Name: characterization of neuronal lysosome interactome with proximity labeling proteomics
Uploaded: 2022-06-23T14:50:00
Description: Watch this Scientific Journal Video about Characterization of Neuronal Lysosome Interactome with Proximity Labeling Proteomics at JoVE.com

This study is supported by the NIH grant (R01NS121608). A.M.F. acknowledges the ARCS-Metro Washington Chapter Scholarship and the Bourbon F. Scribner Endowment Fellowship. We thank the Michael Ward lab at the National Institute for Neurological Disorders and Stroke (NINDS) for molecular biology support and the i3Neuron technology development.

All procedures were approved by the George Washington University biosafety and ethics committee. The compositions of media and buffers used in this protocol are provided in Table 1. The commercial product information used here is provided in the Table of Materials.
1. Human iPSC-derived neuron culture
<ol>
	<li>Human iPSC culture and LAMP1-APEX probe integration (7 days)
	<ol>
		<li>Thaw Matrigel stock solution in an ice bucket at 4 &#176;C ...

<ol>
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Using this LAMP1-APEX probe, proteins on and near the lysosomal membrane are biotinylated and enriched. Given the typical lysosome diameter of 100-1,200 nm, this method provides excellent intracellular resolution with a 10-20 nm labeling radius. LAMP1 is an abundant lysosomal membrane protein and a classical marker for lysosomes, serving as an excellent bait protein for lysosomal APEX labeling at the endogenous expression level. However, limitations also exist when using LAMP1 to target lysosomes, as LAMP1 is also presen...

Lysosomes are catabolic organelles that degrade macromolecules via the lysosomal-autophagy pathway1. Besides degradation, lysosomes are involved in diverse cellular functions such as signaling transduction, nutrient sensing, and secretion2,3,4. Perturbations in lysosomal function have been implicated in lysosomal storage disorders, cancer, aging, and neurodegeneration3,5,6,7. For postmitotic and highly polarized neurons, lysosomes play critical roles in neuronal cellular homeostasis, neurotransmitter release, and long-distance transport along the axons8,9,10,11. However, investigating lysosomes in human neurons has been a challenging task. Recent advancements in induced pluripotent stem cell (iPSC)-derived neuron technologies have enabled the culture of live human neurons that were previously inaccessible, bridging the gap between animal models and human patients to study the human brain12,13. Particularly, the advanced i3Neuron technology stably integrates the neurogenin-2 transcription factor into the iPSC genome under a doxycycline-inducible promoter, driving iPSCs to differentiate into pure cortical neurons in 2 weeks14,15.
Due to the highly dynamic lysosomal activity, capturing lysosomal interactions with other cellular components is technically challenging, particularly in a high-throughput fashion. Proximity labeling technology is well-suited to studying these dynamic interactions because of its capability to capture both stable and transient/weak protein interactions with exceptional spatial specificity16,17. Engineered peroxidase or biotin ligase can be genetically fused to the bait protein. Upon activation, highly reactive biotin radicals are produced to covalently label neighboring proteins, which can then be enriched by streptavidin-coated beads for downstream bottom-up proteomics via liquid chromatography-mass spectrometry (LC-MS) platforms17,18,19,20,21.
An endogenous lysosomal proximity labeling proteomics method was recently developed to capture the dynamic lysosomal microenvironment in i3Neurons22. Engineered ascorbate peroxidase (APEX2) was knocked-in on the C-terminus of the lysosomal associated membrane protein 1 (LAMP1) in iPSCs, which can then be differentiated into cortical neurons. LAMP1 is an abundant lysosomal membrane protein and a classical lysosomal marker23. LAMP1 is also expressed in late endosomes, which mature into lysosomes; these late endosome-lysosomes and nondegradative lysosomes are all referred to as lysosomes in this protocol. This endogenous LAMP1-APEX probe, expressed at the physiological level, can reduce LAMP1 mislocalization and overexpression artifacts. Hundreds of lysosomal membrane proteins and lysosomal interactors can be identified and quantified with excellent spatial resolution in live human neurons.
Here, a detailed protocol for lysosome proximity labeling proteomics in human iPSC-derived neurons is described with further improvements from the recently published method22. The overall workflow is illustrated in Figure 1. The protocol includes hiPSC-derived neuron culture, proximity labeling activation in neurons, validation of APEX activity by fluorescence microscopy, determination of an optimal streptavidin beads-to-input protein ratio, enrichment of biotinylated proteins, on-beads protein digestion, peptide desalting and quantification, LC-MS analysis, and proteomics data analysis. Troubleshooting guidelines and experimental optimizations are also discussed to improve proximity labeling quality control and performance.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10% (w/v) Saponin solution</td>
			<td>Acros Organics</td>
			<td>419231000</td>
			<td>Flourescent Microscopy</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Life Technologies</td>
			<td>A1110501</td>
			<td>cell detachment solution, Cell Culture</td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Fisher Scientific</td>
			<td>17504044</td>
			<td>Cell Culture, Cortical Neuron Medium</td>
		</tr>
		<tr>
			<td>BDNF</td>
			<td>PeproTech</td>
			<td>450-02</td>
			<td>Cell Culture, Cortical Neuron Medium</td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Sigma-Aldrich</td>
			<td>B6768</td>
			<td>Cell Culture, Borate Buffer</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Millipore Sigma</td>
			<td>A8806</td>
			<td>To make standard solutions to measure total protein concentrations</td>
		</tr>
		<tr>
			<td>Brainphys neuronal medium</td>
			<td>STEMCELL Technologies</td>
			<td>5790</td>
			<td>Cell Culture, Cortical Neuron Medium</td>
		</tr>
		<tr>
			<td>CD45R (B220) Antibody Alexa Fluor 561</td>
			<td>Thermo Fisher Scientific</td>
			<td>505-0452-82</td>
			<td>Flourescent Microscopy</td>
		</tr>
		<tr>
			<td>Chroman1 ROCK inhibitor</td>
			<td>Tocris</td>
			<td>716310</td>
			<td>Cell Culture</td>
		</tr>
		<tr>
			<td>cOmplete mini Protease Inhibitor</td>
			<td>Roche</td>
			<td>4693123001</td>
			<td>cocktail inhibitor in Lysis Buffer</td>
		</tr>
		<tr>
			<td>DC Protein Assay Kit II</td>
			<td>Bio-Rad</td>
			<td>5000112</td>
			<td>To determine total protein concentrations of cell lysate</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td>Proximity-labeling Reaction</td>
		</tr>
		<tr>
			<td>DMEM/F12 medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>11320082</td>
			<td>Cell Culture, Dish Coating</td>
		</tr>
		<tr>
			<td>DMEM/F12 medium with HEPES</td>
			<td>Thermo Fisher Scientific</td>
			<td>11330057</td>
			<td>Cell Culture, Induction Medium</td>
		</tr>
		<tr>
			<td>Donkey serum</td>
			<td>Sigma-Aldrich</td>
			<td>D9663</td>
			<td>Flourescent Microscopy</td>
		</tr>
		<tr>
			<td>Doxycycline hyclate, &#8805;98% (HPLC)</td>
			<td>Sigma-Aldrich</td>
			<td>D9891-1G</td>
			<td>Cell Culture, Induction Medium</td>
		</tr>
		<tr>
			<td>Essential 8 Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1517001</td>
			<td>Cell Culture</td>
		</tr>
		<tr>
			<td>Essential 8 Supplement (50x)</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1517101</td>
			<td>Cell Culture</td>
		</tr>
		<tr>
			<td>Extraction plate vacuum manifold kit</td>
			<td>Waters</td>
			<td>WAT097944</td>
			<td>For Peptide desalting</td>
		</tr>
		<tr>
			<td>Formic Acid (FA)</td>
			<td>Fisher Scientific</td>
			<td>A11750</td>
			<td>For LC-MS analysis</td>
		</tr>
		<tr>
			<td>GDNF</td>
			<td>PeproTech</td>
			<td>450-10</td>
			<td>Cell Culture, Cortical Neuron Medium</td>
		</tr>
		<tr>
			<td>Hoechst dye</td>
			<td>Thermo Fisher Scientific</td>
			<td>62239</td>
			<td>Flourescent Microscopy</td>
		</tr>
		<tr>
			<td>HPLC grade methanol</td>
			<td>Fisher Scientific</td>
			<td>A452</td>
			<td>For Peptide desalting</td>
		</tr>
		<tr>
			<td>HPLC grade water</td>
			<td>Fisher Scientific</td>
			<td>W5</td>
			<td>For Peptide desalting</td>
		</tr>
		<tr>
			<td>Human induced pluripotent stem cells</td>
			<td>Corriell Institute</td>
			<td>GM25256</td>
			<td>Cell Culture</td>
		</tr>
		<tr>
			<td>Hydrogen peroxide, ACS, 29-32% w/w aq. soln., stab.</td>
			<td>Thermo Fisher Scientific</td>
			<td>AA33323AD</td>
			<td>Proximity-labeling Reaction</td>
		</tr>
		<tr>
			<td>Iodoacetamide (IAA)</td>
			<td>Millipore Sigma</td>
			<td>I6125</td>
			<td>For Protein Digestion</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Fisher Scientific</td>
			<td>23017015</td>
			<td>Cell Culture, Cortical Neuron Medium</td>
		</tr>
		<tr>
			<td>LC-MS grade Acetonitrile</td>
			<td>Fisher Scientific</td>
			<td>A955</td>
			<td>For LC-MS analysis</td>
		</tr>
		<tr>
			<td>LC-MS grade water</td>
			<td>Fisher Scientific</td>
			<td>W64</td>
			<td>For LC-MS analysis</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Fisher Scientific</td>
			<td>25-030-081</td>
			<td>Cell Culture, Induction Medium</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Thermo Fisher Scientific</td>
			<td>08-774-552</td>
			<td>basement membrane matrix, Cell Culture, Dish Coating</td>
		</tr>
		<tr>
			<td>Mouse anti-human LAMP1 monoclonal antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>h4a3</td>
			<td>Flourescent Microscopy</td>
		</tr>
		<tr>
			<td>N-2 Supplement (100x)</td>
			<td>Fisher Scientific</td>
			<td>17-502-048</td>
			<td>Cell Culture, Induction Medium</td>
		</tr>
		<tr>
			<td>Nitrocellulose Membrane, Precut, 0.45 &#181;m, 7 x 8.5 cm</td>
			<td>Bio-Rad</td>
			<td>1620145</td>
			<td>To conduct dot blot assay for bead titration</td>
		</tr>
		<tr>
			<td>Non-essential amino acids (NEAA)</td>
			<td>Fisher Scientific</td>
			<td>11-140-050</td>
			<td>Cell Culture, Induction Medium</td>
		</tr>
		<tr>
			<td>NT-3</td>
			<td>PeproTech</td>
			<td>450-03</td>
			<td>Cell Culture, Cortical Neuron Medium</td>
		</tr>
		<tr>
			<td>Oasis HLB 96-well solid phase extraction plate</td>
			<td>Waters</td>
			<td>186000309</td>
			<td>For Peptide desalting</td>
		</tr>
		<tr>
			<td>Odyssey Blocking Buffer (TBS)</td>
			<td>LI-COR Biosciences</td>
			<td>927-50000</td>
			<td>To conduct dot blot assay for bead titration</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>Flourescent Microscopy</td>
		</tr>
		<tr>
			<td>Phenol Biotin (1,000x stock)</td>
			<td>Adipogen</td>
			<td>41994-02-9</td>
			<td>Proximity-labeling Reaction</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS) without calcium or magnesium</td>
			<td>Gibco</td>
			<td>10010049</td>
			<td>Cell Culture, Proximity-labeling Reaction, Flourescent Microscopy</td>
		</tr>
		<tr>
			<td>Pierce Quantitative Colorimetric Peptide Assay</td>
			<td>Thermo Fisher</td>
			<td>23275</td>
			<td>Peptide Concentration Assay</td>
		</tr>
		<tr>
			<td>Poly-L-Ornithine (PLO)</td>
			<td>Millipore Sigma</td>
			<td>P3655</td>
			<td>Cell Culture, Dish Coating</td>
		</tr>
		<tr>
			<td>Sodium Ascorbate</td>
			<td>Sigma-Aldrich</td>
			<td>A4034</td>
			<td>Proximity-Labeling Quench Buffer, Lysis Buffer</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S8032</td>
			<td>Proximity-Labeling Quench Buffer, Lysis Buffer, Flourescent Microscopy</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Thermo Fisher Scientific</td>
			<td>S271500</td>
			<td>Cell Culture, Borate Buffer</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP1311220</td>
			<td>Lysis Buffer, Dot blot assay buffer, Beads wash buffer</td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>415413</td>
			<td>Cell Culture, Borate Buffer</td>
		</tr>
		<tr>
			<td>Sodium tetraborate</td>
			<td>Sigma-Aldrich</td>
			<td>221732</td>
			<td>Cell Culture, Borate Buffer</td>
		</tr>
		<tr>
			<td>SpeedVac concentrator</td>
			<td></td>
			<td></td>
			<td>vacuum concentrator</td>
		</tr>
		<tr>
			<td>Streptavidin Magnetic Sepharose Beads</td>
			<td>Cytiva (formal GE)</td>
			<td>28-9857-99</td>
			<td>Enrich biotinylated proteins</td>
		</tr>
		<tr>
			<td>Streptavidin, Alexa Fluor 680 Conjugate</td>
			<td>Thermo Fisher Scientific</td>
			<td>S32358</td>
			<td>To conduct dot blot assay for bead titration</td>
		</tr>
		<tr>
			<td>Thermomixer</td>
			<td></td>
			<td></td>
			<td>temperature-controlled mixer</td>
		</tr>
		<tr>
			<td>Trifluoacetic acid (TFA)</td>
			<td>Millipore Sigma</td>
			<td>302031</td>
			<td>For Peptide desalting</td>
		</tr>
		<tr>
			<td>Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)</td>
			<td>Millipore Sigma</td>
			<td>C4706</td>
			<td>For Protein Digestion</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP152500</td>
			<td>Lysis Buffer, Dot blot assay buffer, Beads wash buffer</td>
		</tr>
		<tr>
			<td>Triton-X</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP151500</td>
			<td>Beads wash buffer</td>
		</tr>
		<tr>
			<td>TROLOX</td>
			<td>Sigma-Aldrich</td>
			<td>648471</td>
			<td>Proximity-Labeling Quench Buffer, Lysis Buffer</td>
		</tr>
		<tr>
			<td>Trypsin/Lys-C Mix, Mass Spec Grade</td>
			<td>Promega</td>
			<td>V5073</td>
			<td>For Protein Digestion</td>
		</tr>
		<tr>
			<td>TWEEN&#160;20</td>
			<td>Millipore Sigma</td>
			<td>P1379</td>
			<td>Dot blot assay buffer</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP169500</td>
			<td>Beads wash and On-Beads Digestion Buffer</td>
		</tr>
		<tr>
			<td>Vitronectin</td>
			<td>STEMCELL Technologies</td>
			<td>7180</td>
			<td>Cell Culture, Dish Coating</td>
		</tr>
		<tr>
			<td>Y-27632 ROCK inhibitor</td>
			<td>Selleck</td>
			<td>S1049</td>
			<td>Cell Culture</td>
		</tr>
	</tbody>
</table>

characterization of neuronal lysosome interactome with proximity labeling proteomics

Lysosomes frequently communicate with a variety of biomolecules to achieve the degradation and other diverse cellular functions. Lysosomes are critical to human brain function, as neurons are postmitotic and rely heavily on the autophagy-lysosome pathway to maintain cellular homeostasis. Despite advancements in the understanding of various lysosomal functions, capturing the highly dynamic communications between lysosomes and other cellular components is technically challenging, particularly in a high-throughput fashion. Here, a detailed protocol is provided for the recently published endogenous (knock-in) lysosome proximity labeling proteomic method in human induced pluripotent stem cell (hiPSC)-derived neurons. 
Both lysosomal membrane proteins and proteins surrounding lysosomes within a 10-20 nm radius can be confidently identified and accurately quantified in live human neurons. Each step of the protocol is described in detail, i.e., hiPSC-neuron culture, proximity labeling, neuron harvest, fluorescence microscopy, biotinylated protein enrichment, protein digestion, LC-MS analysis, and data analysis. In summary, this unique endogenous lysosomal proximity labeling proteomics method provides a high-throughput and robust analytical tool to study the highly dynamic lysosomal activities in live human neurons.

This lysosome proximity labeling proteomics study was conducted in human iPSC-derived neurons to capture the dynamic lysosomal microenvironment in situ in live neurons. Cell morphologies of hiPSCs and hiPSC-derived neurons at different time points are illustrated in Figure 2A. Human iPSCs grow in colonies in E8 medium. Differentiation is initiated by plating iPSCs into doxycycline-containing neuron induction medium. Neurite extensions become more visible each day during the 3 day di...

Watch this Scientific Journal Video about Characterization of Neuronal Lysosome Interactome with Proximity Labeling Proteomics at JoVE.com

Characterization of Neuronal Lysosome Interactome with Proximity Labeling Proteomics

A neuronal lysosome proximity labeling proteomics protocol is described here to characterize the dynamic lysosomal microenvironment in human induced pluripotent stem cell-derived neurons. Lysosomal membrane proteins and proteins that interact with lysosomes (stably or transiently) can be accurately quantified in this method with excellent intracellular spatial resolution in live human neurons.

Lysosomes frequently communicate with a variety of biomolecules to achieve the degradation and other diverse cellular functions. Lysosomes are critical to ...

Lysosomes are the trash disposal systems of the cell. The lysosomal activities are highly dynamic and very difficult to capture. In this study, we developed a proximity labeling proteomics approach to decipher the lysosomal microenvironment in live human neurons.This approach captures the lysosome membrane proteins and the stable and transient interactions with the lysosome and iPSC-derived neurons. Lysosomal dysfunction is heavily implicated in various brain diseases. This technique can be applied to understand various brain diseases and provide potential molecular targets to design new therapies.To begin the procedure for proximity labeling, observe the neurons under a microscope to ensure they are alive and healthy. Take a half-volume of medium from the culture to mix with biotin phenol and add it back to the neurons at a final concentration of 500 micromolar for 30 minutes in a 37 degrees Celsius incubator. Initiate the labeling reaction by adding freshly prepared hydrogen peroxide solution into the neuron culture at one-millimolar final concentration.After a one-minute incubation, aspirate the medium and rinse the culture three times with quench buffer. Tilt the plate to aspirate all the residual buffer. After adding ice-cold lysis buffer, scrape the cell lysates into cold 1.5-milliliter tubes.Sonicate the tubes in an ice-cold water bath at more than 100 watts for 15 minutes with alternate cycles of 40 seconds on and 20 seconds off. After cell lysis, add cold acetone at minus 20 degrees Celsius to the sample at a four-fold volume of the cell-lysate solution. Vortex briefly and incubate at minus 20 degrees Celsius for three hours to precipitate proteins and remove residue biotin phenol.Centrifuge the cell lysate tube at 16, 500 times G for 10 minutes at two degrees Celsius and carefully remove the supernatant without disturbing the protein pellet. Then, wash the pellet twice with one milliliter of acetone at minus 20 degrees Celsius, followed by centrifugation and removal of the supernatant. Next, dry the protein pellet with a vacuum concentrator for one minute.Add cell lysis buffer to completely dissolve the pellet with vortex or sonication. Take 20 microliters of aliquot to determine total protein concentration by the DCA assay. Store the remaining cell solution at minus 80 degrees Celsius.Take the protein lysate sample from minus 80 degrees Celsius freezer and sonicate the sample tube for 30 seconds for quick thawing. Vortex, centrifuge briefly with a benchtop mini centrifuge, and place the sample tube on ice. Next, transfer 250 microliters of streptavidin magnetic bead slurry to each 1.5-milliliter tube.Place these tubes on a magnetic rack for one minute, wash the beads three times with one milliliter of 2%sodium dodecyl sulfate solution, and remove the residual buffer. Based on the cell lysates total protein concentration and the bead titration assay results, add the calculated amount of protein sample needed for 250 microliters of the streptavidin magnetic bead slurry. Then, add cell lysis buffer to the magnetic bead-lysate mixture to the total volume of one microliter and rotate the tube overnight at four degrees Celsius.The next day, centrifuge the sample tube briefly on a benchtop microcentrifuge and place the tube on a magnetic rack for one minute to remove the supernatant. Then, wash the beads twice using one milliliter of wash buffer A with five minutes of rotation at room temperature. Repeat the process with each wash buffer twice, sequentially, with buffer B, buffer C, and buffer D at four degrees Celsius.Place the tube on a magnetic rack for one minute and remove the supernatant. Then, resuspend the beads in 100 microliters of five-millimolar TCEP in 50-millimolar Tris buffer to incubate for 30 minutes on a thermomixer at 37 degrees Celsius, followed by 15-millimolar IAA addition for 30 minutes in the dark and five-millimolar TCEP addition for five minutes to quench residue IAA. Centrifuge the sample tube briefly on a microcentrifuge and place the tube on a magnetic rack for one minute to remove supernatant.Add 200 microliters of five-millimolar TCEP in 50-millimolar Tris buffer to resuspend the beads. Add one microgram of trypsin/Lys-C mix for 14 hours at 1, 200 RPM and 37 degrees Celsius. After 14 hours, add additional 0.2 micrograms of trypsin/Lys-C mix for three hours, spin down the sample tubes, and put them on a magnetic rack for one minute.Then, transfer the peptide supernatant to the clean tubes. Next, wash the beads with 50 microliters of 50-millimolar Tris buffer with shaking for five minutes, combine the peptide supernatants, and add 30 microliters of 10%trifluoroacetic acid to the tube to reduce pH to less than three. After peptide desalting and drying down.resuspend the peptide samples in LC buffer for LC-MS analysis. Transfer the supernatant to the LC sample vial to analyze with nano LC-MS. In the LC-MS method file, Add a custom LC-MS exclusion list with specific masses and retention time ranges for highly abundant contaminant peptide peaks, such as streptavidin, trypsin, and Lys-C.Analyze the LC-MC raw data with proteomics data analysis software. Include two FASTA libraries a Swiss-Prot Homo sapiens reference database, and a newly-built universal contaminant FASTA library with a contaminant prefix in the UniProt ID.Set up the data analysis parameters with a 1%false discovery rate for protein and peptide spectral matching identifications. Select trypsin digestion with three missed cleavages, a fixed modification of cystine carbamidyl methylation, a variable modification of methionine oxidation, and protein end terminal acetylation.For label-free quantification, normalize the peptide MS-I peak intensities to the endogenously biotinylated carboxylase PCCA and reduce the variations in proximity labeling experiments. Export protein level results from the proteomics software as an Excel file. Before the statistical analysis, remove contaminant proteins and proteins with only one PSM or no quantification result.Cell morphologies at different time points of the iPSC neurons illustrated the neurite outgrowth during the three-day differentiation period. After switching to poly-L-ornithine-coated plates in the neuron medium, the neurites formed a network between neurons and axonal extensions became more visible after maturation in two weeks. In the fluorescence imaging, LAMP1 apex activity of biotinylated proteins was stained by streptavidin and co-localized with LAMP1 staining in neurons.The results of the bead titration assay demonstrated a decline of uncaptured biotinylated protein signals when increasing the streptavidin beads amount. Five microliters of streptavidin beads were optimal for 50 micrograms of input proteins from endogenous LAMP1 apex samples. The on-beads digestion with trypsin/Lys-C mix resulted in more protein and peptide identifications and fewer missed cleavages when compared with trypsin alone.Additionally, 1 to 1.5 micrograms of trypsin/Lys-C were found to be optimal for on-beads digestion to obtain the highest number of identified proteins and the lowest percentage of missed cleavages. Known lysosomal membrane proteins were increased in LAMP1 apex versus no apex control. Endogenously biotinylated proteins are highly abundant but remain unchanged.The GO term and protein network analysis suggested that stable lysosomal membrane proteins and transient lysosomal interactors related to endolysosomal trafficking and transport were enriched in LAMP1 apex proteomics. Apex proximity labeling must be quenched at exactly one minute to reduce oxidative stress and experimental variations. We have recently developed a cleavable biotin method that allows us to enrich biotinylated proteins.This method, when combined with our current method, will allow us to reduce the interferences from streptavidin and endogenously biotinylated proteins. We can apply this method to both wild-type and mutant neurons with genetic variants that cause brain diseases. This can help us understand how and why genetic mutations influence lysosomal microenvironment in human neurons.

characterization-neuronal-lysosome-interactome-with-proximity

Research

JoVE Journal

Neuroscience

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

Labeling F-actin Barbed Ends with Rhodamine-actin in Permeabilized Neuronal Growth Cones

The motile tips of growing axons are called growth cones. Growth cones lead navigating axons through developing tissues by interacting with locally expressed molecular guidance cues that bind growth cone receptors and regulate the dynamics and organization of the growth cone cytoskeleton3-6. The main target of these navigational signals is the actin filament meshwork that fills the growth cone periphery and that drives growth cone motility through continual actin polymerization and dynamic remodeling7. Positive or attractive guidance cues induce growth cone turning by stimulating actin filament (F-actin) polymerization in the region of the growth cone periphery that is nearer the source of the attractant cue. This actin polymerization drives local growth cone protrusion, adhesion of the leading margin and axonal elongation toward the attractant.
Actin filament polymerization depends on the availability of sufficient actin monomer and on polymerization nuclei or actin filament barbed ends for the addition of monomer. Actin monomer is abundantly available in chick retinal and dorsal root ganglion (DRG) growth cones. Consequently, polymerization increases rapidly when free F-actin barbed ends become available for monomer addition. This occurs in chick DRG and retinal growth cones via the local activation of the F-actin severing protein actin depolymerizing factor (ADF/cofilin) in the growth cone region closer to an attractant8-10. This heightened ADF/cofilin activity severs actin filaments to create new F-actin barbed ends for polymerization. The following method demonstrates this mechanism. Total content of F-actin is visualized by staining with fluorescent phalloidin. F-actin barbed ends are visualized by the incorporation of rhodamine-actin within growth cones that are permeabilized with the procedure described in the following, which is adapted from previous studies of other motile cells11, 12. When rhodamine-actin is added at a concentration above the critical concentration for actin monomer addition to barbed ends, rhodamine-actin assembles onto free barbed ends. If the attractive cue is presented in a gradient, such as being released from a micropipette positioned to one side of a growth cone, the incorporation of rhodamine-actin onto F-actin barbed ends will be greater in the growth cone side toward the micropipette10.
Growth cones are small and delicate cell structures. The procedures of permeabilization, rhodamine-actin incorporation, fixation and fluorescence visualization are all carefully done and can be conducted on the stage of an inverted microscope. These methods can be applied to studying local actin polymerization in migrating neurons, other primary tissue cells or cell lines.

A method to visualize and quantify F-actin barbed ends in neuronal growth cones is described. After culturing neurons on glass coverslips, cells are permeabilized with a saponin-containing solution. Then, a short incubation with the saponin buffer containing rhodamine-actin incorporates fluorescent actin onto free actin barbed ends.

The motile tips of growing axons are called growth cones. Growth cones lead navigating axons through developing tissues by interacting with locally ...

Recording Large-scale Neuronal Ensembles with Silicon Probes in the Anesthetized Rat

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of behaviors from the spiking activity of its neurons. One of the most effective methods to monitor spiking activity from a large number of neurons in multiple local neuronal circuits simultaneously is by using silicon electrode arrays1-3.
Action potentials produce large transmembrane voltage changes in the vicinity of cell somata. These output signals can be measured by placing a conductor in close proximity of a neuron. If there are many active (spiking) neurons in the vicinity of the tip, the electrode records combined signal from all of them, where contribution of a single neuron is weighted by its 'electrical distance'. Silicon probes are ideal recording electrodes to monitor multiple neurons because of a large number of recording sites (+64) and a small volume. Furthermore, multiple sites can be arranged over a distance of millimeters, thus allowing for the simultaneous recordings of neuronal activity in the various cortical layers or in multiple cortical columns (Fig. 1). Importantly, the geometrically precise distribution of the recording sites also allows for the determination of the spatial relationship of the isolated single neurons4. Here, we describe an acute, large-scale neuronal recording from the left and right forelimb somatosensory cortex simultaneously in an anesthetized rat with silicon probes (Fig. 2).

Extracellular recordings of neuronal activity using silicon probes in the anesthetized rat will be described. This technique allows information to be obtained across multiple brain areas from more than 100 neurons simultaneously. It provides information with single cell resolution about neuronal ensembles dynamics in multiple local circuits.

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

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, ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...