Leflunomide Treatment Does Not Protect Neural Cells following Oxygen-Glucose Deprivation (OGD) In Vitro

Abstract

Neonatal hypoxia-ischemia (HI) affects 2-3 per 1000 live births in developed countries and up to 26 per 1000 live births in developing countries. It is estimated that of the 750,000 infants experiencing a hypoxic-ischemic event during birth per year, more than 400,000 will be severely affected. As treatment options are limited, rapidly identifying new therapeutic avenues is critical, and repurposing drugs already in clinical use offers a fast-track route to clinic. One emerging avenue for therapeutic intervention in neonatal HI is to target mitochondrial dysfunction, which occurs early in the development of brain injury. Mitochondrial dynamics are particularly affected, with mitochondrial fragmentation occurring at the expense of the pro-fusion protein Optic Atrophy (OPA)1. OPA1, together with mitofusins (MFN)1/2, are required for membrane fusion, and therefore, protecting their function may also safeguard mitochondrial dynamics. Leflunomide, an FDA-approved immunosuppressant, was recently identified as an activator of MFN2 with partial effects on OPA1 expression. We, therefore, treated C17.2 cells with Leflunomide before or after oxygen-glucose deprivation, an in vitro mimic of HI, to determine its efficacy as a neuroprotection and inhibitor of mitochondrial dysfunction. Leflunomide increased baseline OPA1 but not MFN2 expression in C17.2 cells. However, Leflunomide was unable to promote cell survival following OGD. Equally, there was no obvious effect on mitochondrial morphology or bioenergetics. These data align with studies suggesting that the tissue and mitochondrial protein profile of the target cell/tissue are critical for taking advantage of the therapeutic actions of Leflunomide.

Keywords: Leflunomide; OPA1; hypoxia-ischemia; mitochondria; mitofusins; neonatal; oxygen-glucose deprivation.

Figure 1

Timeline of Leflunomide experiments. Leflunomide was used as a pretreatment (upper line) or post-treatment following oxygen-glucose deprivation (OGD) in C17.2 cells. Reox: reoxygenation.

Figure 2

High concentrations of Leflunomide are toxic to C17.2 cells. C17.2 cells were treated with varying concentrations of Leflunomide (0–200 µM) for 24 h. (a) Cell nuclei were stained with Hoechst (blue), imaged, and cell number calculated. Inset shows nuclei alone. Scale bar = 25 µm (b) The graph shows fold change in cell number compared with untreated control. Data are expressed as mean ± SD and were analyzed by one-way ANOVA followed by Dunnett’s post hoc test (N = 3, with 3 technical repeats, * p < 0.05).

Figure 3

Leflunomide does not alter cell survival when administered either before or after OGD. (a) C17.2 cells were treated with Leflunomide (50 μM) for 16 h and then subjected to OGD (3 h). Following a subsequent 1 h recovery period, cells were treated with Hoechst, imaged, and quantified. (b) C17.2 cells were exposed to 3 h OGD, followed by Leflunomide treatment (50 μM). After 16 h, the cell number was quantified as above. Both graphs depict fold change in cell number compared with untreated control. Data are expressed as mean ± SD and were analyzed by two-way ANOVA followed by Tukey post hoc test (N = 3, with 3 technical repeats, # p < 0.05 for injury).

Figure 4

Leflunomide pretreatment does not alter mitochondrial morphology in C17.2, regardless of injury. (a) C17.2 cells were treated with Leflunomide (50 μM) for 16 h and/or OGD (3 h). Following a subsequent 1 h recovery period, cells were treated with mitotracker orange and imaged. Scale bar represents 2 μm. (b) Images were quantified for different morphological parameters, including average mitochondrial number, average length, footprint, number of branched networks, and the number of branches per network. Each data point represents a cell color-coded by experiment. Columns represent mean (N = 3 biological replicates, 15–20 technical replicates, * p < 0.05 compared with control).

Figure 5

Leflunomide has limited effects on protein expression in C17.2 cells. HeLa (a) or C17.2 (b–e) cells were treated with Leflunomide (50 μM) for 16 h. (a) HeLa cell lysates were analyzed by Western blot for the expression of MFN2. (b) Lysates from C17.2 cells ± Leflunomide ± OGD were analyzed for the expression of OPA1 and MFN2. (c) MFN2 protein expression was not altered. (d) OPA1 expression increased in control cells (* p < 0.05, one-way ANOVA) after Leflunomide treatment, but this was not maintained following OGD. (e) the ratio of long(L)-OPA1: Short(S)-OPA1 was determined, and although there was a decrease by OGD (two-way ANOVA, * p < 0.05, ** p < 0.01), this was not reversed by Leflunomide treatment (mean ± SD, N = 4).

Figure 6

Leflunomide provokes proton leak in C17.2 cells. C17.2 cells were treated with Leflunomide for 16 h and then subjected to a Seahorse mitochondrial stress test to measure oxygen consumption. (a) Representative traces of treated C17.2 cells in a Seahorse experiment showing mean responses to oligomycin (O), FCCP (F), and rotenone/antimycin A (RA). (b) Graphs of calculated basal respiration, maximal respiration, and proton leak expressed relative to cell number with a scale factor of 200 (N = 3, * p < 0.05, one-way ANOVA).