FULL-LENGTH ORIGINAL RESEARCH
Hippocampal antioxidative system in mesial temporal lobe epilepsy *Aleksandar J. Risti c, †Danijela Savi c, *Dragoslav Sokic, ‡Jelena Bogdanovic Pristov, §Jelena Nestorov, ¶Vladimir Bas carevi c, ¶Savo Raicevic, #Slobodan Savic, and ‡Ivan Spasojevic Epilepsia, **(*):1–11, 2015 doi: 10.1111/epi.12981
SUMMARY
Dr. Aleksandar J. is an assistant Ristic professor at the University of Belgrade School of Medicine.
Objective: To examine antioxidative system in hippocampi of patients with mesial temporal lobe epilepsy associated with hippocampal sclerosis (mTLE-HS). Methods: Activity and levels of antioxidative enzymes—catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), manganese superoxide dismutase (MnSOD), and copper-zinc superoxide dismutase (CuZnSOD)—were assessed in hippocampi of nine pharmacoresistant mTLE-HS patients (mean age 37.7 [standard deviation] 6.6 years) who underwent amygdalohippocampectomy, and in 10 hippocampi obtained via autopsy from five neurologically intact controls (mean age 34.4 9.0 years). Subfield and cellular (neuron/astrocyte) distribution of CAT, GPx, and MnSOD was analyzed in detail using immunohistochemical staining. Results: Sclerotic hippocampi showed drastically increased activity of hydrogen peroxide–removing enzymes, CAT (p < 0.001), GPx (p < 0.001), and GR (p < 0.001), and significantly higher protein levels of CAT (p = 0.006), GPx (p = 0.040), GR (p = 0.024), and MnSOD (p = 0.004), compared to controls. CAT immunofluorescence was located mainly in neurons in both controls and HS. Control hippocampi showed GPx staining in blood vessels and CA neurons. In HS, GPx-rich loci, representing bundles of astrocytes, emerged in different hippocampal regions, whereas the number of GPx-positive vessels was drastically decreased. Neurons with abnormal morphology and strong MnSOD immunofluorescence were present in all neuronal layers in HS. Small autofluorescent deposits, most likely lipofuscin, were observed, along with astrogliosis, in CA1 in HS. Significance: Antioxidative system is upregulated in HS. This documents, for the first time, that epileptogenic hippocampi are exposed to oxidative stress. Our findings provide a basis for understanding the potential involvement of redox alterations in the pathology of epilepsy, and may open new pharmacologic perspectives for mTLE-HS treatment. KEY WORDS: Mesial temporal epilepsy, Hippocampal sclerosis, Glutathione peroxidase, Mitochondria, Superoxide dismutase, Catalase.
The involvement of oxidative stress in the pathology of epilepsy has been largely examined using animal models, whereas studies on human tissues are scarce. It is known
that some inherited epilepsies, such as myoclonic epilepsy with ragged red fibers, are related to dysfunctional mitochondria.1,2 These organelles are the main intracellular
Accepted February 26, 2015. *Center for Epilepsy and Sleep Disorders, Neurology Clinic, Clinical Center of Serbia, Belgrade, Serbia; †Department of Neurobiology, Institute for Biological Research “Sinisa Stankovic,” University of Belgrade, Belgrade, Serbia; ‡Life Sciences Department, Institute for Multidisciplinary Research, University of Belgrade, Belgrade, Serbia; §Department of Biochemistry, Institute for Biological Research “Sinisa Stankovic,” University of Belgrade, Belgrade, Serbia; ¶Institute for Neurosurgery, Clinical Center of Serbia, Belgrade, Serbia; and #Institute of Forensic Medicine, Medical School, University of Belgrade, Belgrade, Serbia Address correspondence to Aleksandar Ristic, Center for Epilepsy and Sleep Disorders, Neurology Clinic, Clinical Center of Serbia, Dr Subotica Starijeg 6, 11000 Belgrade, Serbia. E-mail: [email protected] Wiley Periodicals, Inc. © 2015 International League Against Epilepsy
1
2 A. J. Ristic et al. source of oxidative stress mediators—superoxide and hydrogen peroxide (H2O2).3 Mitochondrial dysfunction appears also to develop in mesial temporal lobe epilepsy associated with hippocampal sclerosis (mTLE-HS), according to different metabolic abnormalities that have been observed in hippocampi of patients with mTLE-HS. These include interictal glucose hypometabolism,4 abnormal nicotinamide adenine dinucleotide phosphate (NAD(P)H) transients provoked by ex vivo stimulation,5 and subfieldspecific decrease of the concentration of mitochondrial metabolite—N-acetyl aspartate.6 Redox alterations in mitochondria are implicated by few available reports that have documented: (1) nonphysiologic activity of mitochondrial complex I, the main site of superoxide production, in epileptic foci in mTLE-HS patients7; (2) low activity of aconitase, a mitochondrial matrix enzyme that is highly susceptible to oxidative damage, in hippocampal CA3 regions of mTLEHS patients8; and (3) altered metabolism of redox-active metals in sclerotic hippocampi.9 Nevertheless, without direct information/evidence that oxidative stress takes place at the “crime scene,” that is, sclerotic hippocampi, the development and nature of prooxidative events in epileptogenic tissues remain elusive. Superoxide and H2O2 show exceptionally short lifetimes in tissues, which practically prevents their measurements ex vivo (and in vivo).10 Therefore, oxidative stress is assessed by measuring the activity/level of enzymes of the antioxidative system (AOS), which is regulated by redox conditions.11,12 In brief, superoxide is dismutated to H2O2 by manganese superoxide dismutase (MnSOD) in mitochondria and by copper-zinc superoxide dismutase (CuZnSOD) in cytosol. Hydrogen peroxide is further broken down to water by catalase (CAT) and glutathione peroxidase (GPx)/glutathione reductase (GR) couple.11,12 In this study, we first assess and compare activity and protein levels of five AOS enzymes in hippocampi of mTLEHS patients and controls. Second, we analyze localization of CAT, GPx, and MnSOD in neurons and astrocytes in different hippocampal regions.
Methods Patients and samples The study was performed on hippocampi of nine patients with pharmacoresistant (≥2 antiepileptic drug failures) mTLE-HS (mean age 37.7 [standard deviation] 6.6 years, range 26–47; mean duration of epilepsy 20.5 12.8 years, range 2–42; five with left HS; m/ f = 3/6). Patients underwent presurgical evaluation at the Neurology Clinic, Clinical Center of Serbia. In all patients, magnetic resonance imaging (MRI) findings were consistent with HS, which was confirmed by histopathologic analysis and classified according to International League Against Epilepsy (ILAE) classification of Epilepsia, **(*):1–11, 2015 doi: 10.1111/epi.12981
HS in patients with TLE.13 All patients were rendered “seizure free” following surgery (median follow-up 30 months). The epileptic hippocampi were removed en bloc, in the course of anterior temporal lobe resection and amygdalohippocampectomy that were conducted at the Neurosurgery Clinic, Clinical Center of Serbia. For further clinical and histologic details see Table S1. Control hippocampi (n = 10) were acquired from five cadavers (mean age 34.4 9.0 years, range 24–51; m/f = 4/1; brain weight 1,596 195 g) that were limited to sudden death without medical intervention, prolonged agonal periods, and secondary diagnosis of drug/alcohol dependency. Control hippocampi were removed en bloc during the autopsy at the Institute of Forensic Medicine, University of Belgrade Medical School, maximally 10 h following death (average time for autopsy was 7.8 h postmortem). The postmortem specimens were free from brain injury. Posterior parts of the head (thickness 0.05); gray, statistical significance for data sets after exclusion of outliers. Epilepsia ILAE
enzymes according to Pearson’s test, taking into account both study groups together, or each group separately. The exclusion of outliers did not affect the outcome of correlation analysis. Distribution of catalase Strong immunofluorescence for CAT was present in neuron somata in gyrus dentatus (GD) and CA1–CA4 in both control (Fig 2A and 2B) and sclerotic hippocampi (Fig 2C, D). In the latter, a decrease in the number of neurons in GD and CA4 was evident (Fig. 2C). Although immunofluorescence is not a quantitative method, it should be mentioned that CAT immunofluorescence in GD neuron somata appears to be stronger in HS compared to controls at the same image acquisition settings. There were no signs of specific CAT staining in CA1 area of total neuronal loss, which was accompanied by astrogliosis (Fig. 2D). Astrocytes in alveus were positive for CAT in both controls and HS (Fig. 2E). Distribution of glutathione peroxidase In controls, a strong immunostaining for GPx was present in the rich network of blood vessels (Fig. 3A–C), judged by Epilepsia, **(*):1–11, 2015 doi: 10.1111/epi.12981
morphologic appearance of stained structures (Fig. 3B). It is noteworthy that GPx staining of vessels could not be related to traces of blood. We base this claim on the fact that erythrocytes contain much higher levels of CAT than GPx, and blood vessels were not positive for CAT. Furthermore, it can be observed that CA4 but not GD neuron somata, were stained for GPx (Fig. 3A). Weak GPx staining was also present in CA1–CA3 neurons (Fig. 3C). Astrocytes in alveus were positive for GPx (Fig. 3D). Sclerotic hippocampi showed a drastic decrease in the number of GPx-positive vessels (Fig. 3E,H). The sums of cross-sectional areas occupied by GPx-positive vessels per mm2 (106 lm2) of GD/CA4 hippocampal preparation (~2.4 mm2 total area) were (mean SD): 20,789 5,825 lm2 in controls and 5,355 254 lm2 in HS (p = 0.029). The sum areas in CA1/alveus hippocampal preparations were: 11,285 3,479 lm2 in controls and 3,055 2,046 lm2 in HS (p = 0.036). Specific new structures—GPx-rich loci, were present in HS. These were observed in GD and CA4 (Fig. 3E,F), CA1 and alveus (Fig. 3G,I), and stratum radiatum (not shown). Loci most likely represent astrocytic bundles, as GPx immunostaining was co-localized with GFAP (Fig. 3E,G), and
5 Antioxidative Enzymes in Epilepsy A
C
D
B
E
Figure 2. Representative micrographs of hippocampi immunostained for catalase. Preparations were stained for CAT (red), neurons (NeuN, green) or astrocytes (GFAP, green), and nuclei (Hoechst, blue). (A) GD and CA4 in control hippocampus. Magnified region of interest (white square) shows granular neuron somata in GD and one large ovoid pyramidal CA4 neuron. Small astrocytic nuclei that are strongly stained with Hoechst can be observed. (B) Area in CA1 in control hippocampus. Magnified region of interest (white square) shows pyramidal CA1 neurons. Alveus is labeled to make the orientation of the preparation clear. (C) GD and CA4 in HS. Magnified region of interest (white square) shows granular neuron somata in GD. (D) Sclerotic area in CA1. GFAP staining demonstrates the presence of astrogliosis in the region of total neuronal loss (demarcated with dashed lines), which does not show CAT immunostaining. (E) Areas in alveus in control (left) and sclerotic (right) hippocampi. Epilepsia ILAE
not with NeuN (Fig. 3F). We noted a trend of positioning of GPx loci around or in the vicinity of blood vessels that were not positive for GPx (probably atrophic vascular structures). The areas of loci varied widely from 777 to 15,302 lm2. In general, neurons in CA regions of HS were not specifically stained for GPx (Fig. 3H). Finally, astrocytes in alveus in HS were positive for GPx (Fig. 3I).
Distribution of manganese superoxide dismutase Weak immunofluorescence for MnSOD was observed in CA4 but not in GD neuron somata (Fig. 4A). CA3– CA1 neurons were also positive for MnSOD (Fig. 4B). This is similar to GPx localization in control hippocampi. The key difference between controls and HS was in the presence of degenerated structures that were positive for Epilepsia, **(*):1–11, 2015 doi: 10.1111/epi.12981
6 A. J. Ristic et al. A
E
B
F
G
C
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I
Figure 3. Representative micrographs of hippocampi immunostained for glutathione peroxidase. Sections were stained for GPx (red), neurons (NeuN, green) or astrocytes (GFAP, green), and nuclei (Hoechst, blue). (A) GD and CA4 in control hippocampus. Large number of GPxpositive blood vessels (some are marked with arrowheads) can be observed. Positive staining of CA4 neurons for GPx can be observed in the region of interest (white square). (B) Blood vessels in control. It can be observed that GPx is located in/on vessels and not in astrocytes. (C) CA1 region in control. Some blood vessels are marked with arrowheads. (D) Area in alveus in control; (E) GD and CA4 in HS. The number of GPx-positive vessels is diminished and those that are left are stained faintly for GPx (arrowheads). GPx-rich loci are marked with arrows. Region of interest (white square) shows a GPx-rich locus. (F) Region of interest (same as from panel C) stained for neurons and GPx. (G) Alveus and CA1 area showing GPx-rich loci (arrows). Magnified region of interest (white square) demonstrates co-localization of GPx and GFAP in the locus. (H) CA1 region in HS. GPx-stained blood vessels are marked with arrowheads. (I) Area in alveus in HS. GPx-rich locus can be observed. Epilepsia ILAE Epilepsia, **(*):1–11, 2015 doi: 10.1111/epi.12981
7 Antioxidative Enzymes in Epilepsy A
D
B
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G
Figure 4. Representative micrographs of hippocampi immunostained for manganese superoxide dismutase. Preparations were stained for MnSOD (red), neurons (NeuN, green) or astrocytes (GFAP, green), and nuclei (Hoechst, blue). (A) GD and CA4 of control hippocampus. Magnified region of interest (white square) shows CA4 neuron somata. (B) Area in CA1 in control hippocampus. (C) GD and CA4 in HS. Magnified region of interest (white square) shows GD neuron somata, and small structures (supposedly degenerated neurons) positive for NeuN and MnSOD (marked with arrowheads). (D) Area in CA1 region in HS. Degenerated neurons are marked with arrowheads in the magnified region of interest (white square). (E) Region of interest in CA1. It can be observed that degenerated cells (arrowheads) are not GFAP positive. Neuronal nuclei are larger and less intensively stained with Hoechst compared to astrocytic nuclei. (F) Region of interest in CA1. It can be observed that degenerated neurons (arrowheads) are not stained for GPx. (G) Alveus in control (left) and sclerotic (right) hippocampus. Epilepsia ILAE
MnSOD and NeuN (Fig. 4C,D), and negative for GFAP (Fig. 4E), GPx (Fig. 4F), and CAT (not shown). These structures most likely represent degenerated neurons. They were present in all neuronal layers, and could not be detected in areas showing total neuronal loss and in areas with no neuron somata, such as alveus. Astrocytes
in alveus in both control and sclerotic hippocampi were positive for MnSOD (Fig. 4G). Autofluorescent aggregates CA1 areas of total neuronal loss showed small number of aggregates that exhibited autofluorescence at a wide range Epilepsia, **(*):1–11, 2015 doi: 10.1111/epi.12981
8 A. J. Ristic et al.
Figure 5. Representative micrographs of autofluorescent deposits in CA1 sclerotic regions. Autofluorescence acquisition excitation (Ex) and emission (Em) wavelengths/band passes (nm) are presented. One filter had Gaussian distribution of wavelengths (G). The deposits can be also observed in preparations stained for neurons (NeuN), astrocytes (GFAP), CAT, and GPx. Epilepsia ILAE
of excitation and emission wavelengths (Fig. 5). These were no larger than one cell and most likely represent lipofuscin deposits. Autofluorescent deposits can be also observed in preparations stained for NeuN, GFAP, CAT, GPx, or MnSOD (not shown).
Discussion We demonstrated here that epileptogenic hippocampi of mTLE-HS patients are exposed to excessive oxidative stress. It is mediated via increased mitochondrial production of superoxide, according to augmented MnSOD level, and via superoxide-derivative, H2O2, as documented by drastic upregulation of CAT, GPx, and GR (the scheme of AOS is presented in Fig. 6). It is noteworthy that changes in cytosolic production of superoxide are not implicated here, since Epilepsia, **(*):1–11, 2015 doi: 10.1111/epi.12981
Figure 6. The main components of AOS. The central site of superoxide (O2 ) production in mitochondria is complex I on the inner membrane of this organelle. Superoxide is dismutated to H2O2 by MnSOD. H2O2 is an uncharged and relatively stable species, thus being capable of crossing the membranes. The diffusion is facilitated by some aquaporins (AQPs). H2O2 leaks from mitochondria to cytosol, where it is removed either by CAT, GPx, or peroxiredoxin/thioredoxin/thioredoxin reductase (Prx/Trx/TrxR) system. GPx uses reduced glutathione (GSH), which is recycled from oxidized glutathione (GSSG) by GR. Alternatively, H2O2 can be decomposed inside mitochondria. This goes mainly via Prx/Trx/ TrxR system (different isoforms compared to cytosol) that contributes to H2O2 removal in mitochondria to 70–80%, and via GPx/GSH/GR system (10–20% contribution). Superoxide that is (non)enzymatically produced in the cytosol is dismutated by CuZnSOD. Finally, H2O2 can enter/leave the cell via cellular membrane. Epilepsia ILAE
the activity/level of CuZnSOD was not increased. Most importantly, sclerotic hippocampi exhibited specific cellular- and subfield-specific distribution of AOS enzymes. Increased MnSOD level in HS supports previous observations of dysfunction of mitochondria in epileptogenic foci of mTLE-HS patients.4–8 It has been reported that mitochondrial complex I shows decreased activity in CA neurons located in the areas with ongoing sclerotic processes.7 Independent of the cause, such setup most likely promotes superoxide production.3 Our findings correlate strongly with this, that is, neurons with ongoing degeneration
9 Antioxidative Enzymes in Epilepsy showed specific immunostaining for MnSOD, an enzyme that handles superoxide in mitochondria. The main feedback loop between mitochondrial dysfunction and epileptogenesis most likely lies in the energy challenge that is posed to neurons by seizures and, on the other side, in the promotion of hyperexcitability and neuronal death via energy depletion, inefficient restoration of ionic gradients including deregulation of calcium metabolism, self-propagating mitochondrial injury, and activation of apoptotic cascades.6,18 Increased superoxide generation in mitochondria may not be only accompaniment but also a direct contributor to these processes. Pertinent to this, it might be of interest to further investigate mitochondrial antioxidative defense in sclerotic hippocampi. It is important to note that we focused here on CAT and GPx/GR because they represent the main sink for H2O2. On the cellular level, CAT shows activity that is at least three orders of magnitude higher compared to peroxiredoxin/thioredoxin/thioredoxin reductase (Prx/Trx/TrxR) system, which is the main route for H2O2 removal in mitochondria.19,20 Namely, it has been shown that the activity of Prx/Trx/TrxR system in isolated brain mitochondria is about 10 nmol of H2O2/min/mg of mitochondrial proteins.19 On the other, CAT activity determined here for control hippocampal samples was approximately 40 lmol of H2O2/min/mg of total proteins. However, Prx/Trx/TrxR system is essential for keeping low levels of mitochondrial H2O2 emission, and acts in concert with glutathione system via glutaredoxin.21,22 In addition, Trx and TrxR are essential for normal redox signaling via regulation of thiol redox switches on different proteins.22 Specific GPx-rich loci were first identified in HS. These loci were observed in GD, CA regions, and alveus, and appear to represent bundles of astrocytes with highly expressed GPx. It has been proposed that astrocytic GPx is essential for H2O2 detoxification in the central nervous system and that astrocytes protect neurons from oxidative stress.23–25 It is important to point out that H2O2 is less reactive and has a larger diffusion radius compared to superoxide. It leaks from mitochondria and cells, which is facilitated by some aquaporins.26 In brief, GPx-rich loci probably represent sites of excessive (neuronal) production of H2O2 that is counteracted by astrocytes. Increased GPx expression was not observed in the areas of astrogliosis, which might explain the lack of correlation between GPx and GFAP levels, that is, the increase in the levels of these two proteins is related to different events—formation of GPx-rich loci and astrogliosis. Furthermore, sclerotic hippocampi were depleted of GPx-positive blood vessels. Pertinent to this, it has been documented that hippocampi of mTLE-HS patients show collapsed vasculature. In fact, blood vessel density is increased, but most of the vessels represent atrophic vascular structures.27 It has been shown using volume fraction analysis that the volume occupied by blood vessels in the CA1 fields in HS samples is about 60% lower compared to controls,28 which corresponds well with
the reduction in the cross-section area occupied by GPx-positive vessels that was observed here (~70%). Downregulation of GPx in endothelial cells might be involved in microvasculature changes that take place in HS. Small autofluorescent aggregates were observed in CA1 regions showing total neuronal loss. These most likely represent lipofuscin deposits. Lipofuscin shows a wide excitation/emission range,29 and has been observed previously in epileptogenic foci in frontal lobes of patients with epilepsys.30 This finding might be relevant for an ongoing debate about the potential involvement of neurodegenerative processes in epilepsy. In general, HS in epilepsy is considered substantially different compared to hippocampal sclerosis in neurodegenerative diseases, in relation to regenerative capacities of hippocampus that are employed in epilepsy.31 However, degenerative component might be present after all, since the accumulation of lipofuscin has been related to neurodegenerative conditions and aging.32 It is still unclear whether lipofuscin has protective (e.g., via iron sequestration) or toxic effects on brain tissue. Finally, it should be mentioned that patients with neuronal ceroid lipofuscinose CLN8 develop epilepsy.32 It appears that GD neuron somata showed increased intensity of CAT immunofluorescence compared to control GD neurons, but, of course, immunohistochemistry could not be applied here as a quantitative method. A large increase in CAT activity/level in HS might be related to some widely present but subtle changes that could not be clarified by immunohistochemistry. It is important to note here that only excessive generation of H2O2 is “malevolent,” whereas a certain physiologic level of H2O2 is obligatory for normal functioning of a wide range of redoxsensitive proteins.22,33 For example, physiologic concentrations of H2O2 keep open KV7.2 channels, which conduct the M-current, a slow K+ current that serves as a brake for neuronal firing.34,35 There are some other H2O2-regulated K+ and Na+ channels, as documented on hippocampal neurons.36 It is tempting to speculate that augmented CAT activity in combination with collapsed vascularization (and probable insufficient delivery of oxygen) might affect redox signaling in some hippocampal regions. Of note, a recent study has found increased CAT level in cerebral hyaline astrocytic inclusions in the brain tissue of three pediatric patients with infantile spasms.37 There was no cell-specific staining for any of the investigated AOS enzymes in CA1 areas showing neuron loss and astrogliosis. Taking also into account the lack of correlation between the levels of astrocyte protein marker and AOS enzymes, this implies that the increase in activity/level of AOS enzymes cannot be attributed to HS-related changes in cell population, i.e., the increased number of astrocytes. This is important to point out because astrocytes might exhibit high AOS activity in some settings.38 Astrocytes in alveus were positive for CAT, GPx, and MnSOD in both control and sclerotic hippocampi. The Epilepsia, **(*):1–11, 2015 doi: 10.1111/epi.12981
FULL-LENGTH ORIGINAL RESEARCH
Hippocampal antioxidative system in mesial temporal lobe epilepsy *Aleksandar J. Risti c, †Danijela Savi c, *Dragoslav Sokic, ‡Jelena Bogdanovic Pristov, §Jelena Nestorov, ¶Vladimir Bas carevi c, ¶Savo Raicevic, #Slobodan Savic, and ‡Ivan Spasojevic Epilepsia, **(*):1–11, 2015 doi: 10.1111/epi.12981
SUMMARY
Dr. Aleksandar J. is an assistant Ristic professor at the University of Belgrade School of Medicine.
Objective: To examine antioxidative system in hippocampi of patients with mesial temporal lobe epilepsy associated with hippocampal sclerosis (mTLE-HS). Methods: Activity and levels of antioxidative enzymes—catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), manganese superoxide dismutase (MnSOD), and copper-zinc superoxide dismutase (CuZnSOD)—were assessed in hippocampi of nine pharmacoresistant mTLE-HS patients (mean age 37.7 [standard deviation] 6.6 years) who underwent amygdalohippocampectomy, and in 10 hippocampi obtained via autopsy from five neurologically intact controls (mean age 34.4 9.0 years). Subfield and cellular (neuron/astrocyte) distribution of CAT, GPx, and MnSOD was analyzed in detail using immunohistochemical staining. Results: Sclerotic hippocampi showed drastically increased activity of hydrogen peroxide–removing enzymes, CAT (p < 0.001), GPx (p < 0.001), and GR (p < 0.001), and significantly higher protein levels of CAT (p = 0.006), GPx (p = 0.040), GR (p = 0.024), and MnSOD (p = 0.004), compared to controls. CAT immunofluorescence was located mainly in neurons in both controls and HS. Control hippocampi showed GPx staining in blood vessels and CA neurons. In HS, GPx-rich loci, representing bundles of astrocytes, emerged in different hippocampal regions, whereas the number of GPx-positive vessels was drastically decreased. Neurons with abnormal morphology and strong MnSOD immunofluorescence were present in all neuronal layers in HS. Small autofluorescent deposits, most likely lipofuscin, were observed, along with astrogliosis, in CA1 in HS. Significance: Antioxidative system is upregulated in HS. This documents, for the first time, that epileptogenic hippocampi are exposed to oxidative stress. Our findings provide a basis for understanding the potential involvement of redox alterations in the pathology of epilepsy, and may open new pharmacologic perspectives for mTLE-HS treatment. KEY WORDS: Mesial temporal epilepsy, Hippocampal sclerosis, Glutathione peroxidase, Mitochondria, Superoxide dismutase, Catalase.
The involvement of oxidative stress in the pathology of epilepsy has been largely examined using animal models, whereas studies on human tissues are scarce. It is known
that some inherited epilepsies, such as myoclonic epilepsy with ragged red fibers, are related to dysfunctional mitochondria.1,2 These organelles are the main intracellular
Accepted February 26, 2015. *Center for Epilepsy and Sleep Disorders, Neurology Clinic, Clinical Center of Serbia, Belgrade, Serbia; †Department of Neurobiology, Institute for Biological Research “Sinisa Stankovic,” University of Belgrade, Belgrade, Serbia; ‡Life Sciences Department, Institute for Multidisciplinary Research, University of Belgrade, Belgrade, Serbia; §Department of Biochemistry, Institute for Biological Research “Sinisa Stankovic,” University of Belgrade, Belgrade, Serbia; ¶Institute for Neurosurgery, Clinical Center of Serbia, Belgrade, Serbia; and #Institute of Forensic Medicine, Medical School, University of Belgrade, Belgrade, Serbia Address correspondence to Aleksandar Ristic, Center for Epilepsy and Sleep Disorders, Neurology Clinic, Clinical Center of Serbia, Dr Subotica Starijeg 6, 11000 Belgrade, Serbia. E-mail: [email protected] Wiley Periodicals, Inc. © 2015 International League Against Epilepsy
1
11 Antioxidative Enzymes in Epilepsy 32. Zhao L, Spassieva SD, Jucius TJ, et al. A deficiency of ceramide biosynthesis causes cerebellar purkinje cell neurodegeneration and lipofuscin accumulation. PLoS Genet 2011;7:e1002063. 33. Spasojevic I, Jones DR, Andrades ME. Hydrogen peroxide in adaptation. Oxid Med Cell Longev 2012;2012:596019. 34. Gamper N, Zaika O, Li Y, et al. Oxidative modification of M-type K (+) channels as a mechanism of cytoprotective neuronal silencing. EMBO J 2006;25:4996–5004. 35. Wuttke TV, Penzien J, Fauler M, et al. Neutralization of a negative charge in the S1-S2 region of the KV7.2 (KCNQ2) channel affects voltage-dependent activation in neonatal epilepsy. J Physiol 2008;586:545–555. 36. Rice ME. H2O2: a dynamic neuromodulator. Neuroscientist 2011;17:389–406. 37. Visanji NP, Wong JC, Wang SX, et al. A proteomic analysis of pediatric seizure cases associated with astrocytic inclusions. Epilepsia 2012;53:e50–e54. 38. Desagher S, Glowinski J, Premont J. Astrocytes protect neurons from hydrogen peroxide toxicity. J Neurosci 1996;16:2553–2562.
39. Duvernoy HM. The human hippocampus. Berlin: Springer-Verlag; 2005.
Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. Clinical and histologic data for patients with mTLE-HS. Figure S1. Representative Western blots for AOS enzymes. Figure S2. GFAP in hippocampal extracts of controls and patients with mTLE-HS.
Epilepsia, **(*):1–11, 2015 doi: 10.1111/epi.12981
Hippocampal antioxidative system in mesial temporal lobe epilepsy *Aleksandar J. Risti c, †Danijela Savi c, *Dragoslav Sokic, ‡Jelena Bogdanovic Pristov, §Jelena Nestorov, ¶Vladimir Bas carevi c, ¶Savo Raicevic, #Slobodan Savic, and ‡Ivan Spasojevic Epilepsia, **(*):1–11, 2015 doi: 10.1111/epi.12981
SUMMARY
Dr. Aleksandar J. is an assistant Ristic professor at the University of Belgrade School of Medicine.
Objective: To examine antioxidative system in hippocampi of patients with mesial temporal lobe epilepsy associated with hippocampal sclerosis (mTLE-HS). Methods: Activity and levels of antioxidative enzymes—catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), manganese superoxide dismutase (MnSOD), and copper-zinc superoxide dismutase (CuZnSOD)—were assessed in hippocampi of nine pharmacoresistant mTLE-HS patients (mean age 37.7 [standard deviation] 6.6 years) who underwent amygdalohippocampectomy, and in 10 hippocampi obtained via autopsy from five neurologically intact controls (mean age 34.4 9.0 years). Subfield and cellular (neuron/astrocyte) distribution of CAT, GPx, and MnSOD was analyzed in detail using immunohistochemical staining. Results: Sclerotic hippocampi showed drastically increased activity of hydrogen peroxide–removing enzymes, CAT (p < 0.001), GPx (p < 0.001), and GR (p < 0.001), and significantly higher protein levels of CAT (p = 0.006), GPx (p = 0.040), GR (p = 0.024), and MnSOD (p = 0.004), compared to controls. CAT immunofluorescence was located mainly in neurons in both controls and HS. Control hippocampi showed GPx staining in blood vessels and CA neurons. In HS, GPx-rich loci, representing bundles of astrocytes, emerged in different hippocampal regions, whereas the number of GPx-positive vessels was drastically decreased. Neurons with abnormal morphology and strong MnSOD immunofluorescence were present in all neuronal layers in HS. Small autofluorescent deposits, most likely lipofuscin, were observed, along with astrogliosis, in CA1 in HS. Significance: Antioxidative system is upregulated in HS. This documents, for the first time, that epileptogenic hippocampi are exposed to oxidative stress. Our findings provide a basis for understanding the potential involvement of redox alterations in the pathology of epilepsy, and may open new pharmacologic perspectives for mTLE-HS treatment. KEY WORDS: Mesial temporal epilepsy, Hippocampal sclerosis, Glutathione peroxidase, Mitochondria, Superoxide dismutase, Catalase.
The involvement of oxidative stress in the pathology of epilepsy has been largely examined using animal models, whereas studies on human tissues are scarce. It is known
that some inherited epilepsies, such as myoclonic epilepsy with ragged red fibers, are related to dysfunctional mitochondria.1,2 These organelles are the main intracellular
Accepted February 26, 2015. *Center for Epilepsy and Sleep Disorders, Neurology Clinic, Clinical Center of Serbia, Belgrade, Serbia; †Department of Neurobiology, Institute for Biological Research “Sinisa Stankovic,” University of Belgrade, Belgrade, Serbia; ‡Life Sciences Department, Institute for Multidisciplinary Research, University of Belgrade, Belgrade, Serbia; §Department of Biochemistry, Institute for Biological Research “Sinisa Stankovic,” University of Belgrade, Belgrade, Serbia; ¶Institute for Neurosurgery, Clinical Center of Serbia, Belgrade, Serbia; and #Institute of Forensic Medicine, Medical School, University of Belgrade, Belgrade, Serbia Address correspondence to Aleksandar Ristic, Center for Epilepsy and Sleep Disorders, Neurology Clinic, Clinical Center of Serbia, Dr Subotica Starijeg 6, 11000 Belgrade, Serbia. E-mail: [email protected] Wiley Periodicals, Inc. © 2015 International League Against Epilepsy
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2 A. J. Ristic et al. source of oxidative stress mediators—superoxide and hydrogen peroxide (H2O2).3 Mitochondrial dysfunction appears also to develop in mesial temporal lobe epilepsy associated with hippocampal sclerosis (mTLE-HS), according to different metabolic abnormalities that have been observed in hippocampi of patients with mTLE-HS. These include interictal glucose hypometabolism,4 abnormal nicotinamide adenine dinucleotide phosphate (NAD(P)H) transients provoked by ex vivo stimulation,5 and subfieldspecific decrease of the concentration of mitochondrial metabolite—N-acetyl aspartate.6 Redox alterations in mitochondria are implicated by few available reports that have documented: (1) nonphysiologic activity of mitochondrial complex I, the main site of superoxide production, in epileptic foci in mTLE-HS patients7; (2) low activity of aconitase, a mitochondrial matrix enzyme that is highly susceptible to oxidative damage, in hippocampal CA3 regions of mTLEHS patients8; and (3) altered metabolism of redox-active metals in sclerotic hippocampi.9 Nevertheless, without direct information/evidence that oxidative stress takes place at the “crime scene,” that is, sclerotic hippocampi, the development and nature of prooxidative events in epileptogenic tissues remain elusive. Superoxide and H2O2 show exceptionally short lifetimes in tissues, which practically prevents their measurements ex vivo (and in vivo).10 Therefore, oxidative stress is assessed by measuring the activity/level of enzymes of the antioxidative system (AOS), which is regulated by redox conditions.11,12 In brief, superoxide is dismutated to H2O2 by manganese superoxide dismutase (MnSOD) in mitochondria and by copper-zinc superoxide dismutase (CuZnSOD) in cytosol. Hydrogen peroxide is further broken down to water by catalase (CAT) and glutathione peroxidase (GPx)/glutathione reductase (GR) couple.11,12 In this study, we first assess and compare activity and protein levels of five AOS enzymes in hippocampi of mTLEHS patients and controls. Second, we analyze localization of CAT, GPx, and MnSOD in neurons and astrocytes in different hippocampal regions.
Methods Patients and samples The study was performed on hippocampi of nine patients with pharmacoresistant (≥2 antiepileptic drug failures) mTLE-HS (mean age 37.7 [standard deviation] 6.6 years, range 26–47; mean duration of epilepsy 20.5 12.8 years, range 2–42; five with left HS; m/ f = 3/6). Patients underwent presurgical evaluation at the Neurology Clinic, Clinical Center of Serbia. In all patients, magnetic resonance imaging (MRI) findings were consistent with HS, which was confirmed by histopathologic analysis and classified according to International League Against Epilepsy (ILAE) classification of Epilepsia, **(*):1–11, 2015 doi: 10.1111/epi.12981
HS in patients with TLE.13 All patients were rendered “seizure free” following surgery (median follow-up 30 months). The epileptic hippocampi were removed en bloc, in the course of anterior temporal lobe resection and amygdalohippocampectomy that were conducted at the Neurosurgery Clinic, Clinical Center of Serbia. For further clinical and histologic details see Table S1. Control hippocampi (n = 10) were acquired from five cadavers (mean age 34.4 9.0 years, range 24–51; m/f = 4/1; brain weight 1,596 195 g) that were limited to sudden death without medical intervention, prolonged agonal periods, and secondary diagnosis of drug/alcohol dependency. Control hippocampi were removed en bloc during the autopsy at the Institute of Forensic Medicine, University of Belgrade Medical School, maximally 10 h following death (average time for autopsy was 7.8 h postmortem). The postmortem specimens were free from brain injury. Posterior parts of the head (thickness 0.05); gray, statistical significance for data sets after exclusion of outliers. Epilepsia ILAE
enzymes according to Pearson’s test, taking into account both study groups together, or each group separately. The exclusion of outliers did not affect the outcome of correlation analysis. Distribution of catalase Strong immunofluorescence for CAT was present in neuron somata in gyrus dentatus (GD) and CA1–CA4 in both control (Fig 2A and 2B) and sclerotic hippocampi (Fig 2C, D). In the latter, a decrease in the number of neurons in GD and CA4 was evident (Fig. 2C). Although immunofluorescence is not a quantitative method, it should be mentioned that CAT immunofluorescence in GD neuron somata appears to be stronger in HS compared to controls at the same image acquisition settings. There were no signs of specific CAT staining in CA1 area of total neuronal loss, which was accompanied by astrogliosis (Fig. 2D). Astrocytes in alveus were positive for CAT in both controls and HS (Fig. 2E). Distribution of glutathione peroxidase In controls, a strong immunostaining for GPx was present in the rich network of blood vessels (Fig. 3A–C), judged by Epilepsia, **(*):1–11, 2015 doi: 10.1111/epi.12981
morphologic appearance of stained structures (Fig. 3B). It is noteworthy that GPx staining of vessels could not be related to traces of blood. We base this claim on the fact that erythrocytes contain much higher levels of CAT than GPx, and blood vessels were not positive for CAT. Furthermore, it can be observed that CA4 but not GD neuron somata, were stained for GPx (Fig. 3A). Weak GPx staining was also present in CA1–CA3 neurons (Fig. 3C). Astrocytes in alveus were positive for GPx (Fig. 3D). Sclerotic hippocampi showed a drastic decrease in the number of GPx-positive vessels (Fig. 3E,H). The sums of cross-sectional areas occupied by GPx-positive vessels per mm2 (106 lm2) of GD/CA4 hippocampal preparation (~2.4 mm2 total area) were (mean SD): 20,789 5,825 lm2 in controls and 5,355 254 lm2 in HS (p = 0.029). The sum areas in CA1/alveus hippocampal preparations were: 11,285 3,479 lm2 in controls and 3,055 2,046 lm2 in HS (p = 0.036). Specific new structures—GPx-rich loci, were present in HS. These were observed in GD and CA4 (Fig. 3E,F), CA1 and alveus (Fig. 3G,I), and stratum radiatum (not shown). Loci most likely represent astrocytic bundles, as GPx immunostaining was co-localized with GFAP (Fig. 3E,G), and
5 Antioxidative Enzymes in Epilepsy A
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Figure 2. Representative micrographs of hippocampi immunostained for catalase. Preparations were stained for CAT (red), neurons (NeuN, green) or astrocytes (GFAP, green), and nuclei (Hoechst, blue). (A) GD and CA4 in control hippocampus. Magnified region of interest (white square) shows granular neuron somata in GD and one large ovoid pyramidal CA4 neuron. Small astrocytic nuclei that are strongly stained with Hoechst can be observed. (B) Area in CA1 in control hippocampus. Magnified region of interest (white square) shows pyramidal CA1 neurons. Alveus is labeled to make the orientation of the preparation clear. (C) GD and CA4 in HS. Magnified region of interest (white square) shows granular neuron somata in GD. (D) Sclerotic area in CA1. GFAP staining demonstrates the presence of astrogliosis in the region of total neuronal loss (demarcated with dashed lines), which does not show CAT immunostaining. (E) Areas in alveus in control (left) and sclerotic (right) hippocampi. Epilepsia ILAE
not with NeuN (Fig. 3F). We noted a trend of positioning of GPx loci around or in the vicinity of blood vessels that were not positive for GPx (probably atrophic vascular structures). The areas of loci varied widely from 777 to 15,302 lm2. In general, neurons in CA regions of HS were not specifically stained for GPx (Fig. 3H). Finally, astrocytes in alveus in HS were positive for GPx (Fig. 3I).
Distribution of manganese superoxide dismutase Weak immunofluorescence for MnSOD was observed in CA4 but not in GD neuron somata (Fig. 4A). CA3– CA1 neurons were also positive for MnSOD (Fig. 4B). This is similar to GPx localization in control hippocampi. The key difference between controls and HS was in the presence of degenerated structures that were positive for Epilepsia, **(*):1–11, 2015 doi: 10.1111/epi.12981
6 A. J. Ristic et al. A
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Figure 3. Representative micrographs of hippocampi immunostained for glutathione peroxidase. Sections were stained for GPx (red), neurons (NeuN, green) or astrocytes (GFAP, green), and nuclei (Hoechst, blue). (A) GD and CA4 in control hippocampus. Large number of GPxpositive blood vessels (some are marked with arrowheads) can be observed. Positive staining of CA4 neurons for GPx can be observed in the region of interest (white square). (B) Blood vessels in control. It can be observed that GPx is located in/on vessels and not in astrocytes. (C) CA1 region in control. Some blood vessels are marked with arrowheads. (D) Area in alveus in control; (E) GD and CA4 in HS. The number of GPx-positive vessels is diminished and those that are left are stained faintly for GPx (arrowheads). GPx-rich loci are marked with arrows. Region of interest (white square) shows a GPx-rich locus. (F) Region of interest (same as from panel C) stained for neurons and GPx. (G) Alveus and CA1 area showing GPx-rich loci (arrows). Magnified region of interest (white square) demonstrates co-localization of GPx and GFAP in the locus. (H) CA1 region in HS. GPx-stained blood vessels are marked with arrowheads. (I) Area in alveus in HS. GPx-rich locus can be observed. Epilepsia ILAE Epilepsia, **(*):1–11, 2015 doi: 10.1111/epi.12981
7 Antioxidative Enzymes in Epilepsy A
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Figure 4. Representative micrographs of hippocampi immunostained for manganese superoxide dismutase. Preparations were stained for MnSOD (red), neurons (NeuN, green) or astrocytes (GFAP, green), and nuclei (Hoechst, blue). (A) GD and CA4 of control hippocampus. Magnified region of interest (white square) shows CA4 neuron somata. (B) Area in CA1 in control hippocampus. (C) GD and CA4 in HS. Magnified region of interest (white square) shows GD neuron somata, and small structures (supposedly degenerated neurons) positive for NeuN and MnSOD (marked with arrowheads). (D) Area in CA1 region in HS. Degenerated neurons are marked with arrowheads in the magnified region of interest (white square). (E) Region of interest in CA1. It can be observed that degenerated cells (arrowheads) are not GFAP positive. Neuronal nuclei are larger and less intensively stained with Hoechst compared to astrocytic nuclei. (F) Region of interest in CA1. It can be observed that degenerated neurons (arrowheads) are not stained for GPx. (G) Alveus in control (left) and sclerotic (right) hippocampus. Epilepsia ILAE
MnSOD and NeuN (Fig. 4C,D), and negative for GFAP (Fig. 4E), GPx (Fig. 4F), and CAT (not shown). These structures most likely represent degenerated neurons. They were present in all neuronal layers, and could not be detected in areas showing total neuronal loss and in areas with no neuron somata, such as alveus. Astrocytes
in alveus in both control and sclerotic hippocampi were positive for MnSOD (Fig. 4G). Autofluorescent aggregates CA1 areas of total neuronal loss showed small number of aggregates that exhibited autofluorescence at a wide range Epilepsia, **(*):1–11, 2015 doi: 10.1111/epi.12981
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Figure 5. Representative micrographs of autofluorescent deposits in CA1 sclerotic regions. Autofluorescence acquisition excitation (Ex) and emission (Em) wavelengths/band passes (nm) are presented. One filter had Gaussian distribution of wavelengths (G). The deposits can be also observed in preparations stained for neurons (NeuN), astrocytes (GFAP), CAT, and GPx. Epilepsia ILAE
of excitation and emission wavelengths (Fig. 5). These were no larger than one cell and most likely represent lipofuscin deposits. Autofluorescent deposits can be also observed in preparations stained for NeuN, GFAP, CAT, GPx, or MnSOD (not shown).
Discussion We demonstrated here that epileptogenic hippocampi of mTLE-HS patients are exposed to excessive oxidative stress. It is mediated via increased mitochondrial production of superoxide, according to augmented MnSOD level, and via superoxide-derivative, H2O2, as documented by drastic upregulation of CAT, GPx, and GR (the scheme of AOS is presented in Fig. 6). It is noteworthy that changes in cytosolic production of superoxide are not implicated here, since Epilepsia, **(*):1–11, 2015 doi: 10.1111/epi.12981
Figure 6. The main components of AOS. The central site of superoxide (O2 ) production in mitochondria is complex I on the inner membrane of this organelle. Superoxide is dismutated to H2O2 by MnSOD. H2O2 is an uncharged and relatively stable species, thus being capable of crossing the membranes. The diffusion is facilitated by some aquaporins (AQPs). H2O2 leaks from mitochondria to cytosol, where it is removed either by CAT, GPx, or peroxiredoxin/thioredoxin/thioredoxin reductase (Prx/Trx/TrxR) system. GPx uses reduced glutathione (GSH), which is recycled from oxidized glutathione (GSSG) by GR. Alternatively, H2O2 can be decomposed inside mitochondria. This goes mainly via Prx/Trx/ TrxR system (different isoforms compared to cytosol) that contributes to H2O2 removal in mitochondria to 70–80%, and via GPx/GSH/GR system (10–20% contribution). Superoxide that is (non)enzymatically produced in the cytosol is dismutated by CuZnSOD. Finally, H2O2 can enter/leave the cell via cellular membrane. Epilepsia ILAE
the activity/level of CuZnSOD was not increased. Most importantly, sclerotic hippocampi exhibited specific cellular- and subfield-specific distribution of AOS enzymes. Increased MnSOD level in HS supports previous observations of dysfunction of mitochondria in epileptogenic foci of mTLE-HS patients.4–8 It has been reported that mitochondrial complex I shows decreased activity in CA neurons located in the areas with ongoing sclerotic processes.7 Independent of the cause, such setup most likely promotes superoxide production.3 Our findings correlate strongly with this, that is, neurons with ongoing degeneration
9 Antioxidative Enzymes in Epilepsy showed specific immunostaining for MnSOD, an enzyme that handles superoxide in mitochondria. The main feedback loop between mitochondrial dysfunction and epileptogenesis most likely lies in the energy challenge that is posed to neurons by seizures and, on the other side, in the promotion of hyperexcitability and neuronal death via energy depletion, inefficient restoration of ionic gradients including deregulation of calcium metabolism, self-propagating mitochondrial injury, and activation of apoptotic cascades.6,18 Increased superoxide generation in mitochondria may not be only accompaniment but also a direct contributor to these processes. Pertinent to this, it might be of interest to further investigate mitochondrial antioxidative defense in sclerotic hippocampi. It is important to note that we focused here on CAT and GPx/GR because they represent the main sink for H2O2. On the cellular level, CAT shows activity that is at least three orders of magnitude higher compared to peroxiredoxin/thioredoxin/thioredoxin reductase (Prx/Trx/TrxR) system, which is the main route for H2O2 removal in mitochondria.19,20 Namely, it has been shown that the activity of Prx/Trx/TrxR system in isolated brain mitochondria is about 10 nmol of H2O2/min/mg of mitochondrial proteins.19 On the other, CAT activity determined here for control hippocampal samples was approximately 40 lmol of H2O2/min/mg of total proteins. However, Prx/Trx/TrxR system is essential for keeping low levels of mitochondrial H2O2 emission, and acts in concert with glutathione system via glutaredoxin.21,22 In addition, Trx and TrxR are essential for normal redox signaling via regulation of thiol redox switches on different proteins.22 Specific GPx-rich loci were first identified in HS. These loci were observed in GD, CA regions, and alveus, and appear to represent bundles of astrocytes with highly expressed GPx. It has been proposed that astrocytic GPx is essential for H2O2 detoxification in the central nervous system and that astrocytes protect neurons from oxidative stress.23–25 It is important to point out that H2O2 is less reactive and has a larger diffusion radius compared to superoxide. It leaks from mitochondria and cells, which is facilitated by some aquaporins.26 In brief, GPx-rich loci probably represent sites of excessive (neuronal) production of H2O2 that is counteracted by astrocytes. Increased GPx expression was not observed in the areas of astrogliosis, which might explain the lack of correlation between GPx and GFAP levels, that is, the increase in the levels of these two proteins is related to different events—formation of GPx-rich loci and astrogliosis. Furthermore, sclerotic hippocampi were depleted of GPx-positive blood vessels. Pertinent to this, it has been documented that hippocampi of mTLE-HS patients show collapsed vasculature. In fact, blood vessel density is increased, but most of the vessels represent atrophic vascular structures.27 It has been shown using volume fraction analysis that the volume occupied by blood vessels in the CA1 fields in HS samples is about 60% lower compared to controls,28 which corresponds well with
the reduction in the cross-section area occupied by GPx-positive vessels that was observed here (~70%). Downregulation of GPx in endothelial cells might be involved in microvasculature changes that take place in HS. Small autofluorescent aggregates were observed in CA1 regions showing total neuronal loss. These most likely represent lipofuscin deposits. Lipofuscin shows a wide excitation/emission range,29 and has been observed previously in epileptogenic foci in frontal lobes of patients with epilepsys.30 This finding might be relevant for an ongoing debate about the potential involvement of neurodegenerative processes in epilepsy. In general, HS in epilepsy is considered substantially different compared to hippocampal sclerosis in neurodegenerative diseases, in relation to regenerative capacities of hippocampus that are employed in epilepsy.31 However, degenerative component might be present after all, since the accumulation of lipofuscin has been related to neurodegenerative conditions and aging.32 It is still unclear whether lipofuscin has protective (e.g., via iron sequestration) or toxic effects on brain tissue. Finally, it should be mentioned that patients with neuronal ceroid lipofuscinose CLN8 develop epilepsy.32 It appears that GD neuron somata showed increased intensity of CAT immunofluorescence compared to control GD neurons, but, of course, immunohistochemistry could not be applied here as a quantitative method. A large increase in CAT activity/level in HS might be related to some widely present but subtle changes that could not be clarified by immunohistochemistry. It is important to note here that only excessive generation of H2O2 is “malevolent,” whereas a certain physiologic level of H2O2 is obligatory for normal functioning of a wide range of redoxsensitive proteins.22,33 For example, physiologic concentrations of H2O2 keep open KV7.2 channels, which conduct the M-current, a slow K+ current that serves as a brake for neuronal firing.34,35 There are some other H2O2-regulated K+ and Na+ channels, as documented on hippocampal neurons.36 It is tempting to speculate that augmented CAT activity in combination with collapsed vascularization (and probable insufficient delivery of oxygen) might affect redox signaling in some hippocampal regions. Of note, a recent study has found increased CAT level in cerebral hyaline astrocytic inclusions in the brain tissue of three pediatric patients with infantile spasms.37 There was no cell-specific staining for any of the investigated AOS enzymes in CA1 areas showing neuron loss and astrogliosis. Taking also into account the lack of correlation between the levels of astrocyte protein marker and AOS enzymes, this implies that the increase in activity/level of AOS enzymes cannot be attributed to HS-related changes in cell population, i.e., the increased number of astrocytes. This is important to point out because astrocytes might exhibit high AOS activity in some settings.38 Astrocytes in alveus were positive for CAT, GPx, and MnSOD in both control and sclerotic hippocampi. The Epilepsia, **(*):1–11, 2015 doi: 10.1111/epi.12981
FULL-LENGTH ORIGINAL RESEARCH
Hippocampal antioxidative system in mesial temporal lobe epilepsy *Aleksandar J. Risti c, †Danijela Savi c, *Dragoslav Sokic, ‡Jelena Bogdanovic Pristov, §Jelena Nestorov, ¶Vladimir Bas carevi c, ¶Savo Raicevic, #Slobodan Savic, and ‡Ivan Spasojevic Epilepsia, **(*):1–11, 2015 doi: 10.1111/epi.12981
SUMMARY
Dr. Aleksandar J. is an assistant Ristic professor at the University of Belgrade School of Medicine.
Objective: To examine antioxidative system in hippocampi of patients with mesial temporal lobe epilepsy associated with hippocampal sclerosis (mTLE-HS). Methods: Activity and levels of antioxidative enzymes—catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), manganese superoxide dismutase (MnSOD), and copper-zinc superoxide dismutase (CuZnSOD)—were assessed in hippocampi of nine pharmacoresistant mTLE-HS patients (mean age 37.7 [standard deviation] 6.6 years) who underwent amygdalohippocampectomy, and in 10 hippocampi obtained via autopsy from five neurologically intact controls (mean age 34.4 9.0 years). Subfield and cellular (neuron/astrocyte) distribution of CAT, GPx, and MnSOD was analyzed in detail using immunohistochemical staining. Results: Sclerotic hippocampi showed drastically increased activity of hydrogen peroxide–removing enzymes, CAT (p < 0.001), GPx (p < 0.001), and GR (p < 0.001), and significantly higher protein levels of CAT (p = 0.006), GPx (p = 0.040), GR (p = 0.024), and MnSOD (p = 0.004), compared to controls. CAT immunofluorescence was located mainly in neurons in both controls and HS. Control hippocampi showed GPx staining in blood vessels and CA neurons. In HS, GPx-rich loci, representing bundles of astrocytes, emerged in different hippocampal regions, whereas the number of GPx-positive vessels was drastically decreased. Neurons with abnormal morphology and strong MnSOD immunofluorescence were present in all neuronal layers in HS. Small autofluorescent deposits, most likely lipofuscin, were observed, along with astrogliosis, in CA1 in HS. Significance: Antioxidative system is upregulated in HS. This documents, for the first time, that epileptogenic hippocampi are exposed to oxidative stress. Our findings provide a basis for understanding the potential involvement of redox alterations in the pathology of epilepsy, and may open new pharmacologic perspectives for mTLE-HS treatment. KEY WORDS: Mesial temporal epilepsy, Hippocampal sclerosis, Glutathione peroxidase, Mitochondria, Superoxide dismutase, Catalase.
The involvement of oxidative stress in the pathology of epilepsy has been largely examined using animal models, whereas studies on human tissues are scarce. It is known
that some inherited epilepsies, such as myoclonic epilepsy with ragged red fibers, are related to dysfunctional mitochondria.1,2 These organelles are the main intracellular
Accepted February 26, 2015. *Center for Epilepsy and Sleep Disorders, Neurology Clinic, Clinical Center of Serbia, Belgrade, Serbia; †Department of Neurobiology, Institute for Biological Research “Sinisa Stankovic,” University of Belgrade, Belgrade, Serbia; ‡Life Sciences Department, Institute for Multidisciplinary Research, University of Belgrade, Belgrade, Serbia; §Department of Biochemistry, Institute for Biological Research “Sinisa Stankovic,” University of Belgrade, Belgrade, Serbia; ¶Institute for Neurosurgery, Clinical Center of Serbia, Belgrade, Serbia; and #Institute of Forensic Medicine, Medical School, University of Belgrade, Belgrade, Serbia Address correspondence to Aleksandar Ristic, Center for Epilepsy and Sleep Disorders, Neurology Clinic, Clinical Center of Serbia, Dr Subotica Starijeg 6, 11000 Belgrade, Serbia. E-mail: [email protected] Wiley Periodicals, Inc. © 2015 International League Against Epilepsy
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11 Antioxidative Enzymes in Epilepsy 32. Zhao L, Spassieva SD, Jucius TJ, et al. A deficiency of ceramide biosynthesis causes cerebellar purkinje cell neurodegeneration and lipofuscin accumulation. PLoS Genet 2011;7:e1002063. 33. Spasojevic I, Jones DR, Andrades ME. Hydrogen peroxide in adaptation. Oxid Med Cell Longev 2012;2012:596019. 34. Gamper N, Zaika O, Li Y, et al. Oxidative modification of M-type K (+) channels as a mechanism of cytoprotective neuronal silencing. EMBO J 2006;25:4996–5004. 35. Wuttke TV, Penzien J, Fauler M, et al. Neutralization of a negative charge in the S1-S2 region of the KV7.2 (KCNQ2) channel affects voltage-dependent activation in neonatal epilepsy. J Physiol 2008;586:545–555. 36. Rice ME. H2O2: a dynamic neuromodulator. Neuroscientist 2011;17:389–406. 37. Visanji NP, Wong JC, Wang SX, et al. A proteomic analysis of pediatric seizure cases associated with astrocytic inclusions. Epilepsia 2012;53:e50–e54. 38. Desagher S, Glowinski J, Premont J. Astrocytes protect neurons from hydrogen peroxide toxicity. J Neurosci 1996;16:2553–2562.
39. Duvernoy HM. The human hippocampus. Berlin: Springer-Verlag; 2005.
Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. Clinical and histologic data for patients with mTLE-HS. Figure S1. Representative Western blots for AOS enzymes. Figure S2. GFAP in hippocampal extracts of controls and patients with mTLE-HS.
Epilepsia, **(*):1–11, 2015 doi: 10.1111/epi.12981
