Introduction

It is widely known that the excitotoxic process is a contributing factor to acute neuronal damage in most chronic neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease and Huntington’s disease [1–3]. Previous reports have demonstrated that the administration of kainic acid (KA) in rodents, an analogue of the excitotoxin glutamate, produces epileptic seizures [4–9]. The greatest abundance of kainate receptors is present in the hippocampal CA3 region, and their activation results in neuronal loss of selective populations in the hippocampus and other brain structures. Moreover, numerous studies have demonstrated that neuronal loss observed in this excitotoxic model involves, at least in part, apoptotic cell death [10, 11]. Likewise, KA can stimulate CA3 neurons directly through stimulation of their own KA receptors and also indirectly by favouring glutamate release secondary to KA stimulation of mossy fibres [4].

One important point is the characterisation of the mechanism involved in KA-induced neurodegeneration. Thus, a large number of studies have been conducted to identify the potential biochemical pathways involved in KA-induced apoptosis, such as oxidative stress, cell cycle re-entry and calpain/cdk5 activation [8, 10–16]. Programmed cell death is primarily mediated by the extrinsic or death receptor pathway, and the intrinsic or mitochondrial pathway, which converge to activate mitochondria [17, 18]. This constitutes a key point because it favours the release of some proteins such as cytochrome c, Smac/Diablo and Omi/HrtA2 into the cytosol, which facilitates the activation of common executioner caspases (caspase-3/7) with the final cleavage of downstream targets that include DNA repair enzymes [19].

It has been previously shown that the extrinsic apoptotic pathway induces apoptosis in lymphoid cells and in neuronal cells deprived of critical survival factors and also in models of excitotoxicity [20]. The Fas receptor is known to induce apoptosis by binding to FasL. Thus, the Fas ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL) are up-regulated following experimental KA administration to rodents [21–23]. In addition, previous studies have shown that the binding of the Fas ligand to FasR triggers the formation of a death-inducing signalling complex (DISC), which consists of FasL, FasR, the adapter protein FADD (Fas-associated death domain protein), and the TNFR1-associated death domain protein (TRADD) to induce the activation of caspase-8, a protease which then activates downstream effector caspase-3, which in turn is responsible for apoptosis. Thus, both extrinsic and intrinsic apoptotic pathways converge to activate common executioner caspases (caspase-3/7) with the final cleavage of downstream targets. Inhibition of Fas-mediated apoptosis is regulated by the FLICE-inhibitory protein (FLIP), the structural homologue of procaspase-8 [24–29].

lpr mice have alterations in the Fas receptor gene due to the insertion of a retrotransposon that causes disruption in its transcription [30]. Attenuation of the Fas signalling system has been related to neurological damage protection in various experimental models of pathophysiological diseases such as ischemia and multiple sclerosis [27, 29–33]. More precisely, lpr mice were protected from the neurotoxicity mediated by the neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), which causes permanent symptoms of Parkinson’s disease [34, 35]. Interestingly, in post-mortem brains of Parkinson’s patients, the levels of Fas are increased in the nigrostriatal region [36]. In addition, deletions of the Fas receptor reduced lesion size and improved brain function after a traumatic brain injury, confirming the role of these signalling pathways in the pathogenesis of delayed brain damage [7, 25]. All these data raised the possibility that Fas signalling activated in different pathologies may render neurons vulnerable to dying [31, 37].

Furthermore, recent studies have specifically exemplified the role of Fas in Alzheimer’s disease (AD) neurodegeneration [28, 38–40]. It has been suggested that β-amyloid can induce neuronal death via processes of apoptosis [40, 41]. Similarly, it has been demonstrated that neurons in the AD brain and neuronal cultures treated with beta-amyloid show a significant increase in Fas ligand expression and immunoreactivity [42]. Moreover, cortical cultures of lpr mice that carry mutations leading to a loss of function of Fas genes are protected from beta-amyloid toxicity. These results, therefore, lend support to the involvement of this pathway in AD apoptosis.

Given the lack of direct evidence linking FasR with the intrinsic apoptotic pathway, we sought to clarify the role of apoptosis mediated by Fas signalling after KA treatment. The present study examined the impact of KA administration in C57BL/6 Fas-deficient (lpr) mice, which exhibit low levels of Fas receptor expression [43, 44]. To investigate whether FasL signalling is also relevant to the neurotoxic process mediated by KA, we analysed the hippocampal apoptotic cell death after intraperitoneal KA administration in Fas receptor-deficient mice (lpr mice). Moreover, a study of apoptotic gene expression was performed in the hippocampus and here we demonstrated that loss of Fas receptor activity (lpr mice) is neuroprotective against excitotoxicity.

Material and Methods

Animals

Wild-type C57BL/6 mice were obtained from Harlan Laboratories or the Jackson Laboratory. Fas-deficient lpr mice (C57BL/6 background) were obtained from Jackson Laboratory (B6.MRL-FasLpr/J, stock number 000482). All mice were housed at the Centro Biología Molecular Severo Ochoa in Madrid. Mice were housed four per cage with food and water available ad libitum and maintained in a temperature-controlled environment on a 12-h light/12-h dark cycle with light onset at 7 am. All experiments were performed under a protocol approved by the CSIC ethical committee.

Kainic Acid Treatment and Sample Preparation

A total of 40 mice were used for the present study. Six-week-old mice were treated with a single intraperitoneal (i.p.) dose (30 mg/kg) of KA (Sigma-Aldrich, St. Louis, MO, USA) or with saline solution for controls. For RNA analysis, 10 wild-type C57BL/6 mice and 10 lpr mice were used. Five mice from each group were used as controls and were treated with KA and killed after 12 h; the hippocampus was then excised and frozen. For immunochemistry and immunofluorescence experiments, we used 10 wild-type C57BL/6 mice and 10 lpr mice. Five from each group served as control and were injected with KA and killed after 24 h.

All mice were anaesthetised by injection (i.p.) of pentobarbital 80 mg/kg. After that, the mice were perfused with 40 g/L paraformaldehyde in 0.1 mol/L phosphate buffer (PB) and the brains were removed and post-fixed for 24 h. The brains were subsequently rinsed in the same solution with 300 g/L sucrose for 24 h and then frozen. Coronal sections of 30 μm were obtained by a Cryostat (Leica Microsystems, Wetzlar, Germany).

RT2 Profiler™ PCR Array

The Mouse Apoptosis RT2 Profiler PCR Array (PAMM-012A, Biosciences, Frederick, MD, USA) profiles the expression of 84 genes involved in apoptosis together with five housekeeping genes. Controls for genomic DNA contamination and for the efficiency of the RT-PCR and PCR were also included in the array and were evaluated after each run. One microgram of total RNA was reverse transcribed in a final reaction mix of 20 μL using the RT2 First Strand Kit according to the manufacturer’s instructions. For one 96-well plate of the PCR array, 1,100 μL of PCR master mix containing 2×SuperArray RT2 qPCR Master Mix and 102 μL of diluted cDNA were prepared, and aliquots of 10 μL were added to each well. The PCR was performed on the LightCycler 480 (Roche Applied Science, Manheim, Germany), and universal cycling conditions (10 min at 95 °C, 15 s at 95 °C, 1 min at 60 °C for 40 cycles) were used. Expression of each gene was normalised to the mean Ct for five housekeeping genes in the PCR array (β-glucuronidase, hypoxanthine phosphoribosyltransferase, heat shock protein 90α, glyceraldehyde-3-phosphate dehydrogenase and β-actin). Relative fold change in expression was calculated using the ΔΔCt method, and the values were expressed as 2-ΔΔCt. The statistical calculation was based on the web-based program of RT2 Profiler™ PCR Array Data Analysis. Significant changes in gene expression between KA-treated mice and untreated controls were assumed as a fold change ≥1.5 and ≤0.6, with a P value < 0.05. The experiments were repeated three times.

RNA Extraction and Real-Time Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)

Total RNA was isolated from mouse hippocampus using TRIzol (Invitrogen, Carlsbad, CA, USA) followed by chloroform, according to the manufacturer’s protocol. RNA concentration was measured using a NanoDrop™ 1000 Spectrophotometer (Thermo Scientific, MA, USA).

As a general procedure, 1 μg of total RNA was reverse transcribed using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Carlsbad, CA, USA). The same amounts of cDNA were subsequently used for quantitative real-time PCR using SYBR Green® PCR Master Mix and performed on the StepOnePlus™ Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA). All samples were run in triplicate, and expression values were normalised to the housekeeping β-actin in the same reaction. Relative normalised mRNA levels were calculated using the ΔΔCt method. Primers used for quantitative real-time PCR are shown in Table 1. Gene-specific primers corresponding to the PCR targets on the PCR RT2 array were designed using Primer Express® Software v2.0 (Applied Biosystems, Carlsbad, CA, USA).

Table 1 Significant changes in transcriptional activity of genes included in the Mouse Apoptosis RT2 Profiler PCR Array, PAMM-012A, and from the hippocampus of KA-treated C57/Bl6 wild-type mice, at 12 h. The administration of this neurotoxin caused significant changes in the expression of 23 genes: 17 genes resulted significantly up-regulated; the most significant up-regulation corresponded to the tnf gene that showed a fold change of 15.95, and the lowest significant gene activation levels corresponded to hells, with a fold change of 1.5. Another six genes resulted down-regulated. Significant changes in gene expression between KA-treated mice and untreated controls were assumed as a fold change ≥1.5 and ≤0.6, with a P value < 0.05. The experiments were repeated three times
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Fluoro-Jade Staining

Neurodegeneration was assessed using Fluoro-Jade B (Chemicon Europe Ltd). Slides were rinsed with phosphate-buffered saline (PBS), followed by two washes in distilled water. Afterwards, slides were immersed in 0.6 g/L potassium permanganate (KMnO4) for 15 min in the dark. Then, after two washes in distilled water, the slides were transferred to the staining solution containing 0.1 mL/L acetic acid and 0.004 mL/L of the fluorochrome Fluoro-Jade B for 30 min in the dark. Slides were rinsed in distilled water, dried, and then submerged directly into xylene and mounted in DPX medium. Slides were analysed using epifluorescence microscopy (Olympus BX61, Olympus, Barcelona, Spain). To determine the positive cell number for Fluoro-Jade B, we counted the cells in each area from serial sections of hippocampus from each mouse brain.

TUNEL Assay

DNA damage was assessed using terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining method. TUNEL staining was performed as described in the In situ Cell Death Detection Kit, TMR red (Roche Molecular Biochemicals, IN, USA) with minor modifications. Sections were rinsed with PBS and immersed in a permeabilisation solution (0.1 % Triton X-100, 0.1 % sodium citrate) for 2 min at 4 °C. Then, slides were washed twice with PBS and incubated with TUNEL reaction mixture in a humidified atmosphere for 60 min at 37 °C in the dark. Afterwards, slides were rinsed three times with PBS and then incubated with 0.1 μg/mL Hoechst 33258 (Sigma-Aldrich, St Louis, MO, USA) nuclear stain for 5 min in the dark. Following a final rinse with PBS, slides were mounted in Fluoromount medium (Sigma-Aldrich, St Louis, MO, USA) and analysed under a confocal fluorescence microscope (Leica TCS SP2 confocal microscope).

Immunohistochemistry

Free-floating coronal sections, 30 μm thick, were rinsed in 0.1 mol/L PB pH 7.2 and treated with 5 mL/L H2O2 and 100 mL/L methanol in PBS. After that, they were pre-incubated in a blocking solution (100 mL/L of foetal bovine serum (FBS), 2.5 g/L of bovine serum albumin and 0.2 mol/L of glycine in PBST, PBS with 5 mL/L of Triton X-100). Then, sections were incubated overnight (O/N) at 4 °C with different primary antibodies: rabbit anti-GFAP (1:2,000; Dako, Glostrup, Denmark) and rabbit anti-Iba1 (1:1,000; Wako, Osaka, Japan), and sequentially incubated for 2 h with Alexa Fluor 594 goat anti-rabbit antibody (1:500; Invitrogen, Eugene, OR, USA). Sections were counterstained with 0.1 μg/mL Hoechst 33258 (Sigma-Aldrich, St Louis, MO, USA) nuclear stain for 5 min in the dark. Immediately, sections were rinsed with PBS and mounted onto gelatinised slides with Fluoromount medium (Sigma-Aldrich, St Louis, MO, USA). Stained sections were examined under a confocal fluorescence microscope (Leica TCS SP2 confocal microscope).

Statistical Analysis

All experiments were conducted in triplicate, and the results were expressed as mean values ± SEM. Differences are considered significant at P < 0.05. P values were determined by Student’s t test.

Results

Identification of the Expression of Apoptotic Genes in the Hippocampus of Wild-Type Mice, After Treatment with Kainic Acid

The administration of KA at a dose of 30 mg/kg in mice favours the onset of seizures and a variety of behavioural changes that have been previously described by a number of laboratories [7–9, 45]. The lpr mice compared to the wild type did not show an improvement in the number of seizures.

To assess whether apoptosis-related genes showed changes in expression in KA-treated mice at 12 h, 84 genes were analysed using the real-time PCR array (Mouse Apoptosis RT2 Profiler PCR Array). The results indicated that administration of this neurotoxin caused significant changes in the expression of 23 genes (Table 1). Among them, a total of 17 genes were significantly up-regulated, whereas the transcriptional activity of six genes was below normal levels. The most significant up-regulation corresponded to the tnf, which showed a fold change of 15, 95 with P value = 0.0017 and the lowest significant gene activation levels corresponded to Hells, with a fold change of 1.5 with P value = 0.0214. In relation to this observation, the exposure of the wild-type strain to KA toxicity significantly activated genes related to the Fas signalling pathway: fas, fasl, and caspase-4, caspase-3 and caspase-8.

Evaluation of Kainic Acid-Induced Neurotoxicity in the Hippocampus of Fas-Deficient lpr Mice

The real-time PCR array results suggested an involvement of the Fas pathway in the apoptotic process occurring in the hippocampus of C57/Bl6 mice, so the next step was to evaluate the implication of this pathway in KA-induced neuronal death. Then, we decided to analyse the neurodegeneration induced in Fas-deficient lpr mice following KA injection. After 24 h of treatment with KA, the neuronal death in hippocampal areas was assessed using the Fluoro-Jade B technique. Interestingly, brain sections from lpr mice exhibited a significant stain reduction compared to the wild type, suggesting the existence of a neuroprotective effect, most probably due to the lack of a functional Fas/CD95 protein (Fig. 1). To clarify whether the identified dying cells were apoptotic, DNA integrity was assessed using TUNEL staining. A decrease in the cell apoptosis in lpr mice treated with KA compared to the wild type (WT) evidenced that the lack of functionality of the Fas receptor plays an antiapoptotic role (Fig. 2).

Fig. 1
figure 1

Neurodegeneration observed by Fluo-Jade B stain in the CA3 and CA1 regions of the hippocampus after 24 h of KA treatment. A decrease of labelled neurons is observed in the CA3 region of kainic acid (KA)-treated lpr mice versus wild-type (wt) mice (b vs a). A decrease of labelled neurons is observed in the CA1 region of kainic acid (KA)-treated lpr mice versus wild-type (wt) mice (d vs c). Scale bar 100 μm. (e) A bar graph shows the quantification of positive neurons for Fluoro-Jade B labelling in the CA3 and CA1 areas of the hippocampus in wild-type and lpr mice. Each point is the mean ± SEM of three independent experiments, determined in five pictures per experiment (***p < 0.001; with respect to kainic acid-treated wild-type mice)

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Fig. 2
figure 2

TUNEL and Hoechst 33342 staining in the CA3 region of the hippocampus from kainic acid-treated wild-type mice (a and c) and kainic acid-treated lpr mice (b and d), after 24 h of treatment. Scale bar 100 μm

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Evaluation of Glial Activation in Fas-Deficient lpr Mice Hippocampus After Kainic Acid Treatment

In the literature, it is widely described that, in addition to neuronal death, the administration of KA produces a glial activation due to excitotoxicity [4, 5]. This glial reactivity is reflected in a change in the morphology of the astrocytes and microglia. In the case of astrocytes, these acquire a star shape because of the growth of their extensions. In the hippocampal brain areas of the KA-treated wild-type mice, a prominent astrogliosis was observed; thus, we detected an increase in GFAP immunoreactivity as well as shape alterations in astrocytes. The astrocyte activation mediated by KA was not abrogated in lpr mice because the pattern of GFAP immunoreactivity was similar to the wild type (Fig. 3). Microglia are a type of glial cell acting as the first and main form of active immune defence in the central nervous system, offering an extremely sensitive reaction when neural damage is induced. Thus, microglial activation was analysed in wild-type mice and lpr mice after KA treatment, with an Iba1 antibody that allows measurement of the inflammatory response by means of changes in the cell shape of microglial cells, characterised by their amoeboid form. KA treatment evidenced microglial reactivity in the hippocampus, insofar as changes in the morphology of the microglial cells were detected in comparison to saline-treated mice (Fig. 3). Likewise, activated microglial morphology was visualised in lpr mice after KA injection, with the data suggesting that the neuroprotection detected in lpr mice after KA is not mediated by a prevention of glial activation.

Fig. 3
figure 3

Microglial and astroglial activation under kainic acid treatment. Iba-1 immunohistochemistry counterstained with Hoechst 33342 in the CA1 region of the hippocampus from saline control wild-type mice (a and e), kainic acid-treated wild-type mice (b and f), saline control lpr mice (c and g) and kainic acid-treated lpr mice (d and h). GFAP immunohistochemistry counterstained with Hoechst 33342 in the CA1 region of the hippocampus from saline control wild-type mice (i), kainic acid-treated wild-type mice (j), saline control lpr mice (k) and kainic acid-treated lpr mice (l). CA1, CA1 field, hippocampus. Scale bars a–d, 100 μm; e–h, 25 μm; i–l, 100 μm

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Expression of Apoptotic Genes in lpr Mice After Treatment with Kainic Acid

We studied the transcriptional activity of genes involved in the apoptotic response to the exposure to KA in the lpr mice strain (Table 2). As a first approach, we determined the basal expression of genes in the hippocampus of both C57/Bl6 and lpr mice strains (Fig. 4). In fact, results show a clear down-regulation of the fas gene in lpr mice, together with trp53bp2, traf2 and nol3 genes. By contrast, a total of six genes were significantly up-regulated: tnf, casp4, casp8, fasl, cflar and cidea. Contrary to what was observed in the C57/Bl6 wild-type strain, treatment of lpr mice with kainic acid does not produce significant changes in the transcriptional activity of genes related to apoptosis (Table 2). In fact, no significant changes were observed either in the fasl gene or in caspase-4 and caspase-8 or the executioner caspase-3 genes. In addition to the lack of activity in cytoplasmic caspase genes, the cell death-inducing DNA fragmentation factor gene, cidea, showed no significant change in transcriptional activity. Nor were any changes observed in the atf5 gene. Therefore, it can be concluded that Fas signalling is involved in kainic acid-induced apoptosis in hippocampal cells (Fig. 5).

Table 2 Significant changes in transcriptional activity of genes included in the Mouse Apoptosis RT2 Profiler PCR Array, PAMM-012A, from the hippocampus of KA-treated lpr mice, at 12 h. Genes that did not prove to be related, directly or indirectly, to apoptotic processes were excluded from the analysis. By contrast to the observation in the C57/Bl6 wild-type strain, treatment of lpr mice with KA, at 12 h, did not produce significant changes in transcriptional activity of genes related to apoptosis, neither in fas ligand gene fasl, nor in caspase-4 and caspase-8 or the executioner caspase-3 genes. Any changes were also not observed in atf5
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Fig. 4
figure 4

Significant changes in the transcriptional activity of genes included in the Mouse Apoptosis RT2 Profiler PCR Array, PAMM-012A, from the hippocampus of both C57/Bl6 wild-type and lpr mice. In lpr mice, a clear down-regulation of the fas, trp53bp2, traf2 and nol3 genes is observed when compared with C57/Bl6. By contrast, a total of six genes were significantly up-regulated: tnf, casp4, casp8, fasl, cflar and cidea

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Fig. 5
figure 5

Drawing of the 23 genes identified by the Mouse Apoptosis RT2 Profiler PCR Array, PAMM-012A, with a significative differential expression (fold change ≥ ±1.5 and P value 0.05) between KA wild-type treated and untreated mice, at 12 h. Fas/FasL interaction induces caspase-8 and caspase-3 activity. Caspase-3 is a key mediator of apoptosis in neuronal cells. This pathway is blocked in the lpr mice fold change ≥±1.5

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Discussion

In the present paper, we report evidence of a novel role for Fas in the apoptosis of hippocampal neurons after treatment with the neurotoxin KA. To provide a mechanistic basis for these findings, we demonstrated that lpr mice treated with this neurotoxin exhibit a significant reduction in neuronal cell death and apoptosis compared with wild-type mice.

Real-time PCR array analysis showed that KA significantly regulated 23 apoptosis-related genes (fold change ≥ ±1.5 and P value 0.05). It is well established that the extrinsic signalling pathway that initiates apoptosis involves death receptor members of the Tnf receptor gene superfamily [21, 23, 26, 42, 46, 47]. Our study demonstrated that Fas, Tnfr1, Fasl and Tnf genes were highly expressed in the hippocampus following KA treatment in wild-type animals. In addition, our findings also prove an overexpression of the Casp8 gene that encodes an initiator caspase and the Casp3 gene that encodes an effector caspase. However, it is noteworthy that KA treatment at 12 h does not promote significant changes in the expression of key genes involved in the intrinsic apoptotic pathway such as caspase-9 and Apaf1, among others. These data suggest that the cell death observed at 24 h of KA treatment may be due mainly to the activation of genes involved in the extrinsic pathway of apoptosis.

Several reports have demonstrated that the expression of the Fas receptor gene is induced after neuropathological lesions including those produced by KA [23–25, 38, 48]. With the aim of reporting more data in the involvement of the Fas signalling pathway in the mechanisms of neuronal death induced by excitotoxicity, we performed a treatment with KA in lpr mice. Previous studies have described that these mice exhibit neuroprotection against traumatic brain injury (TBI), in preclinical models of Parkinson’s disease (MPTP) and amyotrophic lateral sclerosis (ALS), and also models of ischemia, demonstrating the involvement of the Fas pathway in neuronal death [24, 25, 29, 32, 41, 47, 49]. We observed a significant decrease in neurodegeneration in the hippocampus of the KA-treated lpr mice compared to the wild type, so the results strongly suggest that the lack of Fas receptor functionality plays a neuroprotective role following KA administration.

In the model of KA-induced excitotoxicity, it is widely demonstrated that there is clear astroglial and microglial activation [4, 5, 50]. Here, in the present study, we have also observed glial reactivity after KA treatment in the hippocampus of wild-type mice. In the case of the lpr mice, although there was a significant reduction in neuronal death, the glial reactivity induction was similar to that of wild-type mice, indicating that while the Fas pathway prevents neuronal death, it is not involved in the regulation of neuroinflammatory processes. Likewise, KA-induced glial activation may occur through mechanisms independent of Fas [27].

Because reduced neuronal death was observed in lpr mice following KA treatment, we analysed the differentially expressed apoptosis-related genes detected, comparing wild-type KA-treated mice to untreated mice. For this, first of all, we compared the gene expression levels between the two genotypes (wild type and lpr). The lpr mice showed higher expression levels of genes encoding the death receptor ligands (FasL and TNF): caspase-8 that codifies an essential protease for the extrinsic apoptotic pathway, caspase-4 encoding a caspase that plays a role in inflammation, cflar that codifies an apoptotic regulator protein that lacks caspase activity and appears to be itself cleaved into two peptides by Caspase-8 and the pro-apoptotic cidea gene [42, 51]. Furthermore, a down-regulation of the nol3 (nucleolar protein 3) gene was detected, which encodes the antiapoptotic protein ARC (apoptosis repressor with a caspase recruitment domain) that interacts with Caspase-2 and Caspase-8 and inhibits receptor-mediated apoptosis. In addition, nol3 does not play a key role in lpr neuroprotection, because we did not find an increase in gene expression after lpr mice KA treatment.

A down-regulation was also detected in traf2 encoding TRAF2 protein required for TNF-alpha-mediated activation of MAPK8/JNK and NF-κB and in trp53bp2 that codifies a pro-apoptotic member of a family of p53 binding proteins [52]. All these data indicate that the lpr mice genotype does not respond to a neuroprotective profile.

However, after KA administration, we found that some genes implicated in the death pathway did not show significant changes in expression in lpr mice compared to WT, such as fasl, decreasing the FasR/FasL interaction: caspase-3 and caspase-8, the main caspases involved in the extrinsic apoptotic pathway; caspase-4 involved in inflammatory processes; cidea that codifies a apoptotic protein activator and pim2 that codifies an enzyme serine/threonine kinase that has roles in apoptosis and regulation of signal transduction cascades [18, 25, 52–58]. This fact suggests that the expression of these genes could be regulated by the expression of fas in the model of KA-induced neurotoxicity, indicating that they play an important role in the apoptotic cascade in the excitotoxic process.

In agreement with Torres-Peraza et al. [59], we also observed an increase of atf5 gene expression mediated by KA-induced seizures; interestingly, a neuronal increase in atf5 expression has a neuroprotective role [59]. It has been proposed that the neuroprotective effects of ATF5 are mediated by the transcriptional induction of some proteins with a neuroprotective effect, such as Hsp27, Bcl2 and Mcl1 [58–61]. Likewise, in our study, we found a significant up-regulation in mcl1, which was also up-regulated in other models of status epilepticus, such as pilocarpine administration in rodents [62].

Furthermore, in our study, after KA treatment in lpr mice compared to WT, we observed a non-significant increase in atf5, possibly resulting from the decrease in apoptotic neuronal death that occurs in lpr KA-treated mice. At the same time, other genes showed similar changes in gene expression profiles between the two genotypes, including bag3, mcl1, traf2 and bcl10. Interestingly, these genes encode proteins that confer antiapoptotic neuroprotection, which could account for the increase in expression in both WT and lpr mice after KA treatment [62]. Therefore, our results suggest neuroprotective effects in lpr mice after KA administration based on genes that are not activated in this model. Furthermore, these genes could be considered as potential therapeutic drug targets in neurodegenerative diseases. In this line, recent data have demonstrated that a mouse with neuronal caspase-8 deficiency was protected against neuronal injury caused by KA-induced excitotoxicity [63]. The apoptotic prevention observed in lpr mice after KA administration may be at least partly attributable to a decrease in caspase-8, which is also involved in caspase-3 activation. At the same time, the differential gene expression observed in some apoptosis-related genes between untreated genotypes should be intensively studied to determine their functional significance in the process of neuroprotection observed in mice deficient in the Fas receptor.

In conclusion, in the present work, we report evidence of the key role played by the Fas/FasL-mediating apoptosis of neurons in contributing to the process of KA-induced neurodegeneration and affecting neuronal function and survival (Fig. 5). Interestingly, FasR-deficient mice exhibit significantly reduced cell death, although inflammatory cells (microglia/macrophages) and the level of GFAP when compared with the wild type were unchanged. Thus, we propose that targeting the Fas receptor could provide a compelling rationale for therapeutic strategies against neurodegeneration but not against neuroinflammation.