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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 8, 2003 as doi:10.1096/fj.02-0453fje. |
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Institute of Anatomy, Department of Cell Biology and Neurobiology, Humboldt University Medical School Charité Berlin, Germany; and
* Brain Research Institute, Department of Neuromorphology, University of Zurich and Department of Biology, Swiss Federal Institute of Technology Zurich, Switzerland
3Correspondence: Institute of Anatomy, Department of Cell Biology and Neurobiology, Humboldt University Hospital (Charité), Oskar-Hertwig House, Philippstr. 12, Berlin, 10098 Germany. E-mail: nicolai.savaskan{at}charite.de
SPECIFIC AIMS
The Nogo gene encodes for an integral protein responsible for the neurite inhibition properties in myelin. In this study we analyzed the mRNA expression pattern of the Nogo genes (Nogo-A, -B, -C) and their receptor (Nogo-66R) during hippocampal development and postlesional reorganization. We showed that Nogo genes are regulated isoform-specifically during development and after lesion, indicating Nogo functions beyond its known neuronal growth inhibition activity in shaping neuronal circuits.
PRINCIPAL FINDINGS
1. Appearance of myelinated fibers in the hippocampus during development
We first investigated the distribution of myelinated fibers in the developing hippocampal formation by Black Gold, a specific marker for myelin. There are no myelinated fibers present in the hippocampus at postnatal day 0 (P0). Myelinated fibers were first detectable in the stratum lacunosum moleculare of the CA3 region at P17. Conversely, the expression of a prominent myelin component, MBP mRNA, was first found in the hippocampal neuropil at P15 (Fig. 1
). At P30, the most densely populated areas of MBP mRNA expression were the strata oriens and radiatum of the CA regions. Hybridization signals for MBP mRNA were hardly found in any of the hippocampal neuronal cell layers (Fig. 1)
.
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2. Nogo-A, -B, -C and Nogo-66 receptor expression during hippocampal development
Nogo-A, Nogo-B, and Ng66R mRNA expression signals were first detected in the developing brain at P0, the earliest developing point we investigated (Fig. 1)
. At this stage, an intense hybridization signal was found in the pyramidal cells from CA1 to CA3, and a brighter signal was detectable in the presubiculum. Nogo-A, Nogo-B, and Ng66R mRNA were only slightly expressed in the granule cell layer of the dentate gyrus at this time. The mRNA distribution of Nogo-A and Ng66R remained unchanged from P15 to adulthood, with the exception of Ng66R mRNA signals in the hilar region, which declined during adulthood. The Nogo-B transcripts were found in the hippocampus proper until adulthood. However, Nogo-B mRNA levels declined during maturation in the cortex and subiculum (Fig. 1)
.
We barely found a Nogo-C mRNA signal in any of the neuronal cell layers of the hippocampus at P0 and P5. We first found Nogo-C mRNA expression in the neuronal cell layer of the dentate gyrus and the CA1-CA3 region at P15, but could hardly find a hybridization signal in the hilar region and entorhinal cortex (EC). This expression pattern remained unchanged until adulthood (Fig. 1)
.
3. Nogo-A mRNA expression is located in neuronal cell layers of the hippocampal formation
Since the expression pattern of the Nogo gene and its splice variants was distributed in cell layers known for the residence of neurons, we tested the mRNA expression on the cellular level. Using nonradiolabeled probes for Nogo-A transcripts, we revealed its distribution in the pyramidal and granule cells of the hippocampus beside oligodendrocytes in white matter and in entorhinal neurons of layer, II-IV. Granule cells in the dentate gyrus that were Nissl positive gave a strong hybridization signal, but only a few Nogo-A stained cells could be detected in the hippocampus neuropil.
4. Isoform-specific Nogo-A, -B, -C and Nogo-66 receptor mRNA expression following hippocampal deafferentation
In adult nonlesioned control animals, Nogo-A mRNA was predominantly expressed in the pyramidal cell layers of the CA1 to CA3 region, and to a lesser extent in the dentate gyrus (Fig. 1
, Fig. 2
). A massive increase of Nogo-A mRNA levels first occurs 1 day after lesion (1 dal), when a >20-fold increase was visible in the ipsilateral and contralateral cortex. This evident up-regulation peaked at 34-fold of the control levels at 5 dal on the ipsilateral side and at 3 dal on the contralateral side (Fig. 2A, B
). Subsequent Nogo-A mRNA content decreased at longer survival stages and reached control levels at 28 dal.
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In general, the Nogo-B hybridization pattern was less prominent in the hippocampus compared with Nogo-A (Fig. 1)
. A significant up-regulation of the Nogo-B transcript appeared in all cell layers of the hippocampus in the early phase of reorganization and returned to control levels at a time when axonal sprouting processes peak (Fig. 2)
. The granule cell layer and hilar region ipsilateral to the lesion showed the highest hybridization signal 1 dal and declined to control levels at 15 dal.
Nogo-C mRNA was not regulated in cortical regions after entorhinal cortex lesion (ECL). Nogo-C expression of the ipsilateral hippocampal cell layers started to increase during the early lesion stages when glial activity commences and showed a peak at 3 dal (Fig. 2A
). Except for the hilar region, the Nogo-C mRNA later decreased to below control levels at 10 dal and recovered to nonlesioned control levels at 28 dal.
Ng66R mRNA was significantly up-regulated in the cortex following lesion (Fig. 2B, D
). Whereas this alteration reached its maximum at 1 dal on both sides of the lesion, the ipsilateral cortex returned to control levels at 3 dal, while the contralateral side remained highly up-regulated until 28 dal (data not shown). The ipsilateral granule cell layer showed a biphasic time course of Ng66R mRNA, with a maximum peak at 3 dal and a maximum decrease at 15 dal (Fig. 2A
).
5. Nogo-A mRNA expression is up-regulated after seizure
After kainate-induced seizures, we found a significant up-regulation of Nogo-A mRNA in the hippocampus at 5 days postseizure, whereas earlier Nogo-A expression was unaltered.
CONCLUSIONS AND SIGNIFICANCE
During central nervous system (CNS) development axons extend from cells within the CNS and form an intricate and specific web of connections. During hippocampal development, stellate and pyramidal cells from layers II and III of the EC send their main fibers to the outer molecular layer of the dentate gyrus and the stratum lacunosum moleculare via the perforant pathway. First, entorhinal axons arrive at their final target in late embryonic stages in a layer-specific manner, segregating in the outermost part of the dentate molecular layer. Entorhinal terminals are restricted specifically to this layer throughout adulthood and respect termination zones of other afferents, i.e., commissural and septal fiber projections (Fig. 3
). The myelination process of entorhinal afferents in the hippocampal area commences during the second postnatal week and is completed by P60. However, Nogo-A, -B, and Ng66R mRNA expression far precedes myelination in the neuropil of the hippocampus. The first hybridization signals for Nogo-A, -B and Ng66R mRNA could be found in the EC and CA regions of the hippocampus at P0. Nogo-C and MBP mRNA are first significantly expressed in the hippocampus at P15, when the first myelinating fibers appear. However, in contrast to MBP expression, which is located exclusively in white matter regions, Nogo-C mRNA expression was predominantly found in principal cell layers of the hippocampus and to a lesser extent in the neuropil. This observation could be explained by the low metabolic activity of oligodendrocytes in contrast to neurons. This maturation-dependent expression in connection with the lack of neurite growth inhibitory activity of recombinant Nogo-C and its proposed intracellular localization indicate a function different from cell surface signaling.
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All Nogo splice variants were strongly regulated after ECL. Nogo-A, Nogo-B, and Ng66R were up-regulated in the ipsilateral cortex, where lesion-induced cell death and transneuronal alterations occur. However, Ng66R transcripts were also strongly up-regulated in the contralateral cortex.
In the hippocampus, Nogo genes showed an isoform-specific regulation after perforant path lesion. The decreased Ng66R expression correlates well with time of axon ingrowth into the lesioned hippocampus (Fig. 3)
. It was recently shown that Ng66R is also the receptor for oligodendrocyte-myelin glycoprotein, mediating its neurite outgrowth and growth cone collapse activity. Thus, down-regulation of Ng66R is sufficient to attenuate Nogo-mediated neurite growth inhibition since axons that do not express Ng66R are insensitive to growth cone collapse induction by Nogo.
The role of the Nogo genes and their receptor has been not fully elucidated. A cell surface signal with attractive and repulsive functions to other neurons, neurites, or non-neuronal cells has been assumed. The early mRNA expression of all Nogo transcripts during hippocampal development indicates a strong physiological role in addition to axonal inhibition. All Nogo transcripts and their receptor are located predominantly in neurons from postnatal stages on. It could be suggested that because of the complementary expression of the Nogo ligands and the Nogo-66 receptor during hippocampal development, they form a novel receptor-ligand system determining axonal pathfinding leading to a lamina-specific restriction and termination pattern (Fig. 3)
.
Recently, the Strittmatter group has shown that Ng66 receptor inhibition by peptide antagonist results in significant axonal outgrowth promotion and improves functional recovery after spinal cord lesion. However, neutralization of Nogo activity also results in increased plasticity in uninjured CNS fibers. Thus, our data concerning Nogo gene expression under physiological conditions should be significant when considering application of Nogo neutralization antibodies or Nogo receptor inhibitors for therapeutic approaches in the CNS.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0453fje; to cite this article, use FASEB J. (April 8, 2003) 10.1096/fj.02-0453fje 
2 These authors contributed as senior author.
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