The etiology of Parkinson's disease (PD), a neurodegenerative disorder linked to aging, remains unclear. We initially proposed that (N)- methylated beta-carbolinium (MeBC) compounds, metabolic derivatives of physiological indoleamines, may be causative factors. MeBCs structurally mirror N-methyl-4-phenyl-pyridinium (MPP+), the mitochondrially toxic metabolite of N-methyl-4-phenyl-tetrahydro-pyridine, a parkinsonism- inducing contaminant in illicit street drugs. Supporting our proposal, we find that normethyl BCs reported in vivo undergo methylation on the 2- nitrogen and, more surprisingly, the 9(indole)-nitrogen by S- adenosylmethionine-dependent transferase(s) in guinea pig or rat brain. Further, like MPP+, the 7-oxygenated 2-MeBCs and particularly the simple 2,9-Me2BCs are effective mitochondrial respiratory inhibitors, but notably, because they cannot N-deprotonate, only the 2,9-Me2BCs are mitochondrially sequestered like MPP+ (PNAS, 1990). Consistent with these results, certain MeBCs have neurotoxic potencies approaching MPP+ in vitro (PC12 cells) and in vivo (striatal microdialysis or nigral injections in rats); importantly, for simple 2-MeBCs, 9(indole)-methylation markedly enhances toxicity (Science, submitted). The main aim of this application focuses on a key analytical question: are MeBCs -- and especially the unique 2,9-Me2BCs -- present and possibly increased in the human CNS during early or late PD? To answer this we will examine both cerebrospinal fluid and postmortem brain from PD subjects and controls employing HPLC/fluorescence detection; with Dr. Faull's collaboration at UCLA, mass spectrometry will be used for structural and quantitative confirmation. The objective of a 2nd aim is to partially characterize the N-methyltransferase(s) effecting 2-MeBC and 2,9-Me2BC formation in human brain regions, in order to clarify relationships between regional brain MeBC levels and N-methylation activity. In our 3rd aim, to better understand the toxic mechanisms, we will compare selected 2,9-MeBCs, 2-BCs and MPP+ using fetal rat mesencephalic cultures and, in ongoing collaboration with Dr. H. Rollema, in vivo striatal microdialysis in rats. This overall approach should provide definitive answers about the possible involvement of MeBCs in PD.