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Inhibitory activity of myelin-associated glycoprotein on sensory neurons is largely independent of NgR1 and NgR2 and resides within Ig-Like domains 4 and 5.

Wörter V, Schweigreiter R, Kinzel B, Mueller M, Barske C, Böck G, Frentzel S, Bandtlow CE - PLoS ONE (2009)

Bottom Line: Sensory neurons deficient for both NgR proteins displayed only a moderate reduction of MAG-mediated inhibition of neurite growth.Thus, disrupting all known protein and ganglioside receptors for MAG in sensory neurons does not fully abolish its inhibitory activity pointing to the existence of as yet unidentified receptors for MAG.Moreover, by employing a variety of protein mutants, we identified the Ig-like domains 4 or 5 of MAG as necessary and sufficient for growth arrest, whereas abolishing MAG's ability to bind to sialic acid did not interfere with its inhibitory activity.

View Article: PubMed Central - PubMed

Affiliation: Innsbruck Medical University, Biocenter, Division of Neurobiochemistry, Innsbruck, Austria.

ABSTRACT
Myelin-associated glycoprotein (MAG) is a sialic acid binding Ig-like lectin (Siglec) which has been characterized as potent myelin-derived inhibitor of neurite outgrowth. Two members of the Nogo-receptor (NgR) family, NgR1 and NgR2, have been identified as neuronal binding proteins of MAG. In addition, gangliosides have been proposed to bind to and confer the inhibitory activity of MAG on neurons. In this study, we investigated the individual contribution of NgRs and gangliosides to MAG-mediated inhibition of sensory neurons derived from dorsal root ganglia (DRG) of ngr1, ngr2 or ngr1/ngr2 deletion mutants. We found no disinhibition of neurite growth in the absence of either NgR1 or NgR2. Sensory neurons deficient for both NgR proteins displayed only a moderate reduction of MAG-mediated inhibition of neurite growth. If treated with Vibrio cholerae neuraminidase (VCN), inhibition by MAG is further attenuated but still not aned. Thus, disrupting all known protein and ganglioside receptors for MAG in sensory neurons does not fully abolish its inhibitory activity pointing to the existence of as yet unidentified receptors for MAG. Moreover, by employing a variety of protein mutants, we identified the Ig-like domains 4 or 5 of MAG as necessary and sufficient for growth arrest, whereas abolishing MAG's ability to bind to sialic acid did not interfere with its inhibitory activity. These findings provide new insights into the inhibitory function of MAG and suggest similarities but also major differences in MAG inhibition between sensory and central nervous system (CNS) neurons.

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Generation of ngr2 deletion mutant mice.(A) Targeting strategy for the ngr2 mutant. Genomic sequences in intron 1 and 3′UTR of the ngr2 gene were used as targeting sites. (B) Southern analysis of ES cell clones; the expected signal for a targeted clone is at 6.2 kb. Germline transmission after blastocyst injection was obtained for ES cell clone 9H (circled). (C) Real-time-PCR (left) and reverse transcription PCR (right) for NgR2 with mouse brain cDNA confirm the absence of NgR2 in −/− animals. (D) Genotyping of mice using genomic DNA from tail biopsies of wt, ngr1−/−, ngr2−/− and ngr1/2−/− animals. HSVtk, Herpes simplex virus thymidine kinase gene; neo, neomycin resistance gene; polyA, polyadenylation site.
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pone-0005218-g001: Generation of ngr2 deletion mutant mice.(A) Targeting strategy for the ngr2 mutant. Genomic sequences in intron 1 and 3′UTR of the ngr2 gene were used as targeting sites. (B) Southern analysis of ES cell clones; the expected signal for a targeted clone is at 6.2 kb. Germline transmission after blastocyst injection was obtained for ES cell clone 9H (circled). (C) Real-time-PCR (left) and reverse transcription PCR (right) for NgR2 with mouse brain cDNA confirm the absence of NgR2 in −/− animals. (D) Genotyping of mice using genomic DNA from tail biopsies of wt, ngr1−/−, ngr2−/− and ngr1/2−/− animals. HSVtk, Herpes simplex virus thymidine kinase gene; neo, neomycin resistance gene; polyA, polyadenylation site.

Mentions: Inactivation of the murine ngr2 gene was achieved by targeted disruption of exons 2 to 3 with a strategy depicted in Figure 1A. Except for exon 1, which encodes only 10 amino acids, the entire coding sequence was deleted and the mutation was therefore expected to be a allele. The targeted allele was obtained in C57Bl/6 ES cells (B16-III cell line; [26]) as confirmed by Southern Blotting of ES cell genomic DNA (Fig. 1B). ES cells of three targeted clones were injected into Balb/c host blastocysts and germline transmission was achieved with one clone. Real-time and reverse transcription PCR with brain cDNA of homozygous mice confirmed the absence of the NgR2 transcript (Fig. 1C). At genomic level, the mutant allele was detected by PCR-based genotyping of tail biopsies (Fig. 1D). Expression of NgR1 or other known receptor components conferring MAG responsiveness such as p75NTR or TROY/TAJ were unaltered in cultured sensory neurons (data not shown). Heterozygous ngr2 mice appeared phenotypically normal and fertile, and intercrosses between heterozygous mice yielded homozygous ngr2 knock-out animals at a normal Mendelian frequency. Similar to ngr1 deletion mutants [27], homozygous adult ngr2 mice appeared healthy, and histological examination showed no obvious abnormalities in the brain, liver or heart (not shown). ngr1/2 double mice were generated by cross-breeding ngr1 and ngr2 deletion mutants and the absence of both the ngr1 and ngr2 wildtype allele was demonstrated by PCR-based genotyping of tail biopsies (Fig. 1D). ngr1/2 double mice are viable and fertile and morphologically indistinguishable from wildtype animals.


Inhibitory activity of myelin-associated glycoprotein on sensory neurons is largely independent of NgR1 and NgR2 and resides within Ig-Like domains 4 and 5.

Wörter V, Schweigreiter R, Kinzel B, Mueller M, Barske C, Böck G, Frentzel S, Bandtlow CE - PLoS ONE (2009)

Generation of ngr2 deletion mutant mice.(A) Targeting strategy for the ngr2 mutant. Genomic sequences in intron 1 and 3′UTR of the ngr2 gene were used as targeting sites. (B) Southern analysis of ES cell clones; the expected signal for a targeted clone is at 6.2 kb. Germline transmission after blastocyst injection was obtained for ES cell clone 9H (circled). (C) Real-time-PCR (left) and reverse transcription PCR (right) for NgR2 with mouse brain cDNA confirm the absence of NgR2 in −/− animals. (D) Genotyping of mice using genomic DNA from tail biopsies of wt, ngr1−/−, ngr2−/− and ngr1/2−/− animals. HSVtk, Herpes simplex virus thymidine kinase gene; neo, neomycin resistance gene; polyA, polyadenylation site.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2666269&req=5

pone-0005218-g001: Generation of ngr2 deletion mutant mice.(A) Targeting strategy for the ngr2 mutant. Genomic sequences in intron 1 and 3′UTR of the ngr2 gene were used as targeting sites. (B) Southern analysis of ES cell clones; the expected signal for a targeted clone is at 6.2 kb. Germline transmission after blastocyst injection was obtained for ES cell clone 9H (circled). (C) Real-time-PCR (left) and reverse transcription PCR (right) for NgR2 with mouse brain cDNA confirm the absence of NgR2 in −/− animals. (D) Genotyping of mice using genomic DNA from tail biopsies of wt, ngr1−/−, ngr2−/− and ngr1/2−/− animals. HSVtk, Herpes simplex virus thymidine kinase gene; neo, neomycin resistance gene; polyA, polyadenylation site.
Mentions: Inactivation of the murine ngr2 gene was achieved by targeted disruption of exons 2 to 3 with a strategy depicted in Figure 1A. Except for exon 1, which encodes only 10 amino acids, the entire coding sequence was deleted and the mutation was therefore expected to be a allele. The targeted allele was obtained in C57Bl/6 ES cells (B16-III cell line; [26]) as confirmed by Southern Blotting of ES cell genomic DNA (Fig. 1B). ES cells of three targeted clones were injected into Balb/c host blastocysts and germline transmission was achieved with one clone. Real-time and reverse transcription PCR with brain cDNA of homozygous mice confirmed the absence of the NgR2 transcript (Fig. 1C). At genomic level, the mutant allele was detected by PCR-based genotyping of tail biopsies (Fig. 1D). Expression of NgR1 or other known receptor components conferring MAG responsiveness such as p75NTR or TROY/TAJ were unaltered in cultured sensory neurons (data not shown). Heterozygous ngr2 mice appeared phenotypically normal and fertile, and intercrosses between heterozygous mice yielded homozygous ngr2 knock-out animals at a normal Mendelian frequency. Similar to ngr1 deletion mutants [27], homozygous adult ngr2 mice appeared healthy, and histological examination showed no obvious abnormalities in the brain, liver or heart (not shown). ngr1/2 double mice were generated by cross-breeding ngr1 and ngr2 deletion mutants and the absence of both the ngr1 and ngr2 wildtype allele was demonstrated by PCR-based genotyping of tail biopsies (Fig. 1D). ngr1/2 double mice are viable and fertile and morphologically indistinguishable from wildtype animals.

Bottom Line: Sensory neurons deficient for both NgR proteins displayed only a moderate reduction of MAG-mediated inhibition of neurite growth.Thus, disrupting all known protein and ganglioside receptors for MAG in sensory neurons does not fully abolish its inhibitory activity pointing to the existence of as yet unidentified receptors for MAG.Moreover, by employing a variety of protein mutants, we identified the Ig-like domains 4 or 5 of MAG as necessary and sufficient for growth arrest, whereas abolishing MAG's ability to bind to sialic acid did not interfere with its inhibitory activity.

View Article: PubMed Central - PubMed

Affiliation: Innsbruck Medical University, Biocenter, Division of Neurobiochemistry, Innsbruck, Austria.

ABSTRACT
Myelin-associated glycoprotein (MAG) is a sialic acid binding Ig-like lectin (Siglec) which has been characterized as potent myelin-derived inhibitor of neurite outgrowth. Two members of the Nogo-receptor (NgR) family, NgR1 and NgR2, have been identified as neuronal binding proteins of MAG. In addition, gangliosides have been proposed to bind to and confer the inhibitory activity of MAG on neurons. In this study, we investigated the individual contribution of NgRs and gangliosides to MAG-mediated inhibition of sensory neurons derived from dorsal root ganglia (DRG) of ngr1, ngr2 or ngr1/ngr2 deletion mutants. We found no disinhibition of neurite growth in the absence of either NgR1 or NgR2. Sensory neurons deficient for both NgR proteins displayed only a moderate reduction of MAG-mediated inhibition of neurite growth. If treated with Vibrio cholerae neuraminidase (VCN), inhibition by MAG is further attenuated but still not aned. Thus, disrupting all known protein and ganglioside receptors for MAG in sensory neurons does not fully abolish its inhibitory activity pointing to the existence of as yet unidentified receptors for MAG. Moreover, by employing a variety of protein mutants, we identified the Ig-like domains 4 or 5 of MAG as necessary and sufficient for growth arrest, whereas abolishing MAG's ability to bind to sialic acid did not interfere with its inhibitory activity. These findings provide new insights into the inhibitory function of MAG and suggest similarities but also major differences in MAG inhibition between sensory and central nervous system (CNS) neurons.

Show MeSH
Related in: MedlinePlus