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Lysosomal activity regulates Caenorhabditis elegans mitochondrial dynamics through vitamin B12 metabolism

Wei Wei, Gary Ruvkun

Preprint posted on 20 April 2020 https://www.biorxiv.org/content/10.1101/2020.04.20.049502v1

Take your vitamins! Lysosome mediated vitamin B12 metabolism regulates mitochondrial fission in C. elegans.

Selected by Aakriti Jain

Background:

Mitochondrial fission and fusion events are important for both the segregation of damaged mitochondria for degradation and generation of new mitochondria to meet energetic and biosynthetic demands in particular cells or subcellular locations1. Disrupted mitochondrial dynamics by abnormal fusion and fission events are often associated with age-related disorders, such as neurodegenerative conditions. The dynamin family of guanosine triphosphatases (GTPases) facilitate the balance between fusion and fission events2,3. In C. elegans, mitochondrial fission is mediated by the cytosolic protein, dynamin-related protein-1 (DRP-1), which is recruited to the mitochondrial outer membrane where it constricts and severs the organelle3. Indeed, mutation in DRP-1 disrupts mitochondrial fission in C. elegans muscle cells and leads to tangled mitochondria as opposed to mitochondria organised in a normal periodic pattern (Fig. 1A)4. Wei & Ruvkun use an RNAi screen to identify genes that, when inactivated, suppress fission defects in drp-1 mutant C. elegans.

Key Findings:

Using a genome-scale RNAi screen in drp-1 mutant worms, Wei & Ruvkun found that inactivation of spe-5, a component of the lysosomal vacuolar ATPase suppressed both drp-1 mutation-related lethality and the observed mitochondrial fission defects in C. elegans muscle cells, rescuing approximately 25% of the mitochondria back to a parallel tubular morphology (Fig 1A). Inhibition of the vATPase with bafilomycin A1 (BafA1) or concanomycin A (CMA) suppressed drp-1-associated mitochondrial fission defects and lethality as well. Finally, inhibition of lysosomal function through knockdown of either the master regulator of lysosomal biogenesis, HLH-30 [C. elegans orthologue of transcription factor EB (TFEB)], or LMP-1 and LMP-2 (lysosome membrane proteins) elicited the same suppression of mitochondrial fission defects caused by drp-1 mutation as inhibition of vATPase function. Importantly, lysosomal function didn’t impact mitochondrial fission through lysosome-mediated mitophagy because inhibition of components of the mitophagy pathway (such as, PINK-1, PDR-1 or BEC-1) did not suppress lethality or mitochondrial fission defects in drp-1 mutant worms.

In order to elucidate the mechanism through which lysosomal dysfunction impacted mitochondrial fission, Wei & Ruvkun compared drp-1 mutant viability on different diets. They found that when the dynamin defective worms were fed the OP50 E. coli strain (most commonly used C. elegans diet in the lab), they grew faster and had increased viability than when the worms were fed the HT115 E. coli strain (an RNAse III deficient strain used for RNAi screens). One of the key differences between the two E. coli strains is that the OP50 strain supplies lower amounts of vitamin B12, a micronutrient that C. elegans acquire exclusively from diet. Indeed, using a GFP-based reporter for cellular vitamin B12 levels showed that worms grown on OP50 E. coli had a dietary vitamin B12 deficiency when compared to those on HT115. Supplementation of biologically active forms of vitamin B12 to the E. coli OP50 diet was associated with increased lethality and increased mitochondrial fission defects in drp-1 mutant worms. These observations suggested that vitamin B12 deficiency was associated with abrogation of drp-1 mutation-related fission defects. To understand the link between lysosomal dysfunction and vitamin B12 availability, the authors measured vitamin B12 supply using the same GFP-based reporter upon inhibition of the vATPase. They found that both inhibition with CMA or BafA1 or genetic inactivating mutations of the vATPase decreased the induction of GFP suggesting a vitamin B12 deficiency caused by lysosomal dysfunction.

Figure 1. A. Mitochondrial morphology in muscle cells from wild-type (left), drp-1 mutant (middle) and drp-1 mutant + spe-5 deleted (right) C. elegans. Wild-type animals have tubular parallel pattern, drp-1 mutation leads to fission defects and mitochondrial tangling, and spe-5 deletion added to drp-1 mutation rescues some of the fission defects. Figure taken from Figure 1C of preprint. B. Model describing how lysosomal activity affects mitochondrial fission through vitamin B12 uptake and release and MTR-dependent methionine production. Figure taken from Figure 6I of preprint.

 

Finally, the authors investigated how lysosome dysfunction-mediated vitamin B12 deficiency could cause mitochondrial fragmentation by examining metabolic enzymes which use vitamin B12 as a cofactor – the cytosolic enzyme methionine synthase (MTR; metr-1) and the mitochondrial enzyme methylmalonyl-CoA mutase (MCM, mmcm-1). RNAi silencing of metr-1 recapitulated the mitochondrial fission phenotypes caused by vitamin B12 deficiency. Additionally, metr-1 silencing suppressed lethality in drp-1 mutant worms. Since MTR is required for methionine and S-adenosylmethionine (SAM; an important methyl donor) biosynthesis, deletion of MTR was proposed to induce methionine restriction. To test the effects of lower SAM levels, the authors inactivated SAM synthetase (sams-1) and found that not only were mitochondrial fission defects mitigated in drp-1 mutant animals, but also that mitochondrial fission was increased in wild-type animals. Finally, the authors showed that lysosome dysfunction-mediated methionine restriction triggered an increase in mitochondrial biogenesis, as opposed to decreased mitophagy, as the mechanism through which drp-1 mutation defects were mitigated.

Altogether, these results suggest that lysosomal dysfunction cause a vitamin B12 deficiency, which is associated with decreased activity through MTR (Fig. 1B). Decreased MTR activity leads to decreased methionine and SAM biosynthesis, which, ultimately, is associated with increased mitochondrial fission (in the case of wild-type animals) and a rescue of mitochondrial fission defects (in the case of dynamin mutant animals).

What I like about this preprint:

The authors convincingly show a clear association between lysosomal dysfunction and mitochondrial fission and the preprint is well-written. This study proposes an interesting new link between lysosomal and mitochondrial function through vitamin B12 metabolism. Excitingly, there are many questions that open up. For example, this study raises the possibility that there may be other micronutrients that require lysosome-dependent uptake which are important for the proper function of not just mitochondria, but other organelles as well. Additionally, it will be exciting to elucidate the exact mechanisms through which methionine restriction contributes to the mitochondrial fission process. Finally, it would be interesting to discover if there really is a link between human lysosomal storage disorders and vitamin B12 deficiency, and whether this is likely to be a cause for age-related neurodegeneration.

Questions for the authors:

  1. Is there a reason why the dynamin mutation lethality phenotype is better observed at high temperatures in elegans?
  2. If inhibition of lysosomal function through inhibition or knock-down of proteins outside of the vATPase affects mitochondrial fission, why do you think that the vATPase subunit was the main lysosomal protein that came up in the RNAi screen?
  3. Is the transporter or mechanism through which vitamin B12 leaves the lysosome known?
  4. Do you have any hypotheses as to why S-adenosylmethionine levels would affect mitochondrial fission? Is there a known reason why methionine restriction increases mitochondrial biogenesis?

References:

  1. B. Westermann. Mitochondrial fusion and fission in cell life and death. Nature Rev Mol Cell Biol. 11(12):872-884 (2010).
  2. E. Smirnova, D.L. Shurland, S.N. Ryazantsev, A.M. van der Bliek. A human dynamin-related protein controls the distribution of mitochondria. J. Cell Biol. 142(2):351-358 (1998).
  3. A.M. Labrousse, M.D. Zappaterra, D.A. Rube, A.M. van der Bliek. C. elegans dynamin-related protein DRP-1 controls severing of the mitochondrial outer membrane. Mol. Cell. 4(5):815-826 (1999).
  4. W. Wei & G. Ruvkun. Lysosomal activity regulates Caenorhabditis elegans mitochondrial dynamics through vitamin B12 metabolism. BioRxiv. (2020).

 

Posted on: 6 May 2020

doi: https://doi.org/10.1242/prelights.20223

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