Name: human peripheral blood neutrophil isolation for interrogating the parkinson's associated lrrk2 kinase pathway by assessing rab10 phosphorylation
Uploaded: 2020-03-21T13:00:00
Description: Watch this Scientific Journal Video about Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson's Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation at JoVE.com

We thank the healthy volunteers who kindly donated blood for the present study. We thank The Michael J. Fox Foundation for Parkinson&#8217;s Research (MJFF) and the Fox BioNet study leadership (FBN) for their support and input towards the written protocol and the video. We thank Professor Alexander Zimprich from the University of Vienna in Austria for testing our protocol and collaboration. We value the contributions of Paul Davies to the project (general manager of the MRC PPU). We also recognize the excellent technical support of the MRC Protein Phosphorylation and Ubiquitylation Unit (PPU) namely Chemical Synthesis (Natalia Shpiro for synthesising MLi-2), MRC PPU Reagents and Services antibody purification teams (coordinated by Hilary McLauchlan and James Hastie). We thank Mhairi Towler and Fraser Murdoch from Vivomotion for their help with making the videos and animations. We thank Steve Soave from 81 films for assistance with the final edits. Esther Sammler is supported by a Scottish Senior Clinical Academic Fellowship and has received funding from Parkinson&#39;s UK (K-1706).

According to local UK regulation all manipulations and pipetting of human blood are undertaken in a category 2 biological safety cabinet. All procedures were performed in compliance with local ethics review board and all participants have provided informed consent.
1. Preparation
<ol>
	<li>Prepare 0.1 mL of EDTA Stock Solution 1 containing 100 mM EDTA in phosphate-buffered saline (PBS).</li>
	<li>Prepare 60 mL of EDTA Stock Solution 2 containing 1 mM EDTA in PBS.</li>
	<li>Prepare lysis buff...

<ol>
	<li>Paisan-Ruiz, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+of+the+gene+containing+mutations+that+cause+PARK8-linked+Parkinson's+disease.">Cloning of the gene containing mutations that cause PARK8-linked Parkinson's disease.</a> Neuron. 44 (4), 595-600 (2004).</li><li>Fell, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MLi-2,+a+Potent,+Selective,+and+Centrally+Active+Compound+for+Exploring+the+Therapeutic+Potential+and+Safety+of+LRRK2+Kinase+Inhibition.">MLi-2, a Potent, Selective, and Centrally Active Compound for Exploring the Therapeutic Potential and Safety of LRRK2 Kinase Inhibition.</a> Journal of Pharmacology and Experimental Therapeutics. 355 (3), 397-409 (2015).</li><li>Zimprich, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+LRRK2+cause+autosomal-dominant+parkinsonism+with+pleomorphic+pathology.">Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology.</a> Neuron. 44 (4), 601-607 (2004).</li><li>Sardi, S. P., Cedarbaum, J. M., Brundin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+Therapies+for+Parkinson's+Disease:+From+Genetics+to+the+Clinic.">Targeted Therapies for Parkinson's Disease: From Genetics to the Clinic.</a> Journal of Movement Disorders. 33 (5), 684-696 (2018).</li><li>Steger, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphoproteomics+reveals+that+Parkinson's+disease+kinase+LRRK2+regulates+a+subset+of+Rab+GTPases.">Phosphoproteomics reveals that Parkinson's disease kinase LRRK2 regulates a subset of Rab GTPases.</a> Elife. 5, (2016).</li><li>Ito, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phos-tag+analysis+of+Rab10+phosphorylation+by+LRRK2:+a+powerful+assay+for+assessing+kinase+function+and+inhibitors.">Phos-tag analysis of Rab10 phosphorylation by LRRK2: a powerful assay for assessing kinase function and inhibitors.</a> Biochemical Journal. 473 (17), 2671-2685 (2016).</li><li>Alessi, D. R., SammLer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LRRK2+kinase+in+Parkinson's+disease.">LRRK2 kinase in Parkinson's disease.</a> Science. 360 (6384), 36-37 (2018).</li><li>Yue, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+dopaminergic+alterations+and+mitochondrial+abnormalities+in+LRRK2+G2019S+knock-in+mice.">Progressive dopaminergic alterations and mitochondrial abnormalities in LRRK2 G2019S knock-in mice.</a> Neurobiology of Disease. 78, 172-195 (2015).</li><li>Doggett, E. A., Zhao, J., Mork, C. N., Hu, D., Nichols, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+of+LRRK2+serines+955+and+973+is+disrupted+by+Parkinson's+disease+mutations+and+LRRK2+pharmacological+inhibition.">Phosphorylation of LRRK2 serines 955 and 973 is disrupted by Parkinson's disease mutations and LRRK2 pharmacological inhibition.</a> Journal of Neurochemistry. 120 (1), 37-45 (2012).</li><li>Sheng, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ser1292+autophosphorylation+is+an+indicator+of+LRRK2+kinase+activity+and+contributes+to+the+cellular+effects+of+PD+mutations.">Ser1292 autophosphorylation is an indicator of LRRK2 kinase activity and contributes to the cellular effects of PD mutations.</a> Science Translational Medicine. 4 (164), (2012).</li><li>Fan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interrogating+Parkinson's+disease+LRRK2+kinase+pathway+activity+by+assessing+Rab10+phosphorylation+in+human+neutrophils.">Interrogating Parkinson's disease LRRK2 kinase pathway activity by assessing Rab10 phosphorylation in human neutrophils.</a> Biochemical Journal. 475 (1), 23-44 (2018).</li><li>Scott, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+a+3-(4-Pyrimidinyl)+Indazole+(MLi-2),+an+Orally+Available+and+Selective+Leucine-Rich+Repeat+Kinase+2+(LRRK2)+Inhibitor+that+Reduces+Brain+Kinase+Activity.">Discovery of a 3-(4-Pyrimidinyl) Indazole (MLi-2), an Orally Available and Selective Leucine-Rich Repeat Kinase 2 (LRRK2) Inhibitor that Reduces Brain Kinase Activity.</a> Journal of Medicinal Chemistry. 60 (7), 2983-2992 (2017).</li><li>Pham, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+serine+proteases:+specific+regulators+of+inflammation.">Neutrophil serine proteases: specific regulators of inflammation.</a> Nature Reviews Immunology. 6 (7), 541-550 (2006).</li><li>Lis, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+phospho-specific+Rab+protein+antibodies+to+monitor+in+vivo+activity+of+the+LRRK2+Parkinson's+disease+kinase.">Development of phospho-specific Rab protein antibodies to monitor in vivo activity of the LRRK2 Parkinson's disease kinase.</a> Biochemical Journal. 475 (1), 1-22 (2018).</li><li>Mir, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Parkinson's+disease+VPS35[D620N]+mutation+enhances+LRRK2-mediated+Rab+protein+phosphorylation+in+mouse+and+human.">The Parkinson's disease VPS35[D620N] mutation enhances LRRK2-mediated Rab protein phosphorylation in mouse and human.</a> Biochemical Journal. 475 (11), 1861-1883 (2018).</li><li>Rieckmann, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+network+architecture+of+human+immune+cells+unveiled+by+quantitative+proteomics.">Social network architecture of human immune cells unveiled by quantitative proteomics.</a> Nature Immunology. 18 (5), 583-593 (2017).</li><li>Borregaard, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils,+from+marrow+to+microbes.">Neutrophils, from marrow to microbes.</a> Immunity. 33 (5), 657-670 (2010).</li><li>Bain, B., Dean, A., Broom, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+estimation+of+the+lymphocyte+percentage+by+the+Coulter+Counter+Model+S+Plus+III.">The estimation of the lymphocyte percentage by the Coulter Counter Model S Plus III.</a> Clinical &amp; Laboratory Haematology. 6 (3), 273-285 (1984).</li><li>Tomazella, G. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+total+cellular+proteins+of+human+neutrophils.">Proteomic analysis of total cellular proteins of human neutrophils.</a> Proteome Science. 7, 32 (2009).</li></ol>

Compelling clinical, genetic, and biochemical evidence points towards an important role for LRRK2 and in particular its kinase function in Parkinson&#8217;s disease7. LRRK2 kinase inhibitors have been developed and are entering clinical trials2,4,12. As such there is a need for exploiting LRRK2 as a biomarker for target engagement as well as patient stratification. Our protocol describes a robust and easy...

Attempts to slow or stop Parkinson&#8217;s disease (PD) have thus far failed. The discovery of hyperactivating mutations in the leucine rich repeat kinase 2 (LRRK2) that cause and/or increase the risk for PD has led to the development of LRRK2 kinase inhibitors1,2,3. These have now entered clinical trials4. The exact function of LRRK2 is unclear, but a major advancement has been the identification of a subset of Rab GTPase proteins, including Rab10, as the first bona fide physiological substrates of the LRRK2 kinase5,6,7. Key challenges in the era of disease-modifying therapeutics are biochemical markers of LRRK2 kinase activation status and target engagement of LRRK2 kinase inhibitors.
So far, the principal pharmacokinetic marker for LRRK2 inhibitors in vivo has been a cluster of constitutively phosphorylated serine residues of LRRK2, in particular serine 935, that become dephosphorylated in response to diverse LRRK2 inhibitors8,9. However, serine 935 phosphorylation does not correlate with intrinsic cellular LRRK2 kinase activity because it is not directly phosphorylated by LRRK2 and is still phosphorylated in kinase-inactive LRRK210. LRRK2 kinase activity correlates well with autophosphorylation of serine 1292, but it is in practical terms not a suitable readout for endogenous LRRK2 kinase activity by immunoblot analysis of whole cell extracts due to the current lack of reliable and phosphospecific antibodies for this site10,11.
We have developed a robust and easy assay to quantify LRRK2 kinase pathway activity in human peripheral blood cells that measures LRRK2-controlled phosphorylation of its physiological target protein Rab10 at threonine 7311. Peripheral blood is easily accessible by venesection, which is a low risk and quick procedure that causes minimal discomfort. We focus on human peripheral blood neutrophils because they constitute an abundant (37&#8211;80% of all white blood cells) and homogeneous cell population that expresses relatively high levels of both LRRK2 and Rab1011. Furthermore, peripheral blood neutrophils can be isolated quickly and efficiently by employing an immunomagnetic negative approach. To ensure that the subsequent observed Rab10 phosphorylation is mediated by LRRK2, each batch of neutrophils is incubated with or without a potent and selective LRRK2 kinase inhibitor (we use and recommend MLi-2)2,12. This is then followed by cell lysis in a buffer containing the protease inhibitor diisopropyl fluorophosphate (DIFP), which is necessary for suppressing the intrinsic serine protease activity that is known to be high in neutrophils13. For the final analysis by quantitative immunoblotting, we recommend using the MJFF-pRab10 rabbit monoclonal antibody that specifically detects the Rab10 Thr73-phosphoepitope and does not cross-react with other phosphorylated Rab proteins14. Selectivity and specificity of this antibody has been validated in overexpression models of different Rab proteins and a A549 Rab10 knock-out cell line14. Thus, we measure the difference in Rab10 phosphorylation in neutrophil lysates that have been treated with and without a potent and selective LRRK2 kinase inhibitor2. Alternatively, samples could also be analyzed by other methods, such as quantitative mass spectrometry.
In conclusion, LRRK2-controlled Rab10 phosphorylation is a superior marker of LRRK2 kinase activity to LRRK2 phosphorylation at serine 935 and human peripheral blood neutrophils are a valuable resource for PD research into LRRK2. Our protocol provides a robust and easy assay to interrogate LRRK2 pathway activity in peripheral blood neutrophils and allows biochemical stratification of individuals with increased LRRK2 kinase activity15. Importantly, such individuals may benefit from future LRRK2 kinase inhibitor treatment.

<table border="0" cellpadding="0" cellspacing="0" style="width:979px;" width="978">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">1 mL Pipette tips</td>
			<td style="width:132px;">Sarstedt</td>
			<td style="width:95px;">70.762</td>
			<td style="width:432px;">or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">1.5 mL Micro tubes</td>
			<td style="width:132px;">Sarstedt</td>
			<td style="width:95px;">72.690.001</td>
			<td style="width:432px;">or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">10 mL Pipette tips&#160;</td>
			<td style="width:132px;">Sarstedt</td>
			<td style="width:95px;">86.1254.025&#160;</td>
			<td style="width:432px;">or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">10 &#956;L Pipette tips</td>
			<td style="width:132px;">Sarstedt</td>
			<td style="width:95px;">70.113</td>
			<td style="width:432px;">or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">15 mL falcon tube&#160;</td>
			<td style="width:132px;">Cellstar</td>
			<td style="width:95px;">188 271</td>
			<td style="width:432px;">or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">200 &#956;L Pipette tips</td>
			<td style="width:132px;">Sarstedt</td>
			<td style="width:95px;">70.760.002</td>
			<td style="width:432px;">or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">25 mL Pipette tips&#160;</td>
			<td style="width:132px;">Sarstedt</td>
			<td style="width:95px;">86.1685.001</td>
			<td style="width:432px;">or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">50 mL falcon tube&#160;</td>
			<td style="width:132px;">Cellstar</td>
			<td style="width:95px;">227 261</td>
			<td style="width:432px;">or equivalent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">BD Vacutainer Hemogard Closure Plastic K2-EDTA Tube</td>
			<td style="width:132px;">BD&#160;</td>
			<td style="width:95px;">BD 367525</td>
			<td style="width:432px;">or equivalent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Beckman Coulter Allegra X-15R centrifuge</td>
			<td style="width:132px;">Beckman</td>
			<td style="width:95px;"></td>
			<td style="width:432px;">or equivalent centrifuge with swimging bucket rotator for 15 mL and 50 mL falcon tubes at speed 1000-1200 x g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Category 2 biological safety cabinet.</td>
			<td style="width:132px;"></td>
			<td style="width:95px;"></td>
			<td style="width:432px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">cOmplete(EDTA-free) protease inhibitor cocktail</td>
			<td style="width:132px;">Roche</td>
			<td style="width:95px;">11836170001</td>
			<td style="width:432px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">DIFP (Diisopropylfluorophosphate)&#160;</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:95px;">D0879</td>
			<td style="width:432px;">Prepare 0.5M stock solution in isopropanol using special precautions&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethyl sulfoxide&#160;</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:95px;">6250</td>
			<td style="width:432px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Dry ice or liquid nitrogene</td>
			<td style="width:132px;"></td>
			<td style="width:95px;"></td>
			<td style="width:432px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Dulbecco&#39;s phosphate-buffered saline&#160;</td>
			<td style="width:132px;">ThermoFisher</td>
			<td style="width:95px;">14190094</td>
			<td style="width:432px;">or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Easy 50 EasySep Magnet&#160;</td>
			<td style="width:132px;">Stemcell</td>
			<td style="width:95px;">18002</td>
			<td style="width:432px;">for holding 1 x 50ml conical tube</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">EasySep Direct Human Neutrophil Isolation Kit&#160;</td>
			<td style="width:132px;">Stemcell</td>
			<td style="width:95px;">19666</td>
			<td style="width:432px;">This contains Solutions called &#8220;Isolation Cocktail&#8221; and &#8220;RapidSpheres magnetic beads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">EGTA</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:95px;">E3889</td>
			<td style="width:432px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Eppendorf centrifuge 5417R centrifuge</td>
			<td style="width:132px;">Eppendorf</td>
			<td style="width:95px;"></td>
			<td style="width:432px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Ethanol, in spray bottle</td>
			<td style="width:132px;"></td>
			<td style="width:95px;"></td>
			<td style="width:432px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethylenediaminetetraacetic acid&#160;</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:95px;">E6758</td>
			<td style="width:432px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Ice</td>
			<td style="width:132px;"></td>
			<td style="width:95px;"></td>
			<td style="width:432px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Isopropanol (anhydrous grade)&#160;</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:95px;">278475</td>
			<td style="width:432px;"></td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:320px;">Lysis buffer (50 mM Tris-HCl pH 7.5, 1%(v/v) Triton X-100, 1 mM EGTA, 1 mM Na3VO4, 50 mM NaF, 10 mM &#946;-glycerophosphate, 5 mM sodium pyrophosphate, 0.27 M sucrose, 0.1% (v/v) &#946;-mercaptoethanol, 1x cOmplete(EDTA-free) protease inhibitor cocktail (Roche), 1 &#956;g/ml Microcystin-LR, 0.5 mM diisopropylfluorophosphate (DIFP).&#160;</td>
			<td style="width:132px;"></td>
			<td style="width:95px;"></td>
			<td style="width:432px;">alternatively frozen lysis buffer in aliquots without Microcystin-LR, DIFP available from MRC-PPU Reagents (http://mrcppureagents.dundee.ac.uk/)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Merck LRRK2 inhibitor II (MLi-2)</td>
			<td style="width:132px;">Merck</td>
			<td style="width:95px;">438194-10MG</td>
			<td style="width:432px;">or equivalent (potent and selective LRRK2 inhinitor)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Microcystin-LR</td>
			<td style="width:132px;">Enzo Life Sciences</td>
			<td style="width:95px;">ALX-350-012-M001</td>
			<td style="width:432px;">1 mg/ml stock in DMSO and store at -80 oC.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Na3VO4</td>
			<td style="width:132px;">Aldrich</td>
			<td style="width:95px;">450243</td>
			<td style="width:432px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">NaF</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:95px;">S7920</td>
			<td style="width:432px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Odyssey CLx scan Western Blot imaging system</td>
			<td style="width:132px;">Odyssey</td>
			<td style="width:95px;"></td>
			<td style="width:432px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Permanent marker pen</td>
			<td style="width:132px;"></td>
			<td style="width:95px;"></td>
			<td style="width:432px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Personal protection equipment</td>
			<td style="width:132px;"></td>
			<td style="width:95px;"></td>
			<td style="width:432px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">RPMI 1640 Medium&#160;</td>
			<td style="width:132px;">ThermoFisher</td>
			<td style="width:95px;">21875034</td>
			<td style="width:432px;">or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">sodium pyrophosphate</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:95px;">S22</td>
			<td style="width:432px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">sucrose</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:95px;">S0389</td>
			<td style="width:432px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">&#946;-glycerophosphate</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:95px;">50020</td>
			<td style="width:432px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">&#946;-mercaptoethanol</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:95px;">M3148</td>
			<td style="width:432px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Suggested antibodies for Western blotting</td>
			<td style="width:132px;"></td>
			<td style="width:95px;"></td>
			<td style="width:432px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Anti-RAB10 (phospho T73) antibody [MJF-R21]</td>
			<td style="width:132px;">abcam</td>
			<td style="width:95px;">ab230261</td>
			<td style="width:432px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Anti-&#945;-tubulin</td>
			<td style="width:132px;">Cell Signaling Technologies</td>
			<td style="width:95px;">5174</td>
			<td style="width:432px;">used at 1:2000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Goat anti-mouse IRDye 680LT</td>
			<td style="width:132px;">LI-COR</td>
			<td style="width:95px;">926-68020</td>
			<td style="width:432px;">used at 1:10,000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Goat anti-mouse IRDye 800CW</td>
			<td style="width:132px;">LI-COR</td>
			<td style="width:95px;">926-32210</td>
			<td style="width:432px;">used at 1:10,000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Goat anti-rabbit IRDye 800CW</td>
			<td style="width:132px;">LI-COR</td>
			<td style="width:95px;">926-32211</td>
			<td style="width:432px;">used at 1:10,000 dilution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:320px;">MJFF-total Rab10 mouse antibody</td>
			<td style="width:132px;">generated by nanoTools (nanotools.de)</td>
			<td style="width:95px;">not applicable*</td>
			<td style="width:432px;">used at 2 &#956;g/ml final concentration; * The MJFF-total Rab10 antibody generated by nanoTools (www.nanotools.de) [11] will be commercialised by the Michael J Fox Foundation in 2018</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Mouse anti-LRRK2 C-terminus antibody</td>
			<td style="width:132px;">Antibodies Incorporated</td>
			<td style="width:95px;">&#160;75-253</td>
			<td style="width:432px;">used at 1 &#956;g/ml final concentration</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:320px;">pS935-LRRK2</td>
			<td style="width:132px;">MRC PPU Reagents and Services</td>
			<td style="width:95px;">UDD2</td>
			<td style="width:432px;">MJFF-total Rab10 mouse antibody</td>
		</tr>
	</tbody>
</table>

human peripheral blood neutrophil isolation for interrogating the parkinson's associated lrrk2 kinase pathway by assessing rab10 phosphorylation

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson&#8217; disease (PD) and all pathogenic LRRK2 mutations result in hyperactivation of its kinase function. Here, we describe an easy and robust assay to quantify LRRK2 kinase pathway activity in human peripheral blood neutrophils by measuring LRRK2-controlled phosphorylation of one of its physiological substrates, Rab10 at threonine 73. The immunoblotting analysis described requires a fully selective and phosphospecific antibody that recognizes the Rab10 Thr73 epitope phosphorylated by LRRK2, such as the MJFF-pRab10 rabbit monoclonal antibody. It uses human peripheral blood neutrophils, because peripheral blood is easily accessible and neutrophils are an abundant and homogenous constituent. Importantly, neutrophils express relatively high levels of both LRRK2 and Rab10. A potential drawback of neutrophils is their high intrinsic serine protease activity, which necessitates the use of very potent protease inhibitors such as the organophosphorus neurotoxin diisopropylfluorophosphate (DIFP) as part of the lysis buffer. Nevertheless, neutrophils are a valuable resource for research into LRRK2 kinase pathway activity in vivo and should be considered for inclusion into PD biorepository collections.

Our assay allows interrogating the activation of the PD-associated LRRK2 kinase in human peripheral blood neutrophils with LRRK2-dependent Rab10 phosphorylation as a readout. Neutrophils are a homogenous and abundant peripheral white blood cell population that expresses high levels of both the LRRK2 and Rab10 proteins (Figure 1). The only other cell population among the remaining peripheral blood mononuclear cells (PBMCs) with high copy numbers of both proteins are monocytes, but these make ...

Watch this Scientific Journal Video about Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson's Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation at JoVE.com

Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson's Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation

Mutations in the leucine rich repeat kinase 2 gene (LRRK2) cause hereditary Parkinson&#8217;s disease. We have developed an easy and robust method for assessing LRRK2-controlled phosphorylation of Rab10 in human peripheral blood neutrophils. This may help identify individuals with increased LRRK2 kinase pathway activity.

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson&#8217; disease (PD) and all pathogenic LRRK2 ...

The overall goal of this procedure is to provide a method to assess endogenous LRRK2 kinase activity in human peripheral blood. This is achieved by monitoring LRRK2-mediated phosphorylation of Rab proteins, employing Michael J.Fox Foundation phospho-specific Rab monoclonal antibodies. Here, we focus on human peripheral blood neutrophils as a constitute of homogenous site population with high expression levels of both Rab2 and Rab10.The neutrophil isolation is based on a negative selection that within 40 minutes of finished action allows isolation of 99%pure and viable neutrophils while also yielding high amount of protein levels. Collect 10 milliliter of blood into a blood collection tube. Mix gently by inverting tubes seven to eight times.Transfer 10 milliliters of blood into a 50 milliliter conical tube. Add to blood 100 microliters of EDTA stock solution 1, 0.1 molar EDTA PBS Solution, mix gently. Add 500 microliters isolation cocktail, 50 microliters per milliliter from the neutrophil isolation kit to the whole blood sample.Vortex the magnetic beads from the neutrophil isolation kit for 30 seconds before use in order to resuspend very fine magnetic beads. Add 500 microliters magnetic beads to the blood sample and mix gently by inverting the tube several times. Incubate at room temperature for five minutes.Top up tube to 50 milliliter with EDTA stock solution 2. This is one millimolar EDTA PBS Solution. Mix by very gently pipetting up and down two to three times.Place the tube into the magnet and remove the lid to avoid subsequent agitation of the tube. Incubate for 10 minutes at room temperature. Carefully pipette enriched cell suspension that contains neutrophils into a new 50 milliliter conical tube.Do not touch the side of the tube that is in contact with the magnet and avoid collection and perturbation of the red blood cells at the bottom of the tube. Leave approximately 10 milliliters of the red blood cell suspension behind at the bottom of the tube. Vortex magnetic beads for 30 seconds before use and add 0.5 milliliter magnetic beads to the tube containing the enriched neutrophils.Mix gently by inverting the tube. Incubate at room temperature for five minutes. Place the tube into the magnet and remove the lid to avoid subsequent agitation.Incubate for five minutes at room temperature. Carefully pipette the enriched cell suspension that contains neutrophils in a new 50 milliliter conical tube. Do not touch the side of the tube that is in contact with the magnet.Leave approximately five milliliter of the suspension at the bottom of the tube. To ensure complete removal of magnetic beads from cell mixture, place tube containing enriched cells into the magnet. Incubate for 10 minutes at room temperature.Carefully pipette the enriched cell suspension that now contains pure neutrophils into a new 50 milliliter conical tube. Do not touch the side of the tube that is in contact with the magnet. Leave approximately five milliliter of suspension at the bottom of the tube.Top up isolated cells with one millimolar EDTA stock solution 2 to a final volume of approximately 41 milliliters. Pipette up and down to mix. Divide solution equally into two tubes with approximately 20 milliliters in each tube.Centrifuge both tubes at 335G for five minutes. During the centrifugation step, take MLi-2 inhibitor stock, 200 micromolar slash 1000X concentration out of the minus 80 degree freezer and leave at room temperature for subsequent use. Immediately after the centrifugation step and without agitation of the tubes, pour off the supernatant without disturbing the neutrophil pellets.resuspend each cell pellet in 10 milliliters of cell culture medium at room temperature by gently pipetting cells up and down four times. Label one tube DMSO and the other tube MLi-2. To DMSO labeled tube add 10 microliter of DMSO and add 10 microliters of 200 micromolar MLi-2 stock solution by gently pipetting up and down to mix.Incubate samples for 30 minutes at room temperature. Mix gently by inversion every 10 minutes during the incubation period. During the 30 minute LRRK2 kinase inhibitor treatment remove 0.5 molar DIFP stock solution from the minus 80 degree freezer and place in fume hood on ice.Then, move one milligram per milliliter Microcystin-LR stock solution from the minus 80 degree freezer and place at room temperature to thaw. Defrost an aliquot, 0.25 milliliter of the lysis buffer by taking it out of the freezer, allow to defrost at room temperature and then place it on ice for subsequent use. Prepare one milliliter of RPMI medium containing one microliter of DMSO call this DMSO Resuspension Buffer.Prepare one milliliter of RPMI medium containing one microliter of 200 micromolar MLi-2 or alternative LRRK2 kinase inhibitor and call this MLi-2 Resuspension Buffer. After the 30 minute incubation period, centrifuge both tubes at 335G for five minutes, as before. Carefully discard the supernatant in each tube without disturbing the neutrophil pellet.For the DMSO labeled tube, gently resuspend pellet in one milliliter of DMSO Resuspension Buffer. And for the MLi-2 labeled tube, resuspend pellet in one milliliter of MLi-2 Resuspension Buffer. Transfer resuspended cell pellets to corresponding centrifugation tubes labeled DMSO and MLi-2 and centrifuge both tubes at 335G for three minutes.During the centrifugation step, prepare the lysis buffer. In the fume hood, carefully add 0.25 microliters of 0.5 molar DIFP solution, as well as 0.25 microliters of one milligram per milliliter microcystin-LR to the 0.25 milliliter lysis buffer. Mix and leave on ice until use.Immediately after the centrifugation, carefully and completely remove all supernatant with a pipette without disturbing the neutrophil pellet and place tubes on ice. Immediately add 100 microliters of lysis buffer containing DIFP and microcystin-LR to each tube. Using a 100 to 200 microliter pipette, resuspend the cell pellets by pipetting up and down about five to 10 times.Lie cells on ice for 10 minutes. Then, centrifuge tubes at 20, 000 times G for 15 minutes at four degrees celsius to remove cell debris. Transfer DMSO and MLi-2 supernatant containing neutrophil lysates into new centrifugation tubes.Discard debris pellets. Neutrophil lysates are now ready for use or can be snap frozen in liquid nitrogen and stored at minus 80 degrees for future analysis. Using data from the publicly available import database, this graph shows that an abundance of the LRRK2 and Rab10 proteins is particularly high in human peripheral blood neutrophils and monocytes.Isolating human peripheral blood neutrophils with this procedure results in a high impure yield of cells. The table at the top shows the total number of cells isolated from 10 milliliter of peripheral blood from three healthy donors. Also shown are purity and viability of the isolated cells, as well as total protein lysate yield.After treatment with and without the LRRK2 kinase inhibitor, MLi-2, neutrophils are lysed in the presence of the potent protease inhibitor, DIFP. For figure B on the left hand side, 10 micrograms of whole cell extracts per lane were subjected to multiplex quantitative immunoblot analysis with the indicated antibodies. The figure on the right, C, demonstrates the importance of DIFP for prevention of proteolytic degradation, in particular of the large LRRK2 protein.In comparison, high concentrations of PMSF alone are less effective. Here is showed that LRRK2-controlled Rab10 phosphorylation is significantly increased by about threefold in peripheral blood neutrophils derived from patients with hereditary Parkinson's disease harboring a heterozygous VPS35 D620N mutation when compared to controls. The top graph shows the multiplex immunoblot analysis and the bottom, the quantification thereof.This suggests that the VPS35 D620N mutation activates LRRK2 kinase path of activity by a yet unknown mechanism. In summary, we present a facile and robust assay to measure LRRK2 kinase pathway activity by monitoring Rab protein phosphorylation, such as Rab10, in human peripheral-derived neutrophils, this method allows us to assess LRRK2 pathway activity and determine how mutations, disease states or inhibitors modulate LRRK2 pathway activity.

Research

JoVE Journal

Biochemistry

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Oligopeptide Competition Assay for Phosphorylation Site Determination

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Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

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Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

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