Using Live&#45;Cell Imaging to Measure the Effects of Pathological Proteins on Axonal Transport in Primary Hippocampal Neurons

Rebecca L. Mueller; Nicholas M. Kanaan; Benjamin Combs

doi:10.3791/66156

Using Live-Cell Imaging to Measure the Effects of Pathological Proteins on Axonal Transport i...

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JoVE Journal Neuroscience

Using Live-Cell Imaging to Measure the Effects of Pathological Proteins on Axonal Transport in Primary Hippocampal Neurons

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10:38 min

December 22, 2023

DOI: 10.3791/66156-v

Rebecca L. Mueller^1,2, Nicholas M. Kanaan^1,2,3, Benjamin Combs¹

¹Department of Translational Neuroscience, College of Human Medicine,Michigan State University, ²Neuroscience Program,Michigan State University, ³Hauenstein Neuroscience Center, Mercy Health Saint Mary’s

Overview

This study illustrates the combination of transfecting primary hippocampal rodent neurons with live-cell confocal imaging to explore the effects of pathological protein modifications on axonal transport. The aim is to uncover the mechanistic pathways that mediate disruptions caused by tau protein modifications in neurodegenerative diseases.

Key Study Components

Area of Science

Neuroscience
Axonal transport
Neurodegenerative diseases

Background

Neurons perform bidirectional transport of cargo along axons to sustain functionality.
Deficits in transport are linked to several neurodegenerative diseases.
Modified tau protein forms disrupt normal transport mechanisms.
Understanding these mechanisms may reveal pathways of tau-induced neurotoxicity.

Purpose of Study

To identify the signaling pathways altered by pathological tau modifications.
To establish a protocol for analyzing axonal transport in mammalian neurons.
To facilitate the targeting of various proteins affecting transport mechanisms.

Methods Used

Primary hippocampal neurons were transfected and analyzed using live-cell confocal microscopy.
The protocol includes dissection of embryonic mouse hippocampal tissue and subsequent cell culture.
No multiomics analyses are mentioned in the provided text.
Critical steps include preparing slides, transfecting neurons, and imaging two days post-transfection.
Key methodological details include using specific imaging settings to visualize axonal transport.

Main Results

Pathological forms of tau disrupt normal axonal transport dynamics.
Disruption of transport is linked to changes in phosphorylation pathways.
These disruptions could contribute to neurodegeneration.
The study establishes a reproducible approach to investigate transport alterations due to pathological proteins.

Conclusions

This study demonstrates a method to analyze the effects of modified proteins on neuronal transport.
Understanding these mechanisms provides insights into neuronal dysfunction in disease contexts.
The findings may enhance knowledge of axonal transport in neurodegenerative conditions.

Frequently Asked Questions

What are the advantages of using primary hippocampal neurons?

Primary hippocampal neurons provide a relevant biological model to study axonal transport in a disease context, closely mimicking in vivo conditions.

How is the main biological model implemented?

The model is established by isolating and culturing primary hippocampal neurons from embryonic mice, facilitating controlled experimentation on axonal transport.

What types of data are obtained in this study?

Data obtained includes imaging of axonal transport dynamics and identification of the directionality and rate of cargo movement in neurons.

How can this method be adapted for different proteins?

The protocol allows for modification to include various cargo proteins and facilitate the targeting of specific signaling pathway components for investigation.

What are key limitations of this study?

One limitation is the focus on a specific neuronal type, which may not fully represent transport mechanisms across all neuronal populations.

Here, we demonstrate how to combine transfection of primary hippocampal rodent neurons with live-cell confocal imaging to analyze pathological protein-induced effects on axonal transport and identify mechanistic pathways mediating these effects.

Neurons rely on bidirectional transport of cargo along the axon to maintain functional synapsis and neuroconnectivity. Deficits in transport are thought to be critical contributors to the pathogenesis of several neurodegenerative diseases. We aim to identify mechanisms by which disease associated modifications to tau protein disrupt axonal transport.

We established that multiple pathological forms of tau protein disrupt normal axonal transport in mammalian neurons. This effect depends on changes to phosphorylation-based signaling pathways that regulate axonal transport. These disruptions represent a potential mechanism of tau induced neurotoxicity in disease.

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