Feedback-Driven Assembly of the Axon Initial Segment.
Ontology highlight
ABSTRACT:
SUBMITTER: Freal A
PROVIDER: S-EPMC6839619 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The inhibitor of NF-?B alpha (I?B?) protein is an important regulator of the transcription factor NF-?B. In neurons, I?B? has been shown to play a role in neurite outgrowth and cell survival. Recently, a phosphorylated form of I?B? (pI?B? Ser32/36) was reported to be highly enriched at the axon initial segment (AIS) and was proposed to function upstream of ankyrinG in AIS assembly, including ion channel recruitment. However, we report here that the AIS clustering of ankyrinG and Na(+) channels in the brains of I?B? knockout (Nfkbia(-/-)) mice is comparable to that in wild-type littermates. Furthermore, we found that multiple phospho-specific antibodies against pI?B? Ser32/36 non-specifically label AIS in Nfkbia(-/-) cortex and AIS in dissociated Nfkbia(-/-) hippocampal neurons. With the exception of ankyrinG, shRNA-mediated knockdown of known AIS proteins in cultured hippocampal neurons did not eliminate the AIS labeling with pI?B? antibodies. Instead, the pI?B? antibodies cross-react with a phosphorylated epitope of a protein associated with the microtubule-based AIS cytoskeleton that is not integrated into the AIS membrane complex organized by ankyrinG. Our results indicate that pI?B? is neither enriched at the AIS nor required for AIS assembly.
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Project description:Axon initial segments (AISs) initiate action potentials and regulate the trafficking of vesicles between somatodendritic and axonal compartments. However, the mechanisms controlling AIS assembly remain poorly defined. We performed differential proteomics and found nuclear mitotic apparatus protein 1 (NuMA1) is downregulated in AIS-deficient neonatal mouse brains and neurons. NuMA1 is transiently located at the AIS during development where it interacts with the scaffolding protein 4.1B and the dynein regulator lissencephaly 1 (Lis1). Silencing NuMA1 or protein 4.1B by shRNA disrupts AIS assembly, but not maintenance. Silencing Lis1 or overexpressing NuMA1 during AIS assembly increased the density of AIS proteins, including ankyrinG and neurofascin-186 (NF186). NuMA1 inhibits the endocytosis of AIS NF186 by impeding Lis1's interaction with doublecortin, a potent facilitator of NF186 endocytosis. Our results indicate the transient expression and AIS localization of NuMA1 stabilizes the developing AIS by inhibiting endocytosis and removal of AIS proteins.
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Project description:The axon initial segment (AIS) is responsible for both the modulation of action potentials and the maintenance of neuronal polarity. Yet, the molecular mechanisms controlling its assembly are incompletely understood. Our study in single electroporated motor neurons in mouse embryos revealed that AnkyrinG (AnkG), the AIS master organizer, is undetectable in bipolar migrating motor neurons, but is already expressed at the beginning of axonogenesis at E9.5 and initially distributed homogeneously along the entire growing axon. Then, from E11.5, a stage when AnkG is already apposed to the membrane, as observed by electron microscopy, the protein progressively becomes restricted to the proximal axon. Analysis on the global motor neurons population indicated that Neurofascin follows an identical spatio-temporal distribution, whereas sodium channels and β4-spectrin only appear along AnkG(+) segments at E11.5. Early patch-clamp recordings of individual motor neurons indicated that at E12.5 these nascent AISs are already able to generate spikes. Using knock-out mice, we demonstrated that neither β4-spectrin nor Neurofascin control the distal-to-proximal restriction of AnkG.
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Project description:Neuronal maturation requires dramatic morphological and functional changes, but the molecular mechanisms governing this process are not well understood. Here, we studied the role of Rbfox1, Rbfox2, and Rbfox3 proteins, a family of tissue-specific splicing regulators mutated in multiple neurodevelopmental disorders. We generated Rbfox triple knockout (tKO) ventral spinal neurons to define a comprehensive network of alternative exons under Rbfox regulation and to investigate their functional importance in the developing neurons. Rbfox tKO neurons exhibit defects in alternative splicing of many cytoskeletal, membrane, and synaptic proteins, and display immature electrophysiological activity. The axon initial segment (AIS), a subcellular structure important for action potential initiation, is diminished upon Rbfox depletion. We identified an Rbfox-regulated splicing switch in ankyrin G, the AIS "interaction hub" protein, that regulates ankyrin G-beta spectrin affinity and AIS assembly. Our data show that the Rbfox-regulated splicing program plays a crucial role in structural and functional maturation of postmitotic neurons.
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Project description:The axon initial segment (AIS) is the site of action potential generation and a locus of activity-dependent homeostatic plasticity. A multimeric complex of sodium channels, linked via a cytoskeletal scaffold of ankyrin G and beta IV spectrin to submembranous actin rings, mediates these functions. The mechanisms that specify the AIS complex to the proximal axon and underlie its plasticity remain poorly understood. Here we show phosphorylated myosin light chain (pMLC), an activator of contractile myosin II, is highly enriched in the assembling and mature AIS, where it associates with actin rings. MLC phosphorylation and myosin II contractile activity are required for AIS assembly, and they regulate the distribution of AIS components along the axon. pMLC is rapidly lost during depolarization, destabilizing actin and thereby providing a mechanism for activity-dependent structural plasticity of the AIS. Together, these results identify pMLC/myosin II activity as a common link between AIS assembly and plasticity.
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Project description:In most vertebrate neurons, action potentials are initiated in the axon initial segment (AIS), a specialized region of the axon containing a high density of voltage-gated sodium and potassium channels. It has recently been proposed that neurons use plasticity of AIS length and/or location to regulate their intrinsic excitability. Here we quantify the impact of neuron morphology on AIS plasticity using computational models of simplified and realistic somatodendritic morphologies. In small neurons (e.g., dentate granule neurons), excitability was highest when the AIS was of intermediate length and located adjacent to the soma. Conversely, neurons having larger dendritic trees (e.g., pyramidal neurons) were most excitable when the AIS was longer and/or located away from the soma. For any given somatodendritic morphology, increasing dendritic membrane capacitance and/or conductance favored a longer and more distally located AIS. Overall, changes to AIS length, with corresponding changes in total sodium conductance, were far more effective in regulating neuron excitability than were changes in AIS location, while dendritic capacitance had a larger impact on AIS performance than did dendritic conductance. The somatodendritic influence on AIS performance reflects modest soma-to-AIS voltage attenuation combined with neuron size-dependent changes in AIS input resistance, effective membrane time constant, and isolation from somatodendritic capacitance. We conclude that the impact of AIS plasticity on neuron excitability will depend largely on somatodendritic morphology, and that, in some neurons, a shorter or more distally located AIS may promote, rather than limit, action potential generation.
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Project description:AnkyrinG (ankG) is highly enriched in neurons at axon initial segments (AISs) where it clusters Na(+) and K(+) channels and maintains neuronal polarity. How ankG becomes concentrated at the AIS is unknown. Here, we show that as neurons break symmetry, they assemble a distal axonal submembranous cytoskeleton, comprised of ankyrinB (ankB), ?II-spectrin, and ?II-spectrin, that defines a boundary limiting ankG to the proximal axon. Experimentally moving this boundary altered the length of ankG staining in the proximal axon, whereas disruption of the boundary through silencing of ankB, ?II-spectrin, or ?II-spectrin expression blocked AIS assembly and permitted ankG to redistribute throughout the distal axon. In support of an essential role for the distal cytoskeleton in ankG clustering, we also found that ?II and ?II-spectrin-deficient mice had disrupted AIS. Thus, the distal axonal cytoskeleton functions as an intra-axonal boundary restricting ankG to the AIS.
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Project description:The axon initial segment (AIS) is a specialized neuronal compartment required for action potential generation and neuronal polarity. However, understanding the mechanisms regulating AIS structure and function has been hindered by an incomplete knowledge of its molecular composition. Here, using immuno-proximity biotinylation we further define the AIS proteome and its dynamic changes during neuronal maturation. Among the many AIS proteins identified, we show that SCRIB is highly enriched in the AIS both in vitro and in vivo, and exhibits a periodic architecture like the axonal spectrin-based cytoskeleton. We found that ankyrinG interacts with and recruits SCRIB to the AIS. However, loss of SCRIB has no effect on ankyrinG. This powerful and flexible approach further defines the AIS proteome and provides a rich resource to elucidate the mechanisms regulating AIS structure and function.
2024-01-02 | PXD045921 | Pride
Project description:The axon initial segment (AIS) is the site of action potential (AP) initiation in most neurons and is thus a critical site in the regulation of neuronal excitability. Normal function within the discrete AIS compartment requires intricate molecular machinery to ensure the proper concentration and organization of voltage-gated and ligand-gated ion channels; in humans, dysfunction at the AIS due to channel mutations is commonly associated with epileptic disorders. In this review, we will examine the molecular mechanisms underlying the formation of the only synapses found at the AIS: synapses containing γ-aminobutyric type A receptors (GABAARs). GABAARs are heteropentamers assembled from 19 possible subunits and are the primary mediators of fast synaptic inhibition in the brain. Although the total GABAAR population is incredibly heterogeneous, only one specific GABAAR subtype-the α2-containing receptor-is enriched at the AIS. These AIS synapses are innervated by GABAergic chandelier cells, and this inhibitory signaling is thought to contribute to the tight control of AP firing. Here, we will summarize the progress made in understanding the regulation of GABAAR synapse formation, concentrating on post-translational modifications of subunits and on interactions with intracellular proteins. We will then discuss subtype-specific synapse formation, with a focus on synapses found at the AIS, and how these synapses influence neuronal excitation.
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Project description:By assuming that tau protein can be in seven kinetic states, we developed a model of tau protein transport in the axon and in the axon initial segment (AIS). Two separate sets of kinetic constants were determined, one in the axon and the other in the AIS. This was done by fitting the model predictions in the axon with experimental results and by fitting the model predictions in the AIS with the assumed linear increase of the total tau concentration in the AIS. The calibrated model was used to make predictions about tau transport in the axon and in the AIS. To the best of our knowledge, this is the first paper that presents a mathematical model of tau transport in the AIS. Our modeling results suggest that binding of free tau to microtubules creates a negative gradient of free tau in the AIS. This leads to diffusion-driven tau transport from the soma into the AIS. The model further suggests that slow axonal transport and diffusion-driven transport of tau work together in the AIS, moving tau anterogradely. Our numerical results predict an interplay between these two mechanisms: as the distance from the soma increases, the diffusion-driven transport decreases, while motor-driven transport becomes larger. Thus, the machinery in the AIS works as a pump, moving tau into the axon.
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