The reduced metabolic ability of patient-derived SLC24A46 mutant fibroblasts
The mitochondrial respiration function was investigated by examining OCR under both basal conditions and drug-induced mitochondrial stress using the Seahorse assay. The OCR was found to be significantly decreased in patient-derived SLC25A46 mutant fibroblasts compared to normal fibroblasts (Fig. 1A). After a detailed analysis, the basal respiration, oxygen consumption for ATP production, maximum oxygen consumption capacity of mitochondria, proton-leaked oxygen consumption, non-mitochondrial respiration, and the spare respiratory capacity in patient-derived SLC25A46 mutant fibroblasts were all lower than that of normal fibroblasts (Fig. 1B).
The MTT assay reflects the metabolic ability of living cells by measuring the proliferation rates of cells. The results of this assay for the mutant and normal fibroblasts showed no apparent difference in the number of living cells between these two types of fibroblasts on Day 2 after cell seeding. However, subsequent to this time point, the normal fibroblasts showed vigorous metabolism and rapid proliferation rate on Day 4, Day 6, and Day 8 (Fig. 1D). Thus, the metabolic ability and cell proliferation rate of SLC25A46 mutant fibroblasts were significantly lower than that of normal fibroblasts (Fig. 1D). The imaging results showed that the cell density of normal fibroblasts was close to 80–90% on Day 8, while it only reached 40–50% in SLC25A46 mutant fibroblasts (Fig. 1C).
Mitochondrial hyper-fusion in patient-derived SLC24A46 mutant fibroblasts with the live-cell nanoscope-3D-SIM imaging system
The decreased metabolic ability of mutant fibroblasts suggested that the mitochondrial function in SLC25A46 mutant fibroblasts has been disturbed. Sanger sequencing results showed a homozygous, missense point mutation (c.1005A > T; p. Glu335Asp) in SLC25A46 mutant fibroblasts (Fig. 2D). To examine whether this mutation causes any changes in mitochondrial morphology, we used a nanoscope − 3D-SIM imaging approach to observe mitochondrial morphology in those two human cells. The images showed that the normal fibroblasts had round or medium length mitochondria (Fig. 2A), while the SLC25A46 mutant fibroblasts showed slender, hyper-fused mitochondria (Fig. 2B). Imaris software (Nikon, Tokyo, Japan) was used to identify and analyze the mitochondria morphology (Fig. 2C). The results showed that the number of mitochondria in SLC25A46 mutant fibroblasts was significantly lower than what was observed in normal fibroblasts. In contrast, the average area and volume of mitochondria in mutant cells were significantly greater than those in normal fibroblasts (Fig. 2E). The comparative analysis of mitochondrial morphology showed aberrant hyper fusion of mitochondria in the patient-derived SLC24A46 mutant fibroblasts.
Severe damage of mitochondrial cristae in patient-derived SLC24A46 mutant fibroblasts
Previously, mitochondrial fusion was considered to facilitate OXPHOS, and an increase of mitochondrial fusion will improve the mitochondrial OXPHOS level [12, 13]. Mediated mitochondrial fusion was therefore regarded as a new therapeutic target for mitochondrial diseases [14, 15]. However, our group found that the highly-fused mitochondria from SLC25A46 mutant fibroblasts resulted in reduced OXPHOS [16]. What is the underlying cause of this rare condition? One possibility is alterations in the cristae, a most important structures of the inner mitochondrial membrane (IMM), which are deemed as the core of ATP production and mitochondrial respiratory function [17, 18]. Therefore, we decided to investigate whether structural defects of mitochondrial cristae lead to decreased OXPHOS.
Using algorithm-based SIM imaging technology previously developed by our team [8], we identified and extracted cristae first, then quantitatively analyzed the mitochondrial cristae for human-derived normal and patient-derived SLC25A46 mutant fibroblasts. The images showed that the mitochondrial cristae structure was visible and abundant in normal fibroblasts (Fig. 3A). In contrast, the cristae structure was damaged or even vanished in SLC25A46 mutant fibroblasts (Fig. 3B). After quantification analysis, the mean cristae number (Fig. 3C), cristae length (Fig. 3D), and cristae area (Fig. 3E) of SLC25A46 mutant fibroblasts all showed a significant lower values than those observed in normal fibroblasts.
A similar tendency of mitophagy was observed in normal and SLC25A46 mutant fibroblasts
Mitophagy is the general process by which the cell removes severely damaged mitochondria, consequently achieving the purpose of “quality control” of mitochondria within living cells [19, 20]. We observed highly-fused mitochondria with severely damaged cristae structures in SLC25A46 mutant fibroblasts. This raised the obvious questions of whether or not these abnormal mitochondria induce mitophagy? Using the SIM image-based mitochondria-lysosome co-location analysis method in living cells [9], we can observe and quantify mitophagy in normal and SLC25A46 mutant fibroblasts (Fig. 4C).
Our results demonstrate that only slight levels of mitophagy are occurring in both of these cell lines (Fig. 4A, Fig. 4B). After quantitative analysis, there was no statistically significant difference in the value of mitochondrial - lysosome co-location between the two cell lines. Western Blot also confirmed that the values of the LC3-II/LC3-I ratio were comparable between normal and SLC25A46 mutant fibroblasts (Fig. 4d), which was consistent with the results of the SIM image-based analysis method. In addition, with this nanoscope, we can straightforwardly monitor the mitochondrial dynamics and the mitochondria – lysosome interaction dynamics (Fig. 4e).
This novel nanoscope combined with a quantification analysis strategy can not only be used to observe mitochondrial morphology, but also to detect and quantify the damage of structures in sub-mitochondria, assess the extent of mitophagy, and monitor the dynamics of mitochondria and lysosome (Fig. 5). Therefore, this novel approach is a great approach for the observation and etiological diagnosis of mitochondrial damage in patients with mitochondrial disease.