A triplet repeat expansion genetic mouse model of infantile spasms syndrome, Arx(GCG)10+7, with interneuronopathy, spasms in infancy, persistent seizures, and adult cognitive and behavioral impairment

Abstract

Infantile spasms syndrome (ISS) is a catastrophic pediatric epilepsy with motor spasms, persistent seizures, mental retardation, and in some cases, autism. One of its monogenic causes is an insertion mutation [c.304ins (GCG)(7)] on the X chromosome, expanding the first polyalanine tract of the interneuron-specific transcription factor Aristaless-related homeobox (ARX) from 16 to 23 alanine codons. Null mutation of the Arx gene impairs GABA and cholinergic interneuronal migration but results in a neonatal lethal phenotype. We developed the first viable genetic mouse model of ISS that spontaneously recapitulates salient phenotypic features of the human triplet repeat expansion mutation. Arx((GCG)10+7) ("Arx plus 7") pups display abnormal spasm-like myoclonus and other key EEG features, including multifocal spikes, electrodecremental episodes, and spontaneous seizures persisting into maturity. The neurobehavioral profile of Arx mutants was remarkable for lowered anxiety, impaired associative learning, and abnormal social interaction. Laminar decreases of Arx+ cortical interneurons and a selective reduction of calbindin-, but not parvalbumin- or calretinin-expressing interneurons in neocortical layers and hippocampus indicate that specific classes of synaptic inhibition are missing from the adult forebrain, providing a basis for the seizures and cognitive disorder. A significant reduction of calbindin-, NPY (neuropeptide Y)-expressing, and cholinergic interneurons in the mutant striatum suggest that dysinhibition within this network may contribute to the dyskinetic motor spasms. This mouse model narrows the range of critical pathogenic elements within brain inhibitory networks essential to recreate this complex neurodevelopmental syndrome.

Figure 1.

Structure of the ARX protein and gene, and generation of Arx^(GCG)10+7 mice. A, Schematic model of the 564 residue ARX protein showing the exon structure, position, and relative size of known protein domains and the four polyalanine tracts [tract 1 is indicated (arrow)]. B, Schematics of exon 1- and 2-containing (1, 2) region of the Arx gene at Xp21.3 relevant to the (GCG)10+7 knock-in into polyalanine tract 1. The long arrows show the AseI-based Southern blot strategy using a 3′-probe distinguishing wild-type embryonic stem cells from those with the entire knock-in construct recombined (Knock-in + Neo) (C). A second strategy with the 5′-probe used EcoRI fragments (E to E). Probe bars represent 1 kb. The small arrow pairs indicate where diagnostic PCR primers anneal. The knock-in plus Neo schematic demonstrates the left and right homologous arms in the targeting construct, loxP sites flanking the (GCG)10+7 knock-in into polyalanine tract 1, and the frt-flanked neomycin-resistance cassette. The knock-in schematic illustrates the remaining sequence in targeted embryonic stem cells after flp recombinase removed the neomycin-resistance cassette. C, Southern blot of AseI-digested genomic DNA from embryonic stem cells that are WT or contain the knock-in plus neomycin-resistance cassette (KI+Neo), labeled with the 3′-probe. D, Gel of the 2 kb versus 0.335 kb PCR products (small hooked arrow pairs in schematics) from targeted stem cells before (+Neo) and after (Flp-Neo) Flp-recombinase removed the neomycin-resistance cassette. The latter cells were used to produce the chimeras founding the Arx^(GCG)10+7 lines. Molecular weight markers are indicated. E, Gel with the three possible genotyping PCR products. Female heterozygotes show both WT 0.277 kb and 0.335 kb (GCG)10+7 bands. Male hemizygous or female homozygous (GCG)10+7 show a 0.335 kb band (small hooked arrow pairs in B). Molecular weight markers are in 0.2 kb increments.

Figure 2.

Infant Arx^(GCG)10+7 pups display twice as many spontaneous, severe spasm-like movements as do wild-type littermates. A, Still image from a videorecording of a wild-type (no. 1) and three mutant 9-d-old male mouse littermates. Mutant pup no. 9 in lower right quadrant is in the midst of major spasm-like movements. B, Six seconds later, pup no. 9 tonically extends all limbs. C, Eleven seconds later, spasm-like movement in pup no. 9 is complete. D, Spontaneous high-amplitude movements, including startles and displacement of the entire body, occur twice as often in Arx^(GCG)10+7 pups as in nonmutant littermates. Arx^(GCG)10+7 pups (n = 13) are mutant males and females. Nonmutant pups (n = 24) are wild-type males and heterozygous females. ***p < 0.001 by one-way ANOVA. E, Mutants displayed spontaneous low-amplitude movements, including myoclonic twitches and short-distance kicks, at approximately the same rate as did nonmutant littermates (p = 0.24–0.36). Group data shown are average ± SEM.

Figure 3.

EEG abnormalities in Arx^(GCG)10+7 mice. A, Mutants under 21 d of age display sharp spike-slow wave transients followed by attenuation of background activity and an increase in high-frequency background rhythmic activity. The arrow indicates the beginning of a myoclonic twitch associated behaviorally with sudden head drop. This pattern is not seen in older mutants. The record is from temporal electrodes. B, EEG recording of a representative 29 s spontaneous generalized seizure seen in 19-d-old (and older) adult mutants. Slow versive movements of the head accompany these EEG seizures. C, The 6/s spike wave bursts accompanied by behavioral arrest are seen in mutants starting at 14 d through late adulthood. Recording electrode montage: L, left; R, right; F, frontal; T, temporal; P, parietal; O, occipital.

Figure 4.

Arx^(GCG)10+7 mutants behave with abnormally low anxiety, are cognitively impaired, and display an autism-like characteristic. A, The startle response of mice of both genotypes habituates similarly as louder prepulse sounds repeatedly precede the 120 dB startling sound. B, Rotarod tests show that mutant mice perform significantly better than wild-type mice in most trials, and both sets of mice improve their motor skills at a similar rate. C, Mutant mice behave abnormally in the light/dark exploration test, spending twofold longer in the lighted area and making 67% more transitions from dark to light. D, The conditioned fear test reveals impaired associative learning from contextual cues (environment and conditioning sound stimulus) with an aversive stimulus (footshock) in Arx^(GCG)10+7 mice, as assessed by freezing behavior. Data are average ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 versus wild type by one-way ANOVA.

Figure 5.

ARX-positive interneurons are significantly reduced from normal in the neocortex, hippocampus, and striatum of the Arx^(GCG)10+7 mutant. A, B, Low-magnification views of sections of cortex spanning from the pia to the white matter, immunostained with anti-ARX antibody, show a reduction of ARX+ cells throughout the Arx^(GCG)10+7 mutant motor/somatosensory cortex compared with that of Arx^+/+ WT sibling, but more so in the deeper layers. C, D, Higher magnification views (A, B, rectangles) demonstrate that, although ARX is localized in the nuclei of wild-type neurons and some mutant neurons, it is present in the cytoplasm of many mutant cortical neurons (D, large arrows). Note that the selected areas have the highest concentration of ARX+ interneurons for each genotype. E, F, A decrease in ARX+ interneurons in the Arx mutant is also evident in the hilus of the hippocampal dentate gyrus (arrows) compared with that of wild type. G, H, ARX+ interneurons in the mutant caudate–putamen are reduced to approximately one-half the wild-type level; ARX is localized in nuclei (arrows) in both genotypes. Scale bars: A, B, E, F, 200 μm; C, D, G, H, 100 μm.

Figure 6.

Calbindin D_28K-positive interneurons are less abundant in the neocortex, hippocampus, and striatum of the Arx^(GCG)10+7 mutant. A, B, Neocortex of WT and mutant motor/somatosensory cortex immunostained with anti-calbindin D_28K antibody, showing loss of calbindin+ cells throughout the Arx^(GCG)10+7 cortex compared with a WT sibling. Layers V–VI are indicated by the two-headed arrows. C, D, Higher magnification images (A, B, rectangles) show calbindin+ interneurons in layers I–IV. E, F, Calbindin+ interneurons are also decreased in the granule cell layer of the mutant dentate gyrus. G, H, Like the ARX+ interneurons, calbindin+ projection neurons (arrows) in the mutant striatum are reduced to approximately one-half the WT level. Scale bars: A, B, E, F, 200 μm; C, D, G, H, 100 μm.

Figure 7.

Severe reduction of striatal interneurons. The NPY+ interneuron population is reduced in the striatum of the Arx^(GCG)10+7 mutant, although present at WT levels in the neocortex. A, B, Full-thickness views of somatosensory cortex immunostained with anti-NPY antibody show that NPY+ cells (arrows) are not altered in number or location in the Arx^(GCG)10+7 mutant. C, D, NPY+ interneurons (arrows) are decreased by 31% in the mutant caudate–putamen compared with that of a WT sibling. Scale bars: A, B, 200 μm; C, D, 100 μm. The population of cholinergic interneurons in the striatum is reduced in mutant mice to 39% of wild-type values. E, Section of WT striatum showing numerous interneurons labeled with antibodies to ChAT. F, Markedly fewer cholinergic cells are detected in the same region of the Arx^(GCG)10+7 striatum. The arrows indicate immunostained cells. Scale bar, 100 μm.