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In vivo analysis of axonal transport in zebrafish.

ABSTRACT: Intracellular transport of proteins and organelles in neurons plays an essential role in nervous system development and maintenance. Axon outgrowth, synapse formation, and synapse function, among other physiological processes, require active transport of these cargos between the neuronal soma and axon terminals. Abnormalities in this axonal transport are associated with a number of neurodevelopmental and neurodegenerative disorders, such as Charcot-Marie-Tooth disease, Alzheimer disease, and amyotrophic lateral sclerosis. Despite its importance for nervous system development and health, methods for visualizing axonal transport in an intact vertebrate have been lacking. Using the advantages of the zebrafish system, we have developed a straightforward approach to visualize axonal transport of various cargos and motor proteins in intact zebrafish embryos and larvae. Here, we describe this approach in detail and discuss how it can be applied to address questions related to cargo-specific transport regulation and its effects on axon morphology and function in the developing and mature nervous system.

SUBMITTER: Drerup CM

PROVIDER: S-EPMC6337719 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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In vivo analysis of axonal transport in zebrafish.

Drerup Catherine M CM Nechiporuk Alex V AV

Methods in cell biology 20150902

Intracellular transport of proteins and organelles in neurons plays an essential role in nervous system development and maintenance. Axon outgrowth, synapse formation, and synapse function, among other physiological processes, require active transport of these cargos between the neuronal soma and axon terminals. Abnormalities in this axonal transport are associated with a number of neurodevelopmental and neurodegenerative disorders, such as Charcot-Marie-Tooth disease, Alzheimer disease, and amy ...[more]

PMID: 26794521

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Project description:BackgroundAxonal transport is vital for neurons and deficits in this process have been previously reported in a few mouse models of Alzheimer's disease prior to the appearance of plaques and tangles. However, it remains to be determined whether axonal transport is defective prior to the onset of neurodegeneration. The rTg4510 mouse, a fronto-temporal dementia and parkinsonism-17 (FTDP-17) tauopathy model, over-express tau-P301L mutation found in familial forms of FTDP-17, in the forebrain driven by the calcium-calmodulin kinase II promoter. This mouse model exhibits tau pathology, neurodegeneration in the forebrain, and associated behavioral deficits beginning at 4-5 months of age.Animal modelrTg4510 transgenic mice were used in these studies. Mice were given 2 ?L of MnCl2 in each nostril 1 h prior to Magnetic Resonance Imaging (MRI). Following MnCl2 nasal lavage, mice were imaged using Manganese enhanced Magnetic Resonance Imaging (MEMRI) Protocol with TE = 8.5 ms, TR = 504 ms, FOV = 3.0 cm, matrix size = 128 × 128 × 128, number of cycles = 15 with each cycle taking approximately 2 min, 9 s, and 24 ms using Paravision software (BrukerBioSpin, Billerica, MA). During imaging, body temperature was maintained at 37.0 °C using an animal heating system (SA Instruments, Stony Brook, NY).Data analysisResulting images were analyzed using Paravision software. Regions of interest (ROI) within the olfactory neuronal layer (ONL) and the water phantom consisting of one pixel (ONL) and 9 pixels (water) were selected and copied across each of the 15 cycles. Signal intensities (SI) of ONL and water phantom ROIs were measured. SI values obtained for ONL were then normalized the water phantom SI values. The correlation between normalized signal intensity in the ONL and time were assessed using Prism (GraphPad Software, San Diego, CA).ResultsUsing the MEMRI technique on 1.5, 3, 5, and 10-month old rTg4510 mice and littermate controls, we found significant axonal transport deficits present in the rTg4510 mice beginning at 3 months of age in an age-dependent manner. Using linear regression analysis, we measured rates of axonal transport at 1.5, 3, 5, and 10 months of age in rTg4510 and WT mice. Axonal transport rates were observed in rTg4510 mice at 48% of WT levels at 3 months, 40% of WT levels at 5 months, and 30% of WT levels at 10 months of age. In order to determine the point at which tau appears in the cortex, we probed for phosphorylated tau levels, and found that pSer262 is present at 3 months of age, not earlier at 1.5 months of age, but observed no pathological tau species until 6 months of age, months after the onset of the transport deficits. In addition, we saw localization of tau in the ONL at 6 months of age.DiscussionIn our study, we identified the presence of age-dependent axonal transport deficits beginning at 3 months of age in rTg4510 mice. We correlated these deficits at 3 months to the presence of hyperphosphorylated tau in the brain and the presence within the olfactory epithelium. We observed tau pathology not only in the soma of these neurons but also within the axons and processes of these neurons. Our characterization of axonal transport in this tauopathy model provides a functional time point that can be used for future therapeutic interventions.

Dataset Information

In vivo analysis of axonal transport in zebrafish.

Publications

In vivo analysis of axonal transport in zebrafish.

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