Unbiased Automated Quantitation of ROS Signals in Live Retinal Neurons of
            <i>Drosophila</i>
            using Fiji/ImageJ

Deshpande, Prajakta; Gogia, Neha; Chimata, Anuradha Venkatakrishnan; Singh, Amit

doi:10.2144/btn-2021-0006

Cited by 12 publications

(11 citation statements)

References 45 publications

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“…The third instar larval eye-antennal imaginal discs were dissected in cold 1X Schneider’s Drosophila medium (Gibco, Catalogue number #21720024). The samples were incubated in Dihydroethidium (DHE, Life Technologies Catalogue number # D11347) dye solution [(1:300) in 1XPBS] [ 63 , 64 ] for 5 min and were washed three times with cold 1X PBS. DHE is oxidized by superoxide radical to form 2-hydroxyethidium which intercalates with DNA and provides signal at 550 nm in cells where ROS is produced [ 64 – 66 ].…”

Section: Methodsmentioning

confidence: 99%

“…The samples were incubated in Dihydroethidium (DHE, Life Technologies Catalogue number # D11347) dye solution [(1:300) in 1XPBS] [ 63 , 64 ] for 5 min and were washed three times with cold 1X PBS. DHE is oxidized by superoxide radical to form 2-hydroxyethidium which intercalates with DNA and provides signal at 550 nm in cells where ROS is produced [ 64 – 66 ]. The eye discs were then mounted on a slide and were immediately imaged live on Olympus Fluoview 3000, a Laser Scanning Confocal microscope [ 59 ].…”

Section: Methodsmentioning

confidence: 99%

“…All final figures were prepared using Adobe Photoshop software. The number of ROS puncta were quantified from five sets of imaginal discs per genotype by using automated quantification method [ 64 ]. The Interactive H watershed plugin of Fiji/ ImageJ free software was used for automated quantification and the statistical analysis was performed using Microsoft Excel [ 64 ].…”

Section: Methodsmentioning

confidence: 99%

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N-Acetyltransferase 9 ameliorates Aβ42-mediated neurodegeneration in the Drosophila eye

Deshpande,

Chimata,

Snider

et al. 2023

Cell Death Dis

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Alzheimer’s disease (AD), a progressive neurodegenerative disorder, manifests as accumulation of amyloid-beta-42 (Aβ42) plaques and intracellular accumulation of neurofibrillary tangles (NFTs) that results in microtubule destabilization. Targeted expression of human Aβ42 (GMR > Aβ42) in developing Drosophila eye retinal neurons results in Aβ42 plaque(s) and mimics AD-like extensive neurodegeneration. However, there remains a gap in our understanding of the underlying mechanism(s) for Aβ42-mediated neurodegeneration. To address this gap in information, we conducted a forward genetic screen, and identified N-acetyltransferase 9 (Mnat9) as a genetic modifier of GMR > Aβ42 neurodegenerative phenotype. Mnat9 is known to stabilize microtubules by inhibiting c-Jun-N- terminal kinase (JNK) signaling. We found that gain-of-function of Mnat9 rescues GMR > Aβ42 mediated neurodegenerative phenotype whereas loss-of-function of Mnat9 exhibits the converse phenotype of enhanced neurodegeneration. Here, we propose a new neuroprotective function of Mnat9 in downregulating the JNK signaling pathway to ameliorate Aβ42-mediated neurodegeneration, which is independent of its acetylation activity. Transgenic flies expressing human NAT9 (hNAT9), also suppresses Aβ42-mediated neurodegeneration thereby suggesting functional conservation in the interaction of fly Mnat9 or hNAT9 with JNK-mediated neurodegeneration. These studies add to the repertoire of molecular mechanisms that mediate cell death response following accumulation of Aβ42 and may provide new avenues for targeting neurodegeneration.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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N-Acetyltransferase 9 ameliorates Aβ42-mediated neurodegeneration in the Drosophila eye

Deshpande,

Chimata,

Snider

et al. 2023

Cell Death Dis

Self Cite

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“…This versatility of Drosophila model is due to its fully sequenced genome, shorter life cycle, conservation of the basic genetic machinery, less genetic redundancy, presence of orthologs, or homologs of human disease genes etc. Drosophila has been used to model NDs like AD (Tare et al, 2011 ; Cutler et al, 2015 ; Sarkar et al, 2018 ; Deshpande et al, 2019 , 2021 ; Yeates et al, 2019 ; Irwin et al, 2020 ), PD (Dawson et al, 2010 ), ALS (Casci and Pandey, 2015 ; Gogia et al, 2020 ), HD (Marsh et al, 2003 ) etc. Keeping in mind the pathological hallmarks of PD and the importance of using relevant animal models to study PD, Ayajuddin et al , developed and utilized an adult life stage-specific rotenone mediated fly model of PD.…”

Section: Animal Model(s)mentioning

confidence: 99%

Editorial: Protein misfolding, altered mechanisms and neurodegeneration

Gogia

Tare

Kannan

et al. 2023

Front. Mol. Neurosci.

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Editorial on the Research Topic Protein misfolding, altered mechanisms and neurodegenerationNeurodegenerative diseases (NDs) like Alzheimer's disease (AD), Parkinson's disease (PD), Amyotrophic lateral sclerosis (ALS), Frontotemporal lobe degeneration (FTLD), Polyglutamine diseases such as Huntington's disease (HD), Spinocerebellar ataxias (SCAs) etc., are a group of debilitating disorders that affects millions of people worldwide and have no cure to-date. Despite the advancement in our understanding of molecular and genetic mechanisms underlying these NDs, only a limited symptom-based treatment options are available. As the life expectancy increases there is an increase in the number of ND patients, which will seriously challenge the availability of resources and will impact a nation's economy. There is an urgent need to develop an affordable healthcare system and find effective treatment options to provide better clinical regimens to cure these diseases. NDs affect neurons, neuronal connections associated with memory, cognition, thinking, strength, sensation, movements, learning, co-ordination, and other abilities. Although the causative factors of NDs varies from one to another and the differences in the disease symptoms could be many, these diseases share some common features. One of the common pathological hallmarks among the most NDs is aggregation or deposition of misfolded proteins. Compelling evidence from neuropathological, genetic, animal models studies, and other approaches have strongly supported the fact that accumulation of misfolded protein aggregates triggers a series of detrimental events, which results in synaptic alterations, neuronal cell loss, and significantly contributes toward disease pathogenesis.This Research Topic highlights the new approaches employed to develop therapeutics, which can effectively block or slow down the onset or progression of these fatal NDs. This manuscripts collection highlights the current advances in the field of neurodegenerative disorders, which may help in addressing some of the unanswered questions pertaining to this Research Topic. This collection of manuscripts is divided into three vital categories: (1) Disease mechanisms, (2) Therapeutic perspectives, and (3) Animal model(s). We hope that this topic may help discern the gaps, connect the missing links, improve our current understanding, knowledge related to this topic and open new avenues of research focuses to improve current treatments options against these deadly yet incurable disorders.

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“…Laser power, exposure, sensitivity, and resolution were optimized and kept constant across all sections and control slides. Dihydroethidium fluorescence was imaged using the Alexa 555 channel, and elastin autofluorescence was imaged with the Alexa 488 channel (20).…”

Section: Superoxide Staining and Quantificationmentioning

confidence: 99%

Soluble Guanylyl Cyclase Activation Rescues Hyperoxia-Induced Dysfunction of Vascular Relaxation

Mace

Kimlinger

et al. 2022

Shock

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Introduction: Perioperative alterations in perfusion lead to ischemia and reperfusion injury, and supplemental oxygen is administered during surgery to limit hypoxic injury but can lead to hyperoxia. We hypothesized that hyperoxia impairs endothelium-dependent and endothelium-independent vasodilation but not the vasodilatory response to heme-independent soluble guanylyl cyclase activation. Methods: We measured the effect of oxygen on vascular reactivity in mouse aortas. Mice were ventilated with 21% (normoxia), 60% (moderate hyperoxia), or 100% (severe hyperoxia) oxygen during 30 minutes of renal ischemia and 30 minutes of reperfusion. After sacrifice, the thoracic aorta was isolated, and segments mounted on a wire myograph. We measured endothelium-dependent and endothelium-independent vasodilation with escalating concentrations of acetylcholine (ACh) and sodium nitroprusside (SNP), respectively, and we measured the response to heme-independent soluble guanylyl cyclase activation with cinaciguat. Vasodilator responses to each agonist were quantified as the maximal theoretical response (Emax) and the effective concentration to elicit 50% relaxation (EC50) using a sigmoid model and nonlinear mixed-effects regression. Aortic superoxide was measured with dihydroethidium probe and high-performance liquid chromatography quantification of the specific superoxide product 2-hydroxyethidium. Results: Hyperoxia impaired endothelium-dependent (ACh) and endothelium-independent (SNP) vasodilation compared with normoxia and had no effect on cinaciguat-induced vasodilation. The median ACh Emax was 76.4% (95% confidence interval = 69.6 to 83.3) in the normoxia group, 53.5% (46.7 to 60.3) in the moderate hyperoxia group, and 53.1% (46.3 to 60.0) in the severe hyperoxia group (P < 0.001, effect across groups), while the ACh EC50 was not different among groups. The SNP Emax was 133.1% (122.9 to 143.3) in normoxia, 128.3% (118.1 to 138.6) in moderate hyperoxia, and 114.8% (104.6 to 125.0) in severe hyperoxia (P < 0.001, effect across groups), and the SNP EC50 was 0.38 log M greater in moderate hyperoxia than in normoxia (95% confidence interval = 0.18 to 0.58, P < 0.001). Cinaciguat Emax and EC50 were not different among oxygen treatment groups (median range Emax = 78.0% to 79.4% and EC50 = –18.0 to −18.2 log M across oxygen groups). Aorta 2-hydroxyethidium was 1419 pmol/mg of protein (25th–75th percentile = 1178–1513) in normoxia, 1993 (1831–2473) in moderate hyperoxia, and 2078 (1936–2922) in severe hyperoxia (P = 0.008, effect across groups). Conclusions: Hyperoxia, compared with normoxia, impaired endothelium-dependent and endothelium-independent vasodilation but not the response to heme-independent soluble guanylyl cyclase activation, and hyperoxia increased vascular superoxide production. Results from this study could have important implications for patients receiving high concentrations of oxygen and at risk for ischemia reperfusion-mediated organ injury.

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Unbiased Automated Quantitation of ROS Signals in Live Retinal Neurons of Drosophila using Fiji/ImageJ

Cited by 12 publications

References 45 publications

N-Acetyltransferase 9 ameliorates Aβ42-mediated neurodegeneration in the Drosophila eye

N-Acetyltransferase 9 ameliorates Aβ42-mediated neurodegeneration in the Drosophila eye

Editorial: Protein misfolding, altered mechanisms and neurodegeneration

Soluble Guanylyl Cyclase Activation Rescues Hyperoxia-Induced Dysfunction of Vascular Relaxation

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