Measuring Axonal Cargo Transport in Mouse Primary Cortical Cultured Neurons

Summary

The present protocol describes the whole procedure of analysis for axonal transport. In particular, the calculation of transport velocity, not including the pausing, and the visualization method using open-access software "KYMOMAKER" are shown here.

Abstract

Neuronal cells are highly polarized cells that stereotypically harbor several dendrites and an axon. The length of an axon necessitates efficient bidirectional transport by motor proteins. Various reports have suggested that defects in axonal transport are associated with neurodegenerative diseases. Also, the mechanism of the coordination of multiple motor proteins has been an attractive topic. Since the axon has uni-directional microtubules, it is easier to determine which motor proteins are involved in the movement. Therefore, understanding the mechanisms underlying the transport of axonal cargo is crucial for uncovering the molecular mechanism of neurodegenerative diseases and the regulation of motor proteins. Here, we introduce the entire process of axonal transport analysis, including the culturing of mouse primary cortical neurons, transfection of plasmids encoding cargo proteins, and directional and velocity analyses without the effect of pauses. Furthermore, the open-access software "KYMOMAKER" is introduced, which enables the generation of a kymograph to highlight transport traces according to their direction and allow easier visualization of axonal transport.

Introduction

Kinesin family members and cytoplasmic dynein are motor proteins that move along the microtubules in cells to transport their cargo¹. Most kinesins move toward the plus end, whereas dynein moves toward the minus end of a microtubule. The functions and mechanisms of cargo transport in the neuronal axon have been investigated extensively. Because of their length, axons require stable long-distance transport to maintain the health of neurons. Defects in the transport of mitochondria, autophagosomes, and vesicles containing amyloid-β protein precursor (APP) have been reported as causes of neurodegenerative diseases²

Protocol

The experiments were approved by the Animal Studies Committee of Hokkaido University, following the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. Female C57BL/6J mice (pregnant, 15.5 days) were used for the present study.

1. Preparation of mouse primary cortical cultured neurons

1. Add 0.1 mg/mL of poly-L-Lysin in 0.1 M Tris (tris(hydroxymethyl)aminomethane)-HCl (pH 8.5) into an 8-well (No.1.0) glass-bottom chamber (see Table of Materials). Incubate the chamber in a 37 °C incubator for a minimum of 1 h.
2. Rinse the chamber with sterilized water a minimum of thr....

Results

Primary cultured neurons from the E15.5 mouse cortex were cultured in a glass-bottom dish as described. As an example, APP-EGFP, Alcα-EGFP, or Alcβ-EGFP were expressed in the primary cortical neurons. APP and Alcα are known to be transported in the axon by kinesin-1^2,17. APP associates with kinesin-1 via the adapter protein JIP1 (JNK-interacting protein 1), while Alcα directly binds via its double W-acidic motifs. Alcβ is a member of the Alcadei.......

Discussion

An analysis method for axonal transport is described, which includes the calculation of segmented velocity and the generation of kymographs. A critical step during the transfection step is maintaining the health of cultured neurons. The transfection method described by Jiang and Chen²⁹ was followed with minor modifications. Gentle mixing of the DNA/CaCl₂ solution and 2x HBS increased the efficiency of transfection by reducing the size of the precipitations. To prevent the precipitations.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5-Fluoro-2-deoxyuridine	Sigma-Aldrich	F0503
Apo TIRF 100x/1.49 OIL	Nikon
B-27 Supplement (50x), serum free	Thermo fischer scientific	17504044
Bovine serum albumin	Wako	013-25773
CalPhos Mammalian Transfection Kit	Takara	631312
Cell strainer  40 µm Nylon	Falcon	352340
CoolSNAP HQ	Photometrics
Deoxyribonuclease I	Sigma-Aldrich	DN-25
D-Glucose	Wako	041-00595
DMEM/Ham’s F-12	Wako	042-30555
Dumont No. 7 forceps	Dumont	No.7
Feather surgical blade	Feather	No.11
Feather surgical blade handle	Feather	No. 3
Gentamicin	Wako	079-02973
Gentamicin Sulfate	Wako	075-04913
GlutaMAX Supplement	Thermo fischer scientific	35050061
HEPES	DOJINDO	342-01375
Horse Serum, heat inactivated	Thermo fischer scientific	26050088
KCl	Wako	163-03545
KH2PO4	Wako	169-04245
KYMOMAKER			http://www.pharm.hokudai.ac.jp/shinkei/Kymomaker.html
L-15 Medium (Leibovitz)	Sigma-Aldrich	L5520
MetaMorph version 6.2r1	Metamorph
Na2HPO4	Wako	197-02865
NaCl	Wako	197-01667
NaHCO3	Wako	191-01305
Neurobasal Medium	Thermo fischer scientific	21103049
Nikon ECLIPSE TE 2000-E	Nikon
Nunc Lab-Tek  8 well Chambered Coverglass	Thermo fischer scientific	155411
Papain	Worthington	LS003126
Penicillin-Streptomycin (10,000 U/mL)	Thermo Fisher Scientific	15140122
Poly-L-lysine hydrobromide	Sigma-Aldrich	P2636-500MG
Trizma base	Merck	T1194-10PAK	solved with water to make 0.1 M Tris-HCl (pH.8.5)