Folate and anti-folate metabolism in primary cultures of choroid plexus

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Folate receptor alpha (FR) has a limited normal tissue distribution (kidney, placenta and choroid plexus) and likely serves to concentrate and/or prevent folate loss. CSF folate, for example, is 3-4 times plasma folate. Folate deficiency is associated with neural tube defects and many neurological disorders, such as Alzheimer’s disease, chronic fatigue syndrome, and depression. Anti-folates such as methotrexate (MTX) are known to cause both acute and chronic neurotoxic symptoms. In some protocols an estimated 5-15% of pediatric patients receiving IV and/or intrathecal MTX have had severe acute neurological toxicity, such as seizures or stroke-like symptoms. The extent of permanent sequelae is not yet known. Disruption of CSF folate homeostasis and resulting increased homocysteine and/or other excitotoxic amino acids in the brain have been associated with some of these symptoms. The high density of FR expressed in choroid plexus cells (CP) suggests that they may play a major role in CNS folate homeostasis, and disruption of their function by anti-folates or other toxic drugs or conditions may contribute to neurological symptoms. We are therefore studying FR function and folate/anti-folate transport and metabolism in choroid plexus cells, using methodology previously established for studying FR positive kidney proximal tubule cells. Because there are no normal CP cell lines available, we have used primary cultures of rat pup CP to characterize the expression and function of FR and its role in folate uptake and metabolism. Cultures grown to confluence have predominantly epithelial morphology, and form tight junctions as evidenced by staining for ZO-1 and occludin. Folate receptor expression was confirmed by staining with fluorescein-folic acid (FITC-FA). Similar to kidney proximal tubule cells, 3H-folic acid showed tight binding (Kd sub nanomolar) and FR cycling between cell surface and an internal compartment. Using the physiologically relevant folate, 5-methyltetrahydrofolate (5CH3), accumulation over a range of 1 -2000 nM appeared to be mediated by at least two processes with Km values in the 1-5 nM and 500 nM range. Folic acid (FA) could block the majority of 5CH3 accumulation when the latter was in the low physiological range (1-10 nM), indicating that uptake was predominately FR mediated. However, at 30-50 nM 5CH3, only 50% of uptake was blocked by FA, and in the 100-2000 nM range, almost none. This is consistent with uptake predominately mediated via the reduced folate carrier at these higher concentrations. Additional studies will elucidate the effects of anti-folates on CSF folate economy and folate mediated reactions (e.g. homocysteine metabolism) possibly responsible for neurotoxicity.