The long term objective of the proposed research is to develop the experimental and therapeutic use of genetically engineered primary astrocytes as vehicles for neurotrophic factor delivery to the brain.
The specific aims of this proposal are to 1) systematically evaluate the level, stability, and autocrine effects of neurotrophic factor expression by primary astrocytes that are genetically engineered to express two related neurotrophins, beta-nerve growth factor (beta-NGF) and brain- derived neurotrophic factor (BDNF) and 2) evaluate the effects of these transgenic astrocytes on the development, plasticity and regeneration of nigral dopaminergic neurons utilizing a rodent model of Parkinson's disease in which the nigrostriatal pathway is unilaterally lesioned with 6-hydroxydopamine. For the proposed studies, constitutive expression of recombinant beta-NGF or BDNF will be conferred to primary type I rat astrocytes in culture using replication defective retroviral vectors, which allow the efficient and unidirectional transfer of cDNA sequences. The level and stability of recombinant beta-NGF and BDNF expression in culture and following transplantation into the adult rat striatum will be assessed at the messenger RNA level by quantitative RNase protection assays using riboprobes specific for beta-NGF or BDNF mRNA. Expression at the protein level will be assessed using an enzyme-linked immunoassay and a rat retinal ganglion cell bioassay for beta-NGF and BDNF proteins, respectively. Autocrine effects of genomic integration and/or expression of the beta-NGF and BDNF transgenes in astrocytes will be assessed by comparing the expression of proteins that are synthesized by normal versus transgenic astrocytes utilizing high resolution two-dimensional gel electrophoresis. Once characterized, the genetically engineered astrocytes will be tested for their neurotrophic effects on developing dopaminergic neurons in vitro and in vivo. The hypothesis to be tested is that BDNF-producing astrocytes selectively influence the development, plasticity and regeneration of mesencephalic dopaminergic neurons. To this end, the effects of BDNF- and NGF-producing astrocytes on the survival and neuronal differentiation of embryonic dopaminergic neurons will be compared in co-culture and following transplantation into the dopamine-denervated striatum of the unilateral 6-hydroxydopamine lesioned rat. In addition, the ability of BDNF-producing astrocytes to promote regenerative sprouting by adult dopaminergic neurons will be evaluated using a partial lesion model of Parkinson's disease in which a portion of nigrostriatal dopamine neurons remains intact. Analysis of neuronal growth, regeneration and function will include histological, biochemical and behavioral indices. The proposed research is designed to assess the utility as well as the limitations of using transgenic astrocytes to deliver recombinant neurotrophic factors to the brain, and to begin to assess the therapeutic potential of transgenic astrocytes for treating neurodegenerative disease.
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| Saland, L C; Cunningham, L A; Su, C et al. (2000) Glial cell line-derived neurotrophic factor in the rat pituitary gland. Brain Res Bull 52:109-13 |
| Quintana, J G; Lopez-Colberg, I; Cunningham, L A (1998) Use of GFAP-lacZ transgenic mice to determine astrocyte fate in grafts of embryonic ventral midbrain. Brain Res Dev Brain Res 105:147-51 |
| Krobert, K; Lopez-Colberg, I; Cunningham, L A (1997) Astrocytes promote or impair the survival and function of embryonic ventral mesencephalon co-grafts: effects of astrocyte age and expression of recombinant brain-derived neurotrophic factor. Exp Neurol 145:511-23 |
| Cunningham, L A; Short, M P; Breakefield, X O et al. (1994) Nerve growth factor released by transgenic astrocytes enhances the function of adrenal chromaffin cell grafts in a rat model of Parkinson's disease. Brain Res 658:219-31 |
