Ankyrin-G Regulates Forebrain Connectivity and Network Synchronization via Interaction with GABARAP

Abstract

GABAergic circuits are critical for the synchronization and higher order function of brain networks, and defects in this circuitry are linked to neuropsychiatric diseases, including bipolar disorder, schizophrenia, and autism. Work in cultured neurons has shown that ankyrin-G plays a key role in the regulation of GABAergic synapses on the axon initial segment and somatodendritic domain of pyramidal neurons where it interacts directly with the GABAA receptor associated protein (GABARAP) to stabilize cell surface GABAA receptors. Here, we generated a knock-in mouse model expressing a mutation that abolishes the ankyrin-G/GABARAP interaction (Ank3 W1989R) to understand how ankyrin-G and GABARAP regulate GABAergic circuitry in vivo. We found that Ank3 W1989R mice exhibit a striking reduction in forebrain GABAergic synapses resulting in pyramidal cell hyperexcitability and disruptions in network synchronization. In addition, we identified changes in pyramidal cell dendritic spines and axon initial segments consistent with compensation for hyperexcitability. Finally, we identified the ANK3 W1989R variant in a family with bipolar disorder, suggesting a potential role of this variant in disease. Our results highlight the importance of ankyrin-G in regulating forebrain circuitry and provide novel insights into how ANK3 loss-offunction variants may contribute to human disease. molecular mechanisms underlying the subcellular organization of cortical GABAergic synapses remain poorly understood. Abnormalities in GABAergic interneuron circuitry and decreased gamma oscillations have been implicated in many neurodevelopmental and neuropsychiatric disorders 1-8 . Thus, the understanding of the cellular and molecular mechanisms that contribute to the development and function of GABAergic synapses as well as identification of genetic variants that contribute to neuropsychiatric disorders is critical to the discovery of new therapeutic agents for the treatment of diseases involving altered inhibitory circuits.ANK3 encodes ankyrin-G, a fundamental scaffolding protein that organizes critical plasma membrane domains 9, 10 . Alternative splicing of ANK3 in the brain gives rise to three main isoforms of ankyrin-G: the canonical 190 kDa isoform, a 270 kDa isoform, and a giant, 480 kDa isoform. The 190 kDa isoform is expressed in most tissues and cell types throughout the body including brain, heart, skeletal muscle, kidney, and retina. The 270 kDa and 480 kDa isoforms of ankyrin-G are predominantly expressed in the nervous system, and arise from alternative splicing of a 7.8 kb vertebrate-specific exon 9, 11 . The 480 kDa ankyrin-G isoform has been identified as the master organizer of axon initial segments (AIS) and nodes of Ranvier, sites of action potential (AP) initiation and propagation 10 . This splice variant is necessary for the proper clustering of voltage-gated sodium channels, KCNQ2/3 potassium channels, the cell adhesion molecule neurofascin-186, and the cytoskeletal protein βIV-spectrin to excitable domains (reviewed in 12 )....

Structural basis of GABARAP-mediated GABAA receptor trafficking and functions on GABAergic synaptic transmission

“…The Ankyrin-G scaffold is known to be a major organizer of the AIS protein complex. Previous in vivo studies have revealed that giant ankyrin-G functions to stabilize the localization of GABA A R at membrane sectors of both the AIS and the somatodendritic regions of neurons 44,45 . Our demonstration of ankyrin peptides as powerful tools for empirically characterizing GABARAP-GABA A R complex functions implies that further engineering of giant ankyrins or chimeric variants could help to more precisely define the trafficking networks at and beneath the cell membrane micro-domains of neurons.…”

Roles of palmitoylation in structural long-term synaptic plasticity

“…Encoded by the ANK3 gene, Ankyrin-G contains diverse isoforms including small isoforms (100-120 kDa) and large isoforms, in which the larger isoforms (190,270 and 480 kDa) are highly expressed in neurons [281][282][283][284]. Compared with the extensively studied roles of 270 and 480 kDa isoforms in axon initial segment (AIS), nodes of Ranvier (NoR) and inhibitory GABAergic synapses [109,[285][286][287][288][289], the Ankyrin-G 190 kDa isoform (Ankyrin-G-190) was shown to function in regulating the dendritic spine structure and glutamatergic neurotransmission ( [244], Preprint [290]). At synapses, Ankyrin-G-190 forms subsynaptic nanodomains in the spine head surrounding PSDs and within the spine neck to stabilize dendrite and spine architecture both in vitro and in vivo ( [244], Preprint [290]).…”

A Novel Approach to Study Gamma-Band Coherence inEx VivoHippocampal Slices from a Mouse Model of Bipolar Disorder

“…All mice were aged, PND 21-40. C57BL/6J mice were used to develop the assay whereas the knock in Ank3 W1989R model was used as the mouse model of bipolar disorder (Nelson et al, 2018). A subset of the data in this manuscript appeared in Nelson et al 2018.…”

“…Oscillations in the gamma band are crucial for proper execution of many cognitive behaviors including attention, memory and sensory processing (György Buzsáki & Wang, 2012; Cardin et al, 2009; Fries, 2009; Siegle et al, 2014; Tallon-Baudry et al, 1998). Abnormalities in gamma oscillations have been observed in a variety of diseases including schizophrenia, bipolar disorder and Alzheimer’s disease (Goutagny et al, 2013; Gruetzner et al, 2013; Klein et al, 2016; Liu et al, 2012; Nelson et al, 2018; Spencer, 2011; Spencer et al, 2009).…”

“…To our knowledge, this is the first report to demonstrate the ability to generate and sustain gamma oscillations in a submerged ex vivo brain preparations from adult mice with high spatial precision. We demonstrate the power and utility of our approach by characterizing gamma oscillations generated in hippocampal slices from a mouse model of bipolar disorder (Nelson et al, 2018).…”