a-microbiomechanical-system-for-studying-varicosity-formation

A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with ...

The Ohio State University

Biogen

Concerted actions of various ion channels that are precisely targeted along axons are crucial for action potential initiation and propagation, and neurotransmitter release. However, the dynamics of channel protein transport in axons remain unknown. Here, using time-lapse imaging, we found fluorescently tagged Kv1.2 voltage-gated K(+) channels (YFP-Kv1.2) moved bi-directionally in discrete puncta along hippocampal axons. Expressing Kvbeta2, a Kv1 accessory subunit, markedly increased the velocity, the travel distance, and the percentage of moving time of these puncta in both anterograde and retrograde directions. Suppressing the Kvbeta2-associated protein, plus-end binding protein EB1 or kinesin II/KIF3A, by siRNA, significantly decreased the velocity of YFP-Kv1.2 moving puncta in both directions. Kvbeta2 mutants with disrupted either Kv1.2-Kvbeta2 binding or Kvbeta2-EB1 binding failed to increase the velocity of YFP-Kv1.2 puncta, confirming a central role of Kvbeta2. Furthermore, fluorescently tagged Kv1.2 and Kvbeta2 co-moved along axons. Surprisingly, when co-moving with Kv1.2 and Kvbeta2, EB1 appeared to travel markedly faster than its plus-end tracking. Finally, using fission yeast S. pombe expressing YFP-fusion proteins as reference standards to calibrate our microscope, we estimated the numbers of YFP-Kv1.2 tetramers in axonal puncta. Taken together, our results suggest that proper amounts of Kv1 channels and their associated proteins are required for efficient transport of Kv1 channel proteins along axons.

Dynamics of Kv1 channel transport in axons.

Proper axonal and dendritic bundling is essential for the establishment of neuronal connections and the synchronization of synaptic inputs, respectively. Cell adhesion molecules of the L1-CAM (L1-cell adhesion molecule) family regulate axon guidance and fasciculation, neuron migration, dendrite morphology, and synaptic plasticity. It remains unclear how these molecules play so many different roles. Here we show that polarized axon-dendrite targeting of an avian L1-CAM protein, NgCAM (neuron-glia cell adhesion molecule), can regulate the switch of bundling of the two major compartments of rat hippocampal neurons. Using a new in-vitro model for studying neurite-neurite interactions, we found that expressed axonal NgCAM induced robust axonal bundling via the trans-homophilic interaction of immunoglobulin domains. Interestingly, dendritic bundling was induced by the dendritic targeting of NgCAM, caused by either deleting its fibronectin repeats or blocking activities of protein kinases. Consistent with the NgCAM results, expression of mouse L1-CAM also induced axonal bundling and blocking kinase activities disrupted its axonal targeting. Furthermore, the trans-homophilic interaction stabilized the bundle formation, probably through recruiting NgCAM proteins to contact sites and promoting guided axon outgrowth. Taken together, our results suggest that precise localization of L1-CAM is important for establishing proper cell-cell contacts in neural circuits.

Polarized targeting of L1-CAM regulates axonal and dendritic bundling in vitro.

Precise targeting of various voltage-gated ion channels to proper membrane domains is crucial for their distinct roles in neuronal excitability and synaptic transmission. How each channel protein is transported within the cytoplasm is poorly understood. Here, we report that KIF5/kinesin I transports Kv3.1 voltage-gated K(+) (Kv) channels through the axon initial segment (AIS) via direct binding. First, we have identified a novel interaction between Kv3.1 and KIF5, confirmed by immunoprecipitation from mouse brain lysates and by pull-down assays with exogenously expressed proteins. The interaction is mediated by a direct binding between the Kv3.1 N-terminal T1 domain and a conserved region in KIF5 tail domains, in which proper T1 tetramerization is crucial. Overexpression of this region of KIF5B markedly reduces axonal levels of Kv3.1bHA. In mature hippocampal neurons, endogenous Kv3.1b and KIF5 colocalize. Suppressing the endogenous KIF5B level by RNA interference significantly reduces the Kv3.1b axonal level. Furthermore, mutating the Zn(2+)-binding site within T1 markedly decreases channel axonal targeting and forward trafficking, likely through disrupting T1 tetramerization and hence eliminating the binding to KIF5 tail. The mutation also alters channel activity. Interestingly, coexpression of the YFP (yellow fluorescent protein)-tagged KIF5B assists dendritic Kv3.1a and even mutants with a faulty axonal targeting motif to penetrate the AIS. Finally, fluorescently tagged Kv3.1 channels colocalize and comove with KIF5B along axons revealed by two-color time-lapse imaging. Our findings suggest that the binding to KIF5 ensures properly assembled and functioning Kv3.1 channels to be transported into axons.

Kinesin I transports tetramerized Kv3 channels through the axon initial segment via direct binding.

Synaptic inputs received at dendrites are converted into digital outputs encoded by action potentials generated at the axon initial segment in most neurons. Here, we report that alternative splicing regulates polarized targeting of Kv3.1 voltage-gated potassium (Kv) channels to adjust the input-output relationship. The spiking frequency of cultured hippocampal neurons correlated with the level of endogenous Kv3 channels. Expression of axonal Kv3.1b, the longer form of Kv3.1 splice variants, effectively converted slow-spiking young neurons to fast-spiking ones; this was not the case for Kv1.2 or Kv4.2 channel constructs. Despite having identical biophysical properties as Kv3.1b, dendritic Kv3.1a was significantly less effective at increasing the maximal firing frequency. This suggests a possible role of channel targeting in regulating spiking frequency. Mutagenesis studies suggest the electrostatic repulsion between the Kv3.1b N/C termini, created by its C-terminal splice domain, unmasks the Kv3.1b axonal targeting motif. Kv3.1b axonal targeting increased the maximal spiking frequency in response to prolonged depolarization. This finding was further supported by the results of local application of channel blockers and computer simulations. Taken together, our studies have demonstrated that alternative splicing controls neuronal firing rates by regulating the polarized targeting of Kv3.1 channels.

Alternative splicing regulates kv3.1 polarized targeting to adjust maximal spiking frequency.

Zinc, a divalent heavy metal ion and an essential mineral for life, regulates synaptic transmission and neuronal excitability via ion channels. However, its binding sites and regulatory mechanisms are poorly understood. Here, we report that Kv3 channel assembly, localization and activity are regulated by zinc through different binding sites. Local perfusion of zinc reversibly reduced spiking frequency of cultured neurons most likely by suppressing Kv3 channels. Indeed, zinc inhibited Kv3.1 channel activity and slowed activation kinetics, independent of its site in the N-terminal T1 domain. Biochemical assays surprisingly identified a novel zinc-binding site in the Kv3.1 C-terminus, critical for channel activity and axonal targeting, but not for the zinc inhibition. Finally, mutagenesis revealed an important role of the junction between the first transmembrane (TM) segment and the first extracellular loop in sensing zinc. Its mutant enabled fast spiking with relative resistance to the zinc inhibition. Therefore, our studies provide novel mechanistic insights into the multifaceted regulation of Kv3 channel activity and localization by divalent heavy metal ions.

Kv3 channel assembly, trafficking and activity are regulated by zinc through different binding sites.

The conventional kinesin motor transports many different cargos to specific locations in neurons. How cargos regulate motor function remains unclear. Here we focus on KIF5, the heavy chain of conventional kinesin, and report that the Kv3 (Shaw) voltage-gated K(+) channel, the only known tetrameric KIF5-binding protein, clusters and activates KIF5 motors during axonal transport. Endogenous KIF5 often forms clusters along axons, suggesting a potential role of KIF5-binding proteins. Our biochemical assays reveal that the high-affinity multimeric binding between the Kv3.1 T1 domain and KIF5B requires three basic residues in the KIF5B tail. Kv3.1 T1 competes with the motor domain and microtubules, but not with kinesin light chain 1 (KLC1), for binding to the KIF5B tail. Live-cell imaging assays show that four KIF5-binding proteins, Kv3.1, KLC1 and two synaptic proteins SNAP25 and VAMP2, differ in how they regulate KIF5B distribution. Only Kv3.1 markedly increases the frequency and number of KIF5B-YFP anterograde puncta. Deletion of Kv3.1 channels reduces KIF5 clusters in mouse cerebellar neurons. Therefore, clustering and activation of KIF5 motors by Kv3 regulate the motor number in carrier vesicles containing the channel proteins, contributing not only to the specificity of Kv3 channel transport, but also to the cargo-mediated regulation of motor function.

Activation of conventional kinesin motors in clusters by Shaw voltage-gated K+ channels.

Action potentials (APs) propagating along axons require the activation of voltage-gated Na(+) (Nav) channels. How Nav channels are transported into axons is unknown. We show that KIF5/kinesin-1 directly binds to ankyrin-G (AnkG) to transport Nav channels into axons. KIF5 and Nav1.2 channels bind to multiple sites in the AnkG N-terminal domain that contains 24 ankyrin repeats. Disrupting AnkG-KIF5 binding with small interfering RNA or dominant-negative constructs markedly reduced Nav channel levels at the axon initial segment (AIS) and along entire axons, thereby decreasing AP firing. Live-cell imaging showed that fluorescently tagged AnkG or Nav1.2 cotransported with KIF5 along axons. Deleting AnkG in vivo or virus-mediated expression of a dominant-negative KIF5 construct specifically decreased the axonal level of Nav, but not Kv1.2, channels in mouse cerebellum. These results indicate that AnkG functions as an adaptor to link Nav channels to KIF5 during axonal transport before anchoring them to the AIS and nodes of Ranvier. 

Ankyrin-G directly binds to kinesin-1 to transport voltage-gated Na+ channels into axons.

Rapid sensation of mechanical stimuli is often mediated by mechanosensitve ion channels. Their opening results from conformational changes induced by mechanical forces. It leads to membrane permeation of selected ions and thereby to electrical signaling. Newly identified mechanosensitive ion channels are emerging at an astonishing rate, including some that are traditionally assigned for completely different functions. In this review, we first provide a brief overview of ion channels that are known to play a role in mechanosensation. Next, we focus on three representative ones, including the transient receptor potential channel V4 (TRPV4), Kv1.1 voltage-gated potassium (Kv) channel, and Piezo channels. Their structures, biophysical properties, expression and targeting patterns, and physiological functions are highlighted. The potential role of their mechanosensation in related diseases is further discussed. In sum, mechanosensation appears to be achieved in a variety of ways by different proteins and plays a fundamental role in the function of various organs under normal and abnormal conditions. 

Physiological and pathological functions of mechanosensitive ion channels.

Calcium signaling in phagocytes is essential for cellular activation, migration, and the potential resolution of infection or inflammation. The generation of reactive oxygen species (ROS) via activation of NADPH (nicotinamide adenine dinucleotide phosphate)-oxidase activity in macrophages has been linked to altered intracellular calcium concentrations. Because of its role as an oxidative stress sensor in phagocytes, we investigated the function of the cation channel transient receptor potential melastatin 2 (TRPM2) in macrophages during oxidative stress responses induced by Helicobacter pylori infection. We show that Trpm2/ mice, when chronically infected with H. pylori, exhibit increased gastric inflammation and decreased bacterial colonization compared with wild-type (WT) mice. The absence of TRPM2 triggers greater macrophage production of inflammatory mediators and promotes classically activated macrophage M1 polarization in response to H. pylori. TRPM2-deficient macrophages upon H. pylori stimulation are unable to control intracellular calcium levels, which results in calcium overloading. Furthermore, increased intracellular calcium in TRPM2/ macrophages enhanced mitogen-activated protein kinase and NADPH-oxidase activities, compared with WT macrophages. Our data suggest that augmented production of ROS and inflammatory cytokines with TRPM2 deletion regulates oxidative stress in macrophages and consequently decreases H. pylori gastric colonization while increasing inflammation in the gastric mucosa.

TRPM2 ion channels regulate macrophage polarization and gastric inflammation during Helicobacter pylori infection.

Little is known about mechanical regulation of morphological and functional polarity of central neurons. In this study, we report that mechanical stress specifically induces varicosities in the axons but not the dendrites of central neurons by activating TRPV4, a Ca/Na-permeable mechanosensitive channel. This process is unexpectedly rapid and reversible, consistent with the formation of axonal varicosities in vivo induced by mechanical impact in a mouse model of mild traumatic brain injury. In contrast, prolonged stimulation of glutamate receptors induces varicosities in dendrites but not in axons. We further show that axonal varicosities are induced by persistent Ca increase, disassembled microtubules (MTs), and subsequently reversible disruption of axonal transport, and are regulated by stable tubulin-only polypeptide, an MT-associated protein. Finally, axonal varicosity initiation can trigger action potentials to antidromically propagate to the soma in retrograde signaling. Therefore, our study demonstrates a new feature of neuronal polarity: axons and dendrites preferentially respond to physical and chemical stresses, respectively.

Polarity of varicosity initiation in central neuron mechanosensation.

The development of neuroprotective and repair strategies for treating progressive multiple sclerosis (MS) requires new insights into axonal injury. 4-aminopyridine (4-AP), a blocker of voltage-gated K (Kv) channels, is used in symptomatic treatment of progressive MS, but the underlying mechanism remains unclear. Here we report that deleting Kv3.1-the channel with the highest 4-AP sensitivity-reduces clinical signs in experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. In Kv3.1 knockout (KO) mice, EAE lesions in sensory and motor tracts of spinal cord were markedly reduced, and radial astroglia were activated with increased expression of brain derived neurotrophic factor (BDNF). Kv3.3/Kv3.1 and activated BDNF receptors were upregulated in demyelinating axons in EAE and MS lesions. In spinal cord myelin coculture, BDNF treatment promoted myelination, and neuronal firing via altering channel expression. Therefore, suppressing Kv3.1 alters neural circuit activity, which may enhance BNDF signaling and hence protect axons from inflammatory insults.

Article	Year
Dynamics of Kv1 channel transport in axons. PloS one\| PubMed ID: 20694152	2010
Polarized targeting of L1-CAM regulates axonal and dendritic bundling in vitro. The European journal of neuroscience\| PubMed ID: 20964729	2010
Kinesin I transports tetramerized Kv3 channels through the axon initial segment via direct binding. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 21106837	2010
Alternative splicing regulates kv3.1 polarized targeting to adjust maximal spiking frequency. The Journal of biological chemistry\| PubMed ID: 22105078	2012
Kv3 channel assembly, trafficking and activity are regulated by zinc through different binding sites. The Journal of physiology\| PubMed ID: 23420657	2013
Activation of conventional kinesin motors in clusters by Shaw voltage-gated K+ channels. Journal of cell science\| PubMed ID: 23487040	2013
Ankyrin-G directly binds to kinesin-1 to transport voltage-gated Na+ channels into axons. Developmental cell\| PubMed ID: 24412576	2014
Physiological and pathological functions of mechanosensitive ion channels. Molecular neurobiology\| PubMed ID: 24532247	2014
TRPM2 ion channels regulate macrophage polarization and gastric inflammation during Helicobacter pylori infection. Mucosal immunology\| PubMed ID: 27435104	2017
Polarity of varicosity initiation in central neuron mechanosensation. The Journal of cell biology\| PubMed ID: 28606925	2017
Suppression of Inflammatory Demyelinaton and Axon Degeneration through Inhibiting Kv3 Channels. Frontiers in molecular neuroscience\| PubMed ID: 29123469	2017

Yuanzheng Gu

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