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

Summary

Mutations in the leucine rich repeat kinase 2 gene (LRRK2) cause hereditary Parkinson’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.

Abstract

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson’ 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.

Introduction

Attempts to slow or stop Parkinson’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 inhibitors^1,2,3. 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 kinase^5,6,7. 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 inhibitors^8,9. 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 site^10,11.

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 (37–80% 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.

Protocol

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

1. Prepare 0.1 mL of EDTA Stock Solution 1 containing 100 mM EDTA in phosphate-buffered saline (PBS).
2. Prepare 60 mL of EDTA Stock Solution 2 containing 1 mM EDTA in PBS.
3. Prepare lysis buffer containing 50 mM Tris-HCl (pH = 7.5), 1% (v/v) Triton X-100, 1 mM EGTA, 1 mM Na₃VO₄, 50 mM NaF, 10 mM β-glycerophosphate, 5 mM sodium pyrophosphate, 0.27 M sucrose, 0.1% (v/v) β-mercaptoethanol, 1x protease inhibitor cocktail, 1 μg/mL microcystin-LR, and 0.5 mM diisopropyl fluorophosphate (DIFP).
   NOTE: The authors routinely use an EDTA-free product, but an EDTA-containing protease inhibitor cocktail should also work. The lysis buffer can be made in advance without the β-mercaptoethanol, protease inhibitors, microcystin-LR, and DIFP, and stored at 4 °C until use. Ensure that the β-mercaptoethanol, protease inhibitors, microcystin-LR, and DIFP is only added immediately before use.
   CAUTION: DIFP is toxic and should be handled with care in a fume hood following local health and safety risk assessment. DIFP can be added to the lysis buffer and used immediately. Alternatively, the complete lysis buffer containing all other components, including DIFP, can be aliquoted and stored at -80 °C for subsequent use for at least 4 weeks.

2. Neutrophil isolation from whole blood

1. Collect 10 mL of blood into a blood collection tube. Mix gently by inverting tubes 7−8x.
2. Transfer 10 mL of blood into a 50 mL conical tube.
3. Add 100 μL of EDTA Stock Solution 1 to the blood. Mix gently.
4. Add 500 μL of the isolation cocktail (50 μL/mL) from the neutrophil isolation kit (Table of Materials) to the whole blood sample.
5. Vortex the magnetic beads from the neutrophil isolation kit for 30 s before use in order to resuspend the very fine magnetic beads.
6. Add 500 μL of the magnetic beads to the blood sample and mix gently by inverting the tube several times.
7. Incubate at room temperature (RT) for 5 min.
8. Fill the tube to 50 mL with EDTA Stock Solution 2. Mix by very gently pipetting up and down 2–3x.
9. Place the tube into the magnet and remove the lid to avoid subsequent agitation of the tube.
10. Incubate for 10 min at RT.
11. Carefully pipette the enriched cell suspension that contains the neutrophils into a new 50 mL conical tube.
    NOTE: 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 mL of the red blood cell suspension behind at the bottom of the tube.
12. Vortex the magnetic beads for 30 s before use and add 0.5 mL of the magnetic beads to the tube containing the enriched neutrophils. Mix gently by inverting the tube.
13. Incubate at RT for 5 min.
14. Place the tube into the magnet and remove the lid to avoid subsequent agitation.
15. Incubate at RT for 5 min.
16. Carefully pipette the enriched cell suspension that contains the neutrophils into a new 50 mL conical tube.
    NOTE: Do not touch the side of the tube that is in contact with the magnet. Leave approximately 5 mL of the suspension at the bottom of the tube.
17. To ensure the complete removal of magnetic beads from the cell mixture, place the tube containing the enriched cells into the magnet.
18. Incubate for 10 min at RT.
19. Carefully pipette the enriched cell suspension that now contains pure neutrophils into a new 50 mL conical tube.
    NOTE: Do not touch the side of the tube that is in contact with the magnet. Leave approximately 5 mL of the suspension at the bottom of the tube.
20. Mix the isolated cells with 1 mM EDTA Stock Solution 2 to a final volume of approximately 41 mL. Pipette up and down to mix.
21. Divide the solution equally into two tubes with approximately 20 mL in each tube.
22. Centrifuge both tubes at 335 x g for 5 min.
23. During this centrifugation take MLi-2 inhibitor stock (200 µM/1,000x concentration) out of the -80 °C freezer and leave at RT for subsequent use.
24. 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 mL of cell culture media (Table of Materials) at RT by gently pipetting cells up and down 4x.

3. LRRK2 kinase inhibitor treatment of pure neutrophils

1. Label one tube "DMSO" and the other tube "MLi-2".
2. To the "DMSO" labeled tube, add 10 µL of DMSO and mix gently by pipetting up and down 2x with a 10 mL pipette. To the "MLi-2" labeled tube, add 10 μL of 200 μM MLi-2 stock solution (final concentration 200 nM) and mix gently by pipetting up and down 2x with a 10 mL pipette.
3. Incubate the samples for 30 min at RT. Mix gently by inversion every 10 min during the incubation.
4. During the incubation period, remove 0.5 M DIFP stock from the -80 °C freezer and place in a fume hood on ice. Remove 1 mg/mL microcystin-LR stock solution from the -80 °C freezer and leave at RT to thaw. Take an aliquot (0.25 mL) of the lysis buffer out of the freezer, allow it to defrost at RT, and then place it on ice for subsequent use.
5. Prepare 1 mL of cell culture medium containing 1 μL of DMSO and call this DMSO resuspension buffer. Prepare 1 mL of RPMI media containing 1 μL of 200 μM MLi-2 and call this MLi-2 resuspension buffer.
6. After the 30 min incubation period, centrifuge both tubes at 335 x g for 5 min.
7. Carefully discard the supernatant in each tube without disturbing the neutrophil pellet.
8. For the DMSO labeled sample gently resuspend the pellet in 1 mL of the DMSO resuspension buffer and for the MLi-2 labeled tube, resuspend the pellet in 1 mL of the MLi-2 resuspension buffer.
9. Transfer the resuspended cell pellets to corresponding centrifugation tubes labeled "DMSO" and "MLi-2" and centrifuge both tubes at 335 x g for 3 min.
10. During the centrifugation step, prepare the lysis buffer. In the fume hood carefully add 0.25 μL of 0.5 M DIFP solution as well as 0.25 μL of 1 mg/mL microcystin-LR to the 0.25 mL lysis buffer. Mix and leave on ice until use.
    NOTE: Add DIFP to the lysis buffer within 15 min of cell lysis, because DIFP is relatively unstable in an aqueous solution.
11. Immediately after the centrifugation, carefully and completely remove all the supernatant with a pipette without disturbing the neutrophil pellet and place the tubes on ice.
12. Immediately add 100 µL of lysis buffer containing DIFP and microcystine-LR to each tube. Using a 100–200 μL pipette, resuspend the cell pellets by pipetting up and down about 5–10x.
13. Lyse the cells on ice for 10 min.
14. Centrifuge tubes at 20,000 x g for 15 min at 4 °C to remove cell debris.
15. Transfer the "DMSO" and "MLi-2" supernatants containing the neutrophil lysates into new centrifugation tubes. Discard the debris pellet.
    NOTE: The neutrophil lysates are now ready for use or can be snap frozen in liquid nitrogen and stored at -80 °C for future analysis.

Results

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 ...

Discussion

Compelling clinical, genetic, and biochemical evidence points towards an important role for LRRK2 and in particular its kinase function in Parkinson’s disease⁷. LRRK2 kinase inhibitors have been developed and are entering clinical trials^2,4,12. 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...

Materials

Name	Company	Catalog Number	Comments
1 mL Pipette tips	Sarstedt	70.762	or equivalent
1.5 mL Micro tubes	Sarstedt	72.690.001	or equivalent
10 mL Pipette tips	Sarstedt	86.1254.025	or equivalent
10 μL Pipette tips	Sarstedt	70.113	or equivalent
15 mL falcon tube	Cellstar	188 271	or equivalent
200 μL Pipette tips	Sarstedt	70.760.002	or equivalent
25 mL Pipette tips	Sarstedt	86.1685.001	or equivalent
50 mL falcon tube	Cellstar	227 261	or equivalent
BD Vacutainer Hemogard Closure Plastic K2-EDTA Tube	BD	BD 367525	or equivalent
Beckman Coulter Allegra X-15R centrifuge	Beckman		or equivalent centrifuge with swimging bucket rotator for 15 mL and 50 mL falcon tubes at speed 1000-1200 x g
Category 2 biological safety cabinet.
cOmplete(EDTA-free) protease inhibitor cocktail	Roche	11836170001
DIFP (Diisopropylfluorophosphate)	Sigma	D0879	Prepare 0.5M stock solution in isopropanol using special precautions
Dimethyl sulfoxide	Sigma	6250
Dry ice or liquid nitrogene
Dulbecco's phosphate-buffered saline	ThermoFisher	14190094	or equivalent
Easy 50 EasySep Magnet	Stemcell	18002	for holding 1 x 50ml conical tube
EasySep Direct Human Neutrophil Isolation Kit	Stemcell	19666	This contains Solutions called “Isolation Cocktail” and “RapidSpheres magnetic beads
EGTA	Sigma	E3889
Eppendorf centrifuge 5417R centrifuge	Eppendorf
Ethanol, in spray bottle
Ethylenediaminetetraacetic acid	Sigma	E6758
Ice
Isopropanol (anhydrous grade)	Sigma	278475
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 β-glycerophosphate, 5 mM sodium pyrophosphate, 0.27 M sucrose, 0.1% (v/v) β-mercaptoethanol, 1x cOmplete(EDTA-free) protease inhibitor cocktail (Roche), 1 μg/ml Microcystin-LR, 0.5 mM diisopropylfluorophosphate (DIFP).			alternatively frozen lysis buffer in aliquots without Microcystin-LR, DIFP available from MRC-PPU Reagents (http://mrcppureagents.dundee.ac.uk/)
Merck LRRK2 inhibitor II (MLi-2)	Merck	438194-10MG	or equivalent (potent and selective LRRK2 inhinitor)
Microcystin-LR	Enzo Life Sciences	ALX-350-012-M001	1 mg/ml stock in DMSO and store at -80 oC.
Na3VO4	Aldrich	450243
NaF	Sigma	S7920
Odyssey CLx scan Western Blot imaging system	Odyssey
Permanent marker pen
Personal protection equipment
RPMI 1640 Medium	ThermoFisher	21875034	or equivalent
sodium pyrophosphate	Sigma	S22
sucrose	Sigma	S0389
β-glycerophosphate	Sigma	50020
β-mercaptoethanol	Sigma	M3148
Suggested antibodies for Western blotting
Anti-RAB10 (phospho T73) antibody [MJF-R21]	abcam	ab230261
Anti-α-tubulin	Cell Signaling Technologies	5174	used at 1:2000 dilution
Goat anti-mouse IRDye 680LT	LI-COR	926-68020	used at 1:10,000 dilution
Goat anti-mouse IRDye 800CW	LI-COR	926-32210	used at 1:10,000 dilution
Goat anti-rabbit IRDye 800CW	LI-COR	926-32211	used at 1:10,000 dilution
MJFF-total Rab10 mouse antibody	generated by nanoTools (nanotools.de)	not applicable*	used at 2 μg/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
Mouse anti-LRRK2 C-terminus antibody	Antibodies Incorporated	75-253	used at 1 μg/ml final concentration
pS935-LRRK2	MRC PPU Reagents and Services	UDD2	MJFF-total Rab10 mouse antibody