Transgenic Zebrafish Expressing mCherry in the Mitochondria of Dopaminergic Neurons

Noble, Sandra; Godoy, Rafael; Affaticati, Pierre; Ekker, Marc

doi:10.1089/zeb.2015.1085

Cited by 17 publications

(18 citation statements)

References 23 publications

(29 reference statements)

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“…Imaging these superficially-located cell populations is less challenging than the deeply-located CNS dopaminergic neurons that are relevant to PD. Transgenic zebrafish expressing mitochondrially-targeted mCherry in dopaminergic neurons under regulatory elements from the slc6a3 (dopamine transporter) were reported recently (Noble et al, 2015), but dynamic imaging was not carried out. Confocal imaging of CNS axons presents a number of difficulties, including the non-linear vectors of axonal projections, significant tissue thickness and signal attenuation, and embryo pigmentation.…”

Section: Discussionmentioning

confidence: 99%

Live imaging of mitochondrial dynamics in CNS dopaminergic neurons in vivo demonstrates early reversal of mitochondrial transport following MPP+ exposure

Dukes

Bai

Laar

et al. 2016

Neurobiology of Disease

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Extensive convergent evidence collectively suggests that mitochondrial dysfunction is central to the pathogenesis of Parkinson’s disease (PD). Recently, changes in the dynamic properties of mitochondria have been increasingly implicated as a key proximate mechanism underlying neurodegeneration. However, studies have been limited by the lack of a model in which mitochondria can be imaged directly and dynamically in dopaminergic neurons of the intact vertebrate CNS. We generated transgenic zebrafish in which mitochondria of dopaminergic neurons are labeled with a fluorescent reporter, and optimized methods allowing direct intravital imaging of CNS dopaminergic axons and measurement of mitochondrial transport in vivo. The proportion of mitochondria undergoing axonal transport in dopaminergic neurons decreased overall during development between 2 days post-fertilization (dpf) and 5dpf, at which point the major period of growth and synaptogenesis of the relevant axonal projections is complete. Exposure to 0.5 – 1.0mM MPP+ between 4 – 5 dpf did not compromise zebrafish viability or cause detectable changes in the number or morphology of dopaminergic neurons, motor function or monoaminergic neurochemistry. However, 0.5mM MPP+ caused a 300% increase in retrograde mitochondrial transport and a 30% decrease in anterograde transport. In contrast, exposure to higher concentrations of MPP+ caused an overall reduction in mitochondrial transport. This is the first time mitochondrial transport has been observed directly in CNS dopaminergic neurons of a living vertebrate and quantified in a PD model in vivo. Our findings are compatible with a model in which damage at presynaptic dopaminergic terminals causes an early compensatory increase in retrograde transport of compromised mitochondria for degradation in the cell body. These data are important because manipulation of early pathogenic mechanisms might be a valid therapeutic approach to PD. The novel transgenic lines and methods we developed will be useful for future studies on mitochondrial dynamics in health and disease.

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Section: Discussionmentioning

confidence: 99%

Live imaging of mitochondrial dynamics in CNS dopaminergic neurons in vivo demonstrates early reversal of mitochondrial transport following MPP+ exposure

Dukes

Bai

Laar

et al. 2016

Neurobiology of Disease

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“…Adult zebrafish of the AB (Zebrafish International Resource Center), Tg(dlx5a/dlx6a-EGFP) (Noble et al, 2015) x Tg(vglut2a:loxP-RFP-loxP-GFP) (Satou et al, 2012) and Tg(HuC:GCaMP5G) (Ahrens et al, 2013) strain were maintained under standard aquaculture conditions in UV-sterilized water at 28.5 C on a 14 hour light / 10 hour dark cycle.…”

Section: Zebrafish Maintenance and Breedingmentioning

confidence: 99%

“…In vivo imaging of glutamatergic and GABAergic networks Tg(dlx5a/dlx6a-EGFP) x Tg(vglut2a:loxP-RFP-loxP-GFP) zebrafish (Noble et al, 2015;Satou et al, 2012) larvae, injected with control or g3bp1 MO, were anesthetized at 4 dpf with tricaine (MS-222), immobilized in 2% low melting point agarose on a cover glass. For imaging of the glutamatergic and GABAergic networks in the optic tectum a two-photon LSM 780 confocal microscope equipped with an LD LCI Plan Apo 25x/0.8 objective was used.…”

Section: In Vivo Imaging Of Pan-neuronal Activitymentioning

confidence: 99%

G3BPs tether the TSC complex to lysosomes and suppress mTORC1 signaling

Prentzell

Rehbein

Sandoval

et al. 2021

Cell

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Summary Ras GTPase-activating protein-binding proteins 1 and 2 (G3BP1 and G3BP2, respectively) are widely recognized as core components of stress granules (SGs). We report that G3BPs reside at the cytoplasmic surface of lysosomes. They act in a non-redundant manner to anchor the tuberous sclerosis complex (TSC) protein complex to lysosomes and suppress activation of the metabolic master regulator mechanistic target of rapamycin complex 1 (mTORC1) by amino acids and insulin. Like the TSC complex, G3BP1 deficiency elicits phenotypes related to mTORC1 hyperactivity. In the context of tumors, low G3BP1 levels enhance mTORC1-driven breast cancer cell motility and correlate with adverse outcomes in patients. Furthermore, G3bp1 inhibition in zebrafish disturbs neuronal development and function, leading to white matter heterotopia and neuronal hyperactivity. Thus, G3BPs are not only core components of SGs but also a key element of lysosomal TSC-mTORC1 signaling.

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“…Instead, we generated zebrafish embryos expressing Actin:TOM20-mCherry, an mCherry construct localized to the mitochondria and expressed under the actin promoter. 51,52 Introduction of Actin:TOM20-mCherry DNA into the genomic DNA using Tol2 transposase yielded embryos expressing TOM20-mCherry in a mosaic pattern: some cells express the construct while others do not. In this case, a mosaic expression pattern worked to our advantage, allowing us to select retinal tissue where the labelled megamitochondria were spread apart and readily distinguished from one another.…”

Section: Resultsmentioning

confidence: 99%

Multimodal LA-ICP-MS and nanoSIMS imaging enables copper mapping within photoreceptor megamitochondria in a zebrafish model of Menkes disease

et al. 2018

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Copper is essential for eukaryotic life, and animals must acquire this nutrient through the diet and distribute it to cells and organelles for proper function of biological targets. Indeed, mutations in the central copper exporter ATP7A contribute to a spectrum of diseases, including Menkes disease, with symptoms ranging from neurodegeneration to lax connective tissue. As such, a better understanding of the fundamental impacts of ATP7A mutations on in vivo copper distributions is of relevance to those affected by these diseases. Here we combine metal imaging and optical imaging techniques at a variety of spatial resolutions to identify tissues and structures with altered copper levels in the Calamity zebrafish model of Menkes disease. Rapid profiling of tissue slices with LA-ICP-MS identified reduced copper levels in the brain, neuroretina, and liver of Menkes fish compared to control specimens. High resolution nanoSIMS imaging of the neuroretina, combined with electron and confocal microscopies, identified the megamitochondria of photoreceptors as loci of copper accumulation in wildtype fish, with lower levels of megamitochondrial copper observed in Calamity zebrafish. Interestingly, this localized copper decrease does not result in impaired photoreceptor development or altered megamitochondrial morphology, suggesting the prioritization of copper at sufficient levels for maintaining essential mitochondrial functions. Together, these data establish the Calamity zebrafish as an optically transparent in vivo model for the study of neural copper misregulation, illuminate a role for the ATP7A copper exporter in trafficking copper to the neuroretina, and highlight the utility of combining multiple imaging techniques for studying metals in whole organism settings with spatial resolution.

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Transgenic Zebrafish Expressing mCherry in the Mitochondria of Dopaminergic Neurons

Cited by 17 publications

References 23 publications

Live imaging of mitochondrial dynamics in CNS dopaminergic neurons in vivo demonstrates early reversal of mitochondrial transport following MPP+ exposure

Live imaging of mitochondrial dynamics in CNS dopaminergic neurons in vivo demonstrates early reversal of mitochondrial transport following MPP+ exposure

G3BPs tether the TSC complex to lysosomes and suppress mTORC1 signaling

Multimodal LA-ICP-MS and nanoSIMS imaging enables copper mapping within photoreceptor megamitochondria in a zebrafish model of Menkes disease

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