Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia

Yongchao Mou; Sukhada Mukte; Eric Chai; Joshua Dein; Xue-Jun Li

doi:10.3791/60548

Analyse du transport mitochondrial et de la morphologie dans les neurones dérivés de cellules sou...

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JoVE Journal Neuroscience

Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia

Analyse du transport mitochondrial et de la morphologie dans les neurones dérivés de cellules souches pluripotentes induites par l’homme dans la paraplégie spastique héréditaire

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07:32 min

February 9, 2020

DOI: 10.3791/60548-v

Yongchao Mou^1,2, Sukhada Mukte¹, Eric Chai¹, Joshua Dein³, Xue-Jun Li^1,2

¹Department of Biomedical Sciences,University of Illinois College of Medicine Rockford, ²Department of Bioengineering,University of Illinois at Chicago, ³MD Program,University of Illinois College of Medicine Rockford

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Overview

This study investigates mitochondrial transport and morphology using induced pluripotent stem cell-derived forebrain neurons in the context of hereditary spastic paraplegia. The protocol allows for detailed assessment of mitochondrial dynamics along axons, contributing to the understanding of neurodegenerative diseases.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Neurodegenerative Diseases

Background

Mitochondrial dysfunction is a key factor in various neurodegenerative diseases.
Impaired mitochondrial transport and morphology have been linked to axonal degeneration.
The use of induced pluripotent stem cells provides a relevant model for studying human neural processes.

Purpose of Study

To develop a protocol for examining mitochondrial behavior in axons.
To elucidate the relationship between mitochondrial dynamics and neurodegenerative disease mechanisms.
To identify potential therapeutic targets for conditions like hereditary spastic paraplegia.

Methods Used

Cell culture of induced pluripotent stem cell-derived forebrain neurons.
Live cell imaging combined with mitochondrial labeling to assess mitochondrial tracking.
Important steps include dissociation of neurospheres and proper staining with fluorescent dyes.
Image analysis performed using ImageJ, including the generation of kymographs.

Main Results

Characterization of mitochondrial transport revealed significant differences in mitochondrial dynamics.
Quantitation of mitochondrial length and movement showed reduced motility in neurons derived from hereditary spastic paraplegia models.
Findings underscore the importance of mitochondrial function in neural health and disease.

Conclusions

This study provides a vital experimental approach to analyze mitochondrial dynamics in the context of neurodegeneration.
The insights gained can inform future therapeutic strategies targeting mitochondrial dysfunction.
The methodology may enhance our understanding of neuronal mechanisms and disease progression.

Frequently Asked Questions

What are the advantages of using induced pluripotent stem cells for this study?

Induced pluripotent stem cells offer a human-relevant model to explore mitochondrial function and dynamics, allowing for insights directly applicable to human disease.

How is mitochondrial transport assessed in this protocol?

Mitochondrial transport is assessed via live cell imaging, using fluorescent dyes to visualize and track mitochondrial dynamics along the axons.

What outcomes can be measured with this protocol?

Key outcomes include mitochondrial length, area, transport velocity, and motility characteristics, which are crucial for understanding neuronal health.

Can this method be adapted for other types of neurons?

Yes, this methodology can be adapted for other neuronal types by using appropriate differentiation protocols and imaging techniques specific to those neurons.

What are some limitations of this study?

Limitations may include the inability to fully replicate in vivo conditions and potential variability in stem cell differentiation outcomes.

How does this study contribute to the understanding of neurodegenerative diseases?

By using human-derived neurons to study mitochondrial dysfunction, the findings enhance our understanding of the cellular mechanisms underlying neurodegenerative diseases.

Le transport mitochondrial altéré et la morphologie sont impliqués dans diverses maladies neurodegenerative. Le protocole présenté utilise des neurones de cerveaux avant dérivés de cellules souches pluripotentes induits pour évaluer le transport mitochondrial et la morphologie dans la paraplégie spastique héréditaire. Ce protocole permet la caractérisation du trafic mitochondrial le long des axones et l’analyse de leur morphologie, ce qui facilitera l’étude des maladies neurodégénératives.

Le dysfonctionnement mitochondrial sous-tend beaucoup de maladies neurodegenerative. Notre protocole fournit un outil important pour examiner la dynamique mitochondrique dans les axones, facilitant l’étude des maladies neurologiques impliquant la dégénérescence axonale. En combinant l’étiquetage mitochondrial, l’imagerie cellulaire vivante et la technologie induite des cellules souches pluripotentes, notre protocole peut être utilisé pour caractériser le trafic mitochondrial le long des axones humains et analyser leurs morphologies.

Le transport et la morphologie mitochondriques altérés peuvent être observés dans les cultures de cellules souches et les modèles animaux des maladies neurodégénératives fournissant des cibles thérapeutiques potentielles pour traiter ces maladies. Après le jour trente-cinq de la culture, dissocier les neurosphères différenciées des cellules souches pluripotentes induites par l’homme en petits groupes avec 1 mg/ml de solution de détachement cellulaire pendant deux minutes à 37 degrés Celsius. À la fin de l’incubation recueillir les grappes cellulaires par centrifugation et de résuspendre la pastille en un millilitre de NDM.

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