PTENα/PTEN-L and mitophagy

Mitophagy, when defined as the selective removal of damaged mitochondria, is one of the sexiest mitochondrial research areas. At the same time, there is little genetic evidence to support the existence of such phenomenon and most studies are carried out under artificial cell culture conditions (Kauppila et al. 2017). Also, I have already lost count of the number of different mitophagy pathways suggested to exist (Williams et al. 2018). For these reasons I have stopped reading the mitophagy literature.

Nevertheless, two papers in my publication feed caught my eye both describing how a new long form of phosphatase and tensin homolog (PTEN-L or PTENα) regulates mitophagy (Wang et al. 2018, Yin et al. 2018). The funny thing is that while Yin et al describe PTEN-L to be necessary for mitophagy, Wang et al. describe the same protein to be a negative regulator of mitophagy. I think this is a fair reflection of the quality of the contemporary mitophagy research.

How these papers come to different conclusions? Well, both papers have many of the common pitfalls in mitophagy research, which are

• Making a subcellular fractionation experiment using only a cytosolic and mitochondrial fractions. Most likely that mitochondrial fractions will have nuclear and microsome contaminations.
• Using the mRNA levels of Tfam, Nrf1 and Ppargc1α as markers for mitochondrial biogenesis. One should test this either using citrate synthase assay, by western or ideally both.
• Using CCCP, a strong protonophore uncoupling all cellular membranes, as a good reflection of mitochondrial damage.
• Using MitoTracker Red to visualize mitochondria under CCCP treatment because CCCP causes the dye to diffuse out from mitochondria into lysosomes (Padman et al. 2013).

I think I will continue ignoring the mitophagy research also in the future.

PS: Being pedantic here, but there is a difference between cytoplasm and cytosol. Cytoplasm is everything between the plasma membrane and nucleus, so it includes mitochondria. Cytosol is everything outside of cellular membranes/vesicles.

References:

Kauppila TES, Kauppila JHK, Larsson NG. Mammalian Mitochondria and Aging: An Update. Cell Metab. 2017. PMID: 28094012

Padman BS, Bach M, Lucarelli G, Prescott M, Ramm G. The protonophore CCCP interferes with lysosomal degradation of autophagic cargo in yeast and mammalian cells. Autophagy. 2013. PMID: 24150213

Wang L, Cho YL, Tang Y, Wang J, Park JE, Wu Y, Wang C, Tong Y, Chawla R, Zhang J, Shi Y, Deng S, Lu G, Wu Y, Tan HW, Pawijit P, Lim GG, Chan HY, Zhang J, Fang L, Yu H, Liou YC, Karthik M, Bay BH, Lim KL, Sze SK, Yap CT, Shen HM. PTEN-L is a novel protein phosphatase for ubiquitin dephosphorylation to inhibit PINK1-Parkin-mediated mitophagy. Cell Res. 2018. PMID: 29934616

Williams JA, Ding WX. Mechanisms, pathophysiological roles and methods for analyzing mitophagy - recent insights. Biol Chem. 2018. PMID: 28976892

Yin Y, Li G, Yang J, Yang C, Zhu M, Jin Y, McNutt MA. PTENα Regulates Mitophagy and Maintains Mitochondrial Quality Control. Autophagy. 2018. PMID: 29969932

Anti-replicative recombinant 5S rRNA molecules can modulate the mtDNA heteroplasmy in a glucose-dependent manner.

There is plenty of literature suggesting the existence of mitochondrial RNA import in metazoa (Kim et al. 2017). Personally, I am not so convinced by this and it seems only some unicellular eukaryotes and plants have mitochondrial RNA import (Schneider 2011). There was recently an excellent review from Gammage et al. where they discussed the possibility of mitochondrial RNA import in metazoan (Gammage et al. 2018).

From the point of view of mitochondrial RNA import, one of the most hotly debated RNAs is 5S rRNA, a well-known structural component of the cytosolic ribosome. Only some single cell eukaryotes still have it in their mitochondrial DNA (mtDNA) (Janouškovec et al. 2017) but other have lost it. Recent structures of the mitochondrial ribosome showed that it doesn’t possess a 5S rRNA (Greber et al. 2015) but the 5S rRNA has actually been replaced by mtDNA encoded tRNA, the type of which depends on the species in question (Rorbach et al. 2016). So, from the point-of-view of the mitochondrial ribosome, there is no reason to import 5S rRNA.

I collected below (most) of the publications studying 5S rRNA import into mitochondria.

Publication	Method	Result
Yoshionari et al. 1994	Northern using mitoplasts	5S rRNA in mitoplasts is protected by RNAase
Magalhães et al. 1998	Northern and RT-PCR of mitochondrial RNA	5S rRNA in mitoplasts is protected by RNAase
Entelis et al. 2001	Northern of mitochondrial RNA	Different version of 5S rRNA imported with different efficiencies
Smirnov et al. 2008	Northern of mitochondrial RNA	Different version of 5S rRNA imported with different efficiencies
Smirnov et al. 2010	Northern of mitochondrial RNA	5S rRNA has mitochondrial import factors
Smirnov et al. 2011	Northern of mitochondrial RNA	More 5S rRNA import into mitochondria in the presence of pre-mRpL18
Mercer et al. 2011	RNAseq (mitochondria vs mitoplasts)	5S rRNA mildly enriched in mitoplasts vs mitochondria
Comte et al. 2013	Northern of mitochondrial RNA	5S rRNA in mitoplasts is protected by RNAase
Towheed et al. 2014	Northern of mitochondrial RNA	5S rRNA in mitoplasts is protected by RNAase and not imported upon membrane depolarization (FCCP)
Loutre et al. 2018	Northern of mitochondrial RNA	5S rRNA enriched in mitoplasts

I won’t to go to the specifics of each paper but when going through all of them I would have liked to see a few important controls:

• Verification of the mitoplast (mitochondria without the outer membrane) purity on western using antibodies against mitochondrial outer membrane, intermembrane space, inner membrane and matrix proteins.
• Show that the cytosolic ribosomes are not anymore attached to the mitochondrial outer membrane.
• Show that the enriched mitochondria are not contaminated by microsomes or endoplasmic reticulum.
• A RNA control which is not imported into mitochondria but would be digested under the experimental RNAse treatment conditions.
• How the ectopic expression of 5S rRNA or its derivatives affects cytosolic ribosome assembly/function and how much this contributing to mitochondrial changes.

The fact that almost all published mitochondrial 5S rRNA studies come from one lab using always the same approach make me a bit wary. The one published study using RNAseq suggested that 5S rRNA is enriched in mitochondria (Mercer et al. 2011) but some unpublished work from Vamsi Mootha’s lab using similar approach suggests 5S rRNA is just a persistent contaminant in mitochondrial preparations (Wolf 2014).

There was recently a publication suggesting that modified 5S rRNA can targeted into mitochondria and function as an anti-replicative molecule to stop the replication of mtDNA molecule carrying a deletions (Loutre et al. 2018). Figure 3 of this paper immediately raises some concerns as one of the negative control, the cytosolic 5.8S rRNA is also among the mitochondrial RNAs. The authors conclude this amount of contamination to be negligible. Next the authors established cell lines with stable expression of 5S rRNA and its variants. It seems weird to me that they never tested how this ectopic expression affects the main function of 5S rRNA, that is the cytosolic translation. I’m just worried that all the effects they detect are secondary to the changes in cytosolic translation.

These cell lines expressing 5S rRNA had also a heteroplasmic mtDNA deletion and the authors wanted to see whether 5S rRNA expression would affect the heteroplasmy levels of this deletion. Well, 5S rRNA and its derivatives had no effect on the heteroplasmy level of mtDNA deletion in these cell lines under normal glucose but showed some changes in glucose-free media. It should be pointed out though, that they carried out multiple comparisons using Student’s t-test so I wouldn’t trust the results.

Some cell clones with stronger ectopic expression of 5S RNA variants did show decrease in the mtDNA deletion levels. However, this could be also caused by the fact that cytosolic translation is affecting thereby affecting the whole cellular function. Again, they did multiple comparisons using Student’s t-test to analyze these results.

All in all, I find the evidence for mitochondrial 5S rRNA weak at best and people should start controlling better how ectopic 5S rRNA expression affects cytosolic translation. Otherwise everything they see might just be secondary to these cytosolic effects.

Reference:

Comte C, Tonin Y, Heckel-Mager AM, Boucheham A, Smirnov A, Auré K, Lombès A, Martin RP, Entelis N, Tarassov I. Mitochondrial targeting of recombinant RNAs modulates the level of a heteroplasmic mutation in human mitochondrial DNA associated with Kearns Sayre Syndrome. Nucleic Acids Res. 2013. PMID: 23087375

Entelis NS, Kolesnikova OA, Dogan S, Martin RP, Tarassov IA. 5 S rRNA and tRNA import into human mitochondria. Comparison of in vitro requirements. J Biol Chem. 2001. PMID: 11551911

Gammage PA, Moraes CT, Minczuk M. Mitochondrial Genome Engineering: The Revolution May Not Be CRISPR-Ized. Trends Genet. 2017. PMID: 29179920

Greber BJ, Bieri P, Leibundgut M, Leitner A2 Aebersold R, Boehringer D, Ban N. Ribosome. The complete structure of the 55S mammalian mitochondrial ribosome. Science. 2015. PMID: 25837512

Kim KM, Noh JH, Abdelmohsen K, Gorospe M. Mitochondrial noncoding RNA transport. BMB Rep. 2017. PMID: 28115039

Loutre R, Heckel AM, Jeandard D, Tarassov I, Entelis N. Anti-replicative recombinant 5S rRNA molecules can modulate the mtDNA heteroplasmy in a glucose-dependent manner. PLoS One. 2018. PMID: 29912984

Magalhães PJ, Andreu AL, Schon EA. Evidence for the presence of 5S rRNA in mammalian mitochondria. Mol Biol Cell. 1998. PMID: 9725900

Mercer TR, Neph S, Dinger ME, Crawford J, Smith MA, Shearwood AM, Haugen E, Bracken CP, Rackham O, Stamatoyannopoulos JA, Filipovska A, Mattick JS. The human mitochondrial transcriptome. Cell. 2011. PMID: 21854988

Rorbach J, Gao F, Powell CA, D'Souza A, Lightowlers RN, Minczuk M, Chrzanowska-Lightowlers ZM. Human mitochondrial ribosomes can switch their structural RNA composition. Proc Natl Acad Sci U S A. 2016. PMID: 27729525

Schneider A. Mitochondrial tRNA import and its consequences for mitochondrial translation. Annu Rev Biochem. 2011. PMID: 21417719

Smirnov A, Tarassov I, Mager-Heckel AM, Letzelter M, Martin RP, Krasheninnikov IA, Entelis N. Two distinct structural elements of 5S rRNA are needed for its import into human mitochondria. RNA. 2008. PMID: 18314502

Smirnov A, Comte C, Mager-Heckel AM, Addis V, Krasheninnikov IA, Martin RP, Entelis N, Tarassov I. Mitochondrial enzyme rhodanese is essential for 5 S ribosomal RNA import into human mitochondria. J Biol Chem. 2010. PMID: 20663881

Smirnov A, Entelis N, Martin RP, Tarassov I. Biological significance of 5S rRNA import into human mitochondria: role of ribosomal protein MRP-L18. Genes Dev. 2011. PMID: 21685364

Towheed A, Markantone DM, Crain AT, Celotto AM, Palladino MJ. Small mitochondrial-targeted RNAs modulate endogenous mitochondrial protein expression in vivo. Neurobiol Dis. 2014. PMID: 24807207

Wolf AR. Leveraging genomic approaches to characterize mitochondrial RNA biology. Doctoral dissertation, Harvard University.

Yoshionari S, Koike T, Yokogawa T, Nishikawa K, Ueda T, Miura K, Watanabe K. Existence of nuclear-encoded 5S-rRNA in bovine mitochondria. FEBS Lett. 1994. PMID: 7508404

Oxygen in mitochondrial disease: can there be too much of a good thing?

Vamsi Mootha and Patrick Chinnery wrote a short letter to the editors discussing whether or not mitochondrial disease patients should be given high-flow oxygen in medical care (Mootha & Chinnery 2018). A recommended read but I wanted to add few references describing also models where mild hypoxia has been shown to have negative effects.

Indeed, a genome-wide Cas9 screen done in Mootha’s lab identified hypoxia response to be protective against oxidative phosphorylation system (OXPHOS) defects (Jain et al. 2016) and even extend the lifespan of mice lacking a OXPHOS complex I subunit Ndusf4 (Ferrari et al. 2017). They also mentioned in the text that fruit flies carrying a mutation in OXPHOS complex II (succinate dehydrogenase, SDH) are sensitive to high oxygen (Walker et al. 2006) as are the Ndusf4 knockout mice (Jain et al. 2016).

On the other hand, the mitochondrial DNA (mtDNA) mutator mouse suffers from an anemia (Ahlqvist et al. 2015) as do many POLG disease patients (Hikmat et al. 2017) so decreasing oxygen levels is unlikely to be helpful. Also in fruit flies, hypoxia worsens many of the observed phenotypes of mitochondrial mutants (Burman et al. 2014, Whelan et al. 2010).

Clearly, as Mootha and Chinnery advocate, we need more randomized trials to better understand whether some specific mitochondrial disease patients would benefit for not having high-flow oxygen given to them.

References:

Ahlqvist KJ, Leoncini S, Pecorelli A, Wortmann SB, Ahola S, Forsström S, Guerranti R, De Felice C, Smeitink J, Ciccoli L, Hämäläinen RH, Suomalainen A. MtDNA mutagenesis impairs elimination of mitochondria during erythroid maturation leading to enhanced erythrocyte destruction. Nat Commun. 2015. PMID: 25751021

Burman JL, Itsara LS, Kayser EB, Suthammarak W, Wang AM, Kaeberlein M, Sedensky MM, Morgan PG, Pallanck LJ. A Drosophila model of mitochondrial disease caused by a complex I mutation that uncouples proton pumping from electron transfer. Dis Model Mech. 2014. PMID: 25085991

Ferrari M, Jain IH, Goldberger O, Rezoagli E, Thoonen R, Cheng KH, Sosnovik DE, Scherrer-Crosbie M, Mootha VK, Zapol WM. Hypoxia treatment reverses neurodegenerative disease in a mouse model of Leigh syndrome. Proc Natl Acad Sci U S A. 2017. PMID: 28483998

Hikmat O, Charalampos T, Klingenberg C, Rasmussen M, Tallaksen CME, Brodtkorb E, Fiskerstrand T, McFarland R, Rahman S, Bindoff LA. The presence of anaemia negatively influences survival in patients with POLG disease. J Inherit Metab Dis. 2017. PMID: 28865037

Jain IH, Zazzeron L, Goli R, Alexa K, Schatzman-Bone S, Dhillon H, Goldberger O, Peng J, Shalem O, Sanjana NE, Zhang F, Goessling W, Zapol WM, Mootha VK. Hypoxia as a therapy for mitochondrial disease. Science. 2016. PMID: 26917594

Mootha VK, Chinnery PF. Oxygen in mitochondrial disease: can there be too much of a good thing? J Inherit Metab Dis. 2018. PMID: 29948481

Walker DW, Hájek P, Muffat J, Knoepfle D, Cornelison S, Attardi G, Benzer S. Hypersensitivity to oxygen and shortened lifespan in a Drosophila mitochondrial complex II mutant. Proc Natl Acad Sci U S A. 2006. PMID: 17056719

Whelan J, Burke B, Rice A, Tong M, Kuebler D. Sensitivity to seizure-like activity in Drosophila following acute hypoxia and hypercapnia. Brain Res. 2010. PMID: 20034480