Sodium channel endocytosis drives axon initial segment plasticity.

Activity-dependent plasticity of the axon initial segment (AIS) endows neurons with the ability to adapt action potential output to changes in network activity. Action potential initiation at the AIS highly depends on the clustering of voltage-gated sodium channels, but the molecular mechanisms regulating their plasticity remain largely unknown. Here, we developed genetic tools to label endogenous sodium channels and their scaffolding protein, to reveal their nanoscale organization and longitudinally image AIS plasticity in hippocampal neurons in slices and primary cultures. We find that N-methyl-d-aspartate receptor activation causes both long-term synaptic depression and rapid internalization of AIS sodium channels within minutes. The clathrin-mediated endocytosis of sodium channels at the distal AIS increases the threshold for action potential generation. These data reveal a fundamental mechanism for rapid activity-dependent AIS reorganization and suggests that plasticity of intrinsic excitability shares conserved features with synaptic plasticity.

The core PCP protein Prickle2 regulates axon number and AIS maturation by binding to AnkG and modulating microtubule bundling.

Core planar cell polarity (PCP) genes, which are involved in various neurodevelopmental disorders such as neural tube closure, epilepsy, and autism spectrum disorder, have poorly defined molecular signatures in neurons, mostly synapse-centric. Here, we show that the core PCP protein Prickle-like protein 2 (Prickle2) controls neuronal polarity and is a previously unidentified member of the axonal initial segment (AIS) proteome. We found that Prickle2 is present and colocalizes with AnkG480, the AIS master organizer, in the earliest stages of axonal specification and AIS formation. Furthermore, by binding to and regulating AnkG480, Prickle2 modulates its ability to bundle microtubules, a crucial mechanism for establishing neuronal polarity and AIS formation. Prickle2 depletion alters cytoskeleton organization, and Prickle2 levels determine both axon number and AIS maturation. Last, early Prickle2 depletion produces impaired action potential firing.

Neuronal Cytoskeleton: Presynaptic Boutons as Hotspots for Activity-Dependent Microtubule Nucleation.

Local microtubule remodeling plays a crucial role in controlling the transport of neuronal cargo. A new study reveals that excitatory en passant boutons in the axon are hotspots for activity-induced microtubule nucleation and provide tracks for interbouton vesicle trafficking.

Feedback-Driven Assembly of the Axon Initial Segment.

The axon initial segment (AIS) is a unique neuronal compartment that plays a crucial role in the generation of action potential and neuronal polarity. The assembly of the AIS requires membrane, scaffolding, and cytoskeletal proteins, including Ankyrin-G and TRIM46. How these components cooperate in AIS formation is currently poorly understood. Here, we show that Ankyrin-G acts as a scaffold interacting with End-Binding (EB) proteins and membrane proteins such as Neurofascin-186 to recruit TRIM46-positive microtubules to the plasma membrane. Using in vitro reconstitution and cellular assays, we demonstrate that TRIM46 forms parallel microtubule bundles and stabilizes them by acting as a rescue factor. TRIM46-labeled microtubules drive retrograde transport of Neurofascin-186 to the proximal axon, where Ankyrin-G prevents its endocytosis, resulting in stable accumulation of Neurofascin-186 at the AIS. Neurofascin-186 enrichment in turn reinforces membrane anchoring of Ankyrin-G and subsequent recruitment of TRIM46-decorated microtubules. Our study reveals feedback-based mechanisms driving AIS assembly.

TRIM46 Organizes Microtubule Fasciculation in the Axon Initial Segment.

Selective cargo transport into axons and dendrites over the microtubule network is essential for neuron polarization. The axon initial segment (AIS) separates the axon from the somatodendritic compartment and controls the microtubule-dependent transport into the axon. Interestingly, the AIS has a characteristic microtubule organization; it contains bundles of closely spaced microtubules with electron dense cross-bridges, referred to as microtubule fascicles. The microtubule binding protein TRIM46 localizes to the AIS and when overexpressed in non-neuronal cells forms microtubule arrays that closely resemble AIS fascicles in neurons. However, the precise role of TRIM46 in microtubule fasciculation in neurons has not been studied. Here we developed a novel correlative light and electron microscopy approach to study AIS microtubule organization. We show that in cultured rat hippocampal neurons of both sexes, TRIM46 levels steadily increase at the AIS during early neuronal differentiation and at the same time closely spaced microtubules form, whereas the fasciculated microtubules appear at later developmental stages. Moreover, we localized TRIM46 to the electron dense cross-bridges and show that depletion of TRIM46 causes loss of cross-bridges and increased microtubule spacing. These data indicate that TRIM46 has an essential role in organizing microtubule fascicles in the AIS.SIGNIFICANCE STATEMENT The axon initial segment (AIS) is a specialized region at the proximal axon where the action potential is initiated. In addition the AIS separates the axon from the somatodendritic compartment, where it controls protein transport to establish and maintain neuron polarity. Cargo vesicles destined for the axon recognize specialized microtubule tracks that enter the AIS. Interestingly the microtubules entering the AIS form crosslinked bundles, called microtubule fascicules. Recently we found that the microtubule-binding protein TRIM46 localizes to the AIS, where it may organize the AIS microtubules. In the present study we developed a novel correlative light and electron microscopy approach to study the AIS microtubules during neuron development and identified an essential role for TRIM46 in microtubule fasciculation.

Feedback-Driven Mechanisms between Microtubules and the Endoplasmic Reticulum Instruct Neuronal Polarity.

Establishment of neuronal polarity depends on local microtubule (MT) reorganization. The endoplasmic reticulum (ER) consists of cisternae and tubules and, like MTs, forms an extensive network throughout the entire cell. How the two networks interact and control neuronal development is an outstanding question. Here we show that the interplay between MTs and the ER is essential for neuronal polarity. ER tubules localize within the axon, whereas ER cisternae are retained in the somatodendritic domain. MTs are essential for axonal ER tubule stabilization, and, reciprocally, the ER is required for stabilizing and organizing axonal MTs. Recruitment of ER tubules into one minor neurite initiates axon formation, whereas ER retention in the perinuclear area or disruption of ER tubules prevent neuronal polarization. The ER-shaping protein P180, present in axonal ER tubules, controls axon specification by regulating local MT remodeling. We propose a model in which feedback-driven regulation between the ER and MTs instructs neuronal polarity.

Motor axon navigation relies on Fidgetin-like 1-driven microtubule plus end dynamics.

During neural circuit assembly, extrinsic signals are integrated into changes in growth cone (GC) cytoskeleton underlying axon guidance decisions. Microtubules (MTs) were shown to play an instructive role in GC steering. However, the numerous actors required for MT remodeling during axon navigation and their precise mode of action are far from being deciphered. Using loss- and gain-of-function analyses during zebrafish development, we identify in this study the meiotic clade adenosine triphosphatase Fidgetin-like 1 (Fignl1) as a key GC-enriched MT-interacting protein in motor circuit wiring and larval locomotion. We show that Fignl1 controls GC morphology and behavior at intermediate targets by regulating MT plus end dynamics and growth directionality. We further reveal that alternative translation of Fignl1 transcript is a sophisticated mechanism modulating MT dynamics: a full-length isoform regulates MT plus end-tracking protein binding at plus ends, whereas shorter isoforms promote their depolymerization beneath the cell cortex. Our study thus pinpoints Fignl1 as a multifaceted key player in MT remodeling underlying motor circuit connectivity.

Cooperative Interactions between 480 kDa Ankyrin-G and EB Proteins Assemble the Axon Initial Segment.

The axon initial segment (AIS) is required for generating action potentials and maintaining neuronal polarity. Significant progress has been made in deciphering the basic building blocks composing the AIS, but the underlying mechanisms required for AIS formation remains unclear. The scaffolding protein ankyrin-G is the master-organizer of the AIS. Microtubules and their interactors, particularly end-binding proteins (EBs), have emerged as potential key players in AIS formation. Here, we show that the longest isoform of ankyrin-G (480AnkG) selectively associates with EBs via its specific tail domain and that this interaction is crucial for AIS formation and neuronal polarity in cultured rodent hippocampal neurons. EBs are essential for 480AnkG localization and stabilization at the AIS, whereas 480AnkG is required for the specific accumulation of EBs in the proximal axon. Our findings thus provide a conceptual framework for understanding how the cooperative relationship between 480AnkG and EBs induces the assembly of microtubule-AIS structures in the proximal axon. SIGNIFICANCE STATEMENT: Neuronal polarity is crucial for the proper function of neurons. The assembly of the axon initial segment (AIS), which is the hallmark of early neuronal polarization, relies on the longest 480 kDa ankyrin-G isoform. The microtubule cytoskeleton and its interacting proteins were suggested to be early key players in the process of AIS formation. In this study, we show that the crosstalk between 480 kDa ankyrin-G and the microtubule plus-end tracking proteins, EBs, at the proximal axon is decisive for AIS assembly and neuronal polarity. Our work thus provides insight into the functional mechanisms used by 480 kDa ankyrin-G to drive the AIS formation and thereby to establish neuronal polarity.

Dynein Regulator NDEL1 Controls Polarized Cargo Transport at the Axon Initial Segment.

The development and homeostasis of neurons relies heavily on the selective targeting of vesicles into axon and dendrites. Microtubule-based motor proteins play an important role in polarized transport; however, the sorting mechanism to exclude dendritic cargo from the axon is unclear. We show that the dynein regulator NDEL1 controls somatodendritic cargo transport at the axon initial segment (AIS). NDEL1 localizes to the AIS via an interaction with the scaffold protein Ankyrin-G. Depletion of NDEL1 or its binding partner LIS1 results in both cell-wide and local defects, including the non-polarized trafficking of dendritic cargo through the AIS. We propose a model in which LIS1 is a critical mediator of local NDEL1-based dynein activation at the AIS. By localizing to the AIS, NDEL1 facilitates the reversal of somatodendritic cargos in the proximal axon.

In vivo assembly of the axon initial segment in motor neurons.

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 ß4-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 ß4-spectrin nor Neurofascin control the distal-to-proximal restriction of AnkG.