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

Jaimon, Ebsy1 ; Lin, Yu-En1 ; Tonelli, Francesca2 ; Antico, Odetta2 ; Alessi, Dario2 ; Pfeffer, Suzanne1

Research facility: Stanford University School of Medicine

Published Aug 08, 2024; Updated Nov 18, 2024 on Dryad. https://doi.org/10.5061/dryad.q2bvq83tn

Abstract

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.

README

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

https://doi.org/10.5061/dryad.q2bvq83tn

Author/Principal Investigator Information

Name: Suzanne R. Pfeffer

ORCID: 0000-0002-6462-984X

Institution: Stanford University

Address: Beckman

Center Room B413

279 Campus DriveStanford, California 94305-5307

Email: pfeffer@stanford.edu

Author/Principal Investigator Information

Name: Dario R. Alessi

ORCID: 0000-0002-2140-9185

Institution: MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, United Kingdom

Address: MRC PPU

Sir James Black Centre

School of Life Sciences

University of Dundee

Dow St.

Dundee DD1 5EH

Email: d.r.alessi@dundee.ac.uk

Author/Associate or Co-investigator Information

Name: Ebsy Jaimon

ORCID: 0000-0001-6845-2095

Institution: Stanford University

Address: Beckman Center Room B413

279 Campus Drive

Stanford, California 94305-5307

Email: ebsy@stanford.edu

Author/Associate or Co-investigator Information

Name: Yu-En Lin

ORCID: 0000-0002-5848-5405

Institution: Stanford University

Address: Beckman Center Room B413

279 Campus Drive

Stanford, California 94305-5307

Email: yuenlin@stanford.edu

Author/Associate or Co-investigator Information

Name: Francesca Tonelli

ORCID: 0000-0002-4600-6630

Institution: MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, United Kingdom

Address: MRC PPU

Sir James Black Centre

School of Life Sciences

University of Dundee

Dow St.

Dundee DD1 5EH

Email: f.tonelli@dundee.ac.uk

Author/Associate or Co-investigator Information

Name: Odetta Antico

ORCID: 0000-0002-7325-3303

Institution: MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, United Kingdom

Address: MRC PPU

Sir James Black Centre

School of Life Sciences

University of Dundee

Dow St.

Dundee DD1 5EH

Email: o.antico@dundee.ac.uk

Date of data collection: 2023-06-11

Geographic location of data collection: Stanford, CA and University of Dundee, Scotland

Description of the data and file structure

File list:

Fig 1A zip file:

Raw immunoblotting data (.tiff files) and annotated images (.tiff files) for Figure 1A. Littermate- or age-matched LRRK2 R1441C homozygous knock-in mice were fed either a control diet or MLi-2-containing diet for 90 days prior to tissue collection. On the last day of the study, three mice from each group were euthanized by cervical dislocation and tissues (brain and kidneys) collected from these mice were used to monitor inhibition of LRRK2 activity by immunoblotting. 12.5 μg tissue extract was subjected to quantitative immunoblotting analysis with the indicated antibodies and data analyzed using the LI-COR Odyssey CLx imaging and Image Studio software. Each lane represents a tissue sample from a different animal.

Fig 1BCD zip file:

Images of striatal cholinergic interneurons from R1441C LRRK2 homozygous knock-in mice (8 weeks old) fed with MLi-2 inhibitor containing chow or control chow for 3 months. Brains 1, 2, 3, 10, 11, and 12 are from R1441C LRRK2 homozygous knock-in mice fed with control chow and Brains 4, 5, 6, 7, 8, and 9 are from R1441C LRRK2 homozygous knock-in mice fed with MLi-2 inhibitor containing chow.

Fig 1EFG zip file:

Images of striatal astrocytes from R1441C LRRK2 homozygous knock-in mice (8 weeks old) fed with MLi-2 inhibitor containing chow or control chow for 3 months. Brains 1, 2, 3, 10, 11, and 12 are from R1441C LRRK2 homozygous knock-in mice fed with control chow and Brains 4, 5, 6, 7, 8, and 9 are from R1441C LRRK2 homozygous knock-in mice fed with MLi-2 inhibitor containing chow. Note that images of Brains 2 and 9 are split into two files.

Fig 2ABC zip file:

Images of PPN cholinergic neurons from 4-month-old R1441C LRRK2 homozygous knock-in mice. Brains 1, 2, 5, and 6 are WT, and Brains 3, 4, 7, and 8 are R1441C LRRK2 homozygous knock-in. Note that images of Brains 6 are split into two files.

Fig 2DEF zip file:

Images of PPN cholinergic neurons from R1441C LRRK2 homozygous knock-in mice (8 weeks old) fed with MLi-2 inhibitor containing chow or control chow for 3 months. Brains 1, 2, 3, 10, 11, and 12 are from R1441C LRRK2 homozygous knock-in mice fed with control chow and Brains 4, 5, 6, 7, 8, and 9 are from R1441C LRRK2 homozygous knock-in mice fed with MLi-2 inhibitor containing chow. Note that images of Brains 10, 11 and 12 are split into two files.

Fig 3ABC zip file:

Images of striatal cholinergic interneurons from 5-month-old WT and G2019S homozygous knock-in mice. Brains 1,4,7,8 are WT and Brains 2,3,5,6 are G2019S LRRK2 homozygous knock-in. Note that images of all brains are split into two files.

Fig 3DEF zip file:

Fig 4ABC zip file:

Images of striatal astrocytes from 5-month-old WT and G2019S homozygous knock-in mice. Brains 1,4,7,8 are WT and Brains 2,3,5,6 are G2019S LRRK2 homozygous knock-in.

Fig 4DEF zip file:

Fig 5ABC zip file:

Fig 6ABCDE zip file:

Images of striatal parvalbumin interneurons from R1441C LRRK2 homozygous knock-in mice (8 weeks old) fed with MLi-2 inhibitor containing chow or control chow for 3 months. Brains 1, 2, 3, 10, 11, and 12 are from R1441C LRRK2 homozygous knock-in mice fed with control chow and Brains 4, 5, 6, 7, 8, and 9 are from R1441C LRRK2 homozygous knock-in mice fed with MLi-2 inhibitor containing chow.

Fig 7ABCD zip file:

Images from 5-month-old WT and R1441C LRRK2 homozygous knock-in mice. Brains 6,7,8, and 14 are from WT animals, and brains 5,9,12, and 13 are from R1441C LRRK2 homozygous knock-in animals. Note that images of Brain 8 are split into two files.

Fig 7EFGH zip file:

Images from R1441C LRRK2 homozygous knock-in mice (8 weeks old) fed with MLi-2 inhibitor containing chow or control chow for 3 months. Brains 1, 2, 3, 10, 11, and 12 are from R1441C LRRK2 homozygous knock-in mice fed with control chow and Brains 4, 5, 6, 7, 8, and 9 are from R1441C LRRK2 homozygous knock-in mice fed with MLi-2 inhibitor containing chow.

Fig 8AB zip file:

Images of nigral dopaminergic neurons from 10-month-old WT and R1441C LRRK2 homozygous knock-in mice. Brains 7, 12, 22, 25, 34 are WT and Brains 9, 23, 33, 36, 38 are R1441C LRRK2 homozygous knock-in.

Fig 8CD zip file:

Images of nigral dopaminergic neurons from R1441C LRRK2 homozygous knock-in mice (8 weeks old) fed with MLi-2 inhibitor containing chow or control chow for 3 months. Brains 1, 2, 3, 10, 11, and 12 are from R1441C LRRK2 homozygous knock-in mice fed with control chow and Brains 4, 5, 6, 7, 8, and 9 are from R1441C LRRK2 homozygous knock-in mice fed with MLi-2 inhibitor containing chow.

Raw figure files zip file:

Raw image files used to generate all main figures except Fig 1A.

Raw Fig S1 zip file:

Raw image files used to generate figures in Fig S1. Example images of cilia length.

Raw Fig S2 zip file:

Raw image files used to generate figures in Fig S2. Images of control RNAScope reactions carried out for cholinergic interneurons, astrocytes, parvalbumin interneurons, and nigral dopaminergic neurons.

Raw Fig S3 zip file:

Raw image files used to generate figures in Fig S3. Example images of ciliated and unciliated striatal parvalbumin interneurons.

Tables and graphs zip file:

-Tabular data and graphs in Prism that were used to generate graphs shown in all Figures. Note that the cilia length is in µm, and the area measurements by CellProfiler software in Figure 7 are in pixels.

-Tabular data in .csv format that was used to generate graphs shown in all Figures. Note that the cilia length is in µm and the area measurements by CellProfiler software in Figure 7 are in pixels.

Fig 7BCDFGH pipeline zip file:

CellProfiler software pipeline used for quantification in Figure 7BCDFGH.

Raw values.zip:

Raw tabular data in.csv format from quantitation in all figures. The percentage of ciliation and the number of RNA dots were scored manually. Cilia length was measured using FIJI. Automated determination of density and intensity of dopaminergic processes in the striatum were done using CellProfiler. Note that the cilia length is in µm and the area measurements by CellProfiler software in Figure 7 are in pixels.

Key resource table file:

.csv format Key resource table describing the resources used in the study.

Sharing/Access information

Licenses/restrictions placed on the data: None Currently Applicable

Links to publications that cite or use the data: None Currently Applicable

Links to other publicly accessible locations of the data: None Currently Applicable

Links/relationships to ancillary data sets: None Currently Applicable

Was data derived from another source? No

Version change log

1. Figure 7ABCD was newly added.

2. Figure 7ABCD in the previous version is now renamed Figure 7EFGH to reflect their new order.

3. Updated Raw figure files zip file to incorporate updated images of Figure 2D and newly added images from Fig 7A and Fig 7E (7A in the previous version).

4. Added new Raw Fig S1 zip file to incorporate newly added images in Fig S1.

5. Added Raw Fig S2 zip file to incorporate newly added images in Fig S2.

6. Added Raw Fig S3 zip file to incorporate newly added images in Fig S3.

7. Tables and graphs zip file updated to incorporate newly added data from Fig 7BCD, Fig S1, and Fig S4. Renamed data from Fig 7BCD in the previous version to Fig 7FGH to reflect their new order. All the files have now been updated in a tidy format.

8. Renamed Figure 7BCD pipeline zip file to Figure 7BCDFGH pipeline zip file to indicate that the same pipeline is used in all the analyses of Figure 7.

9. Added raw values from newly added Fig 7B-D and renamed previous Fig 7B-D as Fig 7F-H to reflect their new order in the Raw values zip file. All the files have now been updated in a tidy format.

10. Updated Key resource table to add RRID for total Rab12 antibody.

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

Littermate or age-matched 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.

Mouse brain processing

Homozygous LRRK2-mutant (R1441C or G2019S of ages indicated) and age-matched wild type controls 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).

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.

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

Data files

Abstract

README

Methods

Works referencing this dataset