Identification of dopaminergic neuron trophic factor using subtraction hybridization and bioassay

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Using bioassay and subtraction hybridization, we tried to identify the trophic factor for the dopaminergic (DAergic) neurons in the DA-denervated striatum in the rat. Results are following.1. Neural transplantation : Grafted DAergic neurons grew better in the DA-depleted striatum than in the normal striatum. Tyrosine hydroxylase immunoreactivity was higher (x 1.3) and the neurite extension was stronger (x 1.3-1.8) in the DA-depleted striatum.2. Bioassay : Among many fractions, a fraction with molecular weight of 13 to 45 kD in the tissue extract from the DA-depleted striatum enhanced most strongly the survival and neurite extension of the cultured DAergic neurons. This effect was stable with or without astrocyte feeder layers. It also stimulated the neurite genesis in PC12D with the enhancement of the slow activating/long-lasting Ca current. By the treatment of antibodies for NGF and FGF the effect was reduced down to about 50%.3. Antibody : So far antibodies with strong titers have not been obtained yet.4. Gene expression : Using lambdagt 10 as a vector, we got cDNA library from the DA-depleted striatal tissue. After the differential hybridization of these cDNAs with the mRNAs from the intact and DA-depleted striatum we identified 54 plaques whose mRNA expression was much higher in the DA-depleted striatum. The enhancement of mRNA expression was reconfirmed in at least 6 plaques by Sourthern blotting and in situ hybridization. After sequencing these DNAs, three were identified as cytochrome oxidase subunit-I, alpha-globin and ribosomal protein S-8. Other three were found to be unknown substances.Thus, we got several candidates for trophic factor for DAergic neurons. The purification and characterization of these substances are now under the investigation.