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Oxidative stress alters mitochondria at the nodes of Ranvier of ex vivo transected axons
Abstracts
in rodents treated with atomozetine/idazoxan, compared with saline treated animals which displayed an amoeboid morphology, indicating the anti-inflammatory actions of NA leading or contributing ultimately to neuroprotection. doi:10.1016/j.jneuroim.2014.08.326
Neurodegeneration 271 Axonal transport deficits during autoimmune optic neuritis Jovana Bojcevski, Sarah K. Williams, Ricarda Diem, Richard Fairless Neuro-oncology, University Clinic Heidelberg, Heidelberg, Germany Optic neuritis (ON) is an early syndrome present in patients with multiple sclerosis (MS). It is defined as an autoimmune demyelinating disorder that results in axonal loss and visual disturbances. In order to characterize axonal changes during ON, we used an animal model of MS, myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) in brown Norway rats. In this model, retinal ganglion cell (RGC) body loss is visible before inflammation of the optic nerve, suggesting that insult to the RGC body might be a primary event that triggers axonal stress, independent of later axonal loss resulting from inflammatory demyelination. This is supported by observations of disturbed axonal ultrastructure prior to demyelination by electron microscopy. In addition, changes in calcium homeostasis during the preclinical phase of the disease are seen which correlate with calpain-mediated cleavage of spectrin, a protein responsible for actin anchoring to the cell membrane and involved in actin remodeling. Therefore, this study aims to determine the timing of disturbances in axonal transport and cytoskeletal integrity, with focus particularly on the preclinical phase of ON. To achieve this, the following techniques have been employed: (1) immunohistochemistry to assess accumulation of various proteins associated with axonal transport (e.g. kinesin and synaptophysin), including the acute axonal injury marker, beta-APP; (2) intravitreal injections of cholera toxin B-FITC, a protein which is retro- and anterogradely transported along axons, in order to monitor transport timing; and (3) assays to determine filamentous vs globular actin ratios in the optic nerve, to monitor actin destructuralization. Data obtained so far, indicate that despite evidence of changes in the actin cytoskeletal network and axonal ultrastructure during the preclinical disease stage, the microtubule assembly is not grossly affected, with no detectable perturbations in axonal transport. Instead, axonal transport failure is only seen during the clinical disease phase in the vicinity of demyelinating inflammatory lesions. doi:10.1016/j.jneuroim.2014.08.327
156 Oxidative stress alters mitochondria at the nodes of Ranvier of ex vivo transected axons Elena Brosab,c, Jason M. Millwarda,b, Raluca Niesnerd, Friedemann Paulb,c, Carmen Infante-Duartea,b a
Institute for Medical Immunology, Charité-Universitätsmedizin Berlin, Germany; bExperimental and Clinical Research Center, Berlin, Germany; c NeuroCure Clinical Research Center, Charité-Universitätsmedizin Berlin, Germany; dDeutsches Rheuma-Forschungszentrum, Berlin, Germany
123
Axon degeneration is a key feature of multiple sclerosis that contributes to clinical deficit. Because of their unique energy requirements, axons are highly dependent on mitochondrial production of ATP. Even a brief failure of the energetic supply can lead to axonal damage. Here, we investigated the effects of oxidative stress on axonal mitochondria, using an ex vivo model of transected peripheral nerves. We demonstrated that oxidative stress clearly altered mitochondrial morphology, resulting in significantly shorter and rounder organelles. These changes were associated with inhibition of axonal transport. Mitochondria proximal to the nodes of Ranvier were the first to undergo these shape alterations. From this point, mitochondrial alterations progressed bidirectionally along the axon, ultimately reshaping the entire population. Further, we showed that depolarization of mitochondria at the nodes of Ranvier preceded morphological changes. Moreover, exogenous application of nicotinamide adenine dinucleotide and methyl pyruvate, both of which are essential for generating ATP, not only prevented mitochondrial changes at the nodes of Ranvier, but also protected transected axons from degeneration. Together, these results highlight the usefulness of ex vivo transected axons as a simplified model system to monitor mitochondrial dynamics in a degenerative context, and suggest the node of Ranvier as a key location for the initiation of oxidative stress-induced alterations of axonal mitochondria.
doi:10.1016/j.jneuroim.2014.08.328
303 Loss of IGF1R from oligodendrocytes ameliorates neuroinflammation without affecting cell survival Giuseppe Locatellia, Martin Krügerb, Olivia Prazeres da Costac, Barbara Ingold-Heppnerd, Linda Koche, Bettina Schreinerf, Jens Brüninge, Ingo Bechmannb, Burkhard Becherf, Thorsten Buchc a
Institute of Clinical Neuroimmunology, Ludwig-Maximilian Universität München, Munich, Germany; bInstitute of Anatomy, University of Leipzig, Leipzig, Germany; cInstitute for Med. Microbiology, Immunology and Hygiene, Technische Universität München, Munich, Germany; dInstitute of Pathology, Charité–Universitätsmedizin Berlin, Berlin, Germany; e Institute for Genetics, University of Cologne, Cologne, Germany; fInstitute for Exp. Immunology, University of Zurich, Zurich, Germany Signaling through the insulin-like growth factor receptor 1 (IGF1R) regulates differentiation myelination, and survival of oligodendrocyte (ODC) precursor cells. The expression of its ligand IGF-1 is increased at demyelinating lesions in Multiple Sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). However, the role of IGF1R signaling in adult ODCs following experimental demyelination remains unclear. We therefore performed a systematic analysis of the role of IGF1R in mature ODCs in vivo. Absence of IGF1R did not alter ODC density or myelin status, therefore proving that IGF1R does not regulate survival of ODCs in adult mice. Experimental insult to the CNS through cuprizone intoxication or induction of neuroinflammation also showed that absence of IGF1R does not influence survival of ODCs and demyelination. However, IGF1R deficient mice showed a surprising and consistent protection from EAE. Thus, the observed reduction in incidence and severity of EAE highlights the central role of ODCs in the neuroimmunological interplay and the complexity of IGF-1 mediated signaling in the CNS. doi:10.1016/j.jneuroim.2014.08.329
in rodents treated with atomozetine/idazoxan, compared with saline treated animals which displayed an amoeboid morphology, indicating the anti-inflammatory actions of NA leading or contributing ultimately to neuroprotection. doi:10.1016/j.jneuroim.2014.08.326
Neurodegeneration 271 Axonal transport deficits during autoimmune optic neuritis Jovana Bojcevski, Sarah K. Williams, Ricarda Diem, Richard Fairless Neuro-oncology, University Clinic Heidelberg, Heidelberg, Germany Optic neuritis (ON) is an early syndrome present in patients with multiple sclerosis (MS). It is defined as an autoimmune demyelinating disorder that results in axonal loss and visual disturbances. In order to characterize axonal changes during ON, we used an animal model of MS, myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) in brown Norway rats. In this model, retinal ganglion cell (RGC) body loss is visible before inflammation of the optic nerve, suggesting that insult to the RGC body might be a primary event that triggers axonal stress, independent of later axonal loss resulting from inflammatory demyelination. This is supported by observations of disturbed axonal ultrastructure prior to demyelination by electron microscopy. In addition, changes in calcium homeostasis during the preclinical phase of the disease are seen which correlate with calpain-mediated cleavage of spectrin, a protein responsible for actin anchoring to the cell membrane and involved in actin remodeling. Therefore, this study aims to determine the timing of disturbances in axonal transport and cytoskeletal integrity, with focus particularly on the preclinical phase of ON. To achieve this, the following techniques have been employed: (1) immunohistochemistry to assess accumulation of various proteins associated with axonal transport (e.g. kinesin and synaptophysin), including the acute axonal injury marker, beta-APP; (2) intravitreal injections of cholera toxin B-FITC, a protein which is retro- and anterogradely transported along axons, in order to monitor transport timing; and (3) assays to determine filamentous vs globular actin ratios in the optic nerve, to monitor actin destructuralization. Data obtained so far, indicate that despite evidence of changes in the actin cytoskeletal network and axonal ultrastructure during the preclinical disease stage, the microtubule assembly is not grossly affected, with no detectable perturbations in axonal transport. Instead, axonal transport failure is only seen during the clinical disease phase in the vicinity of demyelinating inflammatory lesions. doi:10.1016/j.jneuroim.2014.08.327
156 Oxidative stress alters mitochondria at the nodes of Ranvier of ex vivo transected axons Elena Brosab,c, Jason M. Millwarda,b, Raluca Niesnerd, Friedemann Paulb,c, Carmen Infante-Duartea,b a
Institute for Medical Immunology, Charité-Universitätsmedizin Berlin, Germany; bExperimental and Clinical Research Center, Berlin, Germany; c NeuroCure Clinical Research Center, Charité-Universitätsmedizin Berlin, Germany; dDeutsches Rheuma-Forschungszentrum, Berlin, Germany
123
Axon degeneration is a key feature of multiple sclerosis that contributes to clinical deficit. Because of their unique energy requirements, axons are highly dependent on mitochondrial production of ATP. Even a brief failure of the energetic supply can lead to axonal damage. Here, we investigated the effects of oxidative stress on axonal mitochondria, using an ex vivo model of transected peripheral nerves. We demonstrated that oxidative stress clearly altered mitochondrial morphology, resulting in significantly shorter and rounder organelles. These changes were associated with inhibition of axonal transport. Mitochondria proximal to the nodes of Ranvier were the first to undergo these shape alterations. From this point, mitochondrial alterations progressed bidirectionally along the axon, ultimately reshaping the entire population. Further, we showed that depolarization of mitochondria at the nodes of Ranvier preceded morphological changes. Moreover, exogenous application of nicotinamide adenine dinucleotide and methyl pyruvate, both of which are essential for generating ATP, not only prevented mitochondrial changes at the nodes of Ranvier, but also protected transected axons from degeneration. Together, these results highlight the usefulness of ex vivo transected axons as a simplified model system to monitor mitochondrial dynamics in a degenerative context, and suggest the node of Ranvier as a key location for the initiation of oxidative stress-induced alterations of axonal mitochondria.
doi:10.1016/j.jneuroim.2014.08.328
303 Loss of IGF1R from oligodendrocytes ameliorates neuroinflammation without affecting cell survival Giuseppe Locatellia, Martin Krügerb, Olivia Prazeres da Costac, Barbara Ingold-Heppnerd, Linda Koche, Bettina Schreinerf, Jens Brüninge, Ingo Bechmannb, Burkhard Becherf, Thorsten Buchc a
Institute of Clinical Neuroimmunology, Ludwig-Maximilian Universität München, Munich, Germany; bInstitute of Anatomy, University of Leipzig, Leipzig, Germany; cInstitute for Med. Microbiology, Immunology and Hygiene, Technische Universität München, Munich, Germany; dInstitute of Pathology, Charité–Universitätsmedizin Berlin, Berlin, Germany; e Institute for Genetics, University of Cologne, Cologne, Germany; fInstitute for Exp. Immunology, University of Zurich, Zurich, Germany Signaling through the insulin-like growth factor receptor 1 (IGF1R) regulates differentiation myelination, and survival of oligodendrocyte (ODC) precursor cells. The expression of its ligand IGF-1 is increased at demyelinating lesions in Multiple Sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). However, the role of IGF1R signaling in adult ODCs following experimental demyelination remains unclear. We therefore performed a systematic analysis of the role of IGF1R in mature ODCs in vivo. Absence of IGF1R did not alter ODC density or myelin status, therefore proving that IGF1R does not regulate survival of ODCs in adult mice. Experimental insult to the CNS through cuprizone intoxication or induction of neuroinflammation also showed that absence of IGF1R does not influence survival of ODCs and demyelination. However, IGF1R deficient mice showed a surprising and consistent protection from EAE. Thus, the observed reduction in incidence and severity of EAE highlights the central role of ODCs in the neuroimmunological interplay and the complexity of IGF-1 mediated signaling in the CNS. doi:10.1016/j.jneuroim.2014.08.329