Robert J. Dunn

Identification of myelin-associated glycoprotein as a major myelin-derived inhibitor of neurite growth.

Contact-dependent axon growth inhibitory activity is present in CNS myelin, but the inhibitory proteins have not been fully characterized. We report here that at least two peaks of inhibitory activity can be separated by fractionating solubilized CNS myelin proteins by DEAE chromatography. A major peak of inhibitory activity corresponded to the elution profile of myelin-associated glycoprotein (MAG). Immunodepletion of MAG from these inhibitory fractions removed neurite growth inhibition, whereas recombinant MAG (ectodomain) was a potent inhibitor of neurite outgrowth. Immunodepletion of MAG from total extracts of CNS myelin restored neurite growth up to 63% of control levels. These results establish that MAG is a significant, and possibly the major, inhibitor in CNS myelin; this has broad implications for axonal regeneration in the injured mammalian CNS.

A rat brain na+ channel α subunit with novel gating properties

Abstract We have constructed a full-length rat brain Na + channel α subunit cDNA that differs from the previously reported a subunit of Noda et al. at 6 amino acid positions. Transcription of the cDNA in vitro and injection into Xenopus oocytes resulted in the synthesis of functional Na + channels. Although the single-channel conductance of the channels resulting from cloned cDNA was the same as that of channels resulting from injection of rat brain RNA, we observed two significant differences in the gating properties of the channels. The Na + currents from cloned cDNA displayed much slower macroscopic inactivation compared with those from rat brain mRNA. In addition, the current-voltage relationship for currents from cloned cDNA was shifted 20–25 mV in the depolarizing direction compared with currents from rat brain RNA. Coinjection of low MW rat brain RNA restored normal inactivation of the channels indicating the presence of a component, either a structural subunit of the channel complex or a modifying enzyme, necessary for normal gating of the channel.

Recombinant myelin-associated glycoprotein confers neural adhesion and neurite outgrowth function

Myelin-associated glycoprotein (MAG) cDNA clones for the small (p67) and large (p72) forms were expressed in heterologous cells. Purified recombinant MAG protein was incorporated into fluorescent liposomes, and both forms were shown to bind predominantly to neurites in DRG or spinal cord cultures. This adhesion was completely blocked by Fab fragments of monoclonal anti-MAG antibody. Liposomes prepared with the control protein glycophorin or no protein failed to bind neurites. Small cerebellar neurons, which are not myelinated in vivo, failed to bind MAG liposomes. In a second test of function, p67 MAG-transfected fibroblasts were markedly enhanced in their ability to promote DRG neurite extension over a 2 day culture period compared with control fibroblasts not expressing MAG. Neurite extension was blocked by anti-MAG antibodies. These results show that both forms of MAG can facilitate the interactions between glial cells and neurites that ultimately lead to myelin formation.

Alternative RNA Splicing Generates a Glycosylphosphatidylinositol-anchored Form of Ceruloplasmin in Mammalian Brain

Ceruloplasmin is a copper-containing ferroxidase that is essential for normal iron homeostasis. Whereas ceruloplasmin in plasma is produced and secreted by hepatocytes, in the brain a glycosylphosphatidylinositol (GPI)-anchored form of ceruloplasmin is expressed on the surface of astrocytes. By using a cDNA cloning approach, we have now determined that the GPI-anchored form of ceruloplasmin is generated by alternative RNA splicing. The splicing occurs downstream of exon 18 and replaces the C-terminal 5 amino acids of the secreted form with an alternative 30 amino acids that signal GPI anchor addition. RNase protection analysis demonstrates that the GPI-anchored form is the major form in the brain, whereas the secreted form predominates in the liver. Individuals with aceruloplasminemia, a hereditary deficiency of ceruloplasmin, have severe iron deposition in a number of organs, including the brain where it results in neurodegeneration. Therefore, this novel GPI-anchored form of ceruloplasmin is likely to play an important role in iron metabolism in the central nervous system.

Expression of the gene encoding the β-subunit of S-100 protein in the developing rat brain analyzed by in situ hybridization

To investigate patterns of expression of the gene encoding the beta-subunit of S-100 protein during development of the rat brain we have used Northern blotting and in situ hybridization histochemistry. During late prenatal development beta-S-100 mRNA was observed first in the germinal zone lining the 4th ventricle. In the postnatal cerebellum this mRNA accumulated primarily in Bergmann glia and astrocytes of the deep white matter. In the hindbrain, expression of S-100 mRNA increased steadily in specific regions during the first postnatal week while levels remained low in more anterior brain regions. By the end of the second postnatal week, a dense punctate signal was distributed throughout the midbrain and hindbrain. Expression in forebrain, first observed at E18, was confined to cells lining the ventricle until the second postnatal week when accumulation of mRNA was observed in specific regions of the hippocampus, neocortex and olfactory bulb. The adult brain pattern of beta-S-100 mRNA distribution is attained during the third postnatal week. These results demonstrate a caudal-rostral gradient in expression of the beta-S-100 gene during rat brain development, as well as pronounced regional differences which may reflect the differentiation of subpopulations of astrocytes.

The myelin-associated glycoprotein gene: mapping to human chromosome 19 and mouse chromosome 7 and expression in quivering mice.

Myelin-associated glycoprotein (MAG), a membrane glycoprotein of 100 kDa, is thought to be involved in the process of myelination. A cDNA encoding the amino-terminal half of rat MAG has recently been isolated and sequenced. We have used this cDNA in Southern blot analysis of DNA from 32 somatic cell hybrids to assign the human locus for MAG to chromosome 19 and the mouse locus to chromosome 7. Since the region of mouse chromosome 7-known to contain several other genes that are homologous to genes on human chromosome 19-also carries the quivering (qv) locus, we considered the possibility that a mutation in the MAG gene could be responsible for this neurological disorder. While MAG-specific DNA restriction fragments, mRNA, and protein from qv/qv mice were apparently normal in size and abundance, we have not ruled out the possibility that qv could be caused by a point mutation in the MAG gene.

N-METHYL-D-ASPARTATE RECEPTOR 1 MRNA DISTRIBUTION IN THE CENTRAL NERVOUS SYSTEM OF THE WEAKLY ELECTRIC FISH APTERONOTUS LEPTORHYNCHUS

We have isolated a partial cDNA for the N‐methyl‐D‐aspartate (NMDA) receptor 1 (NMDAR1) subunit from an Apteronotus leptorhynchus brain cDNA library. The A. leptorhynchus cDNA fragment, which corresponds to nucleotides 135–903 within the 5′ region of the rat NR1 mRNA, encodes 252 amino acids that are >80% identical to the homologous segments of the rat, human, and duck NR1 proteins. RNAse protection assays revealed that the A. leptorhynchus NR1 mRNA was highly enriched in the forebrain and hypothalamus, with lesser amounts in the brainstem, and very low levels in the cerebellum. In situ hybridization also demonstrated that neurons in the pallial forebrain were highly enriched in NR1 transcripts. High levels of NR1 mRNA were found in pyramidal cells within the optic tectum and octavolateral regions. Pyramidal cells of the electrosensory lateral line lobe had the highest levels of expression, and the NR1 mRNA was found to be selectively enriched in their apical dendrites. J. Comp. Neurol. 389:65–80, 1997.

SK Channels Provide a Novel Mechanism for the Control of Frequency Tuning in Electrosensory Neurons

One important characteristic of sensory input is frequency, with sensory neurons often tuned to narrow stimulus frequency ranges. Although vital for many neural computations, the cellular basis of such frequency tuning remains mostly unknown. In the electrosensory system of Apteronotus leptorhynchus, the primary processing of important environmental and communication signals occurs in pyramidal neurons of the electrosensory lateral line lobe. Spike trains transmitted by these cells can encode low-frequency prey stimuli with bursts of spikes and high-frequency communication signals with single spikes. Here, we demonstrate that the selective expression of SK2 channels in a subset of pyramidal neurons reduces their response to low-frequency stimuli by opposing their burst responses. Apamin block of the SK2 current in this subset of cells induced bursting and increased their response to low-frequency inputs. SK channel expression thus provides an intrinsic mechanism that predisposes a neuron to respond to higher frequencies and thus specific, behaviorally relevant stimuli.

Inactivation of Kv3.3 potassium channels in heterologous expression systems.

Kv3.3 K+ channels are believed to incorporate an NH2-terminal domain to produce an intermediate rate of inactivation relative to the fast inactivating K+ channels Kv3.4 and Kv1.4. The rate of Kv3.3 inactivation has, however, been difficult to establish given problems in obtaining consistent rates of inactivation in expression systems. This study characterized the properties of AptKv3.3, the teleost homologue of Kv3.3, when expressed in Chinese hamster ovary (CHO) or human embryonic kidney (HEK) cells. We show that the properties of AptKv3.3 differ significantly between CHO and HEK cells, with the largest difference occurring in the rate and voltage dependence of inactivation. While AptKv3.3 in CHO cells showed a fast and voltage-dependent rate of inactivation consistent with N-type inactivation, currents in HEK cells showed rates of inactivation that were voltage-independent and more consistent with a slower C-type inactivation. Examination of the mRNA sequence revealed that the first methionine start site had a weak Kozak consensus sequence, suggesting that the lack of inactivation in HEK cells could be due to translation at a second methionine start site downstream of the NH2-terminal coding region. Mutating the nucleotide sequence surrounding the first methionine start site to one more closely resembling a Kozak consensus sequence produced currents that inactivated with a fast and voltage-dependent rate of inactivation in both CHO and HEK cells. These results indicate that under the appropriate conditions Kv3.3 channels can exhibit fast and reliable inactivation that approaches that more typically expected of “A”-type K+ currents.

Ionic and neuromodulatory regulation of burst discharge controls frequency tuning

Sensory neurons encode natural stimuli by changes in firing rate or by generating specific firing patterns, such as bursts. Many neural computations rely on the fact that neurons can be tuned to specific stimulus frequencies. It is thus important to understand the mechanisms underlying frequency tuning. In the electrosensory system of the weakly electric fish, Apteronotus leptorhynchus, the primary processing of behaviourally relevant sensory signals occurs in pyramidal neurons of the electrosensory lateral line lobe (ELL). These cells encode low frequency prey stimuli with bursts of spikes and high frequency communication signals with single spikes. We describe here how bursting in pyramidal neurons can be regulated by intrinsic conductances in a cell subtype specific fashion across the sensory maps found within the ELL, thereby regulating their frequency tuning. Further, the neuromodulatory regulation of such conductances within individual cells and the consequences to frequency tuning are highlighted. Such alterations in the tuning of the pyramidal neurons may allow weakly electric fish to preferentially select for certain stimuli under various behaviourally relevant circumstances.