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Precisely control mitochondria with light to manipulate cell fate decision

Patrick Ernst, Ningning Xu, Jing Qu, Herbert Chen, Matthew S. Goldberg, Victor Darley-Usmar, Jianyi J. Zhang, Brian O’Rourke, Xiaoguang Liu, Lufang Zhou

Posted on: 14 June 2019

Preprint posted on 29 May 2019

Article now published in Biophysical Journal at http://dx.doi.org/10.1016/j.bpj.2019.06.038

Using optogenetics, the ‘method of the decade’, to depolarise mitochondria.

Selected by Amberley Stephens

Background

The methods used to alter cellular pathways or investigate the role of a protein commonly include overexpression or knock-out of the gene of interest, chemical activation or blocking of a receptor/ion channel/protein-protein interaction. However, these methods can have unknown downstream effects. Optogenetics offers a potentially cleaner method to study certain mechanisms whereby a light-sensitive protein is utilised. These proteins consist of seven transmembrane domains that form ion channels which open and close upon light stimulation.

Results

The correct localisation signal is needed to target channelrhodopsin to mitochondria

Ernst et al., used optogenetics to study the role of depolarisation of mitochondria and its effect on mitophagy, (the ordered degradation of mitochondria). They first investigated different sequences for targeting the channelrhodopsin (Ch2R) to the mitochondria and found that only a mitochondrial leader sequence copied from a large ABCB10 ATPase binding cassette transporter was able to localise the Ch2R.

Channelrhodopsin can depolarise mitochondria

Depolarisation of distinct areas with a 475 nm LED laser lead to the ChR2 ion channels opening and 80% depolarisation of mitochondria in this region compared to unilluminated control areas (Figure 1). Depolarisation was measured using tetramethylrhodamine, methyl ester (TMRM) fluorescence which decreases as the inner mitochondrial membrane potential (ΔΨm) decreases and TMRM is released into the cell. Light levels had to be moderated as high stimulation (7 mW/mm2) lead to increased cell death of the control cells.

Figure 1, taken from preprint Figure 2. Mitochondrial-targeted optogenetics induces selective mitochondrial depolarization in H9C2 cells expressing ChR2-eYFP. A) Confocal image showing eYFP tagged ChR2 and cells in zone 1 which were illuminated by LED (5 mW/mm2 ) and cells in zone 2 were not exposed to illumination. B+C) Confocal images showing mitochondrial membrane potential (ΔΨm), measured by TMRM fluorescent dye (20 nM), of cells in zone 1 and zone 2 before (B) and after (C) light illumination. D) Normalized ΔΨm in cells in the illuminated (i.e. zone 1) and unilluminated (i.e. zone 2) zones before and after light illumination.

Sustained illumination leads to cell death via the apoptotic pathway

Moderate illumination (0.5 mW/mm2) for 24 hrs lead to cell death of the ChR2-eYFP cells, not the control cells. The authors subsequently investigated the mechanisms of this light-induced ChR2 mediated cell death. Cell death was found to be independent of mitochondrial permeability, transition pore opening and independent of reactive oxygen species production, as cell death was not prevented using inhibitors. An apoptosis and necrosis inhibitor were next tested and these experiments showed that the apoptosis inhibitor, which targets caspases, reduces cell death. This showed that light-induced ChR2 mediated cell death was via the apoptotic pathway.

Overexpression of Parkin induces mitophagy upon illumination

The authors then further investigated what occurs when mitochondria become damaged after sustained light activation by studying the PINK1/Parkin-mediated mitophagy pathway. When mitochondria become damaged the ΔΨm is disrupted and PINK1 can not be internalised for processing and is therefore left externally. Parkin, a ubiquitin ligase, recognises the externalised PINK1 and subsequently targets the mitochondria to the mitophagy pathway. Parkin was overexpressed in HeLa cells and over time (0-24 hrs) and under illumination (0.5 mW/mm2) there was an increase in Parkin and Ch2R-eYFP colocalization, an increase in mitochondrial fragmentation and a decrease in mitochondrial mass. This data, combined with the presence of the autophagosome marker, LC3, and lyososome marker, LysoTracker, indicated that mitophagy was occurring, which was not observed in control cells or unilluminated cells. Surprisingly, although pro-apoptotic effects of increased Parkin expression have been previously reported, the overexpression of Parkin did not lead to an increase in cell survival, which may have been expected due to damaged mitochondria being more efficiently removed.

Preconditioning of mitochondria can enhance cell viability

Corroborating previous studies which have shown that preconditioning of mitochondria can increase cell survival during sustained stress, the authors also observed that preconditioning with low-level illumination (2 hours for 0.2 mW/mm2) increased cell viability from ~40% without preconditioning to ~80% viability after illumination for 6 hours at 4 mW/mm2.

Conclusions

Optogenetics can be successfully used to depolarise mitochondria upon light-activation by directing channelrhodopsin to the mitochondrial inner membrane using a mitochondrial leader sequence from the ABCB10 transporter. This technique will allow targeted investigation into the role of the mitochondrial inner membrane potential (ΔΨm) under physiological conditions and under cell stress/death. Overexpression of Parkin leads to the induction of mitophagy with sustained illumination. Further investigation into the role of Parkin in mitophagy may yield potential ways to moderate it to reduce disease burden.

Why I chose this preprint

I am a great fan of techniques that circumvent the use of protein over-expression or chemical inhibition where we don’t really know what downstream effects may be perturbed in the cell. I also liked that the authors explained how they found a mitochondrial leader sequence that worked. There is often a lot of work that goes on behind the scenes which gets left out of papers that may be beneficial to know.

Questions for Authors

How do you know the ChR2 is definitely at the IMM?

Is it possible to block or mutate the ChR2 channel to determine whether it is definitely a transfer of protons through this channel causing depolarisation?

Can you speculate as to what mechanisms induce this protection of cell viability when mitochondria are preconditioned? Has anyone looked at this?

 

doi: https://doi.org/10.1242/prelights.11293

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1 comment

3 years

Patrick Ernst

Hi, first of all thank you for highlighting this, I’m always glad when people take an interest in my work. Sorry to not address your questions sooner – I only came across this now. I hope you find these answers useful, feel free to reach out if you have any more questions. As to your questions:

1) How do you know the ChR2 is definitely at the IMM?

I know that there are some methods to isolate the IMM, but as far as I know those can be difficult. By far THE biggest piece of evidence we have that ChR2 is in the IMM is the fact that it works as intended. We know from fluorescent imaging and western blotting that ChR2-eYFP expression is colocalized with mitochondria, meaning it is either in/attached to the outer membrane (OMM), in the inter-membrane space, in the IMM, or in the mitochondrial matrix. If ChR2 were expressed in the OMM, inter-membrane space, or matrix (or improperly oriented in the IMM), activation via blue light stimulation would have little to no effect on the membrane potential of the IMM. It feels a bit cheap to just say “because it works” as an explanation, but the statistical likelihood of it working and NOT being in the IMM is low enough that it’s a satisfactory answer for me.

2) Is it possible to block or mutate the ChR2 channel to determine whether it is definitely a transfer of protons through this channel causing depolarisation?

A lot of work has been done to mutate ChR2 in order to change its physical properties (e.g. redshifting the absorption spectrum, increasing photocurrent), it has been shown that mutations can affect ion sensitivity/selectability. Cho et al were able to mutate ChR2 to significantly reduce proton conductance by a factor of ~20, so this is definitely within the realm of possibility.

However, when you take into consideration the ion-specific conductivities of ChR2 as well as comparing the intra- and extra-mitochondrial concentrations of those common ions, it follows that the majority of the depolarizing ion flow is due to protons. Schneider et al looked at ion-specific conduction of ChR2 and found that under (mostly) physiological conditions the majority (~65% by my estimate, Figure 6A) of inward current (both initial and steady-state) was due to protons, and that Ca2+ was somewhat suppressed due to competition with protons. It’s not a perfect comparison, as the ion gradients across the cell membrane will differ from those across the IMM, but it does still suggest higher conductivity of protons over other ions and that higher proton current may inhibit conductivity of other ions. Further, it is well-established that the membrane potential of the IMM is predominantly due to the strong proton gradient across the IMM – the ETC utilizes this in ATP production. It follows that taking an ion channel that preferentially conducts H+ (in circumstances where there is a low H+ gradient and large Na+ or Ca2+ gradients) and then placing it in an environment with a very large H+ gradient and much lower Na+ and Ca2+ gradients would elicit conduction almost entirely made up of protons.

Additionally, while not shown in the manuscript we did investigate possible mitochondrial Ca2+ influx (at the request of a reviewer) and found no significant change to intra-mitochondrial Ca2+, suggesting negligible Ca2+ flux through mitochondrial ChR2 activation.

3) Can you speculate as to what mechanisms induce this protection of cell viability when mitochondria are preconditioned? Has anyone looked at this?

I personally don’t know, to such an extent that it would probably be irresponsible of me to guess anything specific. The core concept is straight-forward enough – submit cells to sublethal stress, and they will adapt against that stress, making them more resilient in the near future. But past that my understanding of it is fairly limited. Luckily mitochondrial preconditioning is something that people have been using and studying for years now, typically in the context of ischemic preconditioning in neurons. Correia et al. provide a review of the topic and detail some potential mechanisms involved. Note, there may be some differences in our case, due to the difference in stressors/mechanisms between ischemic preconditioning and our photostimulation.

I recently graduated and have since left that lab/project but I know they’re currently looking into the specific mechanisms behind the cytoprotective effects we’ve seen, hopefully they find something compelling! Since publishing this paper, we have tested the effects of optogenetic mitochondrial preconditioning in the context of other more common stressors and it did have a protective effect against those as well, so the underlying mechanisms may be more similar than I think.

References:

Y.K. Cho, D. Park, A. Yang, F. Chen, A.S. Chuong, N.C. Klapoetke, E.S. Boyden. Multimensional screening yields channelrhodopsin variants having improved photocurrent and order-of-magnitude reductions in calcium and proton currents. J Bio Chem. 2019;294(11):P3806-3821. doi:10.1074/jbc.RA118.006996

F. Schneider, D. Gradmann, P. Hegemann. Ion selectivity and competition in channelrhodopsins. Biophys J. 2013;105(1):91-100. doi:10.1016/j.bpj.2013.05.042

S.C. Correia, C. Carvalho, S. Cardoso, R.X. Santos, M.S. Santos, C.R. Oliveira, G. Perry, X. Zhu, M.A. Smith, P.I. Moreira. Mitochondrial preconditioning: a potential neuroprotective strategy. Front Aging Neurosci. 2010;2:183. doi:10.3389/fnagi.2010.00138

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