Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

Summary

Axonal transport of BDNF, a neurotrophic factor, is critical for the survival and function of several neuronal populations. Some degenerative disorders are marked by disruption of axonal structure and function. We demonstrated the techniques used to examine live trafficking of QD-BDNF in microfluidic chambers using primary neurons.

Abstract

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are increasingly viewed as an early feature of neurodegenerative diseases, including Alzheimer’s disease (AD) and Huntington’s disease (HD). As yet unclear is the mechanism(s) by which axonal injury is induced. We reported the development of a novel technique to produce biologically active, monobiotinylated BDNF (mBtBDNF) that can be used to trace axonal transport of BDNF. Quantum dot-labeled BDNF (QD-BDNF) was produced by conjugating quantum dot 655 to mBtBDNF. A microfluidic device was used to isolate axons from neuron cell bodies. Addition of QD-BDNF to the axonal compartment allowed live imaging of BDNF transport in axons. We demonstrated that QD-BDNF moved essentially exclusively retrogradely, with very few pauses, at a moving velocity of around 1.06 μm/sec. This system can be used to investigate mechanisms of disrupted axonal function in AD or HD, as well as other degenerative disorders.

Introduction

Neurons are highly polarized cells whose long and often highly elaborated processes are fundamental for establishing and maintaining the structure and function of neural circuits. The axon plays a vital role in carrying cargoes to and from synapses. Proteins and organelles synthesized in the cell soma need to be transported through axons to reach the presynaptic terminal to support neuronal function. Correspondingly, signals received at distal axons need to be transduced and conveyed to the soma. These processes are essential for neuronal survival, differentiation, and maintenance. In that axonal transport in some neurons must be conducted through distances more than ....

Protocol

Surgical and animal procedures are carried out strictly according to the NIH Guide for the Care and Use of Laboratory Animals. All experiments involving the use of animals are approved by UCSD Institutional Animal Care and Use Committee.

1. Plasmid Cloning, Expression and Purification of Mono-biotinylated BDNF (mBtBDNF)

NOTE: Construct pre-proBDNFavi and BirA cDNA into pcDNA3.1 vector and coexpress in HEK293FT cells¹⁰. Purify mBtBDNF using Ni-NTA beads according to previously published method of producing mature and biologically active monobiotinylated nerve growth factor (mBtNGF)¹⁰.

Results

Production and Purification of Biologically Active Mono-biotinylated BDNF

The expression vector of BDNF fused with an AviTag sequence (GGGLNDIFEAQKIEWHE) was created according to a previously published protocol¹⁰. The molecular mass of the full length fusion protein was predicted to be ~32 kDa (http://ca.expasy.org/tools/pi_tool.html) Monobiotinylated mature BDNF with a predicted molecular mass of 18 kDa (.......

Discussion

In this study, we report the development of a novel technique to produce biologically active, monobiotinylated BDNF (mBtBDNF) that can be used to trace axonal transport of BDNF. By conjugating the protein to quantum dot streptavidin, and using a microfluidic chamber, the method allows one to detect axonal transport of BDNF in primary neurons with single molecule sensitivity, in real-time and with spatial and temporal resolutions. The tools used herein provide a means by which to study the molecular machines that mediate .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Platinum pfx DNA polymerase	Invitrogen	11708021
EcoRI	Fermentas	FD0274
BamHI	Fermentas	FD0054
HEK293FT cells	Invitrogen	R70007
DMEM-high glucose media	Mediatech	10-013-CV
D-biotin	Sigma	B4639
TurboFect	Fermentas	R0531
PMSF	Sigma	P7626
Aprotinin	Sigma	A6279
Ni-NTA resins	Qiagen	30250
Protease inhibitors cocktail	Sigma	S8820
Silver staining kit	G-Biosciences	786-30
Human recombinant BDNF	Genentech
Microfluidic chambers	Xona	SND450
24 x 40 mm No. 1 glass coverslips	VWR	48393-060
Poly-L-Lysine	Cultrex	3438-100-01
HBSS	Gibco	14185-052
DNase I	Roche	10104159001
Trypsin	Gibco	15090-046
Neurobasal	Gibco	21103-049
FBS	Invitrogen	16000-044
GlutaMax	Invitrogen	35050-061
B27	Gibco	17504-044
QD655-streptavidin conjugates	Invitrogen	Q10121MP
anti-Avi tag antibody	GenScript	A00674
streptavidin-agarose beads	Life Technology	SA100-04
Trichloroacetic acid	Sigma	T6399
HRP-streptavidin	Thermo Scientific	N100
anti-pTrkB antibody	a generous gift from Dr M. Chao of NYU
anti-TrkB antibody	BD Science	610101