Epilepsy: neuroinflammation, neurodegeneration, and APOE genotype
© Aboud et al.; licensee BioMed Central Ltd. 2013
Received: 3 May 2013
Accepted: 28 June 2013
Published: 29 July 2013
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© Aboud et al.; licensee BioMed Central Ltd. 2013
Received: 3 May 2013
Accepted: 28 June 2013
Published: 29 July 2013
Precocious development of Alzheimer-type neuropathological changes in epilepsy patients, especially in APOE ϵ4,4 carriers is well known, but not the ways in which other APOE allelic combinations influence this outcome. Frozen and paraffin-embedded tissue samples resected from superior temporal lobes of 92 patients undergoing temporal lobectomies as a treatment for medication-resistant temporal lobe epilepsy were used in this study. To determine if epilepsy-related changes reflect those in another neurological condition, analogous tissue samples harvested from 10 autopsy-verified Alzheimer brains, and from 10 neurologically and neuropathologically normal control patients were analyzed using immunofluorescence histochemistry, western immunoblot, and real-time PCR to determine genotype effects on neuronal number and size, neuronal and glial expressions of amyloid β (Aβ) precursor protein (βAPP), Aβ, apolipoprotein E (ApoE), S100B, interleukin-1α and β, and α and β secretases; and on markers of neuronal stress, including DNA/RNA damage and caspase 3 expression.
Allelic combinations of APOE influenced each epilepsy-related neuronal and glial response measured as well as neuropathological change. APOE ϵ3,3 conferred greatest neuronal resilience denoted as greatest production of the acute phase proteins and low neuronal stress as assessed by DNA/RNA damage and caspase-3 expression. Among patients having an APOE ϵ2 allele, none had Aβ plaques; their neuronal sizes, like those with APOE ϵ3,3 genotype were larger than those with other genotypes. APOE ϵ4,4 conferred the weakest neuronal resilience in epilepsy as well as in Alzheimer patients, but there were no APOE genotype-dependent differences in these parameters in neurologically normal patients.
Our findings provide evidence that the strength of the neuronal stress response is more related to patient APOE genotype than to either the etiology of the stress or to the age of the patient, suggesting that APOE genotyping may be a useful tool in treatment decisions.
Epilepsy is the third most common cause of neurological disability worldwide  and is associated with precocious development of the neuropathological changes of Alzheimer’s disease (AD) [2–4]. Traumatic brain injury (TBI), which is a major risk factor for the development of epilepsy , is also associated with increased risk for later development of AD, and, in both cases, the risk of development of AD is greater with inheritance of apolipoprotein E ϵ4 alleles (APOE ϵ4) [3, 6]. Exploring possible links between epilepsy-related alterations in neuronal and glial cell responses relative to APOE genotype is potentially important in understanding chronic neurodegenerative sequelae in epilepsy as well as in other forms of brain injury such as TBI [5, 7–10].
In both purified rodent neuronal cell cultures and cultures of the human neuroblastoma cell line NT2, excess glutamate induces marked increases in the expression of the neuronal acute phase response protein βAPP, and in the release of its secreted fragment sAPPα , which is a powerful inducer of glial activation and increased production and release of the proinflammatory cytokine IL-1β . This sAPP-induced glial activation and cytokine expression and release is differentially modulated in the presence of ApoE3 vs ApoE4 , with ApoE3 providing greater protection than ApoE4. In epilepsy, there is overexpression of βAPP and IL-1, as well as the astrocyte-derived, neuritogenic cytokine S100B [13, 14]. Furthermore, a comparison between surgical waste tissues from patients undergoing anterior temporal lobectomy surgery for drug-resistant intractable epilepsy showed that APOE ϵ3,3 and APOE ϵ4,4 genotypes dramatically alter the expression of βAPP and of IL-1 such that the APOE ϵ3 allele is more effective with regard to the maintenance of appropriate neuronal acute phase responses that favor neuronal viability than is APOE ϵ4 . Importantly, several studies have provided evidence that inheritance of an APOE ϵ4 allele is associated with increased risk for Alzheimer neuropathological changes in epilepsy patients . This is particularly relevant to the possibility that the decrease in the ability of ApoE4 compared with ApoE3 to elevate synthesis of the neuronal acute phase protein βAPP  is responsible for less sAPPα release, resulting in diminished neuronal repair and survival .
Epilepsy, in particular, exemplifies the intimate relationship between neuronal stress and triggering of glial activation, as such interactions are self-amplifying in epilepsy. For example, glutamate-induced hyperexcitation in purified primary rat neurons results in increases in βAPP, sAPP, and IL-1β, as well as ApoE, and both IL-1β and ApoE induce βAPP expression and sAPP release. Moreover, IL-1β treatment of neurons results in glutamate release , promoting a proposed self-perpetuating series of events: glutamate → ApoE → βAPP → sAPP → IL-1β → glutamate. Initially, cycle-engendered early acute phase responses may be beneficial, affording neuronal protection and debris clearance. However, because of the self-perpetuating nature of this cycle and the resultant glutamate release from both glia  and neurons  chronic neuronal stress ensues, enhancing the probability of neurodegeneration. Furthermore, the potential of such a cycle to be self-regenerative may explain, at least in part, why even those epilepsy patients with the advantage of an APOE ϵ3,3 genotype may develop Alzheimer-type neuropathological changes.
Evidence of a role for APOE genotype in determining neurodegenerative consequences of epilepsy underscores the need for in-depth analyses of neuronal-glial interactions that may be governed by inheritance of specific APOE allelic combinations. Such analyses provide basic cellular and molecular information regarding pathways involved in neuronal-glial interactions as well as information that may be helpful in clinical decision making.
Comparison between our patient groups – Group 1: patients having one or two alleles of ϵ2 and no ϵ4 [APOE ϵ2,2 (n = 1) and APOE ϵ2,3 (n = 12)] age range = 14 y-73 y, median age = 40 y, average age = 35 y; Group 2: [APOE ϵ3,3 patients (n = 53)], age range = 0.25 y-71 y, median age = 32 y, average age = 30.4 y; Group 3: patients having one ϵ4 allele [APOE ϵ2,4 (n = 2) APOE ϵ3,4 (n = 17)], age range = 10 y-48 y, median age = 30.5 y, average = 29.2 y; Group 4: [APOE ϵ4,4 patients (n = 7)], age range = 10 y-50 y, median age = 34 y, average age = 32 y. Such grouping allowed for investigation of the degree of influence of each of the 6 combinations of the three APOE alleles and provided the following results.
In a given unit area of cortical layers III and IV of superior temporal gyrus in patients in Group 2 (patients having two APOE ϵ3 alleles), the total number of microglia counted was greater than that in other groups (Group 2 = 1137 ± 267 vs Group 1 = 460 ± 112; Group 3 = 534 ± 81; Group 4 = 770 ± 187 microglia/mm2; p < 0.001). Although the number of microglia/mm2 was influenced by APOE genotype, the relative levels of glial cytokine mRNAs IL-1α and IL-1β were not related to genotype (data not shown).
Our results show that, compared to other APOE allelic combinations, neuronal resilience and glial activation were greatest in epilepsy patients with APOE ϵ3,3 genotype. Neuronal resilience correlated with the highest expression of acute phase response proteins βAPP and ApoE, greatest neuronal size, and least indications of DNA fragmentation, oxidation, and potential cell cycling. Similarly, compared to patients with other allelic combinations, patients with APOE ϵ3,3 genotype had more IL-1α-immunoreactive microglia in a given cross-sectional cortical area, more microglia adjacent to neurons, and more astrocytes per given cross-sectional area. In addition, the tissue levels of the astrocyte-derived, neuron-sparing neuritogenic cytokine S100B were highest in patients with APOE ϵ3,3 genotype. These findings are consistent with the idea that APOE genotype influences neural responses to the neuronal stress engendered by the hyperexcitation of epilepsy. Several neural stresses elicit elevation of neuronal acute phase proteins βAPP and ApoE, which, in turn, promote microglial and astrocytic activation with increased expression of glial cytokines such as IL-1β and S100B. These cytokines are known to regulate the expression of each other and both are known to induce the expression of βAPP and ApoE for promotion of neuronal survival and maintenance [15, 17, 22–24]. Together, our findings are consistent with the idea that hyperexcitation first elicits compensatory responses that include overexpression of βAPP, release of sAPP, and glial activation with induction of IL-1 and S100B. The fact that carriers of one or two APOE ϵ3 allele(s) were shown here to be more adept than carriers of other APOE genotypes at eliciting specific neuronal and glial responses that have been associated with neuronal repair and survival [16, 25] suggests that neuronal resilience is, at least in part, dependent on which specific ApoE variant is present.
Our findings that inheritance of two APOE ϵ4 alleles is associated with smaller neurons in both epilepsy and Alzheimer’s disease, but not in neurologically normal controls, together with the dramatic elevation of markers of stress in APOE ϵ4,4 carriers, suggests that such individuals are at greater risk of neuronal damage, regardless of the initiating injury. Elevated expression of ApoE mRNA, in conjunction with a lower expression of ApoE protein in those with APOE ϵ4,4 genotype may represent a failed attempt to increase ApoE expression as a way of increasing βAPP expression, which in itself may be futile as ApoE4 has been shown to be an ineffective stimulant of βAPP expression .
The Aβ plaques and hippocampal atrophy in temporal lobectomy tissues from epilepsy patients have been observed in other studies [4, 26]. Our finding that the incidence of Aβ plaques is dependent on specific APOE allelic subtypes, namely, that having even one APOE ϵ2 allele is associated with an absence of Aβ plaques even in our oldest patient (71y), may in some way be related to a reported decreased risk for AD  and a protective effect of inheritance of APOE ϵ2 against Alzheimer-like neuropathological changes . The Aβ plaques, which were noted in our 10-year old APOE ϵ4,4 patient, appeared to be mature dense core neuritic plaques, while those in carriers of other allelic combinations appeared as diffuse neuritic plaques. These findings support previous studies showing a relationship between the presence of an APOE ϵ4 allele and precocious development of AD in epilepsy patients [3, 29], and suggests the need for further investigation into the role of APOE ϵ4 alleles in Aβ plaque maturation. These findings may have relevance to the fact that plaque maturity is associated with both formation of Aβ dense cores as well as increases in ApoE immunoreactivity as Aβ plaques mature .
The fact that most of the Aβ plaques present in our patients were found in APOE ϵ3,3 carriers may, in principle, be mostly related to increases in βAPP expression, especially in view of concomitant increases in proteins, cytokines, and neurotransmitters that are known to induce increases in neuronal βAPP, viz., ApoE , IL-1 , S100B , and glutamate , perhaps identifying mechanisms by which the advantage of having APOE ϵ3 alleles may be accompanied by the disadvantage of fostering Aβ deposition. Interestingly, with regard to APOE genotype and Alzheimer neuropathological change, carrying even one APOE ϵ2 allele appears to have a protective effect against the formation of Aβ plaques, regardless of age and sex. These interpretations of our results are predicated on the idea that Aβ plaques are the consequence of hyperexcitation-induced, neuronal stress-related cycles that are important in the neuropathological progression observed in epilepsy and which occur as a consequence of disease severity and duration, perhaps regardless of genotype, age, and sex. Our observation regarding greater plaque maturity among those with APOE ϵ4,4 relative to other APOE genotypes is consistent with observations in Alzheimer patients [31, 32] and in mice , which show that ApoE binding to Aβ disrupts Aβ clearance across the blood-brain barrier in an isoform-specific manner, with ApoE4 having a greater disruptive effect than ApoE3 or ApoE2 .
The lower tissue levels of actin in patient carriers of APOE ϵ4,4 compared to those with other APOE allelic combinations may explain, at least in part, the APOE ϵ4,4-related smaller neuronal cell size . Variation in actin expression according to APOE genotype, as we show here, suggests that actin should not be used as a tool for normalizing the relative expression of proteins in situations in which actin varies with specific parameters.
Our finding that even one APOE ϵ2 allele is protective against Aβ plaque deposition is consistent with previous reports associating this genotype with protection against Alzheimer’s disease . The robust neuronal-glial response to the neuronal stress of epilepsy in APOE ϵ3,3 carriers suggests that overall they have an advantage over other genotypes as indicated by an ability to increase neuronal acute phase protein expression, a greater neuronal size, and increased resilience, as indicated by lower levels of markers for RNA and DNA damage, and lower susceptibility to inappropriate cell cycling and death pathways. However, the fact that the percentage of patients with Aβ plaques was highest in APOE ϵ3,3 carriers suggests that these beneficial effects occur at the expense of an increase in the possibility of cleavage of excess βAPP and formation of Aβ, as well as increasing deposition of ApoE in such plaques and in this way perhaps favoring plaque formation. APOE ϵ4,4 carriers in our study were disadvantaged compared to other genotypes, having the smallest neurons among the genotypes, lowest acute phase responses, and highest markers of stress. Taken together, our findings suggest that APOE genotype may be important in decisions regarding timing of surgical intervention for intractable epilepsy, as well as in decisions regarding exposure of individuals to activities with high risk for TBI.
Resected temporal lobe tissues were obtained from 92 epilepsy patients (58 males and 34 females; 1 APOE ϵ2,2, 12 APOE ϵ2,3, 53 APOE ϵ3,3, 2 APOE ϵ2,4, 17 APOE ϵ3,4 and 7 APOE ϵ4,4) with an age at surgery ranging from 0.25 to 73 years (median age = 32 y, average age = 31.7 y). All patients underwent anterior temporal lobectomy for treatment of medication-resistant intractable epilepsy. For more information on patients regarding neuropathological evaluation please see . Surgical waste obtained from the anterior portion of the superior temporal gyrus, an area some distance from sclerotic areas and epileptogenic foci, was dissected at 4 mm intervals, and alternate sections were preserved by flash freezing for molecular analyses and by formalin fixation for histological evaluation. For uniformity, immunohistochemical examination was restricted to cortical layers III and IV of superior temporal gyrus.
Temporal lobe tissues analogous to that collected from epilepsy surgical waste was collected from autopsied brain tissue of neuropathologically diagnosed Alzheimer patients [n = 6 males and n = 4 females; 5 APOE ϵ3,3 (M-60 y; M-73 y; F-76 y; F-80 y; F-88 y) and 5 APOE ϵ4,4 (F-72 y; M-74 y; M-74 y; M-82 y; M-86 y]. Analogous tissue samples were also obtained from neurologically and neuropathologically normal individuals [n = 8 males and n = 2 females; 6 APOE ϵ3,3 (M-69 y; M-78 y; M-78 y; M-81 y; M-83 y; F-87 y) and 4 APOE ϵ4,4 (M-70 y; M-78 y; F-79 y; M-85 y)]. As with the processing of the tissue samples from our epilepsy patients, autopsy tissue samples collected less than eight-hours postmortem from Alzheimer and control patients were identical, i.e., analogous samples to be used for molecular analyses were snap frozen in liquid nitrogen and for immunohistochemical analyses sections of formalin fixed brains were used.
Surgical waste and autopsy tissue are both exempt from IRB review under 46.101 5(b), and this study was approved as an exempt study by the University of Arkansas Institutional Review Board.
For further analysis of data obtained from epilepsy patients, we categorized genotypes into four groups. Group 1: patients having one or two alleles of ϵ2 and no ϵ4 [APOE ϵ2,2 (n = 1) and APOE ϵ2,3 (n = 12)]; Group 2: [APOE ϵ3,3 patients (n = 53)]; Group 3: patients having one ϵ4 allele [APOE ϵ2,4 (n = 2) APOE ϵ3,4 (n = 17)]; Group 4: [APOE ϵ4,4 patients (n = 7)]. This grouping allowed for investigation of the degree of influence of each of the three APOE alleles on observed results.
Reagents: Antibodies: rabbit anti-human IL-1α (Peprotech 4:1000): goat anti-human ApoE (Invitrogen 1:50): mouse anti-human Aβ/βAPP (Covance 1:1000); rabbit anti-human Pan Neuronal Marker (PNM – Millipore ABN 1:500); mouse anti-human DNA/RNA oxidative damage antibody (8-OH-gaunosine – Stress Marq Biosciences 5 μg/ml); rabbit anti-phosphorylated tau (AT8 – Abcam 1:3000); rabbit anti-actin (Santa Cruz 1:1000) were diluted in antibody diluent (DAKO). Secondary antibodies were Alexa Fluor® 488: donkey anti-rabbit or goat anti-rabbit; Alexa Fluor® 594: donkey anti-goat or goat anti-mouse. Mounting media containing Prolong Gold anti-fade reagent with DAPI (Invitrogen) was used to stain nuclei.
Paraffin-embedded tissue was sectioned at 7 μm and processed as previously described . Sections destined for IL-1α, PNM, Stress Marq, and Aβ immunoreaction were pretreated by placing them in boiling sodium citrate buffer (0.01 M, pH 6.0) for 20 minutes; sections for ApoE immunoreaction were placed in trypsin solution for 10 min at 37°C, and all were blocked using protein block (DAKO), and immunoreacted by overnight incubation at room temperature. Appropriate Alexa Fluor-tagged secondary antibodies were diluted in antibody diluent 1:200, and sections were incubated for 60 minutes, washed three times for 5 minutes each in distilled water, and coverslipped with prolong Gold with DAPI.
As previously described [2, 15], a quantitative approach was used to assess numbers of glia and neurons. Three images per slide (40× magnification) were captured at identical exposure settings using a Nikon Eclipse E600 microscope equipped with a Coolsnap monochrome camera. Each of the three images, spanning 37,638.6 μm2, was acquired, analyzed, and thresholded using NIS-Elements BR3 software (Nikon.com). Results regarding neuronal and glial numbers are presented as numbers/mm2. Data were analyzed by ANOVA to assess differences among groups. Significance was provided by p ≤ 0.05.
Human gene sequences for ApoE, βAPP, α-secretase, BACE 1, BACE 2, IL-1α, IL-1β, and S100B, with PCR annealing temperatures and number of amplification cycles
Annealing temp. (Co)
α-secretase (ADAM 10)
Proteins were extracted from brain tissue in a lysis buffer comprised of 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P40, 1 mM EGTA, 1 mM EDTA, and 1% sodium deoxycholate; lysate protein was quantified using a Micro BCA assay reagent kit (Pierce, Rockford IL) as described previously . Aliquots (30 μg each for ApoE and 50 μg for caspase 3) were loaded onto a 4-12% CriterionXT precast Gel Bis-Tris from Bio-Rad (Hercules, CA), subjected to electrophoresis at 80V for 3 h, and transferred to PVDF 0.45 μm Immobilon-FL (Millipore). Blots were blocked in I-Block Buffer (Applied Biosystem Inc., Bedford, MA) for 60 minutes, then incubated overnight at 4°C with either mouse monoclonal anti-ApoE (1:500) (Santa Cruz sc-58242) primary antibody, rabbit anti-human Caspase3\Cleaved Caspase 3 (1:500) (Cell Signaling # 9661-2), or rabbit anti-actin (Santa Cruz 1:1000), and incubated for 1 h at room temperature with alkaline phosphatase-conjugated secondary antibody (1:1000). For protein detection, we used ProteinSimple Multifluor Western Blotting Kit (Santa Clara, CA), and for image capture we used CellBioscience FluorChem Q digital imager (Santa Clara, CA). Autoradiographs were digitized and analyzed using NIH Image software, version 1.60.
Data were analyzed using an unpaired t-test and Wilcoxon distribution score, and results are expressed as mean + SD. In the Wilcoxon distribution plot, the length of the box represents the interquartile range (the distance between the 25th and 75th percentiles), the symbol in the box interior represents the group mean, the horizontal line in the box interior represents the group median, the vertical lines (the whiskers) issuing from the box extend to the group minimum and maximum values. Values were considered significantly different when the p-value was ≤ 0.05.
Amyloid β precursor protein
The authors particularly appreciate the irreplaceable contribution made by the patients and the technical expertise and advice provided by Dr. Ling Liu and Professor Steven Barger, and the technical help and advice provided by Mr. Richard A. Jones and Ms. JoAnn Biedermann, and Dr. Songthip T. Ounpraseuth for his help in statistical analyses. This work was supported in part by a grant from the National Institute on Aging AG12411, The Alexa and William T. Dillard Foundation, and the Windgate Foundation.
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