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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 -
Frontiers in Molecular Neuroscience 2023AnkG, encoded by the gene, is a multifunctional scaffold protein with complex isoform expression: the 480 and 270 kDa isoforms have roles at the axon initial segment...
INTRODUCTION
AnkG, encoded by the gene, is a multifunctional scaffold protein with complex isoform expression: the 480 and 270 kDa isoforms have roles at the axon initial segment and node of Ranvier, whereas the 190 kDa isoform (AnkG-190) has an emerging role in the dendritic shaft and spine heads. All isoforms of AnkG undergo palmitoylation, a post-translational modification regulating protein attachment to lipid membranes. However, palmitoylation of AnkG-190 has not been investigated in dendritic spines. The gene and altered expression of AnkG proteins are associated with a variety of neuropsychiatric and neurodevelopmental disorders including bipolar disorder and are implicated in the lithium response, a commonly used mood stabilizer for bipolar disorder patients, although the precise mechanisms involved are unknown.
RESULT
Here, we showed that Cys70 palmitoylation stabilizes the localization of AnkG-190 in spine heads and at dendritic plasma membrane nanodomains. Mutation of Cys70 impairs AnkG-190 function in dendritic spines and alters PSD-95 scaffolding. Interestingly, we find that lithium reduces AnkG-190 palmitoylation thereby increasing its mobility in dendritic spines. Finally, we demonstrate that the palmitoyl acyl transferase ZDHHC8, but not ZDHHC5, increases AnkG-190 stability in spine heads and is inhibited by lithium.
DISCUSSION
Together, our data reveal that palmitoylation is critical for AnkG-190 localization and function and a potential ZDHHC8/AnkG-190 mechanism linking AnkG-190 mobility to the neuronal effects of lithium.
PubMed: 36969554
DOI: 10.3389/fnmol.2023.1144066 -
The Journal of Physiology May 2023In neocortical layer-5 pyramidal neurons, the action potential (AP) is generated in the axon initial segment (AIS) when the membrane potential (V ) reaches the threshold...
In neocortical layer-5 pyramidal neurons, the action potential (AP) is generated in the axon initial segment (AIS) when the membrane potential (V ) reaches the threshold for activation of the voltage-gated Na channels (VGNCs) Na 1.2 and Na 1.6. Yet, whereas these VGNCs are known to differ in spatial distribution along the AIS and in biophysical properties, our understanding of the functional differences between the two channels remains elusive. Here, using ultrafast Na , V and Ca imaging in combination with partial block of Na 1.2 by the peptide G G -huwentoxin-IV, we demonstrate an exclusive role of Na 1.2 in shaping the generating AP. Precisely, we show that selective block of ∼30% of Na 1.2 widens the AP in the distal part of the AIS and we demonstrate that this effect is due to a loss of activation of BK Ca -activated K channels (CAKCs). Indeed, Ca influx via Na 1.2 activates BK CAKCs, determining the amplitude and the early phase of repolarization of the AP in the AIS. By using control experiments using 4,9-anhydrotetrodotoxin, a moderately selective inhibitor of Na 1.6, we concluded that the Ca influx shaping the early phase of the AP is exclusive of Na 1.2. Hence, we mimicked this result with a neuron model in which the role of the different ion channels tested reproduced the experimental evidence. The exclusive role of Na 1.2 reported here is important for understanding the physiology and pathology of neuronal excitability. KEY POINTS: We optically analysed the action potential generated in the axon initial segment of mouse layer-5 neocortical pyramidal neurons and its associated Na and Ca currents using ultrafast imaging techniques. We found that partial selective block of the voltage-gated Na channel Na 1.2, produced by a recently developed peptide, widens the shape of the action potential in the distal part of the axon initial segment. We demonstrate that this effect is due to a reduction of the Ca influx through Na 1.2 that activates BK Ca -activated K channels. To validate our conclusions, we generated a neuron model that reproduces the ensemble of our experimental results. The present results indicate a specific role of Na 1.2 in the axon initial segment for shaping of the action potential during its generation.
Topics: Mice; Animals; Axon Initial Segment; Action Potentials; Large-Conductance Calcium-Activated Potassium Channels; Pyramidal Cells; Peptides
PubMed: 36946031
DOI: 10.1113/JP283801 -
BioRxiv : the Preprint Server For... Mar 2023Neural circuits are reorganized with specificity during learning. Genetically-defined subgroups of inhibitory interneurons are thought to play distinct roles in...
Neural circuits are reorganized with specificity during learning. Genetically-defined subgroups of inhibitory interneurons are thought to play distinct roles in learning, but heterogeneity within these subgroups has limited our understanding of the scope and nature of their specific contributions to learning. Here we reveal that the chandelier cell (ChC), an interneuron type that specializes in inhibiting the axon-initial segment (AIS) of pyramidal neurons, establishes cortical microcircuits for organizing neural coding through selective axo-axonic synaptic plasticity. We find that organized motor control is mediated by enhanced population coding of direction-tuned premotor neurons, whose tuning is refined through suppression of irrelevant neuronal activity. ChCs are required for learning-dependent refinements via providing selective inhibitory control over pyramidal neurons rather than global suppression. Quantitative analysis on structural plasticity of axo-axonic synapses revealed that ChCs redistributed inhibitory weights to individual pyramidal neurons during learning. These results demonstrate an adaptive logic of the inhibitory circuit motif responsible for organizing distributed neural representations. Thus, ChCs permit efficient cortical computation in a target cell specific manner, which highlights the significance of interneuron diversity.
PubMed: 36945592
DOI: 10.1101/2023.03.10.531767 -
BioRxiv : the Preprint Server For... Mar 2023Axon initial segment (AIS) cell surface proteins mediate key biological processes in neurons including action potential initiation and axo-axonic synapse formation....
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 used 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 used CRISPR-mediated genome editing for insertion of epitope tags in the endogenous proteins. We found Contactin-1 (Cntn1) among the previously unknown AIS proteins we identified. 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 is required for AIS axo-axonic innervation by inhibitory basket cells in the cerebellum and inhibitory chandelier cells in the cortex.
PubMed: 36945454
DOI: 10.1101/2023.03.06.531378 -
Cells Feb 2023Our previous studies demonstrated that enzymatic removal of highly sulfated heparan sulfates with heparinase 1 impaired axonal excitability and reduced expression of...
Our previous studies demonstrated that enzymatic removal of highly sulfated heparan sulfates with heparinase 1 impaired axonal excitability and reduced expression of ankyrin G at the axon initial segments in the CA1 region of the hippocampus ex vivo, impaired context discrimination in vivo, and increased Ca/calmodulin-dependent protein kinase II (CaMKII) activity in vitro. Here, we show that in vivo delivery of heparinase 1 in the CA1 region of the hippocampus elevated autophosphorylation of CaMKII 24 h after injection in mice. Patch clamp recording in CA1 neurons revealed no significant heparinase effects on the amplitude or frequency of miniature excitatory and inhibitory postsynaptic currents, while the threshold for action potential generation was increased and fewer spikes were generated in response to current injection. Delivery of heparinase on the next day after contextual fear conditioning induced context overgeneralization 24 h after injection. Co-administration of heparinase with the CaMKII inhibitor (autocamtide-2-related inhibitory peptide) rescued neuronal excitability and expression of ankyrin G at the axon initial segment. It also restored context discrimination, suggesting the key role of CaMKII in neuronal signaling downstream of heparan sulfate proteoglycans and highlighting a link between impaired CA1 pyramidal cell excitability and context generalization during recall of contextual memories.
Topics: Animals; Mice; Ankyrins; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Heparin Lyase; Heparitin Sulfate; Hippocampus
PubMed: 36899880
DOI: 10.3390/cells12050744 -
Neuroscience Bulletin Jul 2023The axon initial segment (AIS) is a highly specialized axonal compartment where the action potential is initiated. The heterogeneity of AISs has been suggested to occur...
The axon initial segment (AIS) is a highly specialized axonal compartment where the action potential is initiated. The heterogeneity of AISs has been suggested to occur between interneurons and pyramidal neurons (PyNs), which likely contributes to their unique spiking properties. However, whether the various characteristics of AISs can be linked to specific PyN subtypes remains unknown. Here, we report that in the prelimbic cortex (PL) of the mouse, two types of PyNs with axon projections either to the contralateral PL or to the ipsilateral basal lateral amygdala, possess distinct AIS properties reflected by morphology, ion channel expression, action potential initiation, and axo-axonic synaptic inputs from chandelier cells. Furthermore, projection-specific AIS diversity is more prominent in the superficial layer than in the deep layer. Thus, our study reveals the cortical layer- and axon projection-specific heterogeneity of PyN AISs, which may endow the spiking of various PyN types with exquisite modulation.
Topics: Mice; Animals; Axon Initial Segment; Synapses; Pyramidal Cells; Cerebral Cortex; Axons
PubMed: 36849716
DOI: 10.1007/s12264-023-01038-5 -
BioRxiv : the Preprint Server For... Feb 2023encodes the sodium-activated potassium channel Slack (KCNT1, K 1.1), an important mediator of neuronal membrane excitability. Gain-of-function (GOF) mutations in...
UNLABELLED
encodes the sodium-activated potassium channel Slack (KCNT1, K 1.1), an important mediator of neuronal membrane excitability. Gain-of-function (GOF) mutations in humans lead cortical network hyperexcitability and seizures, as well as very severe intellectual disability. Using a mouse model of Slack GOF-associated epilepsy, we found that both excitatory and inhibitory neurons of the cerebral cortex have increased Na -dependent K (K ) currents and voltage-dependent sodium (Na ) currents. The characteristics of the increased K currents were, however, different in the two cell types such that the intrinsic excitability of excitatory neurons was enhanced but that of inhibitory neurons was suppressed. We further showed that the expression of Na channel subunits, particularly that of Na 1.6, is upregulated and that the length of the axon initial segment (AIS) and of axonal Na immunostaining is increased in both neuron types. We found that the proximity of the AIS to the soma is shorter in excitatory neurons than in inhibitory neurons of the mutant animals, potentially contributing to the different effects on membrane excitability. Our study on the coordinate regulation of K currents and the expression of Na channels may provide a new avenue for understanding and treating epilepsies and other neurological disorders.
IN BRIEF
In a genetic mouse model of Na -activated K potassium channel gene -related childhood epilepsy, Wu . show that a disease-causing gain-of-function (GOF) mutation in Slack channel causes opposite effects on excitability of cortical excitatory and inhibitory neurons. In contrast to heterologous expression systems, they find that the increase in potassium current substantially alters the expression of sodium channel subunits, resulting in increased lengths of axonal initial segments.
HIGHLIGHTS
GOF mutations in Slack potassium channel cause elevated outward K currents and inward voltage-dependent Na (Na ) currents in cortical neurons Slack GOF does not alter the expression of Slack channel but upregulates the expression of Na channel Slack GOF enhances the excitability of excitatory neurons but suppresses the firing of inhibitory interneuronsSlack GOF alters the length of AIS in both excitatory and inhibitory neuronsProximity of AIS to the soma is different between excitatory neuron and inhibitory neuron.
PubMed: 36824888
DOI: 10.1101/2023.02.14.528229 -
ELife Feb 2023Voltage-gated sodium channels located in axon initial segments (AIS) trigger action potentials (AP) and play pivotal roles in the excitability of cortical pyramidal...
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 Na1.2 and Na1.6 channels lead to distinct contributions to AP initiation and propagation. While Na1.6 at the distal AIS promotes AP initiation and forward propagation, Na1.2 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 Na1.6, these effects were attributed to SUMOylation of Na1.2. Moreover, SUMO effects were absent in a mouse engineered to express Na1.2-Lys38Gln channels that lack the site for SUMO linkage. Thus, SUMOylation of Na1.2 exclusively controls I generation and AP backpropagation, thereby playing a prominent role in synaptic integration and plasticity.
Topics: Mice; Animals; Action Potentials; Sumoylation; Pyramidal Cells; Neurons; Axon Initial Segment
PubMed: 36794908
DOI: 10.7554/eLife.81463 -
BioRxiv : the Preprint Server For... Feb 2023Na1.7, a membrane-bound voltage-gated sodium channel, is preferentially expressed along primary sensory neurons, including their peripheral & central nerve endings,...
Na1.7, a membrane-bound voltage-gated sodium channel, is preferentially expressed along primary sensory neurons, including their peripheral & central nerve endings, axons, and soma within the dorsal root ganglia and plays an integral role in amplifying membrane depolarization and pain neurotransmission. Loss- and gain-of-function mutations in the gene encoding Na1.7, , are associated with a complete loss of pain sensation or exacerbated pain in humans, respectively. As an enticing pain target supported by human genetic validation, many compounds have been developed to inhibit Na1.7 but have disappointed in clinical trials. The underlying reasons are still unclear, but recent reports suggest that inhibiting Na1.7 in central terminals of nociceptor afferents is critical for achieving pain relief by pharmacological inhibition of Na1.7. We report for the first time that Na1.7 mRNA is expressed in putative projection neurons (NK1R+) in the human spinal dorsal horn, predominantly in lamina 1 and 2, as well as in deep dorsal horn neurons and motor neurons in the ventral horn. Na1.7 protein was found in the central axons of sensory neurons terminating in lamina 1-2, but also was detected in the axon initial segment of resident spinal dorsal horn neurons and in axons entering the anterior commissure. Given that projection neurons are critical for conveying nociceptive information from the dorsal horn to the brain, these data support that dorsal horn Na1.7 expression may play an unappreciated role in pain phenotypes observed in humans with genetic mutations, and in achieving analgesic efficacy in clinical trials.
PubMed: 36778234
DOI: 10.1101/2023.02.04.527110