- Open Access
LRRK2 inhibition does not impart protection from α-synuclein pathology and neuron death in non-transgenic mice
© The Author(s). 2019
- Received: 15 February 2019
- Accepted: 17 February 2019
- Published: 26 February 2019
Mutations in leucine-rich repeat kinase 2 (LRRK2) are one of the most common causes of familial Parkinson’s disease (PD). The most common mutations in the LRRK2 gene induce elevated kinase activity of the LRRK2 protein. Recent studies have also suggested that LRRK2 kinase activity may be elevated in idiopathic PD patients, even in the absence of LRRK2 mutations. LRRK2 is therefore a prime candidate for small molecule kinase inhibitor development. However, it is currently unknown how LRRK2 influences the underlying pathogenesis of PD and how LRRK2 might influence extant pathogenesis. To understand whether LRRK2 inhibition would show some benefit in the absence of LRRK2 mutations, we treated a preclinical mouse model of PD with the potent LRRK2 inhibitor MLi-2. The inhibitor was well-tolerated by mice and dramatically reduced LRRK2 kinase activity. However, LRRK2 inhibition did not reverse motor phenotypes, pathological α-synuclein accumulation or neuron loss. The current study suggests that LRRK2 is not necessary for α-synuclein pathogenesis in this mouse model of PD and that further studies are needed to assess the likely clinical benefit of LRRK2 inhibition in idiopathic PD.
- Leucine-rich repeat kinase 2
Parkinson’s disease (PD) is the second most common neurodegenerative disease, after Alzheimer’s disease (AD), and the most common neurodegenerative movement disorder. PD patients are characterized initially by movement difficulties, but as high as 80% of PD patients will go on to develop dementia as well . Neurons in the olfactory bulb and brainstem are affected first, causing the characteristic motor and olfactory deficits, and over time disease spreads to higher cortical areas concurrent with cognitive decline [4–6]. However, the underlying etiology of this degeneration is still not well understood.
While PD is largely sporadic, insight has been gained through investigation of some of the rare familial mutations that lead to PD. Several of the early mutations identified, as well as whole gene duplications and triplications were in the gene encoding the synaptic protein α-synuclein [2, 7, 18, 20, 21, 27–29, 31, 38]. This is the same protein that forms the characteristic pathology Lewy body aggregates seen in PD [15, 32, 33], suggesting that α-synuclein is a key protein in disease pathogenesis. One of the most commonly mutated genes in PD is leucine-rich repeat kinase 2 (LRRK2) . This large protein has scaffolding, GTPase and kinase domains. Interestingly, the most common mutation in LRRK2 in the kinase domain (p.G2019S), and mutations outside of the kinase domain all seem to elevate kinase activity of the protein [16, 30, 34, 37]. This finding would suggest that elevated LRRK2 kinase activity is playing a role in PD pathogenesis, and has led pharmaceutical companies to develop many highly selective and potent inhibitors of LRRK2 activity for the treatment of PD.
More recently, there has been evidence from urinary exosomes  and the brains of idiopathic PD patients , that even in the absence of LRRK2 mutations, LRRK2 kinase activity may be elevated. If LRRK2 kinase activity is indeed elevated in idiopathic PD patients, it would suggest that LRRK2 is driving some aspect of PD pathogenesis and that LRRK2 inhibitors may be efficacious even in patients that do not carry mutations. Indeed, LRRK2 inhibitor administration in a rat neurotoxin model of degeneration prevented accumulation of pathological α-synuclein . A separate study showed that antisense oligonucleotides (ASOs), which reduce the levels of LRRK2, were also able to reduce the amount of pathological α-synuclein in the vulnerable substantia nigra of mice inoculated with pathological α-synuclein . If inhibiting LRRK2 kinase activity or reducing total LRRK2 levels are efficient at reducing α-synuclein pathology, this may be a viable therapeutic avenue for all patients with PD and even other synucleinopathies.
We have recently developed a mouse model that exhibits α-synuclein pathology throughout the brain and vulnerable neuron death without the overexpression of α-synuclein . Treatment of these mice with the potent LRRK2 inhibitor MLi-2 has allowed us to directly assess the tolerability of LRRK2 inhibition, the extent of LRRK2 kinase inhibition, motor behavior, α-synuclein pathology and neuron death. We report here that MLi-2 is well-tolerated in mice and shows effective inhibition of LRRK2 kinase activity both peripherally and in the central nervous system. However, mice treated with the inhibitor showed no improvement in motor performance, similar development of α-synuclein pathology and similar levels of dopaminergic neuron death compared to control animals. We find that LRRK2 is not essential to α-synuclein pathogenesis in PD and suggest that further studies are necessary to determine whether LRRK2 inhibition will be a viable therapeutic for idiopathic PD.
All housing, breeding, and procedures were performed according to the NIH Guide for the Care and Use of Experimental Animals and approved by the University of Pennsylvania Institutional Animal Care and Use Committee. All mice used in this study were C57BL/6J (JAX 000664, RRID: IMSR_JAX:000664).
Mouse all-limb grip strength was measured using the animal grip strength test (IITC 2200). For this test a rod is attached to a digital force transducer. Mice are moved to a quiet behavioral testing suite and allowed to acclimate for 1 h. Each mouse is held by the base of the tail and allowed to grasp the rod. Once the mouse clasps the rod, the mouse is slowly moved backwards, in line with the force transducer until the mouse releases the rod. The maximum grip force is recorded. The mouse is allowed to rest for several seconds, and then placed on the rod again. The maximum grip strength of 5 tests was recorded. No fatigue was observed during the test period, so the average of all 5 measures is reported.
An accelerating rotarod (MED-Associates) was used to assess motor coordination. Mice received two training sessions and two tests sessions. During the training sessions, mice were placed on a still rod. The rod then began to accelerate from 4 rotations per minute (rpm) to 40 rpm over 5 min. Mice were allowed to rest at least 1 h between training and testing sessions. During the testing sessions, mice were treated as before, and the latency to fall was recorded. The trial was also concluded if a mouse gripped the rod and rotated with it instead of walking. Mice were allowed a maximum of 10 min on the rod.
Mice were assigned to control (n = 7) or MLi-2 (n = 8) groups to equally match sex. Administration of MLi-2 or control diet began 3 days prior to α-synuclein PFF injection. Control diet (Research Diets D01060501) was the same as the MLi-2 diet (Research Diets D17031301) except for the compound and a dye to allow visual discrimination between the two diets. MLi-2 was incorporated at 240 mg/kg diet to achieve approximately 30 mg/kg/day dosing based on approximately 3–4 g diet consumption/day in mice that were approximately 25 g and stayed the same weight for the course of the study. This dose was selected based on previous work with this molecule . Mice were weighed once a week and diet was weighed and replenished every 2–3 days to allow assessment of estimated dosage.
α-synuclein PFF PD mouse model
Purification of recombinant α-synuclein and generation of α-synuclein PFFs was conducted as described elsewhere [24, 35, 36]. All surgery experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Pennsylvania. Mouse α-synuclein PFFs, which were generated at a concentration of 5 mg/mL were vortexed and diluted with Dulbecco’s phosphate-buffered saline (DPBS) to 2 mg/mL. They were then sonicated on high for 10 cycles of 30 s on, 30 s off (Diagenode Biorupter UCD-300 bath sonicator).
Mice were injected when 3–4 months old. Mice were injected unilaterally by insertion of a single needle into the right forebrain (coordinates: + 0.2 mm relative to Bregma, + 2.0 mm from midline) targeting the dorsal striatum (2.6 mm beneath the dura) with 5 μg α-synuclein PFFs (2.5 μL). Injections were performed using a 10 μL syringe (Hamilton, NV) at a rate of 0.4 μL/minute. After 3 months, mice were perfused transcardially with PBS, brains were removed and underwent overnight fixation in 70% ethanol in 150 mM NaCl, pH 7.4. Kidney and lung were removed and underwent overnight fixation in 10% neutral buffered formalin. Spinal cord and liver were snap frozen and stored at − 80 °C for biochemistry.
After perfusion and fixation, brains were embedded in paraffin blocks, cut into 6 μm sections and mounted on glass slides. Slides were then stained using standard immunohistochemistry as described below. Slides were de-paraffinized with 2 sequential 5-min washes in xylenes, followed by 1-min washes in a descending series of ethanols: 100, 100, 95, 80, 70%. Slides were then incubated in deionized water for 1 min prior to antigen retrieval as noted. After antigen retrieval, slides were incubated in 5% hydrogen peroxide in methanol to quench endogenous peroxidase activity. Slides were washed for 10 min in running tap water, 5 min in 0.1 M tris, then blocked in 0.1 M tris/2% fetal bovine serum (FBS). Slides were incubated in primary antibodies overnight. The following primary antibodies were used. For pathologically-phosphorylated α-synuclein, pS129 α-synuclein (EP1536Y; Abcam ab51253, RRID:AB_869973) was used at 1:20,000 with microwave antigen retrieval with citric acid based antigen unmasking solution (Vector H-3300, RRID:AB_2336226). To stain midbrain dopaminergic neurons, Tyrosine hydroxylase (TH-16; Sigma-Aldrich T2928, RRID:AB_477569) was used at 1:5000 with formic acid antigen retrieval. To stain pneumocytes, anti-prosurfactant protein C (proSP-C, Millipore Sigma AB3786, RRID:AB_91588) was used at 1:4000 with microwave antigen retrieval as described above.
Primary antibody was rinsed off with 0.1 M tris for 5 min, then incubated with goat anti-rabbit (Vector BA1000, RRID:AB_2313606) or horse anti-mouse (Vector BA2000, RRID:AB_2313581) biotinylated IgG in 0.1 M tris/2% FBS 1:1000 for 1 h. Biotinylated antibody was rinsed off with 0.1 M tris for 5 min, then incubated with avidin-biotin solution (Vector PK-6100, RRID:AB_2336819) for 1 h. Slides were then rinsed for 5 min with 0.1 M tris, then developed with ImmPACT DAB peroxidase substrate (Vector SK-4105, RRID:AB_2336520) and counterstained briefly with Harris Hematoxylin (Fisher 67–650-01). Slides were washed in running tap water for 5 min, dehydrated in ascending ethanol for 1 min each: 70, 80, 95, 100, 100%, then washed twice in xylenes for 5 min and coversliped in Cytoseal Mounting Media (Fisher 23–244-256).
Hematoxylin and eosin staining was performed on kidney and lung sections using a standard protocol. Briefly, sections were immersed in Harris Hematoxylin (Fisher 67–650-01) for 1 min, rinsed 2 times in distilled water, differentiated in 0.1% acid alcohol solution for 4 s and rinsed in tap water for 15 min. Sections were then immersed in eosin for 1 min, briefly rinsed in tap water, then dehydrated and mounted with Cytoseal Mounting Media (Fisher 23–244-256).
Slides were scanned into digital format on a Lamina scanner (Perkin Elmer) at 20× magnification. Digitized slides were then used for quantitative pathology.
All section selection, annotation and quantification was done blinded to genotype. All quantitation was performed in HALO quantitative pathology software (Indica Labs). Every 10th slide through the midbrain was stained with tyrosine hydroxylase (TH). TH-stained sections were used to annotate the substantia nigra, and cell counting was performed manually in a blinded manner for all sections. The sum of all sections was multiplied by 10 to estimate the total count that would be obtained by counting every section. The substantia nigra annotations drawn onto the TH-stained sections were then transferred to sequential sections that had been stained for pS129 α-synuclein. A single analysis algorithm was then applied equally to all stained sections to quantify the percentage of area occupied by pS129 α-synuclein staining. Specifically, the analysis included all DAB signal that was above a 0.157 optical density threshold, which was empirically determined to not include any background signal. This signal was then normalized to the total tissue area. A minimal tissue optical density of 0.02 was used to exclude any areas where tissue was split. Four other regions were selected for α-synuclein pathology quantification based on their susceptibility to pathology changes in mice expressing G2019S LRRK2 (data not shown). For these regions, only two sections flanking − 2.92 mm relative to Bregma were selected for each brain. The noted regions were annotated on these sections and quantified using the same algorithm as noted above.
Spinal cords and livers were thawed on ice and suspended in 5× volumes/weight of tissue RIPA buffer (50 mM Tris, pH 7.6, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 10 mM NaF) with proteinase and phosphatase inhibitors and 1 mM PMSF. Total protein concentration in each sample was determined by a bicinchoninic acid colorimetric assay (Fisher 23,223 and 23,224), using bovine serum albumin as a standard (Thermo Fisher 23,210). 180 μg of total liver or spinal cord protein was resolved on 5–20% gradient acrylamide gels. Western Blot analysis was performed with primary antibodies targeting LRRK2 (ab133474, Abcam, RRID: AB_2713963, 1:500), pS935 LRRK2 (ab133450, Abcam, RRID:AB_2732035, 1:400) or GAPDH (2-RGM2, Advanced Immunological, RRID:AB_2721282, 1:5000). Primary antibodies were detected using IRDye 800 (Li-cor 925–32,210) or IRDye 680 (Li-cor 925–68,071) secondary antibodies, scanned on Li-cor Odyssey Imaging System and analyzed using Image Studio software. LRRK2 and pS935 LRRK2 values were normalized to GAPDH as an internal loading control, then further normalized to the mean of all control samples.
All statistical analyses were done in GraphPad Prism 7. The analysis used for each data set is described in the figure legends.
MLi-2 is well tolerated and allows for relatively steady dosing in mice
We have recently developed a mouse model of PD that does not rely on the overexpression of α-synuclein or neurotoxins, but rather injection of a small amount of pathogenic α-synuclein seeds in the dorsal striatum of wildtype mice . These mice develop pathological pS129 α-synuclein inclusions that spread throughout the mouse brain to anatomically-interconnected regions, including the vulnerable substantia nigra. Pathology increases over time for 3 months, after which time the dopaminergic neurons of the nigra degenerate and pathology is reduced proportionally . The pathological spread and neuron death in these mice recapitulate important features of PD, and allows the assay of LRRK2 inhibition in a wildtype context. For our studies, we chose to use the potent and specific LRRK2 inhibitor MLi-2 . This inhibitor has a reported IC50 of 0.76 nM and greater than 295-fold selectivity for over 300 kinases . In addition to these properties, MLi-2 is also orally bioavailable, brain penetrant and is stable in mice over prolonged periods of time.
Mice tolerated the diet well and showed no difference in weight compared to control diet animals (Fig. 1b). Diet was weighed every 2–3 days. Combined with mouse weights, diet weight was used to estimate dosage of MLi-2 during the study (Fig. 1c). After initial overconsumption following surgery, mice maintained steady diet consumption for the duration of the study, with an estimated mean dose of 26.5 mg/kg/day.
MLi-2 inhibits LRRK2 kinase activity in the periphery and central nervous system
LRRK2 inhibition leads to mild pneumocyte enlargement
LRRK2 inhibition does not attenuate motor deficits following α-synuclein PFF injection
LRRK2 inhibition does not protect neurons from α-synuclein pathology accumulation or neuron death
Human neuropathology studies suggest that α-synuclein pathology spreads through the brain and that the spread of α-synuclein pathology to higher cortical regions correlates with the progression of cognitive decline seen in many PD patients [4–6, 19]. However, the neurobiology underlying the cell-to-cell spread of α-synuclein pathology is unknown. The predisposition of patients with mutations in LRRK2 to get PD with commensurate α-synuclein pathology suggests that LRRK2 may regulate some aspect of PD pathogenesis. Recent cell biology studies have shown that LRRK2 may phosphorylate a subset of Rab proteins  and thereby regulate vesicular trafficking within the cell [10, 14, 22, 25, 26]. A role in vesicle trafficking would place LRRK2 in an ideal position to regulate cell-to-cell transmission of pathogenic α-synuclein. Further, recent studies have suggested that not only is mutated LRRK2 overactive, LRRK2 in idiopathic PD patients may also be overactive [8, 12]. Together, the putative role of LRRK2 in cellular trafficking events and the hyperactivity of LRRK2 in PD make LRRK2 an ideal candidate for kinase inhibitor development.
Work over the last 10 years has yielded several highly potent, selective and brain-penetrant LRRK2 inhibitors. One of the most potent and selective of these, MLi-2, is also bioavailable in mice and has allowed us to directly assay whether LRRK2 inhibition in vivo can modulate the formation or toxicity of α-synuclein inclusions. In this study we were able to demonstrate that MLi-2 is safe and well-tolerated by mice over a 3-month treatment period. This molecule is also able to dramatically reduce LRRK2 kinase activity. Despite this potency, LRRK2 inhibition showed no effect on the development of α-synuclein pathology in the substantia nigra, or other unrelated regions. Further, LRRK2 inhibition was unable to rescue the progressive death of dopaminergic neurons in the substantia nigra.
This is, to our knowledge, the first study to investigate whether LRRK2 inhibition is able to reverse pathogenic α-synuclein-initiated pathology and neuron death in wildtype animals. Two other reports have suggested that LRRK2 inhibition or knockdown may be efficacious in preventing PD phenotypes. In the first study, α-synuclein PFFs were used to induce α-synuclein pathology in the substantia nigra of mice and LRRK2 ASOs were administered directly into the brain ventricles of mice . LRRK2 ASOs substantially reduced the number of pS129 α-synuclein inclusions and induced mild rescue of TH positive neurons in the substantia nigra. ASOs targeting LRRK2 reduced total protein levels, so it is possible that domains other than the kinase domain of LRRK2 are playing a role in α-synuclein pathogenesis that is not recapitulated by kinase inhibition. In the second study, acute administration of rotenone in rats induced both LRRK2 activation and pS129 α-synuclein accumulation in the substantia nigra . LRRK2 inhibitor PF-360 was able to partially reverse this α-synuclein accumulation, but this model does not exhibit degeneration, so neuron loss was not assayed. It is also not clear if the model of α-synucleinopathy used in our study induces LRRK2 hyperactivation as was observed with rotenone treatment. It will be interesting to see in future studies whether LRRK2 hyperactivation is directly associated with α-synuclein pathogenesis or whether it is generally triggered by degeneration or another concomitant cellular pathway. In neither of these studies was there a robust rescue of dopaminergic neurons, suggesting that there is a LRRK2-independent α-synuclein pathogenesis pathway that eventually leads to neuron loss even when total pathology is reduced.
There is increasing evidence that LRRK2 hyperactivation is present in both familial and sporadic PD, suggesting that LRRK2 plays a role in PD pathogenesis. If so, LRRK2 inhibitors may be therapeutic in idiopathic PD. In a mouse model of idiopathic PD, we found that LRRK2 kinase inhibitor MLi-2 was well-tolerated and showed robust target engagement. However, LRRK2 kinase inhibition in this model did not prevent motor dysfunction, α-synuclein pathology or neuron death. Future work will be important to understand whether LRRK2 inhibition is likely to be therapeutic in idiopathic PD.
We would like to thank members of our laboratory for critical discussions related to this paper.
This study was supported by the Michael J. Fox Foundation and National Institute of Health grants: T32-AG000255, P30-AG10124, P50-NS053488.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Conception and design: MXH, JQT, VMYL; Acquisition and analysis of data: MXH, MS, IM, BZ, MFO, HB; Drafting of the article: MXH, VMYL. All authors read and approved the final manuscript.
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