Light-induced oxidant production by fluorescent proteins

Trewin, Adam J.; Berry, Brandon J.; Wei, Alicia Y.; Bahr, Laura L.; Foster, Thomas H.; Wojtovich, Andrew P.

doi:10.1016/j.freeradbiomed.2018.02.002

Cited by 61 publications

(52 citation statements)

References 115 publications

(180 reference statements)

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“…Here, we also observed a dramatic recruitment of Syt-SN into extrasynaptic eGFP-LC3 puncta, but only modest levels of endogenous Syp (Fig. 10E), implying that ROS-mediated damage caused by SN is rather limited and primarily affects juxtaposed proteins, a concept consistent with the low quantum yield of SN (Shu et al, 2011;Trewin et al, 2018) and the limited damage half-radius of 3-4 nm (Takemoto et al, 2013). Importantly, the inability of photobleaching luminally localized SN to trigger the sorting of Syp-lumSN into autophagy organelles (Fig.…”

Section: Discussionsupporting

confidence: 64%

Light-Activated ROS Production Induces Synaptic Autophagy

et al. 2019

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The regulated turnover of synaptic vesicle (SV) proteins is thought to involve the ubiquitin-dependent tagging and degradation through endo-lysosomal and autophagy pathways. Yet, it remains unclear which of these pathways are used, when they become activated, and whether SVs are cleared en masse together with SV proteins or whether both are degraded selectively. Equally puzzling is how quickly these systems can be activated and whether they function in real-time to support synaptic health. To address these questions, we have developed an imaging-based system that simultaneously tags presynaptic proteins while monitoring autophagy. Moreover, by tagging SV proteins with a light-activated ROS generator, Supernova, it was possible to temporally control the damage to specific SV proteins and assess their consequence to autophagy-mediated clearance mechanisms and synaptic function. Our results show that, in mouse hippocampal neurons of either sex, presynaptic autophagy can be induced in as little as 5-10 min and eliminates primarily the damaged protein rather than the SV en masse. Importantly, we also find that autophagy is essential for synaptic function, as light-activated damage to, for example, Synaptophysin only compromises synaptic function when autophagy is simultaneously blocked. These data support the concept that presynaptic boutons have a robust highly regulated clearance system to maintain not only synapse integrity, but also synaptic function.

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Section: Discussionsupporting

confidence: 64%

Light-Activated ROS Production Induces Synaptic Autophagy

et al. 2019

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“…This concept is supported by a reciprocal performed experiment with neurons expressing Synaptotagmin-SN. Here, we also observed a dramatic recruitment of Synaptotagmin-SN into extrasynaptic eGFP-LC3 puncta, but only modest levels of endogenous Synaptophysin (Figure 8E), implying that ROS mediated damage caused by Supernova is rather limited and primarily affects co-tethered proteins, a concept consistent with the low quantum yield of Supernova (Shu et al, 2011; Trewin et al, 2018) and the limited damage half-radius of 3-4nm (Takemoto et al, 2013). Presumably, using the more potent photosensitizer miniSOG (Lin et al, 2013; Qi, 2012; Shu et al, 2011), more SV proteins could be damaged, causing a much bigger insult perhaps leading to the wholesale removal of SVs.…”

Section: Discussionsupporting

confidence: 64%

Light induced synaptic vesicle autophagy

Hoffmann

Orlando

Andrzejak

et al. 2018

Preprint

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2The regulated turnover of synaptic vesicle (SV) proteins is thought to involve the ubiquitin 3 dependent tagging and degradation through endo-lysosomal and autophagy pathways. Yet, it remains 4 unclear which of these pathways are used, when they become activated and whether SVs are cleared 5 en-mass together with SV proteins or whether both are degraded selectively. Equally puzzling is how 6 quickly these systems can be activated and whether they function in real time to support synaptic 7 health. To address these questions, we have developed an imaging based system that simultaneously 8 tags presynaptic proteins while monitoring autophagy. Moreover, by tagging SV proteins with a light 9 activated reactive oxygen species (ROS) generator, Supernova, it was possible to temporally control 10 the damage to specific SV proteins and assess their consequence to autophagy mediated clearance 11 mechanisms and synaptic function. Our results show that, in mouse hippocampal neurons, presynaptic 12 autophagy can be induced in as little as 5-10 minutes and eliminates primarily the damaged protein 13 rather than the SV en-mass. Importantly, we also find that autophagy is essential for synaptic function, 14as light-induced damage to e.g. Synaptophysin only compromises synaptic function when autophagy is 15 simultaneously blocked. These data support the concept that presynaptic boutons have a robust highly 16 regulated clearance system to maintain not only synapse integrity, but also synaptic function. 17 18 Significance Statement 19 20 The real-time surveillance and clearance of synaptic proteins is thought to be vital to the health, 21 functionality and integrity of vertebrate synapses and is compromised in neurodegenerative 22disorders, yet the fundamental mechanisms regulating these systems remain enigmatic. Our analysis 23 reveals that presynaptic autophagy is a critical part of a real-time clearance system at glutamatergic 24 synapses capable of responding to local damage of synaptic vesicle proteins within minutes and to be 25 critical for the ongoing functionality of these synapses. These data indicate that synapse autophagy is 26 2 not only locally regulated but also crucial for the health and functionality of vertebrate presynaptic 27 boutons. 28 29 75 Moreover, the selective damage of SV proteins allowed us to show that presynaptic autophagy is 76 critical for the real-time maintenance of synaptic transmission. 77 78 79 80 5 Material and Methods 81 82 Construction of vectors: Monitoring of autophagy within presynaptic boutons was achieved by creating 83 a set of lentiviral expression vectors. All vectors are based on the commercially available vector FUGW 84 (Addgene). In order to co-express mCherry-tagged Synaptophysin (Syp) and eGFP-LC3, 85 Synaptophysin-mCherry (Synaptophysin, NM_012664.3) was synthesized by Eurofins Genomics with a 86 downstream glycine linker that was fused to a self-cleaving 2A peptide (Kim et al., 2011). This element 87 was then exchanged with GFP in the FUGW vector by ligation. Subsequently, t...

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“…Blue light can be damaging to biological samples, 49 especially mitochondria, which contain light-sensitive redoxactive flavins that could alter oxidative stress. 50,51 Proton selectivity 52 also avoids caveats of ChR2 moving other cellular ions such as potassium and calcium, 53,54 both ions that are important for other mitochondrial and cellular functions. 55,56 Using a ubiquitously expressed gene promoter, we expressed the light-activated proton pump, Mac, 52 in C. elegans mitochondria.…”

Section: Mitochondria-off (Mtoff) Is Expressed In C Elegans Mitochmentioning

confidence: 99%

Neuronal AMPK coordinates mitochondrial energy sensing and hypoxia resistance inC. elegans

et al. 2020

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Mitochondria are organelles that provide energy to cells. Matching energy supply with energy demand is coordinated through various processes and is critical for cellular adaptation and survival under changing conditions. Therefore, an organism's health depends not only on mitochondrial energy production, but on the ability to sense metabolic status and the ability of mitochondria to signal appropriately to use that energy. 1,2 The mechanisms through which cellular energy-related signaling can occur likely depend on the electrochemical gradient across the mitochondrial inner membrane (IM). Mitochondrial respiration results in a proton gradient across the IM known as the protonmotive force (PMF). Ultimately, respiratory complexes of the electron transport chain (ETC) convert chemical energy into electrical potential energy by pumping protons across the IM, creating this gradient. The PMF can then be consumed at ATP synthase,

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Light-induced oxidant production by fluorescent proteins

Cited by 61 publications

References 115 publications

Light-Activated ROS Production Induces Synaptic Autophagy

Light-Activated ROS Production Induces Synaptic Autophagy

Light induced synaptic vesicle autophagy

Neuronal AMPK coordinates mitochondrial energy sensing and hypoxia resistance inC. elegans

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