Downregulation of Pink1 influences mitochondrial fusion-fission machinery and sensitizes to neurotoxins in dopaminergic cells

Abstract

It is now well established that mitochondria are organelles that, far from being static, are subject to a constant process of change. This process, which has been called mitochondrial dynamics, includes processes of both fusion and fission. Loss of Pink1 (PTEN-induced putative kinase 1) function is associated with early onset recessive Parkinson's disease and it has been proposed that mitochondrial dynamics might be affected by loss of the mitochondrial kinase. Here, we report the effects of silencing Pink1 on mitochondrial fusion and fission events in dopaminergic neuron cell lines. Cells lacking Pink1 were more sensitive to cell death induced by C2-Ceramide, which inhibits proliferation and induces apoptosis. In the same cell lines, mitochondrial morphology was fragmented and this was enhanced by application of forskolin, which stimulates the cAMP pathway that phosphorylates Drp1 and thereby inactivates it. Cells lacking Pink1 had lower Drp1 and Mfn2 expression. Based on these data, we propose that Pink1 may exert a neuroprotective role in part by limiting mitochondrial fission.

Keywords: Ceramide; Mitochondrial dynamics; Parkinson; Pink1; Rotenone.

Fig. 1

Pink1 is necessary for a proper mitochondrial activity; (a) The mean relative Pink1 expression estimated using quantitative RT-PCR, normalized to β-actin expression; (b) Mitochondrial activity measured by MTT assay in shPink1, control shPink1 in CAD cells treated with and without C2-ceramide (25 μM); (c) LDH release was examined for control shPink1 in CAD cells treated with and without C2-ceramide (25 μM) at different times. ***p < 0.0001 compared to control cells, n = 3, 16 data were taken per cell line/treatment.

Fig. 2

Absence of Pink1 causes fragmentation of mitochondria. (a) Mitochondrial morphology analysis. (1) Shows original image (superposition of each of the 20 images that compose the stack); (2) 3D projection of the image obtained in (1); (3) Contrast adjust and application of spatial filters of the image obtained in (2); (4) Conversion of the image to binary (8 bits) for further analysis of particles and shape descriptors, as shown in (5). Analysis was done by using Image J software. (b) Representative images of the mitochondria of the M17 cells. Cells were transfected with mito-YFP and treated with rotenone, FK and CCCP. The arrows point fragmented mitochondria. Scale bar: 5 μm; (c) Quantification of mitochondrial morphology in M17 cells. 280–400 cell per group/treatment were evaluated, n = 3. The values are presented as the percent of the total number of counted cells. Statistical studies were made with two-way ANOVA.

Fig. 3

Pink1 modifies the expression of some proteins involved in mitochondrial fusion and fission. Western blot of some of the proteins involved in fusion/fission processes; (a) Representative images of 3 independent experiments. (b) Bars show the relative levels of Drp1; (b) Fis1; (c) Mfn2, and (d) Opa1 by densitometry. ***p < 0.0001 compared to control cells, n = 3.

Fig. 4

Pink1 deficiency affects the fluorescence intensity of some fusion/fission proteins; (a) Mfn1; (b) Fis1 and (c) p-Drp1 in CAD cells. Representative pictures of shPink1 and control cells in the presence and absence of C2-ceramide. There is a greater green intensity corresponding to Fis1 in shPink1 cells (b), whereas the fluorescence intensity of p-Drp1 and Mfn1 is stronger in control cells (a and c). Mitochondria stained with Mitotracker have mainly a perinuclear localization in shPink1 cells and in cells treated with C2-ceramide. Scale bar: 3.2 μm.

Fig. 5

Model of the effects of the downregulation of Pink1 in CAD and M17 cells at mitochondria level. Pink1 absence produces a lower mitochondrial metabolism, leading to cell death when cells are exposed to the toxic effects of C2-ceramide. It is also proposed that the inhibition of the PI3K/Akt pathway by C2-ceramide could regulate the fusion/fission machinery, but this has to be addressed in neurons. Likewise, there is a decrease in the expression of Drp1 and Mfn2 and an increase of Fis1, related to mitochondrial fragmentation.