Multiple sclerosis drug FTY-720 toxicity is mediated by the heterotypic fusion of organelles in neuroendocrine cells

Abstract

FTY-720 (Fingolimod) was one of the first compounds authorized for the treatment of multiple sclerosis. Among its other activities, this sphingosine analogue enhances exocytosis in neuroendocrine chromaffin cells, altering the quantal release of catecholamines. Surprisingly, the size of chromaffin granules is reduced within few minutes of treatment, a process that is paralleled by the homotypic fusion of granules and their heterotypic fusion with mitochondria, as witnessed by dynamic confocal and TIRF microscopy. Electron microscopy studies support these observations, revealing the fusion of several vesicles with individual mitochondria to form large, round mixed organelles. This cross-fusion is SNARE-dependent, being partially prevented by the expression of an inactive form of SNAP-25. Fused mitochondria exhibit an altered redox potential, which dramatically enhances cell death. Therefore, the cross-fusion of intracellular organelles appears to be a new mechanism to be borne in mind when considering the effect of FTY-720 on the survival of neuroendocrine cells.

Neuroprotective Effects of Fingolimod in a Cellular Model of Optic Neuritis

“…Altered levels of mitochondrial proteins were reported in retinal neurons in models of diabetic retinopathy [ 61 ] and glaucoma [ 62 ]. Results from our study are consistent with studies on neuroprotective properties of FTY in other models where FTY improved mitochondrial stability and restored mitochondrial dynamics under oxidative stress conditions [ 20 , 63 , 64 , 65 ]. Our present study did not investigate the changes in mitochondrial function.…”

Vesicle Fusion as a Target Process for the Action of Sphingosine and Its Derived Drugs

“…These merging organelles then increased in size and roundness at 15 min of initiation of the FTY-720 incubation. The changes were confirmed by electron microscopy that additionally showed a dramatic change in the fusion properties of chromaffin granules in the interior of the cellular cytoplasm [64] as seen in Figure 2. The sequence of events indicated that during FTY-720 treatment (Figure 2): (i) chromaffin granules in the cytosol initiate processes of homotypic fusion between themselves, and fissions to form microvesicles, (ii) with a small delay of minutes chromaffin vesicles started to fuse even with mitochondria increasing the size and roundness of these organelles, (iii) these heterotypic fusions ended in the formation of giant mitochondrial round structures containing multiple vesicular dense cores in their interior.…”

“…Our more recent study revealed that FTY-720 not only mimics sphingosine properties in relation to the fusion of vesicles and the plasma membrane during exocytosis but also exhibits new properties in relation to the fusion and fission of other cellular structures such as mitochondria that had not been reported previously [64].…”

“…In a recent paper, we reported that FTY-720 has a complex effect on secretion from neuroendocrine chromaffin cells because, in addition to modifying the amount of quantal release and the frequency of vesicle fusion in cells stimulated by depolarizations, we also observed that the recruitment of vesicles in response to multiple pulses decreases, indicating that late phases of vesicle transport are also altered [64]. The result agrees well with a report in rat astrocytes in culture of a FTY-720 effect on the transport of the vesicles in the interior of the cytosol, limiting the mobility of different types of vesicles, and therefore their access to secretory sites [65].…”

“…Finally, the dramatic changes in mitochondria lead to a decrease of fluorescence of Mitotracker dye intensity and density revealing the impairment of mitochondrial redox function of around 70% at 15 min. of incubation with the drug [64]. This heavy loss of mitochondrial redox potential has a dramatic effect over chromaffin viability as it would be expected, causing 50% of the cells to die within 2 h after incubation with FTY-720 [64].…”