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INTRODUCTION - There is a need to develop imaging methods sensitive to axonal injury in multiple sclerosis (MS), given the prominent impact of axonal pathology on disability and outcome. Advanced multi-compartmental diffusion models offer novel indices sensitive to white matter microstructure. One such model, neurite orientation dispersion and density imaging (NODDI), is sensitive to neurite morphology, providing indices of apparent volume fractions of axons (v), isotropic water (v) and the dispersion of fibers about a central axis (orientation dispersion index, ODI). NODDI has yet to be studied for its sensitivity to spinal cord pathology. Here, we investigate the feasibility and utility of NODDI in the cervical spinal cord of MS patients.
METHODS - NODDI was applied in the cervical spinal cord in a cohort of 8 controls and 6 MS patients. Statistical analyses were performed to test the sensitivity of NODDI-derived indices to pathology in MS (both lesion and normal appearing white matter NAWM). Diffusion kurtosis imaging (DKI) and diffusion tensor imaging (DTI) analysis were also performed to compare with NODDI.
RESULTS - A decrease in NODDI-derived v was observed at the site of the lesion ( < 0.01), whereas a global increase in ODI was seen throughout white matter ( < 0.001). DKI-derived mean kurtosis (MK) and radial kurtosis (RK) and DTI-derived fractional anisotropy (FA) and radial diffusivity (RD) were all significantly different in MS patients ( < 0.02), however NODDI provided higher contrast between NAWM and lesion in all MS patients.
CONCLUSION - NODDI provides unique contrast that is not available with DKI or DTI, enabling improved characterization of the spinal cord in MS.
Neurite outgrowth is key to the formation of functional circuits during neuronal development. Neurotrophins, including nerve growth factor (NGF), increase neurite outgrowth in part by altering the function and expression of Ca(2+)-permeable cation channels. Here we report that transient receptor potential vanilloid 2 (TRPV2) is an intracellular Ca(2+)-permeable TRPV channel upregulated by NGF via the mitogen-activated protein kinase (MAPK) signaling pathway to augment neurite outgrowth. TRPV2 colocalized with Rab7, a late endosome protein, in addition to TrkA and activated extracellular signal-regulated kinase (ERK) in neurites, indicating that the channel is closely associated with signaling endosomes. In line with these results, we showed that TRPV2 acts as an ERK substrate and identified the motifs necessary for phosphorylation of TRPV2 by ERK. Furthermore, neurite length, TRPV2 expression, and TRPV2-mediated Ca(2+) signals were reduced by mutagenesis of these key ERK phosphorylation sites. Based on these findings, we identified a previously uncharacterized mechanism by which ERK controls TRPV2-mediated Ca(2+) signals in developing neurons and further establish TRPV2 as a critical intracellular ion channel in neuronal function.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Ketamine is an NMDA antagonist and dissociative anesthetic that has been shown to display rapid acting and prolonged antidepressant activity in small-scale human clinical trials. Ketamine also binds to σ receptors, which are believed to be protein targets for a potential new class of antidepressant medications. The purpose of this study was to determine the involvement of σ receptors in the antidepressant-like actions of ketamine. Competition binding assays were performed to assess the affinity of ketamine for σ(1) and σ(2) receptors. The antidepressant-like effects of ketamine were assessed in vitro using a neurite outgrowth model and PC12 cells, and in vivo using the forced swim test. The σ receptor antagonists, NE-100 and BD1047, were evaluated in conjunction with ketamine in these assays to determine the involvement of σ receptors in the antidepressant-like effects of ketamine. Ketamine bound to both σ(1) and σ(2) receptors with μM affinities. Additionally, ketamine potentiated NGF-induced neurite outgrowth in PC12 cells and this effect was attenuated in the presence of NE-100. Ketamine also displayed antidepressant-like effects in the forced swim test; however, these effects were not attenuated by pretreatment with NE-100 or BD1047. Taken together, these data suggest that σ receptor-mediated neuronal remodeling may contribute to the antidepressant effects of ketamine.
Copyright Â© 2011 Elsevier B.V. and ECNP. All rights reserved.
Selenoprotein P (Sepp1) is an important protein involved in selenium (Se) transport and homeostasis. Severe neurologic dysfunction develops in Sepp1 null mice (Sepp1(-/-)) fed a selenium-deficient diet. Sepp1(-/-) mice fed a selenium-deficient diet have extensive degeneration of the brainstem and thalamus, and even when supplemented with selenium exhibit subtle learning deficits and altered basal synaptic transmission and short-term plasticity in the CA1 region of the hippocampus. The goal of this study was to delineate the regional progression of neurodegeneration in the brain, determine the extent of neuronal cell death, and evaluate neurite structural changes within the hippocampus of Sepp1(-/-) mice. Whole brain serial sections of wild-type and Sepp1(-/-) mice maintained on selenium-deficient or supplemented diets over the course of 12 days from weaning were evaluated with amino cupric silver neurodegeneration stain. The neurodegeneration was present in all regions upon weaning and progressed over 12 days in Sepp1(-/-) mice fed selenium-deficient diet, except in the medial forebrain bundle and somatosensory cortex where the neurodegeneration developed post-weaning. The neurodegeneration was predominantly axonal, however the somatosensory cortex and lateral striatum showed silver-stained neurons. Morphologic analysis of the hippocampus revealed decreased dendritic length and spine density, suggesting that loss of Sepp1 also causes subtle changes in the brain that can contribute to functional deficits. These data illustrate that deletion of Sepp1, and presumably selenium deficiency in the brain, produce both neuronal and axonal degeneration as well as more moderate and potentially reversible neurite changes in the developing brain.
Copyright © 2011 Elsevier B.V. All rights reserved.
Axonal regeneration in the central nervous system is prevented, in part, by inhibitory proteins expressed by myelin, including myelin-associated glycoprotein (MAG). Although injury to the corticospinal tract can result in permanent disability, little is known regarding the mechanisms by which MAG affects cortical neurons. Here, we demonstrate that cortical neurons plated on MAG expressing CHO cells, exhibit a striking reduction in process outgrowth. Interestingly, none of the receptors previously implicated in MAG signaling, including the p75 neurotrophin receptor or gangliosides, contributed significantly to MAG-mediated inhibition. However, blocking the small GTPase Rho or its downstream effector kinase, ROCK, partially reversed the effects of MAG on the neurons. In addition, we identified the lipid phosphatase PTEN as a mediator of MAG's inhibitory effects on neurite outgrowth. Knockdown or gene deletion of PTEN or overexpression of activated AKT in cortical neurons resulted in significant, although partial, rescue of neurite outgrowth on MAG-CHO cells. Moreover, MAG decreased the levels of phospho-Akt, suggesting that it activates PTEN in the neurons. Taken together, these results suggest a novel pathway activated by MAG in cortical neurons involving the PTEN/PI3K/AKT axis.
Copyright Â© 2010 Elsevier Inc. All rights reserved.
Studies of mice lacking MHC class I (MHC I)-associated proteins have demonstrated a role for MHC I in neurodevelopment. A central question arising from these observations is whether neuronal recognition of MHC I has specificity for the MHC I allele product and the peptide presented. Using a well-established embryonic retina explant system, we observed that picomolar levels of a recombinant self-MHC I molecule inhibited neurite outgrowth. We then assessed the neurobiological activity of a panel of recombinant soluble MHC Is, consisting of different MHC I heavy chains with a defined self- or nonself-peptide presented, on cultured embryonic retinas from mice with different MHC I haplotypes. We observed that self-MHC I allele products had greater inhibitory neuroactivity than nonself-MHC I molecules, regardless of the nature of the peptide presented, a pattern akin to MHC I recognition by some innate immune system receptors. However, self-MHC I molecules had no effect on retinas from MHC I-deficient mice. These observations suggest that neuronal recognition of MHC I may be coordinated with the inherited MHC I alleles, as occurs in the innate immune system. Consistent with this notion, we show that MHC I and MHC I receptors are coexpressed by precursor cells at the earliest stages of retina development, which could enable such coordination.
Heparan sulfate (HS) plays an essential role in extracellular signaling during development. Biochemical studies have established that HS binding to ligands and receptors is regulated by the fine 6-O-sulfated structure of HS; however, mechanisms that control sulfated HS structure and associated signaling functions in vivo are not known. Extracellular HS 6-O-endosulfatases, SULF1 and SULF2, are candidate enzymatic regulators of HS 6-O-sulfated structure and modulate HS-dependent signaling. To investigate Sulf regulation of developmental signaling, we have disrupted Sulf genes in mouse and identified redundant functions of Sulfs in GDNF-dependent neural innervation and enteric glial formation in the esophagus, resulting in esophageal contractile malfunction in Sulf1(-/-);Sulf2(-/-) mice. SULF1 is expressed in GDNF-expressing esophageal muscle and SULF2 in innervating neurons, establishing their direct functions in esophageal innervation. Biochemical and cell signaling studies show that Sulfs are the major regulators of HS 6-O-desulfation, acting to reduce GDNF binding to HS and to enhance GDNF signaling and neurite sprouting in the embryonic esophagus. The functional specificity of Sulfs in GDNF signaling during esophageal innervation was established by showing that the neurite sprouting is selectively dependent on GDNF, but not on neurotrophins or other signaling ligands. These findings provide the first in vivo evidence that Sulfs are essential developmental regulators of cellular HS 6-O-sulfation for matrix transmission and reception of GDNF signal from muscle to innervating neurons.
Peripheral nerve section promotes regenerative, elongated neuritic growth of adult sensory neurons. Although the role of chloride homeostasis, through the regulation of ionotropic GABA receptors, in the growth status of immature neurons in the CNS begins to emerge, nothing is known of its role in the regenerative growth of injured adult neurons. To analyze the intracellular Cl- variation after a sciatic nerve section in vivo, gramicidin perforated-patch recordings were used to study muscimol-induced currents in mice dorsal root ganglion neurons isolated from control and axotomized neurons. We show that the reversal potential of muscimol-induced current, E(GABA-A), was shifted toward depolarized potentials in axotomized neurons. This was attributable to Cl- influx because removal of extracellular Cl- prevented this shift. Application of bumetanide, an inhibitor of NKCC1 cotransporter and E(GABA-A) recordings in sensory neurons from NKCC1-/- mice, identified NKCC1 as being responsible for the increase in intracellular Cl- in axotomized neurons. In addition, we demonstrate with a phospho-NKCC1 antibody that nerve injury induces an increase in the phosphorylated form of NKCC1 in dorsal root ganglia that could account for intracellular Cl- accumulation. Time-lapse recordings of the neuritic growth of axotomized neurons show a faster growth velocity compared with control. Bumetanide, the intrathecal injection of NKCC1 small interfering RNA, and the use of NKCC1-/- mice demonstrated that NKCC1 is involved in determining the velocity of elongated growth of axotomized neurons. Our results clearly show that NKCC1-induced increase in intracellular chloride concentration is a major event accompanying peripheral nerve regeneration.
Neurons maintain relatively high intracellular concentrations of ascorbic acid, which is achieved primarily by the activity of the sodium-dependent vitamin C transporter SVCT2. In this work, we studied the mechanisms by which neuronal cells in culture transport and maintain ascorbate as well as whether this system contributes to maturation of neuronal function and cellular defense against oxidative stress and excitotoxic injury. We found that the SVCT2 helps to maintain high intracellular ascorbate levels, normal ascorbate transport kinetics, and activity-dependent ascorbate recycling. Immunocytochemistry studies revealed that SVCT2 is expressed primarily in the axons of mature hippocampal neurons in culture. In the absence of SVCT2, hippocampal neurons exhibited stunted neurite outgrowth, less glutamate receptor clustering, and reduced spontaneous neuronal activity. Finally, hippocampal cultures from SVCT2-deficient mice showed increased susceptibility to oxidative damage and N-methyl-D-aspartate-induced excitotoxicity. Our results revealed that maintenance of intracellular ascorbate as a result of SVCT2 activity is crucial for neuronal development, functional maturation, and antioxidant responses.
(c) 2007 Wiley-Liss, Inc.
Alteration of dopamine neurotransmission during development can induce specific changes in neuronal structure and function. Here, we report specific morphological and neurochemical changes of projection neurons and interneurons of the medial frontal cortex of the dopamine D(1) receptor null mouse. Using immunostaining of cytoskeletal proteins and a crossbred D(1) receptor null:YFP transgenic reporter line, we demonstrate that the apical dendrites of pyramidal cells are abnormally organized in the prefrontal and anterior cingulate cortices of mice lacking the D(1) receptor. Neuronal processes exhibit a decrease in bundling and an increase in irregular, tortuous patterning as they weave a course towards the pial surface. In addition, there is increased parvalbumin staining of the dendrites of cortical interneurons in D(1) receptor null mice. Both pyramidal and interneuron alterations are evident by the early postnatal period and persist into adulthood. The alterations show regional specificity, in that dendritic profiles of projection neurons and interneurons in somatosensory and visual cortices develop normally. The abnormalities are reminiscent of those induced by prenatal exposure to cocaine in rabbits, an insult which has been shown to produce an attenuation of D(1) receptor-mediated responses through G(salpha). These results suggest that loss of D(1) receptor-mediated signaling during development produces permanent alterations in the cellular organization of specific cortical areas involved in attention, cognition, and emotion. Pharmacological and behavioral studies in the D(1) null mouse should be interpreted in the context of possible altered circuitry, given the presence of these developmental defects in the organization of dopaminoceptive regions of the cerebral cortex.
Copyright 2005 Wiley-Liss, Inc.