Subject(s)
B-Lymphocytes , Leg/diagnostic imaging , Muscle, Skeletal/diagnostic imaging , Myositis/diagnostic imaging , Myositis/drug therapy , Rituximab/therapeutic use , B-Lymphocytes/drug effects , Humans , Male , Middle Aged , Muscle, Skeletal/drug effects , Rituximab/pharmacology , Severity of Illness Index , Treatment Outcome , UltrasonographyABSTRACT
INTRODUCTION: Miyoshi myopathy, a type of distal myopathy with predominant involvement of the posterior calf muscles, has been assigned to mutations in the dysferlin gene. However, many of the late-onset limb-girdle and distal myopathies that resemble dysferlinopathy or Miyoshi myopathy remain unclassified, even after extensive immunohistological and genetic analysis. CASE PRESENTATION: We report the case of a 59-year-old Caucasian man with distal myopathy and exercise-induced myalgia, preferentially of the leg muscles, closely resembling the Miyoshi phenotype. Magnetic resonance imaging of his calf muscles showed typical fatty replacement of the medial heads of the gastrocnemius muscles and soleus muscles, with progression to the adductor longus muscles over a time course of two years. However, genetic analysis revealed that the phenotype of our patient was not related to a mutation in the dysferlin gene but to a novel homozygous splice mutation in the anoctamin 5 gene. Mutations in the anoctamin 5 gene have so far been identified only in some cases of limb-girdle and distal myopathy. Mutations in the anoctamin 5 gene have been assigned to limb-girdle muscular dystrophy type 2L, while distal Miyoshi-like phenotypes have been classified as Miyoshi myopathy type 3. CONCLUSION: The case presented in this report further strengthens the underlying genetic heterogeneity in Miyoshi myopathy-like phenotypes and adds another family to non-dysferlin, Miyoshi myopathy type 3 of late-onset. Furthermore, our case supports the recent observation that anoctamin 5 mutations are a primary cause of distal non-dysferlin myopathies. Therefore, given the increasing number of anoctamin 5 mutations in Miyoshi-like phenotypes, genetic analysis should include an anoctamin 5 screen in late-onset limb-girdle and distal myopathies.
ABSTRACT
Mutations in SOD1 cause hereditary variants of the fatal motor neuron disease amyotrophic lateral sclerosis (ALS). Pathophysiology of the disease is non-cell-autonomous, with toxicity deriving also from glia. In particular, microglia contribute to disease progression. Methylene blue (MB) inhibits the effect of nitric oxide, which mediates microglial responses to injury. In vivo 2P-LSM imaging was performed in ALS-linked transgenic SOD1(G93A) mice to investigate the effect of MB on microglia-mediated inflammation in the spinal cord. Local superfusion of the lateral spinal cord with MB inhibited the microglial reaction directed at a laser-induced axon transection in control and SOD1(G93A) mice. In vitro, MB at high concentrations inhibited cytokine and chemokine release from microglia of control and advanced clinical SOD1(G93A) mice. Systemic MB-treatment of SOD1(G93A) mice at early preclinical stages significantly delayed disease onset and motor dysfunction. However, an increase of MB dose had no additional effect on disease progression; this was unexpected in view of the local anti-inflammatory effects. Furthermore, in vivo imaging of systemically MB-treated mice also showed no alterations of microglia activity in response to local lesions. Thus although systemic MB treatment had no effect on microgliosis, instead, its use revealed an important influence on motor neuron survival as indicated by an increased number of lumbar anterior horn neurons present at the time of disease onset. Thus, potentially beneficial effects of locally applied MB on inflammatory events contributing to disease progression could not be reproduced in SOD1(G93A) mice via systemic administration, whereas systemic MB application delayed disease onset via neuroprotection.
Subject(s)
Amyotrophic Lateral Sclerosis/pathology , Methylene Blue/pharmacology , Microglia/drug effects , Microglia/pathology , Motor Neurons/drug effects , Motor Neurons/pathology , Superoxide Dismutase/genetics , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/physiopathology , Animals , Animals, Genetically Modified , Cell Survival/drug effects , Cytokines/metabolism , Disease Models, Animal , Disease Progression , Female , Inflammation/metabolism , Inflammation/pathology , Male , Mice , Microglia/metabolism , Motor Activity/drug effects , Superoxide Dismutase-1 , Time FactorsABSTRACT
Pathophysiology of the motoneuron disease amyotrophic lateral sclerosis (ALS) is non-cell-autonomous. In mouse models of familiar ALS, neurotoxicity is derived not only from mutant motor neurons but also from mutant neighbouring glial cells. In vivo imaging by two-photon laser-scanning microscopy was used to study rapid morphological reactions of astroglial cells towards laser-induced axonal transection in ALS-linked transgenic SOD1(G93A) mice. In the affected lateral spinal cord, mutated astroglial cells extended branches towards injured axons within a time frame of minutes to hours post lesion while in control animals astrocytes lack any rapid morphological alteration within the studied time frame. This suggests that astrocytes partially contribute to the rapid response of non-neuronal cells to acute axonal lesions in ALS mice.
Subject(s)
Amyotrophic Lateral Sclerosis/pathology , Astrocytes/pathology , Axons/pathology , Cell Communication/physiology , Motor Neurons/pathology , Amyotrophic Lateral Sclerosis/genetics , Animals , Disease Models, Animal , Mice , Mice, Transgenic , Microglia/pathology , Microscopy, Confocal/methods , Spinal Cord/pathology , Superoxide Dismutase/genetics , Superoxide Dismutase-1ABSTRACT
Kir4.1 channels were found to colocalize with the H(+)/K(+)-ATPase throughout the parietal cell (PC) acid secretory cycle. This study was undertaken to explore their functional role. Acid secretory rates, electrophysiological parameters, PC ultrastructure, and gene and protein expression were determined in gastric mucosae of 7-8-day-old Kir4.1-deficient mice and WT littermates. Kir4.1(-/-) mucosa secreted significantly more acid and initiated secretion significantly faster than WT mucosa. No change in PC number but a relative up-regulation of H(+)/K(+)-ATPase gene and protein expression (but not of other PC ion transporters) was observed. Electron microscopy revealed fully fused canalicular membranes and a lack of tubulovesicles in resting state Kir4.1(-/-) PCs, suggesting that Kir4.1 ablation may also interfere with tubulovesicle endocytosis. The role of this inward rectifier in the PC apical membrane may therefore be to balance between K(+) loss via KCNQ1/KCNE2 and K(+) reabsorption by the slow turnover of the H(+)/K(+)-ATPase, with consequences for K(+) reabsorption, inhibition of acid secretion, and membrane recycling. Our results demonstrate that Kir4.1 channels are involved in the control of acid secretion and suggest that they may also affect secretory membrane recycling.
Subject(s)
Gastric Acid/metabolism , Gene Expression Regulation , Parietal Cells, Gastric/cytology , Potassium Channels, Inwardly Rectifying/physiology , Animals , Biological Transport , Electrophysiology/methods , Endocytosis , Gastric Mucosa/metabolism , H(+)-K(+)-Exchanging ATPase/chemistry , Mice , Mice, Knockout , Microscopy, Electron, Transmission/methods , Microscopy, Fluorescence/methods , Potassium/chemistry , Potassium Channels, Inwardly Rectifying/biosynthesisABSTRACT
Mutations in the enzyme superoxide dismutase-1 (SOD1) cause hereditary variants of the fatal motor neuronal disease Amyotrophic lateral sclerosis (ALS). Pathophysiology of the disease is non-cell-autonomous: neurotoxicity is derived not only from mutant motor neurons but also from mutant neighbouring non-neuronal cells. In vivo imaging by two-photon laser-scanning microscopy was used to compare the role of microglia/macrophage-related neuroinflammation in the CNS and PNS using ALS-linked transgenic SOD1(G93A) mice. These mice contained labeled projection neurons and labeled microglia/macrophages. In the affected lateral spinal cord (in contrast to non-affected dorsal columns), different phases of microglia-mediated inflammation were observed: highly reactive microglial cells in preclinical stages (in 60-day-old mice the reaction to axonal transection was â¼180% of control) and morphologically transformed microglia that have lost their function of tissue surveillance and injury-directed response in clinical stages (reaction to axonal transection was lower than 50% of control). Furthermore, unlike CNS microglia, macrophages of the PNS lack any substantial morphological reaction while preclinical degeneration of peripheral motor axons and neuromuscular junctions was observed. We present in vivo evidence for a different inflammatory activity of microglia and macrophages: an aberrant neuroinflammatory response of microglia in the CNS and an apparently mainly neurodegenerative process in the PNS.
Subject(s)
Amyotrophic Lateral Sclerosis/pathology , Diagnostic Imaging/methods , Inflammation/pathology , Macrophages/pathology , Microglia/pathology , Peripheral Nervous System/pathology , Spinal Cord/pathology , Amyotrophic Lateral Sclerosis/complications , Amyotrophic Lateral Sclerosis/enzymology , Animals , Axons/pathology , Disease Models, Animal , Disease Progression , Inflammation/complications , Macrophages/enzymology , Mice , Mice, Transgenic , Microglia/enzymology , Motor Neurons/pathology , Muscles/innervation , Muscles/pathology , Nerve Degeneration/complications , Nerve Degeneration/pathology , Neuromuscular Junction/pathology , Peripheral Nervous System/enzymology , Spinal Cord/enzymology , Superoxide Dismutase/metabolism , Superoxide Dismutase-1ABSTRACT
As CNS macrophages, microglia show a high spontaneous motility of their processes, continuously surveying their microenvironment. Upon CNS injury, microglia react by immediate cellular polarization and process extension toward the lesion site as well as by subsequent amoeboid lesion-directed migration and phagocytosis. To determine the ability of microglia to fulfill their role within distinctively lesioned tissue in the absence of life support, we investigated microglial activity and responsiveness to laser-induced axonal injuries in the spinal dorsal columns in situ after cardiac and respiratory arrest, i.e., post-mortem, in the progressively degrading nervous tissue. For this purpose, we used time-lapse two-photon laser scanning microscopy in double transgenic mice expressing enhanced green fluorescent protein in microglia and enhanced yellow fluorescent protein in projection neurons. Depending on the premortal condition of the animal, microglial activity and responsiveness remain for up to5-10 hr post-mortem. Thereby, the continuously decreasing glial reaction is independent of oxygen and glucose supply but requires residual ATP, suggesting a parasitic form of energy, such as a transmembrane uptake of ATP released from injured nervous tissue. Even though initially microglia are able to detect axonal injury after disruption of the blood supply, the later aspects of glial reaction, for example amoeboid conversion and migration, are absent post- mortem, corresponding to the failure of microglia to prevent secondary damage after injury of nervous tissue.
Subject(s)
Microglia/physiology , Postmortem Changes , Spinal Cord/cytology , Spinal Cord/physiology , Adenosine Triphosphate/metabolism , Animals , Glucose/metabolism , Image Processing, Computer-Assisted , Luminescent Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microglia/ultrastructure , Microscopy, Confocal , Oxygen Consumption/physiologyABSTRACT
To understand the pathomechanisms of spinal cord injuries will be a prerequisite to develop efficient therapies. By investigating acute lesions of spinal cord white matter in anesthetized mice with fluorescently labeled microglia and axons using in vivo two-photon laser-scanning microscopy (2P-LSM), we identified the messenger nitric oxide (NO) as a modulator of injury-activated microglia. Local tissue damages evoked by high-power laser pulses provoked an immediate attraction of microglial processes. Spinal superfusion with NO synthase and guanylate cyclase inhibitors blocked these extensions. Furthermore, local injection of the NO-donor spermine NONOate (SPNO) or the NO-dependent second messenger cGMP induced efficient migration of microglial cells toward the injection site. High-tissue levels of NO, achieved by uniform superfusion with SPNO and mimicking extended tissue damage, resulted in a fast conversion of the microglial shape from ramified to ameboid indicating cellular activation. When the spinal white matter was preconditioned by increased, ambient ATP (known as a microglial chemoattractant) levels, the attraction of microglial processes to local NO release was augmented, whereas it was abolished at low levels of tissue ATP. Because both signaling molecules, NO and ATP, mediate acute microglial reactions, coordinated pharmacological targeting of NO and purinergic pathways will be an effective mean to influence the innate immune processes after spinal cord injury.
Subject(s)
Adenosine Triphosphate/metabolism , Microglia/physiology , Nitric Oxide/metabolism , Spinal Cord Injuries/physiopathology , Acute Disease , Animals , Axons/drug effects , Axons/physiology , Cell Movement/drug effects , Cell Movement/physiology , Cell Polarity/drug effects , Cell Polarity/physiology , Cyclic GMP/metabolism , Enzyme Inhibitors/pharmacology , Guanylate Cyclase/antagonists & inhibitors , Guanylate Cyclase/metabolism , Mice , Mice, Transgenic , Microglia/cytology , Microglia/drug effects , Nerve Fibers, Myelinated/drug effects , Nerve Fibers, Myelinated/physiology , Nitric Oxide Donors/pharmacology , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/metabolism , Signal Transduction/drug effects , Spermine/analogs & derivatives , Spermine/pharmacology , Spinal Cord/drug effects , Spinal Cord/physiopathologyABSTRACT
Hereditary motor-sensory neuropathy (HMSN) Type 1/CMT 1 is a disorder of the peripheral nervous system. The underlying genetic cause is heterogeneous, and mutations in LITAF (Lipopolysaccharide-induced TNF-alpha factor) represent a rare cause of CMT Type 1. In this report, a novel missense mutation is presented in the LITAF gene (c.430G>A p.V144M) in a German CMT family exhibiting typical electrophysiological features of a demyelinating neuropathy with conduction blocks and variable age at onset. Molecular genetic characterization of demyelinating HMSN should therefore include screening of the LITAF gene if typical signs of a non-homogenous demyelinating neuropathy combined with dominant familial occurrence are evident.
Subject(s)
Charcot-Marie-Tooth Disease/genetics , Mutation, Missense , Nuclear Proteins/genetics , Transcription Factors/genetics , Age of Onset , Charcot-Marie-Tooth Disease/diagnosis , Charcot-Marie-Tooth Disease/physiopathology , Child , Demyelinating Diseases/diagnosis , Demyelinating Diseases/physiopathology , Diagnosis, Differential , Family , Female , Germany , Humans , Male , Middle Aged , Nerve Block , Neural Conduction , Sequence Analysis, DNAABSTRACT
Astrocytes in different brain regions display variable functional properties. In the hippocampus, astrocytes predominantly express time- and voltage-independent currents, but the underlying ion channels are not well defined. This ignorance is partly attributable to abundant intercellular coupling of these cells through gap junctions, impeding quantitative analyses of intrinsic membrane properties. Moreover, distinct types of cells with astroglial properties coexist in a given brain area, a finding that confused previous analyses. In the present study, we investigated expression of inwardly rectifying (Kir) and two-pore-domain (K2P) K+ channels in astrocytes, which are thought to be instrumental in the regulation of K+ homeostasis. Freshly isolated astrocytes were used to improve space-clamp conditions and allow for quantitative assessment of functional parameters. Patch-clamp recordings were combined with immunocytochemistry, Western blot analysis, and semiquantitative transcript analysis. Comparative measurements were performed in different CA1 subregions of astrocyte-targeted transgenic mice. While confirming weak Ba2+ sensitivity in situ, our data demonstrate that in freshly isolated astrocytes, the main proportion of membrane currents is sensitive to micromolar Ba2+ concentrations. Upregulation of Kir4.1 transcripts and protein during the first 10 postnatal days was accompanied by a fourfold increase in astrocyte inward current density. Hippocampal astrocytes from Kir4.1-/- mice lacked Ba2+-sensitive currents. In addition, we report functional expression of K2P channels of the TREK subfamily (TREK1, TREK2), which mediate astroglial outward currents. Together, our findings demonstrate that Kir4.1 constitutes the pivotal K+ channel subunit and that superposition of currents through Kir4.1 and TREK channels underlies the "passive" current pattern of hippocampal astrocytes.
Subject(s)
Astrocytes/physiology , Hippocampus/growth & development , Potassium Channels, Inwardly Rectifying/metabolism , Potassium Channels, Tandem Pore Domain/metabolism , Animals , Animals, Newborn , Astrocytes/drug effects , Barium/pharmacology , Barium Compounds/pharmacology , Carbenoxolone/pharmacology , Chlorides/pharmacology , Connexins/antagonists & inhibitors , Dose-Response Relationship, Drug , Gap Junctions/drug effects , Hippocampus/drug effects , Hippocampus/physiology , Hydrogen-Ion Concentration , Membrane Potentials/drug effects , Membrane Potentials/physiology , Mice , Mice, Knockout , Mice, Transgenic , Potassium Channel Blockers/pharmacology , Potassium Channels, Inwardly Rectifying/genetics , Quinine/pharmacology , RNA, Messenger/metabolismABSTRACT
Multiple sclerosis (MS) is a common inflammatory disease of the central nervous system that results in persistent impairment in young adults. During chronic progressive disease stages, there is a strong correlation between neurodegeneration and disability. Current therapies fail to prevent progression of neurological impairment during these disease stages. Flupirtine, a drug approved for oral use in patients suffering from chronic pain, was used in a rat model of autoimmune optic neuritis and significantly increased the survival of retinal ganglion cells, the neurons that form the axons of the optic nerve. When flupirtine was combined with interferon-beta, an established immunomodulatory therapy for MS, visual functions of the animals were improved during the acute phase of optic neuritis. Furthermore, flupirtine protected retinal ganglion cells from degeneration in a noninflammatory animal model of optic nerve transection. Although flupirtine was shown previously to increase neuronal survival by Bcl-2 up-regulation, this mechanism does not appear to play a role in flupirtine-mediated protection of retinal ganglion cells either in vitro or in vivo. Instead, we showed through patch-clamp investigations that the activation of inwardly rectifying potassium channels is involved in flupirtine-mediated neuroprotection. Considering the few side effects reported in patients who receive long-term flupirtine treatment for chronic pain, our results indicate that this drug is an interesting candidate for further evaluation of its neuroprotective potential in MS.
Subject(s)
Aminopyridines/therapeutic use , Autoimmune Diseases/drug therapy , Neuroprotective Agents/therapeutic use , Optic Neuritis/drug therapy , Aminopyridines/blood , Aminopyridines/pharmacology , Animals , Autoimmune Diseases/pathology , Cell Survival/drug effects , Cells, Cultured , Cytoprotection/drug effects , Disease Progression , Drug Therapy, Combination , Evoked Potentials, Visual/drug effects , Female , Inflammation/pathology , Interferon-beta/therapeutic use , Ion Channel Gating/drug effects , Neuroprotective Agents/blood , Neuroprotective Agents/pharmacology , Optic Nerve/drug effects , Optic Nerve/pathology , Optic Neuritis/pathology , Potassium Channels, Inwardly Rectifying/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Rats , Rats, Inbred BN , Rats, Wistar , Retinal Ganglion Cells/drug effects , Retinal Ganglion Cells/pathologyABSTRACT
Rhythmic activity of respiratory neurons is dependent on the clearance of neurotransmitter by astrocytes. Astrocytes should also be involved in the permanent and rapid clearance of extracellular ions. We analyzed the expression of the weakly inwardly rectifying K+ channel Kir4.1 (KCNJ10) in the respiratory network and studied the possible functions for neuronal activity in the pre-Bötzinger complex.
Subject(s)
Astrocytes/physiology , Potassium Channels, Inwardly Rectifying/physiology , Respiratory Physiological Phenomena , Animals , Gene Deletion , Glial Fibrillary Acidic Protein/genetics , Humans , Mice , Mice, Knockout , Mice, Transgenic , Potassium Channels, Inwardly Rectifying/deficiency , Potassium Channels, Inwardly Rectifying/genetics , Synaptic Transmission/physiologyABSTRACT
In glial cells, inwardly rectifying K(+) channels (Kir) control extracellular [K(+) ](o) homeostasis by uptake of K(+) from the extracellular space and release of K(+) into the microvasculature. Kir channels were also recently implicated in K(+) -associated water influx and cell swelling. We studied the time-dependent expression and functional implication of the glial Kir4.1 channel for astroglial swelling in a spinal cord edema model. In this CNS region, Kir4.1 is expressed on astrocytes from the second postnatal week on and co-localizes with aquaporin 4 (AQP4). Swelling of individual astrocytes in response to osmotic stress and to pharmacological Kir blockade were analyzed by time-lapse-two-photon laser-scanning microscopy in situ. Application of 30% hypotonic solution induced astroglial soma swelling whereas no swelling was observed on astroglial processes or endfeet. Co-application of hypotonic solution and Ba(2+) , a Kir channel blocker, induced prominent swelling of astroglial processes. In Kir4.1-/- mice, however, somatic as well as process swelling was observed upon application of 30% hypotonic solutions. No additional effect was provoked upon co-application with Ba(2+) . Our experiments show that Kir channels prevent glial process swelling under osmotic stress. The underlying Kir channel subunit that controls glial process swelling is Kir4.1, whereas changes of the glial soma are not substantially related to Kir4.1.
Subject(s)
Astrocytes/pathology , Potassium Channels, Inwardly Rectifying/physiology , Spinal Cord Diseases/pathology , Animals , Aquaporin 4/biosynthesis , Aquaporin 4/physiology , Astrocytes/ultrastructure , Blotting, Western , Edema/pathology , Fluorescent Antibody Technique , Glial Fibrillary Acidic Protein/biosynthesis , Glial Fibrillary Acidic Protein/genetics , Humans , Image Processing, Computer-Assisted , Immunohistochemistry , Mice , Mice, Knockout , Microscopy, Confocal , Neuroglia/pathology , Osmotic Pressure , Water/metabolismABSTRACT
In classic neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), the pathogenic concept of a cell-autonomous disease of motor neurons has been challenged increasingly in recent years. Macro- and microglial cells have come to the forefront for their role in multistep degenerative processes in ALS and respective disease models. The activation of astroglial and microglial cells occurs early in the pathogenesis of the disease and seems to greatly influence disease onset and promotion. The role of oligodendrocytes and Schwann cells remains elusive. In this review we highlight the impact of nonneuronal cells in ALS pathology. We discuss diverse glial membrane proteins that are necessary to control neuronal activity and neuronal cell survival, and summarize the contribution of these proteins to motor neuron death in ALS. We also describe recently discovered glial mechanisms that promote motor neuron degeneration using state-of-the-art genetic mouse technology. Finally, we provide an outlook on the extent to which these new pathomechanistic insights may offer novel therapeutic approaches.
Subject(s)
Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/physiopathology , Central Nervous System/physiopathology , Motor Neurons/metabolism , Neuroglia/metabolism , Amyotrophic Lateral Sclerosis/immunology , Animals , Cell Communication/genetics , Cell Communication/immunology , Central Nervous System/immunology , Gliosis/genetics , Gliosis/immunology , Gliosis/physiopathology , Humans , Membrane Proteins/genetics , Membrane Proteins/immunology , Membrane Proteins/metabolism , Motor Neurons/immunology , Nerve Degeneration/genetics , Nerve Degeneration/immunology , Nerve Degeneration/physiopathology , Neuroglia/immunology , Signal Transduction/genetics , Signal Transduction/immunologyABSTRACT
COS-1 cells with heterologeous expression of the Kir4.1 (KCNJ10) channel subunit, possess functional Kir4.1 channels and become capable to generating cytosolic Ca2+ transients, upon lowering of the extracellular K+ concentration to 2 mM or below. These Ca2+ transients are blocked by external Ba2+ (100 microM). Acute brain stem slices from wild-type mice (second post-natal week), which were loaded with the fluorescent Ca2+ indicator Oregon Green BAPTA-1-AM, were exposed to 0.2 mM K+. Under these conditions astrocytes, but not neurons, responded with cytosolic Ca2+ elevations in wild-type mice. This astrocyte-specific response has previously been used to identify astroglial cells type [R. Dallwig, H. Vitten, J.W. Deitmer, A novel barium-sensitive calcium influx into rat astrocytes at low external potassium. Cell Calcium 28 (2000) 247-259]. In Kir4.1 knock-out (Kir4.1-/-) mice, the number of responding cells was dramatically reduced and the Ca2+ transients in responding cells were significantly smaller than in wild-type mice. Our results indicate that Kir4.1 channels are the molecular substrate for the observed Ca2+ influx in astrocytes under conditions of low external K+-concentration.
Subject(s)
Calcium/metabolism , Potassium Channels, Inwardly Rectifying/metabolism , Potassium/metabolism , Aniline Compounds/metabolism , Animals , Astrocytes/cytology , Astrocytes/metabolism , COS Cells , Chlorocebus aethiops , Fluoresceins/metabolism , Fluorescent Dyes/metabolism , Membrane Potentials , Mice , Mice, Knockout , Patch-Clamp Techniques , Potassium Channels, Inwardly Rectifying/genetics , Rats , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolismABSTRACT
Axonal destruction and neuronal loss occur early during multiple sclerosis, an autoimmune inflammatory CNS disease that frequently manifests with acute optic neuritis. Available therapies mainly target the inflammatory component of the disease but fail to prevent neurodegeneration. To investigate the effect of minocycline on the survival of retinal ganglion cells (RGCs), the neurons that form the axons of the optic nerve, we used a rat model of myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis. Optic neuritis in this model was diagnosed by recording visual evoked potentials and RGC function was monitored by measuring electroretinograms. Functional and histopathological data of RGCs and optic nerves revealed neuronal and axonal protection when minocycline treatment was started on the day of immunization. Furthermore, we demonstrate that minocycline-induced neuroprotection is related to a direct antagonism of multiple mechanisms leading to neuronal cell death such as the induction of anti-apoptotic intracellular signalling pathways and a decrease in glutamate excitotoxicity. From these observations, we conclude that minocycline exerts neuroprotective effects independent of its anti-inflammatory properties. This hypothesis was confirmed in a non-inflammatory disease model leading to degeneration of RGCs, the surgical transection of the optic nerve.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental/drug therapy , Encephalomyelitis, Autoimmune, Experimental/immunology , Minocycline/pharmacology , Neuroprotective Agents/pharmacology , Acute Disease , Animals , Anti-Bacterial Agents/blood , Anti-Bacterial Agents/cerebrospinal fluid , Anti-Bacterial Agents/pharmacology , Apoptosis/drug effects , Apoptosis/immunology , Cell Survival/drug effects , Cell Survival/immunology , Encephalomyelitis, Autoimmune, Experimental/physiopathology , Evoked Potentials, Visual , Excitatory Amino Acid Transporter 1/metabolism , Excitatory Amino Acid Transporter 2/metabolism , Excitatory Amino Acid Transporter 3/metabolism , Female , Glutamic Acid/metabolism , Minocycline/blood , Minocycline/cerebrospinal fluid , Myelin Proteins , Myelin-Associated Glycoprotein/immunology , Myelin-Oligodendrocyte Glycoprotein , Neuroprotective Agents/blood , Neuroprotective Agents/cerebrospinal fluid , Optic Nerve/immunology , Optic Nerve/pathology , Optic Nerve/physiopathology , Optic Neuritis/drug therapy , Optic Neuritis/immunology , Optic Neuritis/physiopathology , Rats , Rats, Inbred BN , Retinal Ganglion Cells/immunology , Retinal Ganglion Cells/metabolism , Retinal Ganglion Cells/pathology , Severity of Illness IndexABSTRACT
Transgenic mice expressing the superoxide dismutase G93A mutation (SOD1(G93A)) were used to investigate the role of glial inwardly rectifying K(+) (Kir)4.1 channels, which buffer extracellular K(+) increases in response to neuronal excitation. A progressive decrease in Kir4.1 immunoreactivity was observed predominantly in the ventral horn of SOD1(G93A) mutants. Immunoblotting of spinal cord extracts mirrored these changes by showing a loss of Kir4.1 channels from presymptomatic stages onwards. Kir4.1 channels were found to be expressed in the spinal cord grey matter, targetting astrocytes and clustering around capillaries, supporting their role in clearance of extracellular K(+). To understand the functional implications of extracellular K(+) increases, we challenged the NSC34 motor neurone cell line with increasing extracellular K(+) concentrations. Exposure to high extracellular K(+) induced progressive motor neurone cell death. We suggest that loss of Kir4.1 impairs perineural K(+) homeostasis and may contribute to motor neurone degeneration in SOD1(G93A) mutants by K(+) excitotoxic mechanisms.
Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Neuroglia/metabolism , Potassium Channels, Inwardly Rectifying/metabolism , Spinal Cord/metabolism , Superoxide Dismutase/metabolism , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/pathology , Animals , Aquaporin 4/metabolism , Astrocytes/metabolism , Blotting, Western , Capillaries/cytology , Capillaries/metabolism , Cell Survival/physiology , Cells, Cultured , Extracellular Space/metabolism , Fluorescent Antibody Technique , Glial Fibrillary Acidic Protein/metabolism , Green Fluorescent Proteins/metabolism , Humans , Immunohistochemistry , Mice , Mice, Transgenic , Motor Neurons/metabolism , Motor Neurons/physiology , Potassium/metabolism , Spinal Cord/cytology , Spinal Cord/pathology , Superoxide Dismutase/genetics , Superoxide Dismutase-1ABSTRACT
Ongoing rhythmic neuronal activity in the ventral respiratory group (VRG) of the brain stem results in periodic changes of extracellular K+. To estimate the involvement of the weakly inwardly rectifying K+ channel Kir4.1 (KCNJ10) in extracellular K+ clearance, we examined its functional expression in astrocytes of the respiratory network. Kir4.1 was expressed in astroglial cells of the VRG, predominantly in fine astrocytic processes surrounding capillaries and in close proximity to VRG neurons. Kir4.1 expression was up-regulated during early postnatal development. The physiological role of astrocytic Kir4.1 was studied using mice with a null mutation in the Kir4.1 channel gene that were interbred with transgenic mice expressing the enhanced green fluorescent protein in their astrocytes. The membrane potential was depolarized in astrocytes of Kir4.1-/- mice, and Ba2+-sensitive inward K+ currents were diminished. Brain slices from Kir4.1-/- mice, containing the pre-Bötzinger complex, which generates a respiratory rhythm, did not show any obvious differences in rhythmic bursting activity compared with wild-type controls, indicating that the lack of Kir4.1 channels alone does not impair respiratory network activity. Extracellular K+ measurements revealed that Kir4.1 channels contribute to extracellular K+ regulation. Kir4.1 channels reduce baseline K+ levels, and they compensate for the K+ undershoot. Our data indicate that Kir4.1 channels 1) are expressed in perineuronal processes of astrocytes, 2) constitute the major part of the astrocytic Kir conductance, and 3) contribute to regulation of extracellular K+ in the respiratory network.
Subject(s)
Action Potentials/physiology , Astrocytes/physiology , Biological Clocks/physiology , Medulla Oblongata/physiology , Potassium Channels, Inwardly Rectifying/deficiency , Respiratory Mechanics/physiology , Animals , Biological Transport, Active/physiology , Cells, Cultured , Extracellular Fluid/metabolism , Feedback/physiology , Mice , Mice, Knockout , Potassium , Potassium Channels, Inwardly Rectifying/genetics , Protein SubunitsABSTRACT
It remains poorly understood as to how newly synthesized proteins that are required to act at specific synapses are translocated into only selected subsets of potentiated dendritic spines. Here, we report that F-actin, a major component of the skeletal structure of dendritic spines, may contribute to the regulation of synaptic specificity of protein translocation. We found that the stabilization of F-actin blocked the translocation of GFP-CaMKII and inhibited the diffusion of 3-kDa dextran into spines (in 2-3 weeks cultures). Neuronal activation in hippocampal slices and cultured neurons led to an increase in the activation (decrease in the phosphorylation) of the actin depolymerization factor, cofilin, and a decrease in F-actin. Furthermore, the induction of long-term potentiation by tetanic stimulation induced local transient depolymerization of F-actin both in vivo and in hippocampal slices (8-10 weeks), and this local F-actin depolymerization was blocked by APV, a N-methyl-D-aspartate (NMDA) receptor antagonist. These results suggest that F-actin may play a role in synaptic specificity by allowing protein translocation into only potentiated spines, gated through its depolymerization, which is probably triggered by the activation of NMDA receptors.
Subject(s)
Actins/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Dendritic Spines/metabolism , Neurons/cytology , 2-Amino-5-phosphonovalerate/pharmacology , Actin Depolymerizing Factors/metabolism , Animals , Blotting, Western/methods , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Cells, Cultured , Dendritic Spines/ultrastructure , Depsipeptides/pharmacology , Dextrans/metabolism , Disks Large Homolog 4 Protein , Dose-Response Relationship, Radiation , Electric Stimulation/methods , Embryo, Mammalian , Excitatory Postsynaptic Potentials/drug effects , Excitatory Postsynaptic Potentials/physiology , Excitatory Postsynaptic Potentials/radiation effects , Fluorescent Antibody Technique/methods , Green Fluorescent Proteins/metabolism , Hippocampus/cytology , In Vitro Techniques , Intracellular Signaling Peptides and Proteins/metabolism , Long-Term Potentiation/drug effects , Long-Term Potentiation/physiology , Long-Term Potentiation/radiation effects , Membrane Proteins/metabolism , Microscopy, Immunoelectron/methods , Microtubule-Associated Proteins/metabolism , Neurons/drug effects , Neurons/physiology , Patch-Clamp Techniques/methods , Phosphorylation/drug effects , Phosphorylation/radiation effects , Potassium Chloride/pharmacology , Protein Transport/drug effects , Protein Transport/physiology , Rats , Time Factors , TransfectionABSTRACT
The Kir4.1 gene (KCNJ10) encodes an inwardly rectifying K(+) channel subunit abundantly expressed in the CNS. Its expression in the mammalian inner ear has been suggested but its function in vivo in the inner ear is unknown. Because diverse human hereditary deafness syndromes are associated with mutations in K(+) channels, we examined auditory function and inner ear structure in mice with a genetically inactivated Kir4.1 K(+) channel subunit. Startle response experiments suggest that Kir4.1-/- mice are profoundly deaf, whereas Kir4.1+/- mice react like wild-type mice to acoustic stimuli. In Kir4.1-/- mice, the Reissner membrane is collapsed, the tectorial membrane is swollen, and type I hair cells and spiral ganglion neurons as well as their central processes degenerate over the first postnatal weeks. In the vestibular ganglia, neuronal cell death with apoptotic features is also observed. Immunostaining reveals that Kir4.1 is strongly expressed in stria vascularis of wild-type but not Kir4.1-/- mice. Within the spiral ganglion, Kir4.1 labeling was detected on satellite cells surrounding spiral ganglion neurons and axons. We conclude that Kir4.1 is crucial for normal development of the cochlea and hearing, via two distinct aspects of extracellular K(+) homeostasis: (1). in stria vascularis, Kir4.1 helps to generate the cochlear endolymph; and (2). in spiral and vestibular ganglia, Kir4.1 in surrounding glial cells helps to support the spiral and vestibular ganglion neurons and their projecting axons.