Name: assay to measure nucleocytoplasmic transport in real time within motor neuron-like nsc-34 cells
Uploaded: 2017-05-16T15:00:00.000+00:00
Duration: 8 min 53 s
Description: Watch this Scientific Journal Video about Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells at JoVE.com

We thank all the members of the A.I. laboratory for their helpful comments and suggestions. We would like to thank Prof. Mark Hipp (Max Planck Institute for Biochemistry) for providing us with the shuttle-GFP vector. This work was supported by grants from the Israeli Science Foundation (ISF #124/14), the Binational Science Foundation (BSF #2013325), FP7 Marie Curie Career Integration Grant (CIG # 333794), and The National Institute for Psychobiology in Israel (NIPI #b133-14/15).

1. Cell Line Preparation
<ol>
	<li>Thaw approximately 1,000,000 mouse neuroblastoma spinal cord cells (NSC-34 cells) that were stored in a liquid nitrogen container. Use a 37 &#176;C water incubator until the cells are fully thawed.</li>
	<li>Add fresh, warm medium (DMEM medium containing 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% pen-strep antibiotics).</li>
	<li>Centrifuge thawed cells (SL16R) at 500 x g for 5 min, forming a cell pellet. Discard the medium and add 1 mL of new medium for the cell resuspension.</li>
	<li>Place the resuspended cells in a 15 mL flask and add an extra 5 mL of medium. Incubate the cells overnight in a sterile incubator with 5% CO2 at 37 &#176;C.</li>
	<li>In parallel, place 8 uncoated coverslips (cover glass: 12 mm, 0.13-0.17 mm thick) in a 60-mm culture dish and sterilize using 70% ethanol and exposure to 30 min of UV light until it is dry. Add medium to each dish and dispose to wash off the residual ethanol.</li>
	<li>Add trypsin buffer (2.5 g of trypsin and 0.2 g of EDTA in 1 L of Hanks&#39; Balanced Salt Solution) to the cells after they reach approximately 90% confluence (roughly 6 million cells per flask) to allow adherent cells to dissociate. 
	NOTE: NSC-34 cells are sensitive and only require 15 s of trypsin buffer incubation and 1 additional min of incubation free of trypsin before dissociation using the medium.</li>
	<li>Plate the cells onto the 60 mm culture dishes already containing the coverslips with 3 mL of medium, approximately 350,000 per dish. Leave them in the incubator for 24 h to allow cell attachment onto the coverslips.</li>
</ol>
2. Cell Transfection
NOTE: After approximately 24 h, the NSC-34 cells will reach 40% confluence (approximately 800,000 cells) and will be ready for transfection.
<ol>
	<li>Prepare pure DMEM medium (300 &#181;L of DMEM medium for each 60 mm culture dish) in sterile microcentrifuge tubes.</li>
	<li>Add 2 &#181;g of each plasmid required for transfection to each DMEM-containing microcentrifuge tube. 
	NOTE: The shuttle-GFP plasmid is mandatory in all cell transfections.</li>
	<li>Add transfection reagent to all the microcentrifuge tubes that contain their required plasmids. Here, add 4 &#181;L of the commercial transfection reagent for each 2 &#181;g of DNA.</li>
	<li>Vortex the microcentrifuge tubes containing DMEM, plasmids, and the transfection reagent and incubate them at room temperature for 25 min.</li>
	<li>Add each microcentrifuge tube to a culture dish and stir the dish gently to form a homogenous solution. Place the dish back in the incubator for 48 h. 
	NOTE: Since shuttle-GFP should be expressed in all cells, the shuttle-GFP transfection efficiency can be partially examined and pre-tested using fluorescent microscopy after overnight transfection. A full examination of non-fluorescent expressed proteins can be conducted using an immunoblot analysis.</li>
</ol>
3. Microscopy
<ol>
	<li>Attach the coverslips containing transfected NSC-34 cells to a coverslip holder and wash gently with phosphate-buffered saline (PBS) to remove the detached dead cells.</li>
	<li>Identify a suitable cell patch using a green fluorescent filter with an inverted microscope. 
	NOTE: A suitable cell patch is defined as a microscope field of view showing a large number of cells (20-30 cells in a field) and in which the nuclei are clearly defined in the cells. Cell nuclei within the cell patch are first identified by eye and are manually marked by the imaging software.</li>
	<li>Add 200 &#181;L of an exportin-1 inhibitor Leptomycin B (LMB) buffer containing 10 ng/mL LMB in PBS to the coverslips by dripping the buffer on the coverslips under the microscope. 
	NOTE: This point in time is set as interval zero.</li>
	<li>Quantify the fluorescent intensity of the marked nuclei in 3 s intervals for around 400-500 s.</li>
	<li>Test between 3 and 5 cover slips for each transfection group in each individual experiment.</li>
</ol>
4. Analysis
NOTE: Since different cells express different levels of florescent shuttle-GFP, it is important to normalize the nuclear fluorescent intensity of each cell to its own initial fluorescent intensity.
<ol>
	<li>Normalize the fluorescent intensity of the marked nuclei by dividing 3-s fluorescent interval of each nucleus with the average of the initial six intervals of that nucleus.</li>
	<li>After the normalization of each nucleus, produce a fluorescent intensity curve representing the change in nuclei fluorescence as a function of time for each cell patch. To do this, calculate the average of all the cells in each cell-patch (consisting of approximately 20-30 cells in each patch). 
	NOTE: The nuclei fluorescent intensity curve resembles a Michaelis-Menten saturation curve, which is better observed within increased time periods. This is due to a decrease in the cytosolic amounts of shuttle-GFP with time due to nuclear transport. This decrease statistically lowers the probability of a shuttle-GFP to transport into the nucleus, thus reaching saturation in nuclear fluorescence. The slope derived from the linear initial rate of the curve yields the Vmax entry rate into the nucleus.</li>
	<li>Analyze the Vmax slopes of each cell patch, derived from three to five independent experiments, using the graphing software to produce one slope representing each experimental group. Perform quantification using one-way ANOVA statistical analysis.</li>
</ol>
5. Protein Expression Validation
<ol>
	<li>Place 60 mm culture dishes containing the residual cells (not used in the above experiment) from each experimental group on ice.</li>
	<li>Dispose the medium and wash the cells twice with PBS to dilute and wash the remaining medium.</li>
	<li>Treat the cells with 200-400 &#181;L of lysis buffer containing PBS, 0.5% Triton, and protease inhibitor cocktail tablet (PI). Scrape using a cell scraper.</li>
	<li>Collect the cells in lysis buffer in a microcentrifuge tube and homogenize with a homogenizer at 4,000 rpm for 1 min under ice.</li>
	<li>Centrifuge the homogenized cells at 2,500 x g for 10 min. Collect the supernatant, quantify the protein concentration using a Bradford assay, and store until use at -20 &#176;C.</li>
	<li>Collect between 30 and 70 &#181;g of total protein (depending on the protein) and load it into a SDS-PAGE gel to test for protein expression using an immunoblot assay.</li>
</ol>

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Authors have nothing to disclose.

This protocol demonstrates a highly sensitive and quantitative new assay to evaluate minute changes in nucleocytoplasmic transport. Using this system, it is possible to observe and measure nucleocytoplasmic transport and its dysfunction in real time, not only large defects that result in dramatic changes in protein distribution. This assay not only has a sensitivity advantage, but it also requires little preparation, is inexpensive, and is a very easy and low-engagement assay.
To test the efficiency of this system, C9orf72 proteins, poly(GR) and poly(PR) DPRs, models of ALS and FTD that were previously shown to cause a nucleocytoplasmic transport deficiency, were used5,7,8,10,12. Using this assay, it was possible to demonstrate, in real time, a decrease of about 50% in the nuclear import rate in cells expressing the DPRs as compared to cells transfected with a control vector. Therefore, this system opens the possibility of easily measuring small changes in nucleocytoplasmic transport, which can be caused by these and other ALS or FTD mutants or other diseases in which potential defects in nucleocytoplasmic transport may exist. Although the protocol described here deals with motor neuron-like cells, it could be easily adapted to other cell types. Most importantly, using this system, it is now possible to determine how different molecular factors or drugs may help to prevent or rescue (even partially) nucleocytoplasmic transport deficiencies in ALS, FTD, or other disorders.
One possible limitation in using this assay is the limited ability to observe and mark the cell nuclei in cells that do not possess a clear nucleus or that possess a very small one. To overcome this, a possible solution may be to simply use a greater magnification. However, this will ultimately decrease the amount of cells in each cell patch, thus increasing the amount of experiments needed to reach a significant value.
Another consideration relates to the shuttle-GFP nucleus-to-cytoplasm ratio before the addition of LMB. This ratio may be very different in different cell types and consequently may be too small (close to a 1:1 ratio) to show a visible nucleus. One possible solution is to use a nuclear marker beforehand. This will not affect the final analysis, since each cell is normalized to its original nuclear fluorescence. In addition, it is recommended to calibrate the concentration of LMB used for exportin inhibition before this assay is performed to effectively observe the optimal increase in nuclear fluorescence.
The nucleocytoplasmic transport deficiency is suggested to play a main role in the degeneration of motor neurons in ALS5,6,7,8,9,10,11,12. If that is the case, similar nucleocytoplasmic transport defects as those observed in the C9orf72 model would be predicted to play a role in other ALS models. However, because of the large variability between the different models, the end outcomes and molecular manifestations resulting from nucleocytoplasmic disruption in each case may be unnoticed or phenotypically different. Despite extensive research on different FALS models, linking them with common mechanisms remains a challenge. This assay may now provide the opportunity to overcome, at least partially, this challenge.

The nuclear pore complex (NPC) controls the import and export of large molecules into and out of the cell nucleus. In contrast to small molecules, which can enter the nucleus without regulation1, the transport of larger molecules is strongly controlled by the NPC. These large molecules, such as proteins and RNA, associate with transport factors, including importins and exportins, to be imported and exported from the nucleus2. In order to be imported, proteins must contain a small peptide motif, generally called a nuclear localization signal (NLS), which is bound by importins2. These sequences of amino acids act as a tag and are diverse in composition3,4. Proteins can be exported from the nucleus to the cytoplasm due to their association with exportins, which bind a signal sequence, generally called a nuclear export signal (NES)2. Both importins and exportins are able to transport their cargo due to the regulation of the small Ras related nuclear protein GTPase (Ran)2. Ran has been shown to exist in different conformational states depending on whether it is bound to GTP or GDP. When bound to GTP, Ran can bind to importins or exportins. Upon binding to RanGTP, importins release their cargo, while exportins must bind RanGTP to form a complex with their export cargo. The dominant nucleotide binding state of Ran depends on its location in the nucleus (RanGTP) or the cytoplasm (RanGDP).
Recently, a number of studies have shown a connection between impairments in the nucleocytoplasmic pathway and both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)5,6,7,8,9,10,11,12. ALS is a progressive and fatal neurodegenerative disease affecting both upper and lower motor neurons (MNs)13 and resulting in paralysis and ultimately in death, within 2-5 years on average. Most cases of ALS are classified as sporadic (SALS), lacking any apparent genetic linkage, but 10% are inherited in a dominant manner (familial ALS; FALS). Recently, hexanucleotide repeat expansions (HREs) in the chromosome 9 open reading frame 72 (C9orf72) gene were identified14,15 as a genetic cause of ALS and FTD. These mutants account for 30-40% of FALS cases16, and different studies have contended that they cause toxicity by affecting nucleocytoplasmic transport5,6,7,8,9,10,11,12.
These studies have mostly shown the final outcomes and manifestations of HREs on nucleocytoplasmic transport, but they do not demonstrate nucleocytoplasmic disruption in real time. As a result, only severe nucleocytoplasmic transport deficiency has been evaluated11,17,18.
This protocol describes a new and very sensitive assay to evaluate and quantify nucleocytoplasmic transport dysfunction in real time. The rate of import of a NLS-NES-GFP protein (shuttle-GFP) can be quantified in real time using fluorescent microscopy. This is done using an exportin inhibitor, as described previously18, thus allowing the shuttle-GFP only to enter the nucleus. Using florescent microscopy and a software capable of quantifying fluorescent changes in real time, it is possible to quantify gradual changes in the fluorescence intensity of the shuttle-GFP, located in the nucleus. As a result, a Michaelis-Menten-like saturation curve of the fluorescence-to-time axis, representing the amount of nuclear shuttle-GFP at any given time, can be made. By using the initial linear rate of the curves, it is possible to generate a slope, which represents the rate of entry into the nucleus before fluorescence saturation.
To validate the assay, the translated C9orf72 dipeptide repeats, poly(GR) and poly(PR), which have been previously reported to disrupt nucleocytoplasmic transport, were used5,7,8,10,12. Using this assay, a 50% decrease in the nuclear import rate was observed compared to the control. Using this system, minute changes in nucleocytoplasmic transport can be examined. Moreover, the ability of different factors to rescue a nucleocytoplasmic transport defect can be determined.

<table><tbody><tr><td>Mouse Motor Neuron-Like hybrid Cell Line (NSC-34)</td><td>tebu-bio</td><td>CLU140-A</td><td></td></tr><tr><td>DMEM 4.5 g/L L-glucose</td><td>Biological Industries</td><td>01-055-1A</td><td></td></tr><tr><td>FBS European Grade</td><td>Biological Industries</td><td>04-007-1A</td><td></td></tr><tr><td>SL 16R Centrifuge</td><td>Thermo Scientific</td><td>75004030</td><td></td></tr><tr><td>Thermo Forma Series ii Water Jacketed CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Thermo Scientific</td><td>3111</td><td></td></tr><tr><td>Cover glass 12 mm dia., 0.13-0.17 mm thick</td><td>Bar Naor LTD</td><td></td><td></td></tr><tr><td>TurboFect Transfection Reagent</td><td>Thermo Scientific</td><td>&lt;a href=&quot;https://www.thermofisher.com/order/catalog/product/R0531&quot;&gt;R0531&lt;/a&gt;</td><td></td></tr><tr><td>Axiovert 100 inverted microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Imaging Workbench 2</td><td>Axon Instruments</td><td></td><td></td></tr><tr><td>Leptomycin B</td><td>Sigma</td><td>L2913</td><td></td></tr><tr><td>PBS</td><td>Biological Industries</td><td>02-023-1A</td><td></td></tr><tr><td>Homogenizer motor/control/chuck/90 degree clamp</td><td>Glas-Col</td><td>099C K5424CE</td><td></td></tr><tr><td>MicroCL 17R Centrifuge, Refrigerated</td><td>Thermo Scientific</td><td>75002455</td><td></td></tr></tbody></table>

assay to measure nucleocytoplasmic transport in real time within motor neuron-like nsc-34 cells

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a connection between amyotrophic lateral sclerosis (ALS) and impairments in the nucleocytoplasmic pathway. ALS is a neurodegenerative disease affecting the motor neurons and resulting in paralysis and ultimately in death, within 2-5 years on average. Most cases of ALS are sporadic, lacking any apparent genetic linkage, but 10% are inherited in a dominant manner. Recently, hexanucleotide repeat expansions (HREs) in the chromosome 9 open reading frame 72 (C9orf72) gene were identified as a genetic cause of ALS and frontotemporal dementia (FTD). Importantly, different groups have recently proposed that these mutants affect nucleocytoplasmic transport. These studies have mostly shown the final outcome and manifestations caused by HREs on nucleocytoplasmic transport, but they do not demonstrate nuclear transport dysfunction in real time. As a result, only severe nucleocytoplasmic transport deficiency can be determined, mostly due to high overexpression or exogenous protein insertion. 
This protocol describes a new and very sensitive assay to evaluate and quantify nucleocytoplasmic transport dysfunction in real time. The rate of import of a NLS-NES-GFP protein (shuttle-GFP) can be quantified in real time using fluorescent microscopy. This is performed by using an exportin inhibitor, thus allowing the shuttle GFP only to enter the nucleus. To validate the assay, the C9orf72 HRE translated dipeptide repeats, poly(GR) and poly(PR), which have been previously shown to disrupt nucleocytoplasmic transport, were used. Using the described assay, a 50% decrease in the nuclear import rate was observed compared to the control. Using this system, minute changes in nucleocytoplasmic transport can be examined and the ability of different factors to rescue (even partially) a nucleocytoplasmic transport defect can be determined.

Using the procedure presented here, motor neuron-like NSC-34 cells were transfected with a NLS-NES-GFP (shuttle-GFP) protein. This protein, which has NLS and NES signal sequences (Figure 1), can shuttle between the nucleus and the cytoplasm. Generic GFP can enter or exit the nucleus in the absence of any localization signal tag only by diffusion and is therefore distributed evenly between the nucleus and the cytoplasm (Figure 2A). In contrast, the shuttle-GFP protein is mostly found in the cytoplasm due to the NES and NLS signals affecting its dispersion (Figure 2B). Thus, only the addition of the exportin inhibitor leptomycin B (LMB), which inhibits the protein export, allows for the accumulation of the shuttle-GFP into the nucleus and eventually for the measurement of the shuttle-GFP import rate (Figure 2C). Using a florescent microscope and software to quantify fluorescent changes in real time, gradual changes in the nuclear fluorescence intensity of the shuttle-GFP can be quantified. As a result, a Michaelis-Menten-like saturation curve of the fluorescence-to-time axis (Figure 3), representing the amount of nuclear shuttle-GFP at any given time, can be produced. By using the initial linear rate of the curves (as indicated by the circle in Figure 3), a slope, which represents the rate of entry into the nucleus before fluorescence saturation, can be generated.
Several studies indicate that different C9orf72 proteins are toxic in Drosophila melanogaster models and/or cultured cells6,11. The di-peptide repeats (DPRs), poly(PR) and poly(GR), produced from HREs in the mutated C9orf72 gene, were found to induce nucleolar stress8,19,20 and have been implicated in impaired nucleocytoplasmic transport, making them appear especially harmful. To verify the utility of the described assay, motor neuron-like NSC-34 cells were transfected with these two DPR plasmids, poly(PR) and poly(GR). Since poly(PR) and poly(GR) DPRs are conjugated to GFP as well, it was important to validate that the DPRs were not affected by leptomycin-B as well, which would result in a false-positive result. Poly(PR) expression was observed in the nucleus completely as inclusions; thus, it could not give a false-positive nuclear import signal (Figure 4A). Poly(GR) expression, on the other hand, was observed homogeneously in the cytosol and also as inclusions in the nucleus (Figure 4B). When transfecting NSC-34 cells with poly(GR) only and adding leptomycin-B, no shuttle movement of the expressed poly(GR) was observed (Figure 5).
By analyzing the nucleocytoplasmic transport rate of the shuttle-GFP, NSC-34 cells were found to co-express either poly(PR) or poly(GR) DPRs, showing a decrease in the nuclear import rate of about 50% as compared to cells transfected with an empty plasmid as a control (Figure 5).
<img alt="Figure 1" src="/files/ftp_upload/55676/55676fig1.jpg" /> 
Figure 1: Schematic of the NLS-NES-GFP (shuttle-GFP) construct. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55676/55676fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55676/55676fig2.jpg" /> 
Figure 2: Leptomycin B inhibits the export of shuttle-GFP from the nuclei of NSC-34 cells. Representative images of NSC-34 cells expressing generic GFP (A) or shuttle-GFP protein before (B) and 20 min after the addition of 10 ng/mL Leptomycin B (LMB) (C). Following LMB addition, shuttle-GFP imports into the nucleus, but the export back to the cytoplasm is blocked. Thus, shuttle-GFP accumulates in the nuclei. Scale bar = 15 &#181;m. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55676/55676fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55676/55676fig3.jpg" /> 
Figure 3: Michaelis-Menten-like plot showing an increase in nuclear fluorescence. Representative Michaelis-Menten-like curves portraying an increase in the nuclear fluorescence of two groups, affected and non-affected nucleocytoplasmic transport cells, within 25 min. The initial 5 min show a high difference in increase rate, which is decreased over time.&#160; <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55676/55676fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55676/55676fig4.jpg" /> 
Figure 4: C9orf72 DPRs poly(PR) and poly(GR) expression in NSC-34 cells. Representative images of NSC-34 cells transfected with the C9orf72 DPRs, poly(GR) (A) and poly(PR) (B). poly(GR) is expressed in the nuclei of the cells but is also distributed homogeneously in the cytosol. Poly(PR) is expressed mainly in the nuclei. Scale bar = 30 &#181;m. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55676/55676fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/55676/55676fig5.jpg" /> 
Figure 5: The C9orf72 DPRs, poly(PR) and poly(GR), strongly inhibit the nuclear import rate in NSC-34 cells. Motor neuron-like NSC-34 cells were co-transfected with shuttle-GFP, together with an empty plasmid, poly(PR) or poly(GR). The nuclear import rate was defined as the slope of the initial linear rate of the curve at Vmax. The transport rate obtained with the empty plasmid was define as 100%. Co-transfection with poly(PR) and poly(GR) inhibited the transport rate by about 50% compared to control. Quantitative analysis was performed on three to five independent experiments using one way ANOVA. **** p &#60;0.0001; *** p &#60;0.001. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55676/55676fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

This protocol describes a method to sensitively measure the nucleocytoplasmic transport rate within motor neuron-like NSC-34 cells by quantifying the real-time change in the nuclear import of a NLS-NES-GFP protein.

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

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8.6K Views.  Ben-Gurion University of the Negev. This protocol describes a method to sensitively measure the nucleocytoplasmic transport rate within motor neuron-like NSC-34 cells by quantifying the real-time change in the nuclear import of a NLS-NES-GFP protein.

Video: Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted area of the brain, neural stem cells proliferate and constantly generate neuroblasts that migrate tangentially in chains along the rostral migratory stream (RMS) to reach the olfactory bulb (OB). Once in the OB, neuroblasts switch to radial migration and then differentiate into mature neurons able to incorporate into the preexisting neuronal network. Proper neuroblast migration is a fundamental step in neurogenesis, ensuring the correct functional maturation of newborn neurons. Given the ability of SVZ-derived neuroblasts to target injured areas in the brain, investigating the intracellular mechanisms underlying their motility will not only enhance the understanding of neurogenesis but may also promote the development of neuroregenerative strategies.
This manuscript describes a detailed protocol for the transfection of primary rodent RMS postnatal neuroblasts and the analysis of their motility using a 3D in vitro migration assay recapitulating their mode of migration observed in vivo. Both rat and mouse neuroblasts can be quickly and efficiently transfected via nucleofection with either plasmid DNA, small hairpin (sh)RNA or short interfering (si)RNA oligos targeting genes of interest. To analyze migration, nucleofected cells are reaggregated in &#39;hanging drops&#39; and subsequently embedded in a three-dimensional matrix. Nucleofection per se does not significantly impair the migration of neuroblasts. Pharmacological treatment of nucleofected and reaggregated neuroblasts can also be performed to study the role of signaling pathways involved in neuroblast migration.

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

Neuroblast migration is a crucial step in postnatal neurogenesis. The protocol described here can be used to investigate the role of candidate regulators of neuroblast migration by employing DNA/small hairpin RNA (shRNA) nucleofection and a 3D migration assay with neuroblasts isolated from the rodent postnatal rostral migratory stream.

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted ...

A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic neurotransmitter release. Botulinum neurotoxin serotype A (BoNT/A) is a zinc metalloprotease that blocks exocytosis of neurotransmitter by cleaving the SNAP-25 component of the SNARE complex. Currently there are no licensed medicines to treat BoNT/A poisoning after internalization of the toxin by motor neurons. The development of effective therapeutic measures to counter BoNT/A intoxication has been limited, due in part to the lack of robust high-throughput assays for screening small molecule libraries. Here we describe a high content imaging (HCI) assay with utility for identification of BoNT/A inhibitors. Initial optimization efforts focused on improving the reproducibility of inter-plate results across multiple, independent experiments. Automation of immunostaining, image acquisition, and image analysis were found to increase assay consistency and minimize variability while enabling the multiparameter evaluation of experimental compounds in a murine motor neuron system.

Botulinum neurotoxin is one of the most potent toxins among Category-A biothreat agents, yet a post-exposure therapeutic is not available. The high content imaging approach is a powerful methodology for identifying novel inhibitors as it enables multiparameter screening using biologically relevant motor neurons, the primary target of this toxin.

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic ...

Single-Molecule Imaging of Nuclear Transport

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the last decade. The investigation of molecular interactions with high temporal and spatial resolutions deep within cells has remained challenging due to the inherently weak signals arising from individual molecules. Recent works by Yang et al. demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single molecule level. By the single molecule approach, important kinetics, such as nuclear transport time and efficiency, for signal-dependent and independent cargo molecules have been obtained. Here we described a protocol for the methodological approach with an improved spatiotemporal resolution of 0.4 ms and 12 nm. The improved resolution enabled us to capture transient active transport and passive diffusion events through the nuclear pore complexes (NPC) in semi-intact cells. We expect this method to be used in elucidating other binding and trafficking events within cells.

Single molecule microscopy approch provided novel insights into nuclear transport.

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

Biology

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

A High-content Assay for Monitoring AMPA Receptor Trafficking

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different stimuli. The &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which are responsible for fast excitatory synaptic transmission in neurons, are trafficked to and from the postsynaptic surface to dynamically alter neuronal excitability. AMPA receptor trafficking is essential for synaptic plasticity and can be disrupted in neurological disease. However, prevalent approaches for quantifying receptor trafficking ignore entire receptor pools, are overly time- and labor-intensive, or potentially disrupt normal trafficking mechanisms and therefore complicate the interpretation of resulting data. We present a high-content assay for the quantification of both surface and internal AMPA receptor populations in cultured primary hippocampal neurons using dual fluorescent immunolabeling and a near-infrared fluorescent 96-well microplate scanner. This approach facilitates the rapid screening of bulk internalized and surface receptor densities while minimizing sample material. However, our method has limitations in obtaining single-cell resolution or conducting live cell imaging. Finally, this protocol may be amenable to other receptors and different cell types, provided proper adjustments and optimization.

Neuronal excitability can be modulated through a dynamic process of endo- and exocytosis of excitatory ionotropic glutamate receptors. Described here is an accessible, high-content assay for quantifying surface and internal receptor population pools.

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different ...

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study of this movement in situ was only possible using radioisotopic pulse-labeling, which permits analysis of axonal transport in whole nerves with a temporal resolution of days and a spatial resolution of millimeters. To study neurofilament transport in situ with higher temporal and spatial resolution, we developed a hThy1-paGFP-NFM transgenic mouse that expresses neurofilament protein M tagged with photoactivatable GFP in neurons. Here we describe fluorescence photoactivation pulse-escape and pulse-spread methods to analyze neurofilament transport in single myelinated axons of tibial nerves from these mice ex vivo. Isolated nerve segments are maintained on the microscope stage by perfusion with oxygenated saline and imaged by spinning disk confocal fluorescence microscopy. Violet light is used to activate the fluorescence in a short axonal window. The fluorescence in the activated and flanking regions is analyzed over time, permitting the study of neurofilament transport with temporal and spatial resolution on the order of minutes&#160;and microns, respectively. Mathematical modeling can be used to extract kinetic parameters of neurofilament transport including the velocity, directional bias and pausing behavior from the resulting data. The pulse-escape and pulse-spread methods can also be adapted to visualize neurofilament transport in other nerves. With the development of additional transgenic mice, these methods could also be used to image and analyze the axonal transport of other cytoskeletal and cytosolic proteins in axons.

We describe fluorescence photoactivation methods to analyze the axonal transport of neurofilaments in single myelinated axons of peripheral nerves from transgenic mice that express a photoactivatable neurofilament protein.

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study ...

Nuclear Transport Assays in Permeabilized Mouse Cortical Neurons

Disruption of nucleocytoplasmic transport is increasingly implicated in the pathogenesis of neurodegenerative diseases. Moreover, there is a growing recognition of cell-specific differences in nuclear pore complex structure, prompting a need to adapt nuclear transport methods for use in neurons. Permeabilized cell assays, in which the plasma membrane is selectively perforated by digitonin, are widely used to study passive and active nuclear transport in immortalized cell lines but have not been applied to neuronal cultures. In our initial attempts, we observed the rapid loss of nuclear membrane integrity in primary mouse cortical neurons exposed to even low concentrations of digitonin. We hypothesized that neuronal nuclear membranes may be uniquely vulnerable to the loss of cytoplasmic support. After testing multiple approaches to improve nuclear stability, we observed optimal nuclear integrity following hypotonic lysis in the presence of a concentrated bovine serum albumin cushion. Neuronal nuclei prepared by this approach reliably import recombinant fluorescent cargo in an energy-dependent manner, facilitating analysis of nuclear import by high content microscopy with automated analysis. We anticipate that this method will be broadly applicable to studies of passive and active nuclear transport in primary neurons.

We have developed a reliable method of selective plasma membrane permeabilization of primary mouse cortical neurons for high content automated analysis of neuronal nucleocytoplasmic transport.

Disruption of nucleocytoplasmic transport is increasingly implicated in the pathogenesis of neurodegenerative diseases. Moreover, there is a growing ...

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe dementia (FTLD) is dysfunction of communication between neurons and motor neurons and muscle. The underlying process of synaptic transmission involves membrane depolarization-dependent synaptic vesicle fusion and the release of neurotransmitters into the synapse. This process occurs through localized calcium influx into the presynaptic terminals where synaptic vesicles reside. Here, the protocol describes fluorescence-based live-imaging methodologies that reliably report depolarization-mediated synaptic vesicle exocytosis and presynaptic terminal calcium influx dynamics in cultured neurons.
Using a styryl dye that is incorporated into synaptic vesicle membranes, the synaptic vesicle release is elucidated. On the other hand, to study calcium entry, Gcamp6m is used, a genetically encoded fluorescent reporter. We employ high potassium chloride-mediated depolarization to mimic neuronal activity. To quantify synaptic vesicle exocytosis unambiguously, we measure the loss of normalized styryl dye fluorescence as a function of time. Under similar stimulation conditions, in the case of calcium influx, Gcamp6m fluorescence increases. Normalization and quantification of this fluorescence change are performed in a similar manner to the styryl dye protocol. These methods can be multiplexed with transfection-based overexpression of fluorescently tagged mutant proteins. These protocols have been extensively used to study synaptic dysfunction in models of FUS-ALS and C9ORF72-ALS, utilizing primary rodent cortical and motor neurons. These protocols easily allow for rapid screening of compounds that may improve neuronal communication. As such, these methods are valuable not only for the study of ALS but for all areas of neurodegenerative and developmental neuroscience research.

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe ...

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal transport have devastating consequences for individual neurons and their networks, and contribute to a plethora of neurological disorders. As many of these conditions involve both cell autonomous and non-autonomous mechanisms, and often display a spectrum of pathology across neuronal subtypes, methods to accurately identify and analyze neuronal subsets are imperative.
This paper details protocols to assess in vivo axonal transport of signaling endosomes and mitochondria in sciatic nerves of anesthetized mice. Stepwise instructions are provided to 1) distinguish motor from sensory neurons in vivo, in situ, and ex vivo by using mice that selectively express fluorescent proteins within cholinergic motor neurons; and 2) separately or concurrently assess in vivo axonal transport of signaling endosomes and mitochondria. These complementary intravital approaches facilitate the simultaneous imaging of different cargoes in distinct peripheral nerve axons to quantitatively monitor axonal transport in health and disease.

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Using transgenic fluorescent mice, detailed protocols are described to assess in vivo axonal transport of signaling endosomes and mitochondria within motor and sensory axons of the intact sciatic nerve in live animals.

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal ...

Measuring Axonal Cargo Transport in Mouse Primary Cortical Cultured Neurons

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.

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.

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

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Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors

Single-Molecule Imaging of Nuclear Transport

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

A High-content Assay for Monitoring AMPA Receptor Trafficking

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve

Nuclear Transport Assays in Permeabilized Mouse Cortical Neurons

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Measuring Axonal Cargo Transport in Mouse Primary Cortical Cultured Neurons