Mutations in the Aristaless-related homeobox gene (ARX) have been causally linked to a variety of neurological conditions, particularly, infantile spasms syndrome. ARX is a developmentally regulated homeobox transcription factor with expression both in the ganglionic eminence and the cortical ventricular zone early in development¹. Postnatally, the expression pattern is restricted to GABAergic neurons in the cortex and basal ganglia. During development, ARX functions primarily as a transcriptional repressor²: modulating migration and fate specification of interneurons and controlling ventricular zone proliferation. How loss of function of ARX leads to an epilepsy phenotype is poorly understood. Three genetically modified mice lines have been generated^3–5 to address this issue. These models each develop epilepsy and all have changes in interneuron subtype patterns strongly implicating alterations of interneuron development as a cause of epilepsy. Analysis of these models will both further the molecular understanding of the function of ARX and allow dissection of the pathophysiological properties of the ARX related epilepsies. This chapter will review the current knowledge of the function of Arx, the Arx mouse models, and discuss how these models can lead to a better understanding of the role of interneuron loss in the development of epilepsy during early childhood.

INTRODUCTION

During the first few years of life there exists a spectrum of malignant epileptic disorders. These early epileptic encephalopathies often proceed from one clinical syndrome to another. The named syndromes include Early Infantile Epileptic Encephalopathy (Otahara’s Syndrome), Severe Myoclonic Epilepsy of Infancy (Dravet’s Syndrome), Infantile Spasms (West-Syndrome), Lennox-Gastaut Syndrome and a few others (for review see⁶, as well as various chapters in this book). Up until the last few years, the etiology of these conditions was unknown for many patients. Recently, a number of genes have been linked to early epileptic encephalopathies and often a single gene has been found to be responsible for multiple conditions. The Aristeless-related Homeobox gene (ARX) is, to date, one of the genes most commonly associated with these conditions. A sizeable literature has accumulated describing mutations in ARX that have been reported to cause a variety of neurological disorders, all with epilepsy as a major symptom. ARX was first associated with human disease by three groups in 2002.^7–9 The initial descriptions were of 3 different disorders: X-linked Lissencephaly with ambiguous genitalia (XLAG), X-linked mental retardation (XLMR) with epilepsy and spasticity, and X-linked Infantile Spasms Syndrome (ISSX). The finding of a single gene having significant clinical and genetic heterogeneity has lead to a considerable amount of research, both clinical and basic science, on this gene. In this chapter, we will introduce the clinical and molecular details of ARX, then discuss the molecular mechanisms of the gene, the rodent models of ARX related disease, and end with a discussion of the role of ARX in the pathophysiology of the early epileptic encephalopathies.

ARX IN CLINICAL NEUROLOGY

Mutations in the gene ARX, have been primarily described in boys, and generally result in a clinical phenotype with intractable epilepsy as a predominant feature. The mutations can be broadly classified as those resulting in brain malformations and those without brain malformations. The brain malformation syndromes include XLAG^8,10, Proud syndrome (Agenesis of the Corpus Callosum and ambiguous genitalia)¹¹ and hydranencephaly with abnormal genitalia¹² (OMIM 300215). The ARX disorders without brain malformations include XLMR^7,9, West Syndrome/ISSX^8,13, Partington syndrome (mental retardation with hand dystonia)^14–16 (OMIM 309510), X-linked myoclonic epilepsy with spasticity and intellectual development (XMESID)^17,18, and Ohtahara/Early Infantile Epileptic Encephalopathy Syndrome (EIEE)^19–21, and other cases with epilepsy, intellectual disability, and dystonia/spasticity as part of the syndrome.^22,23 A number of patients with ARX mutations, both with and without clear brain malformations have also been observed to have cysts within the basal ganglia.^4,12,13 Though not a clear malformation, this is a finding that may be typical in patients with ARX mutations.

In females, the only CNS malformation currently described is agenesis of the corpus callosum (ACC) with or without mental retardation (MR) and seizures.^4,12 Females have also been reported to have infantile spasms, epilepsy, and varying degrees of cognitive dysfunction without clear brain malformation.^4,12,24,25 All of these conditions have been linked to a large number of mutations in the gene. From this large group of mutations and conditions, a number of authors have debated the existence of a clear genotype-phenotype correlation.^12,26,27

Ostensibly, a clear genotype-phenotype correlation exists between the malformation phenotypes and the non-malformation phenotypes. Mutations leading to loss of protein function (deletions, non-sense, frame-shift, or splice site mutations) or to changes in the homeodomain (for details see below) lead to malformation syndromes, while missense mutations or expansions of the polyalanine tracts lead to epilepsy phenotypes with normal brain MRIs.¹² However, this genotype-phenotype correlation breaks down when one looks at severity of disease in the non-malformation phenotypes and females.²⁶ For example, the most common mutation reported (45% of all mutations), the expansion of the second polyalanine tract from 12–20 alanines, has been associated with a spectrum of phenotypes from mild MR and seizures to the severe phenotype of Ohtahara Syndrome, suggesting greater genotype-phenotype heterogeneity. Another instance where the genotype-phenotype correlation is less clear is in females with ARX mutations. There are mother-daughter and mother-aunt pairs where one is normal and the other has cognitive disabilities and epilepsy⁴, though this may be due to differences in X-inactivation. Overall, ARX mutations result in a spectrum of disorders all with epilepsy as a major phenotype. A degree of genotype-phenotype correlation exists, but other factors, such as familial modifiers or environmental mediators, must exist to explain the variability that does occur.

MOLECULAR BIOLOGY OF ARX

What Is ARX?

ARX encodes a transcription factor with a role in cortical development. Designated ARX in humans and Arx in mice, the gene codes for a paired class homeodomain protein and is the vertebrate ortholog to the Drosophila aristaless (al) gene. ARX resides at Xp22.11 and produces a 2.8-kb mRNA with significant homology between human and mouse (89.3% sequence and 95% amino acid homology). The gene encodes a 562 amino acid protein, localized to the nucleus, that functions by binding DNA.^7,28,29 Arx is expressed in the developing hypothalamus, thalamus, basal ganglia and cerebral cortex beginning at embryonic day 8 (E8). In the forebrain, Arx is expressed in the anterior neural plate at E8.^7,30 By E10.5, expression is clearly observed in the ventricular zone (VZ) of the dorsal cortex and begins to be in the mantle zone of the emerging ganglionic eminences (GE).³⁰ Ventral telencephalic Arx expression is clearly present in the mantle zone of the GE at E12.5.³⁰ By E14.5 Arx protein can be observed in the dorsal VZ, intermediate zone (IZ) of the GE, and in scattered cells in the marginal zone (MZ) and dorsal IZ and subventricular zone (SVZ)^7,30,31 (Figure 1A). The ventral expression, at E14.5, has settled into the SVZ of the GE, primarily in proliferating neuroblasts³⁰ as determined by co-labeling with Bromodeoxyuridine (BrdU) staining. Arx remains expressed as the cells leave the SVZ to tangentially migrate into the cortex.^8,30,32 The ventral and migratory expression of Arx has almost complete overlap with Gad1 (Glutamic acid decarboxylase 1) expression.³⁰ This expression pattern remains until shortly after birth, when the dorsal VZ and GE is no longer present and Arx expression in the forebrain is observed only in scattered cells in the cortex and basal ganglia (Figure 1B,C). Co-labeling experiments with different interneuron markers has revealed that greater than 75% of all interneurons are Arx positive.^{29–31,33,34} Arx is also expressed in the developing pancreas, testis, and muscle. Data from the pancreas suggests an important role of Arx in fate determination.^35–37 The role of ARX in the brain appears to be more complicated then in the pancreas.

Figure

Ontogeny of Arx protein staining in dorsal neocortex of a wild type mouse. E14.5, E18.5, and P14 coronal sections are presented. A. E14.5 coronal section (4×) with Arx staining (dark brown) in the GE and in the dorsal VZ (asterisk). Two trails (more...)

The functional role of Arx in the telencephalon is an active area of research. Recent studies suggest that Arx is primarily a transcriptional repressor^2,38, and through this mechanism, has important roles in pallial progenitor cell proliferation, non-radial cell migration of interneurons from the ganglionic eminence, and basal ganglia development.^8,32

ARX MODELS

Since the first description of Arx related disorders a number of Arx mouse models have been developed.^3–5,8,36 These models have led to different insights into the disorder. The first mouse was made by homologous recombination of a lacZ containing vector into exon 2, producing an Arx allele with a premature stop codon⁸ and resulting in a truncated protein. These mice died at P0-P2, but were found to have many of the features of XLAG in humans, including a poorly formed small cortex, absent corpus collosum, abnormal tangential migration and differentiation of interneurons, and abnormalities in the male genitalia.⁸ There was a severe alteration in interneuron migration, with a delay in onset and loss of all migrating interneurons except for those along the SVZ.⁸ These authors also uncovered changes in basal ganglia cell populations. Because of the early lethality of the mice, no behavioral or electrophysiological studies could be performed. Shortly afterwards, Collombat and colleagues created a mouse line with targeted knockout of exon 1 and 2 producing a mutant protein with the loss of the first 360 N-terminal amino acids.³⁶ These mice are born but die by P2 and are smaller and appear dehydrated.³⁶ The mice were first developed to study the role of Arx in pancreatic development, but have subsequently been used to study both the down stream targets of Arx and the alterations in interneurons, basal ganglia, and cholinergic neurons, as described in the preceding sections.^30,32,34,43 As these mice were also perinatal lethal, attributable to loss of glucagon producing alpha cells, no phenotypic or physiological analysis could be performed. The functional shortcomings of these two mice lines were solved with a series of 5 Arx model mice lines that were published in 2009.

The latter Arx models took two approaches to developing the mice. The first approach, from our lab, used the Cre-Lox system to stop expression of Arx in cells expressing Dlx5/6, a transcription factor that is expressed in the majority of developing interneurons within the GE.⁴ The Dlx5/6 promoter was chosen to drive the Cre recombinase as loss of interneurons in patients with Arx mutations are believed to be a major contributor to the epilepsy and infantile spasms phenotype.⁵⁶ We were, therefore, attempting to prove the hypothesis that interneuron dysfunction is central to the epilepsy phenotype in various brain malformations. Indeed, the conditional knockout (CKO) mice appear to give credence to this view. The adult CKO male mice had faster, higher voltage EEG tracings with multifocal spikes reminiscent of a hypsarrythmic pattern seen in infants.⁴ The male mice develop convulsive seizures at P14, the earliest time we were able to accurately assess for the presence of electrographic seizures. The male CKO mice, which live past P14, continue to have seizures and develop an infantile spasms like seizure type. The development of the spasm phenotype was found more in the adult animals, most likely due to difference in maturity and neuroanatomy between mice and humans. In addition, we found that approximately half of the female animals also developed seizures at P14 which persisted into adulthood. The loss (or partial loss) of Arx reduced the numbers of calbindin positive interneurons.⁴ Currently, we are determining if there are other interneuronal changes as well as the developmental time course of this loss. Our approach contrasts with the method of the two other laboratories that recently made Arx models.

The approach taken by these labs was to generate mice with known human mutations associated with disease.^3,5 The Noebels’ lab, developed a knock-in Arx mouse with an expansion of the first polyalanine tract, increasing the repeat size from 16 to 23 amino acids.³ These authors found the animals to have possible behavioral spasms between 7–11 days of age, electroclinical spasms between 14–21 days of age, then a development of other seizure types (behavioral arrest and motoric seizures) as the animals mature.³ Behavioral tests on these animals revealed deficits in anxiety, fear conditioning, and social behaviors. Notably, the animals were less anxious and had diminished retention of a conditioned stimulus compared to wild-type mice, suggesting more of an anxiety phenotype, then diminished ability to learn.³ The pathological alteration of the Arx expansion was a regional loss of a population of Arx expressing interneurons. A 50% reduction of Arx was found in the hilus of the hippocampus and straitum and a 68% decrease was discovered in somatosensory and motor cortex, but no change in parietal cortex.³ The authors reported a change in protein localization from nuclear to cytoplasmic, but this contrasts with the other Arx expanded mouse, which found no inclusions or alteration of protein localization.⁵ The partial loss of Arx positive interneurons was due primarily from loss of Calbindin positive cells, similar to the changes found in our Dlx5/6 conditional knockout mouse line. There was no loss of neuropeptide Y or Calretinin positive cells in the cortex. Finally, the authors reported a loss of Calbindin, NPY and cholinergic neurons in the striatum.³ Overall, the features of this mouse model were similar to the Arx conditional knockout.

The third group in the Kitamura laboratory generated three mouse lines. Two were knock-ins of known pathogenic mutations, both disrupting the homeodomain, but one mutation was associated with the XLAG phenotype and the other with the XLMR phenotype.⁵ The third line derived by this group was an expansion of 7 alanines to the first polyalanine tract⁵, a line similar to the mouse made by the Noebels’ lab. The XLAG mutation mouse line was similar in phenotype to the germ line knockout. This mouse was perinatal lethal, had smaller brains, severely diminished numbers of interneurons in the cortex, and had a large loss of cholinergic neurons in the striatum.⁵ The other two lines were viable and recapitulated aspects of the XLMR or ISSX phenotypes. These two lines both develop spontaneous seizures but of differing severities. Only 10% of XLMR point mutation mice were observed to have a seizure and no seizures were captured on video EEG, in the 3 mice that were recorded. In contrast, 70% of the expanded mice developed behavior seizures, and all (3/3) mice with EEG recordings developed seizures, but no interictal EEG abnormalities.⁵ Like the Noebels’ lab expansion mice, the two lines were tested for behavioral deficits. Both lines were observed to have impaired motor coordination and increased anxiety. The two lines were also found to learn poorly in both a passive avoidance task and a radial arm task, which tests hippocampal spatial memory.⁵ The differences in anxiety and motor phenotypes between the Kitamura and Noebels’ expanded mice could be due to the different background strains, C57BL/6J x 129Sv/SvEvBrd mice³ versus pure C57BL/6J mice⁵, or to other differences with strain generation, suggesting that in humans, genetic background and environmental features may modify the phenotypic expression in patients with the expansion mutation. This is indeed what other authors have proposed for the variability of phenotypes observed in patients with expansions of the first and second polyalanine tracts.²⁶

As with the other conditional lines, the two viable lines from the Kitamura lab also had deficiencies in interneuron and striatal neurons. In these animals, interneurons began to migrate at the appropriate time (E12.5) and migrated normally, but with reduced numbers of interneurons entering the cortex. Although the total numbers of interneurons were reduced in postnatal mice, these authors did not find an interneuron subtype-specific difference in either the XLMR or ISSX mutations.⁵ This contrasts with what was found in the basal ganglia of these mice. In the striatum, the XLAG mutation resulted in both a loss of radially and tangential migration, whereas the two less severe mutations only altered tangential migration of interneurons into the basal ganglia. Finally, there was also a loss of cholinergic neurons in the cortex and basal ganglia, but it was greater in the striatum.⁵ Overall, these five mouse lines have a number of similarities, but also a few differences. These lines can now be used to attempt to understand how Arx alters normal cortical circuitry and how these changed neuronal networks result in an epilepsy phenotype and varying degrees of cognitive dysfunction.

FUTURE DIRECTIONS FOR THE STUDY OF ARX

Over the last decade Arx has been linked to over 10 overlapping clinical disorders and a great deal of information regarding the expression pattern, molecular function, and role in cortical development has been generated. This work has answered many questions but inevitably has raised many more. Further study is necessary to understand how different mutations lead to the spectrum of disorders and whether genetic background effect, environment, or changes in the protein due to the mutations are the cause of such variability. Next, a full categorization of the Arx involved gene networks is needed and an understanding of how the gene would have differential effects in the ventral and dorsal telecephalon. Another area for future investigation is the role of Arx in the postnatal brain. Lastly, consideration of how alteration in interneurons leads to a seizure or cognitive phenotype is warranted, and the 4 mouse lines are available to study the pathophysiological changes that occur. The next decade will likely yield the answers to many of these questions.

Disclosure statement: these authors have nothing to disclose.

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