Name: modeling charcot-marie-tooth disease in vitro by transfecting mouse primary motoneurons
Uploaded: 2019-01-07T14:00:00
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...

<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inclusion+body+myopathy+associated+with+Paget+disease+of+bone+and+frontotemporal+dementia+is+caused+by+mutant+valosin-containing+protein.">Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia is caused by mutant valosin-containing protein.</a> Nature Genetics. 36 (4), 377-381 (2004).</li><li>Brown, R. H., Al-Chalabi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyotrophic+Lateral+Sclerosis.">Amyotrophic Lateral Sclerosis.</a> New England Journal of Medicine. 377 (2), 162-172 (2017).</li><li>Taylor, J. P., Brown, R. H., Cleveland, D. 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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GDNF:+a+potent+survival+factor+for+motoneurons+present+in+peripheral+nerve+and+muscle.">GDNF: a potent survival factor for motoneurons present in peripheral nerve and muscle.</a> Science. 266 (5187), 1062-1064 (1994).</li><li>Schaller, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+combinatorial+screening+identifies+neurotrophic+factors+for+selective+classes+of+motor+neurons.">Novel combinatorial screening identifies neurotrophic factors for selective classes of motor neurons.</a> Proceedings of the National Academy of Sciences. 114 (12), E2486-E2493 (2017).</li><li>Jacquier, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alsin/Rac1+signaling+controls+survival+and+growth+of+spinal+motoneurons.">Alsin/Rac1 signaling controls survival and growth of spinal motoneurons.</a> Annals of Neurology. 60 (1), 105-117 (2006).</li><li>Raoul, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+activation+in+presymptomatic+amyotrophic+lateral+sclerosis+(ALS)+mice+of+a+feedback+loop+involving+Fas,+Daxx,+and+FasL.">Chronic activation in presymptomatic amyotrophic lateral sclerosis (ALS) mice of a feedback loop involving Fas, Daxx, and FasL.</a> Proceedings of the National Academy of Sciences. 103 (15), 6007-6012 (2006).</li><li>Madji Hounoum, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wildtype+motoneurons,+ALS-Linked+SOD1+mutation+and+glutamate+profoundly+modify+astrocyte+metabolism+and+lactate+shuttling.">Wildtype motoneurons, ALS-Linked SOD1 mutation and glutamate profoundly modify astrocyte metabolism and lactate shuttling.</a> Glia. 65 (4), 592-605 (2017).</li><li>Magrane, J., Sahawneh, M. A., Przedborski, S., Estevez, A. G., Manfredi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+Dynamics+and+Bioenergetic+Dysfunction+Is+Associated+with+Synaptic+Alterations+in+Mutant+SOD1+Motor+Neurons.">Mitochondrial Dynamics and Bioenergetic Dysfunction Is Associated with Synaptic Alterations in Mutant SOD1 Motor Neurons.</a> Journal of Neuroscience. 32 (1), 229-242 (2012).</li><li>Jacquier, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptic+amyloidogenic+elements+in+mutant+NEFH+causing+Charcot-Marie-Tooth+2+trigger+aggresome+formation+and+neuronal+death.">Cryptic amyloidogenic elements in mutant NEFH causing Charcot-Marie-Tooth 2 trigger aggresome formation and neuronal death.</a> Acta Neuropathologica Communications. 5, (2017).</li><li>Aebischer, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFN&#947;+triggers+a+LIGHT-dependent+selective+death+of+motoneurons+contributing+to+the+non-cell-autonomous+effects+of+mutant+SOD1.">IFN&#947; triggers a LIGHT-dependent selective death of motoneurons contributing to the non-cell-autonomous effects of mutant SOD1.</a> Cell Death and Differentiation. 18 (5), 754-768 (2011).</li><li>Wiese, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+enrichment+of+embryonic+mouse+motoneurons+from+the+lumbar+spinal+cord+of+individual+mouse+embryos.">Isolation and enrichment of embryonic mouse motoneurons from the lumbar spinal cord of individual mouse embryos.</a> Nature Protocols. 5 (1), 31-38 (2010).</li><li>Camu, W., Henderson, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+embryonic+rat+motoneurons+by+panning+on+a+monoclonal+antibody+to+the+low-affinity+NGF+receptor.">Purification of embryonic rat motoneurons by panning on a monoclonal antibody to the low-affinity NGF receptor.</a> Journal of Neuroscience Methods. 44 (1), 59-70 (1992).</li><li>Conrad, R., Jablonka, S., Sczepan, T., Sendtner, M., Wiese, S., Klausmeyer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lectin-based+Isolation+and+Culture+of+Mouse+Embryonic+Motoneurons.">Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons.</a> Journal of Visualized Experiments. (55), (2011).</li><li>Wichterle, H., Lieberam, I., Porter, J. A., Jessell, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+embryonic+stem+cells+into+motor+neurons.">Directed differentiation of embryonic stem cells into motor neurons.</a> Cell. 110 (3), 385-397 (2002).</li><li>Francius, C., Clotman, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+spinal+motor+neuron+diversity:+a+long+quest+for+neuronal+identity.">Generating spinal motor neuron diversity: a long quest for neuronal identity.</a> Cellular and Molecular Life Sciences. 71 (5), 813-829 (2014).</li><li>Neto, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentalized+Microfluidic+Platforms:+The+Unrivaled+Breakthrough+of+In+Vitro+Tools+for+Neurobiological+Research.">Compartmentalized Microfluidic Platforms: The Unrivaled Breakthrough of In Vitro Tools for Neurobiological Research.</a> The Journal of Neuroscience. 36 (46), 11573-11584 (2016).</li><li>Matsuda, T., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+and+RNA+interference+in+the+rodent+retina+in+vivo+and+in+vitro.">Electroporation and RNA interference in the rodent retina in vivo and in vitro.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (1), 16-22 (2004).</li><li>Sleigh, J. N., Weir, G. A., Schiavo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple,+step-by-step+dissection+protocol+for+the+rapid+isolation+of+mouse+dorsal+root+ganglia.">A simple, step-by-step dissection protocol for the rapid isolation of mouse dorsal root ganglia.</a> BMC Research Notes. 9, (2016).</li><li>Yu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+method+for+gene+delivery+into+mouse+dorsal+root+ganglia+neurons.">Highly efficient method for gene delivery into mouse dorsal root ganglia neurons.</a> Frontiers in Molecular Neuroscience. 8 (2), (2015).</li><li>Malin, S. A., Davis, B. M., Molliver, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+dissociated+sensory+neuron+cultures+and+considerations+for+their+use+in+studying+neuronal+function+and+plasticity.">Production of dissociated sensory neuron cultures and considerations for their use in studying neuronal function and plasticity.</a> Nature Protocols. 2 (1), 152-160 (2007).</li></ol>

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>Material</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>&#248; 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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Solutions and mediums</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 Mg2+ Ca2+</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Immuno fluorescence</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, 10x</td>
			<td>ThermoFisher Scientific</td>
			<td>X0515</td>
			<td>without Mg2+ Ca2+</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, ...

This method can help to address key questions in neurobiology, such as motor neuron polarity, axon guidance, axon trafficking, and neurodegenerative mechanisms. The main advantage of this technique is to obtain a highly enriched motor neuron culture while we take to express our knockdown protein by minute affection. To begin this procedure, place one embryo in a dish of dissection media coded with silicon.Under the microscope, hold the embryo with the abdomen against the silicon with forceps. With the second pair of forceps, insert one of the tips into the central canal of the rostrad spinal cord. Then, close the forceps to tear the dorsal tissue slightly.Repeat this operation towards the connell side until the entire spinal cord is opened. Next, position the embryo so that the open spinal cord is against the silicon. Subsequently, detach the rostrad part of the spinal cord from the body.Pinch the rostrad part of the spinal cord, and pull the embryo by the head to extract the spinal cord. Carefully pull away any remaining meninges and DRGs still attached to the spinal cord. Flatten the spinal cord on the silicon and using a scalpel, remove the dorsal half of the cord by cutting along the middle of each side.Cut the middle part into ten pieces, and transfer them to a new tube. To prepare spinal cords cell suspension, collect the spinal cord pieces at the bottom of the tube. Replace HBSS with one milliliter of Ham F-10 medium and add ten microliters of trypsin.Incubate the tube for ten minutes at 37 degrees Celsius. To stop the enzymatic digestion, transfer the fragments to a new 15 milliliter tube, containing 800 microliters of complete L-15 medium, 100 microliters of 4%BSA, and 100 microliters of DNAs and titrate, twice. Let the fragments settle for two minutes before collecting the supernatant in a fresh 15 milliliter tube.Gently add two milliliters of 4%BSA at the bottom of the supernatant tube. Centro fuse it at 470 x gravity for 5 minutes. After 5 minutes, remove the supernatant and re-suspend the cell pallet with 2 milliliters of complete L-15 medium.For motoneuron enrichment, split the cell suspension into two 15 milliliter tubes. Then, add two milliliters of L-15 PH medium to each tube. If starting with six embryos, use the equivalent of three embryos per tube, and three milliliters of medium.Slowly add 2 milliliters of density gradience solution, to the bottom of each tube to obtain a sharp interface. Next, centro fuse it at 830 x gravity for 15 minutes at room temperature. After this step, small cells should be at the bottom of the tube, whereas large cells-such as the motoneuron-should be at the density gradient solution/medium interface.Asperae 1 to 2 milliliters of the cells at the interface and transfer them to a fresh 15 milliliter tube. Adjust the volume to ten milliliters with L-15 PH medium. Subsequently, add two milliliters of the 4%BSA cushion to the bottom of the two tubes.And centro fuse at 470 x gravity for 5 minutes at room temperature. After 5 minutes, remove the supernatant and re-suspend the pallet and 3 milliliters of complete L-15 medium. Repeat the centrifugate at 470 x gravity for 5 minutes at room temperature.Then, remove the supernatant and re-suspend the cell pallet and two milliliters of complete culture medium. To culture motor neurons, dilute them to 5, 000-10, 000 cells in 500 microliters of culture medium per well in a 24 well plate. Then, remove the laminae solution with at P-1, 000 pipet tip.Immediately transfer the motor neurons diluted in culture medium. to the coding plates to avoid drying. To prepare DNA, re-suspend 1 microgram of DNA in 50 microliters of neuronal culture medium.And vortex for 5 seconds. To prepare the B tube, re-suspend 1.5 microliters of beads in 50 microliters of neuronal culture medium. Add 50 microliters of bead solution to 50 microliters of DNA solution and incubate for 20 minutes at room temperature.During this incubation, withdraw 100 microliters of culture medium from the well that will be transfected. Subsequently, transfer 100 microliters of the DNA bead mix to each well. And incubate the 24 well plate, 20-30 minutes on the magnetic plate at 37 degrees Celsius.Shown here, is the modem neuron morphology revealed by standing of endogenous non-phosphorylated neurofilament heavy chain after four days of culture. In these images, show the modem neuron morphology after four days in vitro and transfected for EGFP trans gene by magnetofection at day two. Motto neurons were counter-stained with the marker SMI-32 or the pan-neuro marker TUJ-1 and the arrows indicate the soma of the neurons.These are their confocal images of magneto fected motor neurons, expressing wild type or mutant EGFP NEFH constructs and stained for their autophagic marker LC-3B. Arrows show the mutant EGFP NEFH protein aggregates that are also LC-3B positive Following this procedure, montoholda curcha can be analyzed by video microscopy to visualize axon trafficking and add some guidance. After its initial development to address fundamental questions in neural biology, this technique also prove to be a powerful tool to explore physio pathological mechanisms implicating the spectrum of motoneuron disorders, such as toxic shock syndrome disease, samuel trophic lateral sclerosis, or spinal muscular atrophy.

Research

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Neuroscience

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