Effects of imaging conditions on mitochondrial transport and length in larval motor axons of Drosophila

Louie, Kathryn; Russo, Gary J.; Salkoff, David B.; Wellington, Andrea; Zinsmaier, Konrad E.

doi:10.1016/j.cbpa.2008.06.023

Cited by 23 publications

(21 citation statements)

References 52 publications

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“…Imaging was done in the distal 200 µm of axons innervating muscles 6/7 or 12/13 in segments A3 or A4. Individual organelles moving into the bleached region of an axon were marked with a cursor in the ImageJ plugin MtrackJ (NIH, Bethesda, MD) to indicate x, y, t coordinates and movement parameters were calculated as previously described (Louie, Russo, Salkoff, Wellington, & Zinsmaier, 2008). For flux analysis, individual organelles were tracked with the Volocity Imaging software (Perkin Elmer, Waltham, MA).…”

Section: Methodsmentioning

confidence: 99%

Characterization of axonal transport defects inDrosophilaHuntingtin mutants

Weiß

Littleton

2016

Journal of Neurogenetics

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Polyglutamine (polyQ) expansion within Huntingtin (Htt) causes the fatal neurodegenerative disorder Huntington’s Disease (HD). Although Htt is ubiquitously expressed and conserved from Drosophila to humans, its normal biological function is still being elucidated. Here we characterize a role for the Drosophila Htt homolog (dHtt) in fast axonal transport (FAT). Generation and expression of transgenic dHtt-mRFP and human Htt-mRFP fusion proteins in Drosophila revealed co-localization with mitochondria and synaptic vesicles undergoing FAT. However, Htt was not ubiquitously associated with the transport machinery, as it was excluded from dense-core vesicles and APLIP1 containing vesicles. Quantification of cargo movement in dHtt deficient axons revealed that mitochondria and synaptic vesicles show a decrease in the distance and duration of transport, and an increase in the number of pauses. In addition, the ratio of retrograde to anterograde flux was increased in mutant animals. Densecore vesicles did not display similar defects in processivity, but did show altered retrograde to anterograde flux along axons. Given the co-localization with mitochondria and synaptic vesicles, but not dense-core vesicles, the data suggest dHtt likely acts locally at cargo interaction sites to regulate processivity. An increase in dynein heavy chain expression was also observed in dHtt mutants, suggesting that the altered flux observed for all cargo may represent secondary transport changes occurring independent of dHtt’s primary function. Expression of dHtt in a milton (HAP1) mutant background revealed that the protein does not require mitochondria or HAP1 to localize along axons, suggesting Htt has an independent mechanism for coupling with motors to regulate their processivity during axonal transport.

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Section: Methodsmentioning

confidence: 99%

Characterization of axonal transport defects inDrosophilaHuntingtin mutants

Weiß

Littleton

2016

Journal of Neurogenetics

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“…Live imaging of GFP-SYT was done as described [96]. For imaging of mito-GFP, dissected larvae were bathed in HL-3 solution [97]. Time-lapse images were acquired at 5-s intervals using a Zeiss LSM5 confocal using minimum laser intensity to prevent photobleaching and damage to the tissues.…”

Section: Methodsmentioning

confidence: 99%

Nebula/DSCR1 Upregulation Delays Neurodegeneration and Protects against APP-Induced Axonal Transport Defects by Restoring Calcineurin and GSK-3β Signaling

Shaw

Chang

2013

PLoS Genet

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Post-mortem brains from Down syndrome (DS) and Alzheimer's disease (AD) patients show an upregulation of the Down syndrome critical region 1 protein (DSCR1), but its contribution to AD is not known. To gain insights into the role of DSCR1 in AD, we explored the functional interaction between DSCR1 and the amyloid precursor protein (APP), which is known to cause AD when duplicated or upregulated in DS. We find that the Drosophila homolog of DSCR1, Nebula, delays neurodegeneration and ameliorates axonal transport defects caused by APP overexpression. Live-imaging reveals that Nebula facilitates the transport of synaptic proteins and mitochondria affected by APP upregulation. Furthermore, we show that Nebula upregulation protects against axonal transport defects by restoring calcineurin and GSK-3β signaling altered by APP overexpression, thereby preserving cargo-motor interactions. As impaired transport of essential organelles caused by APP perturbation is thought to be an underlying cause of synaptic failure and neurodegeneration in AD, our findings imply that correcting calcineurin and GSK-3β signaling can prevent APP-induced pathologies. Our data further suggest that upregulation of Nebula/DSCR1 is neuroprotective in the presence of APP upregulation and provides evidence for calcineurin inhibition as a novel target for therapeutic intervention in preventing axonal transport impairments associated with AD.

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“…Wandering third-instar larvae were dissected in chilled HL6 solution supplemented with 4 mM L-glutamate and 0.6 mM CaCl 2 according to published protocols (Louie et al, 2008;Yao et al, 2011). A dissected larva was transferred to a slide, covered with a large coverslip, and immersed in fresh HL6 solution.…”

Section: Methodsmentioning

confidence: 99%

DrosophilaAcyl-CoA Synthetase Long-Chain Family Member 4 Regulates Axonal Transport of Synaptic Vesicles and Is Required for Synaptic Development and Transmission

Liu

Huang²,

Zhang³

et al. 2011

J. Neurosci.

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Acyl-CoA synthetase long-chain family member 4 (ACSL4) converts long-chain fatty acids to acyl-CoAs that are indispensable for lipid metabolism and cell signaling. Mutations in ACSL4 cause nonsyndromic X-linked mental retardation. We previously demonstrated that Drosophila dAcsl is functionally homologous to human ACSL4, and is required for axonal targeting in the brain. Here, we report that Drosophila dAcsl mutants exhibited distally biased axonal aggregates that were immunopositive for the synaptic-vesicle proteins synaptotagmin (Syt) and cysteine-string protein, the late endosome/lysosome marker lysosome-associated membrane protein 1, the autophagosomal marker Atg8, and the multivesicular body marker Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate). In contrast, the axonal distribution of mitochondria and the cell adhesion molecule Fas II (fasciclin II) was normal. Electron microscopy revealed accumulation of prelysomes and multivesicle bodies. These aggregates appear as retrograde instead of anterograde cargos. Live imaging analysis revealed that dAcsl mutations increased the velocity of anterograde transport but reduced the flux, velocity, and processivity of retrograde transport of Syt-enhanced green fluorescent protein-labeled vesicles. Immunohistochemical and electrophysiological analyses showed significantly reduced growth and stability of neuromuscular synapses, and impaired glutamatergic neurotransmission in dAcsl mutants. The axonal aggregates and synaptic defects in dAcsl mutants were fully rescued by neuronal expression of human ACSL4, supporting a functional conservation of ACSL4 across species in the nervous system. Together, our findings demonstrate that dAcsl regulates axonal transport of synaptic vesicles and is required for synaptic development and function. Defects in axonal transport and synaptic function may account, at least in part, for the pathogenesis of ACSL4-related mental retardation.

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Effects of imaging conditions on mitochondrial transport and length in larval motor axons of Drosophila

Cited by 23 publications

References 52 publications

Characterization of axonal transport defects inDrosophilaHuntingtin mutants

Characterization of axonal transport defects inDrosophilaHuntingtin mutants

Nebula/DSCR1 Upregulation Delays Neurodegeneration and Protects against APP-Induced Axonal Transport Defects by Restoring Calcineurin and GSK-3β Signaling

DrosophilaAcyl-CoA Synthetase Long-Chain Family Member 4 Regulates Axonal Transport of Synaptic Vesicles and Is Required for Synaptic Development and Transmission

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