CD36 Deficiency Suppresses Epileptic Seizures

Accepted Manuscript CD36 deficiency suppresses epileptic seizures Fangshuo Zheng, Yong Yang, Shanshan Lu, Qin Yang, Yun Li, Xin Xu, Yanke Zhang, Feng Liu, Xin Tian, Xuefeng Wang PII: DOI: Reference:

S0306-4522(17)30752-2 https://doi.org/10.1016/j.neuroscience.2017.10.024 NSC 18089

To appear in:

Neuroscience

Received Date: Accepted Date:

27 March 2017 18 October 2017

Please cite this article as: F. Zheng, Y. Yang, S. Lu, Q. Yang, Y. Li, X. Xu, Y. Zhang, F. Liu, X. Tian, X. Wang, CD36 deficiency suppresses epileptic seizures, Neuroscience (2017), doi: https://doi.org/10.1016/j.neuroscience. 2017.10.024

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Title: CD36 deficiency suppresses epileptic seizures Authors: Fangshuo Zheng1 , Yong Yang1 ,Shanshan Lu1 ,Qin Yang1 , Yun Li1, Xin Xu1 , Yanke Zhang1 , Feng Liu1, Xin Tian1, Xuefeng Wang1,2. Affiliations: 1Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Neurology, 1 Youyi Road,Chongqing 400016, China 2

Center of Epilepsy, Beijing Institute for Brain Disorders, 10 Xitoutiao,

Youanmen, Fengtai District, Beijing 100069, China.

The Lead Author: Fangshuo Zheng, MD, E-mail: [email protected] The co-authors: Yong Yang, PhD, E-mail: [email protected] Shanshan Lu, MD, E-mail: [email protected] Qin Yang, PhD, E-mail: [email protected] Yun Li, MD, E-mail: [email protected] Xin Xu, PhD, E-mail: [email protected] Yanke Zhang, PhD, E-mail: [email protected] Feng Liu, Phd, E-mail: [email protected] Xin Tian, Phd, E-mail: [email protected] The Corresponding Author: Xuefeng Wang, MD, PhD Correspondence to: X. Wang, Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Neurology, 1 Youyi Road,Chongqing 400016, China.; Tel: +86 136 2835 9876 Fax: +86 023 8901 2878 E-mail: xfyp @163.com -1-

Abbreviations:

AA = arachidonic acid AD = Alzheimer’s disease ACSF = Artificial cerebral spinal fluid ANOVA = Analysis of variance APs = Action potentials Aβ = Amyloid-β peptide CD36 = Cluster of differentiation 36 CNS = Central nervous system KA = Kainic acid LFPs = Local field potentials LPS = lipopolysaccharide Mg2+-free ACSF = ACSF without MgCl2 Nrf2 = Nuclear factor erythroid-derived 2-like factor 2 PD = Parkinson's disease PTZ = Pentylenetetrazol PPARγ = Peroxisome proliferator-activated receptor γ SE = Status epilepticus SRSs = Spontaneous recurrent motor seizures TLE = Temporal lobe epilepsy TLR = Toll-like receptor WT = Wild type

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CD36 deficiency suppresses epileptic seizures

ABSTRACT

Cluster of differentiation 36 (CD36) belongs to the class B scavenger receptor family. CD36 is a glycoprotein found on the surface of various cell types and has been implicated in the mechanism of numerous central nervous system (CNS) diseases. However, the relationship between CD36 and epilepsy remains unknown. In this study, we aimed to detect the expression of CD36 in two different chronic epileptic mouse models and determine whether CD36 deficiency leads to suppressive neuronal hyperexcitability and decreased susceptibility of epileptic seizures. Here, we found that CD36 was expressed in the neurons and that CD36 expression was significantly elevated in epileptic mice induced by pentylenetetrazol (PTZ) and kainic acid (KA). Behavioral studies revealed that CD36 deletion in mice (CD36-/- mice) resulted in an attenuated progression of chronic epilepsy compared with wildtype (WT) mice. Whole-cell patch-clamp technique exhibited a decreased frequency of action potentials (APs) in the hippocampal slices of CD36-/- mice. In addition, local field potentials (LFPs) analysis further indicated that CD36 deletion reduced the frequency and duration of epileptiform-like discharges. These results revealed that CD36 deficiency could produce an antiepileptic effect and could provide new insight into antiepileptic treatment.

Key words: CD36, Epilepsy, Epileptic seizure, Neuronal excitability

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INTRODUCTION

Epilepsy is a common and chronic neurological disorder with a worldwide prevalence of approximately 1–2%. The condition is characterized by recurrent unprovoked seizures(Duncan et al., 2006). Although various antiepileptic drugs are available in clinical settings at present, approximately 20–40% of the newly diagnosed epileptic patients are resistant to pharmacological therapy(French, 2007), and temporal lobe epilepsy (TLE) is the most common drug-refractory epilepsy (Beleza, 2009). Recent studies have revealed that epilepsy can be caused by toxic, metabolic or infectious factors, and these factors cause progressive alterations in brain structure and function(Rakhade and Jensen, 2009, Berg et al., 2010). However, the underlying mechanisms of epilepsy are not clear. CD36 belongs to the class B scavenger receptor family and is a membrane glycoprotein found on the surface of various cell types(Silverstein and Febbraio, 2009). Given its broad ligand specificity, CD36 has been implicated in multiple biological processes, including metabolism of fatty acid, engulfment and

removal

of

cellular

debris

and

production

of

inflammatory

mediators(Febbraio et al., 2001). In particular, CD36 is expressed in numerous cell types throughout the central nervous system (CNS), including cerebrovascular

endothelial

cells,

microglial

cells,

astrocytes

and

neurons(Glezer et al., 2009, Fang et al., 2014). Pharmacological and genetic studies in animal models have demonstrated that CD36 is involved in the pathogenesis of several neurological disorders, such as Parkinson's disease (PD), stroke and Alzheimer’s disease (AD)(Garcia-Bonilla et al., 2014). CD36 functions as a fatty acid translocase and regulates fatty acid levels in several brain phospholipid pools(Song et al., 2010), and previous studies have suggested that fatty acids can directly modulate the activity of specific ion channels and may influence seizure activity(Ordway et al., 1991, Woods and Chiu, 1991). Additionally, the role of CD36 as a key regulator of inflammatory mediator activation in sterile inflammation has been reported(Garcia-Bonilla et al., 2014). Increasing evidence supports the involvement of the inflammatory process in the pathogenesis of epilepsy(Vezzani and Granata, 2005). -4-

Moreover, CD36 is implicated in the recognition of lipopolysaccharide (LPS) in a TLR2/4-independent manner and mediates LPS-induced activation of downstream signaling pathways(Baranova et al., 2008), and a previous study has indicate the powerful proconvulsant effect of LPS in rodents(Sayyah et al., 2003). These present findings suggest that CD36 might play a role in epilepsy. However, there is no direct evidence to determine whether CD36 has an effect on epileptic seizures. Therefore, we hypothesized that CD36 might have a role in the neuronal hyperexcitability observed in epilepsy and may thus affect epileptic seizures. In the current study, we used western blot and immunofluorescent methods to detect CD36 expression in the hippocampus in two chronic epilepsy models, namely, pentylenetetrazol (PTZ)-kindled and kainic acid (KA)-induced chronic epilepsy models. Subsequently, a behavioral study of CD36 knockout (CD36−/−) mice and wild-type (WT) mice was performed in the two chronic epilepsy models. In further analysis, to detect the effect of CD36 deficiency on neuronal excitability, the electrophysiological index by whole-cell patch-clamp and local field potentials (LFPs) were utilized. Our data provided a basic understanding of CD36 deficiency in epilepsy. EXPERIMENTAL PROCEDURES

Experimental Animals Adult male C57BL/6J mice (n=88) were obtained from the Experimental Animal Center of Chongqing Medical University, and adult male CD36 knockout mice (n=27) created on a C57BL/6J background were kindly provided by Dr. Maria Febbraio (Lerner Research Institute, USA). CD36-null mice were generated by deletion of Exon 3 using targeted homologous recombination technique and backcrossed six times to C57Bl/6J (Febbraio et al., 1999). All mice had unlimited access to food and water under standard conditions (12 h light/dark cycle, room temperature of 21–22 °C and relative humidity of 55 ± 5%). All experimental procedures were reviewed and approved by the Commission of Chongqing Medical University for Ethics in Animal Experiments. Chronic Epilepsy Models of Mice -5-

Male C57BL/6J mice (n=60) weighing 24-28 g were subjected to two different chronic epilepsy models for further assessment by western blotting and immunofluorescence analysis. The PTZ-kindled model was established according to the method of Zhu X et al.(Zhu et al., 2016). Thirty healthy C57BL/6J mice (24-28 g) were randomly selected for this model. Mice received intraperitoneal injections of PTZ (35 mg/kg, Sigma–Aldrich, St. Louis, USA) every other day for a total of 15 injections (from Day 1 to 30). Immediately following each injection, behavioral observation was performed for 30 min. Only mice with at least three consecutive seizures scored 4 or 5 were considered to be fully kindled and grouped as the epilepsy group. In contrast, mice not kindled after 15 injections were used as controls. All mice were sacrificed 30 days after the first injection of PTZ. The KA-induced chronic model was established as previously described (Riban et al., 2002, Sada et al., 2015). Thirty healthy C57BL/6J mice (24-28 g) were randomly selected for this model. Mice were deeply anesthetized and unilaterally injected with 1.0 nmol of KA (Sigma-Aldrich Co., St. Louis, USA) in 50 nl saline into the right hippocampus. Stereotaxic injections into the dorsal blade of the CA1 area were performed at the following coordinates with respect to bregma: anteroposterior-1.8 mm, mediolateral-1.5 mm, and 1.5 mm below the dura. We injected KA over a 1-min period with a 0.5-μl microsyringe (Hamilton, Reno, NV) (Sada et al., 2015). At the end of injection, the microsyringe remained in situ for an additional 5 minutes and was finally withdrawn slowly to minimize backflow along the injection trace. Beginning one day after status epilepticus induction and for 4 subsequent weeks, the chronic epilepsy model was confirmed by at least one behavioral spontaneous recurrent motor seizure (SRS). All mice were sacrificed 4 weeks after KA injection. Only mice with observed SRSs during the chronic phase were included in the epilepsy group, and mice that did not exhibit SRSs were used as controls. The intensity of behavioral manifestations was assessed according to a modification of the Racine scale for mice as follows (Wilczynski et al., 2008, Mizoguchi et al., 2011, Yang et al., 2017): Stage 0, no response; Stage 1, -6-

staring and decreased movement; Stage 2, repetitive head and limb locomotions; Stage 3, forelimed clonus and a lordotic posture; Stage 4, sustained rearing with clonus; and Stage 5, generalized tonic-clonic seizures with imbalance or death. Behavioral Study Healthy male CD36-/- mice weighing 24-28 g (n=9) and their corresponding WT C57BL/6J mice (n=10) were subject to the KA-induced model. Animal model was established as described above. During the 4-week period, the occurrence of SRSs and the duration of each SRS in mice were measured by continuous video monitoring for 24 h/day as described previously(Wang et al., 2015, Xiong et al., 2016). Only convulsive stage 4 and 5 seizures were recorded and analyzed. Given that subclinical seizures were typically decribed as mild seizures below stage 4, it is technically difficult to distinguish between these spontaneous episodes and some normal activities of these mice. Thus, we chose this type of assessment to record convulsive motor seizures (i.e., stage 4 and 5 seizures), which is considered to be the most common seizure form in the KA-induced TLE model. These seizures are easily detectable and generally similar to clinical seizures in human patients(Bouilleret et al., 1999, Williams et al., 2009). Only mice with observed SRSs during the chronic phase were included in the final statistical analysis (n=8 in each group). Healthy male CD36-/- mice weighing 24-28 g (n=12) and their corresponding WT C57BL/6J mice (n=12) were subject to the PTZ-kindled model. The animal model was established as described above. Behavioral seizures were observed after each injection, and the highest seizure socre of mice was recorded. Although all mice were included in the final statistical analysis during the PTZ kindling (n=12 in each group), only mice with at least one seizure scored 4 or 5 were used to evaluate group differences of the latency to its first seizure (n=11 in the WT group, and n=9 in the CD36-/- group). Slice Recordings and Analysis We used 6 to 7-week-old male CD36-/- mice (n=6) and their corresponding WT mice (n=6). Coronal slices (30- μm thickness) were obtained using a Vibratome (NVSLM1, Camden Instruments, Loughborough, UK) in a sterile slice solution (containing, in mM: KCl, 2.5; NaH2P04.2H2O, 1.25; MgCl2.H2O, -7-

6; CaCl2, 1; NaHCO3, 26; glucose, 10; and sucrose, 220) at 2 °C. The brain slices were recovered for one hour at room temperature in artificial cerebral spinal fluid (ACSF) saturated with a mixture of 95% O2 and 5% CO2 at pH 7.4 (Wang et al., 2016). To measure the excitability of cells, the whole-cell patch-clamp technique was utilized to measure action potentials (APs) of the pyramidal neurons in the CA1 region as described previously(Wang et al., 2016, Xu et al., 2016). Glass pipettes (3–5 MΩ) were filled with an internal solution (in mM): 60 K2SO4, 60 NMG, 40 HEPES, 4 MgCl2, 0.5 BAPTA, 12 phosphocreatine, 2 Na2ATP and 0.2 Na3GTP (pH 7.2–7.3; 265–270 mOsm). Spontaneous epileptiform discharges were induced by a convulsant bath solution without MgCl2 (Mg2+free ACSF) and were characterized by activity potential manifested as continuous high frequency spike discharges(Sombati and Delorenzo, 1995) A Multi Clamp 700B amplifier (Axon, Sunnyvale, CA, USA) was used to acquiret data, which were filtered at 2 kHz and digitized at 10 kHz. Signals were recorded after electrical activities were stable for at least 5 min using pClamp 9.2 software (Molecular Devices, Sunnyvale, CA, USA). The data collected from the pyramidal neurons of 6 different mice in each group were ultimately analyzed using Mini Analysis 6.0.1 and Clampfit10.3. In vivo Multichannel Electrophysiological Recording After the behavioral observation of the chronic phase of the KA-induced epilepsy model, LFPs analysis was performed as described previously (Maroso et al., 2010, Jiang et al., 2015, Wang et al., 2016). A multichannel microwire array (4 × 4 array of platinum-iridium alloy wire, each with 25 μm diameter, Plexon, Dallas, TX, USA) was implanted into the right dorsal hippocampus (anteroposterior-1.8 mm, mediolateral-1.5 mm, and 1.5 mm below dura). Mice that underwent surgery were allowed to recover for 7 days before LFPs recording. LFPs recording was performed in vivo utilizing OmniPlex® D neural Data Acquisition System (Plexon, Dallas, TX, USA). The LFPs were signals filtered (0.1–1000 Hz), digitized at 4 kHz and preamplified (1000×). NeuroExplorer® v4.0 (Plexon, Dallas, TX, USA) was used for data analysis of LFPs. We continuously recorded the spontaneous paroxysmal events for greater than 60 min and defined the electrophysiological -8-

epileptiform-like discharge as a spike activity lasting longer than 5 s with high frequency (> 5 Hz) and high amplitude (> 2 times the baseline) (JimenezMateos et al., 2012). For each recording session, we determined the frequency and duration of epileptiform-like discharges.The data collected from 7 different mice in each group were ultimately included in the final statistical analysis. Tissue Preparation For western blotting, the collected hippocampal tissues were stored in liquid nitrogen, and then the records were performed on the single sample from each individual mouse. For both models, naive control mice were subjected to similar administration procedures as the treated animals, except for the induction of epileptic seizures. For immunofluorescence labeling, after intracardial perfusion with 50 ml of 0.9% saline and 50 ml of 4% paraformaldehyde, hippocampal tissues were separated immediately and stored in 4% paraformaldehyde followed by 20% sucrose in PBS and 30% sucrose solution. Then, samples were cut into 10-μm slices. Western Blotting Western blotting was performed as previously described(Wang et al., 2016). Total protein was extracted according to the manufacturer's protocol (Keygen Biotech, China). Protein concentrations were determined via the Enhanced BCA Protein Assay Kit (Beyotime, Haimen, China). Extracts were resolved by SDS-PAGE (5% spacer gel; 10% separating gel) electrophoresis and then subjected to western blot analysis. The primary antibodies included rabbit anti-CD36 (1:200, Proteintech, Chicago, USA) and mouse anti-GAPDH (1:5000, Sigma, St. Louis, USA). The secondary antibody was horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000, Zhongshan, Beijing, China). The enhanced ECL chemiluminescence system (Beyotime, Haimen, China) was utilized to detect the protein bands. Then, the resultant OD values were quantified via Quantity One software (Bio-Rad Laboratories, USA). Immunofluorescence labeling Immunofluorescence labeling was performed as previously described(Xiong et al., 2016). Briefly, tissue sections were incubated in the primary antibody mixture. On the second day, sections were incubated in secondary antibodies. -9-

We removed the uncombined primary or secondary antibodies by washing the sections with PBS. The primary antibodies included rabbit anti-CD36 (1:200, Proteintech, Chicago, USA) and mouse anti-Map2 (1:200, Abcam, Cambridge, UK). The secondary antibodies included Alexa Fluor-488 goat anti-rabbit IgG (1:100, Boster Biological Technology, China) and Alexa Fluor-549 goat antimouse (1:100, Zhongshan, Wuhan, China). Laser scanning confocal microscopy was used to detect immunoreactivity. Statistical Analysis All data are expressed as the means±SEM. All statistical analyses were conducted using the statistical software SPSS 19.0. Based on the whether the samples exhibited normal distributions and equal variances (determined by Kolmogorov–Smirnov one-sample and Levene’s test), the experimental results were statistically assessed using parametric or nonparametric tests. Group differences in the mean seizure score during PTZ kindling were evaluated with repeated measures ANOVA (rm-ANOVA) with the withinsubjects factor “Injections” (T1-T15) and the between-subjects factor “Group” (WT versus CD36-/-). When the results of Mauchly's test were violating the sphericity assumption in the rm-ANOVAs, parameter ε was used to adjust the numerator and denominator degrees of freedom. Pairwise comparisons of the the between-subjects factor were performed using Least Significant Difference (LSD) post-hoc analysis. In the pairwise comparisons among the different levels of within-subject factors, considering that using the Bonferroni correction method may lead to conservative statistical results due to excessive times of comparisons, the significance level α‘ was appropriately adjusted to 0.01 and corrected for multiple comparisons. Group differences in the number of seizures and the mean duration of SRSs during the chronic phase of KA-induced epilepsy model were measured using two-way ANOVA with factors “Week” (Week 3 and Week 4) and “Group” (WT versus CD36-/-). χ2-test was performed for calculation of the level of significance in the percentage of seizures during PTZ kindling. For other experimental data were analyzed using Student’s t-test for two-group comparisons. Differences were considered to be significant for values of P<0.05. RESULTS - 10 -

CD36 Expression in PTZ-induced Kindling and KA-induced Epileptic Mice To test whether epilepsy conditions affect CD36 expression, we measured CD36 protein by western blot analysis in fully kindled epileptic mice induced by PTZ. In the present study, we demonstrated that the expression levels of CD36 were significantly increased in the hippocampus (1.04±0.06 in the epilepsy group and 0.51±0.12 in the controls, t=3.99, df=10, P=0.0026 < 0.01; Fig. 1a,b). This area is often associated with epileptic activity in both humans and animal models of epilepsy(Morimoto et al., 2004). In addition, to determine whether CD36 overexpression is a general phenomenon in epilepsy, we employed another widely used epilepsy model. KA-induced epileptic model is recognized as a classical TLE model in academia. Intrahippocampal injections of KA in rodent animals resulted in recurrent epileptic seizures occurring after a latent period, and these recurrent seizures persist for variable periods(Bouilleret et al., 1999, Riban et al., 2002). In accordance with the results of PTZ-kindled epileptogenic tissues, we discovered similar results in the hippocampus of KA-induced epileptic mice (1.06±0.10 in the epilepsy group and 0.46±0.12 in the control group, t=3.90, df=10, P=0.0030 < 0.01; Fig. 1c,d). In the brain, CD36 is mainly expressed in most brain cell types, but its expression in neurons is not well established. In the present study, immunofluorescence labeling of the hippocampus in two chronic models of epilepsy revealed that CD36 was expressed in the neurons (Fig. 2a,b). In addition, staining for the brain slices from CD36-/- mice demonstrated the lack of CD36 protein in the hippocampus of CD36-/- mice and confirmed the specificity of antibodes used (Fig. 3). Effect of CD36 Deficiency on Behavior in Two Mouse Models To examine whether deletion of the CD36 gene may have an effect on epilepsy, we investigated the KA-induced epilepsy model. Generally, this chronic epilepsy model is successfully established in 2-3 weeks after KA injections(Sada et al., 2015). Depending on the observation of behavioral manifestations, we found that the differences in the latent period of SRSs between the CD36-/- group and WT control group were statistically significant - 11 -

(9.75±1.19 days in the WT control group and 13.68±1.31 days in the CD36-/group, t=2.19, df=14, P=0.046 < 0.05, Fig. 4a). After the latent period, CD36-/mice observed decreased number of days of SRSs compared to the WT control group (5.63±0.53 versus 7.38±0.53 days, t=2.32, df=14, P=0.035 < 0.05, Fig. 4b). Additionally, CD36 knockout mice showed significantly reduced SRSs times (two-way ANOVA Group effect: F=23.27, df=1, P=4.48×10-5 < 0.001; Week effect: F=3.32, df=1, P=0.079; Fig. 4c) and mean duration of SRSs (two-way ANOVA Group effect: F=18.25, df=1, P=2.02×10-4 < 0.001; Week effect: F=0.39, df=1, P=0.54; Fig. 4d) after KA injections compared with the WT controls. Then, to clearly determine the effect of CD36 deficiency on epilepsy, we verified the results in the PTZ kindling model. The PTZ-kindled model has the advantages of progressive pathological response and epileptic activity (Bertram, 2007, Zhu et al., 2016). In the chronic epilepsy model, mice were treated with repeated and intermittent intraperitoneal administrations of a subconvulsive dose of PTZ for 30 days. During the course of the experiment, the mice exhibited progressively increasing seizure scores from almost no observable convulsive behavior (Fig. 5a). When we performed PTZ kindling on CD36 knockout mice (n=12) and WT controls (n=12), the first convulsive seizures were both observed after the second injection of PTZ, but CD36 knockout mice exhibited lower intensity of seizures in subsequent evaluation periods compared with the control group (rm-ANOVA Group effect: F=5.69, df=1, P=0.0261 < 0.05; Injections × Group interaction: F=1.66, df=3.47, P=0.176; Injections effect: F=55.02, df=3.47, P=2.22×10-20; Fig. 5a). In addition, the CD36-/- group exhibited prolonged latency to the first seizure score > 3 (12.18±1.54 days in the WT control group and 18.44±2.69 days in the CD36-/- group, t=2.12, df=18, P=0.0482 < 0.05, Fig. 5b) and a reduced number of mice with seizure score > 3 (Fig. 5c). Moreover, the CD36-/- group had a markedly less percentage of stage 4-5 seizures compared with the WT control group (WT: 60.6% versus CD36-/-: 33.9% stage 4-5, χ2=25.68, df=1, P=4.03×10-7 < 0.001, Fig. 5d), demonstrating that CD36 deficiency attenuated the severity of seizures. CD36 Deficiency Inhibits Neuronal Hyperexcitability in Epilepsy - 12 -

To examine whether CD36 deficiency suppresses behavioral activities in chronic epilepsy models and inhibits hyperexcitation in an individual neuron, the whole-cell patch-clamp technique was used to measure APs in hippocampal CA1. In the present study, hippocampal slices were perfused with Mg2+-free ACSF, and we recorded the epileptiform spikes until data were stable for at least 5 min. The typical image of the APs in pyramidal neurons in the CD36-/- group and WT control group are presented in Fig. 6a. The hippocampal slices of WT mice exhibited a rapid firing of APs, whereas slices of CD36 knockout mice exhibited significantly decreased frequencies of APs (2.45±0.51 versus 4.32±0.28 Hz, t=3.22, df=10, P=0.0091 < 0.01; Fig. 6b). Next, an in vivo multichannel electrophysiological recording was used to test whether CD36 deficiency can mitigate the hyperexcitable state in the epileptic brain. LFPs recordings were performed in the KA mouse model during the chronic phase of epilepsy. In all animals, recordings began at least 4 weeks after KA. The typical changes of LFPs in CD36-/- and WT mice are presented in Fig. 6c. We observed that CD36 knockout mice presented decreased frequency of epileptiform-like discharges (10.57±1.13 versus 17.29±1.17, t=4.13, df=12, P=0.0014 < 0.01, Fig. 6d) and reduced total duration of epileptiform-like discharges (346.89±47.23 versus 818.54 ± 101.17 s, t=4.22, df=12, P=0.0012 < 0.01, Fig. 6e). The results observed for LFPs in the KAinduced chronic epilepsy model further indicated that CD36 deficiency could exert an antiepileptic action. DISCUSSION

The principal finding of this study is that CD36 is implicated in epilepsy, and CD36 deficiency has an anti-epilepsy function. First, epilepsy conditions increased the expression of CD36 in the hippocampus. Second, behavioral assays in KA-induced and PTZ-kindled chronic epilepsy models demonstrated that CD36 deficiency alleviated epileptic seizures severity. Third, the electrophysiological index using the whole-cell patch-clamp technique demonstrated that CD36 deficiency inhibits neuronal firing of APs. In addition, the effects of CD36 deficiency on LFPs included a decreased frequency and duration of epileptiform-like discharges in a KA-induced mouse model. This is - 13 -

the first evidence reporting the involvement of CD36 in epilepsy. In the present study, we found that CD36 was located in the neurons in the epileptic hippocampus and that the expression of CD36 is increased in two chronic models of epilepsy. CD36 expression is primarily activated by the transcription factor peroxisome proliferator-activated receptor γ (PPARγ) and the nuclear factor erythroid-derived 2-like factor 2 (Nrf2)(Aubouy et al., 2015). Previous studies indicate that PPARγ and Nrf2 were implicated in mechanisms of epilepsy, and their expression significantly increased in the hippocampus of epileptic animals(Chuang et al., 2012, Wang et al., 2013). These evidence supported that CD36 expression might increase in epilepsy conditions. In addition, it is also possible that the presence of excess CD36 ligands; including amyloid-β peptide (Aβ); thrombospondin-1; and apoptotic cells, which are elevated in epilepsy; is likely to increase the expression of CD36, as CD36 expression occurs in a feed-forward manner(Bengzon et al., 1997, Born, 2015, Mendus et al., 2015). Although it remains unclear which mechanism causes the abnormal expression of CD36 in epileptic brain tissues, the results of our study suggest that CD36 may be involved in epilepsy. In our study, behavioral observations were performed between CD36 knockout mice and WT control mice. We found that CD36 deficiency retarded the progression of chronic epilepsy and decreased the severity of seizure in two chronic models of epilepsy. These findings demonstrate that upregulation of CD36 is not only a concomitant phenomenon of epilepsy, and CD36 deficiency may exert an attenuated action on epileptic seizures. The beneficial effect of CD36 deficiency in epilepsy has been indicated in various previous studies. AD is a neurodegenerative disorder and increases the risk for developing seizures and epilepsy. A high co-occurrence of AD and seizures suggests that they may share common pathological mechanisms(Born, 2015). Aβ is a possible link between AD and seizures. Numerous studies have demonstrated that intraneuronal accumulation of Aβ not only plays a primary role in the etiopathogenesis of AD but also induces hyperexcitation in individual neurons and neural circuits, possibly resulting in seizures(Costa et al., 2016). CD36 is the key player in the binding and internalization of Aβ by - 14 -

neuron cells(Testa et al., 2012). In microglia, the upregulation of CD36 can promote Aβ clearance from the brain parenchyma(Yamanaka et al., 2012). Additionally, several lines of evidence suggest the importance of arachidonic acid (AA) metabolism in the brain and that its dysregulation may be involved in neurologic disorders(Farooqui et al., 2007). Increased concentrations of AA and

its metabolites can enhance

neuronal excitability

and

seizure

activity(Basselin et al., 2003). CD36 plays an important role in the pathogenesis of PD by regulating the levels of AA, and CD36 can facilitate the release of AA by activating membrane calcium channels and cytosolic phospholipase A2(Kim et al., 2011, Kuda et al., 2011). Thus, CD36 deficiency may play an antiepileptic role by alleviating the “neurotoxicity” of these toxic and metabolic factors. Neuronal hyperexcitability is identified as a critical pathological basis of epileptic seizures(Staley, 2015). The possibility that CD36 may regulate neuronal excitability has been indicated by a few studies. Upon interaction with long-chain fatty acids, CD36 induces phosphorylation of protein tyrosine kinases, resulting in recruitment of calcium followed by activation of the calcium channels and membrane depolarization. Thus, neuronal electrical activity is ultimately altered(Le Foll et al., 2009, Dadak et al., 2017). A study also found that OR67d neurons expressing CD36 exhibit obvious increases in spiking frequency after drug stimulation(Gomez-Diaz et al., 2016). In our study, to test the excitability of neurons in CA1 of CD36-/- and WT mice, a Mg2+-free epilepsy model in hippocampal slices was prepared by the whole-cell patchclamp technique for electrophysiological evaluation in vitro. We found that the hippocampal slices of CD36-/- mice showed a decrease in APs frequency compared with WT mice. However, the whole-cell patch-clamp technique can only monitor the intracellular electrical activity of a single neuron, and clinical epileptic seizures are classically characterized by the abnormal electrical activity generated by massive neuronal hypersynchrony in epileptic neural networks(Wu et al., 2013, Truccolo et al., 2014). The LFPs are thought to be complementary to action potential information and reflect synchronized activity in a population of local neurons and their inputs(Weinberger et al., 2006, Pogosyan et al., 2010, Lempka and McIntyre, 2013). Thus, an in vivo - 15 -

multichannel electrophysiological recording was further used to test the effect of CD36 deficiency on LFPs in the hippocampus of KA-induced epileptic mice. Our study revealed a reduced duration and frequency of epileptiform-like discharges in the CD36-/- group. These results reveal that CD36 deficiency may have a role in regulating neuronal excitability and further influencing massive neuronal electrical activity. Given that the primary aim of this study is to investigate whether CD36 affects neuronal hyperexcitability in epilepsy and plays a role in seizure activity, we did not explore the concrete mechanism of CD36 in epilepsy. However, from a pathophysiological standpoint, inflammation may be the most likely potential mechanism for CD36 deficiency to produce anti-epileptic effects. Inflammatory and immune reactions are implicated in the pathogenesis of seizures and epilepsy(Vezzani and Granata, 2005). The proinflammatory cytokine IL-1β can produce powerful proconvulsant functions via a myeloid differentiation factor-88 (MyD88)-dependent signaling pathway in neurons, resulting in the phosphorylation of NMDA receptor-2B (NR2B) and increased NMDAdependent calcium influx. Thus, the neuronal excitability is increased, and ictiogenesis is facilitated(Viviani et al., 2003, Balosso et al., 2008). In addition, Toll-like receptor (TLR), a key receptor of innate immunity, is involved in seizure activity and epileptogenesis by mediating inflammatory reaction(Matin et al., 2015). For CD36, several lines of evidence suggest that upon binding with ligand, CD36 induces the recruitment of TLR heterodimers, which subsequently activate the MyD88-dependent signaling cascades. This action ultimately results in the production of reactive oxygen species, the activation of nuclear factor Kappa B, and the release of pro-inflammatory cytokines. This inflammatory process is associated with several vascular and neurological disorders, and the inflammatory response of CD36-deficient patients or CD36null mice was significantly suppressed in these diseases(Janabi et al., 2000, Abe et al., 2010, Stewart et al., 2010). In conclusion, the present study provided direct evidence that CD36 is overexpressed in drug-induced epilepsy mice models, and CD36 deficiency could produce an anti-epileptic effect and inhibit neuronal hyperexcitability. These data might offer a novel intervention strategy for epileptic seizures. - 16 -

However, further studies are needed to thoroughly clarify the concrete mechanism of CD36 in epilepsy.

Conflict of interest The authors declare no conflict of interest.

Acknowledgments This work was supported by the National Clinical Specialty Construction Foundation of China and National Natural Science Foundation of China (Nos. 81271445, 81471319, 81301109 and 81701279). We are sincerely grateful for Dr. Maria Febbraio, Lerner Research Institute, USA and Dr. Xiongzhong Ruan, Chongqing Key Laboratory of Metabolism on Lipid and Glucose Chongqing Medical University, Chongqing, China for donating CD36-/- mice.

Author Contributions FS.Z. and XF.W. conceived and designed the experiments. Q.Y., YK.Z., F.L., Y.L. and SS.L. performed the experiments and statistical analyses. X.X., Y.Y., X.T. and XF.W. analyzed and collected the data. FS.Z. wrote the manuscript. All authors contributed to preparation of the manuscript and approved the final contributions.

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Figure Captions Fig. 1 CD36 expression levels were increased in two chronic epilepsy models. (a,c). Western blots demonstrating CD36 protein levels in hippocampi from control group or mice with epilepsy in the PTZ kindling model (a) and KAinduced model (c). (b,d). Bar graphs depicting quantification of the hippocampal CD36 in the PTZ kindling model (b) and KA-induced model (d) (**P<0.01, n=6 in each group). Student's t-tests were performed. KA: kainic acid; PTZ: pentylenetetrazol.

Fig. 2 Immunofluorescence of CD36 protein in the hippocampus of mice with chronic epilepsy. (a,b). CD36 (green) was co-expressed with the neuron marker (red) in hippocampus of mice with chronic epilepsy induced by PTZ (a) and KA (b). The white squares show the higher magnification images of positive cells. KA: kainic acid; PTZ: pentylenetetrazol.

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Fig. 3 CD36 labeling in CD36-null mice Representative immunofluorescent images show the lack of CD36 immunoreactivities (green) in CA1 and CA3 stratum lucidum of CD36-null mice. The MAP2 (red) indicates the immunoreactivities of neurons.

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Fig. 4 Effect of CD36 deficiency on the development of epilepsy in the KA-induced chronic epilepsy model. (a). CD36-/- mice exhibited a significantly delayed latent time of SRSs (*P<0.05, n=8 in each group). Student's t-tests were performed. (b). The number of days of SRSs appeared in CD36-/- mice decreased significantly (*P<0.05, n=8). Student's t-tests were performed. (c). CD36-/- mice exhibited significantly reduced SRSs from weeks 3 to 4 post KA injection (***P<0.001, n=8 in each group). Two-way ANOVA was performed. (d). CD36-/- mice exhibiting a significantly reduced mean duration of SRSs from weeks 3 to 4 post KA injection (***P<0.001, n=8 in each group). Twoway ANOVA was performed. KA: kainic acid; SRSs: spontaneous recurrent motor seizures; WT: wild type.

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Fig. 5 Effect of CD36 deficiency on development of epilepsy in the PTZkindled chronic epilepsy model. (a). Mean seizure scores of PTZ-kindled WT and CD36-/- mice during the course of the experiment. Lower seizure scores were noted in CD36 -/- mice (F(1,22) = 55.02, *P < 0.05, n=12 in each group). Repeated measures analysis of variance (ANOVA) was performed. (b). CD36-/- mice exhibited significantly prolonged latency to the first seizure scored > 4 (*P<0.05, n=11 in the WT group, and n=9 in the CD36 -/- group). Student's t-tests were performed. (c). Number of mice with seizures scored > 4 during each day of testing. (d). The percentage of stage 0-3 seizures and stage 4-5 seizures during PTZkindling. (***P<0.001, n=12). χ2-test was performed. PTZ: pentylenetetrazol.

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Fig. 6 Effect of CD36 deficiency on neuronal hyperexcitability. (a). Representative traces of APs of pyramidal neurons from the CD36-/- group and WT control group. (b). Changes in APs frequency between the CD36-/- group and WT control group (**P<0.01, n=6 in each group). Student's t-tests were performed. (c) Typical trace of LFPs on CD36-/- and WT mice. (d) CD36-/- mice exhibited significantly decreased frequency of epileptiformlike discharges compared with WT mice (**P<0.01, n=7 in each group). (e) The duration of epileptiform-like discharges was reduced in CD36-/- mice in the KA-induced chronic epilepsy model compared with WT control. (**P<0.01, n=7 in each group). Student's t-tests were performed. APs: action potentials; LFPs: local field potentials; KA: kainic acid; WT: wild type.

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Highlights •

The expression of CD36 is increased in epileptic mice.

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The brain slices from epileptic mice showed that CD36 is co-expressed with neurons in hippocampus.

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CD36-/- mice showed a attenuated progression of chronic epilepsy

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CD36 deficiency inhibits neuronal firing of APs in the hippocampal slices

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CD36 deficiency on LFPs exhibits attenuated massive neuronal electrical

activity

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