Paul M. Jenkins

Bipolar disorder (BD) is a debilitating neuropsychiatric disorder characterized by recurrent mood perturbations, cognitive and functional impairment, and increased risk of suicide. Genome-wide association studies (GWAS) have repeatedly identified the gene ANK3 , which encodes the neuronal scaffolding protein ankyrin-G, as a risk locus for BD. Ankyrin-G interacts directly with the GABA _A receptor-associated protein (GABARAP) to stabilize inhibitory synapses, providing a potential mechanistic link between ANK3 variation and GABAergic dysfunction observed in BD. Previously, we identified an ANK3 p.W1989R variant in a family with BD and demonstrated that this variant abolishes the ankyrin-G/GABARAP interaction, resulting in inhibitory signaling deficits in mice. Here, we investigated the effects of this variant in human induced pluripotent stem cell (hiPSC)-derived neurons generated from an affected individual heterozygous for the ANK3 p.W1989R variant. CRISPR correction of the variant increased inhibitory synapse density on both glutamatergic and GABAergic patient-derived neurons. Integrated spatial transcriptomics and calcium imaging identified cell-type-dependent changes in gene expression and calcium dynamics. ANK3 p.W1989R somatostatin (SST) positive interneurons displayed altered calcium dynamics and reduced SST expression. To determine whether the variant impacts SST interneuron-mediated inhibition of cortical circuits, we selectively stimulated prefrontal cortical SST interneurons in heterozygous Ank3 p.W1989R mice and recorded optically evoked inhibitory postsynaptic currents (oIPSCs) from cortical pyramidal neurons. SST interneuron-mediated oIPSC peak amplitude, charge transfer, and decay times were reduced in variant mice, without a significant change in the paired-pulse ratio. These findings, using complementary human and mouse models, identify novel roles for ankyrin-G in SST interneuron-mediated inhibitory signaling and demonstrate that altered inhibitory connectivity and SST interneuron function are associated with ANK3 p.W1989R. This work provides mechanistic insight into how disrupted ankyrin-G-dependent synaptic function may contribute to inhibitory signaling deficits relevant to BD.

Recent studies have revealed many alternative exons differentially spliced across diverse neuron types in the mammalian brain, but their links to neuronal physiology remain unclear. Here we characterize a deeply conserved microexon E35a in Ank3 encoding ankyrin-G (AnkG), a multifaceted adaptor protein best known as a master organizer of the axon initial segment (AIS) and as a leading genetic risk factor for bipolar disorder. E35a is predominantly skipped in cortical glutamatergic neurons but included in cortical GABAergic neurons and cerebellar neurons, which is dictated by multiple neuronal splicing factors. In E35a-deletion mice we generated, interneurons show increased excitability and somatic Ca²⁺ activity, without disruption in AIS. Biochemical analyses suggest that E35a inclusion facilitates AnkG interaction with a protein complex involving inositol trisphosphate receptors (InsP3Rs) important for intracellular Ca²⁺ signaling. Alternative splicing therefore allows AnkG to modulate neuron type-specific excitability in addition to its ubiquitous pan-neuronal role in organizing the AIS.

Most neurodevelopmental disorders with single gene diagnoses act via haploinsufficiency, in which only one of the two gene copies is functional¹. SCN2A haploinsufficiency is one of the most frequent causes of neurodevelopmental disorder, often presenting with autism spectrum disorder, intellectual disability and, in a subset of children, refractory epilepsy². Here, using SCN2A haploinsufficiency as a proof-of-concept, we show that upregulation of the existing functional gene copy through CRISPR activation (CRISPRa) can rescue neurological-associated phenotypes in Scn2a haploinsufficient mice. We first show that restoring Scn2a expression in adolescent heterozygous Scn2a conditional knock-in mice rescues electrophysiological deficits associated with Scn2a haploinsufficiency (Scn2a^+/−). Next, using an adeno-associated virus CRISPRa-based treatment in adolescent mice, we show that we can correct intrinsic and synaptic deficits in neocortical pyramidal cells, a major cell type that contributes to neurodevelopmental disorders and seizure aetiology in SCN2A haploinsufficiency. Furthermore, we find that systemic delivery of CRISPRa protects Scn2a^+/− mice against chemoconvulsant-induced seizures. Finally, we also show that adeno-associated virus CRISPRa treatment rescues excitability in SCN2A haploinsufficient human stem-cell-derived neurons. Our results showcase the potential of this therapeutic approach to rescue SCN2A haploinsufficiency and demonstrates that rescue even at adolescent stages can ameliorate neurodevelopmental phenotypes.

Thome C, Janssen JM, Karabulut S, Acuna C, D'Este E, Soyka SJ, Baum K, Bock M, Lehmann N, Roos J, Stevens NA, Hasegawa M, Ganea DA, Benoit CM, Gründemann J, Min LY, Bird KM, Schultz C, Bennett V, Jenkins PM, Engelhardt M. 2025. Live imaging of excitable axonal microdomains in ankyrin-G-GFP mice. eLife 12:RP87078. doi: 10.7554/eLife.87078.

Ankyrins are a family of intracellular scaffolding proteins that control the subcellular localization of a host of critically important signaling proteins within neurons, including many proteins associated with neurological disease. Ankyrin proteins are a vital component of the neuron. These scaffolding proteins must be spatially and temporally arranged to interact with their binding partners and facilitate proper neuronal signaling. Dysfunction of ankyrins is associated with neurodevelopmental disorders such as epilepsy and autism spectrum disorder. Despite the high degree of sequence similarity between ankyrin proteins, they display almost completely nonoverlapping localization and function. How ankyrins localize to the correct subcellular compartments to interact with their binding partners and complete their distinct roles remains poorly understood. Emerging evidence suggests that post-translational modifications may play a key part in this process. Some of the post-translational modifications that have been identified to regulate ankyrins are phosphorylation, ubiquitination, and palmitoylation. These modifications affect proper interactions, function, and localization of ankyrin proteins, which highlights their potential role in disease. This review will give an overview of neuronal ankyrins, and how post-translational modifications could be utilized to regulate protein localization and function in the context of neurological disease.

At the simplest level, neurons are structured to integrate synaptic input and perform computational transforms on that input, converting it into an action potential (AP) code. This process, converting synaptic input into AP output, typically occurs in a specialized region of the axon termed the axon initial segment (AIS). The AIS, as its name implies, is often contained to the first section of axon abutted to the soma and is home to a dizzying array of ion channels, attendant scaffolding proteins, intracellular organelles, extracellular proteins, and, in some cases, synapses. The AIS serves multiple roles as the final arbiter for determining if inputs are sufficient to evoke APs, as a gatekeeper that physically separates the somatodendritic domain from the axon proper, and as a regulator of overall neuronal excitability, dynamically tuning its size to best suit the needs of parent neurons. These complex roles have received considerable attention from experimentalists and theoreticians alike. Here, we review recent advances in our understanding of the AIS and its role in neuronal integration and polarity in health and disease.