Olfactory ensheathing cells promote corticospinal axonal outgrowth by a L1 CAM‐dependent mechanism

Olfactory ensheathing cells (OECs) support the ability of the olfactory neuraxis to continually retarget within the mature central nervous system. This has led many groups to transplant OECS into the lesioned rodent spinal cord (SCd) in vivo, with variable degrees of anatomical, physiological, and behavioral success. Some of the most conflicting results in OEC transplantation have come from the corticospinal tract (CST) which has shown a relatively poor regeneration response. Although spinal neurite sprouting occurs in response to OECs in vivo and in vitro, we do not know if OECs possess the molecular machinery to stimulate outgrowth of functionally important motor tracts like the CST. Here, we assay cultured postnatal day 8 mouse CST neurons expressing yellow fluorescent protein (YFP) for their ability to extend axons and dendrites in response to different glia, and show that CST axons elongate in response to proteins in OEC plasma membrane (PM). In contrast, CST dendritic branching preferentially occurs in response to factors secreted by both OECs and astrocytes. We identify the L1‐neural cell adhesion molecule (L1‐NCAM) as a major component of OEC‐induced corticospinal axon elongation, and have determined that OEC PM factors (including L1), can stimulate CST outgrowth even when inhibition is induced by myelin associated glycoprotein. Together, these results suggest that in the right context, OEC‐derived PM factors could enhance CST axonal regeneration, and potentially contribute to approaches to ameliorate recovery from SCd injury. GLIA 2013;61:1873–1889     

The myelin-associated glycoprotein inhibitor BENZ induces outgrowth and survival of rat dorsal root ganglion cell cultures

The novel myelin-associated glycoprotein (MAG) inhibitor BENZ binds to the N-acetylneuraminic acid (Neu5Ac) portion of the N-terminal Ig-like domain of MAG. Treatment of rat dorsal root ganglion (DRG) cell cultures with BENZ-induced outgrowth of neurofilament 200-positive neurites improved survival of neurons and increased the number of GFAP-positive cells, as determined by fluorescence and confocal laser microscopy and by Western immunoblotting. Furthermore, treatment of DRG cell cultures with BENZ repressed gene and protein expression of the small GTPase RhoA but induced expression of Rho GTP-activating proteins 5 and 24, likely to counteract protein kinase A activity. Specifically, expression of inhibitors of neurite outgrowth, for example, Rock2 and PAK4, was repressed, but cofilin 1, a promoter of axonal growth, was induced. We propose that the MAG inhibitor BENZ abrogates the RhoA-ROCK-cofilin pathway to promote neurite outgrowth. Our findings require confirmation by in vivo animal studies.     

Long‐term efficacy of rituximab in IgM anti‐myelin‐associated glycoprotein neuropathy: RIMAG follow‐up study

The Rituximab vs. Placebo in Polyneuropathy Associated With Anti‐MAG IgM Monoclonal Gammopathy (RIMAG) study showed no improvement using the inflammatory neuropathy cause and treatment sensory score (ISS) as primary outcome in patients with IgM anti‐myelin‐associated glycoprotein neuropathy (IgM anti‐MAG neuropathy) treated with rituximab, when compared with placebo. However, some secondary outcomes seemed to improve in the per protocol analysis. Patients from one participating center in the RIMAG study underwent a new evaluation after a median follow‐up of 6 (interquartile range (IQR) 4.9; 6.5) years, using the same outcome measures used in the original study. Data were recorded in seven rituximab patients (group 1) and in eight placebo patients (group 2). In group 2, six of eight patients received immunotherapy during follow‐up, while only two of seven did in group 1. No significant change was observed in either the ISS or the secondary outcomes in both groups, with the exception of worsening in the 10‐m walk time in group 2 (p = 0.016). The RIMAG follow‐up study failed to find any significant change in most outcome measures in patients from the RIMAG study, some of them having received new immunotherapies. This study stresses the lack of useful clinical scales sensitive enough to capture small, even meaningful, improvement in IgM anti‐MAG neuropathy.     

Exercise decreases myelin-associated glycoprotein expression in the spinal cord and positively modulates neuronal growth

To successfully grow, neurons need to overcome the effects of hostile environments, such as the inhibitory action of myelin. We have evaluated the potential of exercise to overcome the intrinsic limitation of the central nervous system for axonal growth. In line with the demonstrated ability of exercise to increase the regenerative potential of neurons, here we show that exercise reduces the inhibitory capacity of myelin. Cortical neurons grown on myelin from exercised rats showed a more pronounced neurite extension compared with neurons grown on poly-D-lysine, or on myelin extracted from sedentary animals. The activity of cyclin-dependent kinase 5, a kinase involved in neurite outgrowth, was found to be increased in cortical neurons grown on exercise-myelin and in the lumbar spinal cord enlargement of exercised animals. Exercise significantly decreased the levels of myelin-associated glycoprotein (MAG), a potent axonal growth inhibitor, suggesting that downregulation of MAG is part of the mechanism through which exercise reduces growth inhibition. It is known that exercise elevates brain-derived neurotrophic factor (BDNF) spinal cord levels and that BDNF acts to overcome the inhibitory effects of myelin. Accordingly, we blocked the action of BDNF during exercise, which suppressed the exercise-related MAG decrease. Protein kinase A (PKA) has been related to the ability of BDNF to overcome growth inhibition; in agreement, we found that exercise increased PKA levels and this effect was reverted by blocking BDNF. Overall, these results show that exercise promotes a permissive cellular environment for axonal growth in the adult spinal cord requiring BDNF action.     

Laminin-like proteins are differentially regulated during cerebellar development and stimulate granule cell neurite outgrowth in vitro

The basement membrane glycoprotein laminin-1 is a potent stimulator of neurite outgrowth. Although a variety of laminin isoforms have been described in recent years, the role of alternative laminin isoforms in neural development remains largely uncharacterized. We found that a polyclonal antibody raised against the α1, β1, and γ1 chains of laminin-1 and a monoclonal antibody raised against the α2 chain of laminin-2 detect immunoreactive material in neuronal cell bodies in the developing mouse cerebellum. In addition, laminin-1-like immunoreactivity was found in cell types throughout the cerebellum, but laminin-α2-like immunoreactivity was restricted to the Purkinje cells. Purified laminin-1 and laminin-2 stimulated neurite outgrowth in primary cultures of mouse cerebellar granule neurons to a similar extent, whereas the synthetic peptides tested appeared to be active only for cell adhesion and not for stimulation of neurite outgrowth. The E8 proteolytic fragment of laminin-1 contained full neurite outgrowth activity. The identity of laminins expressed in granule neurons was also examined by Western blotting; laminin-like complexes were associated with the cell and appeared to have novel compositions. These results suggest that laminin-like complexes play important roles in cerebellar development. J. Neurosci. Res. 54:233–247, 1998. © 1998 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.     

Molecular basis of interactions between regenerating adult rat thalamic axons and Schwann cells in peripheral nerve grafts II. Tenascin-C

Tenascin-C is a developmentally regulated extracellular matrix component. There is evidence that it may be involved in axon growth and regeneration in peripheral nerves. We have used in situ hybridization and immunocytochemistry to investigate the association of tenascin-C with central nervous system axons regenerating through a peripheral nerve autograft inserted into the thalamus of adult rats. Between 3 days and 4 weeks after implantation, tenascin-C immunoreactivity was increased in the grafts, first at the graft/brain interface, then in the endoneurium of the graft, and finally within the Schwann cell columns of the graft. By electron microscopy, reaction product was present around collagen fibrils and basal laminae in the endoneurium, but the heaviest deposits were found at the surface of regenerating thalamic axons within Schwann cell columns. Schwann cell surfaces were not associated with tenascin-C reaction product except where they faced the tenascin-rich basal lamina or were immediately opposite axons surrounded by tenascin-C. By 8 weeks after graft implantation tenascin-C in the endoneurium and around axons of the graft was decreased. In the brain parenchyma aroundthe proximal part of the graft, axonal sprouts associated with tenascin-C could not be identified earlier than 2 weeks after grafting and were sparse at this stage. Larger numbers of such axons were present at 8–13 weeks after grafting and were located predominantly where the glia limitans between brain and graft appeared to be incomplete, suggesting that the tenascin-C may have penetrated the brain parenchyma from the graft. By in situ hybridization, cells expressing tenascin-C mRNA (probably Schwann cells) appeared first at the brain/graft interface 3 days after grafting and thereafter were mainly located within the grafts. Lightly labelled cells containing tenascin-C mRNA (probably glial cells) were scattered in the thalamic parenchyma both ipsilateral and contralateral to the graft and a few heavily labelled cells were located very close to the tip of the graft. These results show that regenerating adult thalamic axons, unlike regenerating peripheral axons, become intimately associated with peripheral nerve graft-derived tenascin-C, suggesting that they express a tenascin-C receptor, as many neurons do during development, and that tenascin-C derived from Schwann cells may play a role in the regenerative growth of such axons through the grafts. © 1995 Wiley-Liss, Inc.     

S-100 beta and insulin-like growth factor-II differentially regulate growth of developing serotonin and dopamine neurons in vitro.

To study the phenotypic specificity of S-100 beta and insulin-like growth factor II (IGF-II) for developing monoamine neurons, serotonin (5-HT) neurons from the embryonic day 14 (E14) rostral raphe or dopamine (TH) neurons from the substantia nigra/ventral tegmental area were cultured for 3 days in vitro (3 DIV) in the presence of these factors. Neuronotrophic effects were analyzed by computer-assisted morphometry of 5-HT and TH-immunoreactive neurons. S-100 beta and IGF-II differentially regulated the growth of 5-HT and TH neurons but did not affect their survival. S-100 beta significantly increased several parameters of neurite outgrowth by 5-HT neurons but inhibited the spatial extent (field area) of TH neurites. IGF-II promoted growth of cell bodies of both phenotype, but only stimulated neurite outgrowth by TH neurons. S-100 beta and IGF-II differentially affected the number of GFAP immunoreactive cells from raphe and substantia nigra, but these effects did not correlate with the specificity of neuronotrophic effects. S-100 beta and IGF-II immunoreactivities were expressed in glial cultures derived from the same brain regions, raising the possibility that these factors have autocrine effects on glia as well as paracrine actions on neurons. The results of this study suggest that specificity of neurotrophic factors for particular embryonic neurons may be correlated with their neurotransmitter phenotype.     

Methyl 3,4-dihydroxybenzoate promotes neurite outgrowth of cortical neurons cultured in vitro

Cerebral cortical neurons from neonatal rats were cultured in the presence of methyl 3,4-dihydroxybenzoate (MDHB;2,4,and 8 μM).Results showed that MDHB significantly promoted neurite outgrowth and microtubule-associated protein 2 mRNA expression,and increased neuronal survival in a dose-dependent manner.Moreover,MDHB induced brain-derived neurotrophic factor expression.These findings suggest that MDHB has a neurotrophic effect,which may be due to its ability to increase brain-derived neurotrophic factor expression.     

Differential effects of glial cell line-derived neurotrophic factor and neurturin in RET/GFRalpha1-expressing cells

The c-ret protooncogene, RET, encodes a receptor tyrosine kinase. RET is activated by members of the glial cell line-derived neurotrophic factor (GDNF) family of ligands, which include GDNF, neurturin, artemin, and persephin. The ligands bind RET through GDNF family receptor alpha, termed GFRalpha1-4. Despite the importance of RET signaling in the development of the enteric nervous system and the kidney, the differential signaling mechanisms between RET ligands are poorly established. It has been suggested that signal specificity is achieved through binding of the ligand to its preferred GFRalpha. To compare the signaling profiles of GDNF and neurturin, we have identified a cell line, NG108-15, which endogenously expresses RET and GFRalpha1 but not GFRalpha2-4. Immunoblot data showed that GDNF caused a transient activation, whereas neurturin caused a sustained activation, of both p44/p42 MAP kinases and PLCgamma. Under serum starvation, NG108-15 cells differentiate and form neurites. Neurturin but not GDNF stimulated neurite outgrowth, which could be blocked by the selective PLC inhibitor U73122. On the other hand, GDNF but not neurturin promoted cell survival, and this could be blocked by the p44/p42 MAP kinase inhibitor PD98059. Our findings not only show the differential signaling of GDNF and neurturin but also suggest that this can be achieved through binding to the same GFRalpha subtype, leading to distinct biological responses.     

Vagal motor neurons in rats respond to noxious and physiological gastrointestinal distention differentially

Low-pressure gastrointestinal distention modulates gastrointestinal function by a vago-vagal reflex. Noxious visceral distention, as seen in an obstruction of the gastrointestinal tract, causes abdominal pain, vomiting and affective changes. Using single neuron recording and intracellular injection techniques, we characterized the neuronal responses of neurons in the dorsal motor nucleus of the vagus (DMNV) to low- and high-pressure distensions of stomach and duodenum. Low-pressure gastric distention inhibited the mean activity of the DMNV neurons whereas high-pressure gastric distention excited many neurons. Of 47 DMNV neurons, low-pressure gastric distention inhibited 39, excited four, and did not affect four neurons. High-pressure gastric distention inhibited 26, excited 20, and left one unaffected. Thirteen of the 39 DMNV neurons inhibited by low-pressure distention of the stomach reversed their response to excitation during high-pressure gastric distention. Among 47 DMNV neurons, low-pressure duodenal distention inhibited 30, excited 10, and did not affect the remaining seven neurons. High-pressure distention of the duodenum inhibited 25 and excited 22 neurons. Eight DMNV neurons inhibited by low-pressure duodenal distention were excited in early response to high-pressure distention of the duodenum. High-pressure duodenal distention caused an early excitation and late inhibition in the mean activity of the DMNV neurons while low-pressure duodenal distention only produced late inhibition. These results suggest that different reflexes are present between physiological distention and noxious stimulation of gastrointestinal tract.     

Axonal plasticity elicits long‐term changes in oligodendroglia and myelinated fibers

Axons are linked to induction of myelination during development and to the maintenance of myelin and myelinated tracts in the adult CNS. Currently, it is unknown whether and how axonal plasticity in adult CNS impacts the myelinating cells and their precursors. In this article, we report that newly formed axonal sprouts are able to induce a protracted myelination response in adult CNS. We show that newly formed axonal sprouts, induced by lesion of the entorhino–hippocampal perforant pathway, have the ability to induce a myelination response in stratum radiatum and lucidum CA3. The lesion resulted in significant recruitment of newly formed myelinating cells, documented by incorporation of the proliferation marker bromodeoxyuridine into chondroitin sulphate NG2 expressing cells in stratum radiatum and lucidum CA3 early after lesion, and the occurrence of a 28% increase in the number of oligodendrocytes, of which some had incorporated bromodeoxyuridine, 9 weeks post‐lesion. Additionally, a marked increase (41%) in myelinated fibres was detected in silver stained sections. Interestingly, these apparently new fibres achieved the same axon diameter as unlesioned mice but myelin thickness remained thinner than normal, suggesting that the sprouting axons in stratum radiatum and lucidum CA3 were not fully myelinated 9 weeks after lesion. Our combined results show that sprouting axons provide a strong stimulus to oligodendrocyte lineage cells to engage actively in the myelination processes in the adult CNS. © 2009 Wiley‐Liss, Inc.     

Protection by an ACTH4–9 analogue against the toxic effects of cisplatin and taxol on sensory neurons and glial cells in vitro

Sensory neuropathy is a serious side effect of antitumour drugs such as cisplatin and taxol. There are indications that an analogue of the adrenocorticotrophic hormone 4–9 fragment (ACTH_4–9: Met(O₂)-Glu-His-Phe-D-Lys-Phe) can prevent these neurotoxic effects. We studied the potential protective effects of this analogue in cultures of chick dorsal root ganglia and rat Schwann cells treated with cisplatin or taxol to gain insight into the mode of action and characteristics of this neuroprotection. Neurite outgrowth of sensory neurons in vitro was dose-dependently inhibited by cisplatin and taxol; after 48 hr, 10 μg/ml cisplatin reduced outgrowth from 431 ± 17 μm to 220 ± 6 μm and 0.01 μg/ml taxol from 344 ± 3 μm to 200 ± 43 μm. Co-treatment of 10 μg/ml cisplatin with the ACTH_4–9 analogue (0.1 nM–1 nM) resulted in about 35% more outgrowth than cisplatin alone. In contrast, the analogue could not prevent taxol neurotoxicity. Migration of neurons and satellite cells from the DRG-body is completely inhibited by 10 μg/ml cisplatin. Taxol had no effect on the migration of these cells. In addition, cisplatin was more toxic to Schwann cells than taxol; 3–10 μg/ml cisplatin significantly reduced their laminin content, total protein, 2′,3′-Cyclic Nucleotide 3′-Phosphodiesterase activity, and cell division. The ACTH_4–9 analogue (0.01 nM–100 nM) had no effect on the migration of cells out of the DRGs and could not prevent the toxic effect on the Schwann cells. These data support our hypothesis that the neuroprotective effect of ACTH_4–9 analogue is brought about by a direct action on neurons, possibly by replacing a Schwann-/satellite-cell derived trophic factor. Copyright © 1994 Wiley-Liss, Inc.     

Kif1Bbeta isoform is enriched in motor neurons but does not change in a mouse model of amyotrophic lateral sclerosis

The kinesin superfamily motor protein Kif1B is expressed in two isoforms, Kif1Balpha and Kif1Bbeta, with distinct cargo-binding domains. We examined the mRNA distribution of the two isoforms in adjacent sections of brain and spinal cord of adult mice using in situ hybridization analysis. Kif1Bbeta mRNA is enriched in several regions of brain and spinal cord. Its levels are four to five times higher than that of the alpha isoform, which was barely detectable. The highest mRNA levels of Kif1Bbeta were found in the cortex, hippocampus, cerebellum and the grey matter of the spinal cord. At the cellular level the highest signal was found in motor neurons in the motor nuclei of medulla oblongata and the ventral horn of spinal cord. Because expression of other Kif genes is altered in amyotrophic lateral sclerosis (ALS) models, we examined the expression level of Kif1Bbeta mRNA in the spinal cord of transgenic mice carrying the SOD1G93A mutation, a model of familial ALS, at presymptomatic and early stages of the disease. No changes were observed in Kif1Bbeta mRNA in motor neurons or in other regions of the spinal cord. These findings indicate that Kif1Balpha, which modulates the transport of mitochondria, may play a major role in tissues other than the central nervous system. Instead Kif1Bbeta, responsible for the transport of synaptic vesicle precursors, seems to play an important role in the nervous system, particularly in the lower motor neurons. The absence of changes of Kif1Bbeta mRNA in transgenic SOD1G93A mice suggests that other molecular mechanisms may play a role in the disruption of axonal transport occurring in the motor neurons of these mice.     

Specificity of the second messenger pathways involved in basic fibroblast growth factor‐induced survival and neurite growth in chick ciliary ganglion neurons

Basic fibroblast growth factor (bFGF) exerts multiple neurotrophic actions on cultured neurons from the ciliary ganglion of chick embryo, among them promotion of neuronal survival and of neurite outgrowth. To understand the specificity of the signal transduction cascades involved in the control of these processes, we used pharmacological inhibitors of the three main effectors known to act downstream of the bFGF receptor (FGFR): phospholipase Cγ (PLCγ), mitogen‐activated protein kinase (MAPK), and phosphatidylinositol 3‐kinase (PI3‐K). Neuronal survival was assessed at 24 and 48 hr; neurite growth was analyzed both on dissociated neurons and on explants of whole ganglia. Our data show that only the PI3‐K pathway is involved in the survival‐promoting effect of bFGF; on the other hand, all three effectors converge on the enhancement of neurite outgrowth, both on isolated neurons and in whole ganglia. © 2009 Wiley‐Liss, Inc.     

Limitations in daily activities and general perception of quality of life: Long term follow‐up in patients with anti‐myelin‐glycoprotein antibody polyneuropathy

In this study, we assessed the modifications over time of daily activities and quality of life (QoL) in 32 subjects with anti‐myelin‐glycoprotein (MAG) antibody neuropathy. A widespread panel including clinical scores and patient‐reported questionnaires, in compliance of the terms by the International Classification of Functioning, Disability, and Health (ICF) of the World Health Organization (WHO), was employed at enrollment (T0) and at follow‐up evaluation (T1) after a mean interval of 15.4 ± 5.7 months. The Sensory Modality Sum score (SMS) at four limbs showed a significant worsening over time (mean score 27.2 ± 3.9 at T0 vs 25.7 ± 3 at T1 at upper limbs, P = .03; 20.5 ± 4.8 at T0 vs 18.6 ± 5.9 at T1 at lower limbs, P = .04). The Visual Analogue Scale (VAS) for pain significantly worsened at upper limbs at T1 (mean values 0.84 ± 1.95 at T0 vs 1.78 ± 2.6 at T1, P = .03). All the other tests did not show significant differences between T0 and T1. In the subgroup who underwent rituximab (15/32 treated before T0, 3/32 patients treated between T0 and T1 with median interval of 1 year), no significant differences were observed between T0 and T1. Despite the quite long follow‐up, statistical significance was not achieved either for the limited number of patients or for the lack of sensitive outcome measures. In our cohort, the significant worsening of the SMS and VAS after a median of 14 months can be considered as a reliable expression of the natural history of the disease, and suggest that these scales might represent possible outcome measures in anti‐MAG antibody neuropathy.