THE JOURNAL OF COMPARATIVE NEUROLOGY 354:361-376 (1995) Brain-Derived Neurotrophic Factor-Transduced Fibroblasts: Production of BDNF and Effects

Local delivery of brain-derived neurotrophic factor (BDNF) by genetically modified cells provides the unique opportunity to examine the effects of BDNF on adult dopaminergic and cholinergic neurons in vivo. Primary rat fibroblasts were genetically engineered to produce BDNF. Conditioned media from BDNF-transduced fibroblasts supported embryonic chick dorsal root ganglion neurons as well as rat fetal mesencephalic neurons. BDNF-transduced fibroblasts grafted to the rat brain survived and showed continued mRNA production for at least 2 weeks. The effects of BDNF-transduced fibroblast grafts on the dopaminergic and cholinergic systems were then assessed. BDNF-transduced fibroblasts grafted into the normal intact substantia nigra induced sprouting of tyrosine hydroxylaseand neurofilamentimmunoreactive fibers into the graft. Fibroblast grafts implanted into the normal intact striatum and midbrain as well as the 6-hydroxydopamine-lesioned brain did not induce sprouting of dopaminergic fibers; neither did they affect drug-induced rotational behavior. BDNF-transduced fibroblasts did, however, significantly increase the homovanillic acid/ dopamine ratio when grafted into the normal midbrain. Following transection of the fimbriafornix, BDNF-transduced fibroblasts grafted into the septum were unable to rescue the septa1 cholinergic population, as did nerve growth factor-producing fibroblast grafts. Genetically modified fibroblast grafts may provide an effective, localized method of BDNF delivery in vivo to test biological effects of this factor on the central nervous system. Indexing terms: cholinergic, dopaminergic, neurotrophic factor, NGF, transplantation o 1995 Wiley-Liss, Inc. Neurotrophic factors are present in the central nervous system (CNS) and play important roles in neural development, differentiation, and survival (for reviews, see Barde, 1989; Thoenen, 1991). A family of related neurotrophic molecules, called neurotrophins, whose members affect overlapping as well as distinct populations of neurons, has been recently isolated. Members of the neurotrophin family include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophic factor-3 (NT-31, and neurotrophic factors-4/-5 (NT-4/5). Extensive conservation between species and sequence similarity between members (over 50% homology in amino acid identity) suggest important roles for neurotrophins in the CNS (Leibrock et al., 1989; Maisonpierre et al., 1990; Berkemeier et al., 1991). Neurotrophins exert their effects through specific binding to cell surface receptors that exist in both low (Kd N and high (Kd lO-’l)-affinity forms (Meakin and Shooter, 1991). The low-affinity NGF receptor ( ~ 7 5 ~ ~ ~ ~ ) Accepted August 5,1994 Address reprint requests to Dr. Un Jung Kang, Department of Neurology MC 2030, The University of Chicago, 5841 S. Maryland Avenue, Chicago, IL 60637. o 1995 WILEY-LISS. INC. 362 C.A. LUCIDI-PHILLIP1 ET AL. comprises a 75 kD protein (Chao et al., 1986) and binds all known members in the neurotrophin family. Specificity in binding is afforded by high-affinity binding sites on trk protooncogenes: trkA, trkB, and trkC. Neurotrophic factors appear to mediate their effects by inducing phosphorylation of tyrosine residues on trk molecules (Kaplan et al., 1991). However, there exists a great deal of controversy regarding what receptor components are necessary and sufficient to mediate signal transduction (for reviews, see Bothwell, 1991; Meakin and Shooter, 1992). Although the role of neurotrophic factors in shaping the developing nervous system by their presence in limited amounts in target tissues has been best established (Barde, 1989), neurotrophic factors have also been implicated in maintaining adult neuronal populations. Neurotrophin mRNA and protein as well as receptors for neurotrophins are present in the adult nervous system (Korsching et al., 1985; Shelton and Reichardt, 1986), and adult neurons are able to transport retrogradely IZ5I-NGF (Schwab et al., 1979; Seiler and Schwab, 1984) and lZ5I-BDNF (Wiegand et al., 1991; DiStefano et al., 1992). Neurotrophic molecules promote neuronal survival when exogenously administered in amounts that exceed normal physiological levels. NGF, the best-characterized member of the neurotrophin family, has been extensively studied for its role in the cholinergic septohippocampal model, where administration of exogenous NGF to the axotomized septohippocampal projection rescues up to 90% of the transected septa1 cholinergic neurons (Hefti, 1986; Williams et al., 1986; Kromer, 1987; Gage et al., 1988). A decrease in neurotrophic factors during aging has also been hypothesized to predicate degenerative disorders such as Alzheimer’s disease (AD) and Parkinson’s disease (PD; Appel, 1981; Hefti, 19831, where the loss of dopaminergic neurons of the substantia nigra pars compacta (SNC) is correlated with the major motor disability of PD, and the loss of cholinergic neurons in the basal forebrain is associated with memory loss in AD (Bernheimer et al., 1973; Olton, 1990). Although the etiology and mechanisms underlying the selective degeneration of cholinergic and dopaminergic neuronal populations are unclear, the action of neurotrophic substances in protecting and regenerating damaged neurons has recently received attention for its potential therapeutic role (see Phelps, 1989). BDNF promotes the survival and differentiation of fetal cholinergic neurons of the basal forebrain in vitro (Knusel et al., 1991; Nonomura and Hatanaka, 1992) as well as adult cholinergic neurons in vivo following septohippocamCbAT CM-@-gal CM-BDNF CM-FF FF FFI p-gal FFiBDNF FFINGF FN GFAP NF TH p 7 5 K G F R Abbreviations choline acetyltransferase p-gal conditioned media brain-derived neurotrophic factor-conditioned media uninfected fibroblast conditioned media