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 fibroblast growth factor (bFGF, 4 ng/mL).</li>
	<li>After incubation with dispase (1 mg/mL) for 3 min, dissociate the iPSCs into small clumps. Then, culture the iPSC aggregates in suspension in the hESC medium for 4 days. Refer to this timepoint, when the iPSCs are starting as the suspension culture, as day 1 (D1). Change the medium daily for 4 days.</li>
	<li>At D5, collect the iPSC aggregates after centrifugation at 200 x g for 2 min and culture in neural induction media (NIM) for 3 days. Culture the cell aggregates in suspension for 3 days by changing half of the media every 2 days. Add DMH-1 (2 &#956;M) and SB431542 (2 &#956;M) to NIM medium to increase neural induction efficiency.</li>
	<li>At D8, collect the iPSC aggregates after centrifugation at 200 x g for 2 min and let them adhere to 6 well plates (around 20&#8211;30 aggregates per well of the plate) by culturing in NIM with 10% FBS (or with laminin-coated plates) overnight. On the following day, remove the old medium and change to NIM every 2 days until D17. 
	NOTE: The generation of neuroepithelial cells, which is indicated by the formation of columnar cells with rosette structures, is observed in this period.</li>
	<li>At D17, mechanically isolate the neuroepithelial cells in the center of colonies (lifting off directly by applying a small pressure to the center of the colonies) or enzymatically (treated with dispase). Transfer the isolated neuroepithelial cells to non-treated tissue culture T25 flask containing 8 mL of NIM with addition of B27 (1x), cAMP (1 &#181;M), and IGF (10 ng/mL). 
	NOTE: The suspension culture allows cells to form neurospheres that are enriched with cortical projection neural progenitors.</li>
	<li>After D35, plate the neurospheres on the poly-ornithine and LDEV-free reduced growth factor basement membrane matrix (Table of Materials)-coated 35 mm glass bottom dishes to generate telencephalic glutamatergic neurons (cortical projection neurons). Before plating, dissociate the neurospheres to small clusters by incubating with 1 mg/mL cell detachment solution for 2 min at 37 &#176;C.</li>
	<li>Remove the cell detachment solution by centrifugation and resuspend in 1 mL of neural differentiation medium (NDM). Plate cells on the glass bottom dish (around five clusters in 100 &#181;L of medium per dish) to let them attach. Then, add NDM (1 mL per dish) and culture the cells in NDM containing B27 (1x), cAMP (1 &#181;M), IGF (10 ng/mL), hBDNF (10 ng/mL), and GDNF (10 ng/mL). 
	NOTE: Small clusters are plated because they survive well and give rise to long projection neurons. Alternatively, cells can be dissociated and plated at a density around 20,000 cells per dish for better separation.</li>
	<li>Characterize the telencephalic glutamatergic neurons by immunostaining the Tbr1 and &#946;III-tubulin markers.</li>
</ol>
2. Examination of mitochondrial transport along axons of telencephalic glutamatergic neurons
<ol>
	<li>Warm up the NDM and turn on the incubator for the fluorescence microscope set at 37 &#176;C and 5% CO2.</li>
	<li>To visualize mitochondria along the axons of forebrain neurons, incubate the neurons with 50 nM red fluorescent dye to stain mitochondria in live cells (e.g., MitoTracker CMXRos) in NDM for 3 min at 37 &#176;C. Next, wash cells 2x with warmed NDM. The neurons are incubated in NDM.</li>
	<li>In order to record the mitochondrial transport along the axons, take time-lapse images of mitochondrial movement using the 40x objective with a fluorescence microscope. 
	NOTE: To stabilize the culture, perform the live cell imaging when the cells are incubated in the incubator for 20 min following mitochondrial staining.</li>
	<li>Under phase field, identify the axons based on morphological characteristics (directly emerge from neuronal cell body, constant thin long neurites with no branching). Mitochondria move in anterograde (from cell body to distal axon) and retrograde directions (from axonal terminal to cell body). To distinguish the direction of mitochondrial movement along axons, clearly identify the cell bodies of neurons. In this step, focus axons under phase field to reduce photobleaching.</li>
	<li>After distinguishing the cell body and axon, adjust the exposure time and focus of mitochondria in axons. Then, capture the transport of mitochondria within axons every 5 s for a total duration of 5 min, yielding 60 frames. Randomly capture at least five locations for each dish and repeat 3x for each group.</li>
</ol>
3. Data analysis of mitochondrial transport and morphology in cortical neurons
NOTE: Analyze the collected data on mitochondrial transport using an image analysis software (e.g., ImageJ or MetaMorph19). Since ImageJ is readily available, perform the analysis of mitochondrial transport and morphology using ImageJ with the MultiKymograph, Macros, and Analyze particles plugins.
<ol>
	<li>Analyzing mitochondrial transport using ImageJ
	<ol>
		<li>After capturing the time-lapse images of mitochondrial motility, analyze the velocity and movement of mitochondria using the MultiKymograph plugin. The images are saved as .tiff format files, which are analyzed by Fiji software following a previously reported method20.</li>
		<li>Download the Fiji software. Download plugins that will be needed for analyzing mitochondrial moving velocity from kymographs. Download the Bio-formats Package and Kymograph Plugin along with these four .class plugins, including: MultipleKymograph.class, MultipleOverlay.class, StackDifference.class, and WalkingAverage.class (Table of Materials). Move these files to the Fiji plugins folder. Download &#34;tsp050706.txt&#34; plugins to the plugins folder. Restart the Fiji software when the files are moved to the plugins folder.</li>
		<li>Open Fiji software. Choose plugins, then import images of the .tiff series through Bio-Formats Importer under Bio-Formats. Make sure to choose Standard ImageJ in Stack viewing, choose Open all series, check Autoscale, then click OK. Take note of the frame number and size of images in pixels, which are displayed on the top of the image. 
		NOTE: Take 60 frames per image.</li>
		<li>Consider making the images clearer by adjusting brightness/contrast under the Image menu for all 60 frames.</li>
		<li>Right-click the line tool to choose segmented line and draw a segmented line starting from the cell body and ending at the terminal axon. Generate the kymograph by choosing MultipleKymograph under Plugins. The selection of line width is prompted after choosing the MultipleKymograph. Ensure that this is an odd number. Choose 1 for the line width. A kymograph is generated after this step. 
		NOTE: Several important pieces of information can be read from the kymograph. The y-axis of kymograph is the time of duration (5 min) for the 60 frames. The x-axis represents the position of the selected axons.</li>
		<li>Use a diagonal line in the kymograph to determine the movement direction of mitochondria (anterograde, retrograde, or stable). For example, a line going down to the right along the y-axis indicates anterograde movement, and a line going down to the left along the y-axis indicates retrograde movement. A vertical line indicates that there was no movement in the mitochondrion.</li>
		<li>Measure the distance, time values, and velocity for the moving mitochondria using the Macros plugin in Fiji software. Go to Plugins | Macros | Install | tsp050607.txt. Draw a segmented line over the trace of mitochondrial movement on the kymograph, and always draw the line from the superior to inferior region (y-axis).</li>
		<li>After drawing the line, go to Plugins | Macros | read velocities from tsp. Since a segmented line along the trace is drawn, the plugin reads segmented velocities corresponding to the line. 
		NOTE: The unit for all data is pixels. dy sum is the time consumed from the starting point, and dx sum indicates the distance of the mitochondrion moving in the x-axis. dy now and dx now shows the period time and movement distance for every segment, respectively. actual speed shows the speed for every segment (dx now/dy now). average speed indicates the average speed for the mitochondrial moving (dx sum/dy sum).</li>
		<li>Change the unit of dx now from pixel to &#181;m as the ratio of pixel in &#181;m by measuring the scale bar. Convert the unit for distance from pixel to &#181;m by measuring scale bars in images. Use the line tool to draw a line along the scale bar and measure the length of the line by choose Measure&#39; under &#34;Analyze. Change the time from pixel to seconds, as 1 pixel = 5 seconds in this experiment.</li>
		<li>In the spreadsheet file, calculate the average velocity and correspondingly label retrograde or anterograde movement. Average the anterograde or retrograde movement velocity.</li>
		<li>Determine the percentage of stationary and motile mitochondria from the kymograph. The anterograde or retrograde moving mitochondria are defined as moving 5 &#181;m forward or backward from the origin during the entire period21. Mitochondria are considered stationary if they did not move more than 5 &#181;m during the 5 min.</li>
	</ol>
	</li>
	<li>Analysis of mitochondrial morphology using ImageJ 
	NOTE: Determine mitochondrial morphology by measuring mitochondrial length, area, and aspect ratio using ImageJ. To do so, follow the steps below.
	<ol>
		<li>In order to analyze the mitochondrial length and area within axons, download the Straighten_.jar plugin from ImageJ website (see Table of Materials) and move it to the folder of plugins. Restart ImageJ software.</li>
		<li>Open the picture through the open function under File and convert the 32 bit image to 8 bit using Type under Image.</li>
		<li>Draw a segmented line along the axons. Choose Straighten under Plugins and set 50 pixels for Width of Filament/Wide Line. This will generate a straightened axon.</li>
		<li>Adjust the threshold under Image and set the measurement under Analyze by choosing &#34;Area | Perimeter | Fit ellipse | Shape descriptors.</li>
		<li>Measure the scale bar using the line function and set the scale under Analyze by filling the distance in pixel, know distance, and the unit of length. Then, choose Global to set this scale setting.</li>
		<li>Use Analyze Particles under Analyze to determine the area. The prompt parameters are size (pixel^2) = 0.20&#8211;infinity, circularity = 0.00&#8211;1.00, and show = ellipses. Choose Display results. 
		NOTE: The measurement will yield the results of multiple mitochondrial morphology parameters including area, perimeters, length (major), width (minor), and aspect ratio (AR). The corresponding mitochondrial number is also listed.</li>
		<li>Calculate the mitochondrial number per axon (&#181;m) using the mitochondrial number divided by axonal length.</li>
	</ol>
	</li>
</ol>

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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>

The authors declare no competing financial interests.

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 from the dish, it is important to leave some solution in the dish and gently add neurobasal medium. The washing can be performed three or four times to remove the dye. In addition, mitochondria can be labeled with other reporters to measure their transport along axons, such as fluorescent protein fused mitochondria targeting proteins6. In long-term tracking, several probes (i.e., NPA-TPP, 2,1,3-benzothiadiazole [BTD] fluorescent derivatives, and a specific Raman probe) showed great potential in long-term mitochondrial tracking and mitochondrial dynamics analysis23,24,25. The advantages and disadvantages of various probes can be found in the Table 1.
After mitochondrial staining, live cell imaging is performed using a microscope equipped with an incubator. To effectively focus the neurons during imaging, neural samples should be kept in the 37 &#176;C incubator with 5% CO2 and in a humid environment for at least 15 min. To minimize the out-of-focus effects, images at different Z-positions can be taken to make a Z-stack, or the auto-focus function can be utilized. Importantly, to identify the direction of mitochondrial transport (anterograde or retrograde), phase images are taken to distinguish the neuronal cell body and axons. Another critical issue is photobleaching of fluorescent samples, which must be prevented to obtain efficient mitochondrial transport time-lapse images. An effective method to minimize photobleaching is to focus samples through the eyepiece and set all imaging parameters under the phase channel, except for exposure time. Moreover, the automatic scaling pattern can decrease photobleaching of fluorescence.
Mitochondria are highly dynamic organelles and can move in both anterograde and retrograde directions. In neurons, a few mitochondria within axons stay stationary during the recording. Among the moving mitochondria, motion status can vary over time. This phenomenon raises the important question of which mitochondria type is considered stationary or moving. This can be resolved by setting the threshold during mitochondrial axonal transport analysis. To distinguish the static mitochondria, Neumann et al. used the mitochondrial track center, which is defined as the mean of its position coordinates over time, then set the threshold to 350 nm/s so that the maximum deviation distance of the mitochondrion from its track center is in the first frame26. In another study, the authors set 50 nm/s as the threshold to distinguish stationary status from the moving status27. A 300 nm/s threshold was used here to distinguish the microtubule-based transport as done in previous reports28,29. Although the threshold for stationary and moving mitochondria is different, setting the threshold can provide important relative information on mitochondrial movement within axons between wild-type and degenerative neurons.
Most protocols involving mitochondrial transport analysis have used kymograms, which are two-dimensional representation of positions versus time. Multiple automated tools have been developed for the analysis of particle tracking26,30,31,32,33,34. These can accurately separate each frame. In addition to the velocity and motile percentage that ImageJ can measure, this method can measure motile events accurately. However, these are not free to use. Here, the analysis of mitochondrial transport was performed using ImageJ with the &#34;Multikymograph&#34; and &#34;Macros&#34; plugins. These plugins can effectively measure mitochondrial movement. The advantage of these plugins is their ease of use and ability to indicate alterations in mitochondrial axonal transport in the form of kymographs and velocity over time.
Motile mitochondria were analyzed in SPG3A and control neurons. A similar reduction in the percentage of motile mitochondria was observed using two different analysis methods, confirming the usefulness of ImageJ to analyze axonal transport. In addition, mitochondrial morphology can be analyzed using the same set of images, which provides important readouts for studying mitochondrial dysfunction in various neurological diseases. Since axonal degeneration and mitochondrial dysfunction usually occur during earlier stages, before neurons die, this method can be used to examine early pathological changes to help identify molecular pathways and screen therapeutics to rescue neurodegeneration.

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><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&#039;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&#039;&#039; 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 &amp;mu;m Millipore Express&amp;reg; Plus Membrane</td><td>Millipore</td><td>SCGP00525</td><td></td></tr><tr><td>Stericup 500/1000 ml Durapore 0.22 &amp;mu;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>&lt;strong&gt;Plugins for softwares&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Bio-formats Package</td><td>&lt;a href=&quot;http://downloads.openmicroscopy.org/bio-formats/5.1.0/&quot; target=&quot;_blank&quot;&gt;http://downloads.openmicroscopy.org/bio-formats/5.1.0/&lt;/a&gt;</td><td></td><td></td></tr><tr><td>Fiji software</td><td>&lt;a href=&quot;https://fiji.sc/&quot; target=&quot;_blank&quot;&gt;https://fiji.sc/&lt;/a&gt;</td><td></td><td></td></tr><tr><td>Kymograph Plugin</td><td>&lt;a href=&quot;https://www.embl.de/eamnet/html/body_kymograph.html&quot; target=&quot;_blank&quot;&gt;https://www.embl.de/eamnet/html/body_kymograph.html&lt;/a&gt;</td><td></td><td></td></tr><tr><td>MultipleKymograph.class</td><td>&lt;a href=&quot;https://www.embl.de/eamnet/html/body_kymograph.html&quot; target=&quot;_blank&quot;&gt;https://www.embl.de/eamnet/html/body_kymograph.html&lt;/a&gt;</td><td></td><td></td></tr><tr><td>MultipleOverlay.class</td><td>&lt;a href=&quot;https://www.embl.de/eamnet/html/body_kymograph.html&quot; target=&quot;_blank&quot;&gt;https://www.embl.de/eamnet/html/body_kymograph.html&lt;/a&gt;</td><td></td><td></td></tr><tr><td>WalkingAverage.class</td><td>&lt;a href=&quot;https://www.embl.de/eamnet/html/body_kymograph.html&quot; target=&quot;_blank&quot;&gt;https://www.embl.de/eamnet/html/body_kymograph.html&lt;/a&gt;</td><td></td><td></td></tr><tr><td>StackDifference.class</td><td>&lt;a href=&quot;https://www.embl.de/eamnet/html/body_kymograph.html&quot; target=&quot;_blank&quot;&gt;https://www.embl.de/eamnet/html/body_kymograph.html&lt;/a&gt;</td><td></td><td></td></tr><tr><td>Straighten_.jar</td><td>&lt;a href=&quot;https://imagej.nih.gov/ij/plugins/straighten.html&quot; target=&quot;_blank&quot;&gt;https://imagej.nih.gov/ij/plugins/straighten.html&lt;/a&gt;</td><td></td><td></td></tr><tr><td>tsp050706.txt</td><td>&lt;a href=&quot;https://www.embl.de/eamnet/html/body_kymograph.html&quot; target=&quot;_blank&quot;&gt;https://www.embl.de/eamnet/html/body_kymograph.html&lt;/a&gt;</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 plugins, shown in Figure 1.
There are three respective states of mitochondrial motion, including static, anterograde, and retrograde movement (Figure 1B,C). A single mitochondrion can remain static or move in anterograde or retrograde direction within an axon, and here, a segmented line was drawn along the track of mitochondrial movement to determine velocity (Figure 1D). The velocity of mitochondrial movement along the axon is shown in Figure 1E and corresponds to the segmented lines in Figure 1D after reading by &#34;Macros&#34; in ImageJ. Similar to the MetaMorph software, ImageJ can be used to determine the mitochondrial velocity and percentage of motile mitochondria based on the kymograph generated by ImageJ (Figure 1F). Using commercially available analysis software (e.g., MetaMorph), previous data showed that the percentage of motile mitochondria was significantly reduced in SPG3A neurons compared to normal neurons, while velocity was not altered19. To evaluate the ImageJ software, the percentage of motile mitochondria was examined, and a similar reduction in the percentage of motile mitochondria in SPG3A neurons compared to control wild-type (WT) neurons was observed (Figure 1G).
Regarding analysis of mitochondrial morphology, the mitochondrial area, length, and AR were analyzed using the &#34;analyze particles&#34; function in ImageJ. Axons were straightened using the &#34;straighten&#34; plugin (Figure 2A,B). Mitochondria were chosen clearly by adjusting the threshold (Figure 2C,D). Finally, the mitochondrial area, length (major), width (minor), aspect ratio, and perimeter were obtained from the straightened axon using the &#34;Analyze Particles&#34; plugin (Figure 2E,F). Abnormal mitochondrial morphology was (and has previously been) observed in HSP iPSC-derived telencephalic glutamatergic neurons, including SPG15 cells (Figure 2G,H,I)13,22. Both the mitochondrial length and aspect ratio were significantly reduced in SPG15 neuron axons compared to control WT neuron axons (Figure 2G,H,I).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60548/60548fig01.jpg" /> 
Figure 1: Analysis of mitochondrial transport using ImageJ. (A) Immunostaining showing the generation of telencephalic glutamatergic neurons. Tbr1 (red), &#946;III tubulin (green), and Hoechst (blue). Scale bars = 50 &#181;m. This figure has been modified from a previous study19. (B) Mitochondrial transport within axons of neurons. Anterograde mitochondrial movement is defined as the mitochondria moving from the cell body to distal axon, and retrograde mitochondrial movement originates from the distal axon and extends to the neuronal cell body. The segment line is drawn along axons from neuronal cell body to distal axonal terminal in order to generate kymographs. (C) Kymograph is generated using ImageJ; x-axis is the axonal position and y-axis is time. One continuous white line is the mitochondrion moving in anterograde direction (pink arrowhead), retrograde direction (blue arrowhead), or static state (yellow arrowhead). (D) The segmented line is drawn to read the velocity using the Macros plugin. Numbers 1&#8211;8 describe the segment of the line. Anterograde movement (1&#8211;4, 6, 8) and static state (5, 7) can be distinguished from this magnified kymograph. (E) Time and moving distance for each segment, actual and average velocity (in pixels). (F) Kymograph of mitochondrial transport for WT and SPG3A cortical PNs using ImageJ. Scale bar = 10 &#181;m. (G) Motile mitochondrial ratio in WT and SPG3A cortical PNs using ImageJ (*p &#60; 0.05). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60548/60548fig1alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60548/60548fig2a.jpg" /> 
Figure 2: Analyzing mitochondrial morphology using ImageJ. (A) Parameters for straightening the axon. (B) The representative straightened axon. (C,D) Adjustment of threshold to choose mitochondria. (E,F) The measured mitochondrial area, perimeter, length (major), width (minor), and aspect ratio (AR) corresponding to the selected mitochondria in (E). (G) Representative pictures of mitochondria in WT and SPG15 telencephalic glutamatergic neurons. Scale bars = 20 &#181;m. (H) Mitochondrial length in WT and SPG15 neurons. (I) Aspect ratio of mitochondria in WT and SPG15 neurons. **p &#60; 0.01 vs. WT. (G), (H), and (I) are modified from a previous study13. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60548/60548fig2alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Fluorescent Tools</td>
			<td>Advantages</td>
			<td>Disadvantages</td>
			<td>References</td>
		</tr>
		<tr>
			<td>MitoTracker</td>
			<td>MitoTracker is mitochondrial potential-independent and can be used to analyze the colocalization of mitochondria with autophagosome or lysosome.</td>
			<td>MitoTracker is not photostable enough for long-term research.</td>
			<td>35</td>
		</tr>
		<tr>
			<td>NPA-TPP</td>
			<td>This dye is a novel photostable mitochondrial labeling reagent.</td>
			<td>The synthesis and purification process of this dye is time-consuming and costly.</td>
			<td>23</td>
		</tr>
		<tr>
			<td>MitoBADY</td>
			<td>This dye can be used for mitochondrial visualization using Raman microscopy with high sensitivity and specificity.</td>
			<td>Using this dye requires Raman microscope.</td>
			<td>25</td>
		</tr>
		<tr>
			<td>TMRE</td>
			<td>This dye is a non-toxic, specific mitochondrial staining dye with low concentration and no quenching effect.</td>
			<td>TMRE labeling mitochondria depends on the mitochondrial membrane potential.</td>
			<td>36, 37</td>
		</tr>
		<tr>
			<td>Mitochondria-targeted fluorescent proteins</td>
			<td>Mitochondria-targeted fluorescent proteins are more specific and stable.</td>
			<td>This method needs transfection and transfection efficiency is different for different cell types.</td>
			<td>38, 39</td>
		</tr>
	</tbody>
</table>
Table 1: Advantages and disadvantages of some fluorescent tools for mitochondrial labeling.

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 ...

analyzing-mitochondrial-transport-morphology-human-induced

Nanyang Technological University

Research

JoVE Journal

Neuroscience

7.7K Views.  University of Illinois College of Medicine Rockford. Mitochondriale disfunctie ligt ten grondslag aan vele neurodegeneratieve ziekten. Ons protocol biedt een belangrijk hulpmiddel voor het onderzoeken van mitochondriale dynamiek in de axonen, het vergemakkelijken van de studie van neurologische ziekten waarbij axonale degeneratie. Door mitochondriale etikettering, live celbeeldvorming en geïnduceerde pluripotente stamceltechnologie te combineren, kan ons protocol worden gebruikt om mitochondriale handel langs menselijke axonen te karakterisere...

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

In vitro Neuromuscular Junction Induced from Human Induced Pluripotent Stem Cells

The neuromuscular junction (NMJ) is a specialized synapse that transmits action potentials from the motor neuron to skeletal muscle for mechanical movement. The architecture of the NMJ structure influences the functions of the neuron, the muscle and the mutual interaction. Previous studies have reported many strategies by co-culturing the motor neurons and myotubes to generate NMJ in vitro with complex induction process and long culture period but have struggled to recapitulate mature NMJ morphology and function. Our in vitro NMJ induction system is constructed by differentiating human iPSC in a single culture dish. By switching the myogenic and neurogenic induction medium for induction, the resulting NMJ contained pre- and post- synaptic components, including motor neurons, skeletal muscle and Schwann cells in the one month culture. The functional assay of NMJ also showed that the myotubes contraction can be triggered by Ca++ then inhibited by curare, an acetylcholine receptor (AChR) inhibitor, in which the stimulating signal is transmitted through NMJ. This simple and robust approach successfully derived the complex structure of NMJ with functional connectivity. This in vitro human NMJ, with its integrated structures and function, has promising potential for studying pathological mechanisms and compound screening.

Here we provide a protocol to generate in vitro NMJs from human induced pluripotent stem cells (iPSCs). This method can induce NMJs with mature morphology and function in 1 month in a single well. The resulting NMJs could potentially be used to model related diseases, to study pathological mechanisms or to screen drug compounds for therapy.

The neuromuscular junction (NMJ) is a specialized synapse that transmits action potentials from the motor neuron to skeletal muscle for mechanical ...

Developmental Biology

A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases

Post-mortem studies of neurological diseases are not ideal for identifying the underlying causes of disease initiation, as many diseases include a long period of disease progression prior to the onset of symptoms. Because fibroblasts from patients and healthy controls can be efficiently reprogrammed into human induced pluripotent stem cells (hiPSCs), and subsequently differentiated into neural progenitor cells (NPCs) and neurons for the study of these diseases, it is now possible to recapitulate the developmental events that occurred prior to symptom onset in patients. We present a method by which to efficiently differentiate hiPSCs into NPCs, which in addition to being capable of further differentiation into functional neurons, can also be robustly passaged, freeze-thawed or transitioned to grow as neurospheres, enabling rapid genetic screening to identify the molecular factors that impact cellular phenotypes including replication, migration, oxidative stress and/or apoptosis. Patient derived hiPSC NPCs are a unique platform, ideally suited for the empirical testing of the cellular or molecular consequences of manipulating gene expression.

This protocol describes how neural progenitor cells can be differentiated from human induced pluripotent stem cells, in order to yield a robust and replicative neural cell population, which may be used to identify the developmental pathways contributing to disease pathogenesis in many neurological disorders.

Post-mortem studies of neurological diseases are not ideal for identifying the underlying causes of disease initiation, as many diseases include a long ...

Medicine

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to isolate nerve-specific mitochondria and evaluate disease-induced alterations of mitochondria in the distal tip of sensory nerves. The protocol combines fluorescence immunohistochemistry, confocal microscopy and 3D image analysis techniques to visualize and quantify nerve-specific mitochondria. Detailed parameters are defined throughout the procedures in order to provide a concrete example of how to use these techniques to isolate nerve-specific mitochondria. Antibodies were used to label nerve and mitochondrial signals within tissue sections of skin punch biopsies, which was followed by indirect immunofluorescence to visualize nerves and mitochondria with a green and red fluorescent signal respectively. Z-series images were acquired with confocal microscopy and 3D analysis software was used to process and analyze the signals. It is not necessary to follow the exact parameters described within, but it is important to be consistent with the ones chosen throughout the staining, acquisition and analysis steps. The strength of this protocol is that it is applicable to a wide variety of circumstances where one fluorescent signal is used to isolate other signals that would otherwise be impossible to study alone.

This protocol uses three-dimensional (3D) imaging and analysis techniques to visualize and quantify nerve-specific mitochondria. The techniques are applicable to other situations where one fluorescent signal is used to isolate a subset of data from another fluorescent signal.

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to ...

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 motor neuron is obtained by ectopic expression of alternative modules of transcription factors, namely Ngn2, Isl1 and Lhx3 (NIL) or Ngn2, Isl1 and Phox2a (NIP). NIL and NIP specify, respectively, spinal and cranial motor neuron identity. Our protocol starts with the generation of modified iPSC lines in which NIL or NIP are stably integrated in the genome via a piggyBac transposon vector. Expression of the transgenes is then induced by doxycycline and leads, in 5 days, to the conversion of iPSCs into MN progenitors. Subsequent maturation, for 7 days, leads to homogeneous populations of spinal or cranial MNs. Our method holds several advantages over previous protocols: it is extremely rapid and simplified; it does not require viral infection or further MN isolation; it allows generating different MN subpopulations (spinal and cranial) with a remarkable degree of maturation, as demonstrated by the ability to fire trains of action potentials. Moreover, a large number of motor neurons can be obtained without purification from mixed populations. iPSC-derived spinal and cranial motor neurons can be used for in vitro modeling of Amyotrophic Lateral Sclerosis and other neurodegenerative diseases of the motor neuron. Homogeneous motor neuron populations might represent an important resource for cell type specific drug screenings.

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

This protocol allows rapid and efficient conversion of induced pluripotent stem cells into motor neurons with a spinal or cranial identity, by ectopic expression of transcription factors from inducible 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 ...

Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) pathophysiology in vitro. Multi-electrode array (MEA) recordings are a means to record electrical field potentials from large populations of neurons and analyze network activity over time. It was previously demonstrated that the presence of hiPSC-A that are differentiated using techniques to promote a spinal cord astrocyte phenotype improved maturation and electrophysiological activity of regionally specific spinal cord hiPSC-motor neurons (MN) when compared to those cultured without hiPSC-A or in the presence of rodent astrocytes. Described here is a method to co-culture spinal cord hiPSC-A with hiPSC-MN and record electrophysiological activity using MEA recordings. While the differentiation protocols described here are particular to astrocytes and neurons that are regionally specific to the spinal cord, the co-culturing platform can be applied to astrocytes and neurons differentiated with techniques specific to other fates, including cortical hiPSC-A and hiPSC-N. These protocols aim to provide an electrophysiological assay to inform about glia-neuron interactions and provide a platform for testing drugs with therapeutic potential in ALS.

We describe a method for differentiating spinal cord human induced pluripotent-derived astrocytes and neurons and their co-culture for electrophysiological recording.

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) ...

Generation of Human Brain Organoids for Mitochondrial Disease Modeling

Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental defects whose underlying mechanisms remain to be elucidated. A major roadblock is the lack of effective models recapitulating the early-onset neuronal impairment seen in the patients. Advances in the technology of induced pluripotent stem cells (iPSCs) enable the generation of three-dimensional (3D) brain organoids that can be used to investigate the impact of diseases on the development and organization of the nervous system. Researchers, including these authors, have recently introduced human brain organoids to model mitochondrial disorders. This paper reports a detailed protocol for the robust generation of human iPSC-derived brain organoids and their use in mitochondrial bioenergetic profiling and imaging analyses. These experiments will allow the use of brain organoids to investigate metabolic and developmental dysfunctions and may provide crucial information to dissect the neuronal pathology of mitochondrial diseases.

We describe a detailed protocol for the generation of human induced pluripotent stem cell-derived brain organoids and their use in modeling mitochondrial diseases.

Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental ...

Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and Glial Derivatives

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential (MMP), mitochondrial production of reactive oxygen species (ROS), and mitochondrial DNA (mtDNA) copy number are often features of these processes. This report details a novel flow cytometry-based approach to measure multiple mitochondrial parameters in different cell types, including human induced pluripotent stem cells (iPSCs) and iPSC-derived neural and glial cells. This flow-based strategy uses live cells to measure mitochondrial volume, MMP, and ROS levels, as well as fixed cells to estimate components of the mitochondrial respiratory chain (MRC) and mtDNA-associated proteins such as mitochondrial transcription factor A (TFAM). 
By co-staining with fluorescent reporters, including MitoTracker Green (MTG), tetramethylrhodamine ethyl ester (TMRE), and MitoSox Red, changes in mitochondrial volume, MMP, and mitochondrial ROS can be quantified and related to mitochondrial content. Double staining with antibodies against MRC complex subunits and translocase of outer mitochondrial membrane 20 (TOMM20) permits the assessment of MRC subunit expression. As the amount of TFAM is proportional to mtDNA copy number, the measurement of TFAM per TOMM20 gives an indirect measurement of mtDNA per mitochondrial volume. The entire protocol can be carried out within 2-3 h. Importantly, these protocols allow the measurement of mitochondrial parameters, both at the total level and the specific level per mitochondrial volume, using flow cytometry.

This study reports a novel approach to measure multiple mitochondrial functional parameters based on flow cytometry and double staining with two fluorescent reporters or antibodies to detect changes in mitochondrial volume, mitochondrial membrane potential, reactive oxygen species level, mitochondrial respiratory chain composition, and mitochondrial DNA.

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential ...

An In Situ Immunostaining Protocol for Parkinson's Disease Study

Histological Examination of Mitochondrial Morphology in a Parkinson's Disease Model

Mitochondria play a central role in the energy metabolism of cells, and their function is especially important for neurons due to their high energy demand. Therefore, mitochondrial dysfunction is a pathological hallmark of various neurological disorders, including Parkinson's disease. The shape and organization of the mitochondrial network is highly plastic, which allows the cell to respond to environmental cues and needs, and the structure of mitochondria is also tightly linked to their health. Here, we present a protocol to study mitochondrial morphology in situ based on immunostaining of the mitochondrial protein VDAC1 and subsequent image analysis. This tool could be particularly useful for the study of neurodegenerative disorders because it can detect subtle differences in mitochondrial counts and shape induced by aggregates of &#945;-synuclein, an aggregation-prone protein heavily involved in the pathology of Parkinson's disease. This method allows one to report that substantia nigra pars compacta dopaminergic neurons harboring pS129 lesions show mitochondrial fragmentation (as suggested by their reduced Aspect Ratio, AR) compared to their healthy neighboring neurons in a pre-formed fibril intracranial injection Parkinson model.

Author Spotlight: Establishing a New Fluorescence-Based Protocol for In Vivo Mitochondrial Morphology Analysis in Parkinson's Disease

This study presents a method to analyze the morphology of mitochondria based on immunostaining and image analysis in mouse brain tissue in situ. It also describes how this allows one to detect changes in mitochondrial morphology induced by protein aggregation in Parkinson's disease models.

Mitochondria play a central role in the energy metabolism of cells, and their function is especially important for neurons due to their high energy ...

Unveiling Mitochondrial Ultrastructure in Neurons Using STED Microscopy

Using Live Cell STED Imaging to Visualize Mitochondrial Inner Membrane Ultrastructure in Neuronal Cell Models

Mitochondria play many essential roles in the cell, including energy production, regulation of Ca2+ homeostasis, lipid biosynthesis, and production of reactive oxygen species (ROS). These mitochondria-mediated processes take on specialized roles in neurons, coordinating aerobic metabolism to meet the high energy demands of these cells, modulating Ca2+ signaling, providing lipids for axon growth and regeneration, and tuning ROS production for neuronal development and function. Mitochondrial dysfunction is therefore a central driver in neurodegenerative diseases. Mitochondrial structure and function are inextricably linked. The morphologically complex inner membrane with structural infolds called cristae harbors many molecular systems that perform the signature processes of the mitochondrion. The architectural features of the inner membrane are ultrastructural and therefore, too small to be visualized by traditional diffraction-limited resolved microscopy. Thus, most insights on mitochondrial ultrastructure have come from electron microscopy on fixed samples. However, emerging technologies in super-resolution fluorescence microscopy now provide resolution down to tens of nanometers, allowing visualization of ultrastructural features in live cells. Super-resolution imaging therefore offers an unprecedented ability to directly image fine details of mitochondrial structure, nanoscale protein distributions, and cristae dynamics, providing fundamental new insights that link mitochondria to human health and disease. This protocol presents the use of stimulated emission depletion (STED) super-resolution microscopy to visualize the mitochondrial ultrastructure of live human neuroblastoma cells and primary rat neurons. This procedure is organized into five sections: (1) growth and differentiation of the SH-SY5Y cell line, (2) isolation, plating, and growth of primary rat hippocampal neurons, (3) procedures for staining cells for live STED imaging, (4) procedures for live cell STED experiments using a STED microscope&#160;for reference, and (5) guidance for segmentation and image processing using examples to measure and quantify morphological features of the inner membrane.

Author Spotlight: Decoding Mitochondrial Aging 

This protocol presents a workflow for the propagation, differentiation, and staining of cultured SH-SY5Y cells and primary rat hippocampal neurons for mitochondrial ultrastructure visualization and analysis using stimulated emission depletion (STED) microscopy.

Mitochondria play many essential roles in the cell, including energy production, regulation of Ca2+ homeostasis, lipid biosynthesis, and production of ...

Application of Flow Cytometric Analysis for Measuring Multiple Mitochondrial Parameters in 3D Brain Organoids

Mitochondrial dysfunction is a common primary or secondary contributor to many types of neurodegeneration, and changes in mitochondrial mass, mitochondrial respiratory chain (MRC) complexes, and mitochondrial DNA (mtDNA) copy number often feature in these processes. Human brain organoids derived from human induced pluripotent stem cells (iPSCs) recapitulate the brain&#39;s three-dimensional (3D) cytoarchitectural arrangement and offer the possibility to study disease mechanisms and screen new therapeutics in a complex human system. Here, we report a unique flow cytometry-based approach to measure multiple mitochondrial parameters in iPSC-derived cortical organoids. This report details a protocol for generating cortical brain organoids from iPSCs, single-cell dissociation of generated organoids, fixation, staining, and subsequent flow cytometric analysis to assess multiple mitochondrial parameters. Double staining with antibodies against the MRC complex subunit NADH: Ubiquinone Oxidoreductase Subunit B10 (NDUFB10) or mitochondrial transcription factor A (TFAM) together with voltage-dependent anion-selective channel 1 (VDAC 1) permits assessment of the amount of these proteins per mitochondrion. Since the quantity of TFAM corresponds to the amount of mtDNA, it provides an indirect estimation of the number of mtDNA copies per mitochondrial content. This entire procedure can be completed within a span of 2-3 h. Crucially, it allows for the concurrent quantification of multiple mitochondrial parameters, including both total and specific levels relative to the mitochondrial mass.

This protocol reports a unique method of using a streaming cytometer and multiple antibodies for simultaneous assessment of multiple mitochondrial functional parameters, including changes in mitochondrial volume, amounts of the mitochondrial respiratory chain (MRC) complex subunits, and mitochondrial DNA (mtDNA) replication.

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

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