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Bioluminescent Genetically Encoded Glutamate Indicator for Molecular Imaging of Neuronal Activity

E. D. Petersen, E. L. Crespo, G. G. Lambert, A. Torreblanca Zanca, R. Orcutt, U. Hochgeschwender, N. C. Shaner, A. A. Gilad

Posted on: 3 July 2021 , updated on: 10 August 2023

Preprint posted on 17 June 2021

Article now published in ACS Synthetic Biology at http://dx.doi.org/10.1021/acssynbio.2c00687

Always look on the bright side of the brain: @Petersen et al. engineer BLING, the first bioluminescent indicator for neurotransmitter imaging

Selected by Joanna Zell, Kristina Kuhbandner

Updated 10 August 2023 with a postLight by Kristina Kuhbandner

After publication as preprint in June 2021, a peer-reviewed version of the article „Bioluminescent genetically encoded glutamate indicators for molecular imaging of neuronal activity” has now been published in ACS Synthetic Biology. Here, Peterson and colleagues describe a new, first-of-its-kind tool to investigate neuronal activity based on a bioluminescent – instead of a fluorescent – genetically encoded neurotransmitter indicator. This innovation has enormous potential to revolutionize neuroscience research among others by facilitating non-invasive imaging of deep brain structures.

In the peer-reviewed version, the authors added some valuable data which showcase the capacity of the tool: in a proof-of-concept experiment using an acute seizure model, they demonstrated the applicability of their construct in vivo. Seizure induction was accompanied by an increase in bioluminescence that could be measured through the rat brain and skull. With this experiment the authors also addressed two of the questions raised in the preLight, namely if BLING has already been used in vivo and whether the signal is strong enough to be detected through millimetres of tissue. Furthermore, they report about their efforts to improve the tool by creating variants with a range of different properties including varying background luminescence and neurotransmitter response. Lastly, the researchers provide evidence that the bioluminescence-based biosensors are capable to produce sufficient signal which could in the future also be employed to activity-dependently stimulate light-sensitive proteins such as opsins in optogenetics.

Congratulations to the Gilad lab on their fantastic work! We are very excited to follow the future applications of this new bioluminescent indicator.

Background

The development of optical biosensors allows scientists to study neuronal activity by investigating changes in membrane voltage or neurotransmitter concentrations. However, most of these sensors are based on fluorescence and thus require an excitation light source. This presents some problematic constraints on optical biosensor technology, for example (i) limited imaging depth in the brain due to light scattering, (ii) cell damage by the laser beam, (iii) need for invasive hardware implantation such as fibres for excitation, and (iv) photobleaching of fluorescent indicators. One solution to circumvent these problems is the use of bioluminescence (Fig. 1B).

Bioluminescence, or “biologically” induced light, is produced by enzymes such as luciferase, which is found in fireflies, beetles, worms and marine creatures. Luciferases oxidize their substrate luciferin or coelenterazine (CTZ) to a luminescent molecule (coelenteramide) leading to the emission of light (1). In recent years, researchers have developed numerous synthetic luciferase variants. These include split luciferases, which are “turned on” when two halves come together, for example in response to an analyte. Although bioluminescence is already used for imaging purposes, such as visualizing calcium dynamics (2), no bioluminescent neurotransmitter indicator has been described to date.

In this preprint, the authors aimed to develop a genetically encoded bioluminescent neurotransmitter indicator. Petersen et al. chose glutamate as the analyte, since it is one of the most abundant neurotransmitters in the central nervous system, and is important for movement, behaviour, pain perception and mental health. Furthermore, the previously developed glutamate-sensitive construct SuperGluSnFr, which is based on the glutamate binding protein Glt1, is a perfect example of a sensor construct compatible with a split-luciferase, and demonstrates the wide applicability of the genetic optimisation techniques used in this study (3) (Fig. 1A).

Figure 1. (A) Illustration of BLING function: Binding of glutamate to the glutamate sensor (yellow) activates the bioluminescent luciferase variant (blue). (B) Comparison of fluorescence and bioluminescence imaging. In contrast to fluorescence imaging, bioluminescence imaging does not require optical illumination, and thus can be more efficient in deep tissue (taken from Fig. 1, Petersen et al., 2021).

Results

To develop a bioluminescent genetically-encoded neurotransmitter indicator for glutamate, Petersen et al. took a multistep screening approach. Initially, they designed three constructs with three previously described split luciferase variants, which flank the truncated periplasmic Glt1. The constructs have a N-terminal secretion signal domain and a C-terminal membrane anchor (Fig. 1A).

These three variants – named BLING (BioLuminescent Indicator of the Neurotransmitter Glutamate) 0.1, 0.2 and 0.3 – were transfected into HEK cells for testing. After sequential addition of luciferase substrate CTZ and glutamate to the extracellular milieu, the bioluminescent signal was measured with a plate reader. BLING 0.2 was found to be the brightest sensor, and thus its structure was further optimised by varying the 3-amino acid segment around the Glt1 region. For this linker optimization, Petersen et al. created a library consisting of about 400 variants with different linkers containing A, S or P (Ala, Ser, Pro). Screening these constructs revealed a variant they named BLING 1.0 as the sensor with the brightest response to 1 mM glutamate (Fig. 2B), which could efficiently report glutamate concentrations in a dose-dependent manner in a 96-well assay format.

Using live cell bioluminescence microscopy, BLING 1.0 reported physiologically relevant conditions (as low as 1 µM glutamate) at the single cell level. Furthermore, in comparison with established fluorescent neurotransmitter indicators such as iGLuSnFr and GcaMP6m, BLING1.0 as well as BLING0.2 showed a significantly better response to 1 mM glutamate in bulk measurements.

Figure 2. Initial construct designs based on truncated glutamate binding protein Glt1 and bioluminescent Gluc or Nluc luciferase variants with secretion sequence (left) and membrane anchor (right), before (A) and after (B) optimization (taken from Fig. 2 Petersen et al., 2021).

Why we like this preprint

This preprint is a wonderful example of how to use pre-existing resources to create something new. It’s great to see how known constructs can be combined and optimised to help evolve an underdeveloped technique: bioluminescence; and with a pathologically important application: deep brain imaging. Furthermore, BLING 1.0 has multiple possible applications beyond recording neuronal activity, for example in drug screening of fluorescent compounds, where screening with a fluorescent readout is incompatible, or as activators of light-sensitive proteins, with future applications in the treatment of neurological disorders. Above all, it can serve as a model for the development of various other bioluminescent neurotransmitter indicators that use other analytes, such as glycine or GABA. It is also worth mentioning here that the authors have made BLING 1.0 readily available to the scientific community – so if you are interested you can test it in your own setting.

Questions to the authors

  1. Have you already tried to use BLING in vivo?
  2. You describe the use of Furimazine and hCTZ. Are these two different molecules? Which gives the best signal and is the most biocompatible?
  3. What is the time resolution compared to other methods such as iGluSnFr, Dlight or GRAB-DA?
  4. Is this bioluminescence signal strong enough to be observed through several millimetres/ centimetres of tissue?
  5. BLING 1.0 outperforms the fluorescent reporters iGluSnFr and GcaMP6m, which perform less well in bulk measurements. Do you also expect BLING 1.0 to be superior at the single cell level? Is bioluminescence commonly stronger than fluorescence emission? Is the weak signal observed in the fluorescent probes due to their poor adaption to the HEK cell system used (quantum yields are comparable to fluorophores)?
  6. What was the structural change between BLING 0.2 and BLING 1.0? Why do you suspect this improved the activity so much?

References

  • Syed, Aisha J., and James C. Anderson. “Applications of bioluminescence in biotechnology and beyond.” Chemical Society Reviews(2021).
  • Granatiero, Veronica, et al. “Using targeted variants of aequorin to measure Ca2+ levels in intracellular organelles.” Cold Spring Harbor Protocols1 (2014): pdb-prot072843.
  • Hires, Samuel Andrew, Yongling Zhu, and Roger Y. Tsien. “Optical measurement of synaptic glutamate spillover and reuptake by linker optimized glutamate-sensitive fluorescent reporters.”Proceedings of the National Academy of Sciences11 (2008): 4411-4416.

Tags: bioluminescence, biosensor, brain imaging, glutamate, glutamate sensor, luciferase, neurology, neuronal activity, optical biosensor

doi: https://doi.org/10.1242/prelights.29903

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Author's response

Eric Petersen shared

  1. Have you already tried to use BLING in vivo?

We are currently testing BLING in rodents for reporting changes in brain activity.

  1. You describe the use of Furimazine and hCTZ. Are these two different molecules? Which gives the best signal and is the most biocompatible?

These are two different luciferin molecules. Both are derivatives of the marine luciferin Coelenterazine. Both give a very bright signal however the signal from hCTZ decayed very quickly with the perfusion setup we used for microscopy and Furimazine provided a very stable signal over time. There is also a variety of other synthetic luciferins that will also work with BLING.

  1. What is the time resolution compared to other methods such as iGluSnFr, Dlight or GRAB-DA?

The time resolution for microscopy will vary depending on the setup used. Bioluminescence generally produces a dimmer signal than fluorescence. For our experiments we used 1 second exposure times as a starting point, however based on the brightness of BLING we expect to have success with much shorter exposure times allowing for time resolution in the 10s of milliseconds or less. The brightness of the luminescent sensor is also determined by the concentration of luciferin use. In our experiment, the concentration was relatively low, leaving room to increase the brightness, allowing for faster acquisition times.

  1. Is this bioluminescence signal strong enough to be observed through several millimetres/ centimetres of tissue?

Yes. Photons are of course lost along the way as the light travels through tissue. The further the light travels through tissue, the more photons are absorbed by the tissue. So the deeper you go, generally longer exposure times will be required.

  1. BLING 1.0 outperforms the fluorescent reporters iGluSnFr and GcaMP6m, which perform less well in bulk measurements. Do you also expect BLING 1.0 to be superior at the single cell level? Is bioluminescence commonly stronger than fluorescence emission? Is the weak signal observed in the fluorescent probes due to their poor adaption to the HEK cell system used (quantum yields are comparable to fluorophores)?

At this point, I expect BLING and iGluSnFr to perform similarly when recorded at the single cell level with a microscope in terms of their response to changes in glutamate. I believe the reason that bioluminescent reporters perform better with bulk measurements is due to the lack of background or noise that is present with fluorescent imaging modalities such as autofluorescence. With bioluminescence, there is no signal that can be produced by the cells themselves outside of the reporter that we are imaging. Its not that the signals are weak for the fluorescent reporters, more so that the detectable change in brightness is less prominent with bulk measurements in cell cultures which I believe can be largely attributed to autofluorescence from the cells.

  1. What was the structural change between BLING 0.2 and BLING 1.0? Why do you suspect this improved the activity so much?

BLING 1.0 was derived by screening a library containing semi-random linkers which vary in flexibility. The parental construct contained two flexible linkers and the top performer from the library contained one rigid linker and one flexible linker. We also sequenced the next four top performers from the library which were all similar with linker one being rigid and linker two being flexible.

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