Restoration of striatal neuroprotective pathways by kinase inhibitor treatment of Parkinson’s linked-LRRK2 mutant mice

Parkinson’s disease-associated, activating mutations in Leucine Rich Repeat Kinase 2 (LRRK2) block primary cilia formation in cholinergic and parvalbumin interneurons and astrocytes in the striatum, decreasing the production of GDNF and NRTN neuroprotective factors that normally support dopaminergic neuron viability.  We show here that 3 month-dietary administration of the MLi-2 LRRK2 kinase inhibitor restores primary cilia and the Hedgehog-responsive production of neuroprotective GDNF and NRTN by these neurons; cilia are also restored on cholinergic neurons of the pedunculopontine nucleus.  Importantly, we detect recovery of striatal dopaminergic processes and decreased stress-triggered Hedgehog signaling by nigral dopaminergic neurons.  Thus, pathogenic LRRK2-driven cilia loss is reversible in post-mitotic neurons and astrocytes, which suggests that early administration of specific LRRK2 inhibitors may have significant therapeutic benefit for patients in the future.

Methods
Reagents

MLi-2 LRRK2 inhibitor was synthesized by Natalia Shpiro (MRC Reagents and Services, University of Dundee) and was first described to be a selective LRRK2 inhibitor in previous work (M.J Fell et al., 2015). For the MLi-2 in diet study, rodent diet containing MLi-2 at 360 mg per Kg was manufactured by Research diets, Inc.

Research standards for animal studies

Mice were maintained under specific pathogen-free conditions at the University of Dundee (UK). All animal experiments were ethically reviewed and conducted in compliance with the Animals (Scientific Procedures) Act 1986 and guidelines established by the University of Dundee and the U.K. Home Office. Ethical approval for animal studies and breeding was obtained from the University of Dundee ethical committee, and all procedures were performed under a U.K. Home Office project license. The mice were group-housed in an environment with controlled ambient temperature (20–24°C) and humidity (45–55%), following a 12-hour light/12-hour dark cycle, with ad libitum access to food and water. LRRK2 R1441C knock-in mice backcrossed on a C57BL/6J background, were obtained from the Jackson laboratory (Stock number: 009346). LRRK2 G2019S knock-in mice backcrossed on a C57BL/6J background, were obtained from Taconic (Model 13940). Genotyping of mice was performed by PCR using genomic DNA isolated from tail clips or ear biopsies with genotyping confirmation conducted on the day of the experiment.

In-diet MLi-2 administration

 R1441C LRRK2 homozygous knock-in mice (8 weeks of age) were allowed to acclimate to the control rodent diet (Research Diets D01060501; Research Diets, Inc., New Brunswick, NJ) for 14 days before being placed on study at 10 weeks of age. On day 1 of the study, one group (9 mice) received a modified rodent diet targeted to provide a concentration of 60 mg/kg per day of MLi-2 on the basis of an average food intake of 5 g/day (D19012904); the other group (9 mice) received an untreated diet and served as the control group. Bodyweight and food intake were assessed twice weekly. On day 91, mice were culled and tissues collected as described below. For the brain analyses, 9 R1441C LRRK2 litter #1 mates were used (6 MLi-2-fed, 3 controls) plus 3 additional age-matched R1441C LRRK2 controls (from litter #2). For immunoblotting analysis, 6 additional R1441C LRRK2 mice from litter #2 were used: 3 control and 3 MLi-2-fed. For all other experiments, littermates were used.

Mouse brain processing

Mouse brains were fixed by transcardial perfusion using 4% paraformaldehyde (PFA) in PBS as described in dx.doi.org/10.17504/protocols.io.bnwimfce. Whole brain tissue was extracted, post-fixed in 4% PFA for 24 hr and then immersed in 30% (w/v) sucrose in PBS until the tissue settled to the bottom of the tube (~48 hr). The brains were harvested in Dundee and sent with identities blinded until analysis was completed. Prior to cryosectioning, brains were embedded in cubed-shaped plastic blocks with OCT (BioTek, USA) and stored at −80 °C. OCT blocks were allowed to reach −20 °C for ease of sectioning. The brains were oriented to cut coronal sections on a cryotome (Leica CM3050S, Germany) at 16–25 µm thickness and positioned onto SuperFrost plus tissue slides (Thermo Fisher, USA).

Mouse tissue processing for immunoblotting analysis

Three mice from each group (control diet and MLi-2 diet) were euthanized by cervical dislocation. Tissues including brain and kidney were collected and rinsed twice with cold PBS containing phosphatase and protease inhibitors (PhosSTOP, Merck #04906837001, and complete EDTA-free Protease Inhibitor Cocktail, Roche #11836170001) before being snap-frozen. Frozen mouse brains were placed on a stainless steel adult mouse brain slicer matrix (EMS #69090-C) kept on dry ice. Using this setup, 1.0-mm coronal sections were prepared. Landmarks in each slice were aligned with those in a reference atlas and the regions were excised using a cold scalpel or biopsy punch. For the immunoblotting shown in Fig. 1, an entire 1-mm coronal section from each sample was taken at approximately -2.3 mm rostrocaudal from bregma. The sections were then stored at -80 °C until further processing for immunoblotting analysis (https://doi.org/10.17504/protocols.io.bsgrnbv6).

Quantitative immunoblotting analysis

Tissue analysis by immunoblot to measure levels of Rab10, phospho-T73 Rab10, Rab12, phospho-S105 Rab12, LRRK2, and phospho-S935 LRRK2 was performed as described in https://doi.org/10.17504/protocols.io.bsgrnbv6. Briefly, snap-frozen tissues were thawed on ice in a tenfold volume excess of ice-cold lysis buffer containing 50 mM Tris–HCl pH 7.4, 1  mM EGTA, 10 mM 2-glycerophosphate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 270 mM sucrose, supplemented with 1 μg/mL microcystin-LR, 1 mM sodium orthovanadate, cOmplete EDTA-free protease inhibitor cocktail (Roche), and 1% (v/v) Triton X-100 and homogenized using a Precellys Evolution system, employing three cycles of 20 s homogenization (6800 rpm) with 30 s intervals. Lysates were centrifuged at 15,000 × g for 30 min at 4°C, and supernatants were collected for subsequent Bradford protein assay and immunoblot analysis. The following primary antibodies were used: mouse anti-total LRRK2 (Neuromab N241A/34), rabbit anti-LRRK2 pS935 (UDD2 10(12), MRC Reagents and Services), rabbit anti-pT73 Rab10 (ab230261, Abcam), mouse anti-total Rab10 (0680–100/Rab10-605B11, Nanotools), rabbit anti-pS106 Rab12 (ab256487, Abcam), rabbit anti-total Rab12 (A26172, ABclonal). Primary antibody probes were detected using IRDye labeled secondary antibodies (IRDye 680LT Donkey anti-Mouse IgG; IRDye 800CW Donkey-anti-Rabbit IgG). Protein bands were acquired via near-infrared fluorescent detection using the Odyssey CLx imaging system and quantified using Image Studio Lite (Version 5.2.5, RRID:SCR_013715).

Immunoblotting of 25-30µm thick, fixed frozen mouse brain cryosections for tyrosine hydroxylase was performed following the protocol of Krebs et al. (28). Slides were brought from -80°C and placed on a cool platform. Sections were visualized under a dissection microscope, and all regions except the dorsolateral striatum were scraped off using a scalpel. Sections with dorsolateral striatum (roughly 3mm2 per hemisphere) were then lysed by adding 2% SDS, 0.05 M DTT, 10% glycerol, 1 mM EDTA, 60 mM Tris-HCl, pH 7.2 (a total volume of 15µl lysis buffer per section), heating at 95°C for 10 minutes, followed by centrifugation at 10,000 x g for 10 minutes at RT. The supernatant was analyzed by immunoblotting using sheep anti-tyrosine hydroxylase (AB1542, Millipore Sigma) and mouse anti-GAPDH (sc-32233, Santa Cruz Biotechnology) primary antibodies and DyLight™ 800 rabbit anti-sheep IgG (H+L) and IRDye 680RD Donkey anti-Mouse IgG secondary antibodies. Blots were imaged using the Odyssey Infrared scanner (LI-COR) and quantified using FIJI (RRID:SCR_002285).

Immunohistochemical staining

The mouse brain striatum was subjected to immunostaining following a previously established protocol (dx.doi.org/10.17504/protocols.io.bnwimfce). Frozen slides were thawed at room temperature for 15 minutes and then gently washed twice with PBS for 5 minutes each. Antigen retrieval was achieved by incubating the slides in 10 mM sodium citrate buffer pH 6.0, preheated to 95°C, for 15 minutes. Sections were permeabilized with 0.1% Triton X-100 in PBS at room temperature for 15 minutes, followed by blocking with 2% FBS and 1% BSA in PBS for 2 hours at room temperature. Primary antibodies were applied overnight at 4°C, and the next day, sections were exposed to secondary antibodies at room temperature for 2 hours. Secondary antibodies used were donkey highly cross-absorbed H + L antibodies conjugated to Alexa 488, Alexa 568, or Alexa 647, diluted at 1:2000. Nuclei were counterstained with 0.1 μg/ml DAPI (Sigma). Finally, stained tissues were mounted with Fluoromount G and covered with a glass coverslip. All antibody dilutions for tissue staining contained 1% DMSO to facilitate antibody penetration.  Automated determination of the density and intensity of dopaminergic processes in the striatum was carried out as described: dx.doi.org/10.17504/protocols.io.x54v92km4l3e/v1.

Fluorescence in situ hybridization (FISH)

RNAscope fluorescence in situ hybridization was carried out as described (https://bio-protocol.org/exchange/protocoldetail?id=1423&type=3). The RNAscope Multiplex Fluorescent Detection Kit v2 (Advanced Cell Diagnostics) was utilized following the manufacturer's instructions, employing RNAscope 3- plex Negative Control Probe (#320871) or Mm-Ptch1-C2 (#402811-C2), Mm-Gdnf (#421951), Mm-Nrtn-C2 (#441501-C2), Mm-Pvalb-C3 (#421931-C3) and Mm-Shh-C2 (#314361-C2). The Mm-Ptch1-C2, Mm-GDNF, Mm-Pvalb-C3, and Mm-Nrtn-C2 probes were diluted 1:5, 1:20, 1:10, and 1:3, respectively in dilution buffer consisting of 6x saline-sodium citrate buffer (SSC), 0.2% lithium dodecylsulfate, and 20% Calbiochem OmniPur Formamide. Fluorescent visualization of hybridized probes was achieved using Opal 690 or Opal 570 (Akoya Biosciences). Subsequently, brain slices were subjected to blocking with 1% BSA and 2% FBS in TBS (Tris buffered saline) with 0.1% Triton X-100 for 30 minutes. They were then exposed to primary antibodies overnight at 4°C in TBS supplemented with 1% BSA and 1% DMSO. Secondary antibody treatment followed, diluted in TBS with 1% BSA and 1% DMSO containing 0.1 μg/ml DAPI (Sigma) for 2 hours at room temperature. Finally, sections were mounted with Fluoromount G and covered with glass coverslips.

Microscope image acquisition

All images were obtained using a Zeiss LSM 900 confocal microscope (Axio Observer Z1/7) coupled with an Axiocam 705 camera and immersion objective (Plan-Apochromat 63x/1.4 Oil DIC M27). The images were acquired using ZEN 3.4 (blue edition) software, and visualizations and analyses were performed using Fiji and CellProfiler.

In addition to above-mentioned methods, all other statistical analysis was carried out using GraphPad Prism version 10.2.3 for Macintosh, GraphPad Software, Boston, Massachusetts USA, www.graphpad.com.