Candidate autism gene screen identifies critical role for cell-adhesion molecule CASPR2 in dendritic arborization and spine development

Abstract

Mutations in the contactin-associated protein 2 (CNTNAP2) gene encoding CASPR2, a neurexin-related cell-adhesion molecule, predispose to autism, but the function of CASPR2 in neural circuit assembly remains largely unknown. In a knockdown survey of autism candidate genes, we found that CASPR2 is required for normal development of neural networks. RNAi-mediated knockdown of CASPR2 produced a cell-autonomous decrease in dendritic arborization and spine development in pyramidal neurons, leading to a global decline in excitatory and inhibitory synapse numbers and a decrease in synaptic transmission without a detectable change in the properties of these synapses. Our data suggest that in addition to the previously described role of CASPR2 in mature neurons, where CASPR2 organizes nodal microdomains of myelinated axons, CASPR2 performs an earlier organizational function in developing neurons that is essential for neural circuit assembly and operates coincident with the time of autism spectrum disorder (ASD) pathogenesis.dendrite | synaptogenesis A utism spectrum disorders (ASDs) comprise a heterogeneous group of early developmental diseases characterized by repetitive and stereotypic behaviors and impairments in social interactions and language development. ASDs are highly heritable, with recent studies linking mutations at hundreds of genes to ASDs (1-3). These findings raised the fundamental question of how these genes might act in neural circuits without being essential for all brain function. Here, we have attempted to address this question by using a Ca 2+ -imaging screening platform to visualize changes in excitatory network activity following shRNA-mediated knockdown (KD) of prominent cell-adhesion molecules that have been repeatedly implicated in the development of ASDs. We found that molecular manipulation of several ASD candidate genes profoundly influences network activity as monitored by this assay. We observed the biggest effects with the neuronal cell-adhesion molecule contactin-associated protein 2 (CASPR2) that is encoded by the CNTNAP2 gene, leading us to specifically focus on the mechanisms by which this cell-adhesion molecule influences neural circuit development.Mutations in the CNTNAP2 gene have been repeatedly identified in ASD patients (for review, see ref. 4). In addition, mutations in CNTNAP2 also have been linked to epilepsy (5-7), Tourette syndrome (8, 9), schizophrenia (5, 7, 10), attention deficit hyperactivity disorder (ADHD) (11), learning disability (12, 13), and language impairment (14-16). Thus, CNTNAP2 is of central importance for human brain function, as additionally shown by recent in vivo MRI studies in which variations in the CNTNAP2 gene were associated with reduced frontal gray matter and altered functional connectivity (17,18).CASPR2 is a member of the contactin-associated protein family (19). CASPRs are referred to as neurexin IV in Drosophila and are highly homologous to neurexins, which are presynaptic celladhesion molecules (20-23). CASPR2 is best known for its role in mye...

Common genetic variation and schizophrenia polygenic risk influence neurocognitive performance in young adulthood

“…The marker showing the strongest association with sustained attention/vigilance, which surpassed genome‐wide significance cut‐off, is of potential interest since it is located within a brain expressed gene ( CNTNAP5 ) previously implicated in neuropsychiatric diseases with known disturbances in sustained attention, that is bipolar disorder, autism and attention‐deficit hyperactivity disorder [Djurovic S et al, 2010; Pagnamenta AT et al, 2010; Neale BM et al, 2010 ] . CNTNAP5 protein belongs to the neurexin superfamily of cell‐adhesion molecules, which have been shown to constitute key regulators of synapse formation and neurotransmission [Anderson GR et al, 2012; Karayannis T et al, 2014]. Suggestive evidence of association was observed for three additional loci, located within genes whose protein products are involved in glutamatergic signaling ( GRIK3 ) and synaptic vesicle trafficking processes ( ERC2 , BIN1 ).…”

Shining a light on CNTNAP2: complex functions to complex disorders

“…10 Furthermore, in neuronal cultures, transient Caspr2 knockdown produced decreased amplitude (but not frequency) of synaptic responses, suggesting a cell-autonomous impairment of synaptic transmission. 68 However, in a mouse model of Caspr2 loss, firing rate and amplitude were not affected. Instead, a reduction of synchronicity of neuronal firing in the somatosensory cortex was detected.…”

“…CASPR2 knockdown causes decreased dendritic arborisation in mouse cortical neurons, 68 suggesting a normal role in promoting outgrowth and connectivity. Axonal pathfinding defects were also observed when Nrx-IV was absent from Drosophila embryos.…”

Synaptic abnormalities and cytoplasmic glutamate receptor aggregates in contactin associated protein-like 2 /Caspr2 knockout neurons

“…However, CNTNAP2 is also abundant in the brain, where it is enriched in the synaptic plasma membrane fraction (14). Notably, a recent article reported that shRNA-mediated knockdown of CNTNAP2 in young developing cultured cortical neurons causes a significant and cell-autonomous reduction of AMPA and NMDA mEPSCs (miniature excitatory postsynaptic currents) and GABA mIPSCs (miniature inhibitory postsynaptic currents), without affecting presynaptic function (35). Knockdown also resulted in reduced dendritic arborization and reduced spine head size, but not spine density.…”

“…Because CNTNAP2 is at excitatory synapses, and Anderson et al (35) have shown that it regulates synapse development, we wanted to see whether it has synaptogenic properties. We used a coculture assay in which we expressed tagged CNTNAP2 constructs in HEK293 cells, which we then cocultured with hippocampal neurons.…”