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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 1,000 times the diameter of the cell body, the possibility is readily envisioned that even small deficits could markedly impact neuronal and circuit function.

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family of growth factors, is present in many brain regions, including hippocampus, cerebral cortex, and basal forebrain. BDNF plays a crucial role in cognition and memory formation by supporting the survival, differentiation, and function of neurons that participate in cognitive circuits. BDNF binds to its receptor, the tyrosine kinase TrkB, at the axon terminal where it activates TrkB-mediated signaling pathways including the mitogen activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK), phosphatidylinositol-3-kinase (PI3K) and phospholipase C-gamma (PLCγ). The proteins that participate in these signaling pathways are packaged onto endocytic vesicular structures to form the BDNF/TrkB signaling endosome^1-6 that are then retrogradely transported to the neuronal soma.

The microfluidic culture chamber is a very useful platform for studying axonal biology under normal conditions as well as in the setting of injury and disease^7,8. By isolating axons from the cell bodies, the device has allowed one to study transport specifically in axons^8-10. The PDMS based microfluidic platforms with 450 μm microgroove barriers used in this study were commercially purchased (see Materials table). To examine BDNF transport, we developed a novel technology to produce monobiotinylated BDNF (mBtBDNF). We took advantage of the biotin acceptor peptide, AP (also known as AviTag). It is a 15 amino acid sequence that contains a lysine residue that can be specifically ligated to a biotin by the Escherichia coli enzyme biotin ligase, BirA. We fused the AviTag to the C-terminus of the mouse pre-proBDNF cDNA by PCR (Figure 1A). The construct was cloned into the mammalian expression vector, pcDNA3.1 myc-his vector. We also cloned the bacterial BirA DNA into the pcDNA3.1 myc-his vector. The two plasmids were transiently co-transfected into HEK293FT cells to express both proteins. BirA catalyzed the ligation of biotin specifically to the lysine reside within the AviTag at the C-terminus of BDNF at a 1:1 ratio to produce monobiotinylated BDNF monomer. Biotinylated, mature BDNF with a molecular mass of ~18 kDa was recovered and purified from the media using Ni-resin (Figure 1C). The biotinylation of BDNF was complete, as judged by the inability to detect unmodified BDNF by immunoblotting (Figure 1D). Streptavidin conjugated quantum dots, QD 655, were used to label mBtBDNF to make QD-BDNF. The presence of the AviTag did not interfere with activity of BDNF as the mBtBDNF was able to activate phosphorylated TrkB (Figure 1E) and stimulate neurite outgrowth (Figure 1F) to the extent of recombinant human BDNF (rhBDNF). Immunostaining shown that QD-BDNF colocalized with TrkB in hippocampal axons, indicating that QD-BDNF is bioactive (Figure 1G). To study the BDNF transport, QD-BDNF was added to distal axon compartment of microfluidic cultures containing rat E18 hippocampal neurons (Figure 2A). QD-BDNF retrograde transport within axons was captured by real-time live imaging of the red fluorescent tag (Supporting videos S1, S2). By analyzing the kymograph generated, QD-BDNF was observed to be transported retrogradely at a moving velocity of around 1.06 μm/sec (Figure 3A). GFP or mCherry-tagged BDNF have been used to track axonal movement of BDNF. The major drawbacks are that they are not bright enough for single molecule studies. Also, the presence of both anterograde and retrograde BDNF movements makes it difficult to evaluate whether or not the retrogradely transported BDNF was in a neurotrophin/receptor complex.

In this video, we demonstrate the techniques used to examine live trafficking of QD-BDNF in microfluidic chambers using primary neurons. The ultrabrightness and excellent photostability of quantum dots makes it possible to perform long-term tracking of BDNF transport. These techniques can be exploited to enhance studies of axonal function in AD, HD, and other neurodegenerative disorders.

Protokół

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.

2. Preparation of Microfluidic Chambers

Microfluidic neuron culturing device makes it possible to fluidically isolate axons from neuron cell bodies. Assemble chambers with freshly coated coverslips right before each dissection. Microfluidic chambers used in this protocol are commercially purchased (see materials and equipment’s table). Handwash and reused commercially purchased chambers up to 5-6x.

1. Hand wash microfluidic chambers in 1% Alconox. Rinse three times with Milli-Q water for 30 min each. Hand wash chambers again in 70% ethanol.
2. Lay out chambers to dry on Parafilm in the laminar flow hood. Radiate and sterilize both sides of the chambers for 20 min under UV. Store sterilized chambers in a sterile 15 cm dish sealed with Parafilm at room temperature.
3. Place 24 x 40 mm No. 1 glass coverslips in a glass container. Soak the coverslips in 35% hydrochloride acid overnight on a rotator. On the following day, rinse the coverslips three times for 30 min each with water.
4. Sterilize the coverslips one by one by dipping each coverslip into 100% ethanol and flaming over a Bunsen burner. Store dry coverslips in a sterile petri dish and keep it at room temperature until coating.
5. Lay out the coverslips in a 15 cm culture dish. Coat each coverslip with 0.7 ml of 0.01% poly-L-Lysine (PLL) and incubate in the hood at room temperature.
6. After 1 hr, rinse the coverslips three times with sterile water. Dry coverslips with vacuum and place each coverslip into a 6 cm culture dish.
7. To assemble the microfluidic chamber, place the chamber with microgroove side at the bottom onto the PLL coated coverslip with caution not to touch the microgrooves. Gently pressed down the chamber with a pipette tip to ensure the chamber was tightly sealed.

3. Dissection of Neuronal Culture and Plate on Chambers

1. Place two E17-E18 rat hippocampi (one brain) dissected in a 15 ml conical tube containing 2 ml dissection buffer (HBSS, no calcium, no magnesium, with 1% pen/strep and 10 mM HEPES. Rinse the tissues 3x with 5 ml dissection buffer each time. Remove the dissection buffer as much as possible and add 900 μl of fresh dissection buffer.
2. To digest the tissue, add 100 μl of 10x trypsin (2.5%) into the dissection buffer to make 1x working concentration. Place the conical tube in a 37 °C water bath. After 10 min of digestion, add 100 μl of 10 mg/ml DNase I to a final concentration around 1 mg/ml.
3. Using a fire-polished Pasteur glass pipette, gently triturate the tissues by pipetting up and down 5-10x. Right after trituration, quench the trypsin with 2 ml plating medium (Neurobasal with 10% FBS, 2 mM GlutaMax, 2% B27).
4. Leave the sample in the hood for 5 min to allow any debris of the tissues to settle down to the bottom. Carefully remove 2 ml of supernatant into a clean sterile 15 ml conical tube and centrifuge at 200 x g for 5 min to pellet the cells. Resuspend the pellet in 50 μl plating media
5. Count the cells with hemocytometer. Load 15-20 μl cell suspension (~40,000 cells) into one compartment of the microfluidic chamber. Place the chamber in the incubator for 10 min to allow the cells to attach to the coverslip. After 10 min, add more plating media to fill up both compartments of the chamber.
6. On the second day of dissection, completely replace the plating media with maintenance media (Neurobasal with 2 mM GlutaMax, 2% B27) in both the cell body and the axon compartment. Axons from the hippocampal neurons start to cross the microgrooves in day 3 and reach the axon compartment between day 5-7. During this period of time, replace half of the culturing media with fresh maintenance media every 24~48 hr.

4. Axonal Transport of QD-BDNF

1. Prior to live imaging of QD-BDNF axonal transport, deplete BDNF from both the cell body and axon compartments of the microfluidic chamber by thoroughly rinse both compartments with BDNF-free, serum free Neurobasal media every 30 min for 2 hr.
2. During BDNF depletion, prepare the QD-BDNF conjugates. Mix 50 nM of mono-biotinylated BDNF dimer with 50 nM QD655-streptavidin conjugates in neurobasal media and incubate on ice for 60 min.
3. Remove media in the axon compartment and add 300 μl QD-BDNF with a final concentration of 0.25 nM for 4 hr at 37 °C. To minimize the diffusing of QD-BDNF into the cell body compartment, it is very important to always maintain a higher level of media in the cell body compartment than in the axon compartment. Wash off unbound QD-BDNF after incubation before live imaging.
4. Carry out live imaging of QD-BDNF transport using an inverted microscope equipped with a 100X oil objective lens. Warm up the scope and the environmental chamber attached to it to a constant temperature (37 °C) and CO₂ (5%). Use a set of Texas red excitation/emission cubes to visualize the QD655 signal.
5. Acquire and capture time-lapse images within the middle axons at the speed of 1 frame/sec for a total of 2 min using a CCD camera. Use microgrooves with no axons that have no QD and hence no signal as a control for infiltration.
6. Analyze BDNF transport using any image analysis software or NIH ImageJ.

Wyniki

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 (...

Dyskusje

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 ...

Materiały

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