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A hyperexcitability of the motor system is consistently observed in Amyotrophic Lateral Sclerosis (ALS) and has been implicated in the disease pathogenesis. What drives this hyperexcitability in the vast majority of patients is unknown. This is important to know as existing treatments simply reduce all neuronal excitability and fail to distinguish between pathological changes and important homeostatic changes. Understanding what drives the initial pathological changes could therefore provide better treatments. One challenge is that patients represent a heterogeneous population and the vast majority of cases are sporadic. One pathological feature that almost all (~97%) cases (familial and sporadic) have in common are cytoplasmic aggregates of the protein TDP-43 which is normally located in the nucleus. In our experiments we investigated whether this pathology was sufficient to increase neuronal excitability and the mechanisms by which this occurs. We used the TDP-43(ΔNLS) mouse model which successfully recapitulates this pathology in a controllable way. We used in vivo intracellular recordings in this model to demonstrate that TDP-43 pathology is sufficient to drive a severe hyper-excitability of spinal motoneurones. Reductions in soma size and a lengthening and constriction of axon initial segments were observed, which would contribute to enhanced excitability. Resuppression of the transgene resulted in a return to normal excitability parameters by 6-8 weeks. We therefore conclude that TDP-43 pathology itself is sufficient to drive a severe but reversible hyperexcitability of spinal motoneurones., Competing Interests: Declarations. Ethics approval and consent to participate: This animal research was granted by the Danish Animal Experiments Inspectorate (authorization no. 2018-15-0201-01426), and all treatments conformed to the national and European laws on the welfare and protection of animals used for experimentation - i.e., the Danish Animal Welfare Act (2013) and the EU directive 2010/63/EU. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

The axon initial segment (AIS) constitutes not only the site of action potential initiation, but also a hub for activity-dependent modulation of output generation. Recent studies shedding light on AIS function used predominantly post-hoc approaches since no robust murine in vivo live reporters exist. Here, we introduce a reporter line in which the AIS is intrinsically labeled by an ankyrin-G-GFP fusion protein activated by Cre recombinase, tagging the native Ank3 gene. Using confocal, superresolution, and two-photon microscopy as well as whole-cell patch-clamp recordings in vitro, ex vivo , and in vivo, we confirm that the subcellular scaffold of the AIS and electrophysiological parameters of labeled cells remain unchanged. We further uncover rapid AIS remodeling following increased network activity in this model system, as well as highly reproducible in vivo labeling of AIS over weeks. This novel reporter line allows longitudinal studies of AIS modulation and plasticity in vivo in real-time and thus provides a unique approach to study subcellular plasticity in a broad range of applications., Competing Interests: CT, JJ, SK, CA, ED, SS, KB, MB, NL, JR, NS, MH, DG, CB, JG, LM, KB, CS, VB, PJ, ME No competing interests declared, (© 2023, Thome et al.)

In mammalian axon-carrying-dendrite (AcD) neurons, the axon emanates from a basal dendrite, instead of the soma, to create a privileged route for action potential generation at the axon initial segment (AIS). However, it is unclear how such unusual morphology is established and whether the structure and function of the AIS in AcD neurons are preserved. By using dissociated hippocampal cultures as a model, we show that the development of AcD morphology can occur prior to synaptogenesis and independently of the in vivo environment. A single precursor neurite first gives rise to the axon and then to the AcD. The AIS possesses a similar cytoskeletal architecture as the soma-derived AIS and similarly functions as a trafficking barrier to retain axon-specific molecular composition. However, it does not undergo homeostatic plasticity, contains lesser cisternal organelles, and receives fewer inhibitory inputs. Our findings reveal insights into AcD neuron biology and underscore AIS structural differences based on axon onset., (© 2024 Han et al.)

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.

The axon initial segment (AIS) is a critical regulator of neuronal excitability and the initiation site of action potentials. Alterations in the structural features of AIS, such as length and position, have been shown to influence neuronal function, a phenomenon known as activity-dependent AIS plasticity. In addition to their physiological functions, abnormalities in the AIS have been implicated in various neurological disorders. UBE3A is an E3 ubiquitin ligase crucial for protein degradation in neurons. In mature neurons, only the maternal allele of the UBE3A gene is active, and the paternal allele is silenced. However, the role of UBE3A in controlling AIS in the cortical pyramidal neurons has not yet been fully elucidated. In this study, we compared wild-type mice with three different Ube3a-deficient mice and observed specific elongation of the AIS in the prelimbic cortex of the medial prefrontal cortex but not in the somatosensory cortex or motor cortex, as previously reported. Interestingly, we also showed that UBE3A controls AIS length in a cell-autonomous manner using cultured cortical neurons derived from Ube3a-floxed (Ube3a flox/flox ) mice. This study indicates that UBE3A controls AIS length through a cell-autonomous mechanism in vitro. However, non-cell-autonomous mechanisms, including feedback from inhibitory neurons or connections with the hippocampus, may also influence adult AIS length in vivo, thereby helping to maintain the excitability homeostasis of cortical pyramidal neurons in a region-specific manner., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier Inc. All rights reserved.)

Alzheimer´s disease (AD) is characterized by neuronal function loss and degeneration. The integrity of the axon initial segment (AIS) is essential to maintain neuronal function and output. AIS alterations are detected in human post-mortem AD brains and mice models, as well as, neurodevelopmental and mental disorders. However, the mechanisms leading to AIS deregulation in AD and the extrinsic glial origin are elusive. We studied early postnatal differences in AIS cellular/molecular mechanisms in wild-type or APP/PS1 mice and combined neuron-astrocyte co-cultures. We observed AIS integrity alterations, reduced ankyrinG expression and shortening, in APP/PS1 mice from P21 and loss of AIS integrity at 21 DIV in wild-type and APP/PS1 neurons in the presence of APP/PS1 astrocytes. AnkyrinG decrease is due to mRNAs and protein reduction of retinoic acid synthesis enzymes Rdh1 and Aldh1b1, as well as ADNP (Activity-dependent neuroprotective protein) in APP/PS1 astrocytes. This effect was mimicked by wild-type astrocytes expressing ADNP shRNA. In the presence of APP/PS1 astrocytes, wild-type neurons AIS is recovered by inhibition of retinoic acid degradation, and Adnp-derived NAP peptide (NAPVSIPQ) addition or P2X7 receptor inhibition, both regulated by retinoic acid levels. Moreover, P2X7 inhibitor treatment for 2 months impaired AIS disruption in APP/PS1 mice. Our findings extend current knowledge on AIS regulation, providing data to support the role of astrocytes in early postnatal AIS modulation. In conclusion, AD onset may be related to very early glial cell alterations that induce AIS and neuronal function changes, opening new therapeutic approaches to detect and avoid neuronal function loss., (© 2024. The Author(s).)

Vertebrate nervous systems use the axon initial segment (AIS) to initiate action potentials and maintain neuronal polarity. The microtubule-associated protein tripartite motif containing 46 (TRIM46) was reported to regulate axon specification, AIS assembly, and neuronal polarity through the bundling, or fasciculation, of microtubules in the proximal axon. However, these claims are based on TRIM46 knockdown in cultured neurons. To investigate TRIM46 function in vivo, we examined male and female TRIM46 knock-out mice. Contrary to previous reports, we find that TRIM46 is dispensable for axon specification and AIS formation. TRIM46 knock-out mice are viable, have normal behavior, and have normal brain structure. Thus, TRIM46 is not required for AIS formation, axon specification, or nervous system function. However, we confirm that TRIM46 is required for microtubule fasciculation. We also show TRIM46 enrichment in the first ∼100 μm of axon occurs independently of ankyrinG (AnkG) in vivo, although AnkG is required to restrict TRIM46 only to the AIS. Our results highlight the need for further investigation of the mechanisms by which the AIS and microtubules interact to shape neuronal structure and function., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 the authors.)

Developmental and epileptic encephalopathies (DEE) are rare but devastating and largely intractable childhood epilepsies. Genetic variants in ARHGEF9 , encoding a scaffolding protein important for the organization of the postsynaptic density of inhibitory synapses, are associated with DEE accompanied by complex neurological phenotypes. In a mouse model carrying a patient-derived ARHGEF9 variant associated with severe disease, we observed aggregation of postsynaptic proteins and loss of functional inhibitory synapses at the axon initial segment (AIS), altered axo-axonic synaptic inhibition, disrupted action potential generation, and complex seizure phenotypes consistent with clinical observations. These results illustrate diverse roles of ARHGEF9 that converge on regulation of the structure and function of the AIS, thus revealing a pathological mechanism for ARHGEF9 -associated DEE. This unique example of a neuropathological condition associated with multiple AIS dysfunctions may inform strategies for treating neurodevelopmental diseases., Competing Interests: Competing interests statement:The authors declare no competing interest.

Homeostatic plasticity maintains the stability of functional brain networks. The axon initial segment (AIS), where action potentials start, undergoes dynamic adjustment to exert powerful control over neuronal firing properties in response to network activity changes. However, it is poorly understood whether this plasticity involves direct synaptic input to the AIS. Here, we show that changes of GABAergic synaptic input from chandelier cells (ChCs) drive homeostatic tuning of the AIS of principal neurons (PNs) in the prelimbic (PL) region, while those from parvalbumin-positive basket cells do not. This tuning is evident in AIS morphology, voltage-gated sodium channel expression, and PN excitability. Moreover, the impact of this homeostatic plasticity can be reflected in animal behavior. Social behavior, inversely linked to PL PN activity, shows time-dependent alterations tightly coupled to changes in AIS plasticity and PN excitability. Thus, AIS-originated homeostatic plasticity in PNs may counteract deficits elicited by imbalanced ChC presynaptic input at cellular and behavioral levels.

The axon initial segment (AIS) is a critical compartment in neurons. It converts postsynaptic input into action potentials that subsequently trigger information transfer to target neurons. This process relies on the presence of several voltage-gated sodium (Na V ) and potassium (K V ) channels that accumulate in high densities at the AIS. TRAAK is a mechanosensitive leak potassium channel that was recently localized to the nodes of Ranvier. Here, we uncover that TRAAK is also present in AISs of hippocampal and cortical neurons in the adult rat brain as well as in AISs of cultured rat hippocampal neurons. We show that the AIS localization is driven by a C-terminal ankyrin G-binding sequence that organizes TRAAK in a 190 nm spaced periodic pattern that codistributes with periodically organized ankyrin G. We furthermore uncover that while the identified ankyrin G-binding motif is analogous to known ankyrin G-binding motifs in Na V 1 and K V 7.2/K V 7.3 channels, it was acquired by convergent evolution. Our findings identify TRAAK as an AIS ion channel that convergently acquired an ankyrin G-binding motif and expand the role of ankyrin G to include the nanoscale organization of ion channels at the AIS., Competing Interests: Competing interests statement:The authors declare no competing interest.

Changes in mitochondrial distribution are a feature of numerous age-related neurodegenerative diseases. In Drosophila, reducing the activity of Cdk5 causes a neurodegenerative phenotype and is known to affect several mitochondrial properties. Therefore, we investigated whether alterations of mitochondrial distribution are involved in Cdk5-associated neurodegeneration. We find that reducing Cdk5 activity does not alter the balance of mitochondrial localization to the somatodendritic versus axonal neuronal compartments of the mushroom body, the learning and memory center of the Drosophila brain. We do, however, observe changes in mitochondrial distribution at the axon initial segment (AIS), a neuronal compartment located in the proximal axon involved in neuronal polarization and action potential initiation. Specifically, we observe that mitochondria are partially excluded from the AIS in wild-type neurons, but that this exclusion is lost upon reduction of Cdk5 activity, concomitant with the shrinkage of the AIS domain that is known to occur in this condition. This mitochondrial redistribution into the AIS is not likely due to the shortening of the AIS domain itself but rather due to altered Cdk5 activity. Furthermore, mitochondrial redistribution into the AIS is unlikely to be an early driver of neurodegeneration in the context of reduced Cdk5 activity., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Dysregulated neuronal excitability is a hallmark of amyotrophic lateral sclerosis (ALS). We sought to investigate how functional changes to the axon initial segment (AIS), the site of action potential generation, could impact neuronal excitability in ALS human induced pluripotent stem cell (hiPSC) motor neurons. We find that early TDP-43 and C9orf72 hiPSC motor neurons show an increase in the length of the AIS and impaired activity-dependent AIS plasticity that is linked to abnormal homeostatic regulation of neuronal activity and intrinsic hyperexcitability. In turn, these hyperactive neurons drive increased spontaneous myofiber contractions of in vitro hiPSC motor units. In contrast, late hiPSC and postmortem ALS motor neurons show AIS shortening, and hiPSC motor neurons progress to hypoexcitability. At a molecular level, aberrant expression of the AIS master scaffolding protein ankyrin-G and AIS-specific voltage-gated sodium channels mirror these dynamic changes in AIS function and excitability. Our results point toward the AIS as an important site of dysfunction in ALS motor neurons., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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 find 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., (© 2023. The Author(s).)

Axon initial segment (AIS) cell surface proteins mediate key biological processes in neurons including action potential initiation and axo-axonic synapse formation. However, few AIS cell surface proteins have been identified. Here, we use antibody-directed proximity biotinylation to define the cell surface proteins in close proximity to the AIS cell adhesion molecule Neurofascin. To determine the distributions of the identified proteins, we use CRISPR-mediated genome editing for insertion of epitope tags in the endogenous proteins. We identify Contactin-1 (Cntn1) as an AIS cell surface protein. Cntn1 is enriched at the AIS through interactions with Neurofascin and NrCAM. We further show that Cntn1 contributes to assembly of the AIS extracellular matrix, and regulates AIS axo-axonic innervation by inhibitory basket cells in the cerebellum and inhibitory chandelier cells in the cortex., (© 2023. The Author(s).)

Purpose: To characterize a novel neurodevelopmental syndrome due to loss-of-function (LoF) variants in Ankyrin 2 (ANK2), and to explore the effects on neuronal network dynamics and homeostatic plasticity in human-induced pluripotent stem cell-derived neurons., Methods: We collected clinical and molecular data of 12 individuals with heterozygous de novo LoF variants in ANK2. We generated a heterozygous LoF allele of ANK2 using CRISPR/Cas9 in human-induced pluripotent stem cells (hiPSCs). HiPSCs were differentiated into excitatory neurons, and we measured their spontaneous electrophysiological responses using micro-electrode arrays (MEAs). We also characterized their somatodendritic morphology and axon initial segment (AIS) structure and plasticity., Results: We found a broad neurodevelopmental disorder (NDD), comprising intellectual disability, autism spectrum disorders and early onset epilepsy. Using MEAs, we found that hiPSC-derived neurons with heterozygous LoF of ANK2 show a hyperactive and desynchronized neuronal network. ANK2-deficient neurons also showed increased somatodendritic structures and altered AIS structure of which its plasticity is impaired upon activity-dependent modulation., Conclusions: Phenotypic characterization of patients with de novo ANK2 LoF variants defines a novel NDD with early onset epilepsy. Our functional in vitro data of ANK2-deficient human neurons show a specific neuronal phenotype in which reduced ANKB expression leads to hyperactive and desynchronized neuronal network activity, increased somatodendritic complexity and AIS structure and impaired activity-dependent plasticity of the AIS., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The axon initial segment (AIS) is a highly specialized neuronal compartment that regulates the generation of action potentials and maintenance of neuronal polarity. Live imaging of the AIS is challenging due to the limited number of suitable labeling methods. To overcome this limitation, we established a novel approach for live labeling of the AIS using unnatural amino acids (UAAs) and click chemistry. The small size of UAAs and the possibility of introducing them virtually anywhere into target proteins make this method particularly suitable for labeling of complex and spatially restricted proteins. Using this approach, we labeled two large AIS components, the 186 kDa isoform of neurofascin (NF186; encoded by Nfasc) and the 260 kDa voltage-gated Na+ channel (NaV1.6, encoded by Scn8a) in primary neurons and performed conventional and super-resolution microscopy. We also studied the localization of epilepsy-causing NaV1.6 variants with a loss-of-function effect. Finally, to improve the efficiency of UAA incorporation, we developed adeno-associated viral (AAV) vectors for click labeling in neurons, an achievement that could be transferred to more complex systems such as organotypic slice cultures, organoids, and animal models., Competing Interests: Competing interests D.G. is a co-founder of AaviGen GmbH. All the other authors declare no competing interests., (© 2023. Published by The Company of Biologists Ltd.)

Brain channelopathies are a group of neurological disorders that result from genetic mutations affecting ion channels in the brain. Ion channels are specialized proteins that play a crucial role in the electrical activity of nerve cells by controlling the flow of ions such as sodium, potassium, and calcium. When these channels are not functioning properly, they can cause a wide range of neurological symptoms such as seizures, movement disorders, and cognitive impairment. In this context, the axon initial segment (AIS) is the site of action potential initiation in most neurons. This region is characterized by a high density of voltage-gated sodium channels (VGSCs), which are responsible for the rapid depolarization that occurs when the neuron is stimulated. The AIS is also enriched in other ion channels, such as potassium channels, that play a role in shaping the action potential waveform and determining the firing frequency of the neuron. In addition to ion channels, the AIS contains a complex cytoskeletal structure that helps to anchor the channels in place and regulate their function. Therefore, alterations in this complex structure of ion channels, scaffold proteins, and specialized cytoskeleton may also cause brain channelopathies not necessarily associated with ion channel mutations. This review will focus on how the AISs structure, plasticity, and composition alterations may generate changes in action potentials and neuronal dysfunction leading to brain diseases. AIS function alterations may be the consequence of voltage-gated ion channel mutations, but also may be due to ligand-activated channels and receptors and AIS structural and membrane proteins that support the function of voltage-gated ion channels.

The amyloid precursor protein (APP) is linked to the genetics and pathogenesis of Alzheimer's disease (AD). It is the parent protein of the β-amyloid (Aβ) peptide, the main constituent of the amyloid plaques found in an AD brain. The pathways from APP to Aβ are intensively studied, yet the normal functions of APP itself have generated less interest. We report here that glutamate stimulation of neuronal activity leads to a rapid increase in App gene expression. In mouse and human neurons, elevated APP protein changes the structure of the axon initial segment (AIS) where action potentials are initiated. The AIS is shortened in length and shifts away from the cell body. The GCaMP8f Ca 2+ reporter confirms the predicted decrease in neuronal activity. NMDA antagonists or knockdown of App block the glutamate effects. The actions of APP on the AIS are cell-autonomous; exogenous Aβ, either fibrillar or oligomeric, has no effect. In culture, APP Swe (a familial AD mutation) induces larger AIS changes than wild type APP. Ankyrin G and βIV-spectrin, scaffolding proteins of the AIS, both physically associate with APP, more so in AD brains. Finally, in humans with sporadic AD or in the R1.40 AD mouse model, both females and males, neurons have elevated levels of APP protein that invade the AIS. In vivo as in vitro , this increased APP is associated with a significant shortening of the AIS. The findings outline a new role for the APP and encourage a reconsideration of its relationship to AD. SIGNIFICANCE STATEMENT While the amyloid precursor protein (APP) has long been associated with Alzheimer's disease (AD), the normal functions of the full-length Type I membrane protein have been largely unexplored. We report here that the levels of APP protein increase with neuronal activity. In vivo and in vitro , modest amounts of excess APP alter the properties of the axon initial segment. The β-amyloid peptide derived from APP is without effect. Consistent with the observed changes in the axon initial segment which would be expected to decrease action potential firing, we show that APP expression depresses neuronal activity. In mouse AD models and human sporadic AD, APP physically associates with the scaffolding proteins of the axon initial segment, suggesting a relationship with AD dementia., (Copyright © 2023 the authors.)

Voltage-gated sodium channels located in axon initial segments (AIS) trigger action potentials (AP) and play pivotal roles in the excitability of cortical pyramidal neurons. The differential electrophysiological properties and distributions of Na V 1.2 and Na V 1.6 channels lead to distinct contributions to AP initiation and propagation. While Na V 1.6 at the distal AIS promotes AP initiation and forward propagation, Na V 1.2 at the proximal AIS promotes the backpropagation of APs to the soma. Here, we show the small ubiquitin-like modifier (SUMO) pathway modulates Na + channels at the AIS to increase neuronal gain and the speed of backpropagation. Since SUMO does not affect Na V 1.6, these effects were attributed to SUMOylation of Na V 1.2. Moreover, SUMO effects were absent in a mouse engineered to express Na V 1.2-Lys38Gln channels that lack the site for SUMO linkage. Thus, SUMOylation of Na V 1.2 exclusively controls I NaP generation and AP backpropagation, thereby playing a prominent role in synaptic integration and plasticity., Competing Interests: OK, YK, AS, HD, LP, SG, IF No competing interests declared, (© 2023, Kotler et al.)

The structural plasticity of the axon initial segment (AIS) contributes to the homeostatic control of activity and optimizes the function of neural circuits; however, the underlying mechanisms are not fully understood. In this study, we prepared a slice culture containing nucleus magnocellularis from chickens of both sexes that reproduces most features of AIS plasticity in vivo , regarding its effects on characteristics of AIS and cell-type specificity, and revealed that microtubule reorganization via activation of CDK5 underlies plasticity. Treating the culture with a high-K + medium shortened the AIS and reduced sodium current and membrane excitability, specifically in neurons tuned to high-frequency sound, creating a tonotopic difference in AIS length in the nucleus. Pharmacological analyses revealed that this AIS shortening was driven by multiple Ca 2+ pathways and subsequent signaling molecules that converge on CDK5 via the activation of ERK1/2. AIS shortening was suppressed by overexpression of dominant-negative CDK5, whereas it was facilitated by the overexpression of p35, an activator of CDK5. Notably, p35(T138A), a phosphorylation-inactive mutant of p35, did not shorten the AIS. Moreover, microtubule stabilizers occluded AIS shortening during the p35 overexpression, indicating that CDK5/p35 mediated AIS shortening by promoting disassembly of microtubules at distal AIS. This study highlights the importance of microtubule reorganization and regulation of CDK5 activity in structural AIS plasticity and the tuning of AIS characteristics in neurons. SIGNIFICANCE STATEMENT The structural plasticity of AIS has a strong impact on the output of neurons and plays a fundamental role in the physiology and pathology of the brain. However, the mechanisms linking neuronal activity to structural changes in AIS are not well understood. In this study, we prepared an organotypic culture of avian auditory brainstem, reproducing most AIS plasticity features in vivo , and we revealed that activity-dependent AIS shortening occurs through the disassembly of microtubules at distal AIS via activation of CDK5/p35 signals. This study emphasizes the importance of microtubule reorganization and regulation of CDK5 activity in structural AIS plasticity and tonotopic differentiation of AIS structures in the brainstem auditory circuit., (Copyright © 2023 the authors.)

Background: In Alzheimer's disease (AD) brain, neuronal polarity and synaptic connectivity are compromised. A key structure for regulating polarity and functions of neurons is the axon initial segment (AIS), which segregates somatodendritic from axonal proteins and initiates action potentials. Toxic tau species, including extracellular oligomers (xcTauOs), spread tau pathology from neuron to neuron by a prion-like process, but few other cell biological effects of xcTauOs have been described., Objective: Test the hypothesis that AIS structure is sensitive to xcTauOs., Methods: Cultured wild type (WT) and tau knockout (KO) mouse cortical neurons were exposed to xcTauOs, and quantitative western blotting and immunofluorescence microscopy with anti-TRIM46 monitored effects on the AIS. The same methods were used to compare TRIM46 and two other resident AIS proteins in human hippocampal tissue obtained from AD and age-matched non-AD donors., Results: Without affecting total TRIM46 levels, xcTauOs reduce the concentration of TRIM46 within the AIS and cause AIS shortening in cultured WT, but not TKO neurons. Lentiviral-driven tau expression in tau KO neurons rescues AIS length sensitivity to xcTauOs. In human AD hippocampus, the overall protein levels of multiple resident AIS proteins are unchanged compared to non-AD brain, but TRIM46 concentration within the AIS and AIS length are reduced in neurons containing neurofibrillary tangles., Conclusion: xcTauOs cause partial AIS damage in cultured neurons by a mechanism dependent on intracellular tau, thereby raising the possibility that the observed AIS reduction in AD neurons in vivo is caused by xcTauOs working in concert with endogenous neuronal tau.

Mitochondrial Ca 2+ efflux by NCLX is a critical rate-limiting step in mitochondria signaling. We previously showed that NCLX is phosphorylated at a putative Casein Kinase 2 (CKII) site, the serine 271 (S271). Here, we asked if NCLX is regulated by CKII and interrogated the physiological implications of this control. We found that CKII inhibitors down-regulated NCLX-dependent Ca 2+ transport activity in SH-SY5Y neuronal cells and primary hippocampal neurons. Furthermore, we show that the CKII phosphomimetic mutants on NCLX inhibited (S271A) and constitutively activated (S271D) NCLX transport, respectively, rendering it insensitive to CKII inhibition. These phosphomimetic NCLX mutations also control the allosteric regulation of NCLX by mitochondrial membrane potential (ΔΨm). Since the omnipresent CKII is necessary for modulating the plasticity of the axon initial segment (AIS), we interrogated, in hippocampal neurons, if NCLX is required for this process. Similarly to WT neurons, NCLX-KO neurons can exhibit homeostatic plasticity following M-channel block. However, while WT neurons utilize a CKII-sensitive distal relocation of AIS Na + and Kv7 channels to decrease their intrinsic excitability, we did not observe such translocation in NCLX-KO neurons. Thus, our results indicate that NCLX is regulated by CKII and is a crucial link between CKII signaling and fast neuronal plasticity., Competing Interests: The authors declare no conflict of interest.

The axon initial segment (AIS) generates action potentials and maintains neuronal polarity by regulating the differential trafficking and distribution of proteins, transport vesicles, and organelles. Injury and disease can disrupt the AIS, and the subsequent loss of clustered ion channels and polarity mechanisms may alter neuronal excitability and function. However, the impact of AIS disruption on axon regeneration after injury is unknown. We generated male and female mice with AIS-deficient multipolar motor neurons by deleting AnkyrinG, the master scaffolding protein required for AIS assembly and maintenance. We found that after nerve crush, neuromuscular junction reinnervation was significantly delayed in AIS-deficient motor neurons compared with control mice. In contrast, loss of AnkyrinG from pseudo-unipolar sensory neurons did not impair axon regeneration into the intraepidermal nerve fiber layer. Even after AIS-deficient motor neurons reinnervated the neuromuscular junction, they failed to functionally recover because of reduced synaptic vesicle protein 2 at presynaptic terminals. In addition, mRNA trafficking was disrupted in AIS-deficient axons. Our results show that, after nerve injury, an intact AIS is essential for efficient regeneration and functional recovery of axons in multipolar motor neurons. Our results also suggest that loss of polarity in AIS-deficient motor neurons impairs the delivery of axonal proteins, mRNAs, and other cargoes necessary for regeneration. Thus, therapeutic strategies for axon regeneration must consider preservation or reassembly of the AIS. SIGNIFICANCE STATEMENT Disruption of the axon initial segment is a common event after nervous system injury. For multipolar motor neurons, we show that axon initial segments are essential for axon regeneration and functional recovery after injury. Our results may help explain injuries where axon regeneration fails, and suggest strategies to promote more efficient axon regeneration., (Copyright © 2022 the authors.)

Neurons are highly polarized cells with extensive axonal and dendritic projections that send and receive signals over long distances. Neuronal polarity requires sorting and maintaining a unique set of proteins to the neuron's distinct axonal and somatodendritic domains. The axon initial segment (AIS) is a specialized subcellular region located between these two domains and is critical for neuronal polarity. The AIS has a complex and elaborately organized molecular structure that enables its functions in neuronal polarity. Disruption of the AIS is associated with neurodevelopmental and neuropsychiatric disease pathologies, thus highlighting the importance of the AIS in neuronal physiology. This review discusses recent progress toward understanding the molecular architecture of the AIS and its importance in neuronal polarity through regulating protein diffusion and vesicular trafficking., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Neurons are highly polarized cells that face the fundamental challenge of compartmentalizing a vast and diverse repertoire of proteins in order to function properly 1 . The axon initial segment (AIS) is a specialized domain that separates a neuron's morphologically, biochemically and functionally distinct axon and dendrite compartments 2,3 . How the AIS maintains polarity between these compartments is not fully understood. Here we find that in Caenorhabditis elegans, mouse, rat and human neurons, dendritically and axonally polarized transmembrane proteins are recognized by endocytic machinery in the AIS, robustly endocytosed and targeted to late endosomes for degradation. Forcing receptor interaction with the AIS master organizer, ankyrinG, antagonizes receptor endocytosis in the AIS, causes receptor accumulation in the AIS, and leads to polarity deficits with subsequent morphological and behavioural defects. Therefore, endocytic removal of polarized receptors that diffuse into the AIS serves as a membrane-clearance mechanism that is likely to work in conjunction with the known AIS diffusion-barrier mechanism to maintain neuronal polarity on the plasma membrane. Our results reveal a conserved endocytic clearance mechanism in the AIS to maintain neuronal polarity by reinforcing axonal and dendritic compartment membrane boundaries., (© 2022. The Author(s).)

The axon initial segment (AIS) is the site at which action potentials initiate and constitutes a transport filter and diffusion barrier that contribute to the maintenance of neuronal polarity by sorting somato-dendritic cargo. A membrane periodic skeleton (MPS) comprising periodic actin rings provides a scaffold for anchoring various AIS proteins, including structural proteins and different ion channels. Although recent proteomic approaches have identified a considerable number of novel AIS components, details of the structure of the MPS and the roles of its individual components are lacking. The distance between individual actin rings in the MPS (~190 nm) necessitates the employment of super-resolution microscopy techniques to resolve the structural details of the MPS. This protocol describes a method for using cultured rat hippocampal neurons to examine the precise localization of an AIS protein in the MPS relative to sub-membranous actin rings using 3D-structured illumination microscopy (3D-SIM). In addition, an analytical approach to quantitively assess the periodicity of individual components and their position relative to actin rings is also described.

The axon initial segment (AIS) is essential for maintaining neuronal polarity, modulating protein transport into the axon, and action potential generation. These functions are supported by a distinctive actin and microtubule cytoskeleton that controls axonal trafficking and maintains a high density of voltage-gated ion channels linked by scaffold proteins to the AIS cytoskeleton. However, our knowledge of the mechanisms and proteins involved in AIS cytoskeleton regulation to maintain or modulate AIS structure is limited. In this context, formins play a significant role in the modulation of actin and microtubules. We show that pharmacological inhibition of formins modifies AIS actin and microtubule characteristics in cultured hippocampal neurons, reducing F-actin density and decreasing microtubule acetylation. Moreover, formin inhibition diminishes sodium channels, ankyrinG and βIV-spectrin AIS density, and AIS length, in cultured neurons and brain slices, accompanied by decreased neuronal excitability. We show that genetic downregulation of the mDia1 formin by interference RNAs also decreases AIS protein density and shortens AIS length. The ankyrinG decrease and AIS shortening observed in pharmacologically inhibited neurons and neuron-expressing mDia1 shRNAs were impaired by HDAC6 downregulation or EB1-GFP expression, known to increase microtubule acetylation or stability. However, actin stabilization only partially prevented AIS shortening without affecting AIS protein density loss. These results suggest that mDia1 maintain AIS composition and length contributing to the stability of AIS microtubules., (© 2021. The Author(s).)

The axon initial segment (AIS) responsible for action potential initiation is a dynamic structure that varies and changes together with neuronal excitability. Like other neuron types, alpha motoneurons in the mammalian spinal cord express heterogeneity and plasticity in AIS geometry, including length (AISl) and distance from soma (AISd). The present study aimed to establish the relationship of AIS geometry with a measure of intrinsic excitability, rheobase current, that varies by 20-fold or more among normal motoneurons. We began by determining whether AIS length or distance differed for motoneurons in motor pools that exhibit different activity profiles. Motoneurons sampled from the medial gastrocnemius (MG) motor pool exhibited values for average AISd that were significantly greater than that for motoneurons from the soleus (SOL) motor pool, which is more readily recruited in low-level activities. Next, we tested whether AISd covaried with intrinsic excitability of individual motoneurons. In anesthetized rats, we measured rheobase current intracellularly from MG motoneurons in vivo before labeling them for immunohistochemical study of AIS structure. For 16 motoneurons sampled from the MG motor pool, this combinatory approach revealed that AISd, but not AISl, was significantly related to rheobase, as AIS tended to be located further from the soma on motoneurons that were less excitable. Although a causal relation with excitability seems unlikely, AISd falls among a constellation of properties related to the recruitability of motor units and their parent motoneurons., Competing Interests: The authors have declared that no competing interests exist.

The axon initial segment (AIS) is a distinct neuronal domain, which is responsible for initiating action potentials, and therefore of key importance to neuronal signaling. To determine how it functions, it is necessary to establish which proteins reside there, how they are organized, and what the dynamic features are. Great strides have been made in recent years, and it is now clear that several AIS cytoskeletal and membrane proteins interact to form a higher-order periodic structure. Here we briefly describe AIS function, protein composition and molecular architecture, and discuss perspectives for future structural characterization, and if structure predictions will be able to model complex higher-order assemblies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The 20-60 μm axon initial segment (AIS) is proximally located at the interface between the axon and cell body. AIS has characteristic molecular and structural properties regulated by the crucial protein, ankyrin-G. The AIS contains a high density of Na + channels relative to the cell body, which allows low thresholds for the initiation of action potential (AP). Molecular and physiological studies have shown that the AIS is also a key domain for the control of neuronal excitability by homeostatic mechanisms. The AIS has high plasticity in normal developmental processes and pathological activities, such as injury, neurodegeneration, and neurodevelopmental disorders (NDDs). In the first half of this review, we provide an overview of the molecular, structural, and ion-channel characteristics of AIS, AIS regulation through axo-axonic synapses, and axo-glial interactions. In the second half, to understand the relationship between NDDs and AIS, we discuss the activity-dependent plasticity of AIS, the human mutation of AIS regulatory genes, and the pathophysiological role of an abnormal AIS in NDD model animals and patients. We propose that the AIS may provide a potentially valuable structural biomarker in response to abnormal network activity in vivo as well as a new treatment concept at the neural circuit level.

Somatodendritic missorting of the axonal protein TAU is a hallmark of Alzheimer's disease and related tauopathies. Rodent primary neurons and iPSC-derived neurons are used for studying mechanisms of neuronal polarity, including TAU trafficking. However, these models are expensive, time-consuming, and/or require the killing of animals. In this study, we tested four differentiation procedures to generate mature neuron cultures from human SH-SY5Y neuroblastoma cells and assessed the TAU sorting capacity. We show that SH-SY5Y-derived neurons, differentiated with sequential RA/BDNF treatment, are suitable for investigating axonal TAU sorting. These human neurons show pronounced neuronal polarity, axodendritic outgrowth, expression of the neuronal maturation markers TAU and MAP2, and, importantly, efficient axonal sorting of endogenous and transfected human wild-type TAU, similar to mouse primary neurons. We demonstrate that the N-terminal half of TAU is not sufficient for axonal targeting, as a C-terminus-lacking construct (N-term-TAU HA ) is not axonally enriched in both neuronal cell models. Importantly, SH-SY5Y-derived neurons do not show the formation of a classical axon initial segment (AIS), indicated by the lack of ankyrin G (ANKG) and tripartite motif-containing protein 46 (TRIM46) at the proximal axon, which suggests that successful axonal TAU sorting is independent of classical AIS formation. Taken together, our results provide evidence that (i) SH-SY5Y-derived neurons are a valuable human neuronal cell model for studying TAU sorting readily accessible at low cost and without animal need, and that (ii) efficient axonal TAU targeting is independent of ANKG or TRIM46 enrichment at the proximal axon in these neurons., (Copyright © 2021 IBRO. Published by Elsevier Ltd. All rights reserved.)

Monitoring Na + influx in the axon initial segment (AIS) at high spatial and temporal resolution is fundamental to understanding the generation of an action potential (AP). Here, we present protocols to obtain this measurement, focusing on the AIS of layer 5 (L5) somatosensory cortex pyramidal neurons in mouse brain slices. We first outline how to prepare slices for this application, how to select and patch neurons, and how to optimize the image acquisition. Specifically, we describe the preparation of optimal slices, patching and loading of L5 pyramidal neurons with the Na + indicator ING-2, and Na + imaging at 100 µs temporal resolution with a pixel resolution of half a micron. Then, we present a data analysis strategy in order to extract information on the kinetics of activated voltage-gated Na + channels by determining the change in Na + by compensating for bleaching and calculating the time derivative of the resulting fit. In sum, this approach can be widely applied when investigating the function of Na + channels during initiation of an AP and propagation under physiological or pathological conditions in neuronal subtypes. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Preparation of cortical slices Basic Protocol 2: Selection, patching, and Na + fluorescence recording of a neuron Support Protocol: Calibrating Na + fluorescence Basic Protocol 3: Data analysis., (© 2021 Wiley Periodicals LLC.)

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, assembly and activity-dependent plasticity. Giant ankyrin-G, the master organizer of AIS, directly associates with membrane-spanning voltage gated sodium (VSVG) and potassium channels (KCNQ2/3), as well as 186 kDa neurofascin, a L1CAM cell adhesion molecule. Giant ankyrin-G also binds to and recruits cytoplasmic AIS molecules including beta-4-spectrin, and the microtubule-binding proteins, EB1/EB3 and Ndel1. Giant ankyrin-G is sufficient to rescue AIS formation in ankyrin-G deficient neurons. Ankyrin-G also includes a smaller 190 kDa isoform located at dendritic spines instead of the AIS, which is incapable of targeting to the AIS or rescuing the AIS in ankyrin-G-deficient neurons. Here, we described a protocol using cultured hippocampal neurons from ANK3-E22/23-flox mice, which, when transfected with Cre-BFP exhibit loss of all isoform of ankyrin-G and impair the formation of AIS. Combined a modified Banker glia/neuron co-culture system, we developed a method to transfect ankyrin-G null neurons with a 480 kDa ankyrin-G-GFP plasmid, which is sufficient to rescue the formation of AIS. We further employ a quantification method, developed by Salzer and colleagues to deal with variation in AIS distance from the neuronal cell bodies that occurs in hippocampal neuron cultures. This protocol allows quantitative studies of the de novo assembly and dynamic behavior of AIS.

This protocol allows repeated whole-cell patch-clamp recordings from individual rodent CA1 hippocampal neurons, followed by immunohistological labeling of the axon initial segment. This overcomes the need to maintain whole-cell recordings over the timescales required for homeostatic modification to cellular excitability, allowing for correlative analysis of the structure and function of neurons. Moreover, this protocol allows for paired analysis of physiological properties assessed before and after pharmacological treatment, thus providing increased statistical power, despite the relatively low-throughput nature of the recordings. For complete details on the use and execution of this protocol, please refer to Booker et al. (2020a)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

The axon initial segment (AIS) is a critical microdomain for action potential initiation and implicated in the regulation of neuronal excitability during activity-dependent plasticity. While structural AIS plasticity has been suggested to fine-tune neuronal activity when network states change, whether it acts in vivo as a homeostatic regulatory mechanism in behaviorally relevant contexts remains poorly understood. Using the mouse whisker-to-barrel pathway as a model system in combination with immunofluorescence, confocal analysis and electrophysiological recordings, we observed bidirectional AIS plasticity in cortical pyramidal neurons. Furthermore, we find that structural and functional AIS remodeling occurs in distinct temporal domains: Long-term sensory deprivation elicits an AIS length increase, accompanied with an increase in neuronal excitability, while sensory enrichment results in a rapid AIS shortening, accompanied by a decrease in action potential generation. Our findings highlight a central role of the AIS in the homeostatic regulation of neuronal input-output relations.

Voltage gated sodium channels (Na v ) play a crucial role in action potential initiation and propagation. Although the discovery of Na v channels dates back more than 65 years, and great advances in understanding their localization, biophysical properties, and links to disease have been made, there are still many questions to be answered regarding the cellular and molecular mechanisms involved in Na v channel trafficking, localization and regulation. This review summarizes the different trafficking mechanisms underlying the polarized Na v channel localization in neurons, with an emphasis on the axon initial segment (AIS), as well as discussing the latest advances regarding how neurons regulate their excitability by modifying AIS length and location. The importance of Na v channel localization is emphasized by the relationship between mutations, impaired trafficking and disease. While this review focuses on Na v 1.6, other Na v isoforms are also discussed.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In multipolar vertebrate neurons, action potentials (APs) initiate close to the soma, at the axonal initial segment. Invertebrate neurons are typically unipolar with dendrites integrating directly into the axon. Where APs are initiated in the axons of invertebrate neurons is unclear. Voltage-gated sodium (Na V ) channels are a functional hallmark of the axonal initial segment in vertebrates. We used an intronic Minos -Mediated Integration Cassette to determine the endogenous gene expression and subcellular localization of the sole Na V channel in both male and female Drosophila , para Despite being the only Na V channel in the fly, we show that only 23 ± 1% of neurons in the embryonic and larval CNS express para , while in the adult CNS para is broadly expressed. We generated a single-cell transcriptomic atlas of the whole third instar larval brain to identify para expressing neurons and show that it positively correlates with markers of differentiated, actively firing neurons. Therefore, only 23 ± 1% of larval neurons may be capable of firing Na V -dependent APs. We then show that Para is enriched in an axonal segment, distal to the site of dendritic integration into the axon, which we named the distal axonal segment (DAS). The DAS is present in multiple neuron classes in both the third instar larval and adult CNS. Whole cell patch clamp electrophysiological recordings of adult CNS fly neurons are consistent with the interpretation that Na v -dependent APs originate in the DAS. Identification of the distal Na V localization in fly neurons will enable more accurate interpretation of electrophysiological recordings in invertebrates. SIGNIFICANCE STATEMENT The site of action potential (AP) initiation in invertebrates is unknown. We tagged the sole voltage-gated sodium (Na V ) channel in the fly, para , and identified that Para is enriched at a distal axonal segment. The distal axonal segment is located distal to where dendrites impinge on axons and is the likely site of AP initiation. Understanding where APs are initiated improves our ability to model neuronal activity and our interpretation of electrophysiological data. Additionally, para is only expressed in 23 ± 1% of third instar larval neurons but is broadly expressed in adults. Single-cell RNA sequencing of the third instar larval brain shows that para expression correlates with the expression of active, differentiated neuronal markers. Therefore, only 23 ± 1% of third instar larval neurons may be able to actively fire Na V -dependent APs., (Copyright © 2020 the authors.)

The differentiation of neuronal stem cells into polarized neurons is a well-coordinated process which has mostly been studied in classical non-human model systems, but to what extent these findings are recapitulated in human neurons remains unclear. To study neuronal polarization in human neurons, we cultured hiPSC-derived neurons, characterized early developmental stages, measured electrophysiological responses, and systematically profiled transcriptomic and proteomic dynamics during these steps. The neuron transcriptome and proteome shows extensive remodeling, with differential expression profiles of ~1100 transcripts and ~2200 proteins during neuronal differentiation and polarization. We also identified a distinct axon developmental stage marked by the relocation of axon initial segment proteins and increased microtubule remodeling from the distal (stage 3a) to the proximal (stage 3b) axon. This developmental transition coincides with action potential maturation. Our comprehensive characterization and quantitative map of transcriptome and proteome dynamics provides a solid framework for studying polarization in human neurons., Competing Interests: FL, RK, SP, LH, RS, BS, AA, HM, CW No competing interests declared, CH Employee of Genentech, Inc, a member of the Roche group, (© 2020, Lindhout et al.)

Ion channel complexes promote action potential initiation at the mammalian axon initial segment (AIS), and modulation of AIS size by recruitment or loss of proteins can influence neuron excitability. Although endocytosis contributes to AIS turnover, how membrane proteins traffic to this proximal axonal domain is incompletely understood. Neurofascin186 (Nfasc186) has an essential role in stabilising the AIS complex to the proximal axon, and the AIS channel protein Kv7.3 regulates neuron excitability. Therefore, we have studied how these proteins reach the AIS. Vesicles transport Nfasc186 to the soma and axon terminal where they fuse with the neuronal plasma membrane. Nfasc186 is highly mobile after insertion in the axonal membrane and diffuses bidirectionally until immobilised at the AIS through its interaction with AnkyrinG. Kv7.3 is similarly recruited to the AIS. This study reveals how key proteins are delivered to the AIS and thereby how they may contribute to its functional plasticity., Competing Interests: AG, EM, DS, PB No competing interests declared, (© 2020, Ghosh et al.)

Identifying effective treatments for Alzheimer's disease (AD) has proven challenging and has instigated a shift in AD research focus toward the earliest disease-initiating cellular mechanisms. A key insight has been an increase in soluble Aβ oligomers in early AD that is causally linked to neuronal and circuit hyperexcitability. However, other accumulating AD-related peptides and proteins, including those derived from the same amyloid precursor protein, such as Aη or sAPPα, and autonomously, such as tau, exhibit surprising opposing effects on circuit dynamics. We propose that the effects of these on neuronal circuits have profound implications for our understanding of disease complexity and heterogeneity and for the development of personalized diagnostic and therapeutic strategies in AD. Here, we highlight important peptide-specific mechanisms of dynamic pathological disequilibrium of cellular and circuit activity in AD and discuss approaches in which these may be further understood, and theoretically and experimentally leveraged, to elucidate AD pathophysiology., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Previously, we showed that a hierarchy of spectrin cytoskeletal proteins maintains nodal Na + channels (Liu et al., 2020). Here, using mice lacking β1, β4, or β1/β4 spectrins, we show this hierarchy does not function at axon initial segments (AIS). Although β1 spectrin, together with AnkyrinR (AnkR), compensates for loss of nodal β4 spectrin, it cannot compensate at AIS. We show AnkR lacks the domain necessary for AIS localization. Whereas loss of β4 spectrin causes motor impairment and disrupts AIS, loss of β1 spectrin has no discernable effect on central nervous system structure or function. However, mice lacking both neuronal β1 and β4 spectrin show exacerbated nervous system dysfunction compared to mice lacking β1 or β4 spectrin alone, including profound disruption of AIS Na + channel clustering, progressive loss of nodal Na + channels, and seizures. These results further define the important role of AIS and nodal spectrins for nervous system function., Competing Interests: CL, RS, TH, MS, PM, TH, JN, MR No competing interests declared, (© 2020, Liu et al.)

Genetic variations in TMEM106B, coding for a lysosomal membrane protein, affect frontotemporal lobar degeneration (FTLD) in GRN- (coding for progranulin) and C9orf72-expansion carriers and might play a role in aging. To determine the physiological function of TMEM106B, we generated TMEM106B-deficient mice. These mice develop proximal axonal swellings caused by drastically enlarged LAMP1-positive vacuoles, increased retrograde axonal transport of lysosomes, and accumulation of lipofuscin and autophagosomes. Giant vacuoles specifically accumulate at the distal end and within the axon initial segment, but not in peripheral nerves or at axon terminals, resulting in an impaired facial-nerve-dependent motor performance. These data implicate TMEM106B in mediating the axonal transport of LAMP1-positive organelles in motoneurons and axonal sorting at the initial segment. Our data provide mechanistic insight into how TMEM106B affects lysosomal proteolysis and degradative capacity in neurons., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (© 2019 Torii et al.)

Neuronal GABAergic responses switch from excitatory to inhibitory at an early postnatal period in rodents. The timing of this switch is controlled by intracellular Cl- concentrations, but factors determining local levels of cation-chloride cotransporters remain elusive. Here, we report that local abundance of the chloride importer NKCC1 and timely emergence of GABAergic inhibition are modulated by proteasome distribution, which is mediated through interactions of proteasomes with the adaptor Ecm29 and the axon initial segment (AIS) scaffold protein ankyrin G. Mechanistically, both the Ecm29 N-terminal domain and an intact AIS structure are required for transport and tethering of proteasomes in the AIS region. In mice, Ecm29 knockout (KO) in neurons increases the density of NKCC1 protein in the AIS region, a change that positively correlates with a delay in the GABAergic response switch. Phenotypically, Ecm29 KO mice showed increased firing frequency of action potentials at early postnatal ages and were hypersusceptible to chemically induced convulsive seizures. Finally, Ecm29 KO neurons exhibited accelerated AIS developmental positioning, reflecting a perturbed AIS morphological plastic response to hyperexcitability arising from proteasome inhibition, a phenotype rescued by ectopic Ecm29 expression or NKCC1 inhibition. Together, our findings support the idea that neuronal maturation requires regulation of proteasomal distribution controlled by Ecm29., (© 2020 Lee et al.)

Axon initial segments (AISs) generate action potentials and regulate the polarized distribution of proteins, lipids, and organelles in neurons. While the mechanisms of AIS Na + and K + channel clustering are understood, the molecular mechanisms that stabilize the AIS and control neuronal polarity remain obscure. Here, we use proximity biotinylation and mass spectrometry to identify the AIS proteome. We target the biotin-ligase BirA* to the AIS by generating fusion proteins of BirA* with NF186, Ndel1, and Trim46; these chimeras map the molecular organization of AIS intracellular membrane, cytosolic, and microtubule compartments. Our experiments reveal a diverse set of biotinylated proteins not previously reported at the AIS. We show many are located at the AIS, interact with known AIS proteins, and their loss disrupts AIS structure and function. Our results provide conceptual insights and a resource for AIS molecular organization, the mechanisms of AIS stability, and polarized trafficking in neurons.

Dysregulation of neuronal excitability underlies the pathogenesis of tauopathies, including frontotemporal dementia (FTD) with tau inclusions. A majority of FTD-causing tau mutations are located in the microtubule-binding domain, but how these mutations alter neuronal excitability is largely unknown. Here, using CRISPR/Cas9-based gene editing in human pluripotent stem cell (iPSC)-derived neurons and isogenic controls, we show that the FTD-causing V337M tau mutation impairs activity-dependent plasticity of the cytoskeleton in the axon initial segment (AIS). Extracellular recordings by multi-electrode arrays (MEAs) revealed that the V337M tau mutation in human neurons leads to an abnormal increase in neuronal activity in response to chronic depolarization. Stochastic optical reconstruction microscopy of human neurons with this mutation showed that AIS plasticity is impaired by the abnormal accumulation of end-binding protein 3 (EB3) in the AIS submembrane region. These findings expand our understanding of how FTD-causing tau mutations dysregulate components of the neuronal cytoskeleton, leading to network dysfunction., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Giant ankyrin-G (gAnkG) coordinates assembly of axon initial segments (AISs), which are sites of action potential generation located in proximal axons of most vertebrate neurons. Here, we identify a mechanism required for normal neural development in humans that ensures ordered recruitment of gAnkG and β4-spectrin to the AIS. We identified 3 human neurodevelopmental missense mutations located in the neurospecific domain of gAnkG that prevent recruitment of β4-spectrin, resulting in a lower density and more elongated pattern for gAnkG and its partners than in the mature AIS. We found that these mutations inhibit transition of gAnkG from a closed configuration with close apposition of N- and C-terminal domains to an extended state that is required for binding and recruitment of β4-spectrin, and normally occurs early in development of the AIS. We further found that the neurospecific domain is highly phosphorylated in mouse brain, and that phosphorylation at 2 sites (S1982 and S2619) is required for the conformational change and for recruitment of β4-spectrin. Together, these findings resolve a discrete intermediate stage in formation of the AIS that is regulated through phosphorylation of the neurospecific domain of gAnkG., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

The axon initial segment (AIS) comprises a sub-membranous lattice containing periodic actin rings. The overall AIS structure is insensitive to actin-disrupting drugs, but the effects of actin-disrupting drugs on actin rings lack consensus. We examined the effect of latrunculin A and B on the actin cytoskeleton of neurons in culture and actin rings in the AIS. Both latrunculin A and B markedly reduced the overall amount of F-actin in treated neurons in a dose-dependent manner, but the periodicity of actin rings remained unaffected. The insensitivity of AIS actin rings to latrunculin suggests they are relatively stable.