Real-time visualization of oxidative stress-mediated neurodegeneration of individual spinal motor neurons in vivo

Formella, I., Svahn, A.J., Radford, R.A.W., Don, E.K., Cole, N.J., Hogan, A., Lee, A., Chung, R.S., Morsch, M.
Full text @ Redox Biol.

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.

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.

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.

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.

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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