Increased susceptibility to kainic acid–induced seizures in Engrailed-2 knockout mice

doi:10.1016/j.neuroscience.2009.01.007

Neuroscience

Volume 159, Issue 2, 17 March 2009, Pages 842-849

https://doi.org/10.1016/j.neuroscience.2009.01.007 Get rights and content

Abstract

The En2 gene, coding for the homeobox-containing transcription factor Engrailed-2 (EN2), has been associated to autism spectrum disorder (ASD). Due to neuroanatomical and behavioral abnormalities, which partly resemble those observed in ASD patients, En2 knockout (En2^−/−) mice have been proposed as a model for ASD. In the mouse embryo, En2 is involved in the specification of midbrain/hindbrain regions, being predominantly expressed in the developing cerebellum and ventral midbrain, and its expression is maintained in these structures until adulthood. Here we show that in the adult mouse brain, En2 mRNA is expressed also in the hippocampus and cerebral cortex. Hippocampal En2 mRNA content decreased after seizures induced by kainic acid (KA). This suggests that En2 might also influence the functioning of forebrain areas during adulthood and in response to seizures. Indeed, a reduced expression of parvalbumin and somatostatin was detected in the hippocampus of En2^−/− mice as compared to wild-type (WT) mice, indicating an altered GABAergic innervation of limbic circuits in En2^−/− mice. In keeping with these results, En2^−/− mice displayed an increased susceptibility to KA-induced seizures. KA (20 mg/kg) determined more severe and prolonged generalized seizures in En2^−/− mice, when compared to WT animals. Seizures were accompanied by a widespread c-fos and c-jun mRNA induction in the brain of En2^−/− but not WT mice. Long-term histopathological changes (CA1 cell loss, upregulation of neuropeptide Y) also occurred in the hippocampus of KA-treated En2^−/− but not WT mice. These findings suggest that En2^−/− mice might be used as a novel tool to study the link between epilepsy and ASD.

Section snippets

Animals

The generation of En2^−/− mice was previously described (Joyner et al., 1991). The original En2 mutants (mixed 129 Sv×Swiss–Webster genetic background) were crossed at least three times into a C57BL/6 background. En2^+/− heterozygous mice of this background were obtained from the central animal facility of the University of Heidelberg, and used as founder animals to establish our own colony at the University of Pisa. Because En2^−/− mice are viable and fertile (Joyner et al., 1991), two separate

En2 is expressed in the adult mouse hippocampus and cerebral cortex

Neuroanatomical and behavioural studies performed on adult En2^−/− mice suggest that En2 might be expressed also in anterior brain structures during adulthood (Cheh et al 2006, Kuemerle et al 2007). We therefore investigated En2 mRNA expression in different brain areas of the adult mouse brain. To this purpose, we performed quantitative real-time RT-PCR experiments using the mitochondrial ribosomal L41 protein mRNA as a standard for quantification. As expected, L41 amplification gave comparable

Discussion

By using behavioral, gene expression and neuroanatomical analyses, we provided evidence that En2^−/− mice have an increased susceptibility to KA-induced seizures and long-term histopathology. En2^−/− mice displayed more severe and prolonged generalized seizures as compared to WT mice. The occurrence of generalized seizures in En2^−/− mice was accompanied by the widespread mRNA induction of the IEGs c-fos and c-jun, as well as CA1 cell loss and NPY upregulation in mossy fibers.

Systemic KA

Acknowledgments

We thank Giulio Cappagli, Adriano Tacchi and the technical/administrative staff of the Istituto di Neuroscienze del C.N.R. for excellent assistance, and Matteo Caleo, Wolfgang Wurst, Nilima Prakash, Sara Migliarini and Massimo Pasqualetti for helpful discussions and reagents. Y.B. is a recipient of a research grant from the National Research Council (CNR—“Ricerche Spontanee a Tema Libero”—RSTL Program). G.U.C. is a recipient of a research grant from the Italian Ministry of University and

References (47)

F. Antonucci et al.
Botulinum neurotoxin E (Bont/E) reduces CA1 neuron loss and granule cell dispersion, with no effects on chronic seizures, in a mouse model of temporal lobe epilepsy
Exp Neurol
(2008)
R. Benayed et al.
Support for the homeobox transcription factor gene ENGRAILED 2 as an autism spectrum disorder susceptibility locus
Am J Hum Genet
(2005)
Y. Bozzi et al.
Dopamine D2 receptor signaling controls neuronal cell death induced by muscarinic and glutamatergic drugs
Mol Cell Neurosci
(2002)
M.A. Cheh et al.
En2 knockout mice display neurobehavioral and neurochemical alterations relevant to autism spectrum disorder
Brain Res
(2006)
M.R. Cilio et al.
Anticonvulsant action and long-term effects of gabapentin in the immature brain
Neuropharmacology
(2001)
F.E. Dudek et al.
Epileptogenesis in the dentate gyrus: a critical perspective
Prog Brain Res
(2007)
K. Herrup et al.
The genetics of early cerebellar development: networks not pathways
Prog Brain Res
(2005)
M. Hidalgo-Sánchez et al.
Specification of the meso-isthmo-cerebellar region: the Otx2/Gbx2 boundary
Brain Res Brain Res Rev
(2005)
S. Jinno et al.
Cellular architecture of the mouse hippocampus: a quantitative aspect of chemically defined GABAergic neurons with stereology
Neurosci Res
(2006)
A. Joliot et al.
Identification of a signal sequence necessary for the unconventional secretion of engrailed homeoprotein
Curr Biol
(1998)

A.L. Joyner

Engrailed, Wnt and pax genes regulate midbrain–hindbrain development

Trends Genet

(1996)

T.L. Kemper et al.

The contribution of neuropathologic studies to the understanding of autism

Neurol Clin

(1993)

B. Kuemerle et al.

The mouse engrailed genes: a window into autism

Behav Brain Res

(2007)

E.W. Lothman et al.

Kainic acid induced limbic seizures: metabolic, behavioral, electroencephalographic and neuropathological correlates

Brain Res

(1981)

K. Morimoto et al.

Kindling and status epilepticus models of epilepsy: rewiring the brain

Prog Neurobiol

(2004)

J.V. Nadler et al.

Neuropeptide Y in the recurrent mossy fiber pathway

Peptides

(2007)

R.J. Racine

Modification of seizure activity by electrical stimulationII. Motor seizure

Electroencephalogr Clin Neurophysiol

(1972)

P.E. Schauwecker et al.

Genetic dissection of the signals that induce synaptic reorganization

Exp Neurol

(2000)

G. Sperk

Kainic acid seizures in the rat

Prog Neurobiol

(1994)

G. Sperk et al.

Neuropeptide Y in the dentate gyrus

Prog Brain Res

(2007)

T.P. Sutula et al.

Unmasking recurrent excitation generated by mossy fiber sprouting in the epileptic dentate gyrus: an emergent property of a complex system

Prog Brain Res

(2007)

J.O. Willoughby et al.

Fos induction following systemic kainic acid: early expression in hippocampus and later widespread expression correlated with seizure

Neuroscience

(1997)

M. Bauman et al.

Histoanatomic observations of the brain in early infantile autism

Neurology

(1985)

Cited by (65)

Quercetin alleviates kainic acid-induced seizure by inhibiting the Nrf2-mediated ferroptosis pathway
2022, Free Radical Biology and Medicine
Epilepsy is one of the most common neurological disorders in childhood. However, classical antiepileptic drugs are linked with drug toxicity and cognitive function impairment in children. Hence, it is essential to develop a novel therapy to solve this problem. Currently, studies indicate regulating the nuclear factor-erythroid 2-related factor 2 (Nrf2)-mediated ferroptosis pathway represents a potential advanced therapy for seizures. Hence, the present study aimed to explore whether quercetin, a natural polyphenol, could alleviate seizure-induced neuron death and preserve cognitive function by inhibiting Nrf2-mediated ferroptosis.
Kainic acid-induced epileptic mice model, morris water maze (MWM) test, cell counting kit-8 (CCK-8) assays, western blotting analysis, enzyme-linked immunosorbent assay, flow cytometry, quantitative real-time reverse transcription PCR (qRT-PCR), immunofluorescence staining, and RNA sequencing analysis were employed to explore the potential mechanisms by which quercetin exerts protective effects on seizure-induced neuron death in kainic acid-induced epileptic mice model and glutamate-induced HT22 neuronal cell death.
Our findings suggested the association between the Nrf2-mediated ferroptosis pathway and seizures in a clinical setting. Quercetin pretreatment alleviates seizure-like behaviors and cognitive impairment in KA-induced epileptic mice. Additionally, in vitro, co-treatment with quercetin effectively exerts neuroprotective effects in glutamate-induced HT22 neuronal cell death. These protective effects were also closely linked to regulating the Nrf2-mediated ferroptosis pathway. Furthermore, bioinformatic profiling revealed that the SIRT1/Nrf2/SLC7A11/GPX4 pathway plays a crucial role in the Glu-induced HT22 cell death pretreated with quercetin.
These findings indicated that quercetin effectively protects against seizure-induced neuron death in vivo and in vitro and alleviates cognitive function impairment via the SIRT1/Nrf2/SLC7A11/GPX4 pathway.
Effect of thiamazole on kainic acid-induced seizures in mice
2021, Saudi Journal of Biological Sciences
Citation Excerpt :
The outcomes of seizure mainly depend on the regions of brain that are affected by hyper activity (Bozzi and Borrelli 2013). Kainic acid (KA) is known to produce epileptic seizures in rodents as a result of selective degeneration of neurons that causes behavioral abnormalities (Ben-Ari, 1985, McKhann et al 2003, Tripathi et al 2009). As a potent agonist of glutamate receptors, KA produces excitotoxic damage of brain neurons (Chihara et al 2009, Wang et al 2005).
Kainic acid (KA) induced epileptic seizures in mice is a commonly used experimental model of epilepsy. Previous studies have suggested the roles of various neurotransmitters and oxidative stress in KA-induced seizures. An important role of hypothyroidism has also been suggested in epilepsy. Thiamazole (TZ) is an anti-hyperthyroid drug with antioxidant property. This study reports the effect of TZ on KA-induced epileptic seizures in mice, produced by intraperitoneal (IP) injection of KA (18 mg/kg). Prior to KA injection, the animals were treated with TZ (12.5, 25 and 50 mg/kg IP). Our results showed that in KA alone group, about half of the animals developed seizures. Pre-treatment of mice with TZ significantly increased the frequency of seizures in dose-dependent manner. Administration of TZ significantly reduced the latency time and aggravated the severity of seizures. TZ also increased the mortality in KA-treated mice. Striatal dopamine and serotonin levels were markedly increased in KA alone treated mice, which were not significantly affected by TZ treatment. Among the indices of oxidative stress, we observed a significant reduction in cerebral vitamin E whereas the levels of cerebral malondialdehyde and conjugated dienes were significantly increased in animals with high severity of seizures. In conclusion, TZ potentiated the frequency and severity of experimental seizure in mice. There is a possibility of altered metabolism of KA in presence of TZ that might have potentiated the toxicity of KA. These findings suggest a caution while administering anti-hyperthyroid drugs in epileptic seizures.
Neurobiological Mechanisms of Autism Spectrum Disorder and Epilepsy, Insights from Animal Models
2020, Neuroscience
Autism Spectrum Disorder (ASD) and epilepsy are two neurodevelopmental disorders that have a high comorbidity rate, suggesting that a common neurodevelopmental mechanism exists. However, to date there is no conclusive way to predict whether a child will develop either syndrome or both and to what degree associated phenotypes will be affected. Failure to consistently identify predictive patterns of ASD and/or epilepsy diagnosis stems from the fact that they are etiologically heterogeneous conditions and research into their neuropathological mechanisms becomes challenging. Whole genome/exome sequencing has advanced our understanding of the genetic causes of ASD and epilepsy to an extent that currently about half of all ASD as well as epilepsy cases are known to have a genetic basis. In fact, a picture is emerging of both conditions as a collection of distinct genetically defined disorders, although the role of environmental factors has also been established. A plethora of animal models, most of them based on identified human genetic mutations and a few on known environmental causes, have been developed. Animal models provide a major experimental avenue for studying the underlying cellular and molecular mechanisms of human disorders. They also provide invaluable preclinical tools that can be used to test therapeutic approaches. In this review, we first summarize the methods for validating mouse models of ASD and epilepsy. Second, we present the current models validated for the comorbidity and finally, we recapitulate the common pathomechanisms identified in these models with special emphasis on synaptic plasticity.
Retinal defects in mice lacking the autism-associated gene Engrailed-2
2019, Neuroscience
Citation Excerpt :
En2 is widely expressed in the midbrain/hindbrain region (including the cerebellum primordium), starting at embryonic day 8.5 and continuing throughout embryonic and postnatal development (Joyner, 1996; Gherbassi and Simon, 2006). En2 mRNA is also expressed in the hippocampus and cerebral cortex of adult mouse (Tripathi et al., 2009; Sgadò et al., 2013). Mice lacking the homeobox-containing transcription factor En2 (En2−/−) are a reliable model for investigating the neurodevelopmental basis of autism spectrum disorders (ASD).
Defective cortical processing of visual stimuli and altered retinal function have been described in autism spectrum disorder (ASD) patients. In keeping with these findings, anatomical and functional defects have been found in the visual cortex and retina of mice bearing mutations for ASD-associated genes. Here we sought to investigate the anatomy and function of the adult retina of Engrailed 2 knockout (En2^−/−) mice, a model for ASD. Our results showed that En2 is expressed in all three nuclear layers of the adult retina. When compared to age-matched En2^+/+ controls, En2^−/− adult retinas showed a significant decrease in the number of calbindin⁺ horizontal cells, and a significant increase in calbindin⁺ amacrine/ganglion cells. The total number of ganglion cells was not altered in the adult En2^−/− retina, as shown by Brn3a⁺ cell counts. In addition, En2^−/− adult mice showed a significant reduction of photoreceptor (rhodopsin) and bipolar cell (Pcp2, PKCα) markers. Functional defects were also present in the retina of En2 mutants, as indicated by electroretinogram recordings showing a significant reduction in both a-wave and b-wave amplitude in En2^−/− mice as compared to controls. These data show for the first time that anatomical and functional defects are present in the retina of the En2 ASD mouse model.
Behavioral phenotypes and neurobiological mechanisms in the Shank1 mouse model for autism spectrum disorder: A translational perspective
2018, Behavioural Brain Research
Autism spectrum disorder (ASD) is a heterogeneous group of neurodevelopmental disorders, characterized by early-onset deficits in social behavior and communication across multiple contexts, together with restricted, repetitive patterns of behavior, interests, or activities. ASD is among the most heritable neuropsychiatric conditions with heritability estimates higher than 80%, and while available evidence points to a complex set of genetic factors, the SHANK (also known as ProSAP) gene family has emerged as one of the most promising candidates. Several genetic Shank mouse models for ASD were generated, including Shank1 knockout mice. Behavioral studies focusing on the Shank1 knockout mouse model for ASD included assays for detecting ASD-relevant behavioral phenotypes in the following domains: (I) social behavior, (II) communication, and (III) repetitive and stereotyped patterns of behavior. In addition, assays for detecting behavioral phenotypes with relevance to comorbidities in ASD were performed, including but not limited to (IV) cognitive functioning. Here, we summarize and discuss behavioral and neuronal findings obtained in the Shank1 knockout mouse model for ASD. We identify open research questions by comparing such findings with the symptoms present in humans diagnosed with ASD and carrying SHANK1 deletions. We conclude by discussing the implications of the behavioral and neuronal phenotypes displayed by the Shank1 knockout mouse model for the development of future pharmacological interventions in ASD.
Impaired Neuronal Differentiation of Neural Stem Cells Lacking the Engrailed-2 Gene
2018, Neuroscience
Citation Excerpt :
A recent study also showed that GABAergic neurotransmission is altered in the autistic brain (Robertson et al., 2016). Accordingly, a reduced number of GABAergic interneurons as well as abnormalities in GABAergic neurotransmission have been found in the postnatal brain of several mouse models of ASD (Gogolla et al., 2009, 2014; Marin, 2012; Bozzi et al., 2018), including En2 mutant mice (Tripathi et al., 2009; Sgadò et al., 2013b; Allegra et al., 2014; Provenzano et al., 2014a). Both increased and decreased expression of En2 has been associated to ASD (see references in the Introduction), suggesting that a tight control of En2 expression is crucial for correct neural development.
The Engrailed-2 (En2) gene codes for a homeobox-containing transcription factor, involved in midbrain-hindbrain embryonic development. In postnatal brain, En2 is expressed in the ventral mesencephalon, cerebellum, hippocampus and neocortex. Two single-nucleotide polymorphisms (SNPs) that are associated to autism spectrum disorders (ASD) have been identified in the human EN2 gene. Accordingly, mice lacking the En2 homeodomain (En2^hd/hd, referred to as En2^−/−) show molecular, anatomical and behavioral “ASD-like” features. Among these, we previously showed a partial loss of GABAergic interneurons in the En2^−/− postnatal hippocampus and neocortex, accompanied by a marked decrease of brain-derived neurotrophic factor (BDNF) signaling, a crucial determinant of GABAergic differentiation. In order to better investigate the role of En2 in GABAergic interneuron differentiation, we generated and subsequently differentiated neural stem cells (NSCs) from basal ganglia and neocortex of En2^+/+ and En2^−/− mouse embryos. Wild-type NSCs from both basal ganglia and neocortex express En2, while mutant ones do not, as expected. As compared to En2^+/+ NSCs, En2^−/− NSCs derived from basal ganglia show impaired GABAergic differentiation accompanied by a reduced expression of the BDNF receptor trkB. Conversely, En2^−/− NSCs derived from the neocortex expressed high levels of trkB and readily differentiated into neurons, as En2^+/+ NSCs. Our results suggest that En2 contributes to GABAergic neuron differentiation from basal ganglia NSCs through a trkB-dependent BDNF signaling, thus providing a possible explanation for the reduced number of GABAergic interneurons detected in the En2^−/− postnatal forebrain.

View all citing articles on Scopus

¹: Present address: Neurogenetics Laboratory, A. Meyer Children's Hospital, Florence, Italy (P. Sgadò); Centre for Integrative Biology, University of Trento, Italy (S. Casarosa, Y. Bozzi).

View full text

Systems NeuroscienceIncreased susceptibility to kainic acid–induced seizures in Engrailed-2 knockout mice

Abstract

Section snippets

Animals

En2 is expressed in the adult mouse hippocampus and cerebral cortex

Discussion

Acknowledgments

Exp Neurol

Am J Hum Genet

Mol Cell Neurosci

Brain Res

Neuropharmacology

Prog Brain Res

Prog Brain Res

Brain Res Brain Res Rev

Neurosci Res

Curr Biol

Trends Genet

Neurol Clin

Behav Brain Res

Brain Res

Prog Neurobiol

Peptides

Electroencephalogr Clin Neurophysiol

Exp Neurol

Prog Neurobiol

Prog Brain Res

Prog Brain Res

Neuroscience

Histoanatomic observations of the brain in early infantile autism

Neurology

Systems Neuroscience
Increased susceptibility to kainic acid–induced seizures in Engrailed-2 knockout mice