Promoting growth of severed axons after spinal cord injury (SCI) may support restoration of interrupted motor circuits. Damaged axons can grow into an intraspinal Schwann cell (SC) transplant, but they do not become available for integration into circuits because they stall at the sharp SC-astrocyte boundary. We argue that facilitating the intermingling of transplanted SCs with the adjacent astrocytes would render a growth-conducive terrain for axons to exit and grow into the spinal cord. Promoting SC-astrocyte intermingling is addressed in this application. Our pilot data showed that treatment with the antimetabolite, 6-aminonicotinamide (6-AN) results in mingling of SCs with astrocytes. The goal of our proposal is to investigate how 6-AN treatment facilitates SC- astrocyte intermingling. Using a SC-astrocyte confrontation assay, we will test the hypothesis that intermingling of SCs with astrocytes is prevented by inhibition of SC integrin activation by astrocyte aggrecan and ephrinA5 and by strong SC-astrocyte binding via N-cadherin and PSA-NCAM.
In Specific Aim 1, we propose gain-and-loss studies to investigate in astrocytes the role of secreted aggrecan, the surface ligand, ephrin-A5, and the receptors, N-cadherin and FGFR in 6-AN facilitated astrocyte-SC intermingling. The latter two are receptors for the cell-cell binding molecules, N-cadherin (homophyllic binding) and PSA-NCAM, respectively. We will use lentiviral vectors to silence or over express these target molecules. Confocal microscopy will be used to image intermingling cells. Results will be verified in a rat model of spinal cord contusion.
In Specific Aim 2, we propose to investigate the role of SC ?1 integrins in SC-astrocyte intermingling facilitated by 6-AN mediated modulation of astrocyte aggrecan and ephrinA5. We will use lentiviral vectors to silence or over express ?1 integrins and aggrecan and ephrinA5 to determine their relationships and their contributions to SC-astrocyte intermingling. Results will be verified in a rat model of spinal cord contusion. The experiments will expand our understanding of SC-astrocyte intermingling and may reveal potential targets for future approaches to optimize intermingling of transplanted SCs with host spinal cord astrocytes. Also, they may provide fundamental data for developing more effective SC-based repair strategies to build on current clinically used SC transplant-only approaches. In this exploratory proposal, we use a non-viral tool and a model of contusive SCI, the prevalent type of human SCI, to facilitate future translation into the clinic.
A Schwann cell (SC) transplant in the injured spinal cord promotes growth of axons into the transplant but these axons do not exit the transplant environment, preventing their integration in circuits involved in motor function. The axons in a SC transplant stall at a sharp SC-astrocyte boundary. We showed that treatment with 6- aminonicotinamide elicits intermingling of SCs and astrocytes, which could provide the stalled axons a growth- conducive trajectory into the adjacent spinal cord. We propose to investigate mechanisms underlying 6- aminonicotinamide facilitated SC-astrocyte intermingling. The results from our experiments may lead to more effective spinal cord repair approaches building on current clinically tested SC transplant-only approaches.
| Maldonado-Lasunción, Inés; Verhaagen, Joost; Oudega, Martin (2018) Mesenchymal Stem Cell-Macrophage Choreography Supporting Spinal Cord Repair. Neurotherapeutics 15:578-587 |
