Generation of heterozygous D620N-mutant VPS35 SH-SY5Y cells
To date, most studies have investigated the p.D620N variant in VPS35 (VPS35D620N) using stable overexpression of VPS35D620Nin vitro and in vivo models. However, the enhanced VPS35 levels in these models may affect retromer functioning as higher or lower levels of VPS35 are shown to correlate with alterations in mitochondrial fragmentation14,15. This motivated us to use CRISPR-Cas9‒mediated genome editing to introduce the p.D620N variant in VPS35 into the human neuroblastoma SH-SY5Y cells widely used in PD research38,44 (Figure 1A). Restriction fragment length polymorphism analysis using the EcoRI enzyme on a 604-bp genomic DNA region surrounding the variant revealed two putative positive clones (Figure 1B). Sanger sequencing validated the presence of the p.D620N variant, created with a GAT to AAT codon change, in only one of two VPS35 alleles, mimicking the heterozygous carrier status seen in patients (Figure 1C). Additionally, immunoblotting showed that the introduction of the p.D620N variant did not affect the expression levels of VPS35 compared to WT cells (Figure 1D). These cell lines were used for the rest of this study.
PINK1-mediated Parkin recruitment to mitochondria is impaired in CCCP-treated VPS35D620N cells
To investigate whether the p.D620N variant in VPS35 affects PINK1/Parkin-mediated mitophagy, we used the protonophore CCCP to induce mitochondrial stress by dissipating the mitochondrial membrane potential (Δψm) and thereby activate PINK1/Parkin-mediated mitophagy21,24,25. We used immunoblotting to investigate PINK1 accumulation over time in WT and VPS35D620N cells upon 10 µM CCCP treatment. As expected, total PINK1 levels increased slightly after 3 hours of CCCP treatment, and PINK1 accumulation was pronounced after 24 hours of CCCP treatment in whole cell extracts and crude mitochondrial fractions of WT cells (Figure 2A, B). This coincided with a decrease in total Parkin levels (Figure 2A, B), likely due to autoubiquitination and increased proteasomal turnover of mitochondrial-bound Parkin45. Total PINK1 levels, however, were substantially lower in the whole extracts and crude mitochondrial fractions of CCCP-treated VPS35D620N cells at both time points compared to WT cells (Figure 2A‒D). Likewise, total Parkin levels remained similar to those in the untreated condition (Figure 2A, B). Of note, VPS35 levels did not change upon CCCP treatment (Figure 2A, B), and VPS35 was present in the crude mitochondrial fraction (Figure 2B), in line with previous reports14,15.
Previous studies have shown a dose-dependent effect of CCCP and thus we questioned whether a higher dose of CCCP would be able to stabilize PINK1 on mitochondria in the VPS35D620N cells. Indeed, 20 µM CCCP led to higher PINK1 levels compared to 10 µM CCCP in WT cells after 24 hours of treatment and marked PINK1 accumulation was now also seen in the VPS35D620N cells (Figure 2C). However, total PINK1 levels in VPS35D620N cells remained significantly lower than those in WT cells (Figure 2C, E). Consistent with the increase in PINK1 levels upon treatment with 20 µM CCCP, proteasomal degradation of Parkin in WT cells also further increased with 20 µM CCCP, which was not observed in VPS35D620N cells (Supplementary figure 1A, B). Of note, 20 µM CCCP demonstrated increased cytotoxicity compared to 10 µM CCCP (Supplementary figure 1C). This data suggests that 10 µM CCCP induces milder damage to mitochondria then 20 µM CCCP and exposes a not-yet-characterized deficit in the VPS35D620N clones.
Next, we used IF to quantify the translocation of cytosolic EGFP-Parkin to mitochondria upon treatment with CCCP, since endogenous Parkin was not detectable in our cells. WT and VPS35D620N cells were transiently transfected with EGFP-Parkin and subsequently treated with 10 µM CCCP for 6 hours and stained for OMM protein TOM20. As expected, mitochondrial depolarization due to CCCP caused translocation of cytosolic EGFP-Parkin to mitochondria in WT cells, as shown by the colocalization between EGFP-Parkin and TOM20 (Figure 2F). Additionally, less Parkin translocation was seen in VPS35D620N cells (± 23% and ±25%) compared to WT cells (± 63%) (Figure 2G). To complement this observation, the colocalization between endogenous PINK1 and TOM20 was determined using IF in WT and VPS35D620N cells upon CCCP treatment (Figure 3A). CCCP-treated VPS35D620N cells showed less colocalization between PINK1 and TOM20 compared to WT cells, and a dose-dependent effect of CCCP was observed (10 µM CCCP: WT median 0.12 vs. clone 1 median 0.04 and clone 2 0.05; 20 µM CCCP: WT median 0.52 vs. clone 1 median 0.36 and clone 2 median 0.35) (Figure 3B). Together, these data suggest that CCCP-induced PINK1 accumulation is hampered, leading to impaired Parkin recruitment onto mitochondria in VPS35D620N cells.
CCCP-induced mitophagy is impaired in VPS35D620N cells
To prove that the hampered PINK1 and Parkin recruitment onto mitochondria upon CCCP treatment does lead to compromised PINK1/Parkin-mediated mitophagy in VPS35D620N cells, we used previously published dual color fluorescence-quenching EGFP-mCherry mitophagy reporter39, which we stably expressed in WT and VPS35D620N cells. Under normal conditions, mitochondria emit both a red and green fluorescence signal, which results in a yellow color (Figure 4A). Mitochondria damaged by CCCP treatment are transported to lysosomes for degradation, and the EGFP fluorescent signal is quenched within this acidic organelle, leaving mainly a red fluorescent signal (Figure 4A, B). At steady state, both WT and VPS35D620N cells primarily showed a yellow reticulated mitochondrial network, with only a few red-only puncta, probably reflecting mitochondria within lysosomes, i.e. mitolysosomes, and there was no significant difference between the cell lines (Figure 4A, B). In contrast, while WT cells showed a substantial increase in mitochondria with a red-only signal, indicative of an activation of mitophagy39, VPS35D620N cells did not display a shift from yellow to red-only mitochondria after 24 hours of 10 µM CCCP treatment (Figures 4A, B). Interestingly, a punctate rearrangement of the mitochondrial network was observed in VPS35D620N cells after CCCP treatment, in which the mitochondrial clumps seemed larger compared to WT cells (Figure 4A, bottom right panel compared to bottom left panel). This suggests that VPS35D620N cells do react to CCCP but experience impairment in PINK1/Parkin-mediated mitophagy.
To confirm this finding, we investigated mitophagy using a different approach by transiently transfecting WT and VPS35D620N cells with EGFP-LC3, a protein marker for autophagosomes46. Mitophagy was induced by 10 µM CCCP treatment for 6 hours, and we subsequently used IF to examine the colocalization between LC3 puncta, which represent autophagosomes, and TOM20 (Figure 4C, arrowheads). CCCP treatment in WT cells led to approximately twice the amount of LC3- and TOM20-positive mitophagosomes compared to VPS35D620N cells (Figure 4D). Moreover, multiple VPS35D620N cells did not form TOM20-positive autophagosomes, a phenomenon rarely seen in WT cells (Figure 4D). Altogether, these results confirm that CCCP-induced mitophagy is impaired in VPS35D620N cells.
VPS35D620N cells accumulate PINK1 in response to mitochondrial depolarization via antimycin A and oligomycin
Next, we questioned if PINK1/Parkin-mediated mitophagy in VPS35D620N cells would be impaired by treatment with two agents that, like CCCP, also lead to substantial mitochondrial depolarization: subcomplex III inhibitor antimycin A and F1F0 ATPase inhibitor oligomycin47. Antimycin A causes a collapse of the proton gradient across the inner mitochondrial membrane by blocking the mitochondrial electron transport chain, whereas oligomycin inhibits the flow of protons through F1F0 ATPase inhibition, leading to a complete Δψm collapse. As shown using immunoblotting, antimycin A (1 µM, 24 hours) alone was not sufficient to stabilize PINK1 levels in WT cells, while treatment with oligomycin (1 µM, 24 hours) did (Figure 5A). As seen with CCCP, the oligomycin-treated VPS35D620N cells showed less accumulation of PINK1 and higher levels of Parkin compared to WT (Figure 5A). Notably, co-incubation with AO caused high PINK1 accumulation and loss of Parkin in both WT and VPS35D620N cells, and in a similar manner. To corroborate this finding, mitochondrial PINK1 accumulation was determined using IF after 24 hours of 1 µM AO treatment (Figure 5B). In agreement with our immunoblotting data, PINK1 colocalized with TOM20 in almost all WT and VPS35D620N cells, and no differences in the level of colocalization were observed between the different cell lines (Figure 5C). Finally, we monitored AO-induced mitophagy using the dual color mitophagy reporter stably expressed in WT and VPS35D620N cells and observed no differences (Figure 5D). Together, these findings show that PINK1/Parkin recruitment and mitophagy can occur in VPS35D620N cells in response to specific kinds of mitochondrial damage. However, the type and/or severity of insult to the mitochondrial membrane potential determines whether or not PINK1/Parkin-mediated mitophagy is initiated in VPS35D620N cells.
Altered mitochondrial membrane potential and response to CCCP treatment in VPS35D620N cells
To investigate whether AO treatment caused a different type of mitochondrial damage than CCCP treatment, we examined the rearrangement of the mitochondrial network upon exposure to these treatments. To do so, we analyzed the TOM20 distribution using IF to study the morphological characteristics of mitochondria including the number, aspect ratio and length of mitochondria in AO- and CCCP-treated cells (Figure 6A‒C). Both treatments caused mitochondrial fragmentation, as evidenced by a substantial increase in mitochondrial particles (Figure 6A) and decreases in aspect ratio (Figure 6B) and mitochondrial length (Figure 6C). However, AO treatment led to more fragmentation than CCCP treatment, as the number of mitochondrial particles was significantly higher (Figure 6A). Additionally, and in line with our previous results, no differences were observed between WT and VPS35D620N cells upon AO treatment. Interestingly, upon CCCP-treatment, the mitochondrial particles appeared less rounded and longer, as reflected by the increase in aspect ratio and length, respectively, in VPS35D620N cells compared to WT cells (Figure 6A-C). These data suggest that mitochondria respond differently to AO and CCCP treatment and that AO causes more severe mitochondrial damage/fragmentation than CCCP. Additionally, the mitochondria in VPS35D620N cells are affected by the treatments, i.e. they display mitochondrial fragmentation, albeit to a lesser extent than in WT cells.
To further explore why VPS35D620N cells are affected by CCCP-induced damage but do not activate PINK1/Parkin mitophagy, we investigated the Δψm collapse upon CCCP treatment. The Δψm collapse triggers mitochondrial fragmentation, PINK1 accumulation on mitochondria and induction mitophagy21,29. Δψm was measured with the cell-permeant fluorescent dye TMRM in WT and VPS35D620N cells over time upon treatment with 10 µM CCCP. Although CCCP treatment rapidly decreased Δψm in both WT and VPS35D620N cells after 1 minute, and Δψm gradually decreased further during the next 19 minutes (Figure 6D), the collapse in Δψm was significantly lower in VPS35D620N cells compared to WT cells at all measured time points. Additionally, VPS35D620N cells exhibited a lower Δψm at resting condition (±25% less) compared to WT cells (Figure 6E). These data reveal that the mitochondrial membrane potential in the mitochondria of VPS35D620N cells is already altered at steady state and point to an altered mitochondrial susceptibility to CCCP treatment.
VPS35D620N cells exhibit increased mitochondrial fragmentation and damage at steady state
Given that our IF data on mitochondrial distribution showed results inconsistent with previous reports about cells (over)expressing VPS35D620N14,15, likely due to resolution limitations, we used TEM to study WT and VPS35D620N cells at steady state and under CCCP-treated conditions (10 µM CCCP for 6 hours). Here we observed that VPS35D620N cells already had smaller, fragmented mitochondria compared to WT cells at steady state (Figure 7A), something that we had not observed with IF, probably due to the different resolutions of these two experimental approaches. CCCP treatment led to mitochondrial fragmentation in WT cells that resembled the mitochondrial phenotype of VPS35D620N cells at steady state (Figure 7A, B). Notably, no further mitochondrial fragmentation was detected in CCCP-treated VPS35D620N cells compared to the mitochondrial fragmentation seen in VPS35D620N cells at steady state (Figure 7B).
Furthermore, five morphologically distinct categories of mitochondria were observed in the various samples (Figure 7C): I) classical healthy mitochondria with well-defined cristae, II) swollen mitochondria with defined cristae and dark in content, III) mitochondria with unclear, partially visible cristae, IV) mitochondria with very dark content and no visible cristae, and V) aberrant mitochondria with remnants of cristae and light in content. We quantified the proportion of these categories in WT and VPS35D620N cells at steady state and after CCCP treatment. At steady state, most mitochondria (~84%) in WT cells were category I, and the remainder were category II (~9%) and III (~5%). In contrast, in VPS35D620N cells at steady state, a large fraction (~45%) of the mitochondria were in category II, and we observed significantly fewer healthy category I mitochondria compared to WT cells (Figure 7D). Upon CCCP treatment, we observed a shift from category I (~60%) to category II (~26%) mitochondria in WT cells, as well as an increase in category IV mitochondria (from ~1% to ~9%). This suggests that category II mitochondria are damaged. Intriguingly, CCCP treatment did not cause a compositional shift in the mitochondrial population of VPS35D620N cells. These data show that VPS35D620N cells at steady state already contain a population of damaged and fragmented mitochondria and, in agreement with our other results, confirm that this population of mitochondria does not respond further to CCCP treatment.