(J-366) Altered cholesterol trafficking increases glycolytic metabolism and drastically reduces barrier integrity in the blood-brain barrier

Introduction:: : Niemann-Pick Disease Type C (NPC) is a rare autosomal recessive disorder of progressive and ultimately fatal neurodegeneration. NPC is caused by a mutation in the intracellular cholesterol trafficking protein NPC1, which leads to endolysosomal cholesterol accumulation and membrane cholesterol depletion. Despite the devastating nature of NPC, there is no cure for this disease and current treatments are limited [1]. Thus, there is a need to further understand NPC pathophysiology.

Brain microvascular endothelial cell (BMEC) dysfunction has been characterized in several common neurological diseases [2]. However, the role of BMEC in NPC remains poorly understood despite the importance of cholesterol in BMEC transport and metabolic homeostasis. In this study, we investigated how NPC1 inhibition impacts BMEC barrier function and metabolism. We hypothesized that membrane cholesterol depletion in NPC-inhibited BMEC decreases barrier integrity and diminishes glucose metabolism.


Materials and Methods:: Induced-pluripotent stem cell (iPSC)-derived BMECs and primary human BMECs were treated with U18666A, a potent NPC1 inhibitor. Both cell types were used to confirm that metabolic changes occur in BMECs regardless of cell source. Mitochondrial potential and number was assessed by staining live cells with tetramethylrhodamine (TMRM) and MitoTracker-Green. Mitochondrial count and potential was quantified with Nikon Elements T2i software and normalized to nuclei count. Changes in extracellular metabolite concentrations over experimental time points were measured using YSI Bioanalyzer 2900. To further examine changes in metabolic fluxes, we analyzed oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using the Seahorse ATP Rate assay before and after treating cells with oligomycin (to inhibit Complex V) and then rotenone/antimycin A (to inhibit Complex III/IV). BMECs were also labeled with heavy isotope glucose and cell lysate was analyzed with liquid chromatography mass spectrometry to determine changes in glucose utilization in downstream metabolic pathways. We also experimentally assessed barrier integrity in iPSC-BMECs with trans-endothelial resistance (TEER) measurements, followed by sodium fluorescein and dextran permeability assays. Tight junction morphology was also assessed with immunofluorescent staining and confocal microscopy while protein levels were analyzed with western blot. Intracellular calcium was assessed by imaging the calcium ion binding agent Fluo-4-AM in live cells seeded on 96-well plates. Following background correction and thresholding, Flou-4 signal was quantified and normalized to nuclei count.


Results, Conclusions, and Discussions:: Results: U18666A-treated BMECs demonstrated a significant reduction in mitochondrial potential without change in MitoTracker signal (Figure 1A) and a 56% reduction in oxygen consumption rate (Figure 1B). Mass spectrometry also showed decreased incorporation of glucose into TCA cycle metabolism. Contrary to our initial hypothesis, extracellular metabolite measurements revealed that U18666A-treated BMECs consumed 15% more glucose and secreted 30% more lactate (Figure 1C), suggesting an increasingly glycolytic phenotype with the NPC1-deficient phenotype. We also found that NPC1 inhibition increased expression of glycolytic genes as well as extracellular acidification rate, further suggesting a glycolytic shift in these cells. U18666A treatment also decreased barrier integrity by 60% but did not alter tight junction protein continuity at cell-cell borders (Figure 1D). Flou-4-AM staining further demonstrated that NPC1-inhibited cells had elevated intracellular levels of calcium (Figure 1E).

Conclusion: These results suggest that NPC1 inhibition in BMEC decreases vascular barrier integrity, increases glycolytic metabolism, and decreases mitochondrial metabolism. Interestingly, tight junction morphology and protein levels did not appear to change significantly with NPC1-inhibition. These functional changes may occur via increased intracellular calcium. Calcium can modulate both metabolism and barrier function [3-5], suggesting that it may play a critical role in dysfunctional phenotype of NPC1-inhibited BMECs.

Discussion: These data suggest that neurovascular dysfunction occurs in NPC1-deficient BMECs, which could be a potential pathogenic mechanism that contributes to disease progression in NPC1. This could therefore motivate the development of novel neurovascular therapeutics to slow disease progression and treat NPC patients.






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