Name: human peripheral blood neutrophil isolation for interrogating the parkinson's associated lrrk2 kinase pathway by assessing rab10 phosphorylation
Uploaded: 2020-03-21T13:00:00.000+00:00
Duration: 12 min 49 s
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 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 &#946;-glycerophosphate, 5 mM sodium pyrophosphate, 0.27 M sucrose, 0.1% (v/v) &#946;-mercaptoethanol, 1x protease inhibitor cocktail, 1 &#956;g/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 &#946;-mercaptoethanol, protease inhibitors, microcystin-LR, and DIFP, and stored at 4 &#176;C until use. Ensure that the &#946;-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 &#176;C for subsequent use for at least 4 weeks.</li>
</ol>
2. Neutrophil isolation from whole blood
<ol>
	<li>Collect 10 mL of blood into a blood collection tube. Mix gently by inverting tubes 7&#8722;8x.</li>
	<li>Transfer 10 mL of blood into a 50 mL conical tube.</li>
	<li>Add 100 &#956;L of EDTA Stock Solution 1 to the blood. Mix gently.</li>
	<li>Add 500 &#956;L of the isolation cocktail (50 &#956;L/mL) from the neutrophil isolation kit (Table of Materials) to the whole blood sample.</li>
	<li>Vortex the magnetic beads from the neutrophil isolation kit for 30 s before use in order to resuspend the very fine magnetic beads.</li>
	<li>Add 500 &#956;L of the magnetic beads to the blood sample and mix gently by inverting the tube several times.</li>
	<li>Incubate at room temperature (RT) for 5 min.</li>
	<li>Fill the tube to 50 mL with EDTA Stock Solution 2. Mix by very gently pipetting up and down 2&#8211;3x.</li>
	<li>Place the tube into the magnet and remove the lid to avoid subsequent agitation of the tube.</li>
	<li>Incubate for 10 min at RT.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Incubate at RT for 5 min.</li>
	<li>Place the tube into the magnet and remove the lid to avoid subsequent agitation.</li>
	<li>Incubate at RT for 5 min.</li>
	<li>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.</li>
	<li>To ensure the complete removal of magnetic beads from the cell mixture, place the tube containing the enriched cells into the magnet.</li>
	<li>Incubate for 10 min at RT.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Divide the solution equally into two tubes with approximately 20 mL in each tube.</li>
	<li>Centrifuge both tubes at 335 x g for 5 min.</li>
	<li>During this centrifugation take MLi-2 inhibitor stock (200 &#181;M/1,000x concentration) out of the -80 &#176;C freezer and leave at RT for subsequent use.</li>
	<li>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.</li>
</ol>
3. LRRK2 kinase inhibitor treatment of pure neutrophils
<ol>
	<li>Label one tube &#34;DMSO&#34; and the other tube &#34;MLi-2&#34;.</li>
	<li>To the &#34;DMSO&#34; labeled tube, add 10 &#181;L of DMSO and mix gently by pipetting up and down 2x with a 10 mL pipette. To the &#34;MLi-2&#34; labeled tube, add 10 &#956;L of 200 &#956;M MLi-2 stock solution (final concentration 200 nM) and mix gently by pipetting up and down 2x with a 10 mL pipette.</li>
	<li>Incubate the samples for 30 min at RT. Mix gently by inversion every 10 min during the incubation.</li>
	<li>During the incubation period, remove 0.5 M DIFP stock from the -80 &#176;C freezer and place in a fume hood on ice. Remove 1 mg/mL microcystin-LR stock solution from the -80 &#176;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.</li>
	<li>Prepare 1 mL of cell culture medium containing 1 &#956;L of DMSO and call this DMSO resuspension buffer. Prepare 1 mL of RPMI media containing 1 &#956;L of 200 &#956;M MLi-2 and call this MLi-2 resuspension buffer.</li>
	<li>After the 30 min incubation period, centrifuge both tubes at 335 x g for 5 min.</li>
	<li>Carefully discard the supernatant in each tube without disturbing the neutrophil pellet.</li>
	<li>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.</li>
	<li>Transfer the resuspended cell pellets to corresponding centrifugation tubes labeled &#34;DMSO&#34; and &#34;MLi-2&#34; and centrifuge both tubes at 335 x g for 3 min.</li>
	<li>During the centrifugation step, prepare the lysis buffer. In the fume hood carefully add 0.25 &#956;L of 0.5 M DIFP solution as well as 0.25 &#956;L 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.</li>
	<li>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.</li>
	<li>Immediately add 100 &#181;L of lysis buffer containing DIFP and microcystine-LR to each tube. Using a 100&#8211;200 &#956;L pipette, resuspend the cell pellets by pipetting up and down about 5&#8211;10x.</li>
	<li>Lyse the cells on ice for 10 min.</li>
	<li>Centrifuge tubes at 20,000 x g for 15 min at 4 &#176;C to remove cell debris.</li>
	<li>Transfer the &#34;DMSO&#34; and &#34;MLi-2&#34; 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 &#176;C for future analysis.</li>
</ol>

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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. 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The authors have nothing to disclose.

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 assay for analyzing LRRK2 kinase pathway activation as reflected by the phosphorylation of its physiological substrate Rab10 in the homogenous pool of human peripheral blood neutrophils11,14. For analysis by quantitative immunoblotting, we strongly recommend the use of a highly selective phosphospecific Rab10 antibody (MJFF-pRab10 monoclonal antibody)14,17.
We use human neutrophils because they constitute a homogenous subset of peripheral blood cells that make up the dominant leukocyte population17,18. More importantly, neutrophils also have high protein expression levels of LRRK2 and its substate Rab10 (Figure 1)16. In contrast, the remaining subsets of leukocytes that make up the pool of PBMCs are heterogeneous with variable and predominantly low expression of LRRK2 and Rab10. Monocytes and dendritic cells have high expression levels, but are low in overall abundance16.
While we recommend the use of EDTA vacutainer blood collection tubes, an anticoagulant other than EDTA can be used. However, the presence of EDTA is important for the performance of the neutrophil isolation kit. We therefore recommend adding EDTA to the whole blood sample so that a final concentration of 1 mM EDTA is reached even if an alternative anticoagulant is used. The use of the neutrophil isolation kit allows purifying neutrophils directly from human whole blood by immunomagnetic negative selection in a relatively fast and easy way. The number of available magnets determines how many blood samples can be processed in parallel. It is easily feasible to isolate neutrophils from up to six blood samples in parallel using six magnets. A potential drawback is the associated cost for commercial neutrophil isolation kits and the required magnet. Alternative methods for neutrophil isolation from whole blood have been described and rely on density gradient separation or fluorescence activated cell sorting (FACS), which have significant drawbacks. The former is significantly more time and labor intensive and at least in our hands not as reliable and efficient. The latter requires a FACS machine and additional handling steps would need to be introduced, including the depletion of red blood cells and cell staining, adding time to the cell isolation process. Overall, immunomagnetic isolation is fast, generates a highly pure sample, and avoids excessive handling of the cells. With regards to sample processing, we have previously shown that a delay between blood collection and neutrophil isolation of at least up to 24 h does not result in a significant change or variation in the outcome of our assay, which provides flexibility for future clinical exploitation11.
The neutrophil isolation procedure itself is very easy. We recommend vortexing the magnetic beads before each use. It is also important not to disturb the magnet once the tube is inside the magnet to avoid turbulence and dislodgement of the magnetic beads. Neutrophils are being enriched in suspension by several rounds of immunomagnetic removal of all unwanted cells. Care should be taken not to touch the side of the tube that is in touch with the magnet and during the first round of isolation (step 2.11) so as not to perturb the red blood cells at the bottom of the tube. After the final round of pipetting the enriched cell suspension into a new conical tube (step 2.24), neutrophils are immediately available for onward processing. If neutrophils are used to assess LRRK2 activity in cells, neutrophils are then split into two batches and after pelleting by centrifugation, resuspended in either a LRRK2 kinase inhibitor (here, MLi-2) or DMSO containing cell culture medium. As dephosphorylation in the presence and rephosphorylation in the absence of LRRK2 kinase inhibition are relatively rapid events, care needs to be taken to ensure that the MLi-2 treated neutrophil fraction remains exposed to a LRRK2 kinase inhibitor (e.g., MLi-2) up until cell lysis (steps 3.8&#8211;3.10).
There are several centrifugation steps in this protocol using 15 and 50 mL conical tubes. While we routinely use the indicated centrifugation speeds in the protocol, it is possible to increase the centrifugation speed up to 400 x g without adversely affecting the viability of the cells. This results in a slightly firmer neutrophil cell pellet which might help to reduce any potential loss of neutrophil material during the decanting and pipetting off the supernatant steps during this protocol. This will likely increase the yield in terms of total protein lysate obtained. A potential concern is that a higher centrifugation force could activate neutrophils and potentially affect subsequent analysis. Our general recommendation is to handle neutrophils during every step of the protocol as gently as possible.
Our protocol uses the highly potent serine protease inhibitor DIFP (0.5 mM) as part of the neutrophil lysis buffer. DIFP is a potent organophosphorus neurotoxin, and while we have investigated alternative compounds, its addition to the lysis buffer was essential for the analysis of LRRK2-controlled Rab10 phosphorylation in human neutrophils by immunoblotting. For example, replacing DIFP with 1% (w/v) SDS has been used to lyse neutrophils in other studies19 and led to significant protein degradation to the extent that immunoblotting for LRRK2, Rab10, and even the GAPDH loading control did not yield a signal, thus highlighting the importance of including a highly potent protease inhibitor in the lysis buffer (Figure 2C). When replacing DIFP with the less potent, but also less toxic serine protease inhibitor PMSF at 2.5 mM, Rab10 phosporylation was well preserved, but the larger LRRK2 protein underwent significant degradation (Figure 2C). In order to minimize handling of the DIFP stock solution, lysis buffer containing DIFP can be prepared in batches, aliquoted and stored at -20 &#176;C or -80 &#176;C11.With regards to the analysis by quantitative immunoblotting, it is paramount to use a phosphospecific antibody that has been demonstrated to be selective for only a single LRRK2 phosphorylated Rab protein, such as the MJFF-pRab10 antibody used for our studies14.
The protocol can also be scaled up using a maximal volume of whole blood of up to 25 mL, which is the limit that can be processed in one isolation procedure using one magnet, which is able to hold 50 mL conical tubes. The only steps that would need adjustments are step 2.4 (adding 50 &#181;L of isolation cocktail per mL of blood), steps 2.6 and 2.12 (adding 50 &#181;L of magnetic beads per mL of blood), and a proportionate increase in the volume of lysis buffer for neutrophil lysis in step 3.12. All other steps can be kept identical.
In summary, our protocol describes an easy and robust method to isolate human neutrophils from peripheral blood. Neutrophils can then be treated with and without a potent and specific LRRK2 kinase inhibitor to enable the quantification of the LRRK2-controlled phosphorylation of Rab10 in vivo. This can be useful for stratifying individuals according to LRRK2 kinase pathway activity and for identifying those with pathway hyperactivation who might benefit from future LRRK2 kinase inhibitor treatment. While this will be unlikely the case for the majority of people with PD, specifically idiopathic PD, our assay has already been successfully deployed in individuals carrying a rare, heterozygous mutation in another PD-associated gene, VPS35 D620N, where LRRK2 kinase pathway activity is significantly increased by a yet unknown mechanism15. We had previously examined LRRK2-controlled Rab 10 phosphorylation levels in a small number of individuals carrying the more common LRRK2 G2019S mutation that is known to activate LRRK2 kinase activity only modestly by a factor of around two without detecting a significant difference when compared to controls and patients with idiopathic PD with our assay11,14. This and further unpublished data suggest that LRRK2 kinase activity probably requires an increase of &#62;3x in order to yield a significant result using quantitative&#160;immunoblotting. However, the sensitivity for detecting LRRK2 kinase pathway activation may likely be increased if deploying state-of-the-art mass spectrometry technology.
We suggest that neutrophils are a valuable resource for Parkinson&#8217;s disease research into LRRK2 kinase pathway activity and might help identifying individuals who could benefit from future LRRK2 kinase inhibitor treatment.

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><tbody><tr><td>1 mL Pipette tips</td><td>Sarstedt</td><td>70.762</td><td>or equivalent</td></tr><tr><td>1.5 mL Micro tubes</td><td>Sarstedt</td><td>72.690.001</td><td>or equivalent</td></tr><tr><td>10 mL Pipette tips&amp;nbsp;</td><td>Sarstedt</td><td>86.1254.025&amp;nbsp;</td><td>or equivalent</td></tr><tr><td>10 &amp;mu;L Pipette tips</td><td>Sarstedt</td><td>70.113</td><td>or equivalent</td></tr><tr><td>15 mL falcon tube&amp;nbsp;</td><td>Cellstar</td><td>188 271</td><td>or equivalent</td></tr><tr><td>200 &amp;mu;L Pipette tips</td><td>Sarstedt</td><td>70.760.002</td><td>or equivalent</td></tr><tr><td>25 mL Pipette tips&amp;nbsp;</td><td>Sarstedt</td><td>86.1685.001</td><td>or equivalent</td></tr><tr><td>50 mL falcon tube&amp;nbsp;</td><td>Cellstar</td><td>227 261</td><td>or equivalent</td></tr><tr><td>BD Vacutainer Hemogard Closure Plastic K2-EDTA Tube</td><td>BD&amp;nbsp;</td><td>BD 367525</td><td>or equivalent</td></tr><tr><td>Beckman Coulter Allegra X-15R centrifuge</td><td>Beckman</td><td></td><td>or equivalent centrifuge with swimging bucket rotator for 15 mL and 50 mL falcon tubes at speed 1000-1200 x g</td></tr><tr><td>Category 2 biological safety cabinet.</td><td></td><td></td><td></td></tr><tr><td>cOmplete(EDTA-free) protease inhibitor cocktail</td><td>Roche</td><td>11836170001</td><td></td></tr><tr><td>DIFP (Diisopropylfluorophosphate)&amp;nbsp;</td><td>Sigma</td><td>D0879</td><td>Prepare 0.5M stock solution in isopropanol using special precautions&amp;nbsp;</td></tr><tr><td>Dimethyl sulfoxide&amp;nbsp;</td><td>Sigma</td><td>6250</td><td></td></tr><tr><td>Dry ice or liquid nitrogene</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline&amp;nbsp;</td><td>ThermoFisher</td><td>14190094</td><td>or equivalent</td></tr><tr><td>Easy 50 EasySep Magnet&amp;nbsp;</td><td>Stemcell</td><td>18002</td><td>for holding 1 x 50ml conical tube</td></tr><tr><td>EasySep Direct Human Neutrophil Isolation Kit&amp;nbsp;</td><td>Stemcell</td><td>19666</td><td>This contains Solutions called &amp;ldquo;Isolation Cocktail&amp;rdquo; and &amp;ldquo;RapidSpheres magnetic beads</td></tr><tr><td>EGTA</td><td>Sigma</td><td>E3889</td><td></td></tr><tr><td>Eppendorf centrifuge 5417R centrifuge</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Ethanol, in spray bottle</td><td></td><td></td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid&amp;nbsp;</td><td>Sigma</td><td>E6758</td><td></td></tr><tr><td>Ice</td><td></td><td></td><td></td></tr><tr><td>Isopropanol (anhydrous grade)&amp;nbsp;</td><td>Sigma</td><td>278475</td><td></td></tr><tr><td>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 &amp;beta;-glycerophosphate, 5 mM sodium pyrophosphate, 0.27 M sucrose, 0.1% (v/v) &amp;beta;-mercaptoethanol, 1x cOmplete(EDTA-free) protease inhibitor cocktail (Roche), 1 &amp;mu;g/ml Microcystin-LR, 0.5 mM diisopropylfluorophosphate (DIFP).&amp;nbsp;</td><td></td><td></td><td>alternatively frozen lysis buffer in aliquots without Microcystin-LR, DIFP available from MRC-PPU Reagents (http://mrcppureagents.dundee.ac.uk/)</td></tr><tr><td>Merck LRRK2 inhibitor II (MLi-2)</td><td>Merck</td><td>438194-10MG</td><td>or equivalent (potent and selective LRRK2 inhinitor)</td></tr><tr><td>Microcystin-LR</td><td>Enzo Life Sciences</td><td>ALX-350-012-M001</td><td>1 mg/ml stock in DMSO and store at -80 oC.&amp;nbsp;</td></tr><tr><td>Na3VO4</td><td>Aldrich</td><td>450243</td><td></td></tr><tr><td>NaF</td><td>Sigma</td><td>S7920</td><td></td></tr><tr><td>Odyssey CLx scan Western Blot imaging system</td><td>Odyssey</td><td></td><td></td></tr><tr><td>Permanent marker pen</td><td></td><td></td><td></td></tr><tr><td>Personal protection equipment</td><td></td><td></td><td></td></tr><tr><td>RPMI 1640 Medium&amp;nbsp;</td><td>ThermoFisher</td><td>21875034</td><td>or equivalent</td></tr><tr><td>sodium pyrophosphate</td><td>Sigma</td><td>S22</td><td></td></tr><tr><td>sucrose</td><td>Sigma</td><td>S0389</td><td></td></tr><tr><td>&amp;beta;-glycerophosphate</td><td>Sigma</td><td>50020</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Sigma</td><td>M3148</td><td></td></tr><tr><td>&lt;strong&gt;Suggested antibodies for Western blotting&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-RAB10 (phospho T73) antibody [MJF-R21]</td><td>abcam</td><td>ab230261</td><td></td></tr><tr><td>Anti-&amp;alpha;-tubulin</td><td>Cell Signaling Technologies</td><td>5174</td><td>used at 1:2000 dilution</td></tr><tr><td>Goat anti-mouse IRDye 680LT</td><td>LI-COR</td><td>926-68020</td><td>used at 1:10,000 dilution</td></tr><tr><td>Goat anti-mouse IRDye 800CW</td><td>LI-COR</td><td>926-32210</td><td>used at 1:10,000 dilution</td></tr><tr><td>Goat anti-rabbit IRDye 800CW</td><td>LI-COR</td><td>926-32211</td><td>used at 1:10,000 dilution</td></tr><tr><td>MJFF-total Rab10 mouse antibody</td><td>generated by nanoTools (nanotools.de)</td><td>not applicable*</td><td>used at 2 &amp;mu;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><td>Mouse anti-LRRK2 C-terminus antibody</td><td>Antibodies Incorporated</td><td>&amp;nbsp;75-253</td><td>used at 1 &amp;mu;g/ml final concentration</td></tr><tr><td>pS935-LRRK2</td><td>MRC PPU Reagents and Services</td><td>UDD2</td><td>used at 1 &amp;mu;g/ml final concentration</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 up only 2&#8211;12% of white blood cells. This indicates that peripheral blood neutrophils are a more suitable biomatrix for studying LRRK2-controlled Rab10 phosphorylation.
When isolating peripheral blood neutrophils from 10 mL of blood with our procedure, between 0.5&#8211;0.75 mg of total protein lysate per donor was obtained (Figure 2A), which is sufficient for a significant number of immunoblot analysis for which only 10 &#956;g per gel lane are required. While checking purity and viability of cells is not routinely performed, we demonstrated for three healthy donors that the purity of isolated neutrophils is between 94&#8211;98% and the viability of cells ~99% as determined by flow cytometry analysis using the CD66b&#8722;Fluorescein isothiocyanate neutrophil marker and 4&#39;,6-Diamidine-2&#39;-phenylindole dihydrochloride (DAPI) staining for viability (Figure 2A).
While the focus of this publication is the isolation and processing of neutrophils from peripheral blood and not the analysis by quantitative Western blotting, Figure 2B demonstrates that the MJFF-pRab10 monoclonal antibody that specifically detects Rab10 phosphorylated at threonine 73 revealed robust signals in the neutrophil samples, which were markedly suppressed by treatment with a potent and specific LRRK2 kinase inhibitor, in this case MLi-2.
Neutrophils contain high levels of serine proteases that can affect subsequent Western blot analysis. While DIFP effectively suppresses the high protease activity in neutrophils, it is a potent organophosphorus neurotoxin and it would be desirable to replace it with an equally effective but less toxic protease inhibitor, such as phenylmethylsulfonyl fluoride (PMSF). We found that Rab10 phosphorylation was equally well preserved when DIFP was replaced by PMSF at a concentration of 2.5 mM (Figure 2C). However, the integrity of the LRRK2 protein was compromised when using PMSF compared to DIFP, suggesting that the larger LRRK2 protein is more susceptible to degradation (Figure 2C).
We have previously shown that another PD-causing gene mutation VPS35 D620N results in hyperactivation of the LRRK2 kinase by a yet unknown mechanism15. Neutrophils from three people with PD harboring a disease-causing heterozygous VPS35 D620N mutation were isolated using the method described in this article (Figure 3). Neutrophil samples from nine healthy donors were isolated as controls15. Immunoblot analysis using the MJFF-pRab10 monoclonal antibody demonstrates a significant, ~3x increase in Rab10 phosphorylation at Thr 73 in neutrophils from Parkinson&#8217;s patients with a VPS35 D620N mutation compared to the controls. The total Rab10 protein expression is similar in all 12 neutrophil samples.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58956/58956fig1.jpg" /> 
Figure 1: Abundance of LRRK2 and Rab10 proteins in immune cells isolated from human blood using data that is publicly available on the immprot database (http://www.immprot.org)16. The graph shows the number of protein copies per cell for LRRK2 and Rab10 in a range of peripheral blood immune cells, including subsets of T cells, B cells, monocytes, NK cells, dendritic cells, and the granulocytes neutrophils, basophils, and eosinophils. This figure has been modified from Fan et al.11. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58956/58956fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58956/58956fig2.jpg" /> 
Figure 2: Characterization and analysis of human peripheral blood neutrophils and importance of DIFP for the prevention of proteolytic degradation in neutrophils. (A) Neutrophils were isolated from the whole blood of three healthy donors (A,&#160;B, and&#160;C) showing purity, viability, and total protein yield after cell lysis. (B) Neutrophils were treated with or without LRRK2 kinase inhibitor (MLi-2), then lysed and subjected to quantitative immunoblot analysis with the indicated antibodies via near-infrared (NIR) fluorescence imaging. (C) Neutrophils were isolated from two healthy donors and treated with 100 nM MLi-2 for 30 min. Cells were lysed in the presence of either 0.5 mM DIFP or 2.5 mM PMSF to block serine protease activity in neutrophils. A and B have been modified from Mir et al.15, while C has been modified from Fan et al.11. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58956/58956fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58956/58956fig3.jpg" /> 
Figure 3: Increased LRRK2 kinase pathway activity in Parkinson&#8217;s disease patients harboring a heterozygeous VPS35 D620N mutation. Neutrophils were isolated from nine non-age-matched healthy controls and three PD patients with a disease-causing heterozygous VPS35 D620N mutation. The cells were treated with or without 200&#8197;nM MLi-2 for 30&#8197;min before cell lysis. (A) A total of 10&#8197;&#181;g of whole cell extract subjected to quantitative immunoblot analysis with the indicated antibodies via near-infrared (NIR) fluorescence imaging. Immunoblots were quantified for phospho-Thr73 Rab10:total Rab10 ratio (B). Data were analyzed by one-way ANOVA with Tukey&#39;s multiple comparison test. Data presented as means&#8201;&#8201;&#177;&#8201;&#8201;SD; ***p&#8201;&#60;&#8201;0.0001. This figure has been modified from Mir et al.15. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58956/58956fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

human-peripheral-blood-neutrophil-isolation-for-interrogating

Nanyang Technological University

Research

JoVE Journal

Biochemistry

7.4K Views.  University of Dundee. Het algemene doel van deze procedure is om een methode te bieden om endogene LRRK2 kinase activiteit in menselijk perifeer bloed te beoordelen. Dit wordt bereikt door het monitoren van LRRK2-gemedieerde fosforylatie van Rab eiwitten, in dienst Michael J.Fox Foundation fosfo-specifieke Rab monoclonal antilichamen. Hier richten we ons op menselijke perifere bloednefolfielen als een vorm van homogene site bevolking met hoge expressieniveaus van zowel Rab2 en Rab10.De neutrofiele isolatie ...

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

Assessment of Sensorimotor Function in Mouse Models of Parkinson's Disease

Sensitive and reliable behavioral outcome measures are essential to the evaluation of potential therapeutic treatments in preclinical trials for many neurodegenerative diseases. In Parkinson's disease, sensorimotor tests sensitive to varying degrees of nigrostriatal dysfunction are fundamental for testing the efficacy of potential therapeutics. Reliable and quite elegant sensorimotor measures exist for rats, however many of these tests measure sensorimotor asymmetry within the rat and are not entirely suitable for the newer genetic mouse models of PD. We have put together a battery of sensorimotor tests inspired by the sensitive tests in rats and adapted for mice. The test battery highlighted in this study is chosen for a) its sensitivity in a wide variety of mouse models of PD, b) its ease in implementing into a study, and c) its low expense. These tests have proven useful in characterizing novel genetic mouse models of PD as well as in testing potential disease-modifying therapies.

In Parkinson's disease and movement disorders in general, sensitive and reliable behavioral assays are essential for testing novel potential therapeutics. Here, we describe a manageable battery of sensorimotor tests for mice that are sensitive to varying degrees of injury to the nigrostriatal system and useful for preclinical studies.

Sensitive and  reliable behavioral outcome measures are essential to the evaluation of  potential therapeutic treatments in preclinical trials for many  ...

Behavior

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

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of the kinase activity of LRRK2 has been implicated in the pathogenesis of PD, it is essential to establish a method to evaluate the physiological levels of the kinase activity of LRRK2. Recent studies revealed that LRRK2 phosphorylates members of the Rab GTPase family, including Rab10, under physiological conditions. Although the phosphorylation of endogenous Rab10 by LRRK2 in cultured cells could be detected by mass spectrometry, it has been difficult to detect it by immunoblotting due to the poor sensitivity of currently available phosphorylation-specific antibodies for Rab10. Here, we describe a simple method of detecting the endogenous levels of Rab10 phosphorylation by LRRK2 based on immunoblotting utilizing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) combined with a phosphate-binding tag (P-tag), which is N-(5-(2-aminoethylcarbamoyl)pyridin-2-ylmetyl)-N,N&#39;,N&#39;-tris(pyridin-2-yl-methyl)-1,3-diaminopropan-2-ol. The present protocol not only provides an example of the methodology utilizing the P-tag but also enables the assessment of how mutations as well as inhibitor treatment/administration or any other factors alter the downstream signaling of LRRK2 in cells and tissues.

The present study describes a simple method of detecting endogenous levels of Rab10 phosphorylation by leucine-rich repeat kinase 2.

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of ...

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Medicine

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone.&#160;To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e. 5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder.&#160;Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD.&#160;The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates.&#160;This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.

Lipoxygenase (LOX) isozymes can generate products that may increase or decrease neuroinflammation and neurodegeneration.&#160;A gene-environment interaction study could identify LOX isozyme-specific effects. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of nigrostriatal damage in two LOX isozyme-deficient transgenic lines allows for comparison of the contribution of LOX isozymes on dopaminergic integrity and inflammation.

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

Biology

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2, while LRRK2 is implicated in the pathogenesis of Parkinson's disease3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with 32P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, ...

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human islet&#160;samples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...

Applying the RatWalker System for Gait Analysis in a Genetic Rat Model of Parkinson's Disease

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. Gait abnormalities, including decreased arm swing, slower walking speed, and shorter steps are common in PD patients and appear early in the course of disease. Thus, the quantification of motor patterns in animal models of PD will be important for phenotypic characterization during disease course and upon therapeutic treatment. Most cases of PD are idiopathic; however, the identification of hereditary forms of PD uncovered gene mutations and variants, such as loss-of-function mutations in Pink1 and Parkin, two proteins involved in mitochondrial quality control that could be harnessed to create animal models. While mice are resistant to neurodegeneration upon loss of Pink1 and Parkin (single and combined deletion), in rats, Pink1 but not Parkin deficiency leads to nigral DA neuron loss and motor impairment. Here, we report the utility of FTIR imaging to uncover gait changes in freely walking young (2 months of age) male rats with combined loss of Pink1 and Parkin prior to the development of gross visually apparent motor abnormality as these rats age (observed at 4-6 months), characterized by hindlimb dragging as previously reported in Pink1 knockout (KO) rats.

Here we describe the RatWalker system, built by redesigning the MouseWalker apparatus to accommodate for the increased size and weight of rats. This system uses frustrated total internal reflection (FTIR), high-speed video capture, and open-access analysis software to track and quantify gait parameters.

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars ...

Neuroscience

Analyzing the Parkinson's Disease Mouse Model Induced by Adeno-associated Viral Vectors Encoding Human &#945;-Synuclein

Parkinson&#39;s disease is a neurodegenerative disorder that involves the death of the dopaminergic neurons of the nigrostriatal pathway and, consequently, the progressive loss of control of voluntary movements. This neurodegenerative process is triggered by the deposition of protein aggregates in the brain, which are mainly constituted of &#945;-synuclein. Several studies have indicated that neuroinflammation is required to develop the neurodegeneration associated with Parkinson&#39;s disease. Notably, the neuroinflammatory process involves microglial activation as well as the infiltration of peripheral T cells into the substantia nigra (SN). This work analyzes a mouse model of Parkinson&#39;s disease that recapitulates microglial activation, T-cell infiltration into the SN, the neurodegeneration of nigral dopaminergic neurons, and motor impairment. This mouse model of Parkinson&#39;s disease is induced by the stereotaxic delivery of adeno-associated viral vectors encoding the human wild-type &#945;-synuclein (AAV-h&#945;Syn) into the SN. The correct delivery of viral vectors into the SN was confirmed using control vectors encoding green fluorescent protein (GFP). Afterward, how the dose of AAV-h&#945;Syn administered in the SN affected the extent of h&#945;Syn expression, the loss of nigral dopaminergic neurons, and motor impairment were evaluated. Moreover, the dynamics of h&#945;Syn expression, microglial activation, and T-cell infiltration were determined throughout the time course of disease development. Thus, this study provides critical time points that may be useful for targeting synuclein pathology and neuroinflammation in this preclinical model of Parkinson&#39;s disease.

This work analyzes the vector dose and exposure time required to induce neuroinflammation, neurodegeneration, and motor impairment in this preclinical model of Parkinson&#39;s disease. These vectors encoding the human &#945;-synuclein are delivered into the substantia nigra&#160;to recapitulate the synuclein pathology associated with Parkinson&#39;s disease.

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

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