In vitro Metabolic Studies of Dopamine Synthesis and the Toxicity of L-DOPA in Human Cells

This work is divided in two parts. In the first, I investigated the effects of 2,3-
dihydroxy-L-phenylalanine (L-DOPA) on the metabolism of human tyrosine hydroxylase
(TH)-positive neuronal LUHMES cells. L-DOPA is the gold standard treatment
for Parkinson’s disease (PD) and its effects on cellular metabolism are controversial.
It induced a re-routing of intracellular carbon supplies. While glutamine contribution
to tricarboxylic acid (TCA) cycle intermediates increased, glucose contribution to the
same metabolites decreased. Carbon contribution from glucose was decreased in lactate
and was compensated by an increased pyruvate contribution. Pyruvate reacted
with hydrogen peroxide generated during the auto-oxidation of L-DOPA and lead to
an increase of acetate in the medium. In the presence of L-DOPA, this acetate was taken
up by the cells. In combination with an increased glutamate secretion, all these results
seem to point towards a mitochondrial complex II inhibition.
In the second part of this work, I studied and compared dopamine (DA)-producing
in vitro systems. First, I compared gene and protein expression of catecholamine (CA)-
related genes. Then, I performed molecular engineering to increase TH expression in
LUHMES and SH-SY5Y cells. This was sufficient to induce DA production in SH-SY5Y,
but not in LUHMES cells, indicating that TH expression is not sufficient to characterize
dopaminergic neurons. Therefore I used SH-SY5Y cells overexpressing TH to study
substrates for DA production. Upon overexpression of aromatic amino acid decarboxylase
(AADC), LUHMES cells produced DA after L-DOPA supplementation. This model
was useful to study L-DOPA uptake in LUHMES cells and I showed that L-DOPA is
imported via large amino acid transporter (LAT). In conclusion, the expression of TH
is not sufficient to obtain a DA-producing cell system and this work opened many and
answered some questions about DA metabolism.