A bioluminescent probe for longitudinal monitoring of mitochondrial membrane potential
Authors: Umberto De Marchi
Aurélie Hermant
Nicolas Sambiagio
Elena A Goun
Ghyslain Budin
Arkadiy A Bazhin
Tamara Maric
Riccardo Sinisi
Journal: Nature Chemical Biology (2020)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: Mitochondrial membrane potential; ΔΨm; bioluminescent probe; mitochondria-activatable luciferin; aging-related change
Affiliations: Institute of Chemical Sciences and Engineering, School of Basic Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland., Nestlé Research, Lausanne, Switzerland., Institute of Chemical Sciences and Engineering, School of Basic Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland. .

Abstract:  Mitochondrial membrane potential (ΔΨm) is a universal selective indicator of mitochondrial function and is known to play a central role in many human pathologies, such as diabetes mellitus, cancer and Alzheimer's and Parkinson's diseases. Here, we report the design, synthesis and several applications of mitochondria-activatable luciferin (MAL), a bioluminescent probe sensitive to ΔΨm, and partially to plasma membrane potential (ΔΨp), for non-invasive, longitudinal monitoring of ΔΨm in vitro and in vivo. We applied this new technology to evaluate the aging-related change of ΔΨm in mice and showed that nicotinamide riboside (NR) reverts aging-related mitochondrial depolarization, revealing another important aspect of the mechanism of action of this potent biomolecule. In addition, we demonstrated application of the MAL probe for studies of brown adipose tissue (BAT) activation and non-invasive in vivo assessment of ΔΨm in animal cancer models, opening exciting opportunities for understanding the underlying mechanisms and for discovery of effective treatments for many human pathologies. 

Retracted: No

PubPeer comments:
 Authors claim to have prepared molecular wire conductors from pyridine precursors. In abstract: Such a sequence of chemical reactions results in the formation of gel with two distinct morphologies: spheres and fiber-like matrices. In text: This also means that the regions of local order within the gels cannot be restored after irradiation. This phenomenon can be noticed in transmission electron microscopy (TEM) images of the samples before and after irradiation Directed to Figure S4, supporting information: ![file] The  fibers are the carbon supports of the TEM grid. Samples are hard to tell apart except for imaging conditions- rounded particles in size range 0.5 to 1 micron.  Fibers are visible in (b) at lower contrast. NMR is troublesome, Figure 2: ![file] Authors write: The spectrum of the solvent-free P4VP Py gel sample after irradiation for 6 h is shown in Figure 2c. The increased amount of longer oligomers is manifested by the increased intensity of the polyene =C-H group signals and the diminished intensity of the downfield doublet of the O=C-H group. This doublet signal, at about 9.3 ppm, is still discernible after 6 h of irradiation (Figure 2c), but disappears completely after storing the irradiated sample for 24 h in the dark (Figure 2d). At the same time, the intensity of the signal at 8.77 ppm, corresponding to the HC=N group, increases in the dark as compared to the signal of pyridine (Figure 2c,d). 2c,d which shows the disappearance of the aldehyde- it s barely visible in c and has vanished in d, but are the two spectra at different scales? The pyridine peak height is not shown for readers to evaluate the relative intensity of the 8.77ppm peak, and the scale change between 2a and 2b is not given. The C-H signals from ref 10 (for 5-amino 2,4 pentadienal) that appear are 7.06. 6.92 and 9.13, but the peak at 7.21 (from ref 10) is missing (perhaps suggesting the polymer which has no proton at that position is the species that ring-opens), and the spectrum is cut off around 5.9ppm, so the shifts at 5.55 and 5.45 aren t visible. Neither is the result from ref 18, which gives the C2 proton shift as being in the range 5.8-5.9ppm. Ref 18 also gives the HC=N shift (assigned here to the peak at 8.77) as consistently lower for azapolyenealdehydes (8.46, 8.54 in the structures closely corresponding to formula 2 and 3 in the paper). Ref 18 gives UV VIS absorption peaks at 354 and 390 nm for phenyl derivatives of 2 and 3, and ref 10 gives a peak at 339 nm for 1. These are not visible above background in the absorption spectrum (Figure 1). A solution of the pyridinium salt of a derivative of 3 is described as light yellow in ref 18, so the predicted absorption in Figure 3 may not reflect the real-world behaviour of the system. In figure 1, those curves look like [Rayleigh scattering](http: www.fluortools.com software ae documentation tools scatter) from precipitates, and poly(4-vinyl)pyridine is known to photocrosslink ( [Avadanei, 2009] , largely via the alkyl backbone. Could formation of highly crosslinked scatterers, followed by relaxation, explain the nonspecific absorption spectra rather than the presence of molecular wires? Authors write: The intensity of several peaks and shoulders observed in the fluorescence spectrum of irradiated pyridine depend on the excitation wavelength, and each of them belongs to a different soluble oligomer chain length (Supplementary Materials, Figure S2) Figure S2 simply shows the fluorescence emission spectra of pyridine at three excitation wavelengths- it is unclear how the authors have determined the origin to be the chain oligomer structures 2 to 4. Electronic properties: Authors claim to show ionic conductivity, which becomes electronic after irradiation. The standard measurement technique in the study of solid electrolytes is electrochemical impedance spectroscopy and or the use of ion selective electrodes ( [Huggins, 2002] ). The observed hysteresis may also be explained by the reorientation of dipoles in the applied field ( [Dickens, 1992] ), and power-law dependence of a.c. conductivity on frequency (related to the previous) is so ubiquitous as to be called the [ universal dielectric response ] ( [Jonscher, 1977] ). Figure 6: ![file] Authors write: The conductivity of the gels undergoes remarkable changes upon irradiation, as shown in Figure 6. Thus, after one-hour irradiation at 254 nm, and holding the low-MW P4VP Py gel sample for 10 h in the dark, the conductivity and hysteresis typical of ionic conductivity diminished. After repeating the above irradiation holding in the dark treatment two more times, we found that the dependence of the current on the applied voltage becomes linear, following Ohmu2019s law (Figure 6). This is a direct indication of the electronic conductivity mechanism. Lack of (detectable) hysteresis from such a small signal doesn t prove electronic conductivity. It looks like there is a progressive decrease in overall conductivity, rather than a transition from one to the other. It would be better if authors could show (absence of) an electronic conductivity in the initial sample and rule out relaxation effects via EIS analysis of the electrical modulus. It s also a very low conductivity, 3.6x10^-8 S m after the full sequence (black curve). ITO also emits photoelectrons in UVC light- could a photocurrent lead to a measurement artifact? Could the change be explained by drying of the gel? Loss of volatile pyridine from the device over 30 hours and precipitation of the polymer would reduce ionic conductivity, and also reduce any dipole relaxation contributions to the hysteresis. Composition of the gel is not measured or monitored, in particular the water content that is key to the proposed reaction scheme. Pyridines readily oxidize in ambient conditions, and more so under ultraviolet irradiation. Further, studies of poly(4-vinyl)pyridine crosslinking demonstrate backbone-backbone crosslinking (Avadanei, 2009) as a major photoproduct, and have shown that irradiated films retain pyridine chemistry ( [Harnish, 2005] ) In all, the conclusions of the paper are unsupported by the results presented. Critically, nothing has been done to rule out possible confounding effects, and more conventional alternate explanations have not been addressed- from the carbon support of a TEM grid to the universal dielectric response. The experimental methodology is inappropriate for the objective of the investigation- electronic and ionic conductivity is measured with a variety of well-established techniques such as EIS. Theoretical calculations of absorption spectra or reaction energetics are simply insufficient to prove the generation of a specific product where experimental evidence is so scant (a non-specific absorption spectrum, vast numbers of competing reactions in a condensed phase). , 
 The last author is editor for the special-issue to publish this paper with many problems. This is only paper in the special-issue mdpi.com journal molecules special_issues photochem_aromatic No other paper ,