Name: modeling charcot-marie-tooth disease in vitro by transfecting mouse primary motoneurons
Uploaded: 2019-01-07T14:00:00.000+00:00
Duration: 7 min 43 s
Description: Watch this Scientific Journal Video about Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons at JoVE.com

We would like to thank the &#34;Association pour le d&#233;veloppement de la neurog&#233;n&#233;tique&#34; for Dr. Jacquier&#39;s fellowship and AFM-Telethon for its support through MyoNeurAlp strategic plan. We would also like to thank Dr. Chris Henderson, Dr. William Camu, Dr. Brigitte Pettmann, Dr. Cedric Raoul, and Dr. Georg Haase, who participated in developing and improving the technique and spread their knowledge.

All procedures involving animals were accepted by the ethical committee of the institution.
1. Solution Preparation
<ol>
	<li>Prepare 10 mL of 4% BSA dialyzed in L-15 medium.
	<ol>
		<li>Dissolve bovine serum albumin powder at 4% (w/v) in 10 mL of L-15 medium.</li>
		<li>Add the solution to a 20 mL dialysis cassette with a 20 kDa cut-off. Dialyze against 500 mL of L-15 medium for 3 days under agitation at 4 &#176;C. Change the L-15 medium every day.</li>
		<li>Filter the solution through a 0.22 &#181;m membrane and aliquot in 15 mL tubes. Store the tubes at -20 &#176;C. 
		NOTE: This protocol uses the following references: unmodified raw L-15 medium = L-15 medium, acidic L-15 medium = pH L-15, and basic L-15 medium = Bicarbonate L-15.</li>
	</ol>
	</li>
	<li>Prepare 200 mL of pH L-15.
	<ol>
		<li>Adjust the pH of the L-15 medium: First, fill a wash-bottle with some pieces of dry ice. Inject gas (CO2) into the L-15 medium until the color turns orange (~pH 6.4 and ~40 pressures). The pH can be also adjusted with a standard pH meter.</li>
		<li>Filter the medium through a 0.22 &#181;m membrane and store at 4 &#176;C for one month.</li>
	</ol>
	</li>
	<li>Prepare 100 mL of Bicarbonate L-15.
	<ol>
		<li>Add 2.5 mL of 7% sodium bicarbonate to 97.5 mL of L-15 medium and store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare the IPCS (Insulin Putrescin Conalbumin Selenite) supplements.
	<ol>
		<li>Dissolve 2.5 mg of insulin in 0.5 mL of 0.1 M HCl. Add 4.5 mL of double-distilled water.</li>
		<li>Dissolve 8 mg of putrescine in 5 mL of phosphate-buffered saline (PBS).</li>
		<li>Dissolve 100 mg of conalbumin in 10 mL of PBS.</li>
		<li>Dissolve 1 mg of sodium selenite in 19.3 mL of water, adjust the pH to 7.4, and dilute the solution 10-fold.</li>
		<li>Combine 1 mL of insulin solution, 1 mL of putrescine solution, 1 mL of conalbumin, and 0.1 mL of sodium selenite solution to obtain 3.1 mL of IPCS supplement solution. Store the solution at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare a 0.02 mM progesterone solution.
	<ol>
		<li>Dissolve 1 mg of progesterone in 1.6 mL of 80% ethanol.</li>
		<li>Dilute the solution 100-fold in 100% ethanol and store it at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare 100 mL of complete L-15 medium.
	<ol>
		<li>Add 1.8 mL of D-glucose solution (200 mg/mL) to 92 mL of pH L-15 to obtain a final concentration of 3.5 mg/mL glucose.</li>
		<li>Combine 1 mL of 100x Penicillin-Streptomycin (10,000 U/mL), 2 mL of heat-inactivated horse serum, 3.1 mL of IPCS mix, and 0.1 mL of progesterone solution (2 x 10-5 M).</li>
		<li>Filter the medium on a 0.22 &#181;m membrane and store it at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare 5 mL of density gradient solution (final concentration of 5.5%) per experiment.
	<ol>
		<li>Add 476 &#181;L of the commercial 60% density gradient medium to 4524 &#181;L of L-15 (if possible, without red phenol to increase the visual contrast at the interphase; dilution ratio of 1:10.5).</li>
		<li>Store the solution at 4 &#176;C for 2 weeks or freeze it at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare 10 mL of culture media.
	<ol>
		<li>Add 200 &#181;L of supplement medium to 9.6 mL of neuron cell culture medium.</li>
		<li>Add 200 &#181;L of heat-inactivated horse serum (which tends to differentiate glial cell precursors and works against proliferation) to 25 &#181;L of glutamine and 10 &#181;L of 2-mercaptoethanol.</li>
		<li>Filter the media through a 0.22 &#181;m filter.</li>
		<li>Add the following neurotrophic factors: ciliary neurotrophic factor (CNTF) at a final concentration of 10 ng/mL, and brain derived neurotrophic factor (BDNF) and glial cell derived neurotrophic factor (GDNF) at final concentrations of 1 ng/mL.</li>
		<li>Store the media at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare 50 mL of dissection media.
	<ol>
		<li>Add 2.25 mL of glucose (200 mg/mL) to 47.05 mL of HBSS. Add 700 &#181;L of 1 M HEPES with pH 7.4. Store the media at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare a 4% PFA solution.
	<ol>
		<li>Dissolve 20 g of PFA in 500 mL of PBS.</li>
		<li>Under a chemical hood, heat the solution to 60 &#176;C and add a few drops of 1 M NaOH until the solution becomes clear.</li>
		<li>Filter the solution through a filter paper and adjust the pH to 7.2. Store it at -20 &#176;C. 
		NOTE: Alternatively, other commercial PFA solutions can be used and diluted to 4% in PBS.</li>
	</ol>
	</li>
	<li>Clean the dissection tools including the forceps, scissors, and silicone dishes with 70% ethanol before use, and with soap and clear water after use.</li>
</ol>
2. Poly-ornithine/Laminin (Po/L) Dish Coating
NOTE: Volumes are adapted to coat a 24-well plate.
<ol>
	<li>If needed, put a sterile 12 mm coverslip in the well.</li>
	<li>Coat the wells with 500 &#181;L of 10 &#181;g/mL Poly-L-Ornithine solution dissolved in water.</li>
	<li>Let the wells settle at room temperature for 1 h.</li>
	<li>Remove the Poly-L-Ornithine solution and air-dry for 30 min.</li>
	<li>Coat the wells overnight at 37 &#176;C with 500 &#181;L of 3 &#181;g/mL laminin in bicarbonate L-15. 
	NOTE: Wells can be stored at 37 &#176;C for 7 days in the incubator. For long incubations, adding sterile PBS between the wells can help to avoid medium evaporation.</li>
</ol>
3. Dissection
<ol>
	<li>Sacrifice a pregnant mouse when the embryos are at embryonic stage E12.5. Clean the belly with 70% ethanol.</li>
	<li>Remove the womb by cutting the two oviducts and the vagina and put it in a Petri dish filled with cold PBS (without Ca2+ Mg2+).</li>
	<li>Collect all the embryos and transfer them to fresh, cold PBS (without Ca2+ Mg2+) in a Petri dish.</li>
	<li>Put one embryo in a dish coated with silicone and full of dissection media (with HBSS, 4.5 g/L glucose, and 7 mM HEPES pH 7.4). 
	NOTE: Embryo dissection is performed under a microscope using clean fine forceps and scissors. Alternatively, a regular Petri dish without silicone can be used for dissection.</li>
	<li>Remove the head, tail, and viscera, using forceps as scissors.</li>
	<li>Maintain the embryo with the belly against the silicone with forceps.</li>
	<li>With a second pair of forceps, insert one of the tips into the central canal of the rostral spinal cord.</li>
	<li>Close the forceps to tear the dorsal tissue a few millimeters.</li>
	<li>Repeat this operation toward the caudal side until the entire spinal cord is open (Figure 1B).</li>
	<li>Detach the rostral part of the spinal cord (cerebral trunk) from the body.</li>
	<li>Turn the embryo to maintain the open spinal cord against the silicone.</li>
	<li>Pinch the rostral part of the spinal cord and pull the embryo by the head to extract the spinal cord. 
	NOTE: Meninges and dorsal root ganglia (DRGs) should remain attached to the embryo. Otherwise, carefully pull away any remaining meninges and DRGs still attached to the spinal cord. At this step, DRGs could also be collected for sensitive neuron culture (see Discussion).</li>
	<li>Flatten the spinal cord on the silicone and remove the dorsal half of the cord by cutting along the middle of each side using a scalpel. Cut the middle part into 10 pieces using a scalpel, and transfer the pieces to a new tube.</li>
</ol>
4. Spinal Cord Cell Suspension
<ol>
	<li>Repeat this dissection procedure for 6 to 8 spinal cords.</li>
	<li>Let the spinal cord pieces collect at the bottom of the tube and replace the HBSS with 1 mL of Ham-F10 medium.</li>
	<li>Add 10 &#181;L of trypsin (2.5% w/v; final concentration 0.025%). Incubate the tube for 10 min at 37 &#176;C.</li>
	<li>To stop the enzymatic digestion, transfer the fragments to a new 15 mL tube containing 800 &#181;L of complete L-15 medium, 100 &#181;L of 4% (w/v) BSA, and 100 &#181;L of DNase (1 mg/mL in L-15 medium).</li>
	<li>Triturate twice by aspirating 1 mL using a P1000 pipette. Let the fragments settle for 2 min. Collect the supernatant in a fresh 15 mL tube.</li>
	<li>To the fragments, add 900 &#181;L of complete L-15 medium, 100 &#181;L of 4% (w/v) BSA, and 100 &#181;L of DNase (1 mg/mL in L-15 medium).</li>
	<li>Triturate 6 times by aspirating 1 mL using a P1000 pipette. Let the fragments settle for 2 min. Add the supernatant to the 15 mL tube obtained in step 4.5.</li>
	<li>To the fragments, add 900 &#181;L of complete L-15 medium, 100 &#181;L of 4% (w/v) BSA, and 100 &#181;L of DNase (1 mg/mL in L-15 medium).</li>
	<li>Triturate 10 times by aspirating 1 mL using a P1000 pipette. Let the fragments settle for 2 min. Add the supernatant to the 15 mL tube obtained in step 4.5. Proceed with the supernatant.</li>
	<li>Using a P1000 pipet, gently place a 2 mL BSA 4% (w/v) cushion at the bottom of supernatant tube. Centrifuge at 470 x g for 5 min.</li>
	<li>Remove the supernatant and resuspend the cell pellet with 2 mL of complete L-15 medium.</li>
</ol>
5. MN Enrichment by Gradient Density
<ol>
	<li>Split the cell suspension into two 15 mL tubes (1 mL each). Then, add 2 mL of complete L-15 medium. If starting with 6 embryos, use the equivalent of 3 embryos per tube in 3 mL of medium.</li>
	<li>Add 2 mL of density gradient solution by slowly adding the solution to the bottom of each tube to obtain a sharp interface.</li>
	<li>Centrifuge at 830 x g for 15 min at room temperature. After this step, small cells should be at the bottom of the tube, whereas large cells such as MNs should be at the density gradient solution/medium interface.</li>
	<li>Collect the cells at the interface by aspirating 1 or 2 mL and transfer them to a fresh 15 mL tube. Adjust the volume&#160;to 10 mL with L-15 pH medium.</li>
	<li>Add 2 mL of the 4% BSA cushion to the bottoms of the tube&#160;and centrifuge at 470 x g for 5 min at room temperature.</li>
	<li>Remove the supernatant and resuspend the pellet in 3 mL of complete L-15 medium.</li>
	<li>Repeat step 5.5.</li>
	<li>Remove the supernatant and resuspend the cell pellet in 2 mL of complete culture medium. Count the cell number and viability under a phase contrast microscope with a hemocytometer. 
	NOTE: For 6 spinal cords, the cell number is expected to be around 1 x 105 MNs.</li>
</ol>
6. MN Culture
<ol>
	<li>Dilute the MNs to the appropriate density, usually 5,000 to 10,000 cells in 500 &#181;L of culture medium per well in a 24-well plate. 
	NOTE: Seeding density is around 30,000 and 1,500 cells for 6- and 96-well plates, respectively.</li>
	<li>Remove the laminin solution with a P1000.</li>
	<li>Immediately transfer the MNs diluted in culture medium into the coating plates to avoid drying.</li>
	<li>Incubate the culture for 2 days at 37 &#176;C.</li>
</ol>
7. Magnetofection of MNs
NOTE: In the following steps, the quantities used are meant for the transfection of one well of a 24-well plate. Please refer to the manufacturer&#39;s protocol for another format.
<ol>
	<li>For the DNA preparation, resuspend 1 &#181;g of DNA in 50 &#181;L of neuronal culture medium and vortex for 5 s.</li>
	<li>For bead tube preparation, resuspend 1.5 &#181;L of beads in 50 &#181;L of neuronal culture medium.</li>
	<li>Add the 50 &#181;L bead solution to the 50 &#181;L DNA solution and incubate for 20 min at room temperature (total volume = 100 &#181;L).</li>
	<li>During this incubation, withdraw 100 &#181;L of culture medium from the well that will be transfected. 
	NOTE: Put the magnetic plate in the incubator to warm it to 37 &#176;C.</li>
	<li>Transfer 100 &#181;L of the DNA/bead mix to the well, and incubate the plate 20&#8211;30 min on the magnetic plate at 37 &#176;C.</li>
	<li>Wait at least 24 h before detection of cDNA expression.</li>
</ol>
8. Fixation and Staining
<ol>
	<li>Wash the culture 2 times with PBS.</li>
	<li>Incubate the culture for 20 min at room temperature in 4% PFA/PBS.</li>
	<li>Wash the culture 2 times with PBS.</li>
	<li>Incubate the culture in 500 &#181;L of blocking buffer (PBS, 4% BSA, 2% normal serum, 0.3% Triton X-100, 0.1 M glycine) for 1 h at room temperature. 
	NOTE: Normal serum should be derived from the same species as the secondary antibody (e.g., Normal Goat Serum).</li>
	<li>Incubate the culture overnight at 4 &#176;C with primary antibody diluted in 250 &#181;L of blocking buffer. 
	NOTE: For example, TUJ1 is used at 1/1000, SMI32 is used at 1/1,000, and Lc3b is used at 1/200.</li>
	<li>Wash the culture 3 times with PBS.</li>
	<li>Incubate the culture with fluorophore-conjugated secondary antibodies diluted in 250 &#181;L of blocking buffer for 1 hour at room temperature.</li>
	<li>Wash the culture 3 times with PBS.</li>
	<li>Mount on glass slides using mounting medium.</li>
</ol>

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The authors have nothing to disclose.

One of the critical points in this protocol is that the mouse embryos are dissected at a precise time window during development (E12.5) to optimize the amount of MNs obtained at the end. In addition, for optimal yield, the dissection should be performed in the morning or early in the afternoon. Before E12.5 (e.g., at E11.5), dissection is difficult, especially regarding the elimination of the meninges. After E12.5, the number of obtained MNs drops significantly. To control the embryo stage of development, adult ...

Neuromuscular diseases encompass a variety of clinically and genetically distinct pathologies that are characterized by the alteration of muscle and/or the nervous system. Because of advances in sequencing technologies, hundreds of genes responsible for these rare disorders have been identified during the last decade (list available at the Neuromuscular Disease Center, http://neuromuscular.wustl.edu/index.html). The variety of identified mutations indicates that different mutations in a single gene can cause different phenotypes and diseases1,2,3 and that mutations in different genes can produce similar phenotypes4,5. In this context, there are efforts to develop cellular models that can become powerful tools for analyzing mutation consequences and pathological mechanisms.
Spinal MNs have large somas located in the ventral horns of the spinal cord, form long axons to target skeletal muscle fibers, and allow for voluntary movements through the release of acetylcholine at neuromuscular junctions. Since MNs are affected by neuromuscular diseases such as amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease (CMT), and spinal muscular atrophy, Dr. Henderson and colleagues developed the first protocol6 that allowed for cultivation of in vitro spinal MNs and the discovery of neurotrophic factor GDNF7 (glial cell derived neurotrophic factor). Technical refinements since then have allowed for more accurate purification of spinal MNs and their subtypes using FACS8, but enrichment by density gradient remains powerful and widely used in laboratories currently working with primary spinal MNs9,10,11,12,13,14. Subsequently, it is also possible to obtain a higher MN purification grade through immunopanning by taking advantage of surface marker p75(NTR) 15,16,17.
The spinal cord contains different subtypes of cervical, thoracic, and lumbar MNs, as well as median and lateral motor columns that differ among their location in the anterior horn on a dorso-ventral axis and among the targets they innervate8,18. Primary MN cultures can recover all of these MN subtypes in physiological proportions. The main limitation of this technique is the low number of MNs obtained at the end of the procedure; in fact, it can be expected to obtain around 105 MNs from six embryos, which is suitable for microscopy but limiting for biochemistry experiments. To perform experiments with more standardized subtypes and abundant MNs (&#62;106 cells), embryonic stem cell-derived MNs should be considered18.
Transfection of wild-type/mutant transgenes or knockdown endogenous genes into primary MNs is a rapid and helpful tool for deciphering physiopathological pathways, especially when mouse models are unavailable. Magnetofection is one technique for transfecting primary neurons, similar to lipofection without the related neurotoxicity. Furthermore, transfection can be performed on mature neurons after several days in vitro, unlike techniques based on electroporation9. However, one disadvantage of this technique is that the beads bind nucleic acids in the culture, causing noise in DAPI labeling. Viral infection is likely the most efficient technique for transfecting MNs; however, magnetofection does not require certain safety procedures needed for viral production and cellular infection.

<table><tbody><tr><td>&lt;strong&gt;&lt;u&gt;Material&lt;/u&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Silicone dissection dish</td><td>Living systems instrumentation</td><td>DD-90-S-BLK-3PK</td><td>Sylgard</td></tr><tr><td>round coverslip</td><td>NeuVitro, Knittel glass</td><td>GG-12-Pre</td><td>12 mm</td></tr><tr><td>Slide a Lyzer cassettes</td><td>ThermoFisher Scientific</td><td>66030</td><td>20,000 MWCO ; 30 mL</td></tr><tr><td>Filter unit</td><td>Millipore</td><td>SCGVU02RE</td><td></td></tr><tr><td>GP Sterile Syringe Filters</td><td>Millipore</td><td>SLGP033RS</td><td></td></tr><tr><td>4 well plate</td><td>ThermoFisher Scientific</td><td>167063</td><td>Nunclon Delta treated plate</td></tr><tr><td>forceps</td><td>FST by Dumont</td><td>11252-20</td><td>#5 forceps</td></tr><tr><td>scissor</td><td>FST by Dumont</td><td>14060-10</td><td>fine scissors</td></tr><tr><td>scalpel</td><td>FST by Dumont</td><td>10035-20</td><td>curved blade</td></tr><tr><td>scalpel</td><td>FST by Dumont</td><td>10316-14</td><td>micro-knife scalpel</td></tr><tr><td>Petri dish</td><td>Greiner</td><td>663102</td><td>&amp;oslash; x h = 100 x 15 mm</td></tr><tr><td>15 mL polypropylene tube</td><td>Falcon</td><td>352096</td><td></td></tr><tr><td>filter paper</td><td>Watman</td><td>1001125</td><td>circle, 125 mm diameter</td></tr><tr><td>glass chamber slide</td><td>Lab-Tek</td><td>154526</td><td>4 chambers</td></tr><tr><td>Plasmid pCAGEN</td><td>Addgene</td><td>#11160</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;&lt;u&gt;Solutions and mediums&lt;/u&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A9418</td><td></td></tr><tr><td>L-15 medium</td><td>ThermoFisher Scientific</td><td>11415056</td><td></td></tr><tr><td>L-15 medium, no red phenol</td><td>ThermoFisher Scientific</td><td>21083027</td><td></td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>I6634</td><td></td></tr><tr><td>Putrescine</td><td>Sigma-Aldrich</td><td>P5780</td><td></td></tr><tr><td>Conalbumin</td><td>Sigma-Aldrich</td><td>C7786</td><td></td></tr><tr><td>Sodium selenite</td><td>Sigma-Aldrich</td><td>S5261</td><td></td></tr><tr><td>Progesterone</td><td>Sigma-Aldrich</td><td>P8783</td><td></td></tr><tr><td>Poly-DL-Ornithine</td><td>Sigma-Aldrich</td><td>P8638</td><td></td></tr><tr><td>Laminin</td><td>Sigma-Aldrich</td><td>L2020</td><td></td></tr><tr><td>trypsin 2.5%, 10x</td><td>ThermoFisher Scientific</td><td>15090046</td><td></td></tr><tr><td>DNAse</td><td>Sigma-Aldrich</td><td>DN25</td><td></td></tr><tr><td>PBS w/o Ca Mg</td><td>ThermoFisher Scientific</td><td>14190144</td><td>without Mg&lt;sup&gt;2+&lt;/sup&gt; Ca&lt;sup&gt;2+&lt;/sup&gt;</td></tr><tr><td>sodium bicarbonate</td><td>ThermoFisher Scientific</td><td>25080094</td><td></td></tr><tr><td>Neuron cell culture medium</td><td>ThermoFisher Scientific</td><td>A3582901</td><td>Neurobasal Plus medium</td></tr><tr><td>HBSS</td><td>Sigma-Aldrich</td><td>H6648-500ML</td><td></td></tr><tr><td>HEPES buffer 1 M</td><td>ThermoFisher Scientific</td><td>15630056</td><td></td></tr><tr><td>Density gradiant medium</td><td>Sigma-Aldrich</td><td>D1556</td><td>Optiprep</td></tr><tr><td>supplement medium</td><td>ThermoFisher Scientific</td><td>A3582801</td><td>B-27 Plus</td></tr><tr><td>Horse serum heat inactivated</td><td>ThermoFisher Scientific</td><td>26050-088</td><td></td></tr><tr><td>L-Glutamine 200 mM</td><td>ThermoFisher Scientific</td><td>25030024</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>ThermoFisher Scientific</td><td>31350010</td><td></td></tr><tr><td>penicilline/streptomycine</td><td>ThermoFisher Scientific</td><td>15140122</td><td>10,000 U/mL</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;&lt;u&gt;Immuno fluorescence&lt;/u&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PBS, 10x</td><td>ThermoFisher Scientific</td><td>X0515</td><td>without Mg&lt;sup&gt;2+&lt;/sup&gt; Ca&lt;sup&gt;2+&lt;/sup&gt;</td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma-Aldrich</td><td>441244</td><td></td></tr><tr><td>normal goat serum</td><td>Sigma-Aldrich</td><td>G6767</td><td></td></tr><tr><td>glycine</td><td>Sigma-Aldrich</td><td>G7126</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td></td></tr><tr><td>Choline Acetyl Transferase (CHAT)</td><td>Chemicon</td><td>Ab144P</td><td></td></tr><tr><td>Neurofilament H non phosphorylated (SMI32)</td><td>Biolegends</td><td>SMI-32P</td><td>IF at 1/1000</td></tr><tr><td>Islet-1</td><td>DSHB</td><td>40.2D6</td><td></td></tr><tr><td>Islet-2</td><td>DSHB</td><td>39.4D5</td><td></td></tr><tr><td>Hb9</td><td>DSHB</td><td>81.5C10</td><td></td></tr><tr><td>Vectashield mounting medium</td><td>Vector Lab</td><td>H-1000</td><td></td></tr><tr><td>Beta3 tubulin (Tuj1 clone)</td><td>Biolegends</td><td>801201</td><td>IF at 1/1000</td></tr><tr><td>Lc3b</td><td>Cell Signaling Technology</td><td>#2775</td><td>IF at 1/200</td></tr></tbody></table>

modeling charcot-marie-tooth disease in vitro by transfecting mouse primary motoneurons

Neurodegeneration of spinal motoneurons (MNs) is implicated in a large spectrum of neurological disorders including amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, and spinal muscular atrophy, which are all associated with muscular atrophy. Primary cultures of spinal MNs have been used widely to demonstrate the involvement of specific genes in such diseases and characterize the cellular consequences of their mutations. This protocol models a primary MN culture derived from the seminal work of Henderson and colleagues more than twenty years ago. First, we detail a method of dissecting the anterior horns of the spinal cord from a mouse embryo and isolating the MNs from neighboring cells using a density gradient. Then, we present a new way of efficiently transfecting MNs with expression plasmids using magnetofection. Finally, we illustrate how to fix and immunostain primary MNs. Using neurofilament mutations that cause Charcot-Marie-Tooth disease type 2, this protocol demonstrates a qualitative approach to expressing proteins of interest and studying their involvement in MN growth, maintenance, and survival.

After 24 hours in the culture, motoneurons (MNs) should already show significant axonal growth (at least 6 times longer than the soma size). In the following days, axons should continue to grow and display branching (Figure 2). There will be different morphologies due to subtype specificities. For example, median column MNs that innervate axial muscles have shorter and more branched axons than lateral motor column MNs that innervate limb muscles<sup class="xr...

Watch this Scientific Journal Video about Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons at JoVE.com

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

The goal of this technique is to prepare a highly enriched culture of primary motoneurons (MNs) from murine spinal cord. To evaluate the consequences of mutations causing MN diseases, we describe here the isolation of these isolated MNs and their transfection by magnetofection.

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

Neurodegeneration of spinal motoneurons (MNs) is implicated in a large spectrum of neurological disorders including amyotrophic lateral sclerosis, ...

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6.9K Views.  Université Lyon. The goal of this technique is to prepare a highly enriched culture of primary motoneurons (MNs) from murine spinal cord. To evaluate the consequences of mutations causing MN diseases, we describe here the isolation of these isolated MNs and their transfection by magnetofection.

Video: Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing1. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.
Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas2. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.
The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.
Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases3, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.

Cultured muscle cells are an inadequate model to recapitulate innervated muscle in vivo. A functional motor unit can be reproduced in vitro by innervation of differentiated human primary muscle cells using rat embryo spinal cord explants. This article describes how co-cultures of spinal cord explants and muscle cells are established.

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

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

Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are still important because they offer an efficient cellular alternative for studying cellular and molecular events. Though C2C12 myoblast cells have been widely used to study myogenesis, resistance to gene transfection, or viral transduction, hinders research in C2C12 cells. Here, we describe an optimized protocol that includes daily maintenance, transfection and transduction procedures to introduce genes into C2C12 myoblasts and the induction of myocyte differentiation. Collectively, these procedures enable best transfection/transduction efficiencies, as well as consistent differentiation outcomes. The protocol described in establishing DM1 myoblast cell models would benefit the study of myotonic dystrophy, as well as other muscular diseases.

In this protocol, we present the procedures in establishing myotonic dystrophy 1 myoblast models, including optimized C2C12 cell maintenance, gene transfection/transduction, and myocyte differentiation.

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are ...

Medicine

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings provide a sensitive approach to measure nerve conduction in humans and rodent models. To broaden the technical possibilities for electromyography in mice, the measurement of compound muscle action potentials (CMAPs) from the brachial plexus nerve in the forelimb using needle electrodes is described here. CMAP recordings after stimulating the sciatic nerve in hindlimbs have been previously described. The newly introduced method here allows for the evaluation of the nerve conductivity at an additional site, and thus provides a more profound overview of the neuromuscular functionality. The technique provides information on both the relative number of functional axons and the myelination level. Thereby, this method can be applied to assess both axonal diseases as well as demyelinating conditions. This minimally invasive method does not require extraction of the nerve and therefore it is suitable for repeated measurements for longitudinal follow-up in the same animal. Similar recordings are performed in clinical setups to emphasize the translational relevance of the method.

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

The measurement of nerve conduction is a useful tool to assess mouse models of neurodegeneration but it is frequently only applied to stimulate the sciatic nerve in hindlimbs. Here, we describe a technique to measure compound muscle action potential (CMAP) in vivo in the mouse forelimb muscles innervated by the brachial plexus.

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings ...

Engineering and Characterization of an Optogenetic Model of the Human Neuromuscular Junction

Many neuromuscular diseases, such as myasthenia gravis (MG), are associated with dysfunction of the neuromuscular junction (NMJ), which is difficult to characterize in animal models due to physiological differences between animals and humans. Tissue engineering offers opportunities to provide in vitro models of functional human NMJs that can be used to diagnose and investigate NMJ pathologies and test potential therapeutics. By incorporating optogenetic proteins into induced pluripotent stem cells (iPSCs), we generated neurons that can be stimulated with specific wavelengths of light. If the NMJ is healthy and functional, a neurochemical signal from the motoneuron results in muscle contraction. Through the integration of optogenetics and microfabrication with tissue engineering, we established an unbiased and automated methodology for characterizing NMJ function using video analysis. A standardized protocol was developed for NMJ formation, optical stimulation with simultaneous video recording, and video analysis of tissue contractility. Stimulation of optogenetic motoneurons by light to induce skeletal muscle contractions recapitulates human NMJ physiology and allows for repeated functional measurements of NMJ over time and in response to various inputs. We demonstrate this platform&#39;s ability to show functional improvements in neuromuscular connectivity over time and characterize the damaging effects of patient MG antibodies or neurotoxins on NMJ function.

We describe a reproducible, automated, and unbiased imaging system for characterizing neuromuscular junction function using human engineered skeletal muscle tissue and optogenetic motoneurons. This system allows for the functional quantification of neuromuscular connectivity over time and detects diminished neuromuscular function caused by neurotoxins and myasthenia gravis patient serum.

Many neuromuscular diseases, such as myasthenia gravis (MG), are associated with dysfunction of the neuromuscular junction (NMJ), which is difficult to ...

Bioengineering

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 ...

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal transport have devastating consequences for individual neurons and their networks, and contribute to a plethora of neurological disorders. As many of these conditions involve both cell autonomous and non-autonomous mechanisms, and often display a spectrum of pathology across neuronal subtypes, methods to accurately identify and analyze neuronal subsets are imperative.
This paper details protocols to assess in vivo axonal transport of signaling endosomes and mitochondria in sciatic nerves of anesthetized mice. Stepwise instructions are provided to 1) distinguish motor from sensory neurons in vivo, in situ, and ex vivo by using mice that selectively express fluorescent proteins within cholinergic motor neurons; and 2) separately or concurrently assess in vivo axonal transport of signaling endosomes and mitochondria. These complementary intravital approaches facilitate the simultaneous imaging of different cargoes in distinct peripheral nerve axons to quantitatively monitor axonal transport in health and disease.

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Using transgenic fluorescent mice, detailed protocols are described to assess in vivo axonal transport of signaling endosomes and mitochondria within motor and sensory axons of the intact sciatic nerve in live animals.

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal ...

Retroviral Mediated Gene Transduction: A Gene Transfer Technique to Deliver a Transgene in Cultured Cells Using Engineered Retroviral Vectors

Source: Jacquier, A. et al. <a href="https://www.jove.com/v/57988">Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons.</a> J. Vis. Exp. (2019)
In this video, we demonstrate magnetically-guided transfection of plasmid DNA in primary neuronal cell culture. Magnetofection uses an external magnetic field to guide the delivery of plasmids bound to magnetic nanoparticles into cell cytoplasm.

Source: Jacquier, A. et al. Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons. J. Vis. Exp. (2019)
In this video, we ...

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

Summary

Explore More Videos

Chapters in this video

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

In vitro Neuromuscular Junction Induced from Human Induced Pluripotent Stem Cells

Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

Engineering and Characterization of an Optogenetic Model of the Human Neuromuscular Junction

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

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Retroviral Mediated Gene Transduction: A Gene Transfer Technique to Deliver a Transgene in Cultured Cells Using Engineered Retroviral Vectors