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1.
bioRxiv ; 2024 May 10.
Article in English | MEDLINE | ID: mdl-38766050

ABSTRACT

Precise regulation of protein phosphorylation is critical for many cellular processes, and dysfunction in this process has been linked to various neurological disorders and diseases. Protein phosphatase 1 (PP1) is a ubiquitously expressed serine/threonine phosphatase with three major isoforms, (α, ß, γ) and hundreds of known substrates. Previously, we reported that PP1α and PP1γ are essential for the known role of PP1 in synaptic physiology and learning/memory, while PP1ß displayed a surprising opposing function. De novo mutations in PP1ß cause neurodevelopmental disorders in humans, but the mechanisms involved are currently unknown. A Cre-Lox system was used to delete PP1ß specifically in neurons in order to study its effects on developing mice. These animals fail to survive to 3 postnatal weeks, and exhibit deficits in cortical myelination and glutamate release. There was defective compound action potential (CAP) propagation in the optic nerve of the null mice, which was traced to a deficit in the formation of nodes of Ranvier. Finally, it was found that phosphorylation of the PP1ß-specific substrate, myosin light chain 2 (MLC2), is significantly enhanced in PP1ß null optic nerves. Several novel important in vivo roles of PP1ß in neurons were discovered, and these data will aid future investigations in delineating the mechanisms by which de novo mutations in PP1ß lead to intellectual and developmental delays in patients.

2.
bioRxiv ; 2024 Apr 17.
Article in English | MEDLINE | ID: mdl-38496662

ABSTRACT

Upon peripheral nervous system (PNS) injury, severed axons undergo rapid SARM1-dependent Wallerian degeneration (WD). In mammals, the role of SARM1 in PNS regeneration, however, is unknown. Here we demonstrate that Sarm1 is not required for axotomy induced activation of neuron-intrinsic growth programs and axonal growth into a nerve crush site. However, in the distal nerve, Sarm1 is necessary for the timely induction of the Schwann cell (SC) repair response, nerve inflammation, myelin clearance, and regeneration of sensory and motor axons. In Sarm1-/- mice, regenerated fibers exhibit reduced axon caliber, defective nerve conduction, and recovery of motor function is delayed. The growth hostile environment of Sarm1-/- distal nerve tissue was demonstrated by grafting of Sarm1-/- nerve into WT recipients. SC lineage tracing in injured WT and Sarm1-/- mice revealed morphological differences. In the Sarm1-/- distal nerve, the appearance of p75NTR+, c-Jun+ SCs is significantly delayed. Ex vivo, p75NTR and c-Jun upregulation in Sarm1-/- nerves can be rescued by pharmacological inhibition of ErbB kinase. Together, our studies show that Sarm1 is not necessary for the activation of neuron intrinsic growth programs but in the distal nerve is required for the orchestration of cellular programs that underlie rapid axon extension.

3.
Mol Neurobiol ; 59(12): 7486-7494, 2022 Dec.
Article in English | MEDLINE | ID: mdl-36198882

ABSTRACT

Nuclear inhibitor of protein phosphatase 1 (NIPP1) is a known regulator of gene expression and plays roles in many physiological or pathological processes such as stem cell proliferation and skin inflammation. While NIPP1 has many regulatory roles in proliferating cells, its function in the central nervous system (CNS) has not been directly investigated. In the present study, we examined NIPP1 CNS function using a conditional knockout (cKO) mouse model in which the Nipp1 gene is excised from neural precursor cells. These mice exhibited severe developmental impairments that led to premature lethality. To delineate the neurological changes occurring in these animals, we first assessed microtubule-associated protein tau, a known target of NIPP1 activity. We found that phosphorylation of tau is significantly enhanced in NIPP1 cKO mice. Consistent with this, we found altered AKT and PP1 activity in NIPP1 cKO mice, suggesting that increased tau phosphorylation likely results from a shift in kinase/phosphatase activity. Secondly, we observed tremors in the NIPP1 cKO mice which prompted us to explore the integrity of the myelin sheath, an integral structure for CNS function. We demonstrated that in NIPP1 cKO mice, there is a significant decrease in MBP protein expression in the cortex, along with deficits in both the conduction of compound action potentials (CAP) and the percentage of myelinated axons in the optic nerve. Our study suggests that NIPP1 in neural precursor cells regulates phosphorylation of tau and CNS myelination and may represent a novel therapeutic target for neurodegenerative diseases.


Subject(s)
Intracellular Signaling Peptides and Proteins , Neural Stem Cells , Mice , Animals , Protein Phosphatase 1/metabolism , Phosphorylation , Intracellular Signaling Peptides and Proteins/metabolism , Neural Stem Cells/metabolism , Central Nervous System/metabolism , Myelin Sheath/metabolism
5.
Exp Eye Res ; 171: 54-61, 2018 06.
Article in English | MEDLINE | ID: mdl-29526794

ABSTRACT

Optic neuropathies such as glaucoma are characterized by the degeneration of retinal ganglion cells (RGCs) and the irreversible loss of vision. In these diseases, focal axon injury triggers a propagating axon degeneration and, eventually, cell death. Previous work by us and others identified dual leucine zipper kinase (DLK) and JUN N-terminal kinase (JNK) as key mediators of somal cell death signaling in RGCs following axonal injury. Moreover, others have shown that activation of the DLK/JNK pathway contributes to distal axonal degeneration in some neuronal subtypes and that this activation is dependent on the adaptor protein, sterile alpha and TIR motif containing 1 (SARM1). Given that SARM1 acts upstream of DLK/JNK signaling in axon degeneration, we tested whether SARM1 plays a similar role in RGC somal apoptosis in response to optic nerve injury. Using the mouse optic nerve crush (ONC) model, our results show that SARM1 is critical for RGC axonal degeneration and that axons rescued by SARM1 deficiency are electrophysiologically active. Genetic deletion of SARM1 did not, however, prevent DLK/JNK pathway activation in RGC somas nor did it prevent or delay RGC cell death. These results highlight the importance of SARM1 in RGC axon degeneration and suggest that somal activation of the DLK/JNK pathway is activated by an as-yet-unidentified SARM1-independent signal.


Subject(s)
Armadillo Domain Proteins/physiology , Axons/metabolism , Cytoskeletal Proteins/physiology , Disease Models, Animal , Optic Nerve Injuries/metabolism , Receptors, Tumor Necrosis Factor/physiology , Retinal Degeneration/metabolism , Retinal Ganglion Cells/metabolism , Animals , Apoptosis/physiology , Axons/pathology , Cell Count , Cell Survival , Electrophysiology , Immunohistochemistry , Mice , Mice, Inbred C57BL , Mice, Knockout , Nerve Crush , Optic Nerve Injuries/pathology , Retinal Degeneration/pathology , Retinal Ganglion Cells/pathology
6.
Elife ; 62017 12 18.
Article in English | MEDLINE | ID: mdl-29251594

ABSTRACT

Low-density lipoprotein receptor-related protein-1 (LRP1) is a large endocytic and signaling molecule broadly expressed by neurons and glia. In adult mice, global inducible (Lrp1flox/flox;CAG-CreER) or oligodendrocyte (OL)-lineage specific ablation (Lrp1flox/flox;Pdgfra-CreER) of Lrp1 attenuates repair of damaged white matter. In oligodendrocyte progenitor cells (OPCs), Lrp1 is required for cholesterol homeostasis and differentiation into mature OLs. Lrp1-deficient OPC/OLs show a strong increase in the sterol-regulatory element-binding protein-2 yet are unable to maintain normal cholesterol levels, suggesting more global metabolic deficits. Mechanistic studies revealed a decrease in peroxisomal biogenesis factor-2 and fewer peroxisomes in OL processes. Treatment of Lrp1-/- OPCs with cholesterol or activation of peroxisome proliferator-activated receptor-γ with pioglitazone alone is not sufficient to promote differentiation; however, when combined, cholesterol and pioglitazone enhance OPC differentiation into mature OLs. Collectively, our studies reveal a novel role for Lrp1 in peroxisome biogenesis, lipid homeostasis, and OPC differentiation during white matter development and repair.


Subject(s)
Cholesterol/metabolism , Homeostasis , Myelin Sheath/metabolism , Oligodendrocyte Precursor Cells/physiology , Organelle Biogenesis , Peroxisomes/metabolism , Receptors, LDL/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Cell Differentiation , Cells, Cultured , Low Density Lipoprotein Receptor-Related Protein-1 , Mice
7.
Mol Neurodegener ; 12(1): 71, 2017 10 02.
Article in English | MEDLINE | ID: mdl-28969695

ABSTRACT

BACKGROUND: Optic nerve injury is an important pathological component in neurodegenerative diseases such as traumatic optic neuropathies and glaucoma. The molecular signaling pathway(s) critical for retinal ganglion cell (RGC) death after axonal insult, however, is/are not fully defined. RGC death after axonal injury is known to occur by BAX-dependent apoptosis. Two transcription factors JUN (the canonical target of JNK) and DDIT3 (CHOP; a key mediator of the endoplasmic reticulum stress response) are known to be important apoptotic signaling molecules after axonal injury, including in RGCs. However, neither Jun nor Ddit3 deficiency provide complete protection to RGCs after injury. Since Jun and Ddit3 are important apoptotic signaling molecules, we sought to determine if their combined deficiency might provide additive protection to RGCs after axonal injury. METHODS: To determine if DDIT3 regulated the expression of JUN after an axonal insult, mice deficient for Ddit3 were examined after optic nerve crush (ONC). In order to critically test the importance of these genes in RGC death after axonal injury, RGC survival was assessed at multiple time-points after ONC (14, 35, 60, and 120 days after injury) in Jun, Ddit3, and combined Jun/Ddit3 deficient mice. Finally, to directly assess the role of JUN and DDIT3 in axonal degeneration, compound actions potentials were recorded from Jun, Ddit3, and Jun/Ddit3 deficient mice after ONC. RESULTS: Single and combined deficiency of Jun and Ddit3 did not appear to alter gross retinal morphology. Ddit3 deficiency did not alter expression of JUN after axonal injury. Deletion of both Jun and Ddit3 provided significantly greater long-term protection to RGCs as compared to Jun or Ddit3 deficiency alone. Finally, despite the profound protection to RGC somas provided by the deficiency of Jun plus Ddit3, their combined loss did not lessen axonal degeneration. CONCLUSIONS: These results suggest JUN and DDIT3 are independently regulated pro-death signaling molecules in RGCs and together account for the vast majority of apoptotic signaling in RGCs after axonal injury. Thus, JUN and DDIT3 may represent key molecular hubs that integrate upstream signaling events triggered by axonal injury with downstream transcriptional events that ultimately culminate in RGC apoptosis.


Subject(s)
Genes, jun/physiology , Nerve Degeneration/metabolism , Optic Nerve Injuries/metabolism , Retinal Ganglion Cells/pathology , Transcription Factor CHOP/metabolism , Animals , Apoptosis/physiology , Axons/metabolism , Axons/pathology , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Nerve Crush , Nerve Degeneration/pathology , Retinal Ganglion Cells/metabolism , Signal Transduction/physiology
8.
J Neurosci Res ; 95(7): 1402-1414, 2017 07.
Article in English | MEDLINE | ID: mdl-27614087

ABSTRACT

Conduction block by nitric oxide (NO) was examined in myelinated and unmyelinated axons from both the central nervous system and peripheral nervous system. In rat vagus nerves, mouse optic nerves at P12-P23, adult and developing mouse sciatic nerves, and mouse spinal cords, myelinated fibers were preferentially blocked reversibly by concentrations of NO similar to those encountered in inflammatory lesions. The possibility that these differences between myelinated and unmyelinated axons are due to the normal developmental substitution of Na+ channel subtype Nav 1.6 for Nav 1.2 at nodes of Ranvier was tested by repeating experiments on mice null for Nav 1.6. Results were unchanged in this mutant. In shiverer optic nerve, which has only scattered regions with nodes of Ranvier, only the fastest component of the compound action potential was reduced. NO was compared with three other methods of blocking conduction: low Na+ , high K+ , and tetrodotoxin (TTX). In each of these three cases, unmyelinated axons lost conduction simultaneously with myelinated fibers. From changes in conduction velocity in myelinated axons as they were blocked, it was ascertained that NO acted most similarly to TTX. It was concluded that NO likely interacts with axonal Na+ channels through an intermediate that is associated with myelin. © 2016 Wiley Periodicals, Inc.


Subject(s)
Autonomic Nerve Block/methods , Nerve Fibers, Myelinated/physiology , Neural Conduction/physiology , Nitric Oxide/pharmacology , Animals , Mice , Mice, Knockout , Mice, Transgenic , Nerve Fibers, Myelinated/drug effects , Neural Conduction/drug effects , Organ Culture Techniques , Rats , Rats, Inbred Lew , Vagus Nerve/drug effects , Vagus Nerve/physiology
9.
Int J Radiat Oncol Biol Phys ; 96(2): 449-457, 2016 10 01.
Article in English | MEDLINE | ID: mdl-27478169

ABSTRACT

PURPOSE: To determine the late effects of fractionated versus single-dose cranial radiation on murine white matter. METHODS AND MATERIALS: Mice were exposed to 0 Gy, 6 × 6 Gy, or 1 × 20 Gy cranial irradiation at 10 to 12 weeks of age. Endpoints were assessed through 18 months from exposure using immunohistochemistry, electron microscopy, and electrophysiology. RESULTS: Weight gain was temporarily reduced after irradiation; greater loss was seen after single versus fractionated doses. Oligodendrocyte progenitor cells were reduced early and late after both single and fractionated irradiation. Both protocols also increased myelin g-ratio, reduced the number of nodes of Ranvier, and promoted a shift in the proportion of small, unmyelinated versus large, myelinated axon fibers. CONCLUSIONS: Fractionation does not adequately spare normal white matter from late radiation side effects.


Subject(s)
Cell Lineage/radiation effects , Cranial Irradiation/adverse effects , Dose Fractionation, Radiation , Oligodendroglia/radiation effects , Weight Gain/radiation effects , White Matter/radiation effects , Animals , Cells, Cultured , Dose-Response Relationship, Radiation , Mice , Oligodendroglia/pathology , Organ Sparing Treatments/methods , Organs at Risk/radiation effects , Radiation Dosage , Radiation Protection/methods , White Matter/pathology
10.
Elife ; 52016 03 23.
Article in English | MEDLINE | ID: mdl-27008179

ABSTRACT

Proper development of the CNS axon-glia unit requires bi-directional communication between axons and oligodendrocytes (OLs). We show that the signaling lipid phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2] is required in neurons and in OLs for normal CNS myelination. In mice, mutations of Fig4, Pikfyve or Vac14, encoding key components of the PI(3,5)P2 biosynthetic complex, each lead to impaired OL maturation, severe CNS hypomyelination and delayed propagation of compound action potentials. Primary OLs deficient in Fig4 accumulate large LAMP1(+) and Rab7(+) vesicular structures and exhibit reduced membrane sheet expansion. PI(3,5)P2 deficiency leads to accumulation of myelin-associated glycoprotein (MAG) in LAMP1(+)perinuclear vesicles that fail to migrate to the nascent myelin sheet. Live-cell imaging of OLs after genetic or pharmacological inhibition of PI(3,5)P2 synthesis revealed impaired trafficking of plasma membrane-derived MAG through the endolysosomal system in primary cells and brain tissue. Collectively, our studies identify PI(3,5)P2 as a key regulator of myelin membrane trafficking and myelinogenesis.


Subject(s)
Cell Differentiation/drug effects , Myelin Sheath/metabolism , Neurons/metabolism , Oligodendroglia/drug effects , Oligodendroglia/physiology , Phosphatidylinositol Phosphates/biosynthesis , Animals , Gene Deletion , Mice
11.
Neurobiol Dis ; 89: 36-45, 2016 May.
Article in English | MEDLINE | ID: mdl-26807988

ABSTRACT

Mutations of the neuronal sodium channel gene SCN8A are associated with lethal movement disorders in the mouse and with human epileptic encephalopathy. We describe a spontaneous mouse mutation, Scn8a(9J), that is associated with a chronic movement disorder with early onset tremor and adult onset dystonia. Scn8a(9J) homozygotes have a shortened lifespan, with only 50% of mutants surviving beyond 6 months of age. The 3 bp in-frame deletion removes 1 of the 3 adjacent isoleucine residues in transmembrane segment DIVS6 of Nav1.6 (p.Ile1750del). The altered helical orientation of the transmembrane segment displaces pore-lining amino acids with important roles in channel activation and inactivation. The predicted impact on channel activity was confirmed by analysis of cerebellar Purkinje neurons from mutant mice, which lack spontaneous and induced repetitive firing. In a heterologous expression system, the activity of the mutant channel was below the threshold for detection. Observations of decreased nerve conduction velocity and impaired behavior in an open field are also consistent with reduced activity of Nav1.6. The Nav1.6Δ1750 protein is only partially glycosylated. The abundance of mutant Nav1.6 is reduced at nodes of Ranvier and is not detectable at the axon initial segment. Despite a severe reduction in channel activity, the lifespan and motor function of Scn8a(9J/9J) mice are significantly better than null mutants lacking channel protein. The clinical phenotype of this severe hypomorphic mutant expands the spectrum of Scn8a disease to include a recessively inherited, chronic and progressive movement disorder.


Subject(s)
Amino Acids/genetics , Movement Disorders/genetics , NAV1.6 Voltage-Gated Sodium Channel/genetics , Sequence Deletion , Action Potentials , Animals , Axon Initial Segment/metabolism , Behavior, Animal , Cerebral Cortex/metabolism , Cerebral Cortex/physiology , Dystonia/complications , Dystonia/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Movement Disorders/complications , Movement Disorders/veterinary , Muscle Strength , NAV1.6 Voltage-Gated Sodium Channel/physiology , Neural Conduction , Neuromuscular Junction/pathology , Purkinje Cells/metabolism , Purkinje Cells/physiology , Ranvier's Nodes/metabolism , Survival Analysis , Tremor/complications , Tremor/genetics
12.
J Immunol ; 195(6): 2552-9, 2015 Sep 15.
Article in English | MEDLINE | ID: mdl-26238492

ABSTRACT

Multiple sclerosis (MS) is believed to be initiated by myelin-reactive CD4(+) Th cells. IL-12-polarized Th1 cells, IL-23-polarized Th17 cells, and Th17 cells that acquire Th1 characteristics were each implicated in autoimmune pathogenesis. It is debated whether Th cells that can drive the development of demyelinating lesions are phenotypically diverse or arise from a single lineage. In the current study, we assessed the requirement of IL-12 or IL-23 stimulation, as well as Th plasticity, for the differentiation of T cells capable of inducing CNS axon damage. We found that stable murine Th1 and Th17 cells independently transfer experimental autoimmune encephalomyelitis (widely used as an animal model of MS) in the absence of IL-23 and IL-12, respectively. Plastic Th17 cells are particularly potent mediators of demyelination and axonopathy. In parallel studies, we identified MS patients who consistently mount either IFN-γ- or IL-17-skewed responses to myelin basic protein over the course of a year. Brain magnetic resonance imaging revealed that patients with mixed IFN-γ and IL-17 responses have relatively high T1 lesion burden, a measure of permanent axon damage. Our data challenge the dogma that IL-23 and Th17 plasticity are universally required for the development of experimental autoimmune encephalomyelitis. This study definitively demonstrates that autoimmune demyelinating disease can be driven by distinct Th-polarizing factors and effector subsets, underscoring the importance of a customized approach to the pharmaceutical management of MS.


Subject(s)
Encephalomyelitis, Autoimmune, Experimental/immunology , Multiple Sclerosis/immunology , Th1 Cells/immunology , Th17 Cells/immunology , Adoptive Transfer , Animals , Autoimmunity/immunology , Brain/diagnostic imaging , Cell Differentiation/immunology , Demyelinating Diseases/immunology , Humans , Interferon-gamma/biosynthesis , Interferon-gamma/immunology , Interleukin-12/immunology , Interleukin-17/immunology , Interleukin-23/immunology , Magnetic Resonance Imaging , Mice , Mice, Inbred C57BL , Mice, Knockout , Myelin Basic Protein/immunology , Optic Nerve/immunology , Optic Nerve/pathology , Radiography , Th1 Cells/cytology , Th1 Cells/transplantation , Th17 Cells/cytology , Th17 Cells/transplantation
13.
Neurobiol Dis ; 69: 108-16, 2014 Sep.
Article in English | MEDLINE | ID: mdl-24878510

ABSTRACT

Injury to retinal ganglion cell (RGC) axons triggers rapid activation of Jun N-terminal kinase (JNK) signaling, a major prodeath pathway in injured RGCs. Of the multiple kinases that can activate JNK, dual leucine kinase (Dlk) is known to regulate both apoptosis and Wallerian degeneration triggered by axonal insult. Here we tested the importance of Dlk in regulating somal and axonal degeneration of RGCs following axonal injury. Removal of DLK from the developing optic cup did not grossly affect developmental RGC death or inner plexiform layer organization. In the adult, Dlk deficiency significantly delayed axonal-injury induced RGC death. The activation of JUN was also attenuated in Dlk deficient retinas. Dlk deficiency attenuated the activation of the somal pool of JNK but did not prevent activation of the axonal pool of JNK after axonal injury, indicating that JNK activation in different cellular compartments of an RGC following axonal injury is regulated by distinct upstream kinases. In contrast to its robust influence on somal degeneration, Dlk deficiency did not alter RGC axonal degeneration after axonal injury as assessed using physiological readouts of optic nerve function.


Subject(s)
Axons/enzymology , MAP Kinase Kinase Kinases/deficiency , Optic Nerve Injuries/enzymology , Retinal Ganglion Cells/enzymology , Wallerian Degeneration/enzymology , Animals , Axons/pathology , Cell Death/physiology , Cell Survival/physiology , Disease Models, Animal , JNK Mitogen-Activated Protein Kinases/metabolism , MAP Kinase Kinase Kinases/genetics , Mice, Transgenic , Optic Nerve Injuries/pathology , Phosphorylation/physiology , Retina/enzymology , Retina/growth & development , Retina/pathology , Retinal Ganglion Cells/pathology , Signal Transduction , Tissue Culture Techniques , Wallerian Degeneration/pathology
14.
J Nutr ; 144(7): 1058-66, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24744313

ABSTRACT

Gestational iron deficiency (ID) has been associated with a wide variety of central nervous system (CNS) impairments in developing offspring. However, a focus on singular regions has impeded an understanding of the CNS-wide effects of this micronutrient deficiency. Because the developing brain requires iron during specific phases of growth in a region-specific manner, we hypothesized that maternal iron deprivation would lead to region-specific impairments in the CNS of offspring. Female rats were fed an iron control (Fe+) or iron-deficient (Fe-) diet containing 240 or 6 µg/g iron during gestation and lactation. The corpus callosum (CC), hippocampus, and cortex of the offspring were analyzed at postnatal day 21 (P21) and/or P40 using structural and functional measures. In the CC at P40, ID was associated with reduced peak amplitudes of compound action potentials specific to myelinated axons, in which diameters were reduced by ∼20% compared with Fe+ controls. In the hippocampus, ID was associated with a 25% reduction in basal dendritic length of pyramidal neurons at P21, whereas branching complexity was unaffected. We also identified a shift toward increased proximal branching of apical dendrites in ID without an effect on overall length compared with Fe+ controls. ID also affected cortical neurons, but unlike the hippocampus, both apical and basal dendrites displayed a uniform decrease in branching complexity, with no significant effect on overall length. These deficits culminated in significantly poorer performance of P40 Fe- offspring in the novel object recognition task. Collectively, these results demonstrate that non-anemic gestational ID has a significant and region-specific impact on neuronal development and may provide a framework for understanding and recognizing the presentation of clinical symptoms of ID.


Subject(s)
Brain Damage, Chronic/etiology , Cerebral Cortex/diagnostic imaging , Corpus Callosum/diagnostic imaging , Iron Deficiencies , Lactation , Maternal Nutritional Physiological Phenomena , Neurons/diagnostic imaging , Animals , Axons/metabolism , Axons/ultrastructure , Brain Damage, Chronic/congenital , Brain Damage, Chronic/metabolism , Brain Damage, Chronic/pathology , Cerebral Cortex/metabolism , Corpus Callosum/metabolism , Dendrites/metabolism , Dendrites/ultrastructure , Female , Hippocampus/diagnostic imaging , Hippocampus/metabolism , Male , Nerve Fibers, Myelinated/diagnostic imaging , Nerve Fibers, Myelinated/metabolism , Neurogenesis , Neurons/metabolism , Pregnancy , Pyramidal Cells/diagnostic imaging , Pyramidal Cells/metabolism , Random Allocation , Rats , Rats, Inbred F344 , Reproducibility of Results , Ultrasonography
15.
Glia ; 61(2): 240-53, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23109359

ABSTRACT

Myelinated axons are organized into specialized domains critical to their function in saltatory conduction, i.e., nodes, paranodes, juxtaparanodes, and internodes. Here, we describe the distribution and role of the 4.1B protein in this organization. 4.1B is expressed by neurons, and at lower levels by Schwann cells, which also robustly express 4.1G. Immunofluorescence and immuno-EM demonstrates 4.1B is expressed subjacent to the axon membrane in all domains except the nodes. Mice deficient in 4.1B have preserved paranodes, based on marker staining and EM in contrast to the juxtaparanodes, which are substantially affected in both the PNS and CNS. The juxtaparanodal defect is evident in developing and adult nerves and is neuron-autonomous based on myelinating cocultures in which wt Schwann cells were grown with 4.1B-deficient neurons. Despite the juxtaparanodal defect, nerve conduction velocity is unaffected. Preservation of paranodal markers in 4.1B deficient mice is associated with, but not dependent on an increase of 4.1R at the axonal paranodes. Loss of 4.1B in the axon is also associated with reduced levels of the internodal proteins, Necl-1 and Necl-2, and of alpha-2 spectrin. Mutant nerves are modestly hypermyelinated and have increased numbers of Schmidt-Lanterman incisures, increased expression of 4.1G, and express a residual, truncated isoform of 4.1B. These results demonstrate that 4.1B is a key cytoskeletal scaffold for axonal adhesion molecules expressed in the juxtaparanodal and internodal domains that unexpectedly regulates myelin sheath thickness.


Subject(s)
Microfilament Proteins/metabolism , Nerve Fibers, Myelinated/metabolism , Neurons/cytology , Schwann Cells/metabolism , Animals , Ankyrins/metabolism , Axons/metabolism , Axons/ultrastructure , Cell Adhesion Molecule-1 , Cell Adhesion Molecules/metabolism , Cells, Cultured , Electric Stimulation , Embryo, Mammalian , Exploratory Behavior/physiology , Ganglia, Spinal/cytology , Immunoglobulins/metabolism , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Microfilament Proteins/genetics , Microscopy, Electron, Transmission , Microscopy, Immunoelectron , Myelin Basic Protein/metabolism , Myelin P0 Protein/metabolism , Myelin Proteins/metabolism , Neural Conduction/genetics , Neural Conduction/physiology , Ranvier's Nodes/metabolism , Ranvier's Nodes/ultrastructure , Schwann Cells/ultrastructure , Spectrin/metabolism
16.
J Neurosci ; 31(48): 17736-51, 2011 Nov 30.
Article in English | MEDLINE | ID: mdl-22131434

ABSTRACT

The plt (pale tremor) mouse carries a null mutation in the Fig4(Sac3) gene that results in tremor, hypopigmentation, spongiform degeneration of the brain, and juvenile lethality. FIG4 is a ubiquitously expressed phosphatidylinositol 3,5-bisphosphate phosphatase that regulates intracellular vesicle trafficking along the endosomal-lysosomal pathway. In humans, the missense mutation FIG4(I41T) combined with a FIG4 null allele causes Charcot-Marie-Tooth 4J disease, a severe form of peripheral neuropathy. Here we show that Fig4 null mice exhibit a dramatic reduction of myelin in the brain and spinal cord. In the optic nerve, smaller-caliber axons lack myelin sheaths entirely, whereas many large- and intermediate-caliber axons are myelinated but show structural defects at nodes of Ranvier, leading to delayed propagation of action potentials. In the Fig4 null brain and optic nerve, oligodendrocyte (OL) progenitor cells are present at normal abundance and distribution, but the number of myelinating OLs is greatly compromised. The total number of axons in the Fig4 null optic nerve is not reduced. Developmental studies reveal incomplete myelination rather than elevated cell death in the OL linage. Strikingly, there is rescue of CNS myelination and tremor in transgenic mice with neuron-specific expression of Fig4, demonstrating a non-cell-autonomous function of Fig4 in OL maturation and myelin development. In transgenic mice with global overexpression of the human pathogenic FIG4 variant I41T, there is rescue of the myelination defect, suggesting that the CNS of CMT4J patients may be protected from myelin deficiency by expression of the FIG4(I41T) mutant protein.


Subject(s)
Axons/pathology , Charcot-Marie-Tooth Disease/genetics , Flavoproteins/genetics , Myelin Sheath/genetics , Nerve Fibers, Myelinated/pathology , Neurons/metabolism , Phosphoric Monoester Hydrolases/genetics , Animals , Axons/metabolism , Charcot-Marie-Tooth Disease/metabolism , Charcot-Marie-Tooth Disease/pathology , Flavoproteins/metabolism , Mice , Mice, Transgenic , Myelin Sheath/metabolism , Myelin Sheath/pathology , Nerve Fibers, Myelinated/metabolism , Neurons/pathology , Phosphatidylinositols/metabolism , Phosphoinositide Phosphatases , Phosphoric Monoester Hydrolases/metabolism
17.
J Neurosci ; 30(37): 12432-45, 2010 Sep 15.
Article in English | MEDLINE | ID: mdl-20844138

ABSTRACT

In the adult mammalian CNS, the growth inhibitors oligodendrocyte-myelin glycoprotein (OMgp) and the reticulon RTN4 (Nogo) are broadly expressed in oligodendrocytes and neurons. Nogo and OMgp complex with the neuronal cell surface receptors Nogo receptor-1 (NgR1) and paired Ig-like receptor-B (PirB) to regulate neuronal morphology. In the healthy CNS, NgR1 regulates dendritic spine shape and attenuates activity-driven synaptic plasticity at Schaffer collateral-CA1 synapses. Here, we examine whether Nogo and OMgp influence functional synaptic plasticity, the efficacy by which synaptic transmission occurs. In acute hippocampal slices of adult mice, Nogo-66 and OMgp suppress NMDA receptor-dependent long-term potentiation (LTP) when locally applied to Schaffer collateral-CA1 synapses. Neither Nogo-66 nor OMgp influences basal synaptic transmission or paired-pulse facilitation, a form of short-term synaptic plasticity. PirB(-/-) and NgR1(-/-) single mutants and NgR1(-/-);PirB(-/-) double mutants show normal LTP, indistinguishable from wild-type controls. In juvenile mice, LTD in NgR1(-/-), but not PirB(-/-), slices is absent. Mechanistic studies revealed that Nogo-66 and OMgp suppress LTP in an NgR1-dependent manner. OMgp inhibits LTP in part through PirB but independently of p75. This suggests that NgR1 and PirB participate in ligand-dependent inhibition of synaptic plasticity. Loss of NgR1 leads to increased phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), signaling intermediates known to regulate neuronal growth and synaptic function. In primary cortical neurons, BDNF elicited phosphorylation of AKT and p70S6 kinase is attenuated in the presence of myelin inhibitors. Collectively, we provide evidence that mechanisms of neuronal growth inhibition and inhibition of synaptic strength are related. Thus, myelin inhibitors and their receptors may coordinate structural and functional neuronal plasticity in CNS health and disease.


Subject(s)
Down-Regulation/physiology , Myelin Proteins/physiology , Myelin-Associated Glycoprotein/physiology , Neural Inhibition/physiology , Neuronal Plasticity/physiology , Animals , Cell Line , Down-Regulation/genetics , GPI-Linked Proteins , Humans , Long-Term Potentiation/genetics , Long-Term Potentiation/physiology , Mice , Mice, Knockout , Mice, Transgenic , Myelin Proteins/genetics , Myelin Proteins/metabolism , Myelin-Associated Glycoprotein/genetics , Myelin-Associated Glycoprotein/pharmacology , Myelin-Oligodendrocyte Glycoprotein , Neural Inhibition/genetics , Neuronal Plasticity/genetics , Nogo Proteins , Nogo Receptor 1 , Rats , Receptors, Cell Surface/metabolism , Receptors, Cell Surface/physiology , Receptors, Immunologic/deficiency , Receptors, Immunologic/metabolism , Receptors, Immunologic/physiology
18.
Neuron ; 65(4): 490-502, 2010 Feb 25.
Article in English | MEDLINE | ID: mdl-20188654

ABSTRACT

Saltatory conduction requires high-density accumulation of Na(+) channels at the nodes of Ranvier. Nodal Na(+) channel clustering in the peripheral nervous system is regulated by myelinating Schwann cells through unknown mechanisms. During development, Na(+) channels are first clustered at heminodes that border each myelin segment, and later in the mature nodes that are formed by the fusion of two heminodes. Here, we show that initial clustering of Na(+) channels at heminodes requires glial NrCAM and gliomedin, as well as their axonal receptor neurofascin 186 (NF186). We further demonstrate that heminodal clustering coincides with a second, paranodal junction (PNJ)-dependent mechanism that allows Na(+) channels to accumulate at mature nodes by restricting their distribution between two growing myelin internodes. We propose that Schwann cells assemble the nodes of Ranvier by capturing Na(+) channels at heminodes and by constraining their distribution to the nodal gap. Together, these two cooperating mechanisms ensure fast and efficient conduction in myelinated nerves.


Subject(s)
Axons/metabolism , Cell Adhesion Molecules, Neuronal/metabolism , Cell Adhesion Molecules/metabolism , Ranvier's Nodes/metabolism , Schwann Cells/metabolism , Sodium Channels/metabolism , Action Potentials/physiology , Analysis of Variance , Animals , Blotting, Western , Cell Adhesion Molecules, Neuronal/genetics , Cells, Cultured , Electrophysiology , Fluorescent Antibody Technique , Mice , Mice, Knockout , Microscopy, Electron , Myelin Sheath/metabolism , Nerve Fibers, Myelinated/metabolism , Nerve Growth Factors/metabolism , Neural Conduction , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction
19.
J Neurosci ; 28(11): 2753-65, 2008 Mar 12.
Article in English | MEDLINE | ID: mdl-18337405

ABSTRACT

In the mature nervous system, changes in synaptic strength correlate with changes in neuronal structure. Members of the Nogo-66 receptor family have been implicated in regulating neuronal morphology. Nogo-66 receptor 1 (NgR1) supports binding of the myelin inhibitors Nogo-A, MAG (myelin-associated glycoprotein), and OMgp (oligodendrocyte myelin glycoprotein), and is important for growth cone collapse in response to acutely presented inhibitors in vitro. After injury to the corticospinal tract, NgR1 limits axon collateral sprouting but is not important for blocking long-distance regenerative growth in vivo. Here, we report on a novel interaction between NgR1 and select members of the fibroblast growth factor (FGF) family. FGF1 and FGF2 bind directly and with high affinity to NgR1 but not to NgR2 or NgR3. In primary cortical neurons, ectopic NgR1 inhibits FGF2-elicited axonal branching. Loss of NgR1 results in altered spine morphologies along apical dendrites of hippocampal CA1 neurons in vivo. Analysis of synaptosomal fractions revealed that NgR1 is enriched synaptically in the hippocampus. Physiological studies at Schaffer collateral-CA1 synapses uncovered a synaptic function for NgR1. Loss of NgR1 leads to FGF2-dependent enhancement of long-term potentiation (LTP) without altering basal synaptic transmission or short-term plasticity. NgR1 and FGF receptor 1 (FGFR1) are colocalized to synapses, and mechanistic studies revealed that FGFR kinase activity is necessary for FGF2-elicited enhancement of hippocampal LTP in NgR1 mutants. In addition, loss of NgR1 attenuates long-term depression of synaptic transmission at Schaffer collateral-CA1 synapses. Together, our findings establish that physiological NgR1 signaling regulates activity-dependent synaptic strength and uncover neuronal NgR1 as a regulator of synaptic plasticity.


Subject(s)
Dendritic Spines/physiology , Receptors, Cell Surface/physiology , Synapses/physiology , Animals , COS Cells , Cells, Cultured , Chlorocebus aethiops , Dendritic Spines/ultrastructure , Excitatory Postsynaptic Potentials/physiology , GPI-Linked Proteins , Humans , Mice , Mice, Mutant Strains , Nogo Receptor 2 , Protein Binding/physiology , Rats , Synapses/ultrastructure
20.
J Cell Biol ; 178(5): 875-86, 2007 Aug 27.
Article in English | MEDLINE | ID: mdl-17709431

ABSTRACT

Action potential initiation and propagation requires clustered Na(+) (voltage-gated Na(+) [Nav]) channels at axon initial segments (AIS) and nodes of Ranvier. In addition to ion channels, these domains are characterized by cell adhesion molecules (CAMs; neurofascin-186 [NF-186] and neuron glia-related CAM [NrCAM]), cytoskeletal proteins (ankyrinG and betaIV spectrin), and the extracellular chondroitin-sulfate proteoglycan brevican. Schwann cells initiate peripheral nervous system node formation by clustering NF-186, which then recruits ankyrinG and Nav channels. However, AIS assembly of this protein complex does not require glial contact. To determine the AIS assembly mechanism, we silenced expression of AIS proteins by RNA interference. AnkyrinG knockdown prevented AIS localization of all other AIS proteins. Loss of NF-186, NrCAM, Nav channels, or betaIV spectrin did not affect other neuronal AIS proteins. However, loss of NF-186 blocked assembly of the brevican-based AIS extracellular matrix, and NF-186 overexpression caused somatodendritic brevican clustering. Thus, NF-186 assembles and links the specialized brevican-containing AIS extracellular matrix to the intracellular cytoskeleton.


Subject(s)
Axons , Cell Adhesion Molecules/metabolism , Extracellular Matrix/metabolism , Nerve Growth Factors/metabolism , Animals , Ankyrins/genetics , Ankyrins/metabolism , Axons/metabolism , Axons/ultrastructure , Brevican , Cell Adhesion Molecules/genetics , Cells, Cultured , Chondroitin Sulfate Proteoglycans/metabolism , Cytoskeleton/metabolism , Extracellular Matrix/chemistry , Hippocampus/cytology , Lectins, C-Type/metabolism , Nerve Growth Factors/genetics , Nerve Tissue Proteins/metabolism , RNA/genetics , RNA/metabolism , RNA Interference , Ranvier's Nodes/metabolism , Ranvier's Nodes/ultrastructure , Rats , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sodium Channels/genetics , Sodium Channels/metabolism , Spectrin/genetics , Spectrin/metabolism
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