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GEN News Highlights : Sep 29, 2011
Scientists Develop Knockout Mouse that Displays Autism-Related Behaviors and Abnormal Neural Circuitry
Cntnap2-deficient animals exhibited improvements in hyperactivity and repetitive behaviors when treated with risperidone.!--h2>
Scientists have developed a mouse with a single gene knockout that exhibits behaviors and symptoms characteristic of those in humans with autism spectrum disorders (ASD). The David Geffen School of Medicine-led team knocked out the mouse equivalent of the human CNTNAP2 (contactin associated protein-like 2) gene, which has previously been linked with autism.
The resulting Cntnap2-deficient mice showed deficits in the three core aspects of ASD-related behavior and in addition displayed hyperactivity and epileptic seizures, both of which occur in humans with CNTNAP2 mutations. Reporting in Cell, Daniel H. Geschwind, M.D., and Olga Peñagarikano say that the mouse model displayed anomalies in neural circuitry and synchronization in nerve firing but responded positively to the FDA-approved drug risperidone.
The authors claim their data not only demonstrate a functional role for CNTNAP2 in ASD but also provide a new in vivo tool for ASD research, particularly with respect to the formation of neural circuits both during embryogeneisis and postnatally. Their work is described in a paper titled “Absence of CNTNAP2 Leads to Epilepsy, Neuronal Migration Abnormalities, and Core Autism-Related Deficits.”
Among many genes implicated in the genetic basis of ASD, rare and common variants in CNTNAP2 have been linked with disorders such as autism through association, linkage, gene-expression, and imaging analyses. The Cntnap2 gene (also known as Caspr2) is expressed embryonically and encodes a neuronal transmembrane protein member of the neurexin superfamily. However, the authors continue, the fact that myelination takes place postnatally, combined with reports linking the gene with ASD, suggest that CNTNAP2 may also play a role in early brain development.
The authors generated C57BL/6J mice in which the Cntnap2 gene was knocked out (Cntnap2-/- mutants) and confirmed the animals lacked evidence of any CNTNAP2 protein in the brain. The animals appeared normal, and there was no difference in the weight or growth rate of knockouts in comparison with their wild-type littermates.
Cntnap2-/- mutants over six months of age did, however, display spontaneous seizures, which were consistently induced by mild stress during routine handling. Analysis of the knockout animals’ brain tissue showed no gross morphological changes but did detect the presence of ectopic neurons in the corpus callosum at two weeks of age, which persisted through adulthood.
Interestingly, the investigators remark, a previous study has reported the presence of ectopic neurons of unknown origin in the white matter in CDFE syndrome patients together with neuronal migration abnormalities such as abnormal arrangements of neurons in clusters or migratory rows in the deep layers of cortex.
When the team evaluated the laminar positioning of cortical projection neurons in wild-type and Cntnap2 knockout mice, they also found that the deep cortical layers knockouts included significantly increased numbers of neurons that displayed CUX1, a marker for upper cortical layer cells.
Additional neuronal anomalies in the Cntnap2-deficient animals included a reduced number of GABAergic interneurons, indicating a role for CNTNAP2 in GABAergic interneuron development. This finding was of particular note because GABAergic interneurons are widely believed to play a crucial role in the precise timing of neuronal activity, and prior research has suggested that abnormal neural synchrony represents a pathophysiological mechanism in ASD.
Indeed, imaging of neurons from the somatosensory cortex of Cntnap2-/- mice indicated that the neuronal firing pattern was highly asynchronous relative to that in wild-type mice, even though the average firing amplitude and average firing rates weren’t significantly different. This suggests that “the asynchronous firing observed in mutant animals is likely due to a network dysfunction rather than to abnormalities in neuronal activity or conduction per se.”
The Cntnap2 knockouts additionally displayed much greater locomotor activity than wild-type littermates in the open field test and when evaluated for motor coordination and balance using the rotorod: in fact, mutant mice performed significantly better than wild-type animals, the researchers write. This observation supported findings from previous studies in animal models of autism and hyperactivity.
Other differences between Cntnap2-/- mutants and wild-type animals were observed in terms of the knockouts demonstrating higher perseveration and evidence of repetitive behavior. The knockout mice in addition displayed social differences and spent significantly less time interacting with each other than wild-type animals and instead demonstrated increased repetitive behaviors such as grooming and digging. The knockout animals similarly exhibited abnormal vocal communication and impaired nesting behavior, which is mediated by dopaminergic function in mice.
The researchers then treated both Cntnap2-/- animals and their wild-type littermates using risperidone or a control for 7–10 days. The drug therapy decreased activity levels in the knockouts to those of normal wild-type mice and also improved nestbuilding scores. This latter task involves sustained activity, which indicated that the reduced activity levels in knockouts wasn’t due to a sedative effect of risperidone, the researchers point out.
Risperidone therapy also reversed the increased grooming behavior observed in knockout mice and perseveration in the T maze. However, it had no effect on social interaction scores or on sensory hypersensitivity.
“We show that the consequences of CNTNAP2 deficiency in the mouse resemble many of the behavioral and cognitive features observed in patients with idiopathic autism and of the pathological features observed in patients with recessive CNTNAP2 mutations that cause a Mendelian form of syndromic autism,” the authors conclude. “In the current study, neuropathological analysis of Cntnap2-/- mice revealed two major mechanisms that have been shown to lead to epilepsy in humans, cortical neuronal migration abnormalities, and a reduction in the number of GABAergic interneurons.”
These mechanisms, combined with the observation of normal global neuronal activity in terms of firing rate and amplitude, suggest that the asynchronous firing pattern is due to an abnormal neuronal circuit architecture, rather than deficits in neuronal function per se, they add.
“That risperidone also normalized the nesting ability in KO mice provides additional evidence for abnormal striatal dopaminergic function in this model. In addition, the dissociation between repetitive and social behavior with regards to treatment response suggests that Cntnap2-/- mice will be useful for dissecting the distinct circuitries involved in these core components of autistic-related abnormal behavior.”
The team admits that while the neuronal migration abnormalities may have been expected—given previous findings in patients with CDFE syndrome—the reduction in GABAergic neurons was unexpected, “as CNTNAP2 has not been previously associated with GABAergic neuronal function and no such deficit has been demonstrated in CDFE.”
They suggest further studies will be needed to assess interneurons in patients with CDFE and also to discover whether reduction in interneurons in the knockout mice is due to a migration deficit or to a defect in neurogenesis, differentiation, and/or survival.
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