We would like to thank Yue (Pauline) Hu, Rachel Sinit for their technical assistance. The study is supported by NIH grant (PN2 EY016525) and by funding from Down Syndrome Research and Treatment Foundation and the Larry L. Hillblom Foundation.

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 acco...

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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 ...

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 (PLC&#947;). The proteins that participate in these signaling pathways are packaged onto endocytic vesicular structures to form the BDNF/TrkB signaling endosome1-6 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 disease7,8. By isolating axons from the cell bodies, the device has allowed one to study transport specifically in axons8-10. The PDMS based microfluidic platforms with 450 &#956;m microgroove barriers used in this study were commercially purchased (see Materials&#160;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&#160;S1, S2). By analyzing the kymograph generated, QD-BDNF was observed to be transported retrogradely at a moving velocity of around 1.06 &#956;m/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.

<table border="0" cellpadding="0" cellspacing="0" width="641">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:261px;">Name</td>
			<td style="width:205px;">Company</td>
			<td style="width:175px;">Catalog Number</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Platinum pfx DNA polymerase&#160;</td>
			<td>Invitrogen</td>
			<td>11708021</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EcoRI&#160;</td>
			<td>Fermentas</td>
			<td>FD0274</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BamHI&#160;</td>
			<td>Fermentas</td>
			<td>FD0054</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEK293FT cells</td>
			<td>Invitrogen</td>
			<td>R70007</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM-high glucose media</td>
			<td>Mediatech</td>
			<td>10-013-CV</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">d-biotin&#160;</td>
			<td>Sigma</td>
			<td>B4639</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TurboFect&#160;</td>
			<td>Fermentas</td>
			<td>R0531</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PMSF&#160;&#160;</td>
			<td>Sigma</td>
			<td>P7626</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">aprotinin</td>
			<td>Sigma</td>
			<td>A6279</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ni-NTA resins</td>
			<td>Qiagen</td>
			<td>30250</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">protease inhibitors cocktail</td>
			<td>Sigma</td>
			<td>&#160;S8820</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">silver staining kit&#160;</td>
			<td>G-Biosciences</td>
			<td>786-30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human recombinant BDNF</td>
			<td>Genentech</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microfluidic chambers</td>
			<td>Xona</td>
			<td>SND450</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">24x40 mm No. 1 glass coverslips&#160;</td>
			<td>VWR</td>
			<td>48393-060</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">poly-L-Lysine&#160;</td>
			<td>Cultrex</td>
			<td>3438-100-01</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS</td>
			<td>Gibco</td>
			<td>14185-052</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNase I</td>
			<td>Roche</td>
			<td>10104159001</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin</td>
			<td>Gibco</td>
			<td>15090-046</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Neurobasal&#160;</td>
			<td>Gibco</td>
			<td>21103-049</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS&#160;</td>
			<td>Invitrogen</td>
			<td>16000-044</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GlutaMax&#160;</td>
			<td>Invitrogen</td>
			<td>35050-061</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B27&#160;&#160;</td>
			<td>Gibco</td>
			<td>17504-044</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QD655-streptavidin conjugates</td>
			<td>Invitrogen</td>
			<td>&#160;Q10121MP</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-Avi tag antibody</td>
			<td>GenScript</td>
			<td>A00674</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">streptavidin-agarose beads&#160;</td>
			<td>Life Technology</td>
			<td>&#160;SA100-04</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">trichloroacetic acid</td>
			<td>Sigma</td>
			<td>T6399</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HRP-streptavidin&#160;</td>
			<td>Thermo Scientific</td>
			<td>N100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-pTrkB antibody</td>
			<td colspan="2">a generous gift from Dr M. Chao of NYU</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-TrkB antibody</td>
			<td>BD Science</td>
			<td>610101</td>
		</tr>
	</tbody>
</table>

real-time imaging of axonal transport of quantum dot-labeled bdnf in primary neurons

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&#8217;s disease (AD) and Huntington&#8217;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 &#956;m/sec. This system can be used to investigate mechanisms of disrupted axonal function in AD or HD, as well as other degenerative disorders.

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 (<a href="http://ca.expasy.org/tools/pi_tool.html" target="_blank">http://ca.expasy.org/tools/pi_tool.html</a>) Monobiotinylated mature BDNF with a predicted molecular mass of 18 kDa (...

Watch this Scientific Journal Video about Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons at JoVE.com

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

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.

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are ...

real-time-imaging-axonal-transport-quantum-dot-labeled-bdnf-primary

Research

JoVE Journal

Neuroscience

15.0K visualizações.  University of California, San Diego.  O transporte axonal de BDNF, um factor neurotrófico, é crítico para a sobrevivência e função de várias populações neuronais. Algumas desordens degenerativas são marcadas pela ruptura da estrutura e função axonal. Nós demonstramos as técnicas utilizadas para examinar o tráfico ao vivo do QD-BDNF em câmaras microfluídicos usando neurônios primários. 