Biology subjects

Kuba, H.

Publications and source records attributed to Kuba, H..

4 recordsLinked to original sources

bioRxiv2026

CRISPR/Cas9-based knockout screening revealed GSK3β as a key regulator for structural plasticity of axon initial segment

The axon initial segment (AIS) undergoes structural plasticity to tune neuronal excitability, yet the underlying molecular mechanisms remain unclear. Here we developed an in vivo CRISPR/Cas9 knockout platform using an all-in-one triple-guide RNA vector introduced via electroporation and employed this approach to identify molecules that regulate developmental AIS shortening in the chicken nucleus magnocellularis. We targeted fourteen molecules associated with microtubules and found that knockout of either glycogen synthase kinase 3{beta} (GSK3{beta}) or Tau impaired the AIS shortening. Conversely, overexpression of a constitutively active form of GSK3{beta} facilitated the AIS shortening in vivo. This GSK3{beta}-induced shortening was reproduced in slice cultures and suppressed by microtubule stabilization. Together, these findings identify GSK3{beta}-dependent microtubule remodeling as a mechanism underlying developmental AIS shortening and establish an in vivo genetic approach for molecular screening in chick embryos.

neuroscience↗

bioRxiv2025

Twinfilin-1 phosphorylation in reelin signaling regulates actin dynamics and spine development

Reelin is an extracellular glycoprotein essential for neuronal migration, spine development, and synaptic plasticity. Impaired reelin signaling is linked to neurological disorders, including schizophrenia and autism. While reelin mutant (reeler) mice exhibit behavioral deficits associated with impaired spine formation, the underlying molecular mechanisms remain unclear. We identified Twinfilin-1 (Twf1) as a downstream effector of reelin signaling via phosphoproteomic analysis, based on its reduced tyrosine phosphorylation in reeler mice. We found that Src regulated Twf1 phosphorylation at tyrosine 309, and reelin stimulation increased Twf1 phosphorylation in neurons, an effect blocked by the Src inhibitor PP2. A phospho-resistant Twf1 mutant (Twf1 Y309F) showed reduced capping protein binding and a lower F/G-actin ratio. Twf1Y309F mice exhibited cognitive deficits, reduced spine density, smaller spine head size, and a decreased F/G-actin ratio in synaptosomes. These findings highlight Twf1 phosphorylation as a key component of reelin signaling involved in actin remodeling and spine development.

neuroscience↗

bioRxiv2024

Regional heterogeneities of oligodendrocytes determine biased distribution pattern of Ranvier nodes along single axons in sound localization circuit

Spacing of Ranvier nodes along myelinated axons is a critical determinant of conduction velocity, influencing spike arrival timing and hence neural circuit function. In the chick brainstem auditory circuit, the pattern of nodal spacing varies regionally along single axons, enabling precise binaural integration for sound localization. Using this model, we investigated the potential factors underlying the biased nodal spacing pattern. 3D morphometry revealed that these axons were almost fully myelinated by oligodendrocytes exhibiting distinct morphologies and cell densities across regions after hearing onset. The structure of axons did not affect internodal length. Inhibiting vesicular release from the axons did not affect internodal length or oligodendrocyte morphology, but caused unmyelinated segments on the axons by suppressing oligodendrogenesis near the presynaptic terminals. These results suggest that the regional heterogeneity in the intrinsic properties of oligodendrocytes is a prominent determinant of the biased nodal spacing pattern in the sound localization circuit, while activity-dependent signaling supports the pattern by ensuring adequate oligodendrocyte density. Our findings highlight the importance of oligodendrocyte heterogeneity in fine-tuning neural circuit function.

neuroscience↗

bioRxiv2023

Activity-regulated micro-exon splicing programs underlie late-onset plasticity at the axon initial segment

The axon initial segment (AIS) is a specialized neuronal compartment located at the proximal end of axons and initiates action potentials. AIS undergoes plastic changes with aging, disease, and activity levels; however, the molecular mechanisms underlying their plasticity remain unclear. We discovered that depolarization induces diffuse elongation of the AIS in cerebellar granule cells over the span of days via the Ca2+-dependent ERK/MAP kinase pathway. These structural changes were accompanied by a decrease in voltage-gated Na+ channel density, resulting in a homeostatic attenuation in neuronal excitability. Notably, we found that the late-onset AIS plasticity is associated with depolarization-induced alternative splicing of smaller exons (<100 nt) of transcripts encoding AIS-enriched proteins. In addition, depolarization-induced the skipping of the 53-nt exon19 from the transcript of the splicing protein Rbfox1. CRISPR-mediated removal of exon 19 from Rbfox1 promoted its nuclear localization and sequentially induced a series of downstream micro-exon splicing changes in several AIS proteins, recapitulating cerebellar AIS plasticity. In a Rbfox1-independent mechanism, depolarization-induced insertion of the developmentally regulated micro-exon 34 into the key AIS scaffolding protein Ankyrin G (AnkG). The constitutive insertion of exon 34 into AnkG disrupted its interaction with the AIS cytoskeletal protein {beta}IV spectrin and induced plastic changes in the AIS. Our findings provide fundamental mechanistic insights into the activity-mediated late-onset plasticity of AIS, highlighting the power of micro-scale splicing events in the homeostatic regulation of axonal remodeling.

neuroscience↗