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Scientific Reports Feb 2018Local translation of membrane proteins in neuronal subcellular domains like soma, dendrites and axon termini is well-documented. In this study, we isolated the...
Local translation of membrane proteins in neuronal subcellular domains like soma, dendrites and axon termini is well-documented. In this study, we isolated the electrical signaling unit of an axon by dissecting giant axons from mature squids (Dosidicus gigas). Axoplasm extracted from these axons was found to contain ribosomal RNAs, ~8000 messenger RNA species, many encoding the translation machinery, membrane proteins, translocon and signal recognition particle (SRP) subunits, endomembrane-associated proteins, and unprecedented proportions of SRP RNA (~68% identical to human homolog). While these components support endoplasmic reticulum-dependent protein synthesis, functional assessment of a newly synthesized membrane protein in axolemma of an isolated axon is technically challenging. Ion channels are ideal proteins for this purpose because their functional dynamics can be directly evaluated by applying voltage clamp across the axon membrane. We delivered in vitro transcribed RNA encoding native or Drosophila voltage-activated Shaker K channel into excised squid giant axons. We found that total K currents increased in both cases; with added inactivation kinetics on those axons injected with RNA encoding the Shaker channel. These results provide unambiguous evidence that isolated axons can exhibit de novo synthesis, assembly and membrane incorporation of fully functional oligomeric membrane proteins.
Topics: Animals; Axons; Cells, Cultured; Decapodiformes; Drosophila; Drosophila Proteins; Ion Channels; Patch-Clamp Techniques; Protein Biosynthesis; Recombinant Proteins
PubMed: 29396520
DOI: 10.1038/s41598-018-20684-8 -
Cellular & Molecular Immunology Jun 2018Guillain-Barré syndrome (GBS) and transverse myelitis (TM) both represent immunologically mediated polyneuropathies of major clinical importance. Both are thought to... (Review)
Review
Guillain-Barré syndrome (GBS) and transverse myelitis (TM) both represent immunologically mediated polyneuropathies of major clinical importance. Both are thought to have a genetic predisposition, but as of yet no specific genetic risk loci have been clearly defined. Both are considered autoimmune, but again the etiologies remain enigmatic. Both may be induced via molecular mimicry, particularly from infectious agents and vaccines, but clearly host factor and co-founding host responses will modulate disease susceptibility and natural history. GBS is an acute inflammatory immune-mediated polyradiculoneuropathy characterized by tingling, progressive weakness, autonomic dysfunction, and pain. Immune injury specifically takes place at the myelin sheath and related Schwann-cell components in acute inflammatory demyelinating polyneuropathy, whereas in acute motor axonal neuropathy membranes on the nerve axon (the axolemma) are the primary target for immune-related injury. Outbreaks of GBS have been reported, most frequently related to Campylobacter jejuni infection, however, other agents such as Zika Virus have been strongly associated. Patients with GBS related to infections frequently produce antibodies against human peripheral nerve gangliosides. In contrast, TM is an inflammatory disorder characterized by acute or subacute motor, sensory, and autonomic spinal cord dysfunction. There is interruption of ascending and descending neuroanatomical pathways on the transverse plane of the spinal cord similar to GBS. It has been suggested to be triggered by infectious agents and molecular mimicry. In this review, we will focus on the putative role of infectious agents as triggering factors of GBS and TM.
Topics: Communicable Diseases; Guillain-Barre Syndrome; Humans; Immunity; Myelitis, Transverse
PubMed: 29375121
DOI: 10.1038/cmi.2017.142 -
Microscopy and Microanalysis : the... Feb 2018The ramus communicans, neural connection between medial and lateral plantar nerves of the horse, was transected to determine the degree to which medial and lateral...
The ramus communicans, neural connection between medial and lateral plantar nerves of the horse, was transected to determine the degree to which medial and lateral plantar nerves contribute to the plantar ramus. After 2 months, sections of plantar nerves immediately proximal and distal to the communicating branch were collected and processed for electron microscopy. All examined nerves had undergone Wallerian degeneration and contained regenerating and mature fibers. Layers of the myelin sheath were separated by spaces and vacuoles, indicating demyelination of medial and lateral plantar nerves. Shrunken axons varied in diameter and were surrounded by an irregular axolemma. Shrunken axoplasm of both myelinated and non-myelinated fibers contained ruptured mitochondria and cristae, disintegrating cytoskeleton, and vacuoles of various sizes. The cytoplasm of neurolemmocytes contained various-sized vesicles, ruptured mitochondria within a fragile basal lamina and myelin whorls of multilayered structures indicative of Wallerian degeneration. These ultrastructural changes, found proximal and distal to the ramus in medial and lateral plantar nerves, suggest that axonal flow is bi-directional through the ramus communicans of the pelvic limbs of horses, a previously unreported finding. As well, maturity of nerves proximal and distal to the ramus indicates that all nerve fibers do not pass through the ramus.
Topics: Animals; Axons; Horses; Microscopy, Electron; Myelin Sheath; Nerve Fibers; Peripheral Nerves
PubMed: 29362000
DOI: 10.1017/S1431927617012818 -
Glia Apr 2018Glycoprotein M6B and the closely related proteolipid protein regulate oligodendrocyte myelination in the central nervous system, but their role in the peripheral nervous...
Glycoprotein M6B and the closely related proteolipid protein regulate oligodendrocyte myelination in the central nervous system, but their role in the peripheral nervous system is less clear. Here we report that M6B is located at nodes of Ranvier in peripheral nerves where it stabilizes the nodal axolemma. We show that M6B is co-localized and associates with gliomedin at Schwann cell microvilli that are attached to the nodes. Developmental analysis of sciatic nerves, as well as of myelinating Schwann cells/dorsal root ganglion neurons cultures, revealed that M6B is already present at heminodes, which are considered the precursors of mature nodes of Ranvier. However, in contrast to gliomedin, which accumulates at heminodes with or prior to Na channels, we often detected Na channel clusters at heminodes without any associated M6B, indicating that it is not required for initial channel clustering. Consistently, nodal cell adhesion molecules (NF186, NrCAM), ion channels (Nav1.2 and Kv7.2), cytoskeletal proteins (AnkG and βIV spectrin), and microvilli components (pERM, syndecan3, gliomedin), are all present at both heminodes and mature nodes of Ranvier in Gpm6b null mice. Using transmission electron microscopy, we show that the absence of M6B results in progressive appearance of nodal protrusions of the nodal axolemma, that are often accompanied by the presence of enlarged mitochondria. Our results reveal that M6B is a Schwann cell microvilli component that preserves the structural integrity of peripheral nodes of Ranvier.
Topics: Animals; Axons; Cell Adhesion Molecules, Neuronal; Cell Membrane; Cells, Cultured; Ganglia, Spinal; Membrane Glycoproteins; Mice, Knockout; Mitochondria; Nerve Tissue Proteins; Neuroglia; Ranvier's Nodes; Rats; Sciatic Nerve; Sodium Channels; Spinal Cord
PubMed: 29282769
DOI: 10.1002/glia.23285 -
Journal of Controlled Release :... Dec 2017The mechanisms of axonal trafficking and membrane targeting are well established for sodium channels, which are the principle targets for perineurally applied local...
The mechanisms of axonal trafficking and membrane targeting are well established for sodium channels, which are the principle targets for perineurally applied local anaesthetics. However, they have not been thoroughly investigated for G protein coupled receptors such as mu-opioid receptors (MOR). Focusing on these axonal mechanisms, we found that axonal MOR functionality is quite distinct in two different pain states, i.e. hindpaw inflammation and nerve injury. We observed axonal membrane MOR binding and functional G protein coupling exclusively at sites of CCI nerve injury. Moreover at these axonal membrane sites, MOR exhibited extensive co-localization with the membrane proteins SNAP and Na/K-ATPase as well as NGF-dependent enhanced lipid rafts and L1CAM anchoring proteins. Silencing endogenous L1CAM with intrathecal L1CAM specific siRNA, disrupting lipid rafts with the perineurial cholesterol-sequestering agent MβCD, as well as suppressing NGF receptor activation with the perineurial NGF receptor inhibitor K252a abrogated MOR axonal membrane integration, functional coupling, and agonist-elicited antinociception at sites of nerve injury. These findings suggest that local conceptual changes resulting from nerve injury are required for the establishment of functional axonal membrane MOR. Axonal integration and subsequent accessibility of functionally coupled MOR are of great relevance particularly for patients suffering from severe pain due to nerve injury or tumour infiltration.
Topics: Analgesics, Opioid; Animals; Axons; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Fentanyl; Freund's Adjuvant; Inflammation; Male; Naloxone; Narcotic Antagonists; Neuralgia; Rats, Wistar; Receptors, Opioid, mu; Sciatic Nerve
PubMed: 29054370
DOI: 10.1016/j.jconrel.2017.10.016 -
Journal of Integrative Neuroscience 2017The present study investigates the temperature dependence of electrotonic potentials in mathematically-simulated myelinated axons with one of three increasingly-severe...
The present study investigates the temperature dependence of electrotonic potentials in mathematically-simulated myelinated axons with one of three increasingly-severe type of amyotrophic lateral sclerosis (ALS) pathology, termed as ALS1, ALS2 and ALS3, respectively, in the physiological range (30-37∘C). These potentials were elicited by long-lasting (100 ms) subthreshold polarizing current stimuli (±40% of the threshold). Numerical solutions were computed using our temperature-dependent multi-layered model. The results showed the following trends: (i) in ALS1, polarizing electrotonic potentials were normal; (ii) in ALS2 and ALS3, action potentials were elicited in the early parts of the depolarizing electrotonic potentials, and (iii) in ALS3, spontaneous discharges were elicited after the termination of applied hyperpolarizing stimuli (i.e., post-anodal excitation). The ionic currents underlying electrotonic potentials in the ALS1 case were attributable to the activation of potassium fast (Kf+) and slow (Ks+) channels in the nodal and internodal axolemma beneath the myelin sheath. By contrast, in ALS2 and ALS3, the depolarizing stimuli activated the classical "transient" Na+ channels in the nodal and internodal axolemma beneath the myelin sheath eliciting action potential generation. These results obtained were closer to those observed in hypothermia (⩽25∘C) than in hyperthermia (⩾40∘C).
Topics: Amyotrophic Lateral Sclerosis; Axons; Body Temperature; Cations, Monovalent; Computer Simulation; Humans; Membrane Potentials; Models, Neurological; Potassium; Sodium
PubMed: 28891518
DOI: 10.3233/JIN-170022 -
Journal of Cell Science Jul 2017Caspr2 and TAG-1 (also known as CNTNAP2 and CNTN2, respectively) are cell adhesion molecules (CAMs) associated with the voltage-gated potassium channels Kv1.1 and Kv1.2...
Caspr2 and TAG-1 (also known as CNTNAP2 and CNTN2, respectively) are cell adhesion molecules (CAMs) associated with the voltage-gated potassium channels Kv1.1 and Kv1.2 (also known as KCNA1 and KCNA2, respectively) at regions controlling axonal excitability, namely, the axon initial segment (AIS) and juxtaparanodes of myelinated axons. The distribution of Kv1 at juxtaparanodes requires axo-glial contacts mediated by Caspr2 and TAG-1. In the present study, we found that TAG-1 strongly colocalizes with Kv1.2 at the AIS of cultured hippocampal neurons, whereas Caspr2 is uniformly expressed along the axolemma. Live-cell imaging revealed that Caspr2 and TAG-1 are sorted together in axonal transport vesicles. Therefore, their differential distribution may result from diffusion and trapping mechanisms induced by selective partnerships. By using deletion constructs, we identified two molecular determinants of Caspr2 that regulate its axonal positioning. First, the LNG2-EGF1 modules in the ectodomain of Caspr2, which are involved in its axonal distribution. Deletion of these modules promotes AIS localization and association with TAG-1. Second, the cytoplasmic PDZ-binding site of Caspr2, which could elicit AIS enrichment and recruitment of the membrane-associated guanylate kinase (MAGuK) protein MPP2. Hence, the selective distribution of Caspr2 and TAG-1 may be regulated, allowing them to modulate the strategic function of the Kv1 complex along axons.
Topics: Axon Initial Segment; Axons; Cell Adhesion Molecules, Neuronal; Contactin 2; HEK293 Cells; Hippocampus; Humans; Membrane Proteins; Nerve Tissue Proteins; Neuroglia; Neurons; Shaker Superfamily of Potassium Channels
PubMed: 28533267
DOI: 10.1242/jcs.202267 -
Brain : a Journal of Neurology Apr 2017See Saporta and Shy (doi:10.1093/awx048) for a scientific commentary on this article.Effective bidirectional signalling between axons and Schwann cells is essential for...
See Saporta and Shy (doi:10.1093/awx048) for a scientific commentary on this article.Effective bidirectional signalling between axons and Schwann cells is essential for both the development and maintenance of peripheral nerve function. We have established conditions by which human induced pluripotent stem cell-derived sensory neurons can be cultured with rat Schwann cells, and have produced for the first time long-term and stable myelinating co-cultures with human neurons. These cultures contain the specialized domains formed by axonal interaction with myelinating Schwann cells, such as clustered voltage-gated sodium channels at the node of Ranvier and Shaker-type potassium channel (Kv1.2) at the juxtaparanode. Expression of type III neuregulin-1 (TIIINRG1) in induced pluripotent stem cell-derived sensory neurons strongly enhances myelination, while conversely pharmacological blockade of the NRG1-ErbB pathway prevents myelination, providing direct evidence for the ability of this pathway to promote the myelination of human sensory axons. The β-secretase, BACE1 is a protease needed to generate active NRG1 from the full-length form. Due to the fact that it also cleaves amyloid precursor protein, BACE1 is a therapeutic target in Alzheimer's disease, however, consistent with its role in NRG1 processing we find that BACE1 inhibition significantly impairs myelination in our co-culture system. In order to exploit co-cultures to address other clinically relevant problems, they were exposed to anti-disialosyl ganglioside antibodies, including those derived from a patient with a sensory predominant, inflammatory neuropathy with mixed axonal and demyelinating electrophysiology. The co-cultures reveal that both mouse and human disialosyl antibodies target the nodal axolemma, induce acute axonal degeneration in the presence of complement, and impair myelination. The human, neuropathy-associated IgM antibody is also shown to induce complement-independent demyelination. Myelinating co-cultures using human induced pluripotent stem cell-derived sensory neurons thus provide insights into the cellular and molecular specialization of axoglial signalling, how pharmacological agents may promote or impede such signalling and the pathogenic effects of ganglioside antibodies.awx012media15372351982001.
Topics: Adult; Animals; Antibodies, Anti-Idiotypic; Cell Differentiation; Coculture Techniques; ErbB Receptors; Female; Humans; Immunoglobulin G; Mice; Myelin Sheath; Neural Stem Cells; Neuregulin-1; Peripheral Nervous System; Rats; Schwann Cells; Sensory Receptor Cells; Transduction, Genetic
PubMed: 28334857
DOI: 10.1093/brain/awx012 -
Translational Neurodegeneration 2017It is increasingly clear that in addition to myelin disruption, axonal degeneration may also represent a key pathology in multiple sclerosis (MS). Hence, elucidating the...
BACKGROUND
It is increasingly clear that in addition to myelin disruption, axonal degeneration may also represent a key pathology in multiple sclerosis (MS). Hence, elucidating the mechanisms of axonal degeneration may not only enhance our understanding of the overall MS pathology, but also elucidate additional therapeutic targets. The objective of this study is assess the degree of axonal membrane disruption and its significance in motor deficits in EAE mice.
METHODS
Experimental Autoimmune Encephalomyelitis was induced in mice by subcutaneous injection of myelin oligodendrocyte glycoprotein/complete Freud's adjuvant emulsion, followed by two intraperitoneal injections of pertussis toxin. Behavioral assessment was performed using a 5-point scale. Horseradish Peroxidase Exclusion test was used to quantify the disruption of axonal membrane. Polyethylene glycol was prepared as a 30% (w/v) solution in phosphate buffered saline and injected intraperitoneally.
RESULTS
We have found evidence of axonal membrane disruption in EAE mice when symptoms peak and to a lesser degree, in the pre-symptomatic stage of EAE mice. Furthermore, polyethylene glycol (PEG), a known membrane fusogen, significantly reduces axonal membrane disruption in EAE mice. Such PEG-mediated membrane repair was accompanied by significant amelioration of behavioral deficits, including a delay in the emergence of motor deficits, a delay of the emergence of peak symptom, and a reduction in the severity of peak symptom.
CONCLUSIONS
The current study is the first indication that axonal membrane disruption may be an important part of the pathology in EAE mice and may underlies behavioral deficits. Our study also presents the initial observation that PEG may be a therapeutic agent that can repair axolemma, arrest axonal degeneration and reduce motor deficits in EAE mice.
PubMed: 28265351
DOI: 10.1186/s40035-017-0075-7 -
Journal of Integrative Neuroscience Dec 2016Electrotonic potentials allow the accommodative processes to long-lasting subthreshold polarizing stimuli to be assessed. The present study investigates such potentials...
Electrotonic potentials allow the accommodative processes to long-lasting subthreshold polarizing stimuli to be assessed. The present study investigates such potentials in previously simulated cases of amyotrophic lateral sclerosis, termed as ALS1, ALS2 and ALS3, respectively, when the temperature is changed during hypothermia ([Formula: see text]C) and hyperthermia ([Formula: see text]C). The ALS cases are modeled as three progressively severe uniform axonal dysfunctions along the human motor nerve fiber which is simulated by our temperature-dependent multi-layered numerical model. The results show that the polarizing electrotonic potentials in the ALS1 case are quite similar to those in the normal case during hypothermia. Their defining currents are caused by the activation of potassium fast (K[Formula: see text]) and slow (K[Formula: see text]) channels in the nodal and internodal axolemma beneath the myelin sheath. Except in the ALS3 case at 20[Formula: see text]C, where the accommodative processes are blocked by depolarizing stimuli, in the ALS2 and ALS3 cases during hypothermia these stimuli activate the classical "transient" Na[Formula: see text] channels in the nodal and internodal axolemma beneath the myelin sheath. And this leads to action potential generations during the early parts of electrotonic responses in all compartments along the fiber length. Only in the ALS3 case after the termination of long-lasting subthreshold hyperpolarizing stimuli, action potential generations are obtained in the late parts of electrotonic potentials along the fiber length. In comparison to the normal case, in the gradually severe ALS cases, the depolarizing electrotonic potentials gradually increase, while the hyperpolarizing electrotonic potentials gradually decrease during hyperthermia. However, the repetitive firings are not obtained in these polarizing electrotonic potentials. The results show that the accommodative processes to depolarizing stimuli in the ALS3 case are more likely to be blocked during hypothermia than hyperthermia. The results also show that the polarizing electrotonic potentials in the three simulated ALS cases are specific indicators for the motor nerve disease ALS during hypothermia and hyperthermia.
Topics: Action Potentials; Amyotrophic Lateral Sclerosis; Computer Simulation; Fever; Humans; Hypothermia; Models, Neurological; Motor Neurons; Myelin Sheath; Potassium Channels; Sodium Channels; Temperature
PubMed: 28100104
DOI: 10.1142/S0219635216500308