Cellular localization and functional significance of CYP3A4 in the human epileptic brain

Abstract

Purpose: Compelling evidence supports the presence of P450 enzymes (CYPs) in the central nervous system (CNS). However, little information is available on the localization and function of CYPs in the drug-resistant epileptic brain. We have evaluated the pattern of expression of the specific enzyme CYP3A4 and studied its co-localization with MDR1. We also determined whether an association exists between CYP3A4 expression and cell survival.

Methods: Brain specimens were obtained from eight patients undergoing resection to relieve drug-resistant seizures or to remove a cavernous angioma. Each specimen was partitioned for either immunostaining or primary culture of human endothelial cells and astrocytes. Immunostaining was performed using anti-CYP3A4, MDR1, GFAP, or NeuN antibodies. High performance liquid chromatography-ultraviolet (HPLC-UV) analysis was used to quantify carbamazepine (CBZ) metabolism by these cells. CYP3A4 expression was correlated to DAPI) condensation, a marker of cell viability. Human embryonic kidney (HEK) cells were transfected with 4',6-diamidino-2-phenylindole (CYP3A4 to further evaluate the link between CYP3A4 levels, CBZ metabolism, and cell viability.

Key findings: CYP3A4 was expressed by blood-brain barrier (BBB) endothelial cells and by the majority of neurons (75 ± 10%). Fluorescent immunostaining showed coexpression of CYP3A4 and MDR1 in endothelial cells and neurons. CYP3A4 expression inversely correlated with DAPI nuclear condensation. CYP3A4 overexpression in HEK cells conferred resistance to cytotoxic levels of carbamazepine. CYP3A4 levels positively correlated with the amount of CBZ metabolized.

Significance: CYP3A4 brain expression is not only associated with drug metabolism but may also represent a cytoprotective mechanism. Coexpression of CYP3A4 and MDR1 may be involved in cell survival in the diseased brain.

Figure 1

CYP3A4 expression in human temporal lobe epilepsy (TLE) and primary brain cell culture. (A) Examples of a typical brain TLE section and Cresyl violet staining are shown. Dysplastic neurons are indicated by arrowheads, whereas dotted line indicates the boundary between abnormal (double asterisks) and adjacent relative normal region (single asterisk). (B) DAB staining revealed CYP3A4 expression in BBB endothelial cells and neurons. In absence of primary Ab no signal was observed. (C) The majority of neurons (NeuN⁺) in the malformed regions (double asterisks) were CYP3A4 positive (see Fig. 2 for quantification). CYP3A4 colocalized with MDR1 at the BBB (arrows) and neurons (arrowheads). (D) CYP3A4 and MDR1 protein expression was evaluated in primary brain endothelial cells (EPI-EC) and astrocytes (EPI-Astro, see Table 1). Note that, in the patients examined, EPI-EC had the higher levels of CYP3A4. We confirmed overexpression of MDR1 in the same cells. Intensity of the bands was normalized by β-actin. Results are expressed as mean ± standard error of the mean (SEM) [two-way analysis of variance (ANOVA)]. The asterisk indicates p < 0.05, control HBMEC set as 100% of CYP3A4 or MDR1. Epilepsia © ILAE

Figure 2

Expression of CYP3A4 in TS and CA brain specimens. CYP3A4 was colocalized with NEUN, GFAP, and MDR1. (A,B) CYP3A4 was expressed at the BBB of tuberous sclerosis (TS) and cavernous angioma (CA) specimens (see Table 1). We also observed different pattern of CYP3A4 expression in parenchymal cells. In particular, TS and CA brains displayed different extents of glial CYP3A4 staining (arrows and arrowheads in B). CYP3A4 and MDR1 colocalized in neurons and BBB. (C) Quantification of NEUN-CYP3A4 positive cells in TLE, TS, and CA. Note that the majority of neurons were positive for CYP3A4. DAPI staining was used to determine the total number of cells in a given section (*p < 0.05). Epilepsia © ILAE

Figure 3

Pattern of CYP3A4 expression in apoptotic and healthy cells. (A–A1) DAPI nuclear condensation (blue) and levels of CYP3A4 expression (green) were quantified (intensity and number of pixels). Two typical examples of healthy (A) and apoptotic (A1) cells are shown. (B) Note the enlarged nuclei (identified by DAPI) indicating diffuse DNA staining in healthy cells expressing CYP3A4 protein (arrowheads). Note the small condensed nuclei (CYP3A4 negative cells, arrows) reflecting apoptosis or irreversible cell damage. (B1) The extent of DAPI nuclear condensation (luminosity/number of pixel) correlates inversely with CYP3A4 expression. (C) Individual values are shown for each data point. No significant overlap between Gaussian distribution curves was observed among apoptotic and healthy neurons. T-test was used, * = p < 0.05. Epilepsia © ILAE

Figure 4

CYP3A4 transfection results in increased carbamazepine metabolism and cell survival. (A) Western blot confirmed CYP3A4 transfection in HEK cells. (A1) HEK-CYP3A4 cells metabolized CBZ to a greater extent compared to nontransfected HEK. Examples of HPLC traces are provided to show the decrease in CBZ level 72 h after treatment. (B) Cell survival was significantly higher in HEK-CYP3A4⁺ compared to HEK cells. Cells were incubated up to 72 h with a toxic amount of CBZ. Micrographs show the mean cell counting over time. Representative images are shown in B1. Results are expressed as mean ± SEM (t-test). The asterisks indicate p < 0.05. Epilepsia © ILAE

Figure 5

Role of EPI-EC and EPI-Glia in CBZ metabolism. (A) EPI-EC, control HBMEC, and EPI-ASTRO were incubated with CBZ up to 72 h. HPLC-UV analysis revealed that EPI-EC metabolized CBZ to a greater extent compared to the other cell types. No significant changes in CBZ levels were measured in the presence of media alone (not shown). (B) The levels of CYP3A4 expression positively correlated with the amount (μg/mL) of CBZ metabolized. Results are expressed as mean ± SEM (t-test). The asterisks indicate p < 0.05. Epilepsia © ILAE