Name: analyzing mitochondrial transport and morphology in human induced pluripotent stem cell-derived neurons in hereditary spastic paraplegia
Uploaded: 2020-02-09T13:00:00
Description: Watch this Scientific Journal Video about Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia at JoVE.com

This work was supported by the Spastic Paraplegia Foundation, the Blazer Foundation and the NIH (R21NS109837).

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.
<ol>
	<li>Culture iPSCs on mouse embryonic fibroblast (MEF) feeders in human embryonic stem cell (hESC) medium supplemented with ...

<ol>
	<li>Brown, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+transport+of+membranous+and+nonmembranous+cargoes:+a+unified+perspective.">Axonal transport of membranous and nonmembranous cargoes: a unified perspective.</a> Journal of Cell Biology. 160 (6), 817-821 (2003).</li><li>Morris, R. L., Hollenbeck, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+transport+of+mitochondria+along+microtubules+and+F-actin+in+living+vertebrate+neurons.">Axonal transport of mitochondria along microtubules and F-actin in living vertebrate neurons.</a> The Journal of Cell Biology. 131 (5), 1315-1326 (1995).</li><li>Schwarz, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+trafficking+in+neurons.">Mitochondrial trafficking in neurons.</a> Cold Spring Harbor Perspectives in Biology. 5 (6), (2013).</li><li>Magrane, J., Cortez, C., Gan, W. B., Manfredi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+mitochondrial+transport+and+morphology+are+common+pathological+denominators+in+SOD1+and+TDP43+ALS+mouse+models.">Abnormal mitochondrial transport and morphology are common pathological denominators in SOD1 and TDP43 ALS mouse models.</a> Human Molecular Genetics. 23 (6), 1413-1424 (2014).</li><li>Alami, N. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+transport+of+TDP-43+mRNA+granules+is+impaired+by+ALS-causing+mutations.">Axonal transport of TDP-43 mRNA granules is impaired by ALS-causing mutations.</a> Neuron. 81 (3), 536-543 (2014).</li><li>Wang, X., Schwarz, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+axonal+transport+of+mitochondria.">Imaging axonal transport of mitochondria.</a> Methods in Enzymology. 457, 319-333 (2009).</li><li>Course, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Imaging+Mitochondrial+Transport+in+Neurons.">Live Imaging Mitochondrial Transport in Neurons.</a> Neuromethods. 123, 49-66 (2017).</li><li>Chazotte, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labeling+mitochondria+with+fluorescent+dyes+for+imaging.">Labeling mitochondria with fluorescent dyes for imaging.</a> Cold Spring Harbor Protocols. 2009 (6), 4948 (2009).</li><li>Meijering, E., Dzyubachyk, O., Smal, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+cell+and+particle+tracking.">Methods for cell and particle tracking.</a> Methods in Enzymology. 504, 183-200 (2012).</li><li>Bros, H., Hauser, A., Paul, F., Niesner, R., Infante-Duarte, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+Mitochondrial+Movement+Within+Neurons:+Manual+Versus+Automated+Tracking+Methods.">Assessing Mitochondrial Movement Within Neurons: Manual Versus Automated Tracking Methods.</a> Traffic. 16 (8), 906-917 (2015).</li><li>Calkins, M. J., Manczak, M., Mao, P., Shirendeb, U., Reddy, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+mitochondrial+biogenesis,+defective+axonal+transport+of+mitochondria,+abnormal+mitochondrial+dynamics+and+synaptic+degeneration+in+a+mouse+model+of+Alzheimer's+disease.">Impaired mitochondrial biogenesis, defective axonal transport of mitochondria, abnormal mitochondrial dynamics and synaptic degeneration in a mouse model of Alzheimer's disease.</a> Human Molecular Genetics. 20 (23), 4515-4529 (2011).</li><li>Denton, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+mitochondrial+dynamics+underlie+axonal+defects+in+hereditary+spastic+paraplegias.">Impaired mitochondrial dynamics underlie axonal defects in hereditary spastic paraplegias.</a> Human Molecular Genetics. 27 (14), 2517-2530 (2018).</li><li>Kim-Han, J. S., Antenor-Dorsey, J. A., O'Malley, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+parkinsonian+mimetic,+MPP+,+specifically+impairs+mitochondrial+transport+in+dopamine+axons.">The parkinsonian mimetic, MPP+, specifically impairs mitochondrial transport in dopamine axons.</a> Journal of Neuroscience. 31 (19), 7212-7221 (2011).</li><li>Shirendeb, U. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutant+huntingtin's+interaction+with+mitochondrial+protein+Drp1+impairs+mitochondrial+biogenesis+and+causes+defective+axonal+transport+and+synaptic+degeneration+in+Huntington's+disease.">Mutant huntingtin's interaction with mitochondrial protein Drp1 impairs mitochondrial biogenesis and causes defective axonal transport and synaptic degeneration in Huntington's disease.</a> Human Molecular Genetics. 21 (2), 406-420 (2012).</li><li>Lo Giudice, T., Lombardi, F., Santorelli, F. M., Kawarai, T., Orlacchio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hereditary+spastic+paraplegia:+clinical-genetic+characteristics+and+evolving+molecular+mechanisms.">Hereditary spastic paraplegia: clinical-genetic characteristics and evolving molecular mechanisms.</a> Experimental Neurology. 261, 518-539 (2014).</li><li>Blackstone, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+pathways+of+hereditary+spastic+paraplegia.">Cellular pathways of hereditary spastic paraplegia.</a> Annual Review of Neuroscience. 35, 25-47 (2012).</li><li>Boisvert, E. M., Denton, K., Lei, L., Li, X. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+specification+of+telencephalic+glutamatergic+neurons+from+human+pluripotent+stem+cells.">The specification of telencephalic glutamatergic neurons from human pluripotent stem cells.</a> Journal of Visualized Experiments. (74), e50321 (2013).</li><li>Zhu, P. P., Denton, K. R., Pierson, T. M., Li, X. J., Blackstone, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacologic+rescue+of+axon+growth+defects+in+a+human+iPSC+model+of+hereditary+spastic+paraplegia+SPG3A.">Pharmacologic rescue of axon growth defects in a human iPSC model of hereditary spastic paraplegia SPG3A.</a> Human Molecular Genetics. 23 (21), 5638-5648 (2014).</li><li>Marra, M. H., Tobias, Z. J., Cohen, H. R., Glover, G., Weissman, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Time-Lapse+Imaging+in+the+Zebrafish+Lateral+Line:+A+Flexible,+Open-Ended+Research+Project+for+an+Undergraduate+Neurobiology+Laboratory+Course.">In Vivo Time-Lapse Imaging in the Zebrafish Lateral Line: A Flexible, Open-Ended Research Project for an Undergraduate Neurobiology Laboratory Course.</a> Journal of Undergraduate Neuroscience Education. 13 (3), 215-224 (2015).</li><li>Kang, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Docking+of+axonal+mitochondria+by+syntaphilin+controls+their+mobility+and+affects+short-term+facilitation.">Docking of axonal mitochondria by syntaphilin controls their mobility and affects short-term facilitation.</a> Cell. 132 (1), 137-148 (2008).</li><li>Mou, Y., Li, X. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+axonal+defects+by+targeting+mitochondrial+dynamics+in+hereditary+spastic+paraplegias.">Rescue axonal defects by targeting mitochondrial dynamics in hereditary spastic paraplegias.</a> Neural Regeneration Research. 14 (4), 574-577 (2019).</li><li>Huang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+photostable+naphthalimide-based+fluorescent+probe+for+mitochondrial+imaging+and+tracking.">New photostable naphthalimide-based fluorescent probe for mitochondrial imaging and tracking.</a> Biosensors &amp; Bioelectronics. 71, 313-321 (2015).</li><li>Carvalho, P. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designed+benzothiadiazole+fluorophores+for+selective+mitochondrial+imaging+and+dynamics.">Designed benzothiadiazole fluorophores for selective mitochondrial imaging and dynamics.</a> Chemistry. 20 (47), 15360-15374 (2014).</li><li>Yamakoshi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+and+specific+Raman+probe+based+on+bisarylbutadiyne+for+live+cell+imaging+of+mitochondria.">A sensitive and specific Raman probe based on bisarylbutadiyne for live cell imaging of mitochondria.</a> Bioorganic &amp; Medicinal Chemistry Letters. 25 (3), 664-667 (2015).</li><li>Neumann, S., Chassefeyre, R., Campbell, G. E., Encalada, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KymoAnalyzer:+a+software+tool+for+the+quantitative+analysis+of+intracellular+transport+in+neurons.">KymoAnalyzer: a software tool for the quantitative analysis of intracellular transport in neurons.</a> Traffic. 18 (1), 71-88 (2017).</li><li>Chen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+quantifying+mitochondrial+axonal+transport.">A new method for quantifying mitochondrial axonal transport.</a> Protein &amp; Cell. 7 (11), 804-819 (2016).</li><li>De Vos, K. J., Sheetz, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+and+quantification+of+mitochondrial+dynamics+in+living+animal+cells.">Visualization and quantification of mitochondrial dynamics in living animal cells.</a> Methods in Cell Biology. 80, 627-682 (2007).</li><li>Denton, K. R., Xu, C. C., Li, X. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+Axonal+Phenotypes+with+Human+Pluripotent+Stem+Cells.">Modeling Axonal Phenotypes with Human Pluripotent Stem Cells.</a> Methods in Molecular Biology. 1353, 309-321 (2016).</li><li>Andrews, S., Gilley, J., Coleman, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Difference+Tracker:+ImageJ+plugins+for+fully+automated+analysis+of+multiple+axonal+transport+parameters.">Difference Tracker: ImageJ plugins for fully automated analysis of multiple axonal transport parameters.</a> Journal of Neuroscience Methods. 193 (2), 281-287 (2010).</li><li>Reis, G. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+motor+function+in+axonal+transport+in+vivo+probed+by+genetic+and+computational+analysis+in+Drosophila.">Molecular motor function in axonal transport in vivo probed by genetic and computational analysis in Drosophila.</a> Molecular Biology of the Cell. 23 (9), 1700-1714 (2012).</li><li>Broeke, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+quantification+of+cellular+traffic+in+living+cells.">Automated quantification of cellular traffic in living cells.</a> Journal of Neuroscience Methods. 178 (2), 378-384 (2009).</li><li>Welzel, O., Knorr, J., Stroebel, A. M., Kornhuber, J., Groemer, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fast+and+robust+method+for+automated+analysis+of+axonal+transport.">A fast and robust method for automated analysis of axonal transport.</a> European Biophysics Journal : EBJ. 40 (9), 1061-1069 (2011).</li><li>Mukherjee, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+kymograph+analysis+for+profiling+axonal+transport+of+secretory+granules.">Automated kymograph analysis for profiling axonal transport of secretory granules.</a> Medical Image Analysis. 15 (3), 354-367 (2011).</li><li>Klionsky, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+the+use+and+interpretation+of+assays+monitoring+autophagy.">Guidelines for the use and interpretation of assays monitoring autophagy.</a> Autophagy. 12, 1 (2016).</li><li>Metivier, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytofluorometric+detection+of+mitochondrial+alterations+in+early+CD95/Fas/APO-1-triggered+apoptosis+of+Jurkat+T+lymphoma+cells.+Comparison+of+seven+mitochondrion-specific+fluorochromes.">Cytofluorometric detection of mitochondrial alterations in early CD95/Fas/APO-1-triggered apoptosis of Jurkat T lymphoma cells. Comparison of seven mitochondrion-specific fluorochromes.</a> Immunology Letters. 61 (2-3), 157-163 (1998).</li><li>Scaduto, R. C., Grotyohann, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+mitochondrial+membrane+potential+using+fluorescent+rhodamine+derivatives.">Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives.</a> Biophysical Journal. 76, 469-477 (1999).</li><li>Liu, X., Yang, L., Long, Q., Weaver, D., Hajnoczky, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choosing+proper+fluorescent+dyes,+proteins,+and+imaging+techniques+to+study+mitochondrial+dynamics+in+mammalian+cells.">Choosing proper fluorescent dyes, proteins, and imaging techniques to study mitochondrial dynamics in mammalian cells.</a> Biophysics Reports. 3 (4), 64-72 (2017).</li><li>Zhou, B., Lin, M. Y., Sun, T., Knight, A. L., Sheng, Z. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+mitochondrial+transport+in+neurons.">Characterization of mitochondrial transport in neurons.</a> Methods in Enzymology. 547, 75-96 (2014).</li></ol>

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...

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 kinesin and dynein, participate in the fast organelle transport along microtubules to control the movement of mitochondria2,3. Normal neuronal activity requires proper transport of newly assembled mitochondria from neuronal soma to distal axon (anterograde axonal transport) and reverse transport of mitochondria from the distal axon back to the cell body (retrograde transport). Recent studies have indicated that improper mitochondrial allocation is strongly associated with neuronal defects and motor neuron degenerative diseases4,5. Therefore, to dissect the role of mitochondria in neurodegeneration, it is important to establish methods for examining mitochondrial movement along axons in live cultures.
There are two main challenges in examining and analyzing the tracking of mitochondria: (1) identifying mitochondria from the background in every frame, and (2) analyzing and generating the connections between every frame. In resolving the first challenge, a fluorescence labeling approach is used widely to distinguish mitochondria from the background, such as MitoTracker dye or transfection of fluorescence-fused mitochondrial targeting protein (e.g., mito-GFP)6,7,8. To analyze the association between frames, several algorithms and software tools have been described in previous studies9. In a recent paper, researchers compared four different automated tools (e.g., Volocity, Imaris, wrMTrck, and Difference Tracker) to quantify mitochondrial transport. The results showed that despite discrepancies in track length, mitochondrial displacement, movement duration, and velocity, these automated tools are suitable for evaluating transport difference after treatment10. In addition to these tools, an integrated plugin &#34;Macros&#34; for ImageJ (written by Rietdorf and Seitz) has been widely used for analyzing mitochondrial transport11. This method generates kymographs that can be used to analyze mitochondrial movement, including velocity in both anterograde and retrograde directions.
Mitochondria are highly dynamic organelles that constantly change in number and morphology in response to both physiological and pathological conditions. Mitochondrial fission and fusion tightly regulate mitochondrial morphology and homeostasis. The imbalance between mitochondrial fission and fusion can induce extremely short or long mitochondrial networks, which can impair mitochondrial function and result in abnormal neuronal activities and neurodegeneration. Impaired mitochondrial transport and morphology are involved in various neurodegenerative diseases, such as Alzheimer&#39;s disease, Parkinson&#39;s disease, Huntington&#39;s disease, and hereditary spastic paraplegia (HSP)12,13,14,15. HSP is a heterogeneous group of inherited neurological disorders characterized by the degeneration of the corticospinal tract and subsequent failure to control lower limb muscles16,17. In this study, iPSC-derived forebrain neurons are used to assess mitochondrial transport and morphology in HSP. This method provides a unique paradigm for examining mitochondrial dynamics of neuronal axons in live cultures.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Accutase Cell Detachment Solution</td>
			<td>Innovative Cell Technologies</td>
			<td>AT104</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety hood</td>
			<td>Thermo Scientific</td>
			<td>1300 SERIES A2</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A-7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain derived neurotrophic factor (BDNF)</td>
			<td>Peprotech</td>
			<td>450-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>Sorvall Legend X1R/ 75004261</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Chemiglass Life Sciences</td>
			<td>1760-012</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyclic AMP (cAMP)</td>
			<td>Sigma-Aldrich</td>
			<td>D0627</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Gibco</td>
			<td>17105-041</td>
			<td></td>
		</tr>
		<tr>
			<td>Dorsomorphin</td>
			<td>Selleckchem</td>
			<td>S7146</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified eagle medium with F12 nutrient mixture (DMEM/F12)</td>
			<td>Corning</td>
			<td>10-092-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>16141-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibroblast growth factor 2 (FGF2, bFGF)</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix</td>
			<td>Gibco</td>
			<td>A1413201</td>
			<td></td>
		</tr>
		<tr>
			<td>Gem21 NeuroPlex Serum-Free Supplement</td>
			<td>Gemini</td>
			<td>400-160</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bottom Dishes</td>
			<td>MatTek</td>
			<td>P35G-0.170-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>9&#39;&#39; glass pipetes</td>
			<td>VWR</td>
			<td>14673-043</td>
			<td></td>
		</tr>
		<tr>
			<td>Glial derived neurotrophic factor (BDNF)</td>
			<td>Sigma-Aldrich</td>
			<td>D0627</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX-I</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H3149</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin growth factor 1 (IGF1)</td>
			<td>Invitrogen</td>
			<td>M7512</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout Serum Replacer</td>
			<td>Gibco</td>
			<td>A31815</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma</td>
			<td>L-6274</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M3148-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>MitoTracker CMXRos</td>
			<td>Invitrogen</td>
			<td>M7512</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Non Essential Amino Acids</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 NeuroPle Serum-Free Supplement</td>
			<td>Gemini</td>
			<td>400-163</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus microscope IX83</td>
			<td>Olympus</td>
			<td>IX83-ZDC2</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Phase contrast microscope</td>
			<td>Olympus</td>
			<td>CKX41/ IX2-SLP</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plates</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well plates</td>
			<td>Corning</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-ornithine hydrobromide (polyornithine))</td>
			<td>Sigma-Aldrich</td>
			<td>P3655</td>
			<td></td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Stemgent</td>
			<td>04-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 50ml Disposable Vacuum Filtration System 0.22 &#956;m Millipore Express&#174; Plus Membrane</td>
			<td>Millipore</td>
			<td>SCGP00525</td>
			<td></td>
		</tr>
		<tr>
			<td>Stericup 500/1000 ml Durapore 0.22 &#956;M PVDF</td>
			<td>Millipore</td>
			<td>SCGVU10RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Tbr1 antibody (1:2000)</td>
			<td>Chemicon</td>
			<td>AB9616</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin inhibitor</td>
			<td>Gibco</td>
			<td>17075029</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml tubes</td>
			<td>Phenix</td>
			<td>SS-PH50R</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml tubes</td>
			<td>Phenix</td>
			<td>SS-PH15R</td>
			<td></td>
		</tr>
		<tr>
			<td>T25 flasks (untreated)</td>
			<td>VWR</td>
			<td>10861-572</td>
			<td></td>
		</tr>
		<tr>
			<td>Plugins for softwares</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-formats Package</td>
			<td><a href="http://downloads.openmicroscopy.org/bio-formats/5.1.0/" target="_blank">http://downloads.openmicroscopy.org/bio-formats/5.1.0/</a></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji software</td>
			<td><a href="https://fiji.sc/" target="_blank">https://fiji.sc/</a></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kymograph Plugin</td>
			<td><a href="https://www.embl.de/eamnet/html/body_kymograph.html" target="_blank">https://www.embl.de/eamnet/html/body_kymograph.html</a></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MultipleKymograph.class</td>
			<td><a href="https://www.embl.de/eamnet/html/body_kymograph.html" target="_blank">https://www.embl.de/eamnet/html/body_kymograph.html</a></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MultipleOverlay.class</td>
			<td><a href="https://www.embl.de/eamnet/html/body_kymograph.html" target="_blank">https://www.embl.de/eamnet/html/body_kymograph.html</a></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>WalkingAverage.class</td>
			<td><a href="https://www.embl.de/eamnet/html/body_kymograph.html" target="_blank">https://www.embl.de/eamnet/html/body_kymograph.html</a></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>StackDifference.class</td>
			<td><a href="https://www.embl.de/eamnet/html/body_kymograph.html" target="_blank">https://www.embl.de/eamnet/html/body_kymograph.html</a></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Straighten_.jar</td>
			<td><a href="https://imagej.nih.gov/ij/plugins/straighten.html" target="_blank">https://imagej.nih.gov/ij/plugins/straighten.html</a></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>tsp050706.txt</td>
			<td><a href="https://www.embl.de/eamnet/html/body_kymograph.html" target="_blank">https://www.embl.de/eamnet/html/body_kymograph.html</a></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

analyzing mitochondrial transport and morphology in human induced pluripotent stem cell-derived neurons in hereditary spastic paraplegia

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 &#34;MultiKymograph&#34;, &#34;Bioformat importer&#34;, and &#34;Macros&#34; 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.

Here, human iPSCs were differentiated into telencephalic glutamatergic neurons, which were characterized by immunostaining with Tbr1 and &#946;III 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 &#34;MultiKymograph&#34; and &#34;Macros&#34; ImageJ pl...

Watch this Scientific Journal Video about Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia at JoVE.com

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

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 ...

Research

JoVE Journal

Neuroscience

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

Analyzing Dendritic Morphology in Columns and Layers

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and ...

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

Method for High Speed Stretch Injury of Human Induced Pluripotent Stem Cell-derived Neurons in a 96-well Format

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven ...

Compartmentalization of Human Stem Cell-Derived Neurons within Pre-Assembled Plastic Microfluidic Chips

Use of microfluidic devices to compartmentalize cultured neurons has become a standard method in neuroscience. This protocol shows how to use a ...

Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model ...

Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

Conversion of Human Induced Pluripotent Stem Cells iPSCs into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into ...