Scientists headed by a team at the University of North Carolina (UNC) at Chapel Hill School of Medicine used mouse models, cellular assays, and live microscopy to demonstrate that rare variants in the ANK2 gene, which are consistently found in individuals with autism spectrum disorder (ASD), can alter architecture and organization of neurons, potentially contributing to autism and neurodevelopmental comorbidities.

“Our new insights together with our tools and methods will help us assign pathogenicity to other ANK2 variants,” said research lead Damaris Lorenzo, PhD, an assistant professor in the UNC department of cell biology and physiology and member of the UNC Neuroscience Center and the UNC Intellectual and Developmental Disabilities Research Center. “We are certain there is undiscovered biology relevant to brain function and ASD involving this gene and we are pursuing it.”

Lorenzo and colleagues report on their studies in eLife, in a paper titled, “Giant ankyrin-B mediates transduction of axon guidance and collateral branch pruning factor sema 3A.”

ANK2 instructs neurons and other cell types how to make ankyrin-B, a protein with multiple functions in the nervous system. ANK2 encodes for various versions (isoforms) of ankyrin-B through a process known as alternative splicing, whereby portions of the protein are excluded in the final molecules. Mammals, such as mice and humans, express the full-size (giant) ankyrin-B isoform only in neurons. Another highly abundant isoform half the size of the full-size ankyrin-B isoform is found in virtually every other type of cell and organ. “AnkB has emerged as a critical determinant of structural neural connectivity through diverse and divergent roles of its two major, alternatively spliced isoforms in the brain; neuronal-specific 440 kDa (‘giant’) AnkB (AnkB440), and ubiquitously expressed 220 kDa AnkB (AnkB220),” the team noted.

Multiple genetic studies have consistently identified rare variants in ANK2 in individuals with ASD, making it one of the high-confidence risk genes associated with the condition. However, as Lorenzo acknowledged, “Together with its high prevalence and striking clinical presentation, ASD’s uncertain cause is a major limiting step in advancing therapeutic options. The evidence of ASD’s genetic origin is strong but also complex, with at least 100 other high-risk genes linked to the disorder.”

Depending on their type and location in the gene, ANK2 variants can affect giant ankyrin-B exclusively, or both isoforms simultaneously. “… variants in ANK2 have been identified in individuals with ASD and intellectual disability and it is ranked as a top high confidence ASD gene with one of the highest mutability scores,” the authors wrote. “Previous work showed that knock-in mice expressing an ASD-linked Ank2 variant yielding a truncated AnkB440 product exhibit ectopic brain connectivity and behavioral abnormalities.”

Unraveling the cause of ASD is further complicated because single genes such as ANK2 and the isoforms they encode can have multiple cellular functions. However, based on how subgroups of these genes overlap functionally, or work together to enact biological pathways, scientists have proposed convergent mechanisms that may be predominantly affected in individuals with ASD.

One of these common mechanisms is neuronal communication, which is determined in part by alterations in axons—the long extensions that carry signals from neurons to other neurons. Underlying these processes within a single neuron is the axonal cytoskeleton, a complex network of filament-like proteins that play pivotal roles in each neuron’s growth, shape, and plasticity. The axonal cytoskeleton is thought to be another major functional axis affected in ASD. The team explained, “Precise wiring of the neural circuitry relies on a tightly regulated spatiotemporal program of axonal extension and pathfinding to form synapses with their targets. The remarkable task of correctly extending the axon through dense three-dimensional spaces such as the developing mammalian brain is driven by the growth cone (GC), the highly dynamic structure that provides the mechanical force at the tip of the growing axon.”

Previous work by Lorenzo published in JCB showed that simultaneous loss of both major isoforms of ankyrin-B in the brain of mice resulted in profound anatomical defects involving axonal wiring, underscoring the importance of ankyrin-B in brain architecture and function. In a later study published in PNAS, Lorenzo and colleagues from Duke University observed that eliminating only the giant ankyrin-B isoform in neurons cultured in the lab resulted in more axon branches, which implicated deficits in the dynamics of microtubules, an essential cytoskeleton component. “We confirmed the development of axon hyperbranching in cultured neurons selectively lacking AnkB440, but not in neurons from AnkB220 KO mice,” the investigators stated.

In the newly reported study, the Lorenzo lab showed that selective loss of the giant ankyrin-B isoform leads to more axon branches in mouse brains and volumetric increases of multiple axonal bundles including the corpus callosum (CC). In collaboration with co-author Eva Anton, PhD, at the UNC Neuroscience Center, the team found that the giant ankyrin-B isoform is required to maintain the topographic order of callosal axons arising from the somatosensory cortex during brain development, and to ensure the specific targeting and refinement of callosal projections on the opposite side of the brain.

The team did not observe increases in axon branches in a novel mouse knockout (KO) model they engineered that only lacks the shorter ankyrin-B isoform. “Using Golgi staining, we determined that AnkB440 KO, but not AnkB220 KO, cortical neurons grow more collateral branches in the proximal axon, consistent with the in vitro results,” the team stated. “We also found volumetric increases of multiple commissural and other axon tracts in AnkB440 KO mice and deficits in topographic order of midline CC axons arising from the somatosensory cortex and the targeting of their contralateral projections. Lorenzo further stated, “These findings confirm divergent roles of ankyrin-B isoforms and support critical and specialized roles of giant ankyrin-B in axon collateral branch formation, targeting, and refinement.”

“Brain cortical regions have been the most directly linked to ASD pathology,” Lorenzo continued. “The changes we observed in cortical structural connectivity likely result from combined defects in axon branch initiation, guidance, and pruning of misdirected or overabundant projections during development due to giant ankyrin-B deficiency.”

In collaboration with a team led by co-author Meng Meng Fu, PhD, at the National Institute of Neurological Disorders and Stroke, the researchers verified that the observed corpus callosum abnormalities did not involve changes in myelination and in the number and maturation of oligodendrocytes, a non-neuronal brain cell type implicated in similar pathologies.

Cues outside of cells modulate these processes to trigger changes in neurons through attractive and repulsive effects. Lorenzo’s research team showed that cortical neurons require the giant ankyrin-B isoform to make possible the repulsive effects of semaphorin 3A, a molecule that interacts with and collapses the tips of axons and their branches. The team also showed that ANK2 variants exclusively affecting giant ankyrin-B have similar loss of response to the Semaphorin 3A molecule, revealing a possible mechanistic contribution to ASD. “ASD-linked ANK2 variants failed to rescue Sema 3A-induced GC collapse,” they wrote. “The present study sheds new light into the isoform-specific functions of AnkB in modulating axonal architecture, targeting, and refinement in the developing brain and the potential contribution of AnkB deficits to ASD pathology …We propose that impaired response to repellent cues due to AnkB440 deficits leads to axonal targeting and branch pruning defects and may contribute to the pathogenicity of ANK2 variants.”

“Our bottom-up approach of discovery and functional validation contributes to the underdeveloped knowledge database of ASD functional etiology,” Lorenzo added. “This is critical because this heterogeneous and complex disorder likely requires personalized strategies for clinical intervention.”

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