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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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Remyelination potential of Schwann cells grafted into the demyelinated spinal cord. (A and B) Double detection at the lesion site, of P0 (red) and GFP (green) on horizontal sections of the spinal cords grafted with Ct-SC (A) and STX-SC (B) 28 d.p.t. While P0+ myelin profiles were observed in both groups, GFP+/P0+ myelin profiles (arrows, exogenous Schwann cells) were detected in the STX-SC grafted animals only (B). Insert in B illustrates a higher magnification of the boxed area. (C) Quantification of the total P0+ area indicated no difference in global remyelination by Schwann cells between the control and STX groups. Remyelination by the transplanted Schwann cells (P0+/GFP+) occurred in the STX-SC group only, the majority of Schwann cell remyelination being performed by the endogenous cells (P0+/GFP−). At the ultrastructural level, GFP was detected with Bluo-Gal precipitates (D, black precipitates). A close up of the boxed area illustrates the presence of a basal lamina (arrow) around the compact myelin. The poor preservation of myelin is due to the stringent immuno-labelling conditions to detect GFP. Immuno-labelling for MOG (blue), Caspr (red) and GFP (green) shows that STX-SC are associated with Caspr+ internodes (red). (E) Triple immuno-labelling illustrates STX-SC (green) associated with nodal Nav (blue) and paranodal Caspr (red) (F). P0+ myelin profiles (blue) produced by STX-SC (green) are associated with Caspr+ paranodes (red) (G).
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Figure 9: Remyelination potential of Schwann cells grafted into the demyelinated spinal cord. (A and B) Double detection at the lesion site, of P0 (red) and GFP (green) on horizontal sections of the spinal cords grafted with Ct-SC (A) and STX-SC (B) 28 d.p.t. While P0+ myelin profiles were observed in both groups, GFP+/P0+ myelin profiles (arrows, exogenous Schwann cells) were detected in the STX-SC grafted animals only (B). Insert in B illustrates a higher magnification of the boxed area. (C) Quantification of the total P0+ area indicated no difference in global remyelination by Schwann cells between the control and STX groups. Remyelination by the transplanted Schwann cells (P0+/GFP+) occurred in the STX-SC group only, the majority of Schwann cell remyelination being performed by the endogenous cells (P0+/GFP−). At the ultrastructural level, GFP was detected with Bluo-Gal precipitates (D, black precipitates). A close up of the boxed area illustrates the presence of a basal lamina (arrow) around the compact myelin. The poor preservation of myelin is due to the stringent immuno-labelling conditions to detect GFP. Immuno-labelling for MOG (blue), Caspr (red) and GFP (green) shows that STX-SC are associated with Caspr+ internodes (red). (E) Triple immuno-labelling illustrates STX-SC (green) associated with nodal Nav (blue) and paranodal Caspr (red) (F). P0+ myelin profiles (blue) produced by STX-SC (green) are associated with Caspr+ paranodes (red) (G).

Mentions: Remyelination of LPC-induced lesions starts at 7 d.p.t. and is nearly completed by 28 d.p.t. (Gout et al., 1988; Jeffery and Blakemore, 1995). It is achieved by endogenous oligodendrocyte precursors but also by Schwann cells (reviewed in Zujovic et al., 2007). We first investigated whether transplanted Ct-SC and STX-SC participated in the lesion repair by producing newly formed myelin and whether ectopic expression of PSA could promote this process. The relative abundance of peripheral versus CNS remyelination within the margins of the lesion, was evaluated by immunohistochemistry for P0 (Fig. 9A and B) and MOG (Fig. 9E and G) to detect peripheral and central myelin, respectively. Quantification of P0 (Fig. 9F) and MOG immunoreactivity in the lesion showed equal amounts of total peripheral (Ct-SC = 0.09 ± 0.03; STX-SC = 0.13 ± 0.08 µm2/µm2 of lesion) and central (Ct-SC = 0.301 ± 0.07; STX-SC = 0.310 ± 0.08 µm2/µm2 of lesion) myelin for each group. To establish the relative contribution of Ct-SC and STX-SC to exogenous remyelination, the amount of P0 co-localized with GFP was quantified. GFP+/P0+ myelin-like figures were detected in STX-SC grafted animals only (Fig. 9B) and represented 5% of the global Schwann cell remyelination process (Fig. 9C). These data indicate that only STX-SC, which arrived in the lesion faster, were able to out-compete endogenous myelin-forming cells for remyelination. The presence of exogenous newly formed myelin was further confirmed by electron microscopy revealing the presence of GFP with BluoGal precipitates. Figure 9D illustrates the presence of BluoGal precipitates in myelin with specific peripheral myelin features such as the 1:1 association of Schwann cell with axon and the presence of a basal membrane around the compacted myelin (Fig. 9D, inset). Finally, we questioned the functionality of STX-SC-derived myelin focusing on the ability to re-assemble paranodal Caspr and nodal voltage-gated Nav proteins, as endogenous Schwann cells do (Black et al., 2006). Immunodetection of Caspr, and/or Nav in association with P0 and GFP, showed that P0+/GFP+ STX-SC internodes were clearly associated with Nav channels flanked by Caspr+ paranodes at the node level (Fig. 9E–G). STX-SC lead to the reconstruction of well-organized internodes on host axons throughout the lesion with a very similar expression pattern to that observed for endogenous Schwann cells.Figure 9


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)

Remyelination potential of Schwann cells grafted into the demyelinated spinal cord. (A and B) Double detection at the lesion site, of P0 (red) and GFP (green) on horizontal sections of the spinal cords grafted with Ct-SC (A) and STX-SC (B) 28 d.p.t. While P0+ myelin profiles were observed in both groups, GFP+/P0+ myelin profiles (arrows, exogenous Schwann cells) were detected in the STX-SC grafted animals only (B). Insert in B illustrates a higher magnification of the boxed area. (C) Quantification of the total P0+ area indicated no difference in global remyelination by Schwann cells between the control and STX groups. Remyelination by the transplanted Schwann cells (P0+/GFP+) occurred in the STX-SC group only, the majority of Schwann cell remyelination being performed by the endogenous cells (P0+/GFP−). At the ultrastructural level, GFP was detected with Bluo-Gal precipitates (D, black precipitates). A close up of the boxed area illustrates the presence of a basal lamina (arrow) around the compact myelin. The poor preservation of myelin is due to the stringent immuno-labelling conditions to detect GFP. Immuno-labelling for MOG (blue), Caspr (red) and GFP (green) shows that STX-SC are associated with Caspr+ internodes (red). (E) Triple immuno-labelling illustrates STX-SC (green) associated with nodal Nav (blue) and paranodal Caspr (red) (F). P0+ myelin profiles (blue) produced by STX-SC (green) are associated with Caspr+ paranodes (red) (G).
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Figure 9: Remyelination potential of Schwann cells grafted into the demyelinated spinal cord. (A and B) Double detection at the lesion site, of P0 (red) and GFP (green) on horizontal sections of the spinal cords grafted with Ct-SC (A) and STX-SC (B) 28 d.p.t. While P0+ myelin profiles were observed in both groups, GFP+/P0+ myelin profiles (arrows, exogenous Schwann cells) were detected in the STX-SC grafted animals only (B). Insert in B illustrates a higher magnification of the boxed area. (C) Quantification of the total P0+ area indicated no difference in global remyelination by Schwann cells between the control and STX groups. Remyelination by the transplanted Schwann cells (P0+/GFP+) occurred in the STX-SC group only, the majority of Schwann cell remyelination being performed by the endogenous cells (P0+/GFP−). At the ultrastructural level, GFP was detected with Bluo-Gal precipitates (D, black precipitates). A close up of the boxed area illustrates the presence of a basal lamina (arrow) around the compact myelin. The poor preservation of myelin is due to the stringent immuno-labelling conditions to detect GFP. Immuno-labelling for MOG (blue), Caspr (red) and GFP (green) shows that STX-SC are associated with Caspr+ internodes (red). (E) Triple immuno-labelling illustrates STX-SC (green) associated with nodal Nav (blue) and paranodal Caspr (red) (F). P0+ myelin profiles (blue) produced by STX-SC (green) are associated with Caspr+ paranodes (red) (G).
Mentions: Remyelination of LPC-induced lesions starts at 7 d.p.t. and is nearly completed by 28 d.p.t. (Gout et al., 1988; Jeffery and Blakemore, 1995). It is achieved by endogenous oligodendrocyte precursors but also by Schwann cells (reviewed in Zujovic et al., 2007). We first investigated whether transplanted Ct-SC and STX-SC participated in the lesion repair by producing newly formed myelin and whether ectopic expression of PSA could promote this process. The relative abundance of peripheral versus CNS remyelination within the margins of the lesion, was evaluated by immunohistochemistry for P0 (Fig. 9A and B) and MOG (Fig. 9E and G) to detect peripheral and central myelin, respectively. Quantification of P0 (Fig. 9F) and MOG immunoreactivity in the lesion showed equal amounts of total peripheral (Ct-SC = 0.09 ± 0.03; STX-SC = 0.13 ± 0.08 µm2/µm2 of lesion) and central (Ct-SC = 0.301 ± 0.07; STX-SC = 0.310 ± 0.08 µm2/µm2 of lesion) myelin for each group. To establish the relative contribution of Ct-SC and STX-SC to exogenous remyelination, the amount of P0 co-localized with GFP was quantified. GFP+/P0+ myelin-like figures were detected in STX-SC grafted animals only (Fig. 9B) and represented 5% of the global Schwann cell remyelination process (Fig. 9C). These data indicate that only STX-SC, which arrived in the lesion faster, were able to out-compete endogenous myelin-forming cells for remyelination. The presence of exogenous newly formed myelin was further confirmed by electron microscopy revealing the presence of GFP with BluoGal precipitates. Figure 9D illustrates the presence of BluoGal precipitates in myelin with specific peripheral myelin features such as the 1:1 association of Schwann cell with axon and the presence of a basal membrane around the compacted myelin (Fig. 9D, inset). Finally, we questioned the functionality of STX-SC-derived myelin focusing on the ability to re-assemble paranodal Caspr and nodal voltage-gated Nav proteins, as endogenous Schwann cells do (Black et al., 2006). Immunodetection of Caspr, and/or Nav in association with P0 and GFP, showed that P0+/GFP+ STX-SC internodes were clearly associated with Nav channels flanked by Caspr+ paranodes at the node level (Fig. 9E–G). STX-SC lead to the reconstruction of well-organized internodes on host axons throughout the lesion with a very similar expression pattern to that observed for endogenous Schwann cells.Figure 9

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