LRRK2 activity does not dramatically alter α-synuclein pathology in primary neurons

LRRK2 activity does not dramatically alter α-synuclein pathology in primary neurons Mutations in leucine-rich repeat kinase (LRRK2) are the most common cause of heritable Parkinson’s disease (PD), and the most common mutations in LRRK2 lead to elevated kinase activity. For these reasons, inhibitors targeting LRRK2 have been the subject of intense research and development. However, it has been difficult to develop preclinical models that recapitulate PD-relevant LRRK2 phenotypes. The primary pathology in PD is the Lewy body (LB), which is a cytoplasmic aggregate of α-synuclein. The recent demonstration that LB-like aggregates of α-synuclein can be induced in primary neurons has provided a robust model for testing genetic modifiers of PD-relevant aggregation and neurodegeneration. In this study, we test the modulation of α-synuclein pathology by LRRK2 in primary neuron cultures using biochemistry and immunocytochemistry. We find that expression of familial mutant G2019S LRRK2 does not dramatically elevate the pathological burden of α-synuclein or neurodegeneration in neurons. We further test three LRRK2 inhibitors in two strains of wildtype neurons and find that even robust LRRK2 inhibition is insufficient to reduce α-synuclein pathology. LRRK2 inhibitors similarly had no effect in neurons with α-synuclein pathology seeded by human brain-derived pathological α-synuclein. Finally, we find that this lack of pathological modulation by LRRK2 was not confined to hippocampal neurons, but was also absent in midbrain dopaminergic neuron cultures. These data demonstrate that LRRK2 activity does not have more than minor effects on α-synuclein pathology in primary neurons, and more complex models may be needed to evaluate the ability of LRRK2 inhibitors to treat PD. Keywords: LRRK2, Synuclein, pS129, Aggregates, Inhibitor, G2019S Introduction cytoskeletal modeling [17]. LRRK2 mutations are found in Parkinson’s disease (PD) is the most common neurodegen- 4% of hereditary and 1% of sporadic PD patients, and pa- erative movement disorder. Patients with this disease experi- tients with LRRK2 mutations phenocopy sporadic PD pa- ence rigidity, resting tremors, and slowness of movement; tients, with similar onset of disease, α-synuclein 80% of patients will develop dementia throughout the dis- pathology, and responsiveness to dopamine replacement ease course [15]. The clinical diagnosisofPDisconfirmed [11]. The most common mutation (G2019S) in the kinase post-mortem by the presence of intracytoplasmic inclusions domain as well as mutations in the GTPase domain of termed Lewy bodies (LBs), which consist primarily of the LRRK2, elevate kinase activity of LRRK2 [8, 26, 31, 36], synaptic protein α-synuclein [1, 29, 30]. While α-synuclein making LRRK2 an ideal candidate for small molecule in- is thoughttobepathogenicinthislargely sporadic disease, hibitor development. A large number of LRRK2 inhibitors mutations in several genes can increase the lifetime risk of have been developed and refined for increased potency developing disease. The most commonly mutated gene in and low off-target effects over the past decade. However, hereditary PD is leucine-rich repeat kinase 2 (LRRK2, [11]). the lack of a reliable preclinical disease model for LRRK2 LRRK2 is a widely expressed protein with unclear dysfunction has been a major challenge for validating the functions. It has kinase, GTPase and scaffolding domains therapeutic potential of LRRK2 inhibitors. and has been implicated in intracellular trafficking and To understand the relationship between LRRK2 and α-synuclein and develop a model of LRRK2-dependent * Correspondence: vmylee@upenn.edu PD phenotypes, early studies crossed mice expressing Department of Pathology and Laboratory Medicine, Institute on Aging and wildtype or G2019S LRRK2 mice with mice overexpress- Center for Neurodegenerative Disease Research, University of Pennsylvania School of Medicine, 3600 Spruce St, 3rd Floor Maloney, Philadelphia, PA ing α-synuclein with the familial A53T mutation. These 19104-4283, USA © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Henderson et al. Acta Neuropathologica Communications (2018) 6:45 Page 2 of 11 studies all found little or no effect of LRRK2 expression [21, 33, 34]. For treatment of neurons, α-synuclein PFFs, on α-synuclein pathology [4, 14, 20]. However, later stud- which were generated at a concentration of 5 mg/mL were ies which injected adeno-associated virus into the substan- vortexed and diluted with Dulbecco’s phosphate-buffered tia nigra of LRRK2 knockout or G2019S LRRK2 rats found saline (DPBS) to 100 μg/mL. They were then sonicated on that LRRK2 depletion rescued α-synuclein-induced neuro- high for 10 cycles of 30 s on, 30 s off (Diagenode Biorupter degeneration [5] and G2019S LRRK2 exacerbated degener- UCD-300 bath sonicator). α-Synuclein PFFs were then di- ation [3]. Several cell culture studies have found that luted in neuron media to 5 μg/mL and added to neuron LRRK2 may directly interact with α-synuclein [9]orindir- cultures at a concentration of 1 μg/mL for immunocyto- ectly influence α-synuclein expression [23, 28], however chemistry experiments and 2.5 μg/mL for biochemistry none of these cell culture models recapitulate the phos- experiments. phorylated, detergent-insoluble α-synuclein seen in PD patients. The recent finding that α-synuclein pathology can Primary hippocampal cultures be induced in wildtype primary neurons by introduction of Primary hippocampal neuron cultures were prepared small amounts of recombinant α-synuclein pre-formed from postnatal day (P) 1 NTG and G2019S mice and fibrils (PFFs) has allowed the study of α-synuclein path- embryonic day (E) 16–18 CD1 embryos. Dissociated hip- ology in neurons expressing wildtype or mutated LRRK2 pocampal neurons were plated at 17,000 cells/well [32]. A primary neuron model in which LRRK2 activity (96-well plate) or 1,000,000 cells/well (6-well plate) in modulates α-synuclein pathology would be ideal for screen- neuron media (Neurobasal medium (ThermoFisher ing LRRK2 inhibitors and providing biological rationale for 21,103,049) (or Neurobasal A medium (Thermo Fisher moving toward clinical intervention. We used robust im- 10,888–022) for postnatal cultures) supplemented with munocytochemical and biochemical assays for α-synuclein B27 (ThermoFisher 17,504,044), 2 mM GlutaMax pathology in primary hippocampal and midbrain dopamin- (ThermoFisher 35,050,061), and 100 U/mL penicillin/ ergic neurons derived from two strains of wildtype mice streptomycin (ThermoFisher 15,140,122). LRRK2 inhibi- and mice expressing the LRRK2 with the familial G2019S tors PF-475 and PF-360 were synthesized at Pfizer, Inc. mutation to test LRRK2 activity-dependent phenotypes. MLi-2 was obtained from Tocris Bioscience (5756). All We induce α-synuclein pathology with addition of both LRRK2 inhibitors were reconstituted at 10 mM in DMSO recombinant and human-derived fibrillar α-synuclein. In andstoredat − 20 °C. There were further diluted to the order to manipulate LRRK2 activity, we used three final concentration indicated in neuron media with DMSO validated LRRK2 inhibitors. In no system and by no meas- as a vehicle control. Neurons were either fixed with 4% ure did we observe more than very mild LRRK2-dependent paraformaldehyde, 4% sucrose in phosphate-buffered saline changes in α-synuclein pathology. for immunocytochemistry or scraped for biochemistry. Materials and methods Primary midbrain/striatum co-cultures Animals The ventral mesencephalon and striatum were dissected All housing, breeding, and procedures were performed from P2 NTG and G2019S mice in Hibernate A medium according to the NIH Guide for the Care and Use of (ThermoFisher A1247501) with B27 (ThermoFisher Experimental Animals and approved by the University of 17,504,044) and 0.5 mM GlutaMax (ThermoFisher Pennsylvania Institutional Animal Care and Use 35,050,061). The tissue was then digested in papain Committee. C57BL/6 J (NTG, JAX 000664, RRID: (Worthington Biochemical LS003126) for ~ 20 min at IMSR_JAX:000664) and B6.Cg-Tg(Lrrk2*G2019S)2Yue/J 37 °C. Cells were mixed at a ratio of 1:1 and plated at a (G2019S, JAX 012467, RRID: IMSR_JAX:012467) mice density of 34,000 cells/well in 96-well black-walled plates were obtained from Andrew West (University of Alabama, (Perkin Elmer 50–905-1605) in Neurobasal A medium Birmingham). The G2019S mice have multiple inserts of (Thermo Fisher 10,888–022) supplemented with B27 the gene, but were heterozygous at these loci. and 0.4 mM GlutaMax, 50 ng/mL BDNF (PeproTech Subsequently, mice were bred to homozygosity at loci as 450–02) and 30 ng/mL GDNF (Millipore Sigma GF030). determined by quantitative PCR and outbreeding. The expression level of G2019S LRRK2 was thereby stabilized Neuron sequential detergent fractionation in this line of mice. All experiments shown use homozy- Proteins from primary neuronal cultures treated with PBS gous G2019S mice. CD1 (Strain 022, RRID: IMSR_CRL:22) or α-synuclein PFFs were sequentially extracted as de- mice were obtained from Charles River, Wilmington, MA. scribed previously [21, 33, 34]. Briefly, neurons were scraped into 1 volume 1% TX-100 buffer, sonicated and α-Synuclein PFFs spun at 100,000 x g for 30 min. The pellet was sonicated Purification of recombinant α-synuclein and generation of and again spun at 100,000 x g for 30 min in 1 volume 1% α-synuclein PFFs was conducted as described elsewhere TX-100 solution to remove remaining TX-100-soluble Henderson et al. Acta Neuropathologica Communications (2018) 6:45 Page 3 of 11 proteins. This pellet was suspended in 0.5 volumes 2% Immunocyctochemistry SDS solution, sonicated and spun once more at 100,000 x Immunostaining of neuronal cultures was carried out as g for 30 min. The first and final supernatant were kept for described previously [12]. Briefly, cells were perme- immunoblot analysis. Western Blot analysis was per- abilized in 3% BSA + 0.3% TX-100 in PBS for 15 min at formed with primary antibodies targeting α-synuclein room temperature. After a PBS wash, cells were blocked (SNL-4, CNDR, 1:10,000), pS129 α-synuclein (ab168381, for 50 min with 3% BSA in PBS prior to incubation with Abcam, 1:1000), LRRK2 (3514–1, Epitomics, RRID: primary antibodies for 2 h at room temperature. Primary AB_10643781, 1:2000 or ab133474, Abcam, RRID: antibodies used were targeting pS129 α-synuclein (81A, AB_2713963, 1:500), pS935 LRRK2 (ab133450, Abcam, CNDR, 1:5000), MAP2 (17028, CNDR, 1:5000), NeuN 1:500), p62 (H00008878-M01, Abnova, RRID: AB_437085, (MAB377, Millipore, RRID: AB_2298772, 1:1500) or 1:1000) or GAPDH (2-RGM2, Advanced Immunological, tyrosine hydroxylase (TH, T2928, Sigma-Aldrich, RRID: 1:5000). Primary antibodies were detected using IRDye AB_477569, 1:1000). Cells were washed 5× with PBS 800 (Li-cor 925–32,210) or IRDye 680 (Li-cor 925– and incubated with secondary antibodies for 1 h at room 68,071) secondary antibodies, scanned on Li-cor Odyssey temperature. After 5× wash with PBS, cells were incu- Imaging System and analyzed using Image Studio soft- bated in 1:10,000 DAPI in PBS. 96-well plates were im- ware. Values obtained from this program were normalized aged on In Cell Analyzer 2200 (GE Healthcare) and to average PFF alone values. analyzed in the accompanying software. A standard intensity-based threshold was applied to MAP2 and pS129 α-synuclein channels and positive area was quan- Human brain sequential detergent fractionation tified. For NeuN quantification, an object-based analysis Frozen postmortem human cingulate gyrus or frontal cor- was applied to identify objects of specified size and in- tex brain tissue containing abundant α-synuclein-positive tensity. TH+ cell analysis was based on intensity and size inclusions was selected for sequential extraction based on of objects (TH+ cells bodies are much more intense than IHC examination of these samples as described [16]using associated processes). All quantification was optimized previously established methods. These brains were se- and applied equally across all conditions. quentially extracted with increasing detergent strength as previously published [10]. After thawing, meninges were Statistical analysis removed and gray matter was carefully separated from All statistical analyses were done in GraphPad Prism. white matter. Gray matter was weighed and sus- The analysis used for each data set is described in the pended in four volumes (w/v) high salt (HS) buffer accompanying figure legend. More specifically, for ex- (50 mM Tris-HCL (pH 7.4), 750 mM NaCl, 10 mM periments directly comparing only NTG and G2019S NaF, 5 mM EDTA, protease and phosphatase inhibi- neurons (Fig. 1), unpaired t-tests with Welch’s correc- tors), followed by homogenization with a dounce tions were performed. For experiments in which various homogenizer and centrifugation at 100,000 x g for inhibitors were compared, one-way ANOVAs with Dun- 30 min. The HS wash was repeated and the resulting nett’s multiple comparison test (Figs. 3 and 4b, d)or pellet was then homogenized with 9 volumes HS buf- Kruskal-Wallis tests with Dunn’s multiple comparisons fer with 1% TX-100 and centrifuged at 100,000 x g tests (Figs. 4b, e, f and 5) were performed. Experiments for 30 min. The pellet fraction was then homogenized which included both NTG and G2019S neurons and with 9 volumes HS buffer with 1% TX-100 and 30% su- various treatment conditions (Figs. 2 and 6), we per- crose and centrifuged at 100,000 x g for 30 min to float formed two-way ANOVAs with Dunnett’s multiple com- away the myelin, which was discarded. The pellet was then parison test to compare treatment type and Sidak’s homogenized with 9 volumes HS buffer with 1% Sarkosyl, multiple comparison test to evaluate the difference be- rotated for 1 h at room temperature and centrifuged at tween genotypes. 100,000 x g for 30 min. The resulting pellets were washed once with Dulbecco’s PBS and re-suspended in Dulbecco’s Results PBS by brief sonication (QSonica Microson™ XL-2000; 20 G2019S LRRK2 expression does not affect soluble or pulses; setting 2; 0.5 s/pulse). This suspension was termed insoluble α-synuclein in hippocampal neurons the “sarkosyl insoluble fraction” containing pathological To establish a model in which the effect of LRRK2 mu- α-synuclein and used for the cellular assays described tations on α-synuclein could be observed, we used a here. The amounts of α-synuclein in the sarkosyl insoluble transgenic BAC mouse which expresses mouse LRRK2 fractions were determined by sandwich ELISA as de- with the familial G2019S mutation under the endogen- scribed previously [2] using Syn9027 (100 ng/well) as the ous LRRK2 promoter (B6.Cg-Tg(Lrrk2*G2019S)2Yue/J, capture antibody and MJF-R1 (1:1000 dilution) as the re- [19]). This allows overexpression of the mutated LRRK2 porter antibody. in a similar pattern to endogenous LRRK2 [35]. Upon Henderson et al. Acta Neuropathologica Communications (2018) 6:45 Page 4 of 11 1% TX-100 2% SDS PBS PFF PBS PFF Normalized Soluble Protein Levels a b NTG 1.2 35 * ** 60 G2019S ** 30 1.0 -syn 50 0.8 20 20 0.6 15 0.4 0.2 0 0 0.0 pS129 -syn PBS -syn PFF PBS -syn PFF PBS Normalized Insoluble Protein Levels LRRK2 1.5 1.2 High Exp. 1.0 1.0 LRRK2 0.8 1.0 0.8 pS935 LRRK2 0.6 0.6 0.4 High exp. 0.5 0.4 pS935 LRRK2 250 0.2 0.2 0.0 0 .0 0 .0 p62 50 PBS PBS -syn PFF PBS -syn PFF -syn PFF GAPDH Fig. 1 G2019S LRRK2 hippocampal neurons do not have elevated α-synuclein pathology 14 days post-transduction. a Primary hippocampal neurons from NTG or G2019S pups were transduced with 2.5 μg/mL α-synuclein PFFs and allowed to age a further 14 days prior to sequential detergent fractionation. TX-100-insoluble α-synuclein and p62 are similar in both neuron types. b Quantification of soluble proteins shows ~ 25- fold elevation in the expression of LRRK2 and a commensurate ~ 50-fold elevation in pS395 LRRK2, indicative of the elevated LRRK2 kinase activity associated with the G2019S mutation. Soluble α-synuclein levels were equivalent between the cultures. c No significant differences were found between the genotypes in insoluble proteins by an unpaired t-test with Welch’s correction. (N = 3 biological replicates for each protein). Means + s.e.m.; **P < 0.01; *P < 0.05 by an unpaired t-test with Welch’s correction for unequal variances. All values are normalized to NTG neurons treated with α-synuclein PFFs and DMSO initiation of breeding, it was discovered that the mice does not alter pathological α-synuclein accumulation at have multiple inserts of the transgene and were hetero- 14 DPT. zygous at all loci, resulting in non-transgenic pups upon inbreeding in a stochastic manner. To ensure homogen- G2019S LRRK2 hippocampal neurons show mild, eity of cultures and reproducibility of results, the mice reversible elevation in α-synuclein pathology at 21 DPT were bred to stable homozygosity as confirmed by quan- However, it has recently been demonstrated that titative PCR and outbreeding. The genotype of all ani- G2019S neurons may show time-dependent changes in mals used for culture was confirmed by quantitative α-synuclein pathology that don’t become apparent until PCR. We cultured primary hippocampal neurons from 18 DPT [32]. Therefore, we cultured NTG and G2019S the G2019S LRRK2 (G2019S) or C57BL/6 J (NTG) con- neurons, treated at 7 DIV with 1 μg/mL α-synuclein trol animals. G2019S neurons express ~ 25-fold elevated PFFs, and allowed the neurons to develop pathology for LRRK2, resulting in ~ 50-fold increase in phosphorylated 21 DPT. In addition, we treated the neurons 2 days prior LRRK2 at residue Ser935 (pS935), a readout for LRRK2 to transduction with two validated LRRK2 inhibitors, activity [6, 7, 26] (Fig. 1a, b). Despite dramatic elevation PF-06447475 (PF-475) and PF-06685360 (PF-360) at 5, in LRRK2 expression and activity, soluble α-synuclein 30, and 120 nM. At 21 DPT, the neurons were fixed and levels were unchanged (Fig. 1a, b). We induced stained for pS129 α-synuclein, MAP2 (a somatodendritic α-synuclein pathology in these cultures as described pre- marker), and NeuN (a marker of neuronal nuclei). Both viously [12] by adding 2.5 μg/mL recombinant mouse NTG and G2019S neurons develop robust neuritic and α-synuclein pre-formed fibrils (PFFs) to the cultures at cell body pathology in comparison to neurons treated 7 days in vitro (DIV) and allowing the neurons to de- with PBS as a vehicle control which never develop velop pathology for a further 14 days post-transduction pS129 α-synuclein inclusions (Fig. 2a, b). We were able (DPT). The protein from neurons was then sequentially to detect a mild elevation of α-synuclein pathology in extracted in 1% TX-100 followed by 2% SDS, allowing the G2019S neurons, which was reversible with 30 and the separation of pathological, insoluble α-synuclein 120 nM PF-360 (Fig. 2a, b, c). In every other respect from soluble α-synuclein. Both NTG and G2019S neu- measured, NTG and G2019S neurons responded simi- rons accumulated insoluble α-synuclein phosphorylated larly to α-synuclein PFF treatment. The area covered by at residue Ser129 (pS129) to a similar extent (Fig. 1a, c). MAP2 and the number of neurons were not different Additionally, the autophagy receptor p62, which deco- between the two genotypes (Fig. 2d, e), and the mild ele- rates pathological inclusions, including Lewy bodies in vation in pathology did not induce further toxicity. In PD, accumulates to a similar extent in both genotypes addition, at no concentration did the LRRK2 inhibitors (Fig. 1a, c). Thus, a dramatic elevation in LRRK2 activity affect α-synuclein pathology, MAP2 area or neuron NTG G2019S NTG G2019S NTG G2019S NTG G2019S -syn LRRK2 pS129- -syn p-LRRK2 (S935) p62 -syn Henderson et al. Acta Neuropathologica Communications (2018) 6:45 Page 5 of 11 a PBS 1 g/mL -syn PFF PF-475 (nM) PF-360 (nM) 120 nM DMSO DMSO55 30 120 30 120 PF-475 NTG cd ** e G2019S 1.5 **** **** 0.5 0.5 0.5 PF-475 PF-360 PF-475 PF-360 PF-475 PF-360 PF-475 PF-360 PF-475 PF-360 PF-475 PF-360 (nM) (nM) (nM) (nM) (nM) (nM) (nM) (nM) (nM) (nM) (nM) (nM) PBS -syn PFF PBS -syn PFF PBS -syn PFF PBS -syn PFF PBS -syn PFF PBS -syn PFF Fig. 2 G2019S LRRK2 hippocampal neurons show mild, reversible elevation in induced α-synuclein pathology 21 days post-transduction. Primary hippocampal neurons from NTG (a) or G2019S (b) pups were transduced with α-synuclein PFFs and allowed to age a further 21 days prior to fixation and staining for pS129 α-synuclein (magenta), MAP2 (gray) and NeuN (blue). The neurons were additionally treated with LRRK2 inhibitors PF-475 and PF-360 2 days prior to transduction and fed with media containing inhibitors each week thereafter. No large differences can be observed in the type or abundance of α-synuclein pathology. c Quantification of α-synuclein pathology reveals a mild elevation in G2019S neurons, which is reversible with 30 or 120 nM PF-360. *P < 0.05 by Dunnett’s multiple comparison test between NTG and G2019S neurons or Sidak’s multiple comparisons test between G2019S neurons treated with LRRK2 inhibitors. d MAP2 area is reduced with 21 days α-synuclein PFF treatment in both NTG and G2019S neurons, and is not significantly affected by LRRK2 inhibitor treatment. *P < 0.05 by 2-way ANOVA with Dunnett’s multiple comparison test for comparison within genotype. e The number of neurons, as quantified by NeuN number, is reduced with 21 days α-synuclein PFF treatment in both NTG and G2019S neurons, although not significantly by 2-way ANOVA followed by Dunnett’s multiple comparison test and is not significantly affected by LRRK2 inhibitor treatment. (N = 12 biological replicates). Means + s.e.m.; all values are normalized to NTG neurons treated with α-synuclein PFFs and DMSO. Scale bars = 50 μm number in the NTG neurons indicating that there is previous publications showing a reduction of no apparent relationship between LRRK2 activity in α-synuclein pathology in response to both LRRK2 inhib- NTG neurons and α-synuclein pathology. We con- ition [32] and knockdown with anti-sense oligonucleo- clude that, while interesting, the observed elevation in tides [38]. While LRRK2 inhibitors are initially being α-synuclein pathology in the G2019S neurons is too tested in patients with LRRK2 mutations, there is specu- mild to make this a valuable screening tool for lation that if LRRK2 is a fundamental protein in PD LRRK2-dependent phenotypes. pathogenesis, LRRK2 inhibitors may provide clinical benefit for those without LRRK2 mutations. Therefore, LRRK2 inhibitors do not alter α-synuclein pathology in we sought to investigate LRRK2 in wildtype neurons wildtype neurons from a second, distinct strain of mice (CD1). We first The lack of an effect of LRRK2 inhibitors on α-synuclein sought to validate the effect of LRRK2 inhibitors on pathology in NTG neurons was surprising given LRRK2 activity in these neurons and further test these Norm. pS129 -syn Area/MAP2 Area LRRK2*G2019S Hipp Neurons Non-transgenic Hipp Neurons DMSO Merge + NeuN MAP2 pS129 -syn Merge MAP2 pS129 -syn PF-475 DMSO DMSO PF-475 DMSO Normalized MAP2 Area DMSO PF-475 DMSO DMSO PF-475 DMSO Normalized NeuN Number DMSO PF-475 DMSO DMSO PF-475 DMSO 120 Henderson et al. Acta Neuropathologica Communications (2018) 6:45 Page 6 of 11 1% TX-100 2% SDS Normalized Soluble Protein Levels a b 1.2 **** 1.0 1.0 0.8 0.8 0.6 0.6 25 0.4 0.4 -syn 20 0.2 0.2 0.0 0.0 pS129- -syn c Normalized Insoluble Protein Levels 15 * 1.2 1.2 1.25 1.0 1.0 LRRK2 1.00 0.8 0.8 0.75 p-LRRK2 (S935) 0.6 0.6 0.50 0.4 0.4 p62 0.25 0.2 0.2 0.00 0.0 0.0 GAPDH Fig. 3 LRRK2 inhibition does not reduce insoluble α-synuclein in wildtype hippocampal neurons. a Primary hippocampal neurons from CD1 pups were treated with 30 nM LRRK2 inhibitors PF-475, PF-360 or DMSO as a vehicle control, then transduced with 2.5 μg/mL α-synuclein PFFs and allowed to age a further 14 days prior to sequential detergent fractionation. TX-100-insoluble α-synuclein and p62 are similar in both LRRK2 inhibitor treated and untreated neurons. b Quantification of soluble proteins show some reduction of LRRK2 protein levels PF-475 treatment and ~ 75% inhibition of LRRK2 activity (as assayed by pS935 LRRK2) by both PF-475 and PF-360. c Insoluble pS129 α-synuclein was slightly, but significantly elevated by PF-360 treatment, while α-synuclein and p62 were unchanged by one-way ANOVA. (N = 5 biological replicates for each protein). Means + s.e.m.; *P < 0.05, ****p < 0.0001 by one-way ANOVA with Dunnett’s multiple comparison test. All values are normalized first to GAPDH, as a loading control, then to DMSO-treated neurons inhibitors on insoluble, pathological α-synuclein accumula- X-100, sucrose, and sarkosyl buffers, yielding a final pel- tion. CD1 hippocampal neurons were treated with LRRK2 let enriched in LB α-synuclein. This pellet was then sus- inhibitors at 5 DIV and transduced with 2.5 μg/mL pended in phosphate-buffered saline by sonication, α-synuclein PFFs at 7 DIV. Neurons were harvested by se- yielding a final concentration of α-synuclein from 7.5– quential detergent fractionation at 14 DPT. We were able 22.4 μg/mL, Fig. 5a, Table 1). As before, primary hippo- to confirm a robust ~ 75% inhibition of LRRK2 S935 phos- campal neurons were treated with LRRK2 inhibitors at 5 phorylation with 30 nM PF-475 or PF-360 (Fig. 3a, b). DIV followed by treatment with 40 ng/mL LB PF-475 also modestly reduced total LRRK2 levels (Fig. 3a, α-synuclein two days later. This is the maximum con- b). However, inhibition of LRRK2 resulted in no reduction centration that neurons can be treated with due to the in insoluble α-synuclein, pS129 α-synuclein, or p62 (Fig. 3a, relatively low concentration of α-synuclein in these c). Instead, pS129 α-synuclein was slightly,but significantly preps. Neurons were fixed and stained 14 days after the elevated by LRRK2 inhibitor treatment (Fig. 3a, c). addition of LB α-synuclein. The induced pathology is We sought to further validate this finding with a larger sparser than that induced by PFFs due to the lower con- family of LRRK2 inhibitors at increased concentrations centration of α-synuclein (Fig. 5b). The pathology in- that would leave no residual LRRK2 activity. We chose duced by LB α-synuclein and neuron health were not an additional, validated LRRK2 inhibitor (MLi-2) for fur- meaningfully altered by LRRK2 inhibition (Fig. 5b, c, d, ther investigation and added these inhibitors at concen- e), consistent with PFF treatment. trations ranging from 30 to 300 nM to CD1 neurons. Use of these inhibitors resulted in near complete deple- Dopaminergic neurons show no change in α-synuclein tion of pS935 LRRK2 (Fig. 4a, b). The neurons were then pathology in response to G2019S LRRK2 overexpression transduced with α-synuclein PFFs and fixed at 14 DPT. or LRRK2 inhibition No LRRK2 inhibitor, at any concentration, altered All the experiments to this point were performed in pri- α-synuclein pathology (Fig. 4c, d), MAP2 area (Fig. 4c, e) mary hippocampal neuron cultures. These cultures de- or neuron number (Fig. 4c, f). velop robust α-synuclein pathology and can be obtained We then tested whether inhibition of LRRK2 activity in an abundance suitable for both immunocytochemistry can alter α-synuclein pathology induced by a means and biochemistry. However, they are not the neurons other than PFFs. We have recently demonstrated the most affected in PD patients. In order to address ability of LB α-synuclein purified from human brain to whetherweweremissing aphenotype that is induce pS129 α-synuclein pathology in WT neurons dependent on expression of mutant LRRK2 in dopa- [24]. Cortical gray matter from brains with high LB bur- minergic neurons, we developed ventral midbrain and den were sequentially extracted with high salt, Triton striatum primary neuron co-cultures. When treated DMSO PF-475 PF-360 DMSO PF-475 DMSO PF-360 PF-475 PF-360 DMSO PF-475 PF-360 DMSO PF-475 PF-360 DMSO PF-475 PF-360 DMSO PF-475 PF-360 LRRK2 pS129- -syn -syn p-LRRK2 (S935) p62 Henderson et al. Acta Neuropathologica Communications (2018) 6:45 Page 7 of 11 MLi-2 PF-360 PF-475 a d 1.2 **** (nM) (nM) (nM) 1.0 DMSO 30 300 30 300 30 300 kDa 0.8 LRRK2 0.6 pS935 LRRK2 0.4 GAPDH 0.2 0.0 2.0 1.2 PF-475 PF-360 MLi-2 1.5 (nM) (nM) (nM) 0.8 PBS 1 g/mL -syn PFF 1.0 0.4 e 0.5 1.25 ** ** ** ** 0.0 0.0 1.00 PF-475 PF-360 MLi-2 PF-475 PF-360 MLi-2 0.75 (nM) (nM) (nM) (nM) (nM) (nM) 0.50 0.25 PBS 1 g/mL -syn PFF 0.00 300 nM 300 nM 300 nM DMSO DMSO PF-475 PF-360 MLi-2 PF-475 PF-360 MLi-2 (nM) (nM) (nM) PBS 1 g/mL -syn PFF f ** 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 PF-475 PF-360 MLi-2 (nM) (nM) (nM) PBS 1 g/mL -syn PFF Fig. 4 LRRK2 inhibition does not reduce pathological α-synuclein in wildtype hippocampal neurons. a Primary cortical neurons were treated with LRRK2 inhibitors PF-475, PF-360, or MLi-2, at 5 DIV and fed with media containing inhibitors each week for 16 days. Cell lysate was run by Western blot to detect LRRK2 and pS935 LRRK2, which is indicative of LRRK2 activity. Images shown are representative of n =4–12 biological replicates. b Quantification of Western blot of LRRK2 and pS935 LRRK2. LRRK2 levels were not significantly altered by one-way ANOVA, but pS935 levels were reduced to near undetectable levels (*p < 0.05, **p < 0.01, Kruskal-Wallis test with Dunn’s multiple comparison test). c Primary hippocampal neurons from CD1 pups were transduced with α-synuclein PFFs and allowed to age a further 14 days prior to fixation and staining for pS129 α-synuclein (magenta), MAP2 (gray) and NeuN (blue). The neurons were additionally treated with LRRK2 inhibitors PF-475, PF-360, or MLi-2, 2 days prior to transduction and fed with media containing inhibitors each week thereafter. No large differences can be observed in the type or abundance of α-synuclein pathology. d Quantification of α-synuclein pathology reveals no change in response to LRRK2 inhibition, while PBS-treated neurons have no pathology (****p < 0.0001 by Dunnett’s multiple comparison test). MAP2 area (e) and neuron number (f) are also not altered in response to LRRK2 inhibition. No significant response was seen when compared with vehicle-treated neurons by one-way ANOVA with Dunnett’s multiple comparison test (d) or by Kruskal-Wallis test followed by Dunn’s multiple comparison test (e) and (f). (N = 9 biological replicates). Means + s.e.m.; all values are normalized to neurons treated with α-synuclein PFFs and DMSO. Scale bars = 50 μm with α-synuclein PFFs at 7 DIV and allowed to age to G2019S dopaminergic neurons, and LRRK2 inhibi- 14 DPT, dopaminergic neurons positive for TH de- tors did not significantly improve survival of TH+ velop cell body and neuritic pS129 α-synuclein path- neurons (Fig. 6c). ology (Fig. 6a). We treated midbrain/striatum co-cultures from NTG and G2019S pups with 30 nM Discussion LRRK2 inhibitors or vehicle control at 5 DIV. The In this study, we utilized primary neuron cultures to neurons were then treated with α-synuclein PFFs at 7 assess the status of α-synuclein pathology in relation DIV and fixed 14 DPT and stained for pS129 to LRRK2 activity. We performed biochemical and α-synuclein and TH. Both cultures develop a similar immunocytochemical analysis of primary hippocampal amount of α-synuclein pathology in TH+ neurons, and midbrain neurons cultured from two strains of and none of the LRRK2 inhibitors changed the level wildtype mice and a mouse expressing LRRK2 with of pathology in either NTG or G2019S neurons (Fig. the familial G2019S mutation. To further manipulate 6a, b). Further, α-synuclein PFFs were not more toxic LRRK2 activity, we used three validated LRRK2 inhibitors. No r m . L R RK2/ Merge + NeuN MAP2 pS129 -syn GA P D H DMSO N o r m . p S 935 L R R K 2 /G A P D H DMSO N o r m . pS129 -syn A r ea No r m . M AP 2 Ar ea No r m . Neu N Nu m b er /M A P 2 A r ea DMSO DMSO DMSO PF-475 PF-475 PF-475 MLi-2 MLi-2 MLi-2 DMSO DMSO DMSO 30 30 30 100 100 100 300 300 300 30 30 30 100 100 100 300 300 300 30 30 30 100 100 100 300 300 300 Henderson et al. Acta Neuropathologica Communications (2018) 6:45 Page 8 of 11 1.4 Triton Buffer HS Buffer a c 1.2 100,000xg 100,000xg ** 1.0 30 min 30 min 0.8 0.6 Human LB brain 0.4 Sucrose buffer 100,000xg 0.2 +/- LRRK2 30 min 0.0 Inhibitors PBS to Sarkosyl Buffer Sark 100,000xg PBS 40 ng/mL LB -syn Pellet 30 min 1.2 1.0 0.8 0.6 PBS 40 ng/mL -syn PFF 0.4 DMSO DMSO PF-475 PF-360 MLi-2 0.2 0.0 PBS 40 ng/mL LB -syn 1.25 1.00 0.75 0.50 0.25 0.00 PBS 40 ng/mL LB -syn Fig. 5 LRRK2 inhibition does not alter pathology induced by human Lewy body α-synuclein. a A schematic representation of pathological α- synuclein purification from human cortical tissue. b Primary hippocampal neurons from CD1 pups were treated with 100 nM LRRK2 inhibitors or a vehicle control. Two days later, neurons were treated with 40 ng/mL human LB α-synuclein and allowed to age a further 14 days prior to fixation and staining for pS129 α-synuclein (magenta), MAP2 (gray) and NeuN (blue). No large differences can be observed in the type or abundance of α-synuclein pathology. c Quantification of α-synuclein pathology reveals an increase in neurons treated with LB α-synuclein compared to those treated with PBS (**p < 0.01), but no change in response to LRRK2 inhibition. MAP2 area (d) and neuron number (e) are also not altered meaningfully in response to LRRK2 inhibition. (N =11–12 biological replicates treated with LB α-synuclein from 4 separate cases (2 AD, 1 PDD, 1 DLB). Means + s.e.m.; all values are normalized to neurons treated with LB α-synuclein and DMSO. Scale bars = 50 μm In none of these assays did we observe more than a mild LRRK2 [8, 18], but in keeping with the many transgenic LRRK2-dependent alteration in α-synuclein pathology. models of LRRK2 that have since been generated [3, 19, The LRRK2*G019S neurons we describe have ~ 20, 37]. While there is strong support that overexpression 25-fold elevated LRRK2 levels and ~ 50-fold elevated of LRRK2 does not dramatically alter cell health, the effect kinase activity (Fig. 1a, b). Despite this dramatic overex- of LRRK2 on α-synuclein has been unclear. pression of LRRK2, there is no apparent effect on the We induced pathological α-synuclein inclusion forma- neuron health or viability (Figs. 2, 4, 5, and 6). This is in tion in NTG and G2019S neurons through addition of contrast with early research using viral overexpression of recombinant α-synuclein PFFs or human brain-derived Table 1 Clinical Information of the cases used for the Extraction of Pathological alpha-synuclein Case No. Clinical Pathological Race Sex Age of Disease Age at PMI (h) Brain Region Used α-Synuclein Diagnosis Diagnosis onset Death (μg/ml) 1 AD Probable AD/DLB White Female 52 62 11 Middle Frontal Gyrus 20.86 2 CBS AD/DLB White Female 45 55 15 Middle Frontal Gyrus 22.43 3 DLB DLB White Male 76 83 6 Cingulate Gyrus 7.55 4 PDD PDD White Male 60 72 19 Cingulate Gyrus 8.28 PMI postmortem interval DMSO DMSO PF-475 PF-360 MLi-2 DMSO DMSO PF-475 PF-360 MLi-2 DMSO DMSO PF-475 PF-360 MLi-2 Merge + NeuN MAP2 pS129 -syn N o r m . pS129 a-syn A r e a / No r m . Neu N Nu m b er N o r m . M A P 2 A r ea MA P 2 A r ea Henderson et al. Acta Neuropathologica Communications (2018) 6:45 Page 9 of 11 a b PBS 1 g/mL -syn PFF DMSO DMSO PF-475 PF-360 MLi-2 NTG 2.0 G2019S 1.5 *** ** 1.0 0.5 0.0 -syn PFF -syn PFF 2.5 2.0 1.5 1.0 0.5 0.0 -syn PFF -syn PFF Fig. 6 Neither G2019S LRRK2 expression nor LRRK2 inhibition alters α-synuclein pathology in midbrain neurons. a Primary midbrain/striatum cultures from NTG or G2019S pups were transduced with α-synuclein PFFs and allowed to age a further 14 days prior to fixation and staining for pS129 α- synuclein (magenta) and TH (gray). The neurons were additionally treated with 30 nM LRRK2 inhibitors PF-475, PF-360, or MLi-2 2 days prior to transduction and fed with media containing inhibitors each week thereafter. b Quantification of α-synuclein pathology in TH+ neurons shows no effect of G2019S LRRK2 expression or LRRK2 inhibitor treatments by 2-way ANOVA (**p < 0.01, ***p < 0.001 for PBS- compared to PFF-treated neurons by Dunnett’s multiple comparison test). c The number of TH+ neurons showed no significant response to treatment by Kruskal-Wallis test followed by Dunn’s multiple comparison test. (N =6–9 biological replicates). Means + s.e.m.; all values are normalized to NTG neurons treated with α-synuclein LB material and DMSO. Scale bars = 50 μm LB α-synuclein. We designed our initial experiments to LRRK2 protein reduction by anti-sense oligonucleotides test the relationship between LRRK activity and [38] are able to ameliorate α-synuclein pathology in α-synuclein at 14 DPT, before the onset of neurodegen- wildtype neurons. To ensure the validity of our results, eration. We found that there was no alteration of we used both biochemical extraction of pathological α-synuclein pathology in G2019S neurons. A recent α-synuclein as well as immunocytochemistry. Pathology publication using a similar model showed a mild eleva- induced by both recombinant α-synuclein PFFs and hu- tion in α-synuclein in G2019S hippocampal neurons at man LB α-synuclein were resistant to alteration by 18 DPT, while detecting no difference at 7 DPT [32]. LRRK2 inhibition. We also used three validated LRRK2 We found that at 21 DPT, we did observe a mild en- inhibitors, representing different classes of compounds. hancement of α-synuclein pathology in G2019S neurons All immunocytochemical quantification of α-synuclein that was responsive to LRRK2 inhibition (Fig. 2). At this was normalized to MAP2 to ensure no effect of cell timepoint, neurons have begun degenerating, and while density. Our results in wildtype hippocampal neurons we see no LRRK2-dependent difference in degeneration, generated from two strains of mice in addition to dopa- it is unclear if it is the neurodegenerative process that minergic midbrain neurons give us increased confidence results in a mild elevation of α-synuclein pathology in that the lack of LRRK2-dependent phenotypes we see G2019S neurons. are valid. To further explore the role of endogenous LRRK2 in LRRK2 is the most common cause of inherited PD. α-synuclein pathogenesis, we cultured wildtype hippo- Even so, only around 30% of those with G2019S muta- campal and midbrain neurons, and showed using bio- tion in LRRK2 will go on to develop PD [22]. It is there- chemistry and immunocytochemistry that LRRK2 kinase fore probable that LRRK2 mutations exacerbate an inhibition is unable to alter induced α-synuclein path- extant predisposition to PD. Another hypothesized de- ology (Figs. 3, 4, 5, and 6). These findings are in sharp terminant of susceptibility to PD is the misfolding of contrast to recent reports that LRRK2 inhibitors [32]or α-synuclein within aging brains. It is logical to assume LRRK2*G2019S MB/Str Neurons Non-transgenic MB/Str Neurons Merge TH pS129 -syn Merge TH pS129 -syn N o r m . pS129 -syn+ T H N o r m . T H N u m b er Area/T H A r ea PBS DMSO PBS DMSO DMSO DMSO PF-475 PF-475 PF-360 PF-360 Mli-2 Mli-2 PBS DMSO PBS DMSO DMSO DMSO PF-475 PF-475 PF-360 PF-360 Mli-2 Mli-2 Henderson et al. Acta Neuropathologica Communications (2018) 6:45 Page 10 of 11 that challenging neurons with both misfolded Culture Service Center for their assistance procuring CD1 hippocampal neuron cultures. α-synuclein and mutated LRRK2 would be sufficient to drive an enhanced pathogenic process. While there is Funding compelling evidence that this may be true in mouse This study was supported by the Michael J. Fox Foundation and National Institute of Health grants: T32-AG000255, P30-AG10124, P50-NS053488. models [3, 5, 32], we are unable to generate a robust model of LRRK2-dependent α-synuclein pathogenesis in Availability of data and materials primary neuron cultures. There are several possible rea- The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. sons that in vivo models cannot be recapitulated in cell culture. Neuron cultures are inherently closed systems. Authors’ contributions Therefore, if LRRK2 activity can affect the spread of Conception and design: MXH, CP, JQT, VMYL; Acquisition and analysis of data: MXH and CP; Drafting of the article: MXH, VMYL. All authors read and α-synuclein pathology through the brain, this effect approved the final manuscript. would not be captured in a dish. Primary neuron cul- tures also lack all the cell types and complex synaptic Ethics approval and consent to participate All housing, breeding, and procedures were performed according to the NIH organization present in vivo. Indeed, LRRK2 is highly Guide for the Care and Use of Experimental Animals and approved by the expressed in astrocytes [13] and neuroinflammation has University of Pennsylvania Institutional Animal Care and Use Committee. been implicated in LRRK2-dependent phenotypes [3]. It Competing interests is also possible that LRRK2 is not critical in the early The authors declare that they have no competing interests. stages of pathology formation, and only during later stages of disease progression does LRRK2 activity be- Publisher’sNote come important. This is supported by our finding that Springer Nature remains neutral with regard to jurisdictional claims in α-synuclein pathology is only elevated in G2019S neu- published maps and institutional affiliations. rons 21 days after transduction, after the onset of neuro- Received: 20 May 2018 Accepted: 21 May 2018 degeneration. In addition, patients with LRRK2 mutations do not have an earlier onset of disease than References patients with sporadic onset [11], suggesting that LRRK2 1. Baba M, Nakajo S, Tu PH, Tomita T, Nakaya K, Lee VM, Trojanowski JQ, does not speed the disease process, as triplication of Iwatsubo T (1998) Aggregation of alpha-synuclein in Lewy bodies of α-synuclein does [27]. While α-synuclein inclusions are sporadic Parkinson's disease and dementia with Lewy bodies. Am J Pathol 152:879–884 common in PD patients with LRRK2 mutations, not all 2. Covell DJ, Robinson JL, Akhtar RS, Grossman M, Weintraub D, Bucklin HM, LRRK2 patients have α-synuclein pathology and a sub- Pitkin RM, Riddle D, Yousef A, Trojanowski JQ, Lee VM (2017) Novel stantial portion of LRRK2 patients also have tau inclu- conformation-selective alpha-synuclein antibodies raised against different in vitro fibril forms show distinct patterns of Lewy pathology in Parkinson's sions [25]. 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LRRK2 activity does not dramatically alter α-synuclein pathology in primary neurons

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BioMed Central
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Copyright © 2018 by The Author(s).
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Biomedicine; Neurosciences; Pathology; Neurology
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2051-5960
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10.1186/s40478-018-0550-0
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Abstract

Mutations in leucine-rich repeat kinase (LRRK2) are the most common cause of heritable Parkinson’s disease (PD), and the most common mutations in LRRK2 lead to elevated kinase activity. For these reasons, inhibitors targeting LRRK2 have been the subject of intense research and development. However, it has been difficult to develop preclinical models that recapitulate PD-relevant LRRK2 phenotypes. The primary pathology in PD is the Lewy body (LB), which is a cytoplasmic aggregate of α-synuclein. The recent demonstration that LB-like aggregates of α-synuclein can be induced in primary neurons has provided a robust model for testing genetic modifiers of PD-relevant aggregation and neurodegeneration. In this study, we test the modulation of α-synuclein pathology by LRRK2 in primary neuron cultures using biochemistry and immunocytochemistry. We find that expression of familial mutant G2019S LRRK2 does not dramatically elevate the pathological burden of α-synuclein or neurodegeneration in neurons. We further test three LRRK2 inhibitors in two strains of wildtype neurons and find that even robust LRRK2 inhibition is insufficient to reduce α-synuclein pathology. LRRK2 inhibitors similarly had no effect in neurons with α-synuclein pathology seeded by human brain-derived pathological α-synuclein. Finally, we find that this lack of pathological modulation by LRRK2 was not confined to hippocampal neurons, but was also absent in midbrain dopaminergic neuron cultures. These data demonstrate that LRRK2 activity does not have more than minor effects on α-synuclein pathology in primary neurons, and more complex models may be needed to evaluate the ability of LRRK2 inhibitors to treat PD. Keywords: LRRK2, Synuclein, pS129, Aggregates, Inhibitor, G2019S Introduction cytoskeletal modeling [17]. LRRK2 mutations are found in Parkinson’s disease (PD) is the most common neurodegen- 4% of hereditary and 1% of sporadic PD patients, and pa- erative movement disorder. Patients with this disease experi- tients with LRRK2 mutations phenocopy sporadic PD pa- ence rigidity, resting tremors, and slowness of movement; tients, with similar onset of disease, α-synuclein 80% of patients will develop dementia throughout the dis- pathology, and responsiveness to dopamine replacement ease course [15]. The clinical diagnosisofPDisconfirmed [11]. The most common mutation (G2019S) in the kinase post-mortem by the presence of intracytoplasmic inclusions domain as well as mutations in the GTPase domain of termed Lewy bodies (LBs), which consist primarily of the LRRK2, elevate kinase activity of LRRK2 [8, 26, 31, 36], synaptic protein α-synuclein [1, 29, 30]. While α-synuclein making LRRK2 an ideal candidate for small molecule in- is thoughttobepathogenicinthislargely sporadic disease, hibitor development. A large number of LRRK2 inhibitors mutations in several genes can increase the lifetime risk of have been developed and refined for increased potency developing disease. The most commonly mutated gene in and low off-target effects over the past decade. However, hereditary PD is leucine-rich repeat kinase 2 (LRRK2, [11]). the lack of a reliable preclinical disease model for LRRK2 LRRK2 is a widely expressed protein with unclear dysfunction has been a major challenge for validating the functions. It has kinase, GTPase and scaffolding domains therapeutic potential of LRRK2 inhibitors. and has been implicated in intracellular trafficking and To understand the relationship between LRRK2 and α-synuclein and develop a model of LRRK2-dependent * Correspondence: vmylee@upenn.edu PD phenotypes, early studies crossed mice expressing Department of Pathology and Laboratory Medicine, Institute on Aging and wildtype or G2019S LRRK2 mice with mice overexpress- Center for Neurodegenerative Disease Research, University of Pennsylvania School of Medicine, 3600 Spruce St, 3rd Floor Maloney, Philadelphia, PA ing α-synuclein with the familial A53T mutation. These 19104-4283, USA © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Henderson et al. Acta Neuropathologica Communications (2018) 6:45 Page 2 of 11 studies all found little or no effect of LRRK2 expression [21, 33, 34]. For treatment of neurons, α-synuclein PFFs, on α-synuclein pathology [4, 14, 20]. However, later stud- which were generated at a concentration of 5 mg/mL were ies which injected adeno-associated virus into the substan- vortexed and diluted with Dulbecco’s phosphate-buffered tia nigra of LRRK2 knockout or G2019S LRRK2 rats found saline (DPBS) to 100 μg/mL. They were then sonicated on that LRRK2 depletion rescued α-synuclein-induced neuro- high for 10 cycles of 30 s on, 30 s off (Diagenode Biorupter degeneration [5] and G2019S LRRK2 exacerbated degener- UCD-300 bath sonicator). α-Synuclein PFFs were then di- ation [3]. Several cell culture studies have found that luted in neuron media to 5 μg/mL and added to neuron LRRK2 may directly interact with α-synuclein [9]orindir- cultures at a concentration of 1 μg/mL for immunocyto- ectly influence α-synuclein expression [23, 28], however chemistry experiments and 2.5 μg/mL for biochemistry none of these cell culture models recapitulate the phos- experiments. phorylated, detergent-insoluble α-synuclein seen in PD patients. The recent finding that α-synuclein pathology can Primary hippocampal cultures be induced in wildtype primary neurons by introduction of Primary hippocampal neuron cultures were prepared small amounts of recombinant α-synuclein pre-formed from postnatal day (P) 1 NTG and G2019S mice and fibrils (PFFs) has allowed the study of α-synuclein path- embryonic day (E) 16–18 CD1 embryos. Dissociated hip- ology in neurons expressing wildtype or mutated LRRK2 pocampal neurons were plated at 17,000 cells/well [32]. A primary neuron model in which LRRK2 activity (96-well plate) or 1,000,000 cells/well (6-well plate) in modulates α-synuclein pathology would be ideal for screen- neuron media (Neurobasal medium (ThermoFisher ing LRRK2 inhibitors and providing biological rationale for 21,103,049) (or Neurobasal A medium (Thermo Fisher moving toward clinical intervention. We used robust im- 10,888–022) for postnatal cultures) supplemented with munocytochemical and biochemical assays for α-synuclein B27 (ThermoFisher 17,504,044), 2 mM GlutaMax pathology in primary hippocampal and midbrain dopamin- (ThermoFisher 35,050,061), and 100 U/mL penicillin/ ergic neurons derived from two strains of wildtype mice streptomycin (ThermoFisher 15,140,122). LRRK2 inhibi- and mice expressing the LRRK2 with the familial G2019S tors PF-475 and PF-360 were synthesized at Pfizer, Inc. mutation to test LRRK2 activity-dependent phenotypes. MLi-2 was obtained from Tocris Bioscience (5756). All We induce α-synuclein pathology with addition of both LRRK2 inhibitors were reconstituted at 10 mM in DMSO recombinant and human-derived fibrillar α-synuclein. In andstoredat − 20 °C. There were further diluted to the order to manipulate LRRK2 activity, we used three final concentration indicated in neuron media with DMSO validated LRRK2 inhibitors. In no system and by no meas- as a vehicle control. Neurons were either fixed with 4% ure did we observe more than very mild LRRK2-dependent paraformaldehyde, 4% sucrose in phosphate-buffered saline changes in α-synuclein pathology. for immunocytochemistry or scraped for biochemistry. Materials and methods Primary midbrain/striatum co-cultures Animals The ventral mesencephalon and striatum were dissected All housing, breeding, and procedures were performed from P2 NTG and G2019S mice in Hibernate A medium according to the NIH Guide for the Care and Use of (ThermoFisher A1247501) with B27 (ThermoFisher Experimental Animals and approved by the University of 17,504,044) and 0.5 mM GlutaMax (ThermoFisher Pennsylvania Institutional Animal Care and Use 35,050,061). The tissue was then digested in papain Committee. C57BL/6 J (NTG, JAX 000664, RRID: (Worthington Biochemical LS003126) for ~ 20 min at IMSR_JAX:000664) and B6.Cg-Tg(Lrrk2*G2019S)2Yue/J 37 °C. Cells were mixed at a ratio of 1:1 and plated at a (G2019S, JAX 012467, RRID: IMSR_JAX:012467) mice density of 34,000 cells/well in 96-well black-walled plates were obtained from Andrew West (University of Alabama, (Perkin Elmer 50–905-1605) in Neurobasal A medium Birmingham). The G2019S mice have multiple inserts of (Thermo Fisher 10,888–022) supplemented with B27 the gene, but were heterozygous at these loci. and 0.4 mM GlutaMax, 50 ng/mL BDNF (PeproTech Subsequently, mice were bred to homozygosity at loci as 450–02) and 30 ng/mL GDNF (Millipore Sigma GF030). determined by quantitative PCR and outbreeding. The expression level of G2019S LRRK2 was thereby stabilized Neuron sequential detergent fractionation in this line of mice. All experiments shown use homozy- Proteins from primary neuronal cultures treated with PBS gous G2019S mice. CD1 (Strain 022, RRID: IMSR_CRL:22) or α-synuclein PFFs were sequentially extracted as de- mice were obtained from Charles River, Wilmington, MA. scribed previously [21, 33, 34]. Briefly, neurons were scraped into 1 volume 1% TX-100 buffer, sonicated and α-Synuclein PFFs spun at 100,000 x g for 30 min. The pellet was sonicated Purification of recombinant α-synuclein and generation of and again spun at 100,000 x g for 30 min in 1 volume 1% α-synuclein PFFs was conducted as described elsewhere TX-100 solution to remove remaining TX-100-soluble Henderson et al. Acta Neuropathologica Communications (2018) 6:45 Page 3 of 11 proteins. This pellet was suspended in 0.5 volumes 2% Immunocyctochemistry SDS solution, sonicated and spun once more at 100,000 x Immunostaining of neuronal cultures was carried out as g for 30 min. The first and final supernatant were kept for described previously [12]. Briefly, cells were perme- immunoblot analysis. Western Blot analysis was per- abilized in 3% BSA + 0.3% TX-100 in PBS for 15 min at formed with primary antibodies targeting α-synuclein room temperature. After a PBS wash, cells were blocked (SNL-4, CNDR, 1:10,000), pS129 α-synuclein (ab168381, for 50 min with 3% BSA in PBS prior to incubation with Abcam, 1:1000), LRRK2 (3514–1, Epitomics, RRID: primary antibodies for 2 h at room temperature. Primary AB_10643781, 1:2000 or ab133474, Abcam, RRID: antibodies used were targeting pS129 α-synuclein (81A, AB_2713963, 1:500), pS935 LRRK2 (ab133450, Abcam, CNDR, 1:5000), MAP2 (17028, CNDR, 1:5000), NeuN 1:500), p62 (H00008878-M01, Abnova, RRID: AB_437085, (MAB377, Millipore, RRID: AB_2298772, 1:1500) or 1:1000) or GAPDH (2-RGM2, Advanced Immunological, tyrosine hydroxylase (TH, T2928, Sigma-Aldrich, RRID: 1:5000). Primary antibodies were detected using IRDye AB_477569, 1:1000). Cells were washed 5× with PBS 800 (Li-cor 925–32,210) or IRDye 680 (Li-cor 925– and incubated with secondary antibodies for 1 h at room 68,071) secondary antibodies, scanned on Li-cor Odyssey temperature. After 5× wash with PBS, cells were incu- Imaging System and analyzed using Image Studio soft- bated in 1:10,000 DAPI in PBS. 96-well plates were im- ware. Values obtained from this program were normalized aged on In Cell Analyzer 2200 (GE Healthcare) and to average PFF alone values. analyzed in the accompanying software. A standard intensity-based threshold was applied to MAP2 and pS129 α-synuclein channels and positive area was quan- Human brain sequential detergent fractionation tified. For NeuN quantification, an object-based analysis Frozen postmortem human cingulate gyrus or frontal cor- was applied to identify objects of specified size and in- tex brain tissue containing abundant α-synuclein-positive tensity. TH+ cell analysis was based on intensity and size inclusions was selected for sequential extraction based on of objects (TH+ cells bodies are much more intense than IHC examination of these samples as described [16]using associated processes). All quantification was optimized previously established methods. These brains were se- and applied equally across all conditions. quentially extracted with increasing detergent strength as previously published [10]. After thawing, meninges were Statistical analysis removed and gray matter was carefully separated from All statistical analyses were done in GraphPad Prism. white matter. Gray matter was weighed and sus- The analysis used for each data set is described in the pended in four volumes (w/v) high salt (HS) buffer accompanying figure legend. More specifically, for ex- (50 mM Tris-HCL (pH 7.4), 750 mM NaCl, 10 mM periments directly comparing only NTG and G2019S NaF, 5 mM EDTA, protease and phosphatase inhibi- neurons (Fig. 1), unpaired t-tests with Welch’s correc- tors), followed by homogenization with a dounce tions were performed. For experiments in which various homogenizer and centrifugation at 100,000 x g for inhibitors were compared, one-way ANOVAs with Dun- 30 min. The HS wash was repeated and the resulting nett’s multiple comparison test (Figs. 3 and 4b, d)or pellet was then homogenized with 9 volumes HS buf- Kruskal-Wallis tests with Dunn’s multiple comparisons fer with 1% TX-100 and centrifuged at 100,000 x g tests (Figs. 4b, e, f and 5) were performed. Experiments for 30 min. The pellet fraction was then homogenized which included both NTG and G2019S neurons and with 9 volumes HS buffer with 1% TX-100 and 30% su- various treatment conditions (Figs. 2 and 6), we per- crose and centrifuged at 100,000 x g for 30 min to float formed two-way ANOVAs with Dunnett’s multiple com- away the myelin, which was discarded. The pellet was then parison test to compare treatment type and Sidak’s homogenized with 9 volumes HS buffer with 1% Sarkosyl, multiple comparison test to evaluate the difference be- rotated for 1 h at room temperature and centrifuged at tween genotypes. 100,000 x g for 30 min. The resulting pellets were washed once with Dulbecco’s PBS and re-suspended in Dulbecco’s Results PBS by brief sonication (QSonica Microson™ XL-2000; 20 G2019S LRRK2 expression does not affect soluble or pulses; setting 2; 0.5 s/pulse). This suspension was termed insoluble α-synuclein in hippocampal neurons the “sarkosyl insoluble fraction” containing pathological To establish a model in which the effect of LRRK2 mu- α-synuclein and used for the cellular assays described tations on α-synuclein could be observed, we used a here. The amounts of α-synuclein in the sarkosyl insoluble transgenic BAC mouse which expresses mouse LRRK2 fractions were determined by sandwich ELISA as de- with the familial G2019S mutation under the endogen- scribed previously [2] using Syn9027 (100 ng/well) as the ous LRRK2 promoter (B6.Cg-Tg(Lrrk2*G2019S)2Yue/J, capture antibody and MJF-R1 (1:1000 dilution) as the re- [19]). This allows overexpression of the mutated LRRK2 porter antibody. in a similar pattern to endogenous LRRK2 [35]. Upon Henderson et al. Acta Neuropathologica Communications (2018) 6:45 Page 4 of 11 1% TX-100 2% SDS PBS PFF PBS PFF Normalized Soluble Protein Levels a b NTG 1.2 35 * ** 60 G2019S ** 30 1.0 -syn 50 0.8 20 20 0.6 15 0.4 0.2 0 0 0.0 pS129 -syn PBS -syn PFF PBS -syn PFF PBS Normalized Insoluble Protein Levels LRRK2 1.5 1.2 High Exp. 1.0 1.0 LRRK2 0.8 1.0 0.8 pS935 LRRK2 0.6 0.6 0.4 High exp. 0.5 0.4 pS935 LRRK2 250 0.2 0.2 0.0 0 .0 0 .0 p62 50 PBS PBS -syn PFF PBS -syn PFF -syn PFF GAPDH Fig. 1 G2019S LRRK2 hippocampal neurons do not have elevated α-synuclein pathology 14 days post-transduction. a Primary hippocampal neurons from NTG or G2019S pups were transduced with 2.5 μg/mL α-synuclein PFFs and allowed to age a further 14 days prior to sequential detergent fractionation. TX-100-insoluble α-synuclein and p62 are similar in both neuron types. b Quantification of soluble proteins shows ~ 25- fold elevation in the expression of LRRK2 and a commensurate ~ 50-fold elevation in pS395 LRRK2, indicative of the elevated LRRK2 kinase activity associated with the G2019S mutation. Soluble α-synuclein levels were equivalent between the cultures. c No significant differences were found between the genotypes in insoluble proteins by an unpaired t-test with Welch’s correction. (N = 3 biological replicates for each protein). Means + s.e.m.; **P < 0.01; *P < 0.05 by an unpaired t-test with Welch’s correction for unequal variances. All values are normalized to NTG neurons treated with α-synuclein PFFs and DMSO initiation of breeding, it was discovered that the mice does not alter pathological α-synuclein accumulation at have multiple inserts of the transgene and were hetero- 14 DPT. zygous at all loci, resulting in non-transgenic pups upon inbreeding in a stochastic manner. To ensure homogen- G2019S LRRK2 hippocampal neurons show mild, eity of cultures and reproducibility of results, the mice reversible elevation in α-synuclein pathology at 21 DPT were bred to stable homozygosity as confirmed by quan- However, it has recently been demonstrated that titative PCR and outbreeding. The genotype of all ani- G2019S neurons may show time-dependent changes in mals used for culture was confirmed by quantitative α-synuclein pathology that don’t become apparent until PCR. We cultured primary hippocampal neurons from 18 DPT [32]. Therefore, we cultured NTG and G2019S the G2019S LRRK2 (G2019S) or C57BL/6 J (NTG) con- neurons, treated at 7 DIV with 1 μg/mL α-synuclein trol animals. G2019S neurons express ~ 25-fold elevated PFFs, and allowed the neurons to develop pathology for LRRK2, resulting in ~ 50-fold increase in phosphorylated 21 DPT. In addition, we treated the neurons 2 days prior LRRK2 at residue Ser935 (pS935), a readout for LRRK2 to transduction with two validated LRRK2 inhibitors, activity [6, 7, 26] (Fig. 1a, b). Despite dramatic elevation PF-06447475 (PF-475) and PF-06685360 (PF-360) at 5, in LRRK2 expression and activity, soluble α-synuclein 30, and 120 nM. At 21 DPT, the neurons were fixed and levels were unchanged (Fig. 1a, b). We induced stained for pS129 α-synuclein, MAP2 (a somatodendritic α-synuclein pathology in these cultures as described pre- marker), and NeuN (a marker of neuronal nuclei). Both viously [12] by adding 2.5 μg/mL recombinant mouse NTG and G2019S neurons develop robust neuritic and α-synuclein pre-formed fibrils (PFFs) to the cultures at cell body pathology in comparison to neurons treated 7 days in vitro (DIV) and allowing the neurons to de- with PBS as a vehicle control which never develop velop pathology for a further 14 days post-transduction pS129 α-synuclein inclusions (Fig. 2a, b). We were able (DPT). The protein from neurons was then sequentially to detect a mild elevation of α-synuclein pathology in extracted in 1% TX-100 followed by 2% SDS, allowing the G2019S neurons, which was reversible with 30 and the separation of pathological, insoluble α-synuclein 120 nM PF-360 (Fig. 2a, b, c). In every other respect from soluble α-synuclein. Both NTG and G2019S neu- measured, NTG and G2019S neurons responded simi- rons accumulated insoluble α-synuclein phosphorylated larly to α-synuclein PFF treatment. The area covered by at residue Ser129 (pS129) to a similar extent (Fig. 1a, c). MAP2 and the number of neurons were not different Additionally, the autophagy receptor p62, which deco- between the two genotypes (Fig. 2d, e), and the mild ele- rates pathological inclusions, including Lewy bodies in vation in pathology did not induce further toxicity. In PD, accumulates to a similar extent in both genotypes addition, at no concentration did the LRRK2 inhibitors (Fig. 1a, c). Thus, a dramatic elevation in LRRK2 activity affect α-synuclein pathology, MAP2 area or neuron NTG G2019S NTG G2019S NTG G2019S NTG G2019S -syn LRRK2 pS129- -syn p-LRRK2 (S935) p62 -syn Henderson et al. Acta Neuropathologica Communications (2018) 6:45 Page 5 of 11 a PBS 1 g/mL -syn PFF PF-475 (nM) PF-360 (nM) 120 nM DMSO DMSO55 30 120 30 120 PF-475 NTG cd ** e G2019S 1.5 **** **** 0.5 0.5 0.5 PF-475 PF-360 PF-475 PF-360 PF-475 PF-360 PF-475 PF-360 PF-475 PF-360 PF-475 PF-360 (nM) (nM) (nM) (nM) (nM) (nM) (nM) (nM) (nM) (nM) (nM) (nM) PBS -syn PFF PBS -syn PFF PBS -syn PFF PBS -syn PFF PBS -syn PFF PBS -syn PFF Fig. 2 G2019S LRRK2 hippocampal neurons show mild, reversible elevation in induced α-synuclein pathology 21 days post-transduction. Primary hippocampal neurons from NTG (a) or G2019S (b) pups were transduced with α-synuclein PFFs and allowed to age a further 21 days prior to fixation and staining for pS129 α-synuclein (magenta), MAP2 (gray) and NeuN (blue). The neurons were additionally treated with LRRK2 inhibitors PF-475 and PF-360 2 days prior to transduction and fed with media containing inhibitors each week thereafter. No large differences can be observed in the type or abundance of α-synuclein pathology. c Quantification of α-synuclein pathology reveals a mild elevation in G2019S neurons, which is reversible with 30 or 120 nM PF-360. *P < 0.05 by Dunnett’s multiple comparison test between NTG and G2019S neurons or Sidak’s multiple comparisons test between G2019S neurons treated with LRRK2 inhibitors. d MAP2 area is reduced with 21 days α-synuclein PFF treatment in both NTG and G2019S neurons, and is not significantly affected by LRRK2 inhibitor treatment. *P < 0.05 by 2-way ANOVA with Dunnett’s multiple comparison test for comparison within genotype. e The number of neurons, as quantified by NeuN number, is reduced with 21 days α-synuclein PFF treatment in both NTG and G2019S neurons, although not significantly by 2-way ANOVA followed by Dunnett’s multiple comparison test and is not significantly affected by LRRK2 inhibitor treatment. (N = 12 biological replicates). Means + s.e.m.; all values are normalized to NTG neurons treated with α-synuclein PFFs and DMSO. Scale bars = 50 μm number in the NTG neurons indicating that there is previous publications showing a reduction of no apparent relationship between LRRK2 activity in α-synuclein pathology in response to both LRRK2 inhib- NTG neurons and α-synuclein pathology. We con- ition [32] and knockdown with anti-sense oligonucleo- clude that, while interesting, the observed elevation in tides [38]. While LRRK2 inhibitors are initially being α-synuclein pathology in the G2019S neurons is too tested in patients with LRRK2 mutations, there is specu- mild to make this a valuable screening tool for lation that if LRRK2 is a fundamental protein in PD LRRK2-dependent phenotypes. pathogenesis, LRRK2 inhibitors may provide clinical benefit for those without LRRK2 mutations. Therefore, LRRK2 inhibitors do not alter α-synuclein pathology in we sought to investigate LRRK2 in wildtype neurons wildtype neurons from a second, distinct strain of mice (CD1). We first The lack of an effect of LRRK2 inhibitors on α-synuclein sought to validate the effect of LRRK2 inhibitors on pathology in NTG neurons was surprising given LRRK2 activity in these neurons and further test these Norm. pS129 -syn Area/MAP2 Area LRRK2*G2019S Hipp Neurons Non-transgenic Hipp Neurons DMSO Merge + NeuN MAP2 pS129 -syn Merge MAP2 pS129 -syn PF-475 DMSO DMSO PF-475 DMSO Normalized MAP2 Area DMSO PF-475 DMSO DMSO PF-475 DMSO Normalized NeuN Number DMSO PF-475 DMSO DMSO PF-475 DMSO 120 Henderson et al. Acta Neuropathologica Communications (2018) 6:45 Page 6 of 11 1% TX-100 2% SDS Normalized Soluble Protein Levels a b 1.2 **** 1.0 1.0 0.8 0.8 0.6 0.6 25 0.4 0.4 -syn 20 0.2 0.2 0.0 0.0 pS129- -syn c Normalized Insoluble Protein Levels 15 * 1.2 1.2 1.25 1.0 1.0 LRRK2 1.00 0.8 0.8 0.75 p-LRRK2 (S935) 0.6 0.6 0.50 0.4 0.4 p62 0.25 0.2 0.2 0.00 0.0 0.0 GAPDH Fig. 3 LRRK2 inhibition does not reduce insoluble α-synuclein in wildtype hippocampal neurons. a Primary hippocampal neurons from CD1 pups were treated with 30 nM LRRK2 inhibitors PF-475, PF-360 or DMSO as a vehicle control, then transduced with 2.5 μg/mL α-synuclein PFFs and allowed to age a further 14 days prior to sequential detergent fractionation. TX-100-insoluble α-synuclein and p62 are similar in both LRRK2 inhibitor treated and untreated neurons. b Quantification of soluble proteins show some reduction of LRRK2 protein levels PF-475 treatment and ~ 75% inhibition of LRRK2 activity (as assayed by pS935 LRRK2) by both PF-475 and PF-360. c Insoluble pS129 α-synuclein was slightly, but significantly elevated by PF-360 treatment, while α-synuclein and p62 were unchanged by one-way ANOVA. (N = 5 biological replicates for each protein). Means + s.e.m.; *P < 0.05, ****p < 0.0001 by one-way ANOVA with Dunnett’s multiple comparison test. All values are normalized first to GAPDH, as a loading control, then to DMSO-treated neurons inhibitors on insoluble, pathological α-synuclein accumula- X-100, sucrose, and sarkosyl buffers, yielding a final pel- tion. CD1 hippocampal neurons were treated with LRRK2 let enriched in LB α-synuclein. This pellet was then sus- inhibitors at 5 DIV and transduced with 2.5 μg/mL pended in phosphate-buffered saline by sonication, α-synuclein PFFs at 7 DIV. Neurons were harvested by se- yielding a final concentration of α-synuclein from 7.5– quential detergent fractionation at 14 DPT. We were able 22.4 μg/mL, Fig. 5a, Table 1). As before, primary hippo- to confirm a robust ~ 75% inhibition of LRRK2 S935 phos- campal neurons were treated with LRRK2 inhibitors at 5 phorylation with 30 nM PF-475 or PF-360 (Fig. 3a, b). DIV followed by treatment with 40 ng/mL LB PF-475 also modestly reduced total LRRK2 levels (Fig. 3a, α-synuclein two days later. This is the maximum con- b). However, inhibition of LRRK2 resulted in no reduction centration that neurons can be treated with due to the in insoluble α-synuclein, pS129 α-synuclein, or p62 (Fig. 3a, relatively low concentration of α-synuclein in these c). Instead, pS129 α-synuclein was slightly,but significantly preps. Neurons were fixed and stained 14 days after the elevated by LRRK2 inhibitor treatment (Fig. 3a, c). addition of LB α-synuclein. The induced pathology is We sought to further validate this finding with a larger sparser than that induced by PFFs due to the lower con- family of LRRK2 inhibitors at increased concentrations centration of α-synuclein (Fig. 5b). The pathology in- that would leave no residual LRRK2 activity. We chose duced by LB α-synuclein and neuron health were not an additional, validated LRRK2 inhibitor (MLi-2) for fur- meaningfully altered by LRRK2 inhibition (Fig. 5b, c, d, ther investigation and added these inhibitors at concen- e), consistent with PFF treatment. trations ranging from 30 to 300 nM to CD1 neurons. Use of these inhibitors resulted in near complete deple- Dopaminergic neurons show no change in α-synuclein tion of pS935 LRRK2 (Fig. 4a, b). The neurons were then pathology in response to G2019S LRRK2 overexpression transduced with α-synuclein PFFs and fixed at 14 DPT. or LRRK2 inhibition No LRRK2 inhibitor, at any concentration, altered All the experiments to this point were performed in pri- α-synuclein pathology (Fig. 4c, d), MAP2 area (Fig. 4c, e) mary hippocampal neuron cultures. These cultures de- or neuron number (Fig. 4c, f). velop robust α-synuclein pathology and can be obtained We then tested whether inhibition of LRRK2 activity in an abundance suitable for both immunocytochemistry can alter α-synuclein pathology induced by a means and biochemistry. However, they are not the neurons other than PFFs. We have recently demonstrated the most affected in PD patients. In order to address ability of LB α-synuclein purified from human brain to whetherweweremissing aphenotype that is induce pS129 α-synuclein pathology in WT neurons dependent on expression of mutant LRRK2 in dopa- [24]. Cortical gray matter from brains with high LB bur- minergic neurons, we developed ventral midbrain and den were sequentially extracted with high salt, Triton striatum primary neuron co-cultures. When treated DMSO PF-475 PF-360 DMSO PF-475 DMSO PF-360 PF-475 PF-360 DMSO PF-475 PF-360 DMSO PF-475 PF-360 DMSO PF-475 PF-360 DMSO PF-475 PF-360 LRRK2 pS129- -syn -syn p-LRRK2 (S935) p62 Henderson et al. Acta Neuropathologica Communications (2018) 6:45 Page 7 of 11 MLi-2 PF-360 PF-475 a d 1.2 **** (nM) (nM) (nM) 1.0 DMSO 30 300 30 300 30 300 kDa 0.8 LRRK2 0.6 pS935 LRRK2 0.4 GAPDH 0.2 0.0 2.0 1.2 PF-475 PF-360 MLi-2 1.5 (nM) (nM) (nM) 0.8 PBS 1 g/mL -syn PFF 1.0 0.4 e 0.5 1.25 ** ** ** ** 0.0 0.0 1.00 PF-475 PF-360 MLi-2 PF-475 PF-360 MLi-2 0.75 (nM) (nM) (nM) (nM) (nM) (nM) 0.50 0.25 PBS 1 g/mL -syn PFF 0.00 300 nM 300 nM 300 nM DMSO DMSO PF-475 PF-360 MLi-2 PF-475 PF-360 MLi-2 (nM) (nM) (nM) PBS 1 g/mL -syn PFF f ** 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 PF-475 PF-360 MLi-2 (nM) (nM) (nM) PBS 1 g/mL -syn PFF Fig. 4 LRRK2 inhibition does not reduce pathological α-synuclein in wildtype hippocampal neurons. a Primary cortical neurons were treated with LRRK2 inhibitors PF-475, PF-360, or MLi-2, at 5 DIV and fed with media containing inhibitors each week for 16 days. Cell lysate was run by Western blot to detect LRRK2 and pS935 LRRK2, which is indicative of LRRK2 activity. Images shown are representative of n =4–12 biological replicates. b Quantification of Western blot of LRRK2 and pS935 LRRK2. LRRK2 levels were not significantly altered by one-way ANOVA, but pS935 levels were reduced to near undetectable levels (*p < 0.05, **p < 0.01, Kruskal-Wallis test with Dunn’s multiple comparison test). c Primary hippocampal neurons from CD1 pups were transduced with α-synuclein PFFs and allowed to age a further 14 days prior to fixation and staining for pS129 α-synuclein (magenta), MAP2 (gray) and NeuN (blue). The neurons were additionally treated with LRRK2 inhibitors PF-475, PF-360, or MLi-2, 2 days prior to transduction and fed with media containing inhibitors each week thereafter. No large differences can be observed in the type or abundance of α-synuclein pathology. d Quantification of α-synuclein pathology reveals no change in response to LRRK2 inhibition, while PBS-treated neurons have no pathology (****p < 0.0001 by Dunnett’s multiple comparison test). MAP2 area (e) and neuron number (f) are also not altered in response to LRRK2 inhibition. No significant response was seen when compared with vehicle-treated neurons by one-way ANOVA with Dunnett’s multiple comparison test (d) or by Kruskal-Wallis test followed by Dunn’s multiple comparison test (e) and (f). (N = 9 biological replicates). Means + s.e.m.; all values are normalized to neurons treated with α-synuclein PFFs and DMSO. Scale bars = 50 μm with α-synuclein PFFs at 7 DIV and allowed to age to G2019S dopaminergic neurons, and LRRK2 inhibi- 14 DPT, dopaminergic neurons positive for TH de- tors did not significantly improve survival of TH+ velop cell body and neuritic pS129 α-synuclein path- neurons (Fig. 6c). ology (Fig. 6a). We treated midbrain/striatum co-cultures from NTG and G2019S pups with 30 nM Discussion LRRK2 inhibitors or vehicle control at 5 DIV. The In this study, we utilized primary neuron cultures to neurons were then treated with α-synuclein PFFs at 7 assess the status of α-synuclein pathology in relation DIV and fixed 14 DPT and stained for pS129 to LRRK2 activity. We performed biochemical and α-synuclein and TH. Both cultures develop a similar immunocytochemical analysis of primary hippocampal amount of α-synuclein pathology in TH+ neurons, and midbrain neurons cultured from two strains of and none of the LRRK2 inhibitors changed the level wildtype mice and a mouse expressing LRRK2 with of pathology in either NTG or G2019S neurons (Fig. the familial G2019S mutation. To further manipulate 6a, b). Further, α-synuclein PFFs were not more toxic LRRK2 activity, we used three validated LRRK2 inhibitors. No r m . L R RK2/ Merge + NeuN MAP2 pS129 -syn GA P D H DMSO N o r m . p S 935 L R R K 2 /G A P D H DMSO N o r m . pS129 -syn A r ea No r m . M AP 2 Ar ea No r m . Neu N Nu m b er /M A P 2 A r ea DMSO DMSO DMSO PF-475 PF-475 PF-475 MLi-2 MLi-2 MLi-2 DMSO DMSO DMSO 30 30 30 100 100 100 300 300 300 30 30 30 100 100 100 300 300 300 30 30 30 100 100 100 300 300 300 Henderson et al. Acta Neuropathologica Communications (2018) 6:45 Page 8 of 11 1.4 Triton Buffer HS Buffer a c 1.2 100,000xg 100,000xg ** 1.0 30 min 30 min 0.8 0.6 Human LB brain 0.4 Sucrose buffer 100,000xg 0.2 +/- LRRK2 30 min 0.0 Inhibitors PBS to Sarkosyl Buffer Sark 100,000xg PBS 40 ng/mL LB -syn Pellet 30 min 1.2 1.0 0.8 0.6 PBS 40 ng/mL -syn PFF 0.4 DMSO DMSO PF-475 PF-360 MLi-2 0.2 0.0 PBS 40 ng/mL LB -syn 1.25 1.00 0.75 0.50 0.25 0.00 PBS 40 ng/mL LB -syn Fig. 5 LRRK2 inhibition does not alter pathology induced by human Lewy body α-synuclein. a A schematic representation of pathological α- synuclein purification from human cortical tissue. b Primary hippocampal neurons from CD1 pups were treated with 100 nM LRRK2 inhibitors or a vehicle control. Two days later, neurons were treated with 40 ng/mL human LB α-synuclein and allowed to age a further 14 days prior to fixation and staining for pS129 α-synuclein (magenta), MAP2 (gray) and NeuN (blue). No large differences can be observed in the type or abundance of α-synuclein pathology. c Quantification of α-synuclein pathology reveals an increase in neurons treated with LB α-synuclein compared to those treated with PBS (**p < 0.01), but no change in response to LRRK2 inhibition. MAP2 area (d) and neuron number (e) are also not altered meaningfully in response to LRRK2 inhibition. (N =11–12 biological replicates treated with LB α-synuclein from 4 separate cases (2 AD, 1 PDD, 1 DLB). Means + s.e.m.; all values are normalized to neurons treated with LB α-synuclein and DMSO. Scale bars = 50 μm In none of these assays did we observe more than a mild LRRK2 [8, 18], but in keeping with the many transgenic LRRK2-dependent alteration in α-synuclein pathology. models of LRRK2 that have since been generated [3, 19, The LRRK2*G019S neurons we describe have ~ 20, 37]. While there is strong support that overexpression 25-fold elevated LRRK2 levels and ~ 50-fold elevated of LRRK2 does not dramatically alter cell health, the effect kinase activity (Fig. 1a, b). Despite this dramatic overex- of LRRK2 on α-synuclein has been unclear. pression of LRRK2, there is no apparent effect on the We induced pathological α-synuclein inclusion forma- neuron health or viability (Figs. 2, 4, 5, and 6). This is in tion in NTG and G2019S neurons through addition of contrast with early research using viral overexpression of recombinant α-synuclein PFFs or human brain-derived Table 1 Clinical Information of the cases used for the Extraction of Pathological alpha-synuclein Case No. Clinical Pathological Race Sex Age of Disease Age at PMI (h) Brain Region Used α-Synuclein Diagnosis Diagnosis onset Death (μg/ml) 1 AD Probable AD/DLB White Female 52 62 11 Middle Frontal Gyrus 20.86 2 CBS AD/DLB White Female 45 55 15 Middle Frontal Gyrus 22.43 3 DLB DLB White Male 76 83 6 Cingulate Gyrus 7.55 4 PDD PDD White Male 60 72 19 Cingulate Gyrus 8.28 PMI postmortem interval DMSO DMSO PF-475 PF-360 MLi-2 DMSO DMSO PF-475 PF-360 MLi-2 DMSO DMSO PF-475 PF-360 MLi-2 Merge + NeuN MAP2 pS129 -syn N o r m . pS129 a-syn A r e a / No r m . Neu N Nu m b er N o r m . M A P 2 A r ea MA P 2 A r ea Henderson et al. Acta Neuropathologica Communications (2018) 6:45 Page 9 of 11 a b PBS 1 g/mL -syn PFF DMSO DMSO PF-475 PF-360 MLi-2 NTG 2.0 G2019S 1.5 *** ** 1.0 0.5 0.0 -syn PFF -syn PFF 2.5 2.0 1.5 1.0 0.5 0.0 -syn PFF -syn PFF Fig. 6 Neither G2019S LRRK2 expression nor LRRK2 inhibition alters α-synuclein pathology in midbrain neurons. a Primary midbrain/striatum cultures from NTG or G2019S pups were transduced with α-synuclein PFFs and allowed to age a further 14 days prior to fixation and staining for pS129 α- synuclein (magenta) and TH (gray). The neurons were additionally treated with 30 nM LRRK2 inhibitors PF-475, PF-360, or MLi-2 2 days prior to transduction and fed with media containing inhibitors each week thereafter. b Quantification of α-synuclein pathology in TH+ neurons shows no effect of G2019S LRRK2 expression or LRRK2 inhibitor treatments by 2-way ANOVA (**p < 0.01, ***p < 0.001 for PBS- compared to PFF-treated neurons by Dunnett’s multiple comparison test). c The number of TH+ neurons showed no significant response to treatment by Kruskal-Wallis test followed by Dunn’s multiple comparison test. (N =6–9 biological replicates). Means + s.e.m.; all values are normalized to NTG neurons treated with α-synuclein LB material and DMSO. Scale bars = 50 μm LB α-synuclein. We designed our initial experiments to LRRK2 protein reduction by anti-sense oligonucleotides test the relationship between LRRK activity and [38] are able to ameliorate α-synuclein pathology in α-synuclein at 14 DPT, before the onset of neurodegen- wildtype neurons. To ensure the validity of our results, eration. We found that there was no alteration of we used both biochemical extraction of pathological α-synuclein pathology in G2019S neurons. A recent α-synuclein as well as immunocytochemistry. Pathology publication using a similar model showed a mild eleva- induced by both recombinant α-synuclein PFFs and hu- tion in α-synuclein in G2019S hippocampal neurons at man LB α-synuclein were resistant to alteration by 18 DPT, while detecting no difference at 7 DPT [32]. LRRK2 inhibition. We also used three validated LRRK2 We found that at 21 DPT, we did observe a mild en- inhibitors, representing different classes of compounds. hancement of α-synuclein pathology in G2019S neurons All immunocytochemical quantification of α-synuclein that was responsive to LRRK2 inhibition (Fig. 2). At this was normalized to MAP2 to ensure no effect of cell timepoint, neurons have begun degenerating, and while density. Our results in wildtype hippocampal neurons we see no LRRK2-dependent difference in degeneration, generated from two strains of mice in addition to dopa- it is unclear if it is the neurodegenerative process that minergic midbrain neurons give us increased confidence results in a mild elevation of α-synuclein pathology in that the lack of LRRK2-dependent phenotypes we see G2019S neurons. are valid. To further explore the role of endogenous LRRK2 in LRRK2 is the most common cause of inherited PD. α-synuclein pathogenesis, we cultured wildtype hippo- Even so, only around 30% of those with G2019S muta- campal and midbrain neurons, and showed using bio- tion in LRRK2 will go on to develop PD [22]. It is there- chemistry and immunocytochemistry that LRRK2 kinase fore probable that LRRK2 mutations exacerbate an inhibition is unable to alter induced α-synuclein path- extant predisposition to PD. Another hypothesized de- ology (Figs. 3, 4, 5, and 6). These findings are in sharp terminant of susceptibility to PD is the misfolding of contrast to recent reports that LRRK2 inhibitors [32]or α-synuclein within aging brains. It is logical to assume LRRK2*G2019S MB/Str Neurons Non-transgenic MB/Str Neurons Merge TH pS129 -syn Merge TH pS129 -syn N o r m . pS129 -syn+ T H N o r m . T H N u m b er Area/T H A r ea PBS DMSO PBS DMSO DMSO DMSO PF-475 PF-475 PF-360 PF-360 Mli-2 Mli-2 PBS DMSO PBS DMSO DMSO DMSO PF-475 PF-475 PF-360 PF-360 Mli-2 Mli-2 Henderson et al. Acta Neuropathologica Communications (2018) 6:45 Page 10 of 11 that challenging neurons with both misfolded Culture Service Center for their assistance procuring CD1 hippocampal neuron cultures. α-synuclein and mutated LRRK2 would be sufficient to drive an enhanced pathogenic process. While there is Funding compelling evidence that this may be true in mouse This study was supported by the Michael J. Fox Foundation and National Institute of Health grants: T32-AG000255, P30-AG10124, P50-NS053488. models [3, 5, 32], we are unable to generate a robust model of LRRK2-dependent α-synuclein pathogenesis in Availability of data and materials primary neuron cultures. There are several possible rea- The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. sons that in vivo models cannot be recapitulated in cell culture. Neuron cultures are inherently closed systems. Authors’ contributions Therefore, if LRRK2 activity can affect the spread of Conception and design: MXH, CP, JQT, VMYL; Acquisition and analysis of data: MXH and CP; Drafting of the article: MXH, VMYL. All authors read and α-synuclein pathology through the brain, this effect approved the final manuscript. would not be captured in a dish. Primary neuron cul- tures also lack all the cell types and complex synaptic Ethics approval and consent to participate All housing, breeding, and procedures were performed according to the NIH organization present in vivo. Indeed, LRRK2 is highly Guide for the Care and Use of Experimental Animals and approved by the expressed in astrocytes [13] and neuroinflammation has University of Pennsylvania Institutional Animal Care and Use Committee. been implicated in LRRK2-dependent phenotypes [3]. It Competing interests is also possible that LRRK2 is not critical in the early The authors declare that they have no competing interests. stages of pathology formation, and only during later stages of disease progression does LRRK2 activity be- Publisher’sNote come important. This is supported by our finding that Springer Nature remains neutral with regard to jurisdictional claims in α-synuclein pathology is only elevated in G2019S neu- published maps and institutional affiliations. rons 21 days after transduction, after the onset of neuro- Received: 20 May 2018 Accepted: 21 May 2018 degeneration. 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Published: May 31, 2018

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