Transplants of Cells Genetically Modified To Express Neurotrophin-3 Rescue Axotomized Clarke’s Nucleus Neurons After Spinal Cord Hemisection in Adult Rats B. Timothy Himes, 1,2 * Yi Liu, 1 Joanna M. Solowska, 1 Evan Y. Snyder, 3 Itzhak Fischer, 1 and Alan Tessler 1,2 1 Department of Neurobiology and Anatomy, MCP Hahnemann University, Philadelphia, Pennsylvania 2 Philadelphia Veterans Administration Medical Center, Philadelphia, Pennsylvania 3 Departments of Neurology, Pediatrics and Neurosurgery, Harvard Medical School, Children’s Hospital, Boston, Massachusetts To test the idea that genetically engineered cells can rescue axotomized neurons, we transplanted fibroblasts and immortalized neural stem cells (NSCs) modified to express neurotrophic factors into the injured spinal cord. The neurotrophin-3 (NT-3) or nerve growth factor (NGF) transgene was introduced into these cells using recom- binant retroviral vectors containing an internal ribosome entry site (IRES) sequence and the -galactosidase or alkaline phosphatase reporter gene. Bioassay confirmed biological activity of the secreted neurotrophic factors. Clarke’s nucleus (CN) axons, which project to the rostral spinal cord and cerebellum, were cut unilaterally in adult rats by T8 hemisection. Rats received transplants of fibroblasts or NSCs genetically modified to express NT-3 or NGF and a reporter gene, only a reporter gene, or no transplant. Two months postoperatively, grafted cells survived at the hemisection site. Grafted fibroblasts and NSCs expressed a reporter gene and immunoreactivity for the NGF or NT-3 transgene. Rats receiving no trans- plant or a transplant expressing only a reporter gene showed a 30% loss of CN neurons in the L1 segment on the lesioned side. NGF-expressing transplants produced partial rescue compared with hemisection alone. There was no significant neuron loss in rats receiving grafts of either fibroblasts or NSCs engineered to express NT-3. We postulate that NT-3 mediates survival of CN neurons through interaction with trkC receptors, which are ex- pressed on CN neurons. These results support the idea that NT-3 contributes to long-term survival of axoto- mized CN neurons and show that genetically modified cells rescue axotomized neurons as efficiently as fetal CNS transplants. J. Neurosci. Res. 65:549 –564, 2001. © 2001 Wiley-Liss, Inc. Key words: neurotransplantation; gene therapy; spinal cord injury; neurotrophin-3; Clarke’s nucleus Administration of neurotrophic factors is one prom- ising strategy for promoting the survival and regeneration of injured spinal and supraspinal neurons. The neurotro- phin family includes brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), and neurotrophin 4/5 (NT-4/5; Lindsay et al., 1994). All neurotrophins bind to a low-affinity neurotro- phin receptor (Rodriguez-Tebar et al., 1990; Squinto et al., 1991) but exert many of their biological effects through high-affinity binding to a family of receptor ty- rosine kinases (trk; Cordon-Cardo et al., 1991; Klein et al., 1991; Ip et al., 1993). Supply of neurotrophic factors appears to be one mechanism by which transplants of embryonic spinal cord enable axotomized brainstem (Bregman and Reier, 1986; Bregman and Kunkel-Bagden, 1988) and spinal cord neu- rons to survive. Injury at the T8 segment in newborn rats causes 40% of Clarke’s nucleus (CN) neurons at L1 to be lost, and the same lesion in adults reduces the number by 30% (Himes et al., 1994). Transplants of embryonic spinal cord, cerebellum, and neocortex enable the axotomized neurons to survive, whereas transplants of embryonic stri- atum are ineffective (Himes et al., 1994). The three tissue types that rescue CN neurons express high levels of NT-3 mRNA at the time of transplantation (Maisonpierre et al., 1990a), but embryonic striatum does not express detect- able NT-3. Several systems have been described in which the selectivity of the trk receptors for their corresponding neurotrophin ligands allows exogenous administration of neurotrophic factors to enhance survival and regeneration of specific CNS neurons. Red nucleus neurons express trkB mRNA (Berkemeier et al., 1991); trkB and trkC, but Contract grant sponsor: Department of Veteran Affairs; Contract grant sponsor: NIH; Contract grant number: NS24707. *Correspondence to: Dr. B. Timothy Himes, Department of Neurobiology and Anatomy, MCP Hahnemann University, 2900 Queen Lane, Philadel- phia, PA 19129. E-mail: bthimes@drexel.edu Received 27 November 2000; Revised 25 April 2001; Accepted 27 April 2001 Journal of Neuroscience Research 65:549 –564 (2001) © 2001 Wiley-Liss, Inc.