Epilepsy-Associated Reelin Dysfunction Induces Granule Cell Dispersion in the Dentate Gyrus&Z.Star;

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Epilepsy-Associated Reelin Dysfunction Induces Granule Cell Dispersion in the Dentate Gyrusq M Frotscher, Center for Molecular Neurobiology Hamburg, University Medical Center Hamburg-Eppendorf, Hamburg, Germany CA Haas, Medical Center – University of Freiburg, Faculty of Medicine, University of Freiburg, Germany

Introduction 1 BackgrounddGranule Cell Dispersion in Epilepsyda Migration Defect? 1 Methods 2 Recent Results 3 Unilateral Injection of Kainate, an Agonist of the Excitatory Transmitter Glutamate, Results in the Development of GCD in Mice 3 Reelin’s Role in the Development of GCD 4 Reelin Keeps Adult Granule Cells in Register 5 Summary and Conclusions 5 Future Directions 5 Further Reading 7

Introduction

Ammon’s horn sclerosis (AHS) is a hallmark of temporal lobe epilepsy. AHS is characterized by neuronal loss in hippocampal regions CA3 and CA1 and in the hilus of the dentate gyrus, and by granule cell dispersion (GCD). The term sclerosis describes a glial hypertrophy accompanying the neuronal loss. The neuron loss is likely to represent excitotoxic cell death associated with increased calcium entry into the cells. This mechanism of cell death is supported by studies showing that intracellular application of calcium buffers prevents excitotoxic cell death of hilar neurons. Neurons in hippocampal region CA2, which is located in between CA1 and CA3, are less vulnerable and are particularly rich in calcium-binding proteins. The factors that induce a broadening of the granule cell layerdie, granule cell dispersiondare less clear. Does increased neuronal activity force the granule cells to leave their normally tightly packed layer? Does seizure activity stimulate neurogenesis in the dentate gyrus, leading to an increase in neuron number and thus to a broadening of the granule cell layer? Does glial hypertrophy in AHS play a role in the development of granule cell dispersion? In this article, we summarize our recent studies which addressed these questions. In these studies, we used tissue samples from temporal lobe epilepsy (TLE) patients subjected to hippocampal resection for therapeutical reasons. In addition, we used tissue from mice that had received a unilateral hippocampal injection of kainate. By comparing our results in human tissue and in experimental animals, we obtained new insights into the processes leading to the development of granule cell dispersion in epilepsy.

BackgrounddGranule Cell Dispersion in Epilepsyda Migration Defect?

Granule cell dispersion is best described as an abnormal broadening of the granule cell layer, with many granule cells migrating far into the molecular layer (Fig. 1). GCD is regularly associated with Ammon’s horn sclerosis, but there are also cases of Ammon’s horn sclerosis lacking a prominent GCD. There is strong evidence that Ammon’s horn sclerosis, the degeneration of neurons in hippocampal regions CA1 and CA3 and in the hilus of the dentate gyrus, represents excitotoxic changes resulting from increased neuronal activity. Does this also hold true for GCD? The neurons that migrate out into the molecular layer do not show signs of neuronal degeneration. Alternatively, is GCD a migration defect of the granule cells during development that eventually leads to epilepsy? It would not be too far-fetched to assume a developmental migration defect, as there are mouse mutants that show a structural phenotype reminiscent of GCD and a functional phenotype of seizures/epilepsy. A more scattered distribution of the granule cells leads to an overlap of the normally segregated input and output sides of the granule cells. In a densely packed granule cell layer, the input side (ie, the granule cell dendritic arbors in the molecular layer) is normally separated from the output side (the mossy ﬁber axons invading the hilus). A loss of this clear-cut lamination will allow more superﬁcially located granule cells to contact dendrites of deeply located neurons, resulting in an increased granule cell-to-granule cell connectivity. Would this aberrant circuitry contribute to increased excitability of the granule cells, resulting in epileptic activity?

q Change history: December 2015. Author Carola Haas and author Michael Frotscher made minor corrections to the text with a major change to the paragraph “Future directions” (the changes are on page 12). In addition, a few references were added. They were not cited in the text since all other references of the original manuscript were also not cited in the text but added as “Further Reading”.

Reference Module in Neuroscience and Biobehavioral Psychology http://dx.doi.org/10.1016/B978-0-12-809324-5.00032-8 1 2 Epilepsy-Associated Reelin Dysfunction Induces Granule Cell Dispersion in the Dentate Gyrus

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Figure 1 Morphology of normal and epileptic human hippocampus stained with cresyl violet. (A) Human control hippocampus with normal distribution of neurons in hippocampal subfields and in the dentate gyrus, where the granule cells are located in a dense layer. (B) Epileptic human hippocampus with characteristic features of Ammon’s horn sclerosis. Selective cell loss is obvious in hippocampal subfields CA1 and CA3. The granule cell layer is dispersed. (C) Granule cell layer of a normal human dentate gyrus. The granule cells are arranged in a densely packed layer. (D) Loss of dense granule cell packing (granule cell dispersion) in temporal lobe epilepsy. Scale bars A, B: 600 mm; C, D: 75 mm. CA1, CA2, CA3, hippocampal subfields; GCL, granule cell layer. Reproduced from Haas, A., Frotscher, M., 2004. Migration disorders and epilepsy. In: Herdegen, T., Delgado- García, J.M. (Eds.), Brain. Damage and Repair. Kluwer Academic Publishers, Dordrecht, pp. 391–402; copyright Kluwer Academic Publishers.

One mutant that shows a migration defect similar to GCD is the reeler mouse lacking the extracellular matrix protein reelin. Reelin is known to be synthesized and secreted by Cajal-Retzius cells in the marginal zones of cortex and hippocampus, and there are severe migration defects in these regions in the reeler mutant. Could it be that GCD in epileptic patients is associated with an altered reelin expression? As a first step towards a better understanding of GCD, we looked at reelin expression in tissue samples from epileptic patients with GCD and in samples from autopsy controls. These control patients did not suffer from epileptic seizures and did not show Ammon’s horn sclerosis and GCD. We measured reelin expression by real-time PCR and quantified reelin-expressing cells using in situ hybridization for reelin mRNA. We found a significant decrease in reelin expression in patients with granule cell dispersion with both methods. Moreover, there was an inverse correlation between the number of reelin mRNA-expressing cells in the dentate gyrus and the extent of granule cell dispersion, quantified by measuring the width of the granule cell layer. While these findings pointed to a role of reelin in the development of granule cell dispersion, they could not clarify whether decreased reelin expression during development had caused granule cell dispersion which in turn led to epilepsy or, alternatively, epileptic seizures interfered with reelin expression, leading to GCD. For obvious reasons, these questions could not be addressed in studies of tissue samples from patients.

Methods

We used tissue samples from TLE patients and a mouse epilepsy model that recapitulates the main histopathological and electro- physiological features of human TLE. A single unilateral injection of the glutamate agonist kainate (KA) into the hippocampus of adult mice induced spontaneous, focal epileptic seizures and Ammon’s horn sclerosis, including GCD. We used cresyl violet staining, immunohistochemistry, in situ hybridization and real-time PCR analysis to monitor cell death, GCD development, neurogenesis, glial cell proliferation, and reelin expression in human tissue and tissue obtained from kainate-injected mice. Epilepsy-Associated Reelin Dysfunction Induces Granule Cell Dispersion in the Dentate Gyrus 3

Recent Results Unilateral Injection of Kainate, an Agonist of the Excitatory Transmitter Glutamate, Results in the Development of GCD in Mice Increased neuronal activity is associated with an increased release of the excitatory neurotransmitter, glutamate. Thus, glutamate receptor agonists, such as kainate, are often injected into animals to induce epileptic activity. Unilateral injection of kainate into one hippocampus (in mice) was not only found to induce seizure activity but also to lead to Ammon’s horn sclerosis with neuronal degeneration in CA1 and CA3 and granule cell dispersion in the dentate gyrus. Interestingly, these degenerative changes are not found on the contralateral, non-injected side. After a latency period of 2 weeks, the animals developed seizure activity as monitored by EEG recordings. They also developed a prominent granule cell dispersion on the side of kainate injection but not on the contralateral side, indicating that GCD resulted from the injection of the excitotoxin. The broadening of the granule cell layer developed progressively; GCD was evident as early as 1 week post injection and was fully expressed after 6 weeks (Fig. 2). What is the nature of kainate-induced GCD? Is it a migration of fully differentiated granule cells, or does seizure activity increase neurogenesis and aberrant migration of the newly generated granule cells? It is generally accepted that post-migratory neuroblasts that are about to differentiate their dendritic and axonal arbors are no longer able to migrate. How, then, could fully differentiated granule cells migrate out of a densely packed granule cell layer? In our experimental animals, we were in the position to address these questions by injecting them with bromodeoxyuridine (BrdU), a marker of newly generated cells. We found that neurogenesis was significantly decreased on the side of kainate injection (where GCD occurs) when compared to the non-injected control side (where no GCD was observed). Given the paucity of neurogenesis on the side that shows GCD, we concluded that adult, differentiated granule cells have “migrated”, and that GCD does not represent an abnormal migration of newly generated granule cells. The cessation of neurogenesis on the side of kainate injection came as a surprise, since studies of other epilepsy models had shown epileptic activity to induce neurogenesis. Further, while neurogenesis had ceased on the side of kainate injection, we noticed an increased gliogenesis, as revealed by double labeling for the astro- cytic marker GFAP (glial fibrillary acidic protein) and BrdU. It is reasonable to assume that this increased formation of astrocytes, particularly in the hilar region and in CA1 and CA3, is associated with the excitotoxic neurodegenerative changes observed in these regions. There was also an increase in activated microglial cells, as demonstrated by labeling for the microglial marker Iba1. Next, an attempt was made to determine whether or not increased neurogenesis is involved in the formation of GCD in human epileptic patients. To this end we used immunostaining for Ki67, a marker of the cell cycle, in tissue samples from epileptic patients. We were unable to find stained cells among the neurons that had migrated out of the granule cell layer, suggesting that in human epileptic patients, like in kainate-injected mice, granule cell dispersion is not an aberrant migration of young neurons.

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Figure 2 Development of granule cell dispersion induced by unilateral intrahippocampal kainate injection in the adult mouse. Coronal sections of hippocampus were stained with cresyl violet. (A) Contralateral, non-injected hippocampus displaying the normal histology of the hippocampal formation. (B–D) Ipsilateral hippocampus 7 days (B), 21 days (C) and 6 weeks (D) after kainate injection. A progressive dispersion of the granule cells (arrows) is observed. Note neuronal loss in CA1 (arrowheads), CA3, and hilus. Scale bar: 100 mm; GCL, granule cell layer. Reproduced from Heinrich, C., Nitta, N., Flubacher, A., et al., 2006. Reelin deﬁciency and displacement of mature neurons, but not neurogenesis, underlie the formation of granule cell dispersion in the epileptic hippocampus. J. Neurosci. 26, 4701–4713; copyright by the Society for Neuroscience. 4 Epilepsy-Associated Reelin Dysfunction Induces Granule Cell Dispersion in the Dentate Gyrus

Reelin’s Role in the Development of GCD As mentioned above, reelin expression was found to be decreased in tissue samples from epileptic patients. This finding pointed to a role of reelin in granule cell lamination, reminiscent of the lamination defects seen in the mouse mutant lacking reelin. However, the dispersed granule cells in the reeler mouse represent a maldevelopment of the granule cell layer, whereas GCD in epileptic patients appears to be a secondary effect associated with seizure activity. Numerous studies have shown that reelin is a molecule important for the normal development of cell and fiber layers in the hippocampus. However, it is less clear how one might explain a decreased reelin expression in tissue samples of adult epileptic patients. Animals with unilateral injections of kainate appeared useful in studies of reelin expression associated with GCD. On the kainate-injected side, where GCD developed, we found a dramatic decrease in reelin expression in quantitative real-time RT- PCR studies and by counting reelin mRNA-expressing cells using in situ hybridization (Fig. 3). On the contralateral side, where no granule cell dispersion developed, reelin expression was normal when compared with naïve control animals. The findings point to a close relation between reelin expression and the development of GCD, but did not elucidate the underlying mechanisms.

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Figure 3 Time course of reelin mRNA expression in the mouse hippocampus after unilateral intrahippocampal kainate injection. Reelin mRNA expressing neurons were localized by in situ hybridization histochemistry. (A) Contralateral hippocampus. Many reelin mRNA-positive neurons are present in CA1, CA3, in the hilus, and along the hippocampal fissure. (B) Ipsilateral hippocampus of a saline-injected control mouse 2 days after injection. The distribution of reelin mRNA-positive neurons is similar to that in (a). (C–F) Ipsilateral hippocampus 2 days (C), 7 days (D), 2 weeks (E) and 6 weeks (F) after kainate injection. (C) A dramatic reduction of reelin mRNA expression is evident all over the hippocampus 2 days after kainate injection. Except a few labeled cells, which are still present along the hippocampal fissure, no reelin mRNA-positive neurons can be detected in CA1 and in the hilus. (D) Seven days after kainate injection, a few reelin mRNA-positive neurons can be observed along the hippocampal fissure (arrows). (E, F) Numbers of reelin mRNA expressing cells remain low along the hippocampal fissure after two (E) and six (F) weeks following kainate injection, in parallel with a progressive development of GCD. GCL, granule cell layer. Scale bars: A–F, 100 mm. Reproduced from Heinrich, C., Nitta, N., Flubacher, A., et al., 2006. Reelin deficiency and displacement of mature neurons, but not neurogenesis, underlie the formation of granule cell dispersion in the epileptic hippocampus. J. Neurosci. 26, 4701–4713; copyright by the Society for Neuroscience. Epilepsy-Associated Reelin Dysfunction Induces Granule Cell Dispersion in the Dentate Gyrus 5

Reelin Keeps Adult Granule Cells in Register The CR-50 antibody against reelin has proven its usefulness in blocking reelin function in a variety of experimental paradigms. Working with the concept that a decreased reelin expression induces an aberrant migration of granule cells (similar to the aberrant migration of these neurons in reeler mutants), we reasoned that blocking reelin function should lead to granule cell dispersion. We thus used adult naïve mice that received unilateral infusions of CR-50 antibody into one hippocampus via osmotic minipumps for 1 week. For controls, unspecific IgG was infused. On the side of CR-50 infusion, we observed a significant development of GCD near the infusion site; no development of GCD was observed when unspecific IgG was injected. The results were quantified by measuring the thickness of the granule cell layer and comparing the infused hippocampus with the contralateral one. While no statistically significant differences were observed between the contralateral hippocampus and the ipsilateral hippocampus when unspecific IgG was infused, the thickness of the granule cell layer had significantly increased on the side of CR-50 injection (Fig. 4). These results clearly show that the expression of reelin is not only required for the normal development of dentate lamination but for the maintenance of a compact granule cell layer in the adult brain.

Summary and Conclusions

Correlating the results of studies on tissue samples from epileptic patients and in experimental animals and mouse mutants sheds light on the mechanisms leading to granule cell dispersion in epilepsy. A similar loss in granule cell lamination in reeler mutants and in human GCD prompted our study of reelin expression in tissue samples from patients. The decreased reelin expression we observed in human GCD led us to study reelin expression in an animal model of epilepsy. We found both a decreased reelin expression and GCD only on the side of kainate injection (not on the contralateral control side). Interestingly, there was ongoing postnatal neurogenesis on the contralateral side, but a cessation of neurogenesis on the kainate-injected side. From these findings, we concluded that injection of the glutamate agonist kainate induced seizure activity associated with GCD, paralleled by a decreased reelin expression. A causal link between the development of GCD and decreased reelin expression was found in our experiments with antibodies neutralizing reelin. Infusion of CR-50 antibody, but not of unspecific IgG, was sufficient to induce GCD in adult, naïve mice. In summary, we propose the following scenario for the development of GCD: 1. Seizure activity in epileptic patients, or in experimental animals induced by unilateral kainate injections, interferes with reelin expression. 2. The decreased reelin expression in adult animals or human patients allows mature granule cells to invade the molecular layer and the hilus. This result is consistent with a “stop signal function” of reelin. Reelin-expressing Cajal-Retzius cells in the marginal zone of the neocortex and in the molecular layer of the dentate gyrus prevent migrating neurons from invading these zones during development; in the hilus of the dentate gyrus reelin-expressing interneurons might be involved. In reeler mutants lacking reelin, but not in wild-type animals, the marginal zone of the neocortex is densely populated by neurons, and in the dentate gyrus the granule cells are dispersed and their characteristic dense packing is lost. Reelin appears to have a similar function in the adult organism: Decreased reelin expression, and thus a loss of the “stop signal”, allows granule cells to change their positions. These aberrantly “migrating” cells were not, in our studies, newly generated neurons but differentiated granule cells. 3. A more general conclusion from these studies is that there are molecules, such as reelin, which are important for the maintenance of the structural organization of the brain. Their malexpression may result in secondary structural changes such as granule cell dispersion. It is tempting to speculate that not only seizure activity, but also a variety of other noxious stimuli, may interfere with reelin expression (or with the expression of other control genes) and lead to structural changes in the adult brain that are reminiscent of developmental defects and contribute to neurological or psychiatric diseases.

Future Directions

Our results have shown that reelin is important for the maintenance of the laminated organization of the dentate gyrus. In epileptic patients with decreased reelin expression, in kainate-injected mice, and in the reelin-deﬁcient mutant reeler, dentate granule cells do not form a compact cell layer but are dispersed. Can the application of reelin rescue granule cell dispersion? Using slice cultures of reeler mutants as a model, we were able to show that the scattered distribution of the granule cells can be rescued by wildtype cocultures. Based on these ﬁndings, we have treated kainate-injected mice with infusions of recombinant reelin. As described, mice with unilateral injections of kainate develop epileptic seizures and show decreased reelin expression and granule cell dispersion on the injected side but not on the contralateral side. Following treatment with recombinant reelin we indeed observed a reduced granule cell dispersion, but there is a long way ahead before we will know whether or not reelin treatment may open up a new therapeutic avenue. The results reported here have shown that reelin expression is sensitive to epileptic activity. There may be other noxious factors that interfere with the expression of reelin or other molecules important for the maintenance of normal network structure and function. Along this line, it needs to be determined by molecular approaches in which way reelin expression is regulated. Moreover, our recent studies using a conditional reelin knockout mouse revealed that the mere loss of reelin in the adult hippocampus is not 6 Epilepsy-Associated Reelin Dysfunction Induces Granule Cell Dispersion in the Dentate Gyrus

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Figure 4 Effect of chronic CR-50 antibody infusion into the dentate gyrus of adult mice. Antibodies were chronically administered by osmotic pumps inserted in the right hippocampus over a period of 7 days followed by another 7 days of survival. Morphological changes were visualized by cresyl violet staining. Infusion of CR-50 resulted in a massive increase in the width of the granule cell layer (B, D) as compared to the contralateral side (A, C). The widening of the granule cell layer was confined to the site of injection, indicating that a local neutralization of reelin altered the lamination of adult granule cells. No change in lamination was observed after infusion of mouse control IgG (E, F). (E) Contralateral side. (F) Side of IgG infusion. Scale bars: A, B, E, F: 150 mm; C, D: 40 mm. (G) Quantitative evaluation of GCL width in CR-50 (n ¼ 6) and IgG-infused hippocampi (n ¼ 5). The width of the GCL was measured in the ipsi- and contralateral hippocampus in five sections/animal, and five measurements were taken per hippocampus. Mean values S.E.M. are shown. In the CR-50-injected animals, the width of the GCL was significantly increased as compared to the contralateral side (p < 0.0277) using the two sample Wilcoxon rank-sum test. Comparisons were of the unpaired two-sided type. Reproduced from Heinrich, C., Nitta, N., Flubacher, A., et al., 2006. Reelin deficiency and displacement of mature neurons, but not neurogenesis, underlie the formation of granule cell dispersion in the epileptic hippocampus. J. Neurosci. 26, 4701–4713; copyright by the Society for Neuroscience. Epilepsy-Associated Reelin Dysfunction Induces Granule Cell Dispersion in the Dentate Gyrus 7 sufficient to induce GCD in a dentate gyrus that underwent normal development but was then subjected to reelin deficiency. Addi- tional mechanisms might be involved such as yet unknown signaling cascades activated by neuronal overexcitation in human epilepsy and experimental epilepsy in animals. Similarly, changes in the proteolytic processing of reelin might play a role. In fact, there is recent evidence from our laboratories showing that proteolytic processing of reelin is impaired under epileptic condi- tions and contributes to the development of GCD. Finally, future studies need to address the effects of reelin signaling on cytoskel- etal reorganization which is required for the actual migration process. In spite of increasing knowledge about the signaling cascade activated by reelin binding to its receptors, very little is known about the ways in which reelin acts on the cytoskeleton to control cell movement and arrest, respectively.