Chemoptogenetic ablation of neuronal mitochondria in vivo with spatiotemporal precision and controllable severity

Abstract

Mitochondrial dysfunction is implicated in the pathogenesis of multiple neurological diseases, but elucidation of underlying mechanisms is limited experimentally by the inability to damage specific mitochondria in defined neuronal groups. We developed a precision chemoptogenetic approach to target neuronal mitochondria in the intact nervous system in vivo. MG2I, a chemical fluorogen, produces singlet oxygen when bound to the fluorogen-activating protein dL5** and exposed to far-red light. Transgenic zebrafish expressing dL5** within neuronal mitochondria showed dramatic MG2I- and light-dependent neurobehavioral deficits, caused by neuronal bioenergetic crisis and acute neuronal depolarization. These abnormalities resulted from loss of neuronal respiration, associated with mitochondrial fragmentation, swelling and elimination of cristae. Remaining cellular ultrastructure was preserved initially, but cellular pathology downstream of mitochondrial damage eventually culminated in neuronal death. Our work provides powerful new chemoptogenetic tools for investigating mitochondrial homeostasis and pathophysiology and shows a direct relationship between mitochondrial function, neuronal biogenetics and whole-animal behavior.

α-Synuclein amplifies cytoplasmic peroxide flux and oxidative stress provoked by mitochondrial inhibitors in CNS dopaminergic neurons in vivo

“…Since the transgenic lines were made using Gal4/UAS genetics, the redox biosensors can be expressed in any other tissue of interest by crossing the responder lines with an appropriate Gal4 driver, expanding the utility of these lines for future studies in other organ systems. Coupled with other new transgenic lines that allow targeted induction of chemoptogenetic damage to mitochondria in specific cells of a live zebrafish [ 73 ], these resources will be valuable for future studies to define sources, targets, and consequences of ROS signals in vivo .…”

“…Embryos were raised at 28.5 °C in cycles of 14 h light – 10 h dark, with illumination in white light at 200 Lux, color temperature 3700 K, in E3 buffer (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl 2 , 0.33 mM MgSO 4 ; all chemicals were supplied by Sigma, St. Louis, MO). Transgenic Tg( UAS:hsa.SNCA-p2A-nls-mCherry ) Pt423 , Tg( UAS:p2A-nls-mCherry ) Pt424 , Tg( UAS:roGFP2-Orp1 ) Pt428 and Tg( UAS:Grx1-roGFP2 ) Pt429 lines were generated using the Tol2 transposon method as in our previous work [ 73 ]. Embryos were injected at the 1-cell stage with 1 – 2 nL of transgene solution (25 μg/μL transgene plasmid; 0.5% phenol red; 240 mM KCl; 40 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.4; 50 ng/μL tol2 transposase mRNA; nuclease-free H 2 O).…”

“…Zebrafish locomotor behavioral testing was performed exactly as described in our previous work [ 73 , 77 ]. 5dpf zebrafish larvae were placed in the wells of a black-walled 96-well plate with an optical glass bottom in an incubator at 28.5 °C.…”

“…Zebrafish were anesthetized in tricaine and fixed in 4% PFA at 4 °C for 16hr, then rinsed with cold PBS prior to preparation of frozen sections or dissection to remove the brain for whole-mount analysis. Sections were labeled for immunofluorescence as described previously [ 73 ] using primary antibodies to TH (#MAB318, 1:500, Millipore) and human α-Synuclein (#sc7011 R, 1:400, Santa Cruz). Whole mount immunofluorescence labeling was carried out as described previously [ 18 ], using primary antibodies to TH (rabbit polyclonal, Millipore, # AB152; 1:1000) and GFP (chicken polyclonal, Abcam, Cat # AB13970; 1:5000).…”

Subcellular Singlet Oxygen and Cell Death: Location Matters

“…Genetically targeted PS Killer Red (Bulina et al, 2006 ), MiniSOG (Ruiz-González et al, 2013 ), and SuperNova (Takemoto et al, 2013 ) have all been used to target intracellular organelles, with varying results on efficacy in directed cell killing, depending on the organelle targeted, expression level, and illumination parameters [reviews: (Wojtovich and Foster, 2014 ; Trewin et al, 2018 )]. Targeted and activated photosensitizers (TAPs) using a genetically encoded fluorogen-activating protein (FAP) enable selective photosensitization directed to targeted cellular sites within genetically or molecularly targeted cells (Wang et al, 2017 ; Ackerman et al, 2019 ), as recently illustrated in transgenic zebrafish cardiovascular and nervous systems (He et al, 2016 ; Xie et al, 2020 ), and subcellular locations including mitochondria and telomeres, among others (Fouquerel et al, 2019 ; Jang et al, 2019 ; Qian et al, 2019 ). Due to the short range of action of 1 O 2 in living systems (Kuimova et al, 2009 ), this targeting and activation of the PS protects adjacent normal cells and reduces damage to nearby tissues, because photoactivity requires coincidence of the dye, the activating protein, and the illumination source (Lovell et al, 2010 ; He et al, 2016 ).…”