Name: an improved protocol to purify and directly mono-biotinylate recombinant bdnf in a tube for cellular trafficking studies in neurons
Uploaded: 2020-07-11T13:00:00
Description: Watch this Scientific Journal Video about An Improved Protocol to Purify and Directly Mono-Biotinylate Recombinant BDNF in a Tube for Cellular Trafficking Studies in Neurons at JoVE.com

The authors gratefully acknowledge financial support from Fondecyt (1171137) (FCB), the Basal Center of Excellence in Science and Technology (AFB 170005) (FCB), Millenium-Nucleus (P07/011-F) (FCB), the Wellcome Trust Senior Investigator Award (107116/Z/15/Z) (GS) and a UK Dementia Research Institute Foundation award (GS). This work was supported by the Unidad de Microscop&#237;a Avanzada UC (UMA UC).

All experiments were carried out in accordance with the approved guidelines of CONICYT (Chilean National Commission for Scientific and Technological Research). The protocols used in this study were approved by the Biosecurity and Bioethical and Animal Welfare Committees of the Pontificia Universidad Cat&#243;lica de Chile. Experiments involving vertebrates were approved by the Bioethical and Animal Welfare Committee of the Pontificia Universidad Cat&#243;lica de Chile.
NOTE: The following prot...

<ol>
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In this article, an optimized methodology for the production and purification of mbtBDNF in an affinity chromatography-based procedure is described, based on the work of Sung and collaborators17. The optimizations include the use of a cost-effective transfection reagent (PEI) while maintaining the efficiency of more expensive transfection methods such as lipofectamine. This optimization translates into a significant cost reduction in the protocol, allowing for scalability while maintaining high co...

Neurons are the functional units of the nervous system possessing a complex and specialized morphology that allows synaptic communication, and thus, the generation of coordinated and complex behavior in response to diverse stimuli. Neuronal projections such as dendrites and axons are critical structural features involved in neuronal communication, and neurotrophins are crucial players in determining their morphology and function1. Neurotrophins are a family of secreted growth factors that include NGF, NT-3, NT-4, and brain-derived neurotrophic factor (BDNF)2. In the central nervous system (CNS), BDNF participates in diverse biological processes including neurotransmission, dendritic arborization, maturation of dendritic spines, long-term potentiation, among others3,4. Therefore, BDNF plays a critical role in regulating neuronal function.
Diverse cellular processes regulate BDNF dynamics and function. On the neuronal surface, BDNF binds the tropomyosin receptor kinase B (TrkB) and/or the p75 neurotrophin receptor (p75). BDNF-TrkB and BDNF-p75 complexes are endocytosed and sorted in different endocytic organelles5,6,7,8. Correct intracellular trafficking of the BDNF/TrkB complex is required for proper BDNF signaling in different neuronal circuits9,10,11. For this reason, a deep understanding of BDNF trafficking dynamics and its alterations found in pathophysiological processes is essential to understand BDNF signaling in health and disease. The development of novel and specific molecular tools to monitor this process will help to drive this field forward and allow a better grasp of the regulatory mechanisms involved.
There are several tools available for the study of BDNF trafficking in neurons. A commonly used methodology involves the transfection of recombinant BDNF tagged with fluorescent molecules such as green fluorescent protein (GFP) or the monomeric fluorescent red-shifted variant of GFP mCherry12,13. However, a major shortcoming of BDNF overexpression is that it eliminates the possibility of delivering known concentrations of this neurotrophin. Also, it may result in cellular toxicity, obscuring the interpretation of results14. An alternative strategy is the transfection of an epitope-tagged TrkB, such as Flag-TrkB. This methodology allows the study of TrkB internalization dynamics15, but it also involves transfection, which might result in altered TrkB function and cellular toxicity. To overcome these methodological hurdles, recombinant variants of NGF and BDNF containing an Avi sequence (BDNFAvi), which can be mono-biotinylated by the biotin-ligase enzyme BirA, were developed16,17. Biotinylated recombinant BDNF can be coupled to different streptavidin-bound tools, which include fluorophores, beads, paramagnetic nanoparticles among others for detection. In terms of live-cell imaging, quantum dots (QD) have become frequently used fluorophores, as they have desirable characteristics for single-particle tracking, such as increased brightness and resistance to photobleaching when compared to small molecule fluorophores18.
The production of mono-biotinylated BDNF (mbtBDNF) using BDNFAvi has been achieved by co-transfection of plasmids driving the expression of BDNFAvi and BirA, followed by the purification of the recombinant protein by affinity chromatography with a yield of 1-2 &#956;g of BDNF per 20 mL of HEK293-conditioned culture media17. Here, we propose a modification of this protocol that allows for BDNFAvi purification from 500 mL of HEK293-conditioned media, which seeks to maximize protein recovery in a chromatography-column based protocol for ease of manipulation. The used transfection agent, polyethyleneimine (PEI), ensures a cost-effective method without sacrificing transfection yield. The mono-biotinylation step has been adapted to an in vitro reaction to avoid the complications associated with co-transfections and to ensure homogeneous labeling of BDNF. The biological activity of the mbtBDNF was demonstrated by western blot and fluorescence microscopy experiments, including activation of pCREB and live cell imaging to study retrograde axonal transport of BDNF in microfluidic chambers. The use of this protocol allows for optimized, high-yield production of homogenous mono-biotinylated and biologically active BDNF.

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">2 way stopcock</td>
			<td style="width:121px;">BioRad</td>
			<td style="width:161px;">7328102</td>
			<td style="width:167px;">Chromatography apparatus component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-mercaptoethanol</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:161px;">M6250</td>
			<td style="width:167px;">BDNF elution buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Acrylamide/Bisacrylamide</td>
			<td style="width:121px;">BioRad</td>
			<td style="width:161px;">1610154</td>
			<td style="width:167px;">SDS-PAGE gel preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Amicon Ultra-15 10K</td>
			<td style="width:121px;">Millipore</td>
			<td style="width:161px;">UFC901024</td>
			<td style="width:167px;">BDNF concentration</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ammonium Persulfate</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:161px;">A9164</td>
			<td style="width:167px;">SDS-PAGE gel preparation</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">anti B-III-Tubulin antibody</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:161px;">T8578</td>
			<td style="width:167px;">Western blot assays for BDNF biological activity detection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti BDNF antibody</td>
			<td style="width:121px;">Alomone</td>
			<td style="width:161px;">AGP-021</td>
			<td style="width:167px;">Western blot assays for BDNF quantification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti BDNF antibody</td>
			<td style="width:121px;">Alomone</td>
			<td style="width:161px;">ANT-010</td>
			<td style="width:167px;">Western blot assays for BDNF quantification</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Anti ERK antibody</td>
			<td style="width:121px;">Cell Signaling</td>
			<td style="width:161px;">9102</td>
			<td style="width:167px;">Western blot assays for BDNF biological activity detection</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">anti pCREB antibody (S133)</td>
			<td style="width:121px;">Cell Signaling</td>
			<td style="width:161px;">9198</td>
			<td style="width:167px;">Western blot assays for BDNF biological activity detection</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">anti pERK antibody (T202, Y204)</td>
			<td style="width:121px;">Cell Signaling</td>
			<td style="width:161px;">4370</td>
			<td style="width:167px;">Western blot assays for BDNF biological activity detection</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">anti pTrkB antibody (Y515)</td>
			<td style="width:121px;">Abcam</td>
			<td style="width:161px;">ab109684</td>
			<td style="width:167px;">Western blot assays for BDNF biological activity detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Antibiotic/Antimycotic</td>
			<td style="width:121px;">Gibco</td>
			<td>15240-062</td>
			<td style="width:167px;">HEK293 maintenance</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ATP</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:161px;">A26209</td>
			<td style="width:167px;">BDNF monobiotinylation buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">B-27 Supplement</td>
			<td style="width:121px;">Gibco</td>
			<td>17504-044</td>
			<td style="width:167px;">Neuron maintenance</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bicine</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:161px;">B3876</td>
			<td style="width:167px;">BDNF monobiotinylation buffer</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">BirA-GST</td>
			<td style="width:121px;">BPS Bioscience</td>
			<td style="width:161px;">70031</td>
			<td style="width:167px;">Enzyme for BDNF AviTag monobiotinylation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bovine Fetal Serum</td>
			<td style="width:121px;">HyClone</td>
			<td>HC.SH30396.02</td>
			<td style="width:167px;">HEK293 maintenance</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">Bovine Serum Albumin</td>
			<td style="width:121px;">Jackson ImmunoResearch</td>
			<td>001-000-162</td>
			<td style="width:167px;">BDNF buffer modification component, blocking buffer for western blot and immunofluorescence</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">D-Biotin</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:161px;">B4639</td>
			<td style="width:167px;">BDNF monobiotinylation buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dithiothreitol</td>
			<td style="width:121px;">Invitrogen</td>
			<td style="width:161px;">15508-013</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM High Glucose Medium</td>
			<td style="width:121px;">Gibco</td>
			<td>11965-092</td>
			<td style="width:167px;">Neuron seeding</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM Medium</td>
			<td style="width:121px;">Gibco</td>
			<td>11995-081</td>
			<td style="width:167px;">HEK293 maintenance</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Econo Column Funnel</td>
			<td style="width:121px;">BioRad</td>
			<td>7310003</td>
			<td style="width:167px;">Chromatography apparatus component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td style="width:121px;">Merck</td>
			<td style="width:161px;">108418</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">EZ-ECL Kit</td>
			<td style="width:121px;">Biological Industries</td>
			<td style="width:161px;">1633664</td>
			<td style="width:167px;">Protein detection by western blotting</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glutamax</td>
			<td style="width:121px;">Gibco</td>
			<td>35050-061</td>
			<td style="width:167px;">Neuron and HEK293 maintenance</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Glycerol</td>
			<td style="width:121px;">Merck</td>
			<td style="width:161px;">104094</td>
			<td style="width:167px;">BDNF elution buffer, lysis buffer for western blot assays</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Hettich Rotina 46R Centrifuge</td>
			<td style="width:121px;">Hettich</td>
			<td style="width:161px;">Discontinued</td>
			<td style="width:167px;">Centrifuge used for clearing the medium of debris</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Hettich Universal 32R Centrifuge</td>
			<td style="width:121px;">Hettich</td>
			<td style="width:161px;">Discontinued</td>
			<td style="width:167px;">Centrifuge used for protein concentrator centrifugation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Horse Serum</td>
			<td style="width:121px;">Gibco</td>
			<td>16050-122</td>
			<td style="width:167px;">Neuron seeding</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ImageQuant LAS 500</td>
			<td style="width:121px;">GE Healthcare Life Sciences</td>
			<td style="width:161px;">29005063</td>
			<td style="width:167px;">Western blot image acquisition</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Imidazole</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:161px;">I55513</td>
			<td style="width:167px;">BDNF buffer modification component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KCl</td>
			<td style="width:121px;">Winkler</td>
			<td style="width:161px;">BM-1370</td>
			<td style="width:167px;">PBS component</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">KH2PO4</td>
			<td style="width:121px;">Merck</td>
			<td style="width:161px;">104873</td>
			<td style="width:167px;">PBS component</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Laminin</td>
			<td style="width:121px;">Invitrogen</td>
			<td style="width:161px;">23017-015</td>
			<td style="width:167px;">Cover coating for compartmentalized neurons</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Luer Tubing Adaptor</td>
			<td style="width:121px;">BioRad</td>
			<td>7323245</td>
			<td style="width:167px;">Chromatography apparatus component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Luminata&#8482; Forte Western HRP Substrate</td>
			<td style="width:121px;">Millipore</td>
			<td style="width:161px;">WBLUF0100</td>
			<td style="width:167px;">Protein detection by western blotting</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;">Mg(CH3COO)2</td>
			<td style="width:121px;">Merck</td>
			<td style="width:161px;">105819</td>
			<td style="width:167px;">BDNF monobiotinylation buffer</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Mowiol 4-88</td>
			<td style="width:121px;">Calbiochem</td>
			<td style="width:161px;">475904</td>
			<td style="width:167px;">Mounting reagent for immunofluorescence assays</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MyOne C1 Streptavidin Magnetic Beads</td>
			<td style="width:121px;">Invitrogen</td>
			<td style="width:161px;">65001</td>
			<td style="width:167px;">Biotinylation verification</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;">Na2HPO4</td>
			<td style="width:121px;">Merck</td>
			<td style="width:161px;">106586</td>
			<td style="width:167px;">BDNF buffer modification component</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">NaCl</td>
			<td style="width:121px;">Winkler</td>
			<td style="width:161px;">BM-1630</td>
			<td style="width:167px;">PBS component, BDNF buffer modification component</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;">NaH2PO4</td>
			<td style="width:121px;">Merck</td>
			<td style="width:161px;">106346</td>
			<td style="width:167px;">BDNF buffer modification component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Neurobasal Medium</td>
			<td style="width:121px;">Gibco</td>
			<td>21103-049</td>
			<td style="width:167px;">Neuron maintenance</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ni-NTA Agarose Beads</td>
			<td style="width:121px;">Qiagen</td>
			<td style="width:161px;">30210</td>
			<td style="width:167px;">BDNF AviTag purification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nikon Ti2-E</td>
			<td style="width:121px;">Nikon</td>
			<td style="width:161px;"></td>
			<td style="width:167px;">Microscope for fluorescence imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nitrocellulose Membrane</td>
			<td style="width:121px;">BioRad</td>
			<td style="width:161px;">1620115</td>
			<td style="width:167px;">Protein transfer for western blotting</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">ORCA-Flash4.0 V3 Digital CMOS camera</td>
			<td style="width:121px;">Hamamatsu</td>
			<td style="width:161px;">C13440-20CU</td>
			<td style="width:167px;">Camera for epifluorescence imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">P8340 Protease Inhibitor Cocktail</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:161px;">P8340</td>
			<td style="width:167px;">BDNF buffer modification component</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Paraformaldehyde</td>
			<td style="width:121px;">Merck</td>
			<td style="width:161px;">104005</td>
			<td style="width:167px;">Fixative for immunofluorescence assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin/Streptomycin</td>
			<td style="width:121px;">Gibco</td>
			<td>15140-122</td>
			<td style="width:167px;">Neuron maintenance</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Poli-D-Lysine</td>
			<td style="width:121px;">Corning</td>
			<td style="width:161px;">DLW354210</td>
			<td style="width:167px;">Cover coating for compartmentalized neurons</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Poli-L-Lysine</td>
			<td style="width:121px;">Millipore</td>
			<td style="width:161px;">P2363</td>
			<td style="width:167px;">Cover coating for non-compartmentalized neurons</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Poly-Prep Chromatography Column</td>
			<td style="width:121px;">BioRad</td>
			<td>7311550</td>
			<td style="width:167px;">Chromatography apparatus component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Polyethyleneimine 25K</td>
			<td style="width:121px;">Polysciences Inc.</td>
			<td>PLY-0296</td>
			<td style="width:167px;">HEK293 transfection</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Quantum Dots 655 streptavidin conjugate</td>
			<td style="width:121px;">Invitrogen</td>
			<td style="width:161px;">Q10121MP</td>
			<td style="width:167px;">Monobiotinylated BDNF AviTag label for live and fixed cell experiments</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Saponin</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:161px;">S4521</td>
			<td style="width:167px;">Detergent for immunofluorescence assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sucrose</td>
			<td style="width:121px;">Merck</td>
			<td style="width:161px;">107687</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Syldgard 184 silicone elastomer base</td>
			<td style="width:121px;">Poirot</td>
			<td style="width:161px;">4019862</td>
			<td style="width:167px;">Microfluidic chamber preparation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TEMED</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:161px;">T9281</td>
			<td style="width:167px;">SDS-PAGE gel preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris</td>
			<td style="width:121px;">Winkler</td>
			<td style="width:161px;">BM-2000</td>
			<td style="width:167px;">Lysis buffer component</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Triton X100</td>
			<td style="width:121px;">Merck</td>
			<td style="width:161px;">108603</td>
			<td style="width:167px;">Cell permeabilization in immunofluorescence and western blot assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin-EDTA 0.5%</td>
			<td style="width:121px;">Gibco</td>
			<td>15400-054</td>
			<td style="width:167px;">HEK293 passaging</td>
		</tr>
	</tbody>
</table>

an improved protocol to purify and directly mono-biotinylate recombinant bdnf in a tube for cellular trafficking studies in neurons

Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells and then cost-effectively purified by affinity chromatography. A reproducible protocol was developed to directly&#160;mono-biotinylate BDNFAvi with the enzyme BirA in a tube. In this reaction, mono-biotinylated BDNFAvi retains its biological activity.
Neurotrophins are target-derived growth factors playing a role in neuronal development and maintenance. They require rapid transport mechanisms along the endocytic pathway to allow long-distance signaling between different neuronal compartments. The development of molecular tools to study the trafficking of neurotrophins has enabled the precise tracking of these proteins in the cell using in vivo recording. In this protocol, we developed an optimized and cost-effective procedure for the production of mono-biotinylated BDNF. A recombinant BDNF variant containing a biotinylable avi sequence (BDNFAvi) is produced in HEK293 cells in the microgram range and then purified in an easily scalable procedure using affinity chromatography. The purified BDNF can then be homogeneously mono-biotinylated by a direct in vitro reaction with the enzyme BirA in a tube. The biological activity of the mono-biotinylated BDNF (mbtBDNF) can be conjugated to streptavidin-conjugated to different fluorophores. BDNFAvi and mbtBDNF retain their biological activity demonstrated through the detection of downstream phosphorylated targets using western blot and activation of the transcription factor CREB, respectively. Using streptavidin-quantum dots, we were able to visualize mbtBDNF internalization concomitant with activation of CREB, which was detected with a phospho-CREB specific antibody. In addition, mbtBDNF conjugated to streptavidin-quantum dots was suitable for retrograde transport analysis in cortical neurons grown in microfluidic chambers. Thus, in tube produced mbtBDNF is a reliable tool to study physiological signaling endosome dynamics and trafficking in neurons.

The use of a chromatographic column-based protocol allows the processing of significant volumes of HEK293 conditioned media. In Figure 1, the results of the purification of BDNFAvi from 500 mL of conditioned media are shown. Consecutive elutions of BDNFAvi from the Ni-NTA agarose beads yield decreasing concentrations of BDNFAvi (Figure 1A). After four consecutive elutions (each lasting 15 min), the majority of the BDNF captured by the beads is recovered. The con...

Watch this Scientific Journal Video about An Improved Protocol to Purify and Directly Mono-Biotinylate Recombinant BDNF in a Tube for Cellular Trafficking Studies in Neurons at JoVE.com

An Improved Protocol to Purify and Directly Mono-Biotinylate Recombinant BDNF in a Tube for Cellular Trafficking Studies in Neurons

Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells in a cost-effective manner and is purified by affinity chromatography. BDNFavi is then directly mono-biotinylated with the enzyme BirA in a tube. BDNFavi and mono-biotinylated BDNFavi retain their biological activity when compared to commercially available BDNF.

Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells and then cost-effectively purified by affinity chromatography. A ...

The overall goal of this procedure is to produce and purify BDNF oviduct in an optimized protocol involving transfection of HEK293 cells and purification in a chromatography column. The BDNF oviduct is then mono-biotinylated by an in vitro reaction for posterior labeling and detection by fluorescence microscopy. The main advantage of this procedure is that it allows the production, purification, and mono-biotinylation of BDNF in an optimized manner.The gestation parameters have been optimized for maximum BDNF production using a cost-effective transfection reagent and the purification process is performed in a chromatography setup that facilitates manipulation and allows to escalate the procedure. Begin by diluting 60 micrograms of polyethylenimine in 500 microliters of unsupplemented DMEM and 20 micrograms of BDNF oviduct plasmid in 500 microliters of unsupplemented DMEM per 15 centimeter dish to be transfected. Incubate for five minutes at room temperature and then carefully pipette the DNA solution into a polyethylenimine tube, mixing once by up/down motion.Incubate for 25 minutes at room temperature and then drip one milliliter of the PEI/DNA mixture throughout each 15 centimeter dish. Incubate the cells with the PEI/DNA mixture for three hours and then change the medium to incubation buffer. 48 hours after the transfection of the cells, collect the medium from all the dishes.Then add the BDNF medium components to the HEK293 supernatant including sodium chloride, phosphate buffered components, imidazole, and protease inhibitors. Incubate in ice for 15 minutes. Then aliquot the medium into centrifuge tubes.Centrifuge for 45 minutes at 10, 000 g in a four degree Celsius gold centrifuge. This step allows the elimination of cell debris and dead cells suspended in the media. Then collect the supernatants.Add BSA at a final concentration of 0.1%and store at minus 20 degrees Celsius for up to two months. To purify the BDNF, begin by thawing the media in a 37 degree Celsius thermoregulated bath. Then aliquot the media into centrifuge tubes.Centrifuge for one hour at 3, 500 g in a four degree Celsius gold centrifuge. This step allows the elimination of remaining debris to ensure adequate flow through the chromatography column. Then use the protein concentrators to reduce the media from 500 milliliters to 100 milliliters.Follow the manufacturer's recommended centrifugation parameters for optimal concentration. Then add 500 microliters of nickel-NTA agarose beads to the concentrated media and incubate overnight in a rocker at four degrees Celsius. The next day, assemble the chromatography apparatus and pour the media into it.Let it rest for five minutes and then open the two-way stop cock to let the medium flow through. Wash with five milliliters of wash buffer for five minutes making sure to resuspend the beads in the column. Repeat three times.Finally, add one milliliter of elution buffer to the column making sure to resuspend the beads. Incubate for 15 minutes and then collect the eluent in a microcentrifuge tube. Repeat this step three times for complete elution of BDNF.To biotinylate the BDNF, take an aliquot containing 800 nanograms of the protein and add the biotinylation buffer reagent and then some BirA-GST in a one-to-one molar relation to BDNF. Incubate in a hybridization oven at 30 degrees Celsius for 60 minutes, mixing the contents of the tube every 15 minutes by inversion. Then add the same volume of ATP and BirA-GST as in the first step and incubate for 60 minutes, mixing the contents of the tube every 15 minutes.The biotinylated BDNF can then be left in ice for immediate biotinylation verification or stored at minus 80 degrees Celsius for posterior analysis. To verify the biotinylation of the BDNF oviduct, begin by blocking 30 microliters of streptavidin magnetic beads per BDNF sample in a blocking buffer. Incubate at room temperature for one hour and then precipitate the magnetic beads in a magnetic rack separator and then discard the blocking buffer.Add 50 microliters of fresh blocking buffer and the BDNF sample to the beads, making sure to resuspend them completely. Homogenize the contents of the tube and incubate at four degrees Celsius for one hour in a microcentrifuge tube rotator. Prepare the sample for western blotting as described in the text to assess the efficiency of the biotinylation reaction.To conjugate the BDNF oviduct to the quantum dots, begin by adding the necessary volume of quantum dots to achieve a one-to-one molar relation to BDNF and then dilute to a final volume of 20 microliters. Homogenize the contents of the tube and then wrap it in aluminum foil to protect it from the light. Incubate at room temperature for 30 minute in a rocker.Then dilute the BDNF oviduct to the desired final concentration and add it to the axonal compartment of a microfluidic chamber, incubating for 210 minutes for posterior live or fixed cell imaging. The use of a chromatography column-based protocol allows the processing of significant volumes of HEK293 conditioned media. In this experiment, 500 milliliters of conditioned media were processed to purify BDNF oviduct.Four consecutive elutions lasting 15 minutes each yielded decreasing concentrations of BDNF oviduct ranging from six to 28 nanograms per microliter. The total yield amounted to approximately 60 micrograms of BDNF oviduct. This purified BDNF was then biotinylated by an in vitro reaction mediated by BirA-GST.The sample was homogeneously biotinylated as demonstrated by the lack of non-biotinylated BDNF oviduct in the supernatant of the magnetic bead cleared buffer. These results show that the proposed protocol allows for the purification of significant amounts of BDNF oviduct and homogenous in vitro mono-biotinylation. Then the biological activity of the mono-biotinylated BDNF was evaluated using two different experimental approaches.First, cortical neurons seeded in 60 millimeter plates were stimulated with 50 nanograms per milliliter of mono-biotinylated BDNF for 30 minutes and then proteins were prepared for western blot analysis. The biological activity of the mono-biotinylated BDNF was quantified by detecting phospho-TrkB and phospho-ERK. Binding of BDNF to TrkB triggers the auto-phosphorylation of the receptor and the activation of several downstream kinases including ERK.A negative control condition which wasn't stimulated with BDNF and a positive control condition which was stimulated with commercially available BDNF were included for the analysis of BDNF oviduct biological activity. The bands were both phosphorylated proteins, had a similar intensity in neurons treated with commercial BDNF and mono-biotinylated BDNF, and both showed a stronger signal than the negative control condition. Then the biological activity of mono-biotinylated BDNF coupled to streptavidin quantum dots was evaluated to demonstrate that they can be used in live imaging experiments.Cortical neurons were seeded in 10 millimeter covers and treated with a final concentration of 200 picomolar or two nanomolar BDNF quantum dots for 30 minutes before fixing and staining for phospho-CREB. CREB is a transcription factor which is targeted by activated ERK12 in cortical neurons. Stimulating neurons with increasing concentrations of BDNF quantum dots resulted in a dose-dependent increase of phosphorylation of CREB and presence of quantum dot particles surrounding the nucleus indicating that the BDNF quantum dot particles were endocytosed and triggered the activation of signaling pathways associated with BDNF-mediated TrkB activation.A two-fold increase in phospho-CREB signal was detected when stimulating neurons with 200 picomolar BDNF quantum dots whereas stimulating with two nanomolar BDNF quantum dots resulted in a 3.5-fold increase in the phospho-CREB signal. Therefore, the biotinylated BDNF oviduct is biologically active and it doesn't lose its activity when coupled to streptavidin quantum dots making it suitable for immunofluorescence and live cell imaging. Finally, the imaging potential of BDNF quantum dots was evaluated in compartmentalized cultures using microfluidic chambers.Cortical neurons were seeded in microfluidic chambers to separate the axonal and somatodendritic compartments and were stimulated with two nanomolar BDNF quantum dots for 3.5 hours. Live cell microscopy was performed and the resulting kymographs were used to quantify the speed of BDNF quantum dot containing organelles. An average moving speed of 0.91 micrometers per second was detected which is in line with previous analysis of cytoplasmic thymine-mediated transport.Microfluidic chambers treated with two nanomolar streptavidin quantum dots didn't show moving quantum dots in the micro groups as shown by the kymograph. Cells grown under the same conditions were stimulated with 500 picomolar or two nanomolar BDNF quantum dots for 210 minutes and then fixed and labeled with a nuclear stain. Neurons treated with BDNF quantum dots show a dose-dependent accumulation of the labeled protein in all the analyzed subcompartments including the proximal and distal portions of the micro group and the somatodendritic compartment.In contrast, control neurons showed almost no quantum dot signal throughout the chamber. Therefore, the BDNF quantum dot can be detected in live and fixed cells in microfluidic chambers. After watching this video, you should have a good understanding of how to produce mono-biotinylated BDNF oviduct in a chromatography column-based cost effective procedure.

an-improved-protocol-to-purify-directly-mono-biotinylate-recombinant

Research

JoVE Journal

Neuroscience

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and subsequently digests this material, commonly through lysosomal degradation. Microglia are the resident immune cells of the central nervous system (CNS) whose phagocytic function has been described in a broad range of conditions from neurodegenerative disease (e.g., beta-amyloid clearance in Alzheimer&#8217;s disease) to development of the healthy brain (e.g., synaptic pruning)1-6. The following protocol is an engulfment assay developed to visualize and quantify microglia-mediated engulfment of presynaptic inputs in the developing mouse retinogeniculate system7. While this assay was used to assess microglia function in this particular context, a similar approach may be used to assess other phagocytes throughout the brain (e.g., astrocytes) and the rest of the body (e.g., peripheral macrophages) as well as other contexts in which synaptic remodeling occurs (e.g. ,brain injury/disease).

Microglia are the resident immune cells of the central nervous system (CNS) with a high capacity to phagocytose or engulf material in their extracellular environment. Here, a broadly applicable, reliable, and highly quantitative assay for visualizing and measuring microglia-mediated engulfment of synaptic components is described.

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and ...

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.

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 ...

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 &#181;m2). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta1-40 (A&#946;1-40) with or without a therapeutic&#160;intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.

Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of these afferent neurons are located within the sensory ganglia. Sensory ganglia innervate a specific organ or portion of the body. For instance, the dorsal root ganglia (DRG) are located in the vertebral column and extend processes throughout the body and limbs. The trigeminal ganglia are located in the skull and innervate the face, and upper airways. Vagal afferents of the nodose ganglia extend throughout the gut, heart, and lungs. The nodose neurons control a diverse array of functions such as: respiratory rate, airway irritation, and cough reflexes. Thus, to understand and manipulate their function, it is critical to identify and isolate airway specific neuronal sub-populations. In the mouse, the airways are exposed to a fluorescent tracer dye, Fast Blue, for retrograde tracing of airway-specific nodose neurons. The nodose ganglia are dissociated and fluorescence activated cell (FAC) sorting is used to collect dye positive cells. Next, high quality ribonucleic acid (RNA) is extracted from dye positive cells for next generation sequencing. Using this method airway specific neuronal gene expression is determined.

Organ specific sensory neurons are difficult to identify. Fast Blue tracing is used to identify nodose neurons innervating the airways for cell sorting. Sorted nodose neurons are used to extract high quality ribonucleic acid (RNA) for sequencing. Using this protocol, gene expression of airway specific neurons is determined.

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...