These data show that interneuron-specific loss of Arx
in mice results in a developmental form of epilepsy with characteristics similar to the phenotypes observed in humans with ARX
mutations. Importantly, the mouse model recapitulates both the human male and female conditions with all the males and approximately half of the females presenting with epilepsy. The finding that an interneuron specific loss of ARX
recapitulates many key characteristics of the human condition, with Arx
expression left intact in the developing neocortex, suggests a critical role for interneurons in the pathogenesis of epilepsy in these patients and strongly support the concept of an ‘interneuronopathy’ (Kato and Dobyns, 2005
), as the cause of some forms of developmental epilepsies, specifically including infantile spasms. This study is not the only model to endorse the interneuronopathy concept. Recent work in the Scn1a heterozygous knockout mice shows the pathophysiological mechanism for the seizures in those animals is an interneuron specific loss of the channel resulting in an overall reduction in inhibition (Yu et al.
The discovery of epilepsy in about half of female mice with heterozygous loss of Arx in interneurons is novel, and strongly supports our observation of epilepsy in about half of heterozygous or ‘carrier’ human females, at least among human females with unbiased ascertainment. An even higher proportion of females—about two-thirds—have abnormal development. The first reports of human ARX mutations described asymptomatic mothers as healthy carrier of the mutations. Our larger dataset confirms these reports, which we now attribute to a bias of ascertainment. Specifically, we hypothesize that heterozygous mothers of affected male probands have a bias of ascertainment toward a normal phenotype, which fits well with their having lived to adulthood and demonstrated high reproductive fitness. We also hypothesize the corollary that female probands have a bias of ascertainment toward an abnormal phenotype. These data are also consistent with many other X-linked disorders in humans, in which a substantial and sometimes large proportion of heterozygous females are affected, although they are typically less severely affected than hemizygous males (Dobyns et al., 2004).
One potential explanation for the variability in the female phenotype in this X-linked condition is X-inactivation. We tested the XCI status of the females by performing DNA methylation studies of the human AR
locus (Allen et al.
). There are two possible issues with these studies. First, a potential difference in X-inactivation between blood and brain exists due to mosaicism at the time X-inactivation is established (Novelli et al.
; Young and Zoghbi, 2004
), although at least one study in Rett syndrome has suggested that the XCI pattern in blood is an accurate indicator of XCI patterns in the brain in a majority of patients (Shahbazian et al.
). The other potential confounding issue with our X-inactivation studies, particularly the comparison of skewing within families is the possibility of crossovers between the ARX and AR loci. This data needs to be confirmed with either an XCI assay located close to the ARX gene or testing of polymorphic markers between the two genes. With these potential limitations, our data does not show clear evidence for increased skewing of XCI in either symptomatic or asymptomatic females, but the number of females tested is too low to draw firm conclusions. We found some differences in direction of XCI among female relatives in some families, but cannot yet conclude whether these differences are enough to impact the phenotype. We will need to study larger numbers of heterozygous females and test XCI status of the ARX gene itself in both humans and mouse, which will require development of new XCI assays in both groups, to settle the questions we raise here.
The epilepsy phenotype in the mice resembles an infantile spasms type seizure in several important ways. As in humans, the immature mice develop partial seizures (Racine Stage 5 partial seizures with secondary generalization) early in life that evolve into different seizure types, including an epileptic spasm seizure associated with an electrodecrement of EEG, and persist into adulthood. We recognize that evolution of the seizure phenotype in these animals differs from the human condition and appears to be ‘reversed’ with the epileptic spasms occurring earlier in humans and later in mouse. However, tonic or tonic–clonic focal seizures often precede or occur concurrently with the onset of infantile spasms in children (Carrazana et al.
; Ohtahara and Yamatogi, 2001
). Therefore, the apparent ‘reversal’ of the seizure phenotype that we observe may simply represent occurrence of the same early phenotype that is often observed in humans, rather than a reversal. Whether the sequence of seizures is ‘reversed’ or not, we do not believe that the minor discrepancies observed between human and mouse invalidate the Arx conditional knockout mouse as a viable model for the developmental epilepsies. The mouse and human central nervous systems differ significantly in development, structure and function, which could result in the persistence of a more immature cortical network. This in turn could explain any differences in evolution of the seizure phenotype.
Does this new Arx
conditional mutant mouse serve as an animal model of infantile spasm syndrome? Specific criteria for animal models of infantile spasm syndrome have been proposed (Stafstrom and Holmes, 2002
; Baram, 2007
), and this model meets most of these criteria, importantly including an appropriate age parallel. Several other animal models of infantile spasm syndrome have been proposed in the past (Baram and Schultz, 1995
; Velisek et al.
), but each lacks more than one feature of a valid animal model of infantile spasm syndrome, particularly the age parallel and spontaneity of seizures. Our model has the added strength of recapitulating one of the known genetic causes of human infantile spasm syndrome. Overall, the Arx
conditional knockout mouse will prove to be a useful model to study the underlying pathogenesis of infantile spasm syndrome and related early epileptic encephalopathies, as affected animals develop spontaneous seizures at the appropriate age including ‘spasm-like’ seizures, the seizures evolve as the animal matures, and the mice recapitulate the carrier state found in humans.
The proposed pathophysiological mechanism of the observed phenotype in these animals is a specific loss of interneurons resulting in an overall increase in excitation. Towards this end, a subtype specific loss of interneurons was found in this model, consisting of a primary loss of calbindin expressing interneurons. This subtype specific loss was not unexpected due to the use of this particular Dlx5/6CIG
line as the Cre. This Dlx5/6CIG
line employs the Dlx5/6 enhancer element I56i that has been shown to be more strongly expressed in Calbindin positive neurons compared to Calretinin positive cells (Ghanem et al.
; Potter et al.
). The Calretinin positive cells are under the control of a different enhancer element, URE2. The data from these studies suggest that 85–95% of Calbindin interneurons co-express with the I56i element but only 75–85% co-express with Calretinin (Ghanem et al.
; Potter et al.
). This difference likely explains the greater loss of Calbindin than Calretinin interneurons in our study. A second possible explanation for the seizure phenotype would involve pan-interneuronal abnormalities in these mice, but with the exception of the Calbindin subclass, the phenotype is not one of cell loss. Instead an abnormal network, mis-specification or subtle alterations in location could account for the observed functional defect.
Another issue with the interneuron changes found in these studies are that Arx−/+;Dlx5/6CIG females have greater cell loss than the Arx−/Y;Dlx5/6CIG males. We found increased Calbindin cell loss in the Arx−/+;Dlx5/6CIG females, while Calbindin cell loss was more variable in males; one had an equivalent loss and the others less. This is an interesting and unexpected finding. One possible explanation is variability of the Cre-recombinase activity. This could be addressed in vitro but would not directly answer the question. Future work will address this interesting finding.
In summary, the epilepsy phenotypes in the Arx−/y;Dlx5/6CIG male and corresponding female mice provide a new genetic mouse model of a human genetic developmental epilepsy, with many features of infantile spasms, and provides a useful model to study the underlying pathophysiology in this condition. Our results also significantly change the counselling regarding female fetuses or children with severe mutations of ARX. Further studies in these mice will permit a better understanding of the multiple roles of interneurons in establishing normal network properties in the developing brain. Understanding the pathophysiologic mechanism as to how these mice develop an infantile spasms-like phenotype may facilitate the development of more specific therapies against this malignant developmental epilepsy.