-
Neuronal Signaling Dec 2021In neurons, the axon and axon initial segment (AIS) are critical structures for action potential initiation and propagation. Their formation and function rely on tight... (Review)
Review
In neurons, the axon and axon initial segment (AIS) are critical structures for action potential initiation and propagation. Their formation and function rely on tight compartmentalisation, a process where specific proteins are trafficked to and retained at distinct subcellular locations. One mechanism which regulates protein trafficking and association with lipid membranes is the modification of protein cysteine residues with the 16-carbon palmitic acid, known as S-acylation or palmitoylation. Palmitoylation, akin to phosphorylation, is reversible, with palmitate cycling being mediated by substrate-specific enzymes. Palmitoylation is well-known to be highly prevalent among neuronal proteins and is well studied in the context of the synapse. Comparatively, how palmitoylation regulates trafficking and clustering of axonal and AIS proteins remains less understood. This review provides an overview of the current understanding of the biochemical regulation of palmitoylation, its involvement in various neurological diseases, and the most up-to-date perspective on axonal palmitoylation. Through a palmitoylation analysis of the AIS proteome, we also report that an overwhelming proportion of AIS proteins are likely palmitoylated. Overall, our review and analysis confirm a central role for palmitoylation in the formation and function of the axon and AIS and provide a resource for further exploration of palmitoylation-dependent protein targeting to and function at the AIS.
PubMed: 34659801
DOI: 10.1042/NS20210005 -
Proceedings of the National Academy of... Mar 2022SignificanceChandelier cells (ChCs) are a unique type of GABAergic interneuron that form axo-axonic synapses exclusively on the axon initial segment (AIS) of neocortical...
SignificanceChandelier cells (ChCs) are a unique type of GABAergic interneuron that form axo-axonic synapses exclusively on the axon initial segment (AIS) of neocortical pyramidal neurons (PyNs), allowing them to exert powerful yet precise control over PyN firing and population output. The importance of proper ChC function is further underscored by the association of ChC connectivity defects with various neurological conditions. Despite this, the cellular mechanisms governing ChC axo-axonic synapse formation remain poorly understood. Here, we identify microglia as key regulators of ChC axonal morphogenesis and AIS synaptogenesis, and show that disease-induced aberrant microglial activation perturbs proper ChC synaptic development/connectivity in the neocortex. In doing so, such findings highlight the therapeutic potential of manipulating microglia to ensure proper brain wiring.
Topics: Animals; Axon Initial Segment; GABAergic Neurons; Mice; Microglia; Pyramidal Cells; Synapses
PubMed: 35263225
DOI: 10.1073/pnas.2114476119 -
Cells Sep 2020Actin and non-muscle myosins have long been known to play important roles in growth cone steering and neurite outgrowth. More recently, novel functions for non-muscle... (Review)
Review
Actin and non-muscle myosins have long been known to play important roles in growth cone steering and neurite outgrowth. More recently, novel functions for non-muscle myosin have been described in axons and dendritic spines. Consequently, possible roles of actomyosin contraction in organizing and maintaining structural properties of dendritic spines, the size and location of axon initial segment and axonal diameter are emerging research topics. In this review, we aim to summarize recent findings involving myosin localization and function in these compartments and to discuss possible roles for actomyosin in their function and the signaling pathways that control them.
Topics: Actin Cytoskeleton; Actins; Axons; Calcium; Calcium Signaling; Calpain; Cell Compartmentation; Dendritic Spines; Humans; Myosins; Neuronal Plasticity; Spectrin
PubMed: 32882840
DOI: 10.3390/cells9092006 -
Journal of Cell Science Jun 2023The axon initial segment (AIS) is a highly specialized neuronal compartment that regulates the generation of action potentials and maintenance of neuronal polarity. Live...
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.
Topics: Animals; Action Potentials; Amino Acids; Axon Initial Segment; Click Chemistry; Neurons; Mice; Rats
PubMed: 37288813
DOI: 10.1242/jcs.260600 -
Brain Communications 2022Grey matter damage has been established as a key contributor to disability progression in multiple sclerosis. Aside from neuronal loss and axonal transections, which...
Grey matter damage has been established as a key contributor to disability progression in multiple sclerosis. Aside from neuronal loss and axonal transections, which predominate in cortical demyelinated lesions, synaptic alterations have been detected in both demyelinated plaques and normal-appearing grey matter, resulting in functional neuronal damage. The axon initial segment is a key element of neuronal function, responsible for action potential initiation and maintenance of neuronal polarity. Despite several reports of profound axon initial segment alterations in different pathological models, among which experimental auto-immune encephalomyelitis, whether the axon initial segment is affected in multiple sclerosis is still unknown. Using immunohistochemistry, we analysed axon initial segments from control and multiple sclerosis tissue, focusing on layer 5/6 pyramidal neurons in the neocortex and Purkinje cells in the cerebellum and performed analysis on the parameters known to control neuronal excitability, i.e. axon initial segment length and position. We found that the axon initial segment length was increased only in pyramidal neurons of inactive demyelinated lesions, compared with normal appearing grey matter tissue. In contrast, in both cell types, the axon initial segment position was altered, with an increased soma-axon initial segment gap, in both active and inactive demyelinated lesions. In addition, using a computational model, we show that this increased gap between soma and axon initial segment might increase neuronal excitability. Taken together, these results show, for the first time, changes of axon initial segments in multiple sclerosis, in active as well as inactive grey matter lesions in both neocortex and cerebellum, which might alter neuronal function.
PubMed: 36451656
DOI: 10.1093/braincomms/fcac284 -
Molecular Psychiatry Apr 2023Copy number variations (CNVs) are associated with psychiatric and neurodevelopmental disorders (NDDs), and most, including the recurrent 15q13.3 microdeletion disorder,...
Copy number variations (CNVs) are associated with psychiatric and neurodevelopmental disorders (NDDs), and most, including the recurrent 15q13.3 microdeletion disorder, have unknown disease mechanisms. We used a heterozygous 15q13.3 microdeletion mouse model and patient iPSC-derived neurons to reveal developmental defects in neuronal maturation and network activity. To identify the underlying molecular dysfunction, we developed a neuron-specific proximity-labeling proteomics (BioID2) pipeline, combined with patient mutations, to target the 15q13.3 CNV genetic driver OTUD7A. OTUD7A is an emerging independent NDD risk gene with no known function in the brain, but has putative deubiquitinase function. The OTUD7A protein-protein interaction network included synaptic, axonal, and cytoskeletal proteins and was enriched for ASD and epilepsy risk genes (Ank3, Ank2, SPTAN1, SPTBN1). The interactions between OTUD7A and Ankyrin-G (Ank3) and Ankyrin-B (Ank2) were disrupted by an epilepsy-associated OTUD7A L233F variant. Further investigation of Ankyrin-G in mouse and human 15q13.3 microdeletion and OTUD7A models revealed protein instability, increased polyubiquitination, and decreased levels in the axon initial segment, while structured illumination microscopy identified reduced Ankyrin-G nanodomains in dendritic spines. Functional analysis of human 15q13.3 microdeletion and OTUD7A models revealed shared and distinct impairments to axonal growth and intrinsic excitability. Importantly, restoring OTUD7A or Ankyrin-G expression in 15q13.3 microdeletion neurons led to a reversal of abnormalities. These data reveal a critical OTUD7A-Ankyrin pathway in neuronal development, which is impaired in the 15q13.3 microdeletion syndrome, leading to neuronal dysfunction. Furthermore, our study highlights the utility of targeting CNV genes using cell type-specific proteomics to identify shared and unexplored disease mechanisms across NDDs.
Topics: Humans; Mice; Animals; Ankyrins; DNA Copy Number Variations; Epilepsy; Neurons
PubMed: 36604605
DOI: 10.1038/s41380-022-01937-5 -
Channels (Austin, Tex.) Dec 2020Voltage gated sodium channels (Na) play a crucial role in action potential initiation and propagation. Although the discovery of Na channels dates back more than... (Review)
Review
Voltage gated sodium channels (Na) play a crucial role in action potential initiation and propagation. Although the discovery of Na 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 channel trafficking, localization and regulation. This review summarizes the different trafficking mechanisms underlying the polarized Na 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 channel localization is emphasized by the relationship between mutations, impaired trafficking and disease. While this review focuses on Na1.6, other Na isoforms are also discussed.
Topics: Action Potentials; Animals; Axon Initial Segment; Female; Humans; NAV1.6 Voltage-Gated Sodium Channel; Neurons; Protein Transport; Voltage-Gated Sodium Channels
PubMed: 31841065
DOI: 10.1080/19336950.2019.1700082 -
Neurology(R) Neuroimmunology &... May 2023Autoantibodies to leucine-rich glioma inactivated protein 1 (LGI1) cause an autoimmune limbic encephalitis with frequent focal seizures and anterograde memory...
BACKGROUND AND OBJECTIVES
Autoantibodies to leucine-rich glioma inactivated protein 1 (LGI1) cause an autoimmune limbic encephalitis with frequent focal seizures and anterograde memory dysfunction. LGI1 is a neuronal secreted linker protein with 2 functional domains: the leucine-rich repeat (LRR) and epitempin (EPTP) regions. LGI1 autoantibodies are known to interfere with presynaptic function and neuronal excitability; however, their epitope-specific mechanisms are incompletely understood.
METHODS
We used patient-derived monoclonal autoantibodies (mAbs), which target either LRR or EPTP domains of LGI1 to investigate long-term antibody-induced alteration of neuronal function. LRR- and EPTP-specific effects were evaluated by patch-clamp recordings in cultured hippocampal neurons and compared with biophysical neuron modeling. K1.1 channel clustering at the axon initial segment (AIS) was quantified by immunocytochemistry and structured illumination microscopy techniques.
RESULTS
Both EPTP and LRR domain-specific mAbs decreased the latency of first somatic action potential firing. However, only the LRR-specific mAbs increased the number of action potential firing together with enhanced initial instantaneous frequency and promoted spike-frequency adaptation, which were less pronounced after the EPTP mAb. This also led to an effective reduction in the slope of ramp-like depolarization in the subthreshold response, suggesting K1 channel dysfunction. A biophysical model of a hippocampal neuron corroborated experimental results and suggests that an isolated reduction of the conductance of K1-mediated K currents largely accounts for the antibody-induced alterations in the initial firing phase and spike-frequency adaptation. Furthermore, K1.1 channel density was spatially redistributed from the distal toward the proximal site of AIS under LRR mAb treatment and, to a lesser extant, under EPTP mAb.
DISCUSSION
These findings indicate an epitope-specific pathophysiology of LGI1 autoantibodies. The pronounced neuronal hyperexcitability and SFA together with dropped slope of ramp-like depolarization after LRR-targeted interference suggest disruption of LGI1-dependent clustering of K channel complexes. Moreover, considering the effective triggering of action potentials at the distal AIS, the altered spatial distribution of K1.1 channel density may contribute to these effects through impairing neuronal control of action potential initiation and synaptic integration.
Topics: Humans; Antibodies, Monoclonal; Autoantibodies; Epitopes; Intracellular Signaling Peptides and Proteins; Leucine; Nerve Tissue Proteins; Neurons
PubMed: 37028941
DOI: 10.1212/NXI.0000000000200096 -
Translational Vision Science &... Apr 2023Axons depend on long-range transport of proteins and organelles which increases susceptibility to metabolic stress in disease. The axon initial segment (AIS) is...
PURPOSE
Axons depend on long-range transport of proteins and organelles which increases susceptibility to metabolic stress in disease. The axon initial segment (AIS) is particularly vulnerable due to the high bioenergetic demand of action potential generation. Here, we prepared retinal ganglion cells derived from human embryonic stem cells (hRGCs) to probe how axonal stress alters AIS morphology.
METHODS
hRGCs were cultured on coverslips or microfluidic platforms. We assayed AIS specification and morphology by immunolabeling against ankyrin G (ankG), an axon-specific protein, and postsynaptic density 95 (PSD-95), a dendrite-specific protein. Using microfluidic platforms that enable fluidic isolation, we added colchicine to the axon compartment to lesion axons. We verified axonopathy by measuring the anterograde axon transport of cholera toxin subunit B and immunolabeling against cleaved caspase 3 (CC3) and phosphorylated neurofilament H (SMI-34). We determined the influence of axon injury on AIS morphology by immunolabeling samples against ankG and measuring AIS distance from soma and length.
RESULTS
Based on measurements of ankG and PSD-95 immunolabeling, microfluidic platforms promote the formation and separation of distinct somatic-dendritic versus axonal compartments in hRGCs compared to coverslip cultures. Chemical lesioning of axons by colchicine reduced hRGC anterograde axon transport, increased varicosity density, and enhanced expression of CC3 and SMI-34. Interestingly, we found that colchicine selectively affected hRGCs with axon-carrying dendrites by reducing AIS distance from somas and increasing length, thus suggesting reduced capacity to maintain excitability.
CONCLUSIONS
Thus, microfluidic platforms promote polarized hRGCs that enable modeling of axonopathy.
TRANSLATIONAL RELEVANCE
Microfluidic platforms may be used to assay compartmentalized degeneration that occurs during glaucoma.
Topics: Humans; Retinal Ganglion Cells; Microfluidics; Axons; Action Potentials
PubMed: 37010860
DOI: 10.1167/tvst.12.4.1 -
Cellular and Molecular Neurobiology Apr 2022Studies from postmortem and animal models have revealed altered synapse morphology and function in the brain of posttraumatic stress disorder (PTSD). And the effects of...
Studies from postmortem and animal models have revealed altered synapse morphology and function in the brain of posttraumatic stress disorder (PTSD). And the effects of PTSD on dendrites and spines have been reported, however, the effection on axon include microtubule (MT) and synaptic vesicles of presynaptic elements remains unknown. Hippocampus is involved in abnormal memory in PTSD. In the present study, we used the single prolonged stress (SPS) model to mimic PTSD. Quantitative real-time polymerase chain reaction (RT-qPCR) and high-throughput sequencing (GSE153081) were utilized to analyze differentially expressed genes (DEGs) in the hippocampus of control and SPS rats. Immunofluorescence and western blotting were performed to examine change in axon-related proteins. Synaptic function was evaluated by measuring miniature excitatory postsynaptic currents (mEPSCs). RNA-sequencing analysis revealed 230 significantly DEGs between the control and SPS groups. Gene Ontology analysis revealed upregulation in axonemal assembly, MT formation, or movement, but downregulation in axon initial segment and synaptic vesicles fusion in the hippocampus of SPS rats. Increased expression in tau, β-tubulin MAP1B, KIF9, CCDC40, DNAH12 and decreased expression in p-tau, stathmin suggested SPS induced axon extension. Increased protein expression in VAMP, STX1A, Munc18-1 and decreased expression in synaptotagmin-1 suggested SPS induced more SNARE complex formation but decreased ability in synaptic vesicle fusion to presynaptic active zone membrane in the hippocampus of SPS rats. Further, low mEPSC frequency in SPS rats indicated dysfunction in presynaptic membrane. These results suggest that axon extension and synaptic vesicles fusion abnormality are involved in dysfunction of PTSD.
Topics: Animals; Axons; Disease Models, Animal; Hippocampus; Rats; Stress Disorders, Post-Traumatic
PubMed: 32930942
DOI: 10.1007/s10571-020-00960-w