To assess the metabolic adaptation of human neurons to paraquat, we used a foetal human mesencephalic cell line (LUHMES, Lund human mesencephalic) as a model system. These conditionally immortali. The culturing of LUHMES cells in differentiation medium for 7 days resulted in expression of the neuronal marker β3-tubulin in a subset of cells (Figure 1A)
of tyrosine hydroxylase (Figure 1B), a dopaminergic neuronal marker associated
with the differentiation of these cells. We have next analysed the global metabolic changes in differentiated LUHMES cells. The levels of several intermediates in glycolysis were increased in the differentiated cells, suggesting that their glycolytic activity was decreased compared with that in non-differentiated cells (Figure 1C). In addition, the level of pyruvate, a product of glycolysis, was decreased, consistent with the attenuation of glycolysis. This may reflect a decreased energy demand of these cells. Further, the levels of many building blocks of membranes and intermediates in membrane synthesis and degradation, including precursors for lipids synthesi ed by mitochondria, such as ethanolamine and phosphoethanolamine, as well as precursors for endoplasmic reticulum lipid synthesis, such as choline and choline phosphate, were significantly altered in the differentiated LUHMES cells (Figure 1D). Taken together, these findings suggest that membrane remodelling was more prevalent in the differentiated LUHMES cells. This may be a consequence of the speciali ation process and function of the differentiated cells. Further, the differentiation of mesencephalic cells into post-mitotic neurons resulted in increased levels of the neurotransmitter acetylcholine, consistent with the speciali ation of these cells (Figure 1D). Even though we have detected an increase in dopaminergic
lineage markers in differentiated LUHMES cells (Figure 1B), metabolic analysis
failed to detect neurotransmitters such as dopamine that are present in
ed cells can be differentiated in vitro into post-mitotic neuronal cells
We have next investigated the metabolic consequences of the exposure of differentiated LUHMES cells to paraquat. Exposure to increasing concentrations of paraquat for 24 h resulted in decreased cell viability (Figure 1E)
an increase in the expression of heat shock protein 60 (HSP60), a mitochondrial
stress marker (Figure 1F). However, at the minimum paraquat concentration employed in our study (1 μM), no significant effect on cell viability was observed. We have next analysed the metabolic changes associated with the exposure of differentiated cells to paraquat for 24 h. Decreased glycolytic activity was observed following treatment with 1 µM and 10 µM paraquat, as indicated by significantly increased levels of glucose, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate, dihydroxyacetone phosphate, 3-phosphoglycerate and pyruvate. The build-up of glycolytic intermediates may have reflected decreased pyruvate fuelling the TCA cycle, which appeared to be inhibited
as a consequence of mitochondrial impairment. Moreover, decreased levels of NAD+ or direct inhibition of glycolysis by paraquat may have been contributing factors to the decreased glycolytic activity. At 500 µM paraquat, and to a lesser degree, at 100 µM, only minor changes in glycolysis were observed, possibly indicating that the cells were metabolically compromised and had limited glycolytic capacity (Figure 1G). The levels of many TCA cycle intermediates were significantly decreased, suggesting that the cycle was less active
1H). The effects of PQ on both glycolysis and TCA cycle suggests a compromise in
the energy-generating capacity of differentiated LUHMES. A loss of ATP, the
intracellular energy currency, has also been reported following exposure of
differentiated LUHMES to the MPP+, the active metabolite of MPTP. Paraquat has been suggested to interfere with the electron transfer chain, resulting in the formation of O2-. Aconitase, an iron cluster (Fe4S4)-containing enzyme responsible for the conversion of citrate to iso-citrate is readily damaged by oxidative stress Increased citrate levels were observed at 10 µM paraquat, consistent with aconitase inhibition. In addition, the levels of glutamine and many other amino acids were decreased (also see Supplementary Table 1), suggesting an attempt by anaplerotic reactions to replenish TCA cycle intermediates.
We also detected significant decreases in the levels of the redox couple NAD and NADH following paraquat treatment, as well as a decrease in the level of the phosphorylated equivalent, NADP (Figure 1I). NADPH is important for paraquat toxicity by cellular redox cycling, and it drives the formation of superoxide ions (O). Oxidative stress results in DNA damage and activation of poly(ADP-ribose) polymerases (PARPs), enzymes that deplete intracellular NAD+ stores, resulting in the formation of nicotinamide. Nicotinamide levels were not altered, suggesting that either PARP activity or the rate of NAD+ synthesis from nicotinamide was low. It is possible that paraquat directly inhibits NAD+ formation from either tryptophan or nicotinamide. However, there was no indication that either pathway was inhibited. Alternatively, the decreased NAD+ level may have been a consequence of adaptation to an altered energy status (described further below). Depletion of NAD+ was strongly associated with decreased energy metabolism.
We next focused on the induction of oxidative stress markers following paraquat treatment. Cholesterol is an important lipid component of cellular membranes, and it is readily oxidi ed by the reactive oxygen species (ROS) to 7-α/β-hydrocholesterol or 7-ketocholesterol. We observed increases in the levels of oxidi ed cholesterol derivatives, consistent with paraquat-induced oxidative stress to cell membranes. As a consequence of cholesterol degradation by ROS, the levels of markers for cholesterol synthesis, such as lanosterol, lathosterol and 7-dehydrocholesterol, were significantly increased; these changes may represent a cellular response designed to replenish the cholesterol pool (Figure 1J). In addition, levels of the abundant small-molecular-weight antioxidants glutathione (GSH/reduced) and ascorbate were decreased following paraquat treatment, suggesting a reduction in the antioxidant buffering capacity of the paraquat-treated cells (Figure 1K). Next, we examined the effects of paraquat on neurotransmitter levels in LUHMES cells. The levels of several neurotransmitters were altered following paraquat treatment, with the strongest effect observed at the 10 µM concentration (Figure 1L). NAA, NAAG and acetylcholine are excitatory, while GABA is inhibitory. The results of this study suggests that paraquat elicits complex changes favouring an excitatory state. Interestingly, synaptic activity has been shown to promote resistance to oxidative stress, suggesting that these changes may be the part of the antioxidant response to paraquat.