We show that the W1989R mutation causes decreases in GABAergic synapses in layer II/III of somatosensory cortex and CA1 of hippocampus, while sparing inhibitory synapses on cerebellar Purkinje neurons and thalamic neurons. reduction in forebrain GABAergic synapses resulting in pyramidal cell hyperexcitability and disruptions in network QNZ (EVP4593) synchronization. In addition, we identified changes in pyramidal cell dendritic spines and axon initial segments consistent with compensation for hyperexcitability. Finally, we identified the 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 loss-of-function variants may contribute to human disease. INTRODUCTION GABAergic interneurons are essential for the proper synchronization and function of neuronal networks that underlie normal cognition, mood, and behavior. GABAergic interneurons target to unique postsynaptic domains on excitatory neurons; however, the molecular mechanisms underlying the subcellular organization of forebrain GABAergic synapses remain poorly understood. Abnormalities in GABAergic interneuron circuitry and decreased gamma oscillations have been implicated QNZ (EVP4593) in many neurodevelopmental and neuropsychiatric disorders1C8. 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. encodes ankyrin-G, a fundamental scaffolding protein that organizes critical plasma membrane domains9, 10. Alternative splicing of 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 QNZ (EVP4593) 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 Rabbit Polyclonal to U51 the nervous system, and arise from alternative splicing of a single 7.8 kb giant exon9, 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 propagation10. 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 IV-spectrin to excitable domains (reviewed in 12). Importantly, the 480 kDa ankyrin-G isoform has also been shown to stabilize GABAergic synapses on the soma and AIS of excitatory pyramidal neurons by interacting with the GABAA receptor-associated protein (GABARAP) to inhibit GABAA receptor endocytosis 13. GABARAP and GABARAP-like 1, members of the ubiquitin-like LC3 family of microtubule-associated proteins, mediate GABAA receptor QNZ (EVP4593) trafficking between the cell surface and intracellular compartments14. GABARAP and other members of the LC3 family interact with LC3-interacting region (LIR) motifs15. The giant exon that encodes the 480 kDa ankyrin-G isoform contains an LIR motif, which includes residue W198913, 16. Mutation of W1989 to arginine (W1989R) completely abolished the binding between ankyrin-G and GABARAP13. Deletion of wild-type (WT) ankyrin-G and replacement with W1989R 480 kDa ankyrin-G failed to rescue GABAA receptors to the soma and AIS or restore miniature inhibitory postsynaptic currents (mIPSCs) in cultured mouse hippocampal neurons13. Taken together, these findings suggested that 480 kDa ankyrin-G plays a critical role in stabilizing GABAergic synapses has not been investigated. Here, we have generated a novel knock-in mouse model expressing W1989R. This allowed us to study, for the first time, the relationship between the 480 kDa ankyrin-G isoform and GABAergic synapse formation and function in a model that survives to adulthood and is capable of forming the AIS and nodes of Ranvier. We show that the W1989R mutation causes decreases in GABAergic synapses in layer II/III of somatosensory cortex and CA1 of hippocampus, while sparing inhibitory synapses on cerebellar Purkinje neurons and thalamic neurons. The decreases in inhibitory synapses cause hyperexcitability of cortical and hippocampal pyramidal neurons and decreases in gamma oscillations. Interestingly, we also detect changes consistent with compensation for the loss of inhibitory tone, including shortening of the AIS and decreases in dendritic spine density and excitatory post synaptic currents. Finally, we report the identification of a family with bipolar disorder (BD) that carries the W1989R human variant (rs372922084, c.5965T C (p.Trp1989Arg)), which may contribute to the pathophysiology of psychiatric disease. RESULTS W1989, located within the giant exon of ankyrin-G, is necessary for binding to a hydrophobic pocket in GABARAP. The 480 kDa splice variant interacts with GABARAP to inhibit GABAA receptor endocytosis and stabilize GABAergic synapses13. Here, we explored the molecular basis governing this interaction by resolving the crystal structure of the ankyrin-G/GABARAP complex. Crystallography data show that the LIR motif within the.