   behavior in candidate gene association studies (CGAS) mentioned
   above.^[214]36 Overall, the down-regulation of PSD95 protein was
   correlated with aggressive behavior, supporting a common mechanism of
   aggressive behavior.

   In addition, PER treatment induced the increasing levels of AMPA-type
   glutamate receptors in mice. AMPA receptors are especially intriguing
   since they have direct and lasting effects on aggression.^[215]47 A
   growing body of experimental and clinical evidence suggests that AMPA
   receptors have gradually been valued for their role in aggressive
   behavior.^[216]1,[217]48 Previous studies have confirmed that genetic
   modification of the AMPA receptor leads to changes in aggressive
   behavior; blocking the AMPA receptor can reduce or increase aggressive
   behavior.^[218]6,[219]49,[220]50 It has been suggested that mice
   lacking the AMPAR subunit and GluA1 mutations, resulting in a
   significantly reduced AMPAR function, are less aggressive than
   wild-type littermates.^[221]36 Furthermore, several recent studies
   demonstrated that chronic ultrasonic exposure induces aggressive
   behavior in mice, resulting in decreased GluA1 expression and increased
   GluA2 expression in the hippocampus and prefrontal cortex.^[222]51 The
   aggressiveness of intermale mice with decreased AMPAR function and
   AMPAR knockout mice is significantly lower than that of wild-type (WT)
   littermates.^[223]52 AMPA receptors mainly mediate fast excitatory
   synaptic transmission, which plays an important role in synaptic
   transmission efficiency, neuron integration function, and synaptic
   plasticity.^[224]53 Synaptic plasticity largely depends on the amount
   of AMPA receptors and the adjustment of gating characteristics.^[225]54
   Accumulating evidence suggests that dysfunctional synaptic
   rearrangement of AMPA receptors may play a critical role in
   aggression.^[226]55 AMPA receptors are highly expressed in the CA1 and
   CA3 subregions and the dentate gyrus of the hippocampus, which is also
   believed to play a significant role in determining whether to attack or
   not. Compared with the non-aggressive control group, the levels of AMPA
   receptors in the isolation-induced aggressive mice were significantly
   higher in the amygdala and prefrontal cortex.^[227]56 Studies have
   shown that AMPA receptors are expressed throughout the brain, and their
   normal activation is essential for modulation of synaptic plasticity
   (such as long-term potential enhancement (LTP) and long-term depression
   (LTD)).^[228]57 LTP and LTD can occur on the hypothalamic synapses,
   which are important for attacking.^[229]58 LTP is part of the causal
   mechanism and increases aggressiveness through repeated successful
   attacks.^[230]59 Taken together, our results reveal that PER-induced
   aggression is involved in synaptic plasticity.

   Proteomic data showed that PER treatment activated Ras pathway-related
   proteins in mice. The extracellular signal-regulated kinase (ERK) is a
   key member in the Ras intracellular signaling pathway and could
   regulate hippocampal synaptic transmission and plasticity.^[231]60 In
   this study, it was clear that the phosphorylation of ERK1/2 expression
   was activated in PER-treated mice in contrast with the control group.
   Our results are consistent with the previous studies stating that PER
   could affect the phosphorylation of ERK1/2 expression in the
   hippocampus.^[232]61 Enrichment analysis studies showed that aggression
   genes are involved in Ras–ERK signaling. ERK1/2 signaling forms
   intracellular signaling cascades and the phosphorylation of ERK1/2
   coordinates cellular responses that are usually associated with
   aggression.^[233]62 There is growing evidence that the ERK1/2 kinases
   trigger cascade phosphorylation events to regulate AMPAR subunit gene
   expression and synaptic plasticity.^[234]63Calotropis gigantea
   significantly increased the p-ERK1/2 expression in hippocampal neurons
   to promote neurite outgrowth and synaptogenesis.^[235]64 In particular,
   Ras initiates extracellular signal-regulated kinase (ERK) signaling to
   drive synaptic delivery of AMPA receptors containing subunits with long
   cytoplasmic termini.^[236]65 These results suggest that abnormal
   Ras/ERK signaling and phosphorylation of ERK1/2 may be involved in the
   aggressive behavior induced by PER treatment.

5. Limitations and Considerations

   Some limitations related to the current study should be considered
   here. Although we were able to validate our findings from the proteomic
   comparison study, it is possible that some important synaptic protein
   level changes may have been missed due to the relatively small number
   of sample size in the LC–MS analyses; thus, these findings should be
   interpreted with caution and need to be confirmed in larger samples.
   Aggressive behavior is a complex phenomenon that may involve different
   emotional states in humans, such as increased anger, high emotional
   responses, or decreased control. Animal models only reflect the
   specific aggressive manifestations and cannot fully explain how
   perampanel induced aggressive behavior. Therefore, further proteomic
   research should be performed with cerebrospinal fluid or blood samples
   from aggressive behavior individuals. Neuroimaging studies help
   quantify the activity of specific brain regions or circuits associated
   with aggressive behavior. The study of proteomics combined with
   neuroimaging data can more effectively determine the cause of
   perampanel-induced aggressive behavior.

6. Conclusions

   This is the first report of the whole proteomic changes in the
   hippocampus for perampanel-induced aggressive behavior. Chronic PER
   exposure induces aggressive behavior in mice, accompanied by changes in
   certain hippocampal proteins, particularly those involved in synaptic
   plasticity and the Ras–ERK signaling pathway. Changes in hippocampal
   synapses and AMPA receptor proteins may be involved in PER-induced
   aggression. Our results will contribute to the understanding of the
   roles of proteins in pathophysiological mechanisms of aggressive
   behavior induced by PER. The disturbance of these proteins in the
   hippocampus could be related to the aberrant aggressive behavior. The
   identified biological functions and pathways in the hippocampus are one
   of the mechanisms of PER-induced aggressive behavior in humans and thus
   must be further investigated.

Acknowledgments