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. 2018 Oct;19:226-234.
doi: 10.1016/j.redox.2018.08.011. Epub 2018 Aug 23.

Real-time Visualization of Oxidative Stress-Mediated Neurodegeneration of Individual Spinal Motor Neurons in Vivo

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Free PMC article

Real-time Visualization of Oxidative Stress-Mediated Neurodegeneration of Individual Spinal Motor Neurons in Vivo

Isabel Formella et al. Redox Biol. .
Free PMC article

Abstract

Generation of reactive oxygen species (ROS) has been shown to be important for many physiological processes, ranging from cell differentiation to apoptosis. With the development of the genetically encoded photosensitiser KillerRed (KR) it is now possible to efficiently produce ROS dose-dependently in a specific cell type upon green light illumination. Zebrafish are the ideal vertebrate animal model for these optogenetic methods because of their transparency and efficient transgenesis. Here we describe a zebrafish model that expresses membrane-targeted KR selectively in motor neurons. We show that KR-activated neurons in the spinal cord undergo stress and cell death after induction of ROS. Using single-cell resolution and time-lapse confocal imaging, we selectively induced neurodegeneration in KR-expressing neurons leading to characteristic signs of apoptosis and cell death. We furthermore illustrate a targeted microglia response to the induction site as part of a physiological response within the zebrafish spinal cord. Our data demonstrate the successful implementation of KR mediated ROS toxicity in motor neurons in vivo and has important implications for studying the effects of ROS in a variety of conditions within the central nervous system, including aging and age-related neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis.

Keywords: Microscopy; Motor neurons; Optogenetics; Oxidative stress; Zebrafish.

Figures

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Fig. 1
Fig. 1
Motor neuron (MN) specific expression of KillerRed (KR). (A) A membrane localization signal (MLS) targets the photosensitizer protein KillerRed (KR) to the intracellular cell membrane of MNs (mnx1 promoter). Upon green light illumination (LI,), KR induces lipid oxidization generating reactive oxygen species (ROS) alongside photo-bleaching of KR. (B) Synthetic transposase mRNA and a Tol2 transposon plasmid DNA construct containing the Tol2 element, the mnx1 promoter and the sequence encoding MLS-KR were co-injected into one cell stage zebrafish eggs. The Tol2 construct is excised from the plasmid DNA and integrated into the genomic DNA. Tol2 insertions in germ cells are transmitted to the F1 generation (modified after Kawakami et al., 2007). (C) PCR analysis of genomic DNA extracted from 24hpf F1 generation zebrafish embryos, confirmed germ line transmission of KR. Expected product size for MLS-KR was 531 bp, b-actin served as a positive control (housekeeping gene). (D-E) MN specific MLS-KR expression (red) at 3 dpf (Tg[mnx1:MLS-KillerRed]). Images are lateral views, anterior to the left, dorsal to the top. Scale bar 25 µm.
Fig. 2
Fig. 2
Generation of mosaic KR expression utilising GAL4-UAS regulation. (A) The GAL4/upstream activating sequence (UAS) system is a powerful method for analysing cell function in vivo. The yeast GAL4 transcription factor activates the transcription of target genes by binding to UAS cis-regulatory sites. The Gal4/UAS system can be used as two-component gene expression system carried in separate lines. The driver line (Tg[met:Gal4; UAS:EGFP]) provides tissue-specific GAL4 expression and the responder line (Tg[4xnrUAS:MLS-KR, cryaa:EGFP]) carries the coding sequence for the gene of interest under the control of the UAS site. In the double transgenic F1 embryos, Gal4 expressing cells are visualized by fluorescent reporters, providing mosaic expression of the gene of interest (modified after Asakawa & Kawakami, 2008). (B) Zebrafish embryo at 3 dpf expressing both met:EGFP (green) and KR (red) (Tg[met:Gal4;UAS:EGFP;4xnrUAS:MLS-KillerRed, cryaa:EGFP]). (C-E) 5 dpf zebrafish larvae show mosaic expression for both KR-ve/EGFP+ve (C, E (green)), KR+ve/EGFP-ve (D, E (red)) and KR+ve/EGFP+ve (E (yellow)) motor neurons. All images are lateral views, anterior to the left, dorsal to the top. Scale bar 50 µm.
Fig. 3
Fig. 3
KR-activated ROS generation is rescued by the antioxidant N-acetyl Cysteine (NAC). The cell-permeable reporter CM-H2DCFDA was used to quantify ROS production 30 min after the OS event. (A) Intracellular ROS accumulation was quantified via microplate reader detection of the oxidized fluorescent reporter in control larvae (white bar, DMSO 0.1%, 1 h), H2O2 treated fish (grey bar, 5 mM, 1 h) and light-illuminated KR+ve zebrafish (black bar, LI, 2 h light-illumination). (B) The potent ROS scavenging compound Nacetylcysteine (NAC) rescued H2O2 induced ROS production. Zebrafish embryos (3 dpf) were pre-treated with DMSO or NAC before subsequent exposure to H2O2. (C) Light-illumination of KR+ve embryos resulted in an increase in ROS detection that was partially rescued by NAC pre-incubation. The arbitrary units of fluorescence measured in duplicate were normalized to blank readings. *P < 0.05, **P < 0.01; ***P < 0.001; ****P < 0.0001; one-way ANOVA and Tukey's post-hoc test (n = 3, SEM).
Fig. 4
Fig. 4
KR-mediated ROS production leads to MN degeneration. (A) A transgenic zebrafish (Tg[met:Gal4;UAS:EGFP];Tg[UAS:MLS-KR]) mosaically expressing EGFP (green; ai) and KR (red, aii) in individual neurons prior to light illumination and KR-activation (aiii). (B) Light illumination of fluorescent neurons leads to bleaching of KR fluorescence, evident 60 min post-illumination (bii), while EGFP fluorescence intensities remained unchanged (bi). (C) Live imaging of KR activated neurons for up to 10 h revealed a series of morphological changes including progressive anterograde degeneration (ci-ciii, arrowheads), soma shrinkage (arrows) and MN death (asterisks). Scale bar 25 µm.
Fig. 5
Fig. 5
KR activation lead to ANNEXINV (A5) accumulation and degeneration of MNs. (A) Triple fluorescent zebrafish (2 dpf) positive for KR (red) and TagBFP (blue) selectively in MNs, as well ubiquitous expression of A5 (yellow), were used to visualise apoptotic processes after oxidative stress induction through KR illumination. (B) Prior to light illumination within a restricted area (green dotted line) of the zebrafish spinal cord, MNs showed high intensities of KR and TagBFP expression (bi). (C) Following light illumination for 75 min the fluorescence intensity of KR (red) was markedly reduced in the light-exposed region while no changes in TagBFP-intensities could be observed. (D) Time-lapse imaging following KR activation revealed A5 accumulation (di, arrowheads) along the axon and cell soma selectively within the light-activated area (2 h post-illumination). Scale bars 25 µm.
Fig. 6
Fig. 6
Microglia migrate towards the site of KR activation. (A-H) Time lapse imaging of a zebrafish expressing green fluorescent microglia (mpeg1:EGFP), and MNs labelled in blue (mnx1:mTagBFP) and expressing red KR (mnx1:MLS-KillerRed) throughout the spinal cord. Post-illumination (pI, B, white dotted line) KR fluorescence was significantly reduced while TagBFP fluorescence remained unaffected. Green fluorescent microglia within close proximity extended its processes towards to the KR activation site within the first two hours (C-D), seemingly inspecting light-targeted MN-bodies. Microglia subsequently moved away from the illumination site (E). Approximately 9 h post-illumination, microglia were again observed at the illumination site (F). These microglia underwent characteristic morphological changes (amoeboid body) and remained at the KR activation site for several hours (G-H). Notably, near the site of microglia activity a TagBFP+ve MN disappeared, conceivably indicating its death due to KR activation (Supplementary Video 4). Scale bar 50 µm.

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References

    1. Yoshikawa T. Free radicals and their scavengers in Parkinson's disease. Eur. Neurol. 1993;33(Suppl 1):S60–S68. - PubMed
    1. Perry G., Cash A.D., Smith M.A. Alzheimer disease and oxidative stress. J. Biomed. Biotechnol. 2002;2:120–123. - PMC - PubMed
    1. Blasco H., Garcon G., Patin F., Veyrat-Durebex C., Boyer J., Devos D., Vourc'h P., Andres C.R., Corcia P. Panel of oxidative stress and inflammatory biomarkers in ALS: a pilot study. Can. J. Neurol. Sci. J. Can. Des. Sci. Neurol. 2016:1–6. - PubMed
    1. Ferrante R.J., Browne S.E., Shinobu L.A., Bowling A.C., Baik M.J., MacGarvey U., Kowall N.W., Brown R.H., Jr., Beal M.F. Evidence of increased oxidative damage in both sporadic and familial amyotrophic lateral sclerosis. J. Neurochem. 1997;69:2064–2074. - PubMed
    1. Qin B., Cartier L., Dubois-Dauphin M., Li B., Serrander L., Krause K.H. A key role for the microglial NADPH oxidase in APP-dependent killing of neurons. Neurobiol. Aging. 2006;27:1577–1587. - PubMed

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