Targeted expression of a multifunctional chimeric neurotrophin in the lesioned sciatic nerve accelerates regeneration of sensory and motor axons (nerve growth factorybrain-derived neurotrophic factorynerve regenerationysite-directed mutagenesisytransgenic mice)

Peripheral nerve injury markedly regulates expression of neurotrophins and their receptors in the lesioned nerve. However, the role of endogenously produced neurotrophins in the process of nerve regeneration is unclear. Expression of a multifunctional neurotrophin, panneurotrophin-1 (PNT-1), was targeted to the peripheral nerves of transgenic mice by using a gene promoter that is specifically activated after nerve lesion but that is otherwise silent in all other tissues and during development. PNT-1 is a chimeric neurotrophin that combines the active sites of the neurotrophins nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 and binds and activates all known neurotrophin receptors. In adult transgenic mice, PNT-1 was highly expressed in transected but not in intact sciatic nerve. Morphometric analyses at the electron microscopy level showed increased and accelerated recovery of axon diameter of myelinated fibers in crushed peripheral nerves of transgenic mice compared with wild type. Examination of nerve bundles in target tissues indicated accelerated reinnervation of foot pad dermis and flexor plantaris muscle in transgenic mice. Moreover, transected sensory and motor axons of transgenic mice showed faster and increased return of neurophysiological responses, suggesting an accelerated rate of axonal elongation. Importantly, transgenic mice also showed a markedly ameliorated loss of skeletal muscle weight, indicating functional regeneration of motor axons. Together, these data provide evidence, at both the anatomical and functional levels, that neurotrophins endogenously produced by the lesioned nerve are capable of significantly accelerating the regeneration of both sensory and motor axons after peripheral nerve damage. In addition, our results indicate that exogenous PNT-1 administration may be an effective therapeutic treatment of peripheral nerve injuries. Lesion of peripheral nerves may cause permanent denervation with paralysis and disability in humans and represents a challenging problem in neurosurgery. A slow rate of nerve regeneration conspires together with atrophy and degeneration of denervated organs to increase the risk of permanent disability after peripheral nerve injury, emphasizing the importance of rapid and timely reinnervation to optimize organ viability. Axonal repair of mature neurons involves complex molecular and cellular interactions. Neurotrophic factors are secreted polypeptides that control the survival, differentiation, and maintenance of vertebrate neurons (1, 2). These activities of neurotrophic factors have elicited great interest in these molecules as possible therapeutic agents in neurodegenerative diseases and nerve damage. Despite intense research in the neurotrophic factor field, relatively little is known about their physiological roles during peripheral nerve regeneration, although the possible utility of exogenously applied neurotrophic factors to promote and enhance axonal regrowth has been documented. Part of the difficulty in assessing the role of individual neurotrophic factors in nerve regeneration is because of the complex trophic requirements of sensory and motor neurons, which limit the efficacy achieved by any single factor. The neurotrophins are a family of structurally and functionally related neurotrophic factors that play important roles in the development, maintenance, and functional plasticity of many neuronal populations in the central and peripheral nervous systems (3, 4). In vitro and in vivo studies, including gene knock-out experiments, have shown that different subpopulations of peripheral sensory neurons require different neurotrophins for survival and differentiation. Thus, nociceptive and thermoceptive sensory neurons in dorsal root ganglia (DRG) require nerve growth factor (NGF) (5), whereas muscle sensory neurons require neurotrophin-3 (NT-3) (6, 7). Although mice lacking any of the neurotrophins do not show deficits in motor neuron number during development, brainderived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4) are potent survival and trophic factors for intact as well as injured motor neurons in vivo (8–12). Thus, sensory and motor neurons have heterogeneous neurotrophin requirements, and because their axons extend through the same peripheral nerves, functional regeneration after nerve injury is likely to require the concomitant action of different neurotrophins. Expression of both neurotrophins and their receptors is regulated in response to peripheral nerve transection (13), suggesting that they form part of an endogenous mechanism of nerve repair. The functional importance of neurotrophins endogenously produced by the injured nerve in the process of nerve regeneration is, however, unclear. Much effort, on the other hand, has been devoted to study the effects of exogenously applied neurotrophins in lesioned peripheral nerves. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424y98y955269-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: PNT-1, pan-neurotrophin-1; DRG, dorsal root ganglia; NGF, nerve growth factor; NT, neurotrophin; BDNF, brain-derived neurotrophic factor; CAP, compound action potential; CAT, chloramphenicol acetyltransferase. †Present address: Division of Biochemistry, Biomedical Research Center, Osaka University Medical School, Osaka 565, Japan. ‡Present address: The PNI Unit, The Royal National Orthopedic Hospital, Brockley Hill, Stanmore, Middlesex HA7 4LP, United Kingdom. \Present address: Department of Developmental Biology, Uppsala University, Uppsala, Sweden. ††To whom reprint requests should be addressed. e-mail: carlos@ cajal.mbb.ki.se.