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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 -
European Journal of Translational... Jul 2023Chronic Ataxic Neuropathy with anti-Disialosyl IgM Antibodies (CANDA) is a rare form of immune-mediated sensory ataxic neuropathy. We describe the case of a 45-year-old...
Chronic Ataxic Neuropathy with anti-Disialosyl IgM Antibodies (CANDA) is a rare form of immune-mediated sensory ataxic neuropathy. We describe the case of a 45-year-old man, who was diagnosed with CANDA in October 2018. Since then, he has been treated with monthly courses of intravenous immunoglobulin administration (IV Ig) and, in October 2022, he underwent plasmapheresis, reporting a sudden worsening of clinical and motor picture. After a new IV Ig cycle admission, the patient was hospitalized to perform intensive rehabilitation, involving two individual sessions per day (90 minutes each) for 5 days a week. During hospitalization it was registered a relevant improvement in the muscle strength of the lower limbs (LLs). Furthermore, progressive improvements were recorded both in patient's motor performance and in his level of autonomy in activities of daily living. These results had a positive impact on his quality of life and made it possible to reduce the frequency of IV Ig treatments. This is the first case in literature reporting the combined effect of rehabilitation treatment and medical therapy in CANDA neuropathy.
PubMed: 37522810
DOI: 10.4081/ejtm.2023.11557 -
Journal of Neuroimmunology Jun 2016Interferon-gamma (IFN-γ) upregulates major histocompatibility complex class II (MHC class II) antigens and intercellular adhesion molecule-1 (ICAM-1) on Schwann cells...
Interferon-gamma (IFN-γ) upregulates major histocompatibility complex class II (MHC class II) antigens and intercellular adhesion molecule-1 (ICAM-1) on Schwann cells (SC) in vitro, but in nerves of animals and patients MHC class II is primarily expressed on inflammatory cells. We investigated whether SC maturation influences their expression. IFN-γ induced MHC class II and upregulated ICAM-1; the axolemma-like signal 8-bromo cyclic adenosine monophosphate (8 Br cAMP) with IFN-γ inhibited expression. Delaying addition of 8 Br cAMP to SC already exposed to IFN-γ inhibited ongoing expression; addition of IFN-γ to SC already exposed to 8 Br cAMP resulted in minimal expression. Variability of cytokine-induced MHC class II and ICAM-1 expression by SC in vivo may represent the variability of signals from axolemma.
Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Animals, Newborn; Cell Differentiation; Cells, Cultured; Dose-Response Relationship, Drug; Drug Interactions; Gene Expression Regulation; Histocompatibility Antigens Class II; Intercellular Adhesion Molecule-1; Interferon-gamma; Rats; Schwann Cells; Time Factors
PubMed: 27235355
DOI: 10.1016/j.jneuroim.2016.03.013 -
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 Structural Biology Apr 2015Myelin - the multilayer membrane that envelops axons - is a facilitator of rapid nerve conduction. Oligodendrocytes form CNS myelin; the prevailing hypothesis being that...
Myelin - the multilayer membrane that envelops axons - is a facilitator of rapid nerve conduction. Oligodendrocytes form CNS myelin; the prevailing hypothesis being that they do it by extending a process that circumnavigates the axon. It is pertinent to ask how myelin is built because oligodendrocyte plasma membrane and myelin are compositionally different. To this end, we examined oligodendrocyte cultures and embryonic avian optic nerves by electron microscopy, immuno-electron microscopy and three-dimensional electron tomography. The results support three novel concepts. Myelin membranes are synthesized as tubules and packaged into "myelinophore organelles" in the oligodendrocyte perikaryon. Myelin membranes are matured in and transported by myelinophore organelles within an oligodendrocyte process. The myelin sheath is generated by myelin membrane fusion inside an oligodendrocyte process. These findings abrogate the dogma of myelin resulting from a wrapping motion of an oligodendrocyte process and open up new avenues in the quest for understanding myelination in health and disease.
Topics: Animals; Axons; Cell Membrane; Cells, Cultured; Central Nervous System; Chick Embryo; Myelin Sheath; Oligodendroglia; Organelles; Sheep, Domestic; Stochastic Processes
PubMed: 25682762
DOI: 10.1016/j.jsb.2015.01.015 -
Journal of Brachial Plexus and... 2016Our previous experiments demonstrated modulation of the amplitude of the axonal compound action potential (CAP) by electrical stimulation. To verify assumption that...
Our previous experiments demonstrated modulation of the amplitude of the axonal compound action potential (CAP) by electrical stimulation. To verify assumption that glutamate released from axons could be involved in this phenomenon, the modification of the axonal CAP induced by glutamate was investigated. The major objective of this research is to verify the hypothesis that axonal activity would trigger the release of glutamate, which in turn would interact with specific axonal receptors modifying the amplitude of the action potential. Segments of the sciatic nerve were exposed to exogenous glutamate in vitro, and CAP was recorded before and after glutamate application. In some experiments, the release of radioactive glutamate analog from the sciatic nerve exposed to exogenous glutamate was also evaluated. The glutamate-induced increase in CAP was blocked by different glutamate receptor antagonists. The effect of glutamate was not observed in Ca-free medium, and was blocked by antagonists of calcium channels. Exogenous glutamate, applied to the segments of sciatic nerve, induced the release of radioactive glutamate analog, demonstrating glutamate-induced glutamate release. Immunohistochemical examination revealed that axolemma contains components necessary for glutamatergic neurotransmission. The proteins of the axonal membrane can under the influence of electrical stimulation or exogenous glutamate change membrane permeability and ionic conductance, leading to a change in the amplitude of CAP. We suggest that increased axonal activity leads to the release of glutamate that results in changes in the amplitude of CAPs.
PubMed: 28077958
DOI: 10.1055/s-0036-1593441 -
The Journal of Physiology Jan 2016In peripheral myelinated axons of mammalian spinal motor neurons, Ca(2+) influx was thought to occur only in pathological conditions such as ischaemia. Using Ca(2+)...
In peripheral myelinated axons of mammalian spinal motor neurons, Ca(2+) influx was thought to occur only in pathological conditions such as ischaemia. Using Ca(2+) imaging in mouse large motor axons, we find that physiological stimulation with trains of action potentials transiently elevates axoplasmic [C(2+)] around nodes of Ranvier. These stimulation-induced [Ca(2+)] elevations require Ca(2+) influx, and are partially reduced by blocking T-type Ca(2+) channels (e.g. mibefradil) and by blocking the Na(+)/Ca(2+) exchanger (NCX), suggesting an important contribution of Ca(2+) influx via reverse-mode NCX activity. Acute disruption of paranodal myelin dramatically increases stimulation-induced [Ca(2+)] elevations around nodes by allowing activation of sub-myelin L-type (nimodipine-sensitive) Ca(2+) channels. The Ca(2+) that enters myelinated motor axons during normal activity is likely to contribute to several signalling pathways; the larger Ca(2+) influx that occurs following demyelination may contribute to the axonal degeneration that occurs in peripheral demyelinating diseases. Activity-dependent Ca(2+) signalling is well established for somata and terminals of mammalian spinal motor neurons, but not for their axons. Imaging of an intra-axonally injected fluorescent [Ca(2+)] indicator revealed that during repetitive action potential stimulation, [Ca(2+)] elevations localized to nodal regions occurred in mouse motor axons from ventral roots, phrenic nerve and intramuscular branches. These [Ca(2+)] elevations (∼ 0.1 μm with stimulation at 50 Hz, 10 s) were blocked by removal of Ca(2+) from the extracellular solution. Effects of pharmacological blockers indicated contributions from both T-type Ca(2+) channels and reverse mode Na(+)/Ca(2+) exchange (NCX). Acute disruption of paranodal myelin (by stretch or lysophosphatidylcholine) increased the stimulation-induced [Ca(2+)] elevations, which now included a prominent contribution from L-type Ca(2+) channels. These results suggest that the peri-nodal axolemma of motor axons includes multiple pathways for stimulation-induced Ca(2+) influx, some active in normally-myelinated axons (T-type channels, NCX), others active only when exposed by myelin disruption (L-type channels). The modest axoplasmic peri-nodal [Ca(2+)] elevations measured in intact motor axons might mediate local responses to axonal activation. The larger [Ca(2+) ] elevations measured after myelin disruption might, over time, contribute to the axonal degeneration observed in peripheral demyelinating neuropathies.
Topics: Action Potentials; Animals; Axons; Calcium Channels; Calcium Signaling; Mice; Mice, Inbred C57BL; Motor Neurons; Ranvier's Nodes; Sodium-Calcium Exchanger
PubMed: 26365250
DOI: 10.1113/JP271207 -
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 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 -
Journal of Integrative Neuroscience Sep 2014To expand our studies on accommodation in human motor nerve axons, the effects of temperature on polarizing nodal and internodal electrotonic potentials and their...
To expand our studies on accommodation in human motor nerve axons, the effects of temperature on polarizing nodal and internodal electrotonic potentials and their current kinetics are investigated. The computations use our temperature dependent multi-layered model of the myelinated human motor nerve fiber and the temperature is increased from 20°C to 42°C. The results show that for temperatures from 28°C to 37°C, the polarizing electrotonic potentials almost coincide, as the kinetics of their ionic currents is changed a little. The normal (at 37°C) resting membrane potential is further depolarized or hyperpolarized during hypothermia (≤ 25°C) or hyperthermia (≥ 40°C), respectively and its change is determined by the flow of ionic currents through the internodal axolemma during the polarizing current stimuli. The polarizing electrotonic potentials are more altered during hypothermia and are most altered during hyperthermia. During hyperthermia, the depolarizing nodal and internodal electrotonic potentials are determined by the nodal slow (I Ks ) and internodal fast (I Kf ) and slow (I Ks ) potassium currents. The hyperpolarizing internodal electrotonic potentials are determined by the activation of internodal channels, which are different during hyperthermia at 40°C and 42°C. These potentials are determined by the internodal I Ks current at 40°C and by the internodal inward rectifier (I IR ) and leakage (I Lk ) currents at 42°C. The difference in accommodation to hyperpolarizing currents during focal and uniform hyperthermia at 42°C is discussed. The present results are essential for the interpretation of mechanisms of threshold electrotonus measurements in subjects with symptoms of cooling, warming and fever, which can result from alterations in body temperature.
Topics: Axons; Computer Simulation; Humans; Kinetics; Membrane Potentials; Models, Neurological; Motor Neurons; Nerve Fibers, Myelinated; Potassium; Temperature
PubMed: 25164353
DOI: 10.1142/S0219635214500095