FBXL4 disease is a unique form of Mitochondrial Disease involving "healthy" mitochondria. FBXL4 disease is further unique in that the mechanism of disease is largely understood, involving excessive BNIP3/NIX-mediated mitophagy, and methods of disease rescue are known.

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 Mitochondrial Diseases in that the mitochondria are suspected functional [5], but consistently undergoing excessive mitophagy [2] [3] [4]. The disease involves reduced mitochondrial fusion capability and fragmented networks which likely cause the mtDNA depletion and mtDNA nucleoid abnormalities [6]. Knockout of BNIP3 and NIX has been shown to rescue FBXL4 disease in pre-clinical models [2] [3] [4].

Clinically, FBLX4 disease can be one of the more severe forms of Mitochondrial Disease. The FBLX4 parent organization has collected a registry of 250+ children diagnosed with the condition showcasing a large spectrum of clinical presentation. The majority of children pass away in the first years of life, but there are also individuals living into their 40's. Common presentation includes lactic acidemia, hypotonia, and developmental delay caused by severe encephalomyopathy associated with progressive cerebral atrophy and variable involvement of the white matter, deep gray nuclei, and brainstem structures [7] [8]. Craniofacial dysmorphism is commonly present [7] [8]. Many individuals are G-tube dependent, yet few require tracheotomy.

FBXL4 disease models are well established and offer a unique environment to manipulate mitochondrial dynamics and morphology with "healthy" mitochondria.

Existing animal models include:

• US-based mice colony with homozygous FBXL4 mutations with correlation to human patients [unpublished]

• Mouse models: FBXL4−/− [2] [9], FBXL4 mutation knock-in [10], and related gene PPTC7−/− [11] [12]

• FBLX4sa12470 zebrafish [12] and fbxl-1(ok3741) C. elegans [13]

Existing human models include:

• Patient fibroblasts of many allele combinations

• iPSC, neural progenitor cells, and cortical neurons [10]

[See citations at bottom of page]

Key FBXL4 Cellular Publications

(Contact hopeforFBXL4@gmail.com for publications detailing clinical case studies)

• 2013 - FBXL4-gene loss of function associated with human mtDNA depletion; Cells involve severe respiratory chain deficiency, reduced mitochondrial membrane potential, hyperfragmented mito networks, and enlarged nucleoids with perinuclear clustering.

  • The American Society of Human Genetics (ASHG) - Mutations in FBXL4 Cause Mitochondrial Encephalopathy and a Disorder of Mitochondrial DNA Maintenance

  • The American Society of Human Genetics (ASHG) - Mutations in FBXL4, Encoding a Mitochondrial Protein, Cause Early-Onset Mitochondrial Encephalomyopathy 

• 2019 - FBXL4 protein associated with promoting mitochondrial fusion; mtDNA depletion and nucleoid abnormalities theorized as result of network fragmentation.

  • Biochimica et Biophysica Acta (BBA) - Characterization of the C584R variant in the mtDNA depletion syndrome gene FBXL4, reveals a novel role for FBXL4 as a regulator of mitochondrial fusion

• 2020 - FBXL4-gene identified as modulator of mitophagy; First knock-out mouse model.

  • EMBO Molecular Medicine - FBXL4 deficiency increases mitochondrial removal by autophagy

• 2022 - Treatment of Dichloroacetate, a PDHc activator, is shown to improve neurologic/muscular function and improve mitochondrial dysfunction and morphology in FBXL4 disease models.

  • Disclaimer - DCA can cause toxic neuropathy with some mitigation found via adjusting dose on the basis of glutathione transferase ζ-1 genotypes.

  • Journal of Clinical Investigation (JCI): Insight - Dichloroacetate improves mitochondrial function, physiology, and morphology in FBXL4 disease models

• 2023 - FBXL4-gene identified as suppressor of BNIP3/NIX receptor-mediated mitophagy.

  • EMBO Journal - A mitochondrial SCF‐FBXL4 ubiquitin E3 ligase complex degrades BNIP3 and NIX to restrain mitophagy and prevent mitochondrial disease

  • EMBO Journal - FBXL4 ubiquitin ligase deficiency promotes mitophagy by elevating NIX levels

  • EMBO Journal - FBXL4 suppresses mitophagy by restricting the accumulation of NIX and BNIP3 mitophagy receptors  

  • Nature: Cell Death and Differentiation - FBXL4 mutations cause excessive mitophagy via BNIP3/BNIP3L accumulation leading to mitochondrial DNA depletion syndrome 

• 2024 - PPTC7 identified as the scaffold by which FBXL4 regulates BNIP3/NIX mitophagy receptors.

  • Life Science Alliance - Dual-localized PPTC7 limits mitophagy through proximal and dynamic interactions with BNIP3 and NIX 

  • Molecular Cell - A mitophagy sensor PPTC7 controls BNIP3 and NIX degradation to regulate mitochondrial mass 

  • EMBO Reports - PPTC7 antagonizes mitophagy by promoting BNIP3 and NIX degradation via SCFFBXL4  

  • Nature: Cell Death and Disease -PPTC7 acts as an essential co-factor of the SCFFBXL4 ubiquitin ligase complex to restrict BNIP3/BNIP3L-dependent mitophagy 

• 2024 - AMPK activation associated with upregulation of PINK1/Parkin mitophagy and downregulation of BNIP3/NIX; Further demonstration mitochondria targeted by the BNIP3/NIX mitophagy pathway may be "functional".

  • Cell Press: Molecular Cell - Opposing roles for AMPK in regulating distinct mitophagy pathways 

• 2024 - A study of HFpEF (Heart failure with preserved ejection fraction) demonstrates FBXL4 inhibits Drp1, preventing mitochondrial fragmentation; FBXL4 overexpression had no noted impact in mice control population.

  • Redox Biology -FBXL4 protects against HFpEF through Drp1-Mediated regulation of mitochondrial dynamics and the downstream SERCA2a 

• 2025 - Theory proposed for how ubiquitylation and mitochondrial import may provide a "switch" between PINK1/Parkin and BNIP3/NIX pathways.

  • Cell Press: Trends in Cell Biology -Diverse routes to mitophagy governed by ubiquitylation and mitochondrial import 

• 2025 - Theory proposed that PINK1/Parkin mitophagy provides "Quality Control" while BNIP3/NIX mitophagy provides "Quantity Control".

  • Cell Press: Molecular Cell - AMPK: Balancing mitochondrial quality and quantity through opposite regulation of mitophagy pathways

Key Tangential Publications

Immune System Involvement in Mitochondrial Diseases / Leigh Syndrome

• 2023 - Leigh Syndrome lesions shown enriched with macrophages of peripheral origin in addition to microglia, brain resident macrophages;  Csf1r inhibition suppresses neurodegenerative lesions (in Ndufs4−/− mice).

  • International Society of Neuropathology - Peripheral macrophages drive CNS disease in the Ndufs4(−/−) model of Leigh syndrome 

• 2025 - Leigh Syndrome Central Nervous System pathology shown (in Ndufs4−/− mice) driven by the monocyte/macrophage innate immune system, not T cells, B cells, or NK cells.

  • Public Library of Science (PLOS) - Disruption of adaptive immunity does not attenuate disease in the Ndufs4(-/-) model of Leigh syndrome 

• 2025 - Demonstration that Mitochondrial Diseases (POLG-mutated mice) have excessive immune responses to infection causing widespread inflammation and tissue damage.

  • Nature: Communications - Caspase-11 drives macrophage hyperinflammation in models of Polg-related mitochondrial disease 

  • Jackson Lab Media Release 

Mitochondrial Transplantation in Nuclear DNA mutations of Mitochondrial Disease

• 2024 - Mitochondrial Transplantation shown to extend lifespan, improve neurological function, and increase energy expenditure in Leigh Syndrome (Ndufs4−/− mice)

  • Nature: Metabolism - Mitochondria transfer-based therapies reduce the morbidity and mortality of Leigh syndrome 

  • Luca Science Media Release 

Hypoxia Treatment for Leigh Syndrome

• Disclaimer: Hypoxia is also known to increase BNIP3/NIX-mediated mitophagy and may be contraindicated in FBXL4 disease.

• 2020 - Leigh Syndrome mice (Ndufs4−/−) exhibit brain tissue hyperoxia which is normalized by hypoxic breathing which has indications to reverse neurological disease; Activation of Hypoxia Inducible Factors (HIFs) is insufficient.

  • Cell Press: Cell Metabolism - Leigh Syndrome Mouse Model Can Be Rescued by Interventions that Normalize Brain Hyperoxia, but Not HIF Activation 

• 2025 - A small molecule drug is shown to counter Leigh Syndrome brain hyperoxia and delay/reverse disease progression (in Ndufs4−/− mice). 

  • Cell Press - HypoxyStat, a small-molecule form of hypoxia therapy that increases oxygen-hemoglobin affinity 

  • News Medical Media Release 

Citations

[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] Sabouny, R., Wong, R., Lee-Glover, L., Greenway, S. C., Sinasac, D. S., Khan, A., & Shutt, T. E. (2019). Characterization of the C584R variant in the mtDNA depletion syndrome gene FBXL4, reveals a novel role for FBXL4 as a regulator of mitochondrial fusion. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 1865(11), 165536. https://doi.org/10.1016/j.bbadis.2019.165536 

[7] Gai, X., Ghezzi, D., Johnson, M. A., Biagosch, C. A., Shamseldin, H. E., Haack, T. B., Reyes, A., Tsukikawa, M., Sheldon, C. A., Srinivasan, S., Gorza, M., Kremer, L. S., Wieland, T., Strom, T. M., Polyak, E., Place, E., Consugar, M., Ostrovsky, J., Vidoni, S., … Zeviani, M. (2013). Mutations in FBXL4, Encoding a Mitochondrial Protein, Cause Early-Onset Mitochondrial Encephalomyopathy. The American Journal of Human Genetics, 93(3), 482–495. https://doi.org/10.1016/j.ajhg.2013.07.016 

[8] Bonnen, P. E., Yarham, J. W., Besse, A., Wu, P., Faqeih, E. A., Al-Asmari, A. M., Saleh, M. A. M., Eyaid, W., Hadeel, A., He, L., Smith, F., Yau, S., Simcox, E. M., Miwa, S., Donti, T., Abu-Amero, K. K., Wong, L.-J., Craigen, W. J., Graham, B. H., … Taylor, R. W. (2013). Mutations in FBXL4 Cause Mitochondrial Encephalopathy and a Disorder of Mitochondrial DNA Maintenance. The American Journal of Human Genetics, 93(3), 471–481. https://doi.org/10.1016/j.ajhg.2013.07.017

[9] 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). <scp>FBXL</scp> 4 deficiency increases mitochondrial removal by autophagy. EMBO Molecular Medicine, 12(7). https://doi.org/10.15252/emmm.201911659 

[10] Chen, Y., Jiao, D., Liu, Y., Xu, X., Wang, Y., Luo, X., Saiyin, H., Li, Y., Gao, K., Chen, Y., Zhao, S.-M., Ma, L., & Wang, C. (2023). FBXL4 mutations cause excessive mitophagy via BNIP3/BNIP3L accumulation leading to mitochondrial DNA depletion syndrome. Cell Death &amp; Differentiation, 30(10), 2351–2363. https://doi.org/10.1038/s41418-023-01205-1  

[11] Niemi, N. M., Serrano, L. R., Muehlbauer, L. K., Balnis, C. E., Wei, L., Smith, A. J., Kozul, K.-L., Forny, M., Connor, O. M., Rashan, E. H., Shishkova, E., Schueler, K. L., Keller, M. P., Attie, A. D., Friedman, J. R., Pagan, J. K., Coon, J. J., & Pagliarini, D. J. (2023). PPTC7 maintains mitochondrial protein content by suppressing receptor-mediated mitophagy. Nature Communications, 14(1). https://doi.org/10.1038/s41467-023-42069-w

[12] Xu, X., Chen, Y., Fei, S., Jiang, X., Zhou, X., Xue, Y., Li, Y., Zhao, S.-M., Huang, Y., & Wang, C. (2025). PPTC7 acts as an essential co-factor of the SCFFBXL4 ubiquitin ligase complex to restrict BNIP3/3L-dependent mitophagy. Cell Death &amp; Disease, 16(1). https://doi.org/10.1038/s41419-025-07463-w 

[13] Lavorato, M., Nakamaru-Ogiso, E., Mathew, N. D., Herman, E., Shah, N., Haroon, S., Xiao, R., Seiler, C., & Falk, M. J. (2022). Dichloroacetate improves mitochondrial function, physiology, and morphology in FBXL4 disease models. JCI Insight, 7(16). https://doi.org/10.1172/jci.insight.156346