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Ectopic expression of polysialylated neural cell adhesion molecule in adult macaque Schwann cells promotes their migration and remyelination potential in the central nervous system.

Bachelin C, Zujovic V, Buchet D, Mallet J, Baron-Van Evercooren A - Brain (2009)

Bottom Line: In vitro, we found that ectopic expression of polysialylate promoted adult macaque Schwann cell migration and improved their integration among astrocytes in vitro without modifying their antigenic properties as either non-myelinating or pro-myelinating.These greater performances of sialyltransferase expressing Schwann cell correlated with their sustained expression of polysialylated neural cell adhesion molecule at early times when migrating from the graft to the lesion, and its progressive downregulation at later times during remyelination.These results underline the potential therapeutic benefit to genetically modify Schwann cells to overcome their poor migration capacity and promote their repair potential in demyelinating disorders of the central nervous system.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche de l'Institut du Cerveau et de la Moelle Epiniere, Universite Pierre et Marie Curie-Paris 6, UMR-S975, Paris, France.

ABSTRACT
Recent findings suggested that inducing neural cell adhesion molecule polysialylation in rodents is a promising strategy for promoting tissue repair in the injured central nervous system. Since autologous grafting of Schwann cells is one potential strategy to promote central nervous system remyelination, it is essential to show that such a strategy can be translated to adult primate Schwann cells and is of interest for myelin diseases. Adult macaque Schwann cells were transduced with a lentiviral vector encoding sialyltransferase, an enzyme responsible for neural cell adhesion molecule polysialylation. In vitro, we found that ectopic expression of polysialylate promoted adult macaque Schwann cell migration and improved their integration among astrocytes in vitro without modifying their antigenic properties as either non-myelinating or pro-myelinating. In addition, forced expression of polysialylate in adult macaque Schwann cells decreased their adhesion with sister cells. To investigate the ability of adult macaque Schwann cells to integrate and migrate in vivo, focally induced demyelination was targeted to the spinal cord dorsal funiculus of nude mice, and both control and sialyltransferase expressing Schwann cells overexpressing green fluorescein protein were grafted remotely from the lesion site. Analysis of the spatio-temporal distribution of the grafted Schwann cells performed in toto and in situ, showed that in both groups, Schwann cells migrated towards the lesion site. However, migration of sialyltransferase expressing Schwann cells was more efficient than that of control Schwann cells, leading to their accelerated recruitment by the lesion. Moreover, ectopic expression of polysialylated neural cell adhesion molecule promoted adult macaque Schwann cell interaction with reactive astrocytes when exiting the graft, and their 'chain-like' migration along the dorsal midline. The accelerated migration of sialyltransferase expressing Schwann cells to the lesion site enhanced their ability to compete for myelin repair with endogenous cells, while control Schwann cells were unable to do so. Finally, remyelination by the exogenous sialyltransferase expressing Schwann cells restored the normal distribution of paranodal and nodal elements on the host axons. These greater performances of sialyltransferase expressing Schwann cell correlated with their sustained expression of polysialylated neural cell adhesion molecule at early times when migrating from the graft to the lesion, and its progressive downregulation at later times during remyelination. These results underline the potential therapeutic benefit to genetically modify Schwann cells to overcome their poor migration capacity and promote their repair potential in demyelinating disorders of the central nervous system.

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In vitro expression of NCAM and PSA-NCAM by adult macaque Schwann cells. Ct-SC express NCAM (A) but not PSA-NCAM (B). STX-SC strongly express PSA-NCAM (C, red) on their membrane, while STX-GFP (C, green) expression is restricted to the Golgi, the location of sialyltransferase activity. After treatment with endoN, which specifcally cleaves PSA on NCAM, PSA is no longer expressed on the Schwann cell membrane (red, D) while STX-GFP is still present in the Golgi (green, D).
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Figure 1: In vitro expression of NCAM and PSA-NCAM by adult macaque Schwann cells. Ct-SC express NCAM (A) but not PSA-NCAM (B). STX-SC strongly express PSA-NCAM (C, red) on their membrane, while STX-GFP (C, green) expression is restricted to the Golgi, the location of sialyltransferase activity. After treatment with endoN, which specifcally cleaves PSA on NCAM, PSA is no longer expressed on the Schwann cell membrane (red, D) while STX-GFP is still present in the Golgi (green, D).

Mentions: Immunocytochemistry for NCAM and PSA-NCAM showed that like in the neonate rodent, adult macaque Schwann cells expressed NCAM (Fig. 1A) but not PSA-NCAM (Fig. 1B). To force expression of PSA in Schwann cells, they were purified and transduced with the STX or control lentiviral vector. Schwann cells were transduced with an efficiency of 73 ± 3%. This was confirmed by immunohistochemistry for PSA-NCAM, which showed that 75 ± 4% of the STX-SC expressed PSA-NCAM (Fig. 1C) while none of the Ct-SC did (Fig. 1B). Transduced STX-SC expressed PSA-NCAM strongly on their membrane, while expression of STX-GFP protein was restricted to the Golgi, the expected location of the sialyltransferase. Thus, the enzyme activity and localization were not affected by its fusion with GFP. This expression was stable since PSA and STX expression lasted in vitro for up to 6 months in the majority of transduced Schwann cells. Treatment with endoN showed that PSA was no longer expressed on the Schwann cells membrane while STX-GFP remained expressed in the Golgi (Fig. 1D). Since EndoN cleaves PSA specifically on NCAM, these results confirm that STX specifically sialylates NCAM in transduced macaque Schwann cells.Figure 1


Ectopic expression of polysialylated neural cell adhesion molecule in adult macaque Schwann cells promotes their migration and remyelination potential in the central nervous system.

Bachelin C, Zujovic V, Buchet D, Mallet J, Baron-Van Evercooren A - Brain (2009)

In vitro expression of NCAM and PSA-NCAM by adult macaque Schwann cells. Ct-SC express NCAM (A) but not PSA-NCAM (B). STX-SC strongly express PSA-NCAM (C, red) on their membrane, while STX-GFP (C, green) expression is restricted to the Golgi, the location of sialyltransferase activity. After treatment with endoN, which specifcally cleaves PSA on NCAM, PSA is no longer expressed on the Schwann cell membrane (red, D) while STX-GFP is still present in the Golgi (green, D).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2822629&req=5

Figure 1: In vitro expression of NCAM and PSA-NCAM by adult macaque Schwann cells. Ct-SC express NCAM (A) but not PSA-NCAM (B). STX-SC strongly express PSA-NCAM (C, red) on their membrane, while STX-GFP (C, green) expression is restricted to the Golgi, the location of sialyltransferase activity. After treatment with endoN, which specifcally cleaves PSA on NCAM, PSA is no longer expressed on the Schwann cell membrane (red, D) while STX-GFP is still present in the Golgi (green, D).
Mentions: Immunocytochemistry for NCAM and PSA-NCAM showed that like in the neonate rodent, adult macaque Schwann cells expressed NCAM (Fig. 1A) but not PSA-NCAM (Fig. 1B). To force expression of PSA in Schwann cells, they were purified and transduced with the STX or control lentiviral vector. Schwann cells were transduced with an efficiency of 73 ± 3%. This was confirmed by immunohistochemistry for PSA-NCAM, which showed that 75 ± 4% of the STX-SC expressed PSA-NCAM (Fig. 1C) while none of the Ct-SC did (Fig. 1B). Transduced STX-SC expressed PSA-NCAM strongly on their membrane, while expression of STX-GFP protein was restricted to the Golgi, the expected location of the sialyltransferase. Thus, the enzyme activity and localization were not affected by its fusion with GFP. This expression was stable since PSA and STX expression lasted in vitro for up to 6 months in the majority of transduced Schwann cells. Treatment with endoN showed that PSA was no longer expressed on the Schwann cells membrane while STX-GFP remained expressed in the Golgi (Fig. 1D). Since EndoN cleaves PSA specifically on NCAM, these results confirm that STX specifically sialylates NCAM in transduced macaque Schwann cells.Figure 1

Bottom Line: In vitro, we found that ectopic expression of polysialylate promoted adult macaque Schwann cell migration and improved their integration among astrocytes in vitro without modifying their antigenic properties as either non-myelinating or pro-myelinating.These greater performances of sialyltransferase expressing Schwann cell correlated with their sustained expression of polysialylated neural cell adhesion molecule at early times when migrating from the graft to the lesion, and its progressive downregulation at later times during remyelination.These results underline the potential therapeutic benefit to genetically modify Schwann cells to overcome their poor migration capacity and promote their repair potential in demyelinating disorders of the central nervous system.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche de l'Institut du Cerveau et de la Moelle Epiniere, Universite Pierre et Marie Curie-Paris 6, UMR-S975, Paris, France.

ABSTRACT
Recent findings suggested that inducing neural cell adhesion molecule polysialylation in rodents is a promising strategy for promoting tissue repair in the injured central nervous system. Since autologous grafting of Schwann cells is one potential strategy to promote central nervous system remyelination, it is essential to show that such a strategy can be translated to adult primate Schwann cells and is of interest for myelin diseases. Adult macaque Schwann cells were transduced with a lentiviral vector encoding sialyltransferase, an enzyme responsible for neural cell adhesion molecule polysialylation. In vitro, we found that ectopic expression of polysialylate promoted adult macaque Schwann cell migration and improved their integration among astrocytes in vitro without modifying their antigenic properties as either non-myelinating or pro-myelinating. In addition, forced expression of polysialylate in adult macaque Schwann cells decreased their adhesion with sister cells. To investigate the ability of adult macaque Schwann cells to integrate and migrate in vivo, focally induced demyelination was targeted to the spinal cord dorsal funiculus of nude mice, and both control and sialyltransferase expressing Schwann cells overexpressing green fluorescein protein were grafted remotely from the lesion site. Analysis of the spatio-temporal distribution of the grafted Schwann cells performed in toto and in situ, showed that in both groups, Schwann cells migrated towards the lesion site. However, migration of sialyltransferase expressing Schwann cells was more efficient than that of control Schwann cells, leading to their accelerated recruitment by the lesion. Moreover, ectopic expression of polysialylated neural cell adhesion molecule promoted adult macaque Schwann cell interaction with reactive astrocytes when exiting the graft, and their 'chain-like' migration along the dorsal midline. The accelerated migration of sialyltransferase expressing Schwann cells to the lesion site enhanced their ability to compete for myelin repair with endogenous cells, while control Schwann cells were unable to do so. Finally, remyelination by the exogenous sialyltransferase expressing Schwann cells restored the normal distribution of paranodal and nodal elements on the host axons. These greater performances of sialyltransferase expressing Schwann cell correlated with their sustained expression of polysialylated neural cell adhesion molecule at early times when migrating from the graft to the lesion, and its progressive downregulation at later times during remyelination. These results underline the potential therapeutic benefit to genetically modify Schwann cells to overcome their poor migration capacity and promote their repair potential in demyelinating disorders of the central nervous system.

Show MeSH
Related in: MedlinePlus