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FBXL4 disease is excessive mitophagy resulting in too few mitochondria, so the primary biomarker is mitochondrial mass - Mitochondrial transplantation could demonstrate disease rescue!
When mutated, the FBXL4 gene causes one of many forms of Mitochondrial Disease which can realize with Leigh Syndrome symptoms [1]. Unlike most genes causing Leigh Syndrome, the mechanism of FBXL4 disease is defined and has recently spurred significant interest worldwide [2] [3] [4]. FBXL4 disease is unique from other forms of Leigh Syndrome in that the mitochondria are suspected functional [5], there are just too few total mitochondria due to excessive mitophagy [2] [3] [4].
While FBXL4-deficient cells would require higher dosing and more frequent treatments of mitochondrial transplantation than other Mitochondrial Disease forms, it is conceivable that optimal transplantation could demonstrate full rescue of diseased cells, at least for brief periods of time. As mitochondrial mass is the primary marker for FBXL4 disease, measurement of treatment success is clear and easy to obtain. Given FBXL4-deficient cells have functional mitochondria [5], disease rescue may be more attainable than other forms of Mitochondrial Disease where dysfunctional host mitochondria may still persist.
Studying mitochondrial transplantation within the context of FBXL4 deficiency is likely to unlock breakthroughs in core understanding of basal mitophagy.
Questions which FBXL4-deficient cells may answer following mitochondrial transplantation:
In an environment of high basal mitophagy (due to excessive BNIP3/NIX mitophagy receptors), would FBXL4-deficient cells accept the transfer?
What dosing and frequency of delivery is required to normalize mitochondrial mass in FBXL4-deficient cells?
Would the influx of mitochondria alter the ratio of BNIP3 and/or NIX at the outer mitochondrial membrane? Would the rate of mitophagy accelerate or remain constant as a response to introducing more mitochondria?
Are other mitochondrial stress responses, such as the unfolded protein response, activated or suppressed after transfer?
Do cells have an upper-limit on mitophagy (i.e. limit to dispose of mass)? What are the ramifications of reaching that limit?
Given FBXL4-deficient cells have healthy mitochondria, would BNIP3/NIX-mediated mitophagy target the transplanted mitochondria more or less than native mitochondria?
How do above responses vary by tissue type?
While high perinatal mortality rates have previously complicated FBXL4 knockout mice studies, inducible gene knockout techniques would enable a high-yield of adults who then survive several months.
When the trans-placental nutrient supply rapidly stops at birth, newborn mice upregulate autophagy for the first day of life [6]. Given that FBXL4 pathogenic mutations result in loss-of-function [2] [3] [4], FBXL4 Knockout (KO) mice serve as a model of FBXL4 deficiency. FBXL4 KO mice are born with lower mitochondrial mass which make the first 1-2 days of life especially volatile [7]. In triple KO mice models of FBXL4, BNIP3 and NIX, 80% of the population survive into adulthood with no evidence of developmental delay [2]. FBXL4 KO mice which do manage to survive the perinatal period tend to live at least 8 months [7]. If FBXL4 KO was induced in young adult mice, the majority would be expected to survive long enough to sufficiently demonstrate mitochondrial mass improvement following transplantation.
[1] https://umdf.org/leigh-syndrome/
[2] Cao, Y., Zheng, J., Wan, H., Sun, Y., Fu, S., Liu, S., He, B., Cai, G., Cao, Y., Huang, H., Li, Q., Ma, Y., Chen, S., Wang, F., & Jiang, H. (2023). A mitochondrial SCF‐FBXL4 ubiquitin E3 ligase complex degrades BNIP3 and NIX to restrain mitophagy and prevent mitochondrial disease. In The EMBO Journal (Vol. 42, Issue 13). Springer Science and Business Media LLC. https://doi.org/10.15252/embj.2022113033
[3] Elcocks, H., Brazel, A. J., McCarron, K. R., Kaulich, M., Husnjak, K., Mortiboys, H., Clague, M. J., & Urbé, S. (2023). FBXL4 ubiquitin ligase deficiency promotes mitophagy by elevating NIX levels. In The EMBO Journal (Vol. 42, Issue 13). Springer Science and Business Media LLC. https://doi.org/10.15252/embj.2022112799
[4] Nguyen‐Dien, G. T., Kozul, K., Cui, Y., Townsend, B., Kulkarni, P. G., Ooi, S. S., Marzio, A., Carrodus, N., Zuryn, S., Pagano, M., Parton, R. G., Lazarou, M., Millard, S. S., Taylor, R. W., Collins, B. M., Jones, M. J., & Pagan, J. K. (2023). FBXL4 suppresses mitophagy by restricting the accumulation of NIX and BNIP3 mitophagy receptors. In The EMBO Journal (Vol. 42, Issue 13). Springer Science and Business Media LLC. https://doi.org/10.15252/embj.2022112767
[5] Longo, M., Bishnu, A., Risiglione, P., Montava-Garriga, L., Cuenco, J., Sakamoto, K., MacKintosh, C., & Ganley, I. G. (2024). Opposing roles for AMPK in regulating distinct mitophagy pathways. In Molecular Cell (Vol. 84, Issue 22, pp. 4350-4367.e9). Elsevier BV. https://doi.org/10.1016/j.molcel.2024.10.025
[6] Kuma, A., Hatano, M., Matsui, M., Yamamoto, A., Nakaya, H., Yoshimori, T., Ohsumi, Y., Tokuhisa, T., & Mizushima, N. (2004). The role of autophagy during the early neonatal starvation period. In Nature (Vol. 432, Issue 7020, pp. 1032–1036). Springer Science and Business Media LLC. https://doi.org/10.1038/nature03029
[7] Alsina, D., Lytovchenko, O., Schab, A., Atanassov, I., Schober, F. A., Jiang, M., Koolmeister, C., Wedell, A., Taylor, R. W., Wredenberg, A., & Larsson, N. (2020). FBXL4 deficiency increases mitochondrial removal by autophagy. In EMBO Molecular Medicine (Vol. 12, Issue 7). Springer Science and Business Media LLC. https://doi.org/10.15252/emmm.201911659
The FBLX4 gene suppresses basal receptor-mediated mitophagy. Consequently, loss-of-function FBXL4 mutations result in excessive BNIP3/NIX-mediated mitophagy and a form of Mitochondrial Disease characterized by mitochondrial mass reduction.
"Mitochondria targeted via NIX-mediated mitophagy show a much higher degree of functionality compared with mitochondria targeted via Parkin-mediated mitophagy"
"Knockout (KO) of either BNIP3 or NIX restores mitochondrial mass and rescues perinatal lethality of Fbxl4−/− mice, establishing excessive mitophagy as a cause of human disease"
Key Supporting Research
2013 - FBXL4-gene loss of function associated with human mt-DNA depletion
2020 - FBXL4-gene identified as modulator of mitophagy
2023 - FBXL4-gene associated with BNIP3/NIX receptor-mediated mitophagy
2024 - PPTC7 identified as the scaffold by which FBXL4 regulates BNIP3/NIX mitophagy receptors
2024 - Further demonstration that the mitochondria targeted by the BNIP3/NIX mitophagy pathway may be fully "functional"
2025 - Theory proposed for how ubiquitylation and mitochondrial import may provide a "switch" between PINK1/Parkin and NIX/BNIP3 pathways
2025 - Theory proposed that PINK1/Parkin mitophagy provides "Quality Control" while BNIP3/NIX mitophagy provides "Quantity Control"