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

Summary

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.

Abstract

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.

Introduction

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 diseases^1,2,3 and that mutations in differe....

Protocol

All procedures involving animals were accepted by the ethical committee of the institution.

1. Solution Preparation

1. Prepare 10 mL of 4% BSA dialyzed in L-15 medium.
   1. Dissolve bovine serum albumin powder at 4% (w/v) in 10 mL of L-15 medium.
   2. 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 °C. Change the L-15 medium every day.
   3. Filter the solution through a 0.22 µm membrane and aliquot in 15 mL tubes. Store the tubes at -20 °C.
      NOTE: This protocol uses the following references: unmodified....

Results

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

Discussion

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Material
Silicone dissection dish	Living systems instrumentation	DD-90-S-BLK-3PK	Sylgard
round coverslip	NeuVitro, Knittel glass	GG-12-Pre	12 mm
Slide a Lyzer cassettes	ThermoFisher Scientific	66030	20,000 MWCO ; 30 mL
Filter unit	Millipore	SCGVU02RE
GP Sterile Syringe Filters	Millipore	SLGP033RS
4 well plate	ThermoFisher Scientific	167063	Nunclon Delta treated plate
forceps	FST by Dumont	11252-20	#5 forceps
scissor	FST by Dumont	14060-10	fine scissors
scalpel	FST by Dumont	10035-20	curved blade
scalpel	FST by Dumont	10316-14	micro-knife scalpel
Petri dish	Greiner	663102	ø x h = 100 x 15 mm
15 mL polypropylene tube	Falcon	352096
filter paper	Watman	1001125	circle, 125 mm diameter
glass chamber slide	Lab-Tek	154526	4 chambers
Plasmid pCAGEN	Addgene	#11160
Name	Company	Catalog Number	Comments
Solutions and mediums
Bovine serum albumin	Sigma-Aldrich	A9418
L-15 medium	ThermoFisher Scientific	11415056
L-15 medium, no red phenol	ThermoFisher Scientific	21083027
Insulin	Sigma-Aldrich	I6634
Putrescine	Sigma-Aldrich	P5780
Conalbumin	Sigma-Aldrich	C7786
Sodium selenite	Sigma-Aldrich	S5261
Progesterone	Sigma-Aldrich	P8783
Poly-DL-Ornithine	Sigma-Aldrich	P8638
Laminin	Sigma-Aldrich	L2020
trypsin 2.5%, 10x	ThermoFisher Scientific	15090046
DNAse	Sigma-Aldrich	DN25
PBS w/o Ca Mg	ThermoFisher Scientific	14190144	without Mg2+ Ca2+
sodium bicarbonate	ThermoFisher Scientific	25080094
Neuron cell culture medium	ThermoFisher Scientific	A3582901	Neurobasal Plus medium
HBSS	Sigma-Aldrich	H6648-500ML
HEPES buffer 1 M	ThermoFisher Scientific	15630056
Density gradiant medium	Sigma-Aldrich	D1556	Optiprep
supplement medium	ThermoFisher Scientific	A3582801	B-27 Plus
Horse serum heat inactivated	ThermoFisher Scientific	26050-088
L-Glutamine 200 mM	ThermoFisher Scientific	25030024
2-mercaptoethanol	ThermoFisher Scientific	31350010
penicilline/streptomycine	ThermoFisher Scientific	15140122	10,000 U/mL
Name	Company	Catalog Number	Comments
Immuno fluorescence
PBS, 10x	ThermoFisher Scientific	X0515	without Mg2+ Ca2+
Paraformaldehyde (PFA)	Sigma-Aldrich	441244
normal goat serum	Sigma-Aldrich	G6767
glycine	Sigma-Aldrich	G7126
Triton X-100	Sigma-Aldrich	T8787
Choline Acetyl Transferase (CHAT)	Chemicon	Ab144P
Neurofilament H non phosphorylated (SMI32)	Biolegends	SMI-32P	IF at 1/1000
Islet-1	DSHB	40.2D6
Islet-2	DSHB	39.4D5
Hb9	DSHB	81.5C10
Vectashield mounting medium	Vector Lab	H-1000
Beta3 tubulin (Tuj1 clone)	Biolegends	801201	IF at 1/1000
Lc3b	Cell Signaling Technology	#2775	IF at 1/200