BIOTRANSFORMATION OF THE MPTP ANALOG THYLAMINOPHENYLETHENYL]-1,2,3,6-TETRAHYDROPYRIDINE TO A FLUORESCENT PYRIDINIUM METABOLITE BY INTACT NEUROBLASTOMA-CELLS

The possibility of imaging monoamine oxidase (MAO) containing neurons through the MAO-mediated conversion of the nonfluorescent tetrahydropy ridine compound thylaminophenylethenyl]-1,2,3,6-tetrahydropyridine (t- THP) to the corresponding fluorescent -methyl-4-[4-dimethylaminophenyl ethenyl]pyridinium species (t-P+) was examined with the aid of human n euroblastoma cells (SH-SY5Y). Fluorescence microscopy and fluorescence measurements established the intracellular formation of a fluorescent species with maximal excitation/emission wavelengths of 485/620 and 5 30/620 nm corresponding to the fluorescence characteristics of synthet ic t-P+. An independent assay confirmed the presence of both MAO-A and MAO-B in these cells. As expected, the development of the fluorescenc e was inhibited by both clorgyline (an MAO-A inhibitor) and deprenyl ( an MAO-B inhibitor). Cytotoxic effects, as determined by trypan blue d ye exclusion for viability and by the MTT 4,5-dimethylthiazole-2-yl)-2 ,5-diphenyltetrazolium bromide] assay for mitochondrial integrity, wer e not observed in cells incubated with concentrations of t-THP as high as 10(-3) M for 4 hr. The results from these studies with a neuronal cell line of human origin suggest: (1) that SH-SY5Y cells metabolize a nd, therefore, can be used for study of tetrahydropyridine compounds i n vitro, and (2) that t-THP may be a useful agent to monitor neurodege nerative processes in MAO-rich neurons, including the dopaminergic nig rostriatal neurons that are damaged by the parkinsonian-inducing tetra hydropyrridine MPTP. The potential advantage of using t-THP over relat ed imaging techniques is the possibility of assessing neuronal functio n by an in vivo processing of the reporter molecule rather than by pos tmortem immunofluorescent or formaldehyde-based procedures. (C) 1996 A cademic Press, Inc.