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The CNTF-derived peptide mimetic Cintrofin attenuates spatial-learning deficits in a rat post-status epilepticus model
Neuroscience Letters 556 (2013) 170–175
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
The CNTF-derived peptide mimetic Cintrofin attenuates spatial-learning deficits in a rat post-status epilepticus model Vera Russmann a , Natalie Seeger a , Christina Zellinger a , Martin Hadamitzky a , Stanislava Pankratova b , Hannes Wendt a , Elisabeth Bock b , Vladimir Berezin b , Heidrun Potschka a,∗ a b
Institute of Pharmacology, Toxicology, and Pharmacy, Ludwig-Maximilians-University Munich, Germany Department of Neuroscience and Pharmacology, Protein Laboratory, Panum Institute, University of Copenhagen, Copenhagen N, Denmark
h i g h l i g h t s • • • • •
We report first in vivo efficacy data for the novel CNTF peptide mimetic Cintrofin. Impact of Cintrofin was evaluated in a rat post-status epilepticus model. Cintrofin prevented long-term alterations in number of neuronal progenitor cells. Cintrofin significantly attenuated the persistence of basal dendrites. Cintrofin exerted beneficial effects on disease-associated cognitive impairment.
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Article history: Received 5 August 2013 Received in revised form 10 September 2013 Accepted 1 October 2013 Keywords: Status epilepticus Epileptogenesis Mimetic peptide Ciliary neurotrophic factor Neurogenesis Learning
a b s t r a c t Ciliary neurotrophic growth factor is considered a potential therapeutic agent for central nervous system diseases. We report first in vivo data of the ciliary neurotrophic growth factor peptide mimetic Cintrofin in a rat post-status epilepticus model. Cintrofin prevented long-term alterations in the number of doublecortin-positive neuronal progenitor cells and attenuated the persistence of basal dendrites. In contrast, Cintrofin did neither affect acute status epilepticus-associated alterations in hippocampal cell proliferation and neurogenesis nor reveal any relevant effect on seizure activity. Whereas status epilepticus caused a significant disturbance in spatial learning in reversed peptide-treated rats, the performance of Cintrofin-treated rats did not differ from controls. The study confirms that Cintrofin comprises an active sequence mimicking effects of its parent molecule. While the data argue against an antiepileptogenic effect, they indicate a putative disease-modifying impact of Cintrofin. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The ciliary neurotrophic factor (CNTF) possesses various beneficial effects demonstrated in experimental models and clinical trials. It proved to promote neurite outgrowth, ameliorate learning and memory deficits, enhance glial glutamate uptake and thereby protect from excitotoxic cell damage [1–3,6,7,9,11]. Considering that neuronal plasticity and network reorganization as well as neuronal cell loss characterize the process of epilepsy development following an initial brain injury [12,15], treatment with CNTF might be
∗ Corresponding author at: Institute of Pharmacology, Toxicology, and Pharmacy, Ludwig-Maximilians-University, Koeniginstr. 16, D-80539 Munich, Germany. Tel.: +49 89 21802662; fax: +49 89 218016556. E-mail address: [email protected] (H. Potschka). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.10.003
an attractive prophylactic compound to interfere with epileptogenesis. In support of this hypothesis intrahippocampal injections of CNTF significantly attenuated kainate mediated cell death [8]. Translational development of respective non-invasive approaches is hampered by limitations in stability and brain distribution of large complex molecules [1]. Therefore, the most important work in this context relates to the development of small molecules that mimic or potentiate the activity of neurotrophic factors [1]. The region corresponding to the A helix–AB loop, and CD loop–D helix of CNTF has been shown to be important for high affinity interactions of CNTF with leukemia inhibitory factor receptor (LIFR) [10]. Recently, a peptide derived from this region termed Cintrofin has been demonstrated to induce STAT3, Akt and ERK phosphorylation in primary neurons, induce neuronal differentiation and promote neuronal survival in vitro [17]. Based on its promising in vitro effects, we decided to evaluate the disease-modifying and
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antiepileptogenic potential of Cintrofin in a rat post-status epilepticus (SE) model. 2. Materials and methods 2.1. Animals Female Sprague Dawley rats (200–224 g) were purchased from Harlan Winkelmann, An Venray, The Netherlands, or Charles River, Sulzfeld, Germany. All experiments were done in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). All efforts were made to minimize pain or discomfort of the animals used. 2.2. Determination of Cintrofin concentration in plasma and CSF Biotinylated Cintrofin (Schafer-N, Copenhagen, Denmark) was administered to 18 rats (Charles River, Sulzfeld, Germany; 10 mg/kg s.c.). At 15 min, 30 min, 1 h, 2 h, 4 h, and 8 h after injection blood samples were taken under fentanyl/droperidol/midazolam anesthesia. Cerebrospinal fluid (CSF) was collected 1 h after administration as described previously [18]. Cintrofin concentrations were measured using a competitive ELISA described recently [13] (for details see Data S1). Peptide concentration peaked in plasma 1 h after administration and was detected in CSF with ratio 8 (plasma) to 1 (CSF). Thus, the pharmacokinetic analyses confirmed that Cintrofin crosses the BBB (Supplementary Fig. 1). 2.3. Post-status epilepticus model Electrodes were stereotactically implanted into the right basolateral amygdala (BLA) of 60 rats (Harlan Winkelmann, An Venray, Netherlands) as described previously [14] (stereotactic coordinates in mm relative to Bregma AP – 2.2; L – 4.7; DV – 8.5). Bupivacain 0.5% was used for local anesthesia. For induction of a SSSE, rats (n = 34) were electrically stimulated via the BLA electrode as described previously [19]. 2.4. Treatment with the CNTF-derived peptide mimetic Cintrofin and BrdU labeling The sequence of Cintrofin was derived from human CNTF (148-DGGLFEKKLWGLKV-161; UniProtKB entry P26441). Cintrofin and a respective control peptide (amino acids order reversed) were synthesized by GL Biochem Ltd. (Shanghai, China). Rats were treated with either reversed peptide or Cintrofin (10 mg/kg s.c.). Five minutes following SSSE animals received the first administration, which was repeated at days 1–4 following SSSE. Furthermore, rats received a total of ten i.p. injections of 50 mg/kg 5 Bromodeoxyuridine (BrdU; Sigma–Aldrich, Taufkirchen, Germany), which was injected twice daily at days 5–9 following SSSE (interval 8 h). 2.5. Monitoring and behavioral evaluation Seven weeks after SSSE, animals were monitored for 19 days as described elsewhere [4,14]. Since only 16 rats could be EEGmonitored at the same time, we randomly chose rats from each group (n = 8/treatment group) for monitoring of spontaneous seizures. For rating severity of seizures, Racine’s scale was used [16]. Eleven weeks after SSSE, behavior of the animals was evaluated in groups of n = 10 in open field (OF), elevated plus maze (EPM), Black-white box (BWB) and Morris Water Maze (MWM). During the MWM testing phase one animal was euthanized reducing the
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number of Cintrofin-treated SE rats to n = 9. Behavioral paradigms were performed as described previously [4,14,19]. 2.6. Histological and immunohistological evaluation Following behavioral analyses, rats were perfused as reported previously [19]. Nissl staining and immunohistochemistry was performed as described earlier [14,20] (for details see Data S2). One series was Nissl-stained with thionin to verify the electrode localization and to visualize neurodegeneration. Cell proliferation in the early phase following SE was assessed by BrdU staining, whereby neurons were identified by BrdU/NeuN double-staining. Neuronal progenitor cells and early postmitotic neurons were evaluated based on doublecortin expression. Activated microglia were identified based on expression of the surface marker ED1. Evaluation of neurodegeneration was performed in CA1, CA2, CA3a, CA3c, hilus, dentate gyrus, parietal and piriform cortex of the hippocampal formation. Severity of neuronal damage was assessed by a grading system used in previous studies [4,5]. The number of doublecortin-labeled cells and hilar basal dendrites was quantified by unbiased stereological counting as described previously [21]. In the CA1, CA3a, CA3c, hilus and dentate gyrus activated microglia were assessed by a grading system described previously [19]. Double-labeled cells were counted in at least six sections per animal as reported in former studies [19,20]. Due to damage of some brain sections we were not able to analyze data from each rat. BrdU-, BrdU/NeuN-, and ED1-labeling were analyzed in reversed peptide-treated control rats (n = 10), reversed peptide-treated SE rats (n = 10), Cintrofin-treated control rats (n = 10), and Cintrofintreated SE rats (n = 9). Doublecortin-labeled cells were analyzed in reversed peptide-treated control rats (n = 10), reversed peptidetreated SE rats (n = 10), Cintrofin-treated control rats (n = 10), and Cintrofin-treated SE rats (n = 8). 2.7. Statistics Seizure frequency and duration were analyzed by Mann–Whitney U-test. Kruskal–Wallis test followed by Mann–Whitney U-test was used for the analysis of neurodegeneration and microglia activation. Behavioral parameters and differences in proliferation rates and neurogenesis were analyzed by two-way analysis of variance, followed by post hoc comparison with Bonferroni. All tests were used two-sided and p < 0.05 was considered significant. 3. Results 3.1. Impact of Cintrofin on neurogenesis, hippocampal neurodegeneration and microglia activation In the granule cell layer and the hilus of reversed peptidetreated rats with SE the number of BrdU-labeled cells significantly exceeded that in reversed peptide-treated control rats without SE by 86% (Fig. 1A). In Cintrofin-treated rats the SE-associated increase in the cell proliferation rate reached comparable levels to reversed peptide-treated SE rats exceeding that in control rats without SE by 88% (p < 0.0001). In rats treated with the reversed control peptide analysis revealed the typical SE-associated increase in hippocampal neurogenesis, which was assessed based on BrdU/NeuN double-labeling (p < 0.0001) (Fig. 1B). Cintrofin did not alter this effect of SE. In the reversed peptide-treated group, SE resulted in longterm effects on the number of doublecortin-expressing neuronal progenitor cells with a significant increase as compared to control animals without SE (p < 0.05; Fig. 1D). The number of doublecortin-positive cells with basal dendrites was
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Fig. 1. Impact of Cintrofin on neurogenesis. Number of BrdU-positive cells (A) and BrdU/NeuN (B) double-labeled cells in the rat granule cell layer and the hilus of reversed peptide-treated controls (n = 10), reversed peptide-treated SE rats (n = 10), Cintrofin-treated controls (n = 10) and Cintrofin-treated SE rats (n = 9). A significant increase in the number of BrdU-positive cells was observed in the two SE groups in comparison to control rats (A). Newborn cells were further analyzed based on BrdU/NeuN doublelabeling (B). The number of BrdU/NeuN double-labeled cells was significantly increased in the two SE groups in comparison to control rats without SE. Double-labeling was verified by careful analysis of confocal z-series. In (C) two representative double-labeled cells of a Cintrofin-treated SE rat within the dentate gyrus are shown in high magnification. Scale bar: 10 m. Number of doublecortin-labeled cells (D) and hilar basal dendrites (E) in the hippocampus of reversed peptide-treated controls (n = 10), reversed peptide-treated SE rats (n = 10), Cintrofin-treated controls (n = 10) and Cintrofin-treated SE rats (n = 8). A significant increase in the number of doublecortin-labeled cells and hilar basal dendrites was observed in the reversed peptide-treated SE rats in comparison to reversed peptide-treated controls. Data are given as mean ± SEM (ANOVA, followed by Bonferroni, p < 0.05). Significant differences are indicated by asterisks. A high magnification photograph of a reversed peptide-treated SE rat is given in (F). A doublecortin-labeled cell body is positioned at the border of the granule cell layer (GCL) and the subgranular zone (SGZ). This cell has a long basal dendrite traveling deep into the hilus (H) (black arrows) and an apical dendrite extending into the GCL. Scale bar: 100 m.
increased by 3014% in reversed peptide-treated rats with SE as compared to respective controls (p < 0.01; Fig. 1E). Cintrofin treatment in the early phase following SE efficaciously prevented the SE-associated expansion of neuronal progenitor cells. Moreover, Cintrofin attenuated the persistence of basal dendrites.
Neurodegeneration scores in the CA2 (p = 0.0054), CA3a (p < 0.0001) and CA3c (p < 0.0001) region of the hippocampus as well as in the piriform cortex (p < 0.0001) revealed a typical SEassociated neuronal cell loss in reversed peptide treated rats. No evidence for neuronal cell loss (score 0) was found in control rats in the CA2, CA3a and CA3c region of the hippocampus
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as well as in the piriform cortex. In Cintrofin-treated SE rats neurodegeneration scores were comparable to those in rats receiving the reversed peptide [median: CA2: reversed peptide-treated 0.50 (range 0.00–1.50), Cintrofin-treated 1.00 (range 0.00–3.00); CA3a: reversed peptide-treated 1.00 (range 0.00–1.00), Cintrofintreated 1.00 (range 0.00–1.00); CA3c: reversed peptide-treated 1.00 (range 0.00–2.00), Cintrofin-treated 1.00 (range 1.00–2.00), piriform cortex: reversed peptide-treated 2.00 (range 0.00–3.00), Cintrofin-treated 2.00 (range 0.00–3.00); Supplementary Fig. 2]. In reversed peptide treated rats, SE resulted in significant increases in the number of activated microglia reaching a median score of 1.00 with a range of 1.00–2.50 in the CA1 (p = 0.0002), and a range of 1.00–2.00 in CA2 (p = 0.0002), CA3a (p = 0.0002) and CA3c (p < 0.0001) region of the hippocampus as well as the dentate gyrus (p = 0.0002). Cintrofin treatment did not counteract the SE-associated increase in activated microglia cells in a relevant manner. The score amounted to a median of 1.00 in all groups with a range of 0.00–3.00 in CA1, and a range of 1.00–2.00 in all remaining regions. In rats without SE, Cintrofin tended to expand the population of microglia cells reaching a significant difference to rats treated with the reversed control peptide in the CA3c region (p < 0.0001) [median: CA1 0.50 (range 0.00–1.00); CA2 0.50 (range 0.00–1.00); CA3a 1.00 (range 0.00–1.00); CA3c 1.00 (0.00–1.00); dentate gyrus 1.00 (range 0.00–1.00)]. Reversed peptide-treated control rats reached scores of 0.00 (range 0.00–1.00) in the CA1, CA2, CA3a and CA3c region of the hippocampus as well as the dentate gyrus. 3.2. Development of spontaneous seizures and behavioral analysis During the monitoring phase, spontaneous seizures were detected in all reversed peptide-treated and six out of 8 Cintrofintreated rats. The number of seizures recorded and observed in the two treatment groups did not differ in a significant manner (mean ± SEM; number of seizures recorded and observed: reversed
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peptide-treated 33.75 ± 30.63; Cintrofin-treated 3.63 ± 1.94). In the group of the reversed peptide-treated rats one animal exhibited an extremely high seizure number of 248 seizures. Without this outlier, the mean number of seizures amounted to 3.14 ± 1.37 in the group treated with the reversed control peptide. No group difference was evident in the mean duration of spontaneous seizures recorded during the monitoring phase (mean ± SEM: reversed peptide-treated 32.28 ± 8.87 s; Cintrofin-treated 48.06 ± 19.44 s). In the OF, rats with SE exhibited a significant increase in the locomotion parameters distance moved (p < 0.05) and velocity (p < 0.05) regardless of treatment (mean ± SEM; distance without SE: reversed peptide-treated 5393 ± 337, Cintrofintreated 5344 ± 488; distance with SE: reversed peptide-treated 6937 ± 588, Cintrofin-treated 7120 ± 358; velocity without SE: reversed peptide-treated 9.00 ± 0.56, Cintrofin-treated 8.91 ± 0.81; velocity with SE: reversed peptide-treated 11.57 ± 0.98, Cintrofintreated 11.87 ± 0.60). Cintrofin treatment reduced the time spend in the periphery of the OF (p < 0.05) and increased the time spend in the middle ring (p < 0.01) in rats with SE. No comparable effect of Cintrofin treatment was observed in Cintrofin-treated electrodeimplanted controls (data not shown). In the EPM paradigm, SE increased the locomotor activity of Cintrofin-treated rats (data not shown). Neither SE nor Cintrofintreatment affected the time spent on the periphery of the open arms and the frequency to enter the periphery of the open arms (data not shown). No group difference was evident in the behavior of the rats in the Black-white box paradigm (data not shown). In reversed peptide-treated SE rats, the mean latency to find the platform position in the MWM paradigm significantly exceeded that in the control group without SE at most testing days, i.e. days 2 (p < 0.05), 4 (p < 0.01) and 5 (p < 0.05) (Fig. 2A). In contrast, respective data did not differ in a significant manner between Cintrofin-treated rats with SE and Cintrofin-treated rats without SE (Fig. 2B). In the spatial probe trial, the performance of SE rats treated with Cintrofin did not differ in a significant manner from
Fig. 2. MWM. Acquisition learning of platform location of reversed peptide-treated animals (A) and Cintrofin-treated animals [reversed peptide-treated controls (n = 10), reversed peptide-treated SE rats (n = 10), Cintrofin-treated controls (n = 10), Cintrofin-treated SE rats (n = 9)]. (A and B) Illustrate the mean ± SEM of the time to reach the hidden platform during the five days MWM testing. On days 2, 4 and 5 reversed peptide-treated SE animals differ significantly in comparison to the corresponding control group. In the spatial probe trial the latency to the former platform position (C) and the frequency to cross the platform position (D) differ significantly in reversed peptide-treated SE rats in comparison to the corresponding control group. Significant differences are indicated by asterisks (ANOVA, followed by Bonferroni, p < 0.05).
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that of controls (Fig. 2C and D). In contrast, the performance was negatively affected by SE in animals receiving the reversed control peptide with significantly less crossings of the former platform position (p < 0.01) and significantly more time needed to find the former position (p < 0.05).
4. Discussion Testing of Cintrofin in a post-SE model and comparison with data from a group receiving a control reversed peptide demonstrated that Cintrofin can efficaciously prevent epileptogenesis-associated long-term alterations in the neuronal progenitor cell population and in spatial learning. CNTF seems to function as an endogenous regulator of hippocampal neurogenesis with enhancement of cell proliferation and neuronal differentiation [22]. In the present study, treatment with Cintrofin did not affect proliferation, neuronal differentiation and survival of neurons generated at days 5–9 following SE. However, analysis of doublecortin-expressing neuronal progenitor cells 13 weeks later indicated that Cintrofin treatment results in long-term cellular effects. In particular, the data confirmed a normalization of the proliferation rate and neuroblast formation in the chronic phase. Moreover, Cintrofin pretreatment efficaciously prevented the seizure-associated persistence of basal dendrites. The data suggest that Cintrofin promotes the normal development of newly formed dendrites into the direction of the molecular layer. Considering their network integration granule cell basal dendrites are discussed as a potential route for recurrent excitation between granule cells [23]. However, the fact that Cintrofin treatment counteracted basal dendrite persistence but did not affect the development of spontaneous recurrent seizures in the chronic phase rather argues against a critical contribution of hilar basal dendrites to hyperexcitability in the epileptic brain. Recent studies revealed that CNTF affects the activation state of microglial cells [24,25]. Considering these data we analyzed whether Cintrofin treatment exerted long-term effects on microglial cells. As expected, SE resulted in an expansion of the number of activated microglial cells in hippocampal sub-regions. Cintrofin treatment in the early phase following SE did not exert consistent effects on the population of ED1-positive microglia cells. Thus, it is unlikely that the long-term effects of Cintrofin on the neuronal progenitor population are linked to alterations in the functional state of microglial cells. In sham-implanted controls without SE the CNTF-peptide Cintrofin tended to promote microglia activation. These data further support in vitro studies describing effects of CNTF on the activity of microglia [24,25]. Moreover, the results suggest that Cintrofin comprises sequences of CNTF mediating the effects on microglia. Based on its neuroprotective effects CNTF is discussed as a putative therapeutic for various neurodegenerative CNS disorders [1,2,26] Intrahippocampal injection of the neurotrophic factor prior to induction of a SE by local kainate administration proved to reduce cell death [8]. In the post-SE model used in the present study the CNTF-derived peptide failed to protect from excitotoxic damage. The lack of neuroprotective effects might suggest that Cintrofin does not include relevant sequences of CNTF. However, the results might also reflect differences between the animal models as well as differences between the treatment schemes e.g. onset of Cintrofin administration following SE vs. onset of CNTF administration prior to SE. Whereas our data do not suggest an antiepileptogenic effect of Cintrofin, testing in a spatial learning paradigm indicated a diseasemodifying effect of the peptide mimetic. Beneficial effects of CNTF on spatial learning have been described following sub-chronic or
chronic treatment with the neurotrophic factor based on a cellular delivery approach or intraventricular administration in a focal ischemia and a Huntington’s model [11,6,7]. Moreover, the CNTF peptides 6 and 6c improved the performance in the MWM [3,27]. Our findings with Cintrofin in the post-SE model are in line with these data and additionally suggest that a short-term treatment with a CNTF-derived peptide can exert long-term beneficial effects on disease-associated cognitive impairment. Most importantly, the data confirm that Cintrofin efficaciously mimics the cognitionpreserving effects of its parent molecule. Testing in further behavioral paradigms revealed a reduction in anxiety-associated behavior in epileptic rats which was further promoted by Cintrofin treatment. However, considering the activity data of the epileptic rats, this alteration might rather reflect a hyperactivity and attention deficit syndrome, which occurs in epileptic patients [28] but has been rarely studied in rodent epilepsy models. In conclusion, the study confirms that the Cintrofin peptide mimetic comprises an active sequence mimicking effects of its parent molecule. In particular, Cintrofin can prevent disease-associated long-term alterations of hippocampal neuronal progenitor cells. Along with this effect Cintrofin treatment proved to partially counteract long-term cognitive deficits. Even though the data argue against an antiepileptogenic effect, they indicate a putative disease-modifying effect of Cintrofin.
Acknowledgements This research was supported by a grant of the Deutsche Forschungsgemeinschaft (DFG FOR 1103; PO-681/5-1). The authors thank Marion Fisch, Sieglinde Fischlein, Carmen Meyer, Angela Vicidomini, Andrea Wehmeyer and Heidrun Zankl for their technical assistance. The authors declare that they do not have any conflict of interest.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.neulet.2013.10.003.
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
The CNTF-derived peptide mimetic Cintrofin attenuates spatial-learning deficits in a rat post-status epilepticus model Vera Russmann a , Natalie Seeger a , Christina Zellinger a , Martin Hadamitzky a , Stanislava Pankratova b , Hannes Wendt a , Elisabeth Bock b , Vladimir Berezin b , Heidrun Potschka a,∗ a b
Institute of Pharmacology, Toxicology, and Pharmacy, Ludwig-Maximilians-University Munich, Germany Department of Neuroscience and Pharmacology, Protein Laboratory, Panum Institute, University of Copenhagen, Copenhagen N, Denmark
h i g h l i g h t s • • • • •
We report first in vivo efficacy data for the novel CNTF peptide mimetic Cintrofin. Impact of Cintrofin was evaluated in a rat post-status epilepticus model. Cintrofin prevented long-term alterations in number of neuronal progenitor cells. Cintrofin significantly attenuated the persistence of basal dendrites. Cintrofin exerted beneficial effects on disease-associated cognitive impairment.
a r t i c l e
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Article history: Received 5 August 2013 Received in revised form 10 September 2013 Accepted 1 October 2013 Keywords: Status epilepticus Epileptogenesis Mimetic peptide Ciliary neurotrophic factor Neurogenesis Learning
a b s t r a c t Ciliary neurotrophic growth factor is considered a potential therapeutic agent for central nervous system diseases. We report first in vivo data of the ciliary neurotrophic growth factor peptide mimetic Cintrofin in a rat post-status epilepticus model. Cintrofin prevented long-term alterations in the number of doublecortin-positive neuronal progenitor cells and attenuated the persistence of basal dendrites. In contrast, Cintrofin did neither affect acute status epilepticus-associated alterations in hippocampal cell proliferation and neurogenesis nor reveal any relevant effect on seizure activity. Whereas status epilepticus caused a significant disturbance in spatial learning in reversed peptide-treated rats, the performance of Cintrofin-treated rats did not differ from controls. The study confirms that Cintrofin comprises an active sequence mimicking effects of its parent molecule. While the data argue against an antiepileptogenic effect, they indicate a putative disease-modifying impact of Cintrofin. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The ciliary neurotrophic factor (CNTF) possesses various beneficial effects demonstrated in experimental models and clinical trials. It proved to promote neurite outgrowth, ameliorate learning and memory deficits, enhance glial glutamate uptake and thereby protect from excitotoxic cell damage [1–3,6,7,9,11]. Considering that neuronal plasticity and network reorganization as well as neuronal cell loss characterize the process of epilepsy development following an initial brain injury [12,15], treatment with CNTF might be
∗ Corresponding author at: Institute of Pharmacology, Toxicology, and Pharmacy, Ludwig-Maximilians-University, Koeniginstr. 16, D-80539 Munich, Germany. Tel.: +49 89 21802662; fax: +49 89 218016556. E-mail address: [email protected] (H. Potschka). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.10.003
an attractive prophylactic compound to interfere with epileptogenesis. In support of this hypothesis intrahippocampal injections of CNTF significantly attenuated kainate mediated cell death [8]. Translational development of respective non-invasive approaches is hampered by limitations in stability and brain distribution of large complex molecules [1]. Therefore, the most important work in this context relates to the development of small molecules that mimic or potentiate the activity of neurotrophic factors [1]. The region corresponding to the A helix–AB loop, and CD loop–D helix of CNTF has been shown to be important for high affinity interactions of CNTF with leukemia inhibitory factor receptor (LIFR) [10]. Recently, a peptide derived from this region termed Cintrofin has been demonstrated to induce STAT3, Akt and ERK phosphorylation in primary neurons, induce neuronal differentiation and promote neuronal survival in vitro [17]. Based on its promising in vitro effects, we decided to evaluate the disease-modifying and
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antiepileptogenic potential of Cintrofin in a rat post-status epilepticus (SE) model. 2. Materials and methods 2.1. Animals Female Sprague Dawley rats (200–224 g) were purchased from Harlan Winkelmann, An Venray, The Netherlands, or Charles River, Sulzfeld, Germany. All experiments were done in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). All efforts were made to minimize pain or discomfort of the animals used. 2.2. Determination of Cintrofin concentration in plasma and CSF Biotinylated Cintrofin (Schafer-N, Copenhagen, Denmark) was administered to 18 rats (Charles River, Sulzfeld, Germany; 10 mg/kg s.c.). At 15 min, 30 min, 1 h, 2 h, 4 h, and 8 h after injection blood samples were taken under fentanyl/droperidol/midazolam anesthesia. Cerebrospinal fluid (CSF) was collected 1 h after administration as described previously [18]. Cintrofin concentrations were measured using a competitive ELISA described recently [13] (for details see Data S1). Peptide concentration peaked in plasma 1 h after administration and was detected in CSF with ratio 8 (plasma) to 1 (CSF). Thus, the pharmacokinetic analyses confirmed that Cintrofin crosses the BBB (Supplementary Fig. 1). 2.3. Post-status epilepticus model Electrodes were stereotactically implanted into the right basolateral amygdala (BLA) of 60 rats (Harlan Winkelmann, An Venray, Netherlands) as described previously [14] (stereotactic coordinates in mm relative to Bregma AP – 2.2; L – 4.7; DV – 8.5). Bupivacain 0.5% was used for local anesthesia. For induction of a SSSE, rats (n = 34) were electrically stimulated via the BLA electrode as described previously [19]. 2.4. Treatment with the CNTF-derived peptide mimetic Cintrofin and BrdU labeling The sequence of Cintrofin was derived from human CNTF (148-DGGLFEKKLWGLKV-161; UniProtKB entry P26441). Cintrofin and a respective control peptide (amino acids order reversed) were synthesized by GL Biochem Ltd. (Shanghai, China). Rats were treated with either reversed peptide or Cintrofin (10 mg/kg s.c.). Five minutes following SSSE animals received the first administration, which was repeated at days 1–4 following SSSE. Furthermore, rats received a total of ten i.p. injections of 50 mg/kg 5 Bromodeoxyuridine (BrdU; Sigma–Aldrich, Taufkirchen, Germany), which was injected twice daily at days 5–9 following SSSE (interval 8 h). 2.5. Monitoring and behavioral evaluation Seven weeks after SSSE, animals were monitored for 19 days as described elsewhere [4,14]. Since only 16 rats could be EEGmonitored at the same time, we randomly chose rats from each group (n = 8/treatment group) for monitoring of spontaneous seizures. For rating severity of seizures, Racine’s scale was used [16]. Eleven weeks after SSSE, behavior of the animals was evaluated in groups of n = 10 in open field (OF), elevated plus maze (EPM), Black-white box (BWB) and Morris Water Maze (MWM). During the MWM testing phase one animal was euthanized reducing the
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number of Cintrofin-treated SE rats to n = 9. Behavioral paradigms were performed as described previously [4,14,19]. 2.6. Histological and immunohistological evaluation Following behavioral analyses, rats were perfused as reported previously [19]. Nissl staining and immunohistochemistry was performed as described earlier [14,20] (for details see Data S2). One series was Nissl-stained with thionin to verify the electrode localization and to visualize neurodegeneration. Cell proliferation in the early phase following SE was assessed by BrdU staining, whereby neurons were identified by BrdU/NeuN double-staining. Neuronal progenitor cells and early postmitotic neurons were evaluated based on doublecortin expression. Activated microglia were identified based on expression of the surface marker ED1. Evaluation of neurodegeneration was performed in CA1, CA2, CA3a, CA3c, hilus, dentate gyrus, parietal and piriform cortex of the hippocampal formation. Severity of neuronal damage was assessed by a grading system used in previous studies [4,5]. The number of doublecortin-labeled cells and hilar basal dendrites was quantified by unbiased stereological counting as described previously [21]. In the CA1, CA3a, CA3c, hilus and dentate gyrus activated microglia were assessed by a grading system described previously [19]. Double-labeled cells were counted in at least six sections per animal as reported in former studies [19,20]. Due to damage of some brain sections we were not able to analyze data from each rat. BrdU-, BrdU/NeuN-, and ED1-labeling were analyzed in reversed peptide-treated control rats (n = 10), reversed peptide-treated SE rats (n = 10), Cintrofin-treated control rats (n = 10), and Cintrofintreated SE rats (n = 9). Doublecortin-labeled cells were analyzed in reversed peptide-treated control rats (n = 10), reversed peptidetreated SE rats (n = 10), Cintrofin-treated control rats (n = 10), and Cintrofin-treated SE rats (n = 8). 2.7. Statistics Seizure frequency and duration were analyzed by Mann–Whitney U-test. Kruskal–Wallis test followed by Mann–Whitney U-test was used for the analysis of neurodegeneration and microglia activation. Behavioral parameters and differences in proliferation rates and neurogenesis were analyzed by two-way analysis of variance, followed by post hoc comparison with Bonferroni. All tests were used two-sided and p < 0.05 was considered significant. 3. Results 3.1. Impact of Cintrofin on neurogenesis, hippocampal neurodegeneration and microglia activation In the granule cell layer and the hilus of reversed peptidetreated rats with SE the number of BrdU-labeled cells significantly exceeded that in reversed peptide-treated control rats without SE by 86% (Fig. 1A). In Cintrofin-treated rats the SE-associated increase in the cell proliferation rate reached comparable levels to reversed peptide-treated SE rats exceeding that in control rats without SE by 88% (p < 0.0001). In rats treated with the reversed control peptide analysis revealed the typical SE-associated increase in hippocampal neurogenesis, which was assessed based on BrdU/NeuN double-labeling (p < 0.0001) (Fig. 1B). Cintrofin did not alter this effect of SE. In the reversed peptide-treated group, SE resulted in longterm effects on the number of doublecortin-expressing neuronal progenitor cells with a significant increase as compared to control animals without SE (p < 0.05; Fig. 1D). The number of doublecortin-positive cells with basal dendrites was
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Fig. 1. Impact of Cintrofin on neurogenesis. Number of BrdU-positive cells (A) and BrdU/NeuN (B) double-labeled cells in the rat granule cell layer and the hilus of reversed peptide-treated controls (n = 10), reversed peptide-treated SE rats (n = 10), Cintrofin-treated controls (n = 10) and Cintrofin-treated SE rats (n = 9). A significant increase in the number of BrdU-positive cells was observed in the two SE groups in comparison to control rats (A). Newborn cells were further analyzed based on BrdU/NeuN doublelabeling (B). The number of BrdU/NeuN double-labeled cells was significantly increased in the two SE groups in comparison to control rats without SE. Double-labeling was verified by careful analysis of confocal z-series. In (C) two representative double-labeled cells of a Cintrofin-treated SE rat within the dentate gyrus are shown in high magnification. Scale bar: 10 m. Number of doublecortin-labeled cells (D) and hilar basal dendrites (E) in the hippocampus of reversed peptide-treated controls (n = 10), reversed peptide-treated SE rats (n = 10), Cintrofin-treated controls (n = 10) and Cintrofin-treated SE rats (n = 8). A significant increase in the number of doublecortin-labeled cells and hilar basal dendrites was observed in the reversed peptide-treated SE rats in comparison to reversed peptide-treated controls. Data are given as mean ± SEM (ANOVA, followed by Bonferroni, p < 0.05). Significant differences are indicated by asterisks. A high magnification photograph of a reversed peptide-treated SE rat is given in (F). A doublecortin-labeled cell body is positioned at the border of the granule cell layer (GCL) and the subgranular zone (SGZ). This cell has a long basal dendrite traveling deep into the hilus (H) (black arrows) and an apical dendrite extending into the GCL. Scale bar: 100 m.
increased by 3014% in reversed peptide-treated rats with SE as compared to respective controls (p < 0.01; Fig. 1E). Cintrofin treatment in the early phase following SE efficaciously prevented the SE-associated expansion of neuronal progenitor cells. Moreover, Cintrofin attenuated the persistence of basal dendrites.
Neurodegeneration scores in the CA2 (p = 0.0054), CA3a (p < 0.0001) and CA3c (p < 0.0001) region of the hippocampus as well as in the piriform cortex (p < 0.0001) revealed a typical SEassociated neuronal cell loss in reversed peptide treated rats. No evidence for neuronal cell loss (score 0) was found in control rats in the CA2, CA3a and CA3c region of the hippocampus
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as well as in the piriform cortex. In Cintrofin-treated SE rats neurodegeneration scores were comparable to those in rats receiving the reversed peptide [median: CA2: reversed peptide-treated 0.50 (range 0.00–1.50), Cintrofin-treated 1.00 (range 0.00–3.00); CA3a: reversed peptide-treated 1.00 (range 0.00–1.00), Cintrofintreated 1.00 (range 0.00–1.00); CA3c: reversed peptide-treated 1.00 (range 0.00–2.00), Cintrofin-treated 1.00 (range 1.00–2.00), piriform cortex: reversed peptide-treated 2.00 (range 0.00–3.00), Cintrofin-treated 2.00 (range 0.00–3.00); Supplementary Fig. 2]. In reversed peptide treated rats, SE resulted in significant increases in the number of activated microglia reaching a median score of 1.00 with a range of 1.00–2.50 in the CA1 (p = 0.0002), and a range of 1.00–2.00 in CA2 (p = 0.0002), CA3a (p = 0.0002) and CA3c (p < 0.0001) region of the hippocampus as well as the dentate gyrus (p = 0.0002). Cintrofin treatment did not counteract the SE-associated increase in activated microglia cells in a relevant manner. The score amounted to a median of 1.00 in all groups with a range of 0.00–3.00 in CA1, and a range of 1.00–2.00 in all remaining regions. In rats without SE, Cintrofin tended to expand the population of microglia cells reaching a significant difference to rats treated with the reversed control peptide in the CA3c region (p < 0.0001) [median: CA1 0.50 (range 0.00–1.00); CA2 0.50 (range 0.00–1.00); CA3a 1.00 (range 0.00–1.00); CA3c 1.00 (0.00–1.00); dentate gyrus 1.00 (range 0.00–1.00)]. Reversed peptide-treated control rats reached scores of 0.00 (range 0.00–1.00) in the CA1, CA2, CA3a and CA3c region of the hippocampus as well as the dentate gyrus. 3.2. Development of spontaneous seizures and behavioral analysis During the monitoring phase, spontaneous seizures were detected in all reversed peptide-treated and six out of 8 Cintrofintreated rats. The number of seizures recorded and observed in the two treatment groups did not differ in a significant manner (mean ± SEM; number of seizures recorded and observed: reversed
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peptide-treated 33.75 ± 30.63; Cintrofin-treated 3.63 ± 1.94). In the group of the reversed peptide-treated rats one animal exhibited an extremely high seizure number of 248 seizures. Without this outlier, the mean number of seizures amounted to 3.14 ± 1.37 in the group treated with the reversed control peptide. No group difference was evident in the mean duration of spontaneous seizures recorded during the monitoring phase (mean ± SEM: reversed peptide-treated 32.28 ± 8.87 s; Cintrofin-treated 48.06 ± 19.44 s). In the OF, rats with SE exhibited a significant increase in the locomotion parameters distance moved (p < 0.05) and velocity (p < 0.05) regardless of treatment (mean ± SEM; distance without SE: reversed peptide-treated 5393 ± 337, Cintrofintreated 5344 ± 488; distance with SE: reversed peptide-treated 6937 ± 588, Cintrofin-treated 7120 ± 358; velocity without SE: reversed peptide-treated 9.00 ± 0.56, Cintrofin-treated 8.91 ± 0.81; velocity with SE: reversed peptide-treated 11.57 ± 0.98, Cintrofintreated 11.87 ± 0.60). Cintrofin treatment reduced the time spend in the periphery of the OF (p < 0.05) and increased the time spend in the middle ring (p < 0.01) in rats with SE. No comparable effect of Cintrofin treatment was observed in Cintrofin-treated electrodeimplanted controls (data not shown). In the EPM paradigm, SE increased the locomotor activity of Cintrofin-treated rats (data not shown). Neither SE nor Cintrofintreatment affected the time spent on the periphery of the open arms and the frequency to enter the periphery of the open arms (data not shown). No group difference was evident in the behavior of the rats in the Black-white box paradigm (data not shown). In reversed peptide-treated SE rats, the mean latency to find the platform position in the MWM paradigm significantly exceeded that in the control group without SE at most testing days, i.e. days 2 (p < 0.05), 4 (p < 0.01) and 5 (p < 0.05) (Fig. 2A). In contrast, respective data did not differ in a significant manner between Cintrofin-treated rats with SE and Cintrofin-treated rats without SE (Fig. 2B). In the spatial probe trial, the performance of SE rats treated with Cintrofin did not differ in a significant manner from
Fig. 2. MWM. Acquisition learning of platform location of reversed peptide-treated animals (A) and Cintrofin-treated animals [reversed peptide-treated controls (n = 10), reversed peptide-treated SE rats (n = 10), Cintrofin-treated controls (n = 10), Cintrofin-treated SE rats (n = 9)]. (A and B) Illustrate the mean ± SEM of the time to reach the hidden platform during the five days MWM testing. On days 2, 4 and 5 reversed peptide-treated SE animals differ significantly in comparison to the corresponding control group. In the spatial probe trial the latency to the former platform position (C) and the frequency to cross the platform position (D) differ significantly in reversed peptide-treated SE rats in comparison to the corresponding control group. Significant differences are indicated by asterisks (ANOVA, followed by Bonferroni, p < 0.05).
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that of controls (Fig. 2C and D). In contrast, the performance was negatively affected by SE in animals receiving the reversed control peptide with significantly less crossings of the former platform position (p < 0.01) and significantly more time needed to find the former position (p < 0.05).
4. Discussion Testing of Cintrofin in a post-SE model and comparison with data from a group receiving a control reversed peptide demonstrated that Cintrofin can efficaciously prevent epileptogenesis-associated long-term alterations in the neuronal progenitor cell population and in spatial learning. CNTF seems to function as an endogenous regulator of hippocampal neurogenesis with enhancement of cell proliferation and neuronal differentiation [22]. In the present study, treatment with Cintrofin did not affect proliferation, neuronal differentiation and survival of neurons generated at days 5–9 following SE. However, analysis of doublecortin-expressing neuronal progenitor cells 13 weeks later indicated that Cintrofin treatment results in long-term cellular effects. In particular, the data confirmed a normalization of the proliferation rate and neuroblast formation in the chronic phase. Moreover, Cintrofin pretreatment efficaciously prevented the seizure-associated persistence of basal dendrites. The data suggest that Cintrofin promotes the normal development of newly formed dendrites into the direction of the molecular layer. Considering their network integration granule cell basal dendrites are discussed as a potential route for recurrent excitation between granule cells [23]. However, the fact that Cintrofin treatment counteracted basal dendrite persistence but did not affect the development of spontaneous recurrent seizures in the chronic phase rather argues against a critical contribution of hilar basal dendrites to hyperexcitability in the epileptic brain. Recent studies revealed that CNTF affects the activation state of microglial cells [24,25]. Considering these data we analyzed whether Cintrofin treatment exerted long-term effects on microglial cells. As expected, SE resulted in an expansion of the number of activated microglial cells in hippocampal sub-regions. Cintrofin treatment in the early phase following SE did not exert consistent effects on the population of ED1-positive microglia cells. Thus, it is unlikely that the long-term effects of Cintrofin on the neuronal progenitor population are linked to alterations in the functional state of microglial cells. In sham-implanted controls without SE the CNTF-peptide Cintrofin tended to promote microglia activation. These data further support in vitro studies describing effects of CNTF on the activity of microglia [24,25]. Moreover, the results suggest that Cintrofin comprises sequences of CNTF mediating the effects on microglia. Based on its neuroprotective effects CNTF is discussed as a putative therapeutic for various neurodegenerative CNS disorders [1,2,26] Intrahippocampal injection of the neurotrophic factor prior to induction of a SE by local kainate administration proved to reduce cell death [8]. In the post-SE model used in the present study the CNTF-derived peptide failed to protect from excitotoxic damage. The lack of neuroprotective effects might suggest that Cintrofin does not include relevant sequences of CNTF. However, the results might also reflect differences between the animal models as well as differences between the treatment schemes e.g. onset of Cintrofin administration following SE vs. onset of CNTF administration prior to SE. Whereas our data do not suggest an antiepileptogenic effect of Cintrofin, testing in a spatial learning paradigm indicated a diseasemodifying effect of the peptide mimetic. Beneficial effects of CNTF on spatial learning have been described following sub-chronic or
chronic treatment with the neurotrophic factor based on a cellular delivery approach or intraventricular administration in a focal ischemia and a Huntington’s model [11,6,7]. Moreover, the CNTF peptides 6 and 6c improved the performance in the MWM [3,27]. Our findings with Cintrofin in the post-SE model are in line with these data and additionally suggest that a short-term treatment with a CNTF-derived peptide can exert long-term beneficial effects on disease-associated cognitive impairment. Most importantly, the data confirm that Cintrofin efficaciously mimics the cognitionpreserving effects of its parent molecule. Testing in further behavioral paradigms revealed a reduction in anxiety-associated behavior in epileptic rats which was further promoted by Cintrofin treatment. However, considering the activity data of the epileptic rats, this alteration might rather reflect a hyperactivity and attention deficit syndrome, which occurs in epileptic patients [28] but has been rarely studied in rodent epilepsy models. In conclusion, the study confirms that the Cintrofin peptide mimetic comprises an active sequence mimicking effects of its parent molecule. In particular, Cintrofin can prevent disease-associated long-term alterations of hippocampal neuronal progenitor cells. Along with this effect Cintrofin treatment proved to partially counteract long-term cognitive deficits. Even though the data argue against an antiepileptogenic effect, they indicate a putative disease-modifying effect of Cintrofin.
Acknowledgements This research was supported by a grant of the Deutsche Forschungsgemeinschaft (DFG FOR 1103; PO-681/5-1). The authors thank Marion Fisch, Sieglinde Fischlein, Carmen Meyer, Angela Vicidomini, Andrea Wehmeyer and Heidrun Zankl for their technical assistance. The authors declare that they do not have any conflict of interest.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.neulet.2013.10.003.
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