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Impaired mitochondrial transport and morphology are involved in various neurodegenerative diseases. The presented protocol uses induced pluripotent stem cell-derived forebrain neurons to assess mitochondrial transport and morphology in hereditary spastic paraplegia. This protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology, which will facilitate the study of neurodegenerative disease.
Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human neurons, which may contribute to neurodegeneration in various disease states. Although it is challenging to examine mitochondrial dynamics in live human nerves, such paradigms are critical for studying the role of mitochondria in neurodegeneration. Described here is a protocol for analyzing mitochondrial transport and mitochondrial morphology in forebrain neuron axons derived from human induced pluripotent stem cells (iPSCs). The iPSCs are differentiated into telencephalic glutamatergic neurons using well-established methods. Mitochondria of the neurons are stained with MitoTracker CMXRos, and mitochondrial movement within the axons are captured using a live-cell imaging microscope equipped with an incubator for cell culture. Time-lapse images are analyzed using software with "MultiKymograph", "Bioformat importer", and "Macros" plugins. Kymographs of mitochondrial transport are generated, and average mitochondrial velocity in the anterograde and retrograde directions is read from the kymograph. Regarding mitochondrial morphology analysis, mitochondrial length, area, and aspect ratio are obtained using the ImageJ. In summary, this protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology to facilitate studies of neurodegenerative diseases.
Mitochondrial motility and distribution play a vital role in fulfilling variable and specialized energetic demands in polarized neurons. Neurons can extend extremely long axons to connect with targets through the formation of synapses, which demand high levels of energy for Ca2+ buffering and ion currents. Transport of mitochondria from soma to axon is critical for supporting axonal and synaptic function of neurons. Spatially and temporally dynamic mitochondrial movement is conducted by fast axonal transport at rates of several micrometers per second1.
Specifically, motor or adaptor proteins, such as kines....
1. Generation of telencephalic glutamatergic neurons from iPSCs
NOTE: The detailed protocol for maintaining iPSCs and their differentiation into telencephalic glutamatergic neurons are similar to those described previously18. Here, the critical process during the differentiation of human pluripotent stem cells is introduced and highlighted.
Here, human iPSCs were differentiated into telencephalic glutamatergic neurons, which were characterized by immunostaining with Tbr1 and βIII tubulin markers (Figure 1A). To examine the axonal transport of mitochondria, these cells were stained with red fluorescent dye, and time-lapse imaging was performed. Since ImageJ is readily available and easier to obtain, mitochondrial transport was further analyzed with the "MultiKymograph" and "Macros" ImageJ pl.......
This article describes a method to analyze mitochondrial transport and morphology in neuronal axons using red fluorescent dye and ImageJ software, both of which provide a unique platform to study axonal degeneration and mitochondrial morphology in neurodegenerative disease. There are several critical steps in the protocol, including staining of mitochondria, live cell imaging, and analyzing the images. In this method, a fluorescent dye was used to stain mitochondria. Since human iPSC-derived neurons are easily detached f.......
This work was supported by the Spastic Paraplegia Foundation, the Blazer Foundation and the NIH (R21NS109837).
....Name | Company | Catalog Number | Comments |
Accutase Cell Detachment Solution | Innovative Cell Technologies | AT104 | |
Biosafety hood | Thermo Scientific | 1300 SERIES A2 | |
Bovine serum albumin (BSA) | Sigma | A-7906 | |
Brain derived neurotrophic factor (BDNF) | Peprotech | 450-02 | |
Centrifuge | Thermo Scientific | Sorvall Legend X1R/ 75004261 | |
Coverslips | Chemiglass Life Sciences | 1760-012 | |
Cyclic AMP (cAMP) | Sigma-Aldrich | D0627 | |
Dispase | Gibco | 17105-041 | |
Dorsomorphin | Selleckchem | S7146 | |
Dulbecco's modified eagle medium with F12 nutrient mixture (DMEM/F12) | Corning | 10-092-CV | |
FBS | Gibco | 16141-002 | |
Fibroblast growth factor 2 (FGF2, bFGF) | Peprotech | 100-18B | |
Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix | Gibco | A1413201 | |
Gem21 NeuroPlex Serum-Free Supplement | Gemini | 400-160 | |
Glass Bottom Dishes | MatTek | P35G-0.170-14-C | |
9'' glass pipetes | VWR | 14673-043 | |
Glial derived neurotrophic factor (BDNF) | Sigma-Aldrich | D0627 | |
GlutaMAX-I | Gibco | 35050-061 | |
Heparin | Sigma | H3149 | |
Insulin growth factor 1 (IGF1) | Invitrogen | M7512 | |
Knockout Serum Replacer | Gibco | A31815 | |
Laminin | Sigma | L-6274 | |
2-Mercaptoethanol | Sigma | M3148-100ML | |
MitoTracker CMXRos | Invitrogen | M7512 | |
Neurobasal medium | Gibco | 21103-049 | |
Non Essential Amino Acids | Gibco | 11140-050 | |
N2 NeuroPle Serum-Free Supplement | Gemini | 400-163 | |
Olympus microscope IX83 | Olympus | IX83-ZDC2 | |
PBS | Corning | 21-031-CV | |
Phase contrast microscope | Olympus | CKX41/ IX2-SLP | |
6 well plates | Corning | 353046 | |
24 well plates | Corning | 353047 | |
Poly-L-ornithine hydrobromide (polyornithine)) | Sigma-Aldrich | P3655 | |
SB431542 | Stemgent | 04-0010 | |
Sterile 50ml Disposable Vacuum Filtration System 0.22 μm Millipore Express® Plus Membrane | Millipore | SCGP00525 | |
Stericup 500/1000 ml Durapore 0.22 μM PVDF | Millipore | SCGVU10RE | |
Tbr1 antibody (1:2000) | Chemicon | AB9616 | |
Trypsin inhibitor | Gibco | 17075029 | |
50 ml tubes | Phenix | SS-PH50R | |
15 ml tubes | Phenix | SS-PH15R | |
T25 flasks (untreated) | VWR | 10861-572 | |
Plugins for softwares | |||
Bio-formats Package | http://downloads.openmicroscopy.org/bio-formats/5.1.0/ | ||
Fiji software | https://fiji.sc/ | ||
Kymograph Plugin | https://www.embl.de/eamnet/html/body_kymograph.html | ||
MultipleKymograph.class | https://www.embl.de/eamnet/html/body_kymograph.html | ||
MultipleOverlay.class | https://www.embl.de/eamnet/html/body_kymograph.html | ||
WalkingAverage.class | https://www.embl.de/eamnet/html/body_kymograph.html | ||
StackDifference.class | https://www.embl.de/eamnet/html/body_kymograph.html | ||
Straighten_.jar | https://imagej.nih.gov/ij/plugins/straighten.html | ||
tsp050706.txt | https://www.embl.de/eamnet/html/body_kymograph.html |
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