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Exp Neurobiol 2021; 30(3): 232-243
Published online June 30, 2021
© The Korean Society for Brain and Neural Sciences
Hyejung Kim1†, Hyuna Sim2,3†, Joo-Eun Lee2, Mi Kyoung Seo4, Juhee Lim5‡, Yeojin Bang5, Daleum Nam1, Seo-Young Lee 6, Sun-Ku Chung7, Hyun Jin Choi5, Sung Woo Park4,8, Ilhong Son1,9, Janghwan Kim2,3* and Wongi Seol1*
1InAm Neuroscience Research Center, Sanbon Medical Center, College of Medicine, Wonkwang University, Gunpo 15865, 2Stem Cell Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, 3Department of Functional Genomics, KRIBB School of Bioscience, University of Science and Technology, Daejeon 34141, 4Paik Institute for Clinical Research, Inje University College of Medicine, Busan 47392, 5College of Pharmacy, CHA University, Seongnam 13496, 6Division of Clinical Medicine, Korea Institute of Oriental Medicine, Daejeon 34054, 7Division of Herbal Medicine Research, Korea Institute of Oriental Medicine, Daejeon 34054, 8Department of Convergence Biomedical Science, Inje University College of Medicine, Busan 47392, 9Department of Neurology, Sanbon Medical Center, College of Medicine, Wonkwang University, Gunpo 15865, Korea
Correspondence to: *To whom correspondence should be addressed.
Janghwan Kim, TEL: 82-42-860-4478, FAX: 82-42-860-4608
Wongi Seol, TEL: 82-31-390-2411, FAX: 82-31-390-2414
†These authors contributed equally to this work.
‡Present address: College of Pharmacy, Woosuk University, Wanju 55338, Korea.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Mutations in the Leucine-rich repeat kinase 2 (
Keywords: Primary cilia, Neuron, LRRK2 kinase, Ciliogenesis, Parkinson’s disease
Several mutations in the leucine-rich repeat kinase 2 (
Cilia are cellular appendages that protrude from the cell body and are found in most cells including neurons. Primary cilia in neurons play roles in modulating neurogenesis such as axonal guidance and cell polarity . Functional cilia are known to be critical for the development of midbrain dopaminergic neurons via Sonic hedgehog (Shh)-mediated signaling . Serum starvation is one of the major cues inducing ciliogenesis, the formation of primary cilia .
LRRK2 specifically phosphorylates a subset of Rab proteins, major regulators of vesicle trafficking [10, 11], which is essential for ciliogenesis. In addition, Rab8 and Rab10 are crucial regulators of ciliogenesis [12, 13]. Recent studies reported that the kinase activity increased by LRRK2 pathogenic R1441C/G and G2019S mutations impaired ciliogenesis via Rab8/10 phosphorylation [11, 14]. However, an earlier study showed that only Rab10 knockout (KO), but not Rab8 KO, increased ciliogenesis although both Rab8 and 10 are substrates of LRRK2 kinase . Further studies disclosed that PPM1H specifically dephosphorylated Rab proteins phosphorylated by LRRK2, and PPM1H knockdown suppressed ciliogenesis . Sobu et al.  reported that treatment of pathogenic LRRK2 R1441C MEF cells with MLi-2, an LRRK2 kinase inhibitor, significantly increased ciliogenesis at least 3-fold. Another study reported that the proportion of ciliated cells in HEK293T cells expressing
The SH-SY5Y cell line is a widely used cellular model of dopaminergic neurons . Bae et al.  reported that treatment of SH-SY5Y and RPE cells with mitochondrial respiratory complex-1 inhibitors, rotenone and 1-methyl-4-phenylpyridinium (MPP+), which are also PD-causing chemicals, increased ciliogenesis and cilium length. They further showed that a decrease in mitochondrial fission by siDrp1 or fusion by siOPA1, decreased or increased ciliogenesis and cilium length, respectively . They concluded that primary cilia modulate mitochondrial stress to enhance dopaminergic neuron survival. Because both an increase in LRRK2 kinase activity and cellular exposure to MPP+ and rotenone are major causes of PD development, the results of these studies are somewhat contradictory [11, 14, 15, 17, 20]. Except for the study by Bae et. al, most of these studies were carried out in non-neuronal cells such as MEF, hTERT-RPE, iPSCs, A549, and HEK-293T cells, although the immunostainings of cilia in in brain tissue neurons  and primary astrocytes  have been reported.
Therefore, we decided to test the effect of LRRK2 kinase activity on ciliogenesis in cell models such as primary neurons and neural stem cells (NSCs) differentiated from human induced pluripotent stem cells (iPSCs) derived from fibroblasts obtained from a PD patient with the G2019S mutation as well as differentiated SH-SY5Y cells. Our results suggest that the relationship between LRRK2 kinase activity and ciliogenesis is dependent upon the cell type or genetic background, and is much more complicated than presented in previous reports.
Human fibroblasts from a PD patient harboring the
Human iPSCs were generated from WT (MRC5, ATCC® CCL-171) and G2019S+/- (ND38262, Coriell) fibroblasts using episomal vectors and electroporation with the Neon transfection system (Invitrogen, Carlsbad, CA, USA) as described previously . Established human iPSCs were fed TeSRTM-E8TM (STEMCELL Technologies, Vancouver, BC, Canada) according to the manufacturer’s recommendations. Neural stem cells (NSCs) were differentiated from human iPSCs as previously described  with some modifications . Human iPSCs were seeded on GeltrexTM LDEV-free Reduced Growth Factor Basement Membrane Matrix-coated dishes at about 20% confluency. For neural induction, Neural Induction Basal Medium 1 [NIM: 50% Advanced DMEM/F12, 50% Neurobasal medium, N-2 supplement (100X), B-27 supplement (50X) minus vitamin A (B27), 1% GlutamaxTM, and 1% P/S] supplemented with 10 ng/ml human LIF (PeproTech, Inc., Rocky Hill, NJ, USA), 4 μM CHIR99021, 3 μM SB431542 (Tocris Bioscience, Bristol, UK), 2 μM Dorsomorphin, and 0.1 μM Compound E (Sigma-Aldrich, St. Louis, MO, USA) were used to treat the cells for two days. Then, the differentiating cells were fed with the same medium without Dorsomorphin for another five days. On day 7 of differentiation, the cells were passaged with Accutase cell detachment solution (Sigma-Aldrich) and maintained in NIM supplemented with 10 ng/ml human LIF, 3 μM CHIR99021, and 2 μM SB431542. NSCs were passaged every week using Accutase cell detachment solution. The NSCs were treated with 1 μM MLi-2 (MedChemExpress, NJ, USA) or dimethyl sulfoxide (DMSO, Sigma-Aldrich) for control cells for three days before the detection of ciliogenesis.
The NSCs were further differentiated into neuronal cells as previously described  with some modifications. The NSCs were plated on poly-L-ornithine (PLO)/laminin (Sigma-Aldrich)-coated dishes. Two days after seeding, the medium was changed to neuronal differentiation medium (NDM: Neurobasal medium, B-27, 1% GlutamaxTM supplement, and 1% P/S) supplemented with 20 ng/ml brain-derived neurotrophic factor (BDNF, Research and Diagnostic Systems, Inc., Minneapolis, MN, USA), 20 ng/ml glial cell-derived neurotrophic factor (GDNF, Research and Diagnostic Systems, Inc.), 0.2 mM 2-phospho ascorbic acid, 400 μM dibutyryl-cAMP (Enzo Life Sciences, NY, USA) and 1 ng/mL transforming growth factor β-3 (TGF-β3, PeproTech, Inc., Rocky Hill, NJ, USA). Half of the medium volume was changed every three days. On day 14 of the differentiation, the cells were re-plated on PLO/laminin-coated dishes. On day 18 of the differentiation, the cells were treated with 1 μM MLi-2 or DMSO control for 24 h before the detection of ciliogenesis.
The PD patient’s B-lymphocytes with an LRRK2 G2019S mutation (ND14317, Coriell) were reprogrammed into G2019S iPSCs  and then, the G2019S mutation was corrected to generate the KIOMi002-A WT iPSC line to prepare an isogenic WT/G2019S pair . The WT/G2019S pair of iPSCs were incubated and treated with 400 nM MLi-2 for 24 h. The same WT/G2019S pair of iPSCs were differentiated to NSCs as above, and cultured with or without B27 for 24 h. Both cell types were stained with anti-Arl13b (Proteintech, # 17711-1-AP) to detect cilia as described below.
Pregnant Sprague-Dawley rats were obtained from Orient Bio (Seongnam, South Korea) and the experimental procedures were approved by the Inje Medical College Committee for Animal Experimentation and the Institutional Animal Laboratory Review Board (Approval no. 2018-016).
Cortical or hippocampal neuronal cultures were prepared from the cortex or hippocampus, respectively, of fetuses on embryonic day 19 as previously described . To test for ciliogenesis, the cells were plated on 12-well dishes at a density of 1×105 cells per well and the culture medium with 1X anti-mitotic agent (80 μM 5’fluoro-2’ deoxyuridine and 40 μM uridine) was changed every two days. After incubation for 14 days, the cells were treated with 1 μM GSK-2578215A (GSK) or 100 nM MLi-2 for one day before immunostaining for ciliogenesis analysis.
G2019S+/- mice were generated from crossings G2019S [FVB/N-Tg(LRRK2*G2019S)1Cjli/J; The Jackson Lab. No: 009609] and WT mice [FVB/NJ; The Jackson Lab. No: 001800]. We were able to obtain only two G2019S+/- mice by two independent crossings. After genotyping, murine cortical neurons were prepared from G2019S+/- and their WT littermate mice using a method similar to that used for rat primary neuronal culture (IACUC approval no. 190095) and grown in Neurobasal medium with 1% L-glutamine, 2% B27, and 1% P/S for nine days. Then, the cells were incubated with or without B27, for 24 h and subjected to immunostaining.
SH-SY5Y cells were cultured in DMEM (Corning, #10-013-CV) with 10% FBS and 1% P/S, and differentiated with 13 μM retinoic acid for six days. The differentiated SH-SH5Y (dSH-SY5Y) cells were treated with MLi-2 for 24 h before immunostaining. SN4741 dopaminergic neuronal cells were cultured in DMEM with 10% FBS and 1% P/S at 34°C.
The pEGFP vector was used to construct pGFP-Rab10 expressing human Rab10 protein fused to GFP. SN4741 cells were transfected with pEGFP or GFP-Rab10 using Lipo-D293 (SignaGen Lab. Frederick, MD, USA). One day after transfection, the cells were serum-starved for 24 h, fixed, and immunostained with the Arl13B antibody.
The cells were washed with Dulbecco's Phosphate-Buffered Saline (DPBS, Welgene, Korea) and fixed in 4% formaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) for 10 min at room temperature. After washing with DPBS, the cells were blocked and permeabilized with 3% BSA and 0.3% TritonX-100 (Sigma-Aldrich) in DPBS for 1 h at room temperature.
The cells were stained with anti-Arl13b (1:100~250) in 1% BSA in DPBS and washed with 0.1% BSA in DPBS, followed by incubation in the proper secondary antibody. If needed, anti-βIII tubulin (Merck Millipore, AB9354) was used for staining. Hoechst33342 was used to visualize the nuclei. Cell images were taken with a confocal microscope or a FLoid cell imaging station (Thermo Fisher Scientific) and the ciliated cells were counted by two persons blinded to the treatments. We used Arl13B as a cilia marker for the entire study. To obtain the percentage of ciliated cells, the number of Arl13B+ cells was counted as ciliated cells and divided by the total number of cells.
To compare LRRK2 kinase activity, pT73-Rab10 antibody (Abcam, #ab230261) was used after gel electrophoresis of cell lysates, as described previously . In addition, β-actin (Santa-Cruz, #sc-47778) antibodies were used to normalize the loading amounts.
The Prism5 program (GraphPad Software, La joplla, CA, USA) was used to perform the statistical analyses. The data were analyzed by one-way ANOVA with Tukey’s or Sidak’s multiple comparison test. For experiments with two kinds of samples, the Student’s
To investigate the effect of LRRK2 kinase activity on ciliogenesis in neurons, we first tested ciliogenesis in the dSH-SY5Y human dopaminergic neuronal cell line with or without serum starvation and treatment with MLi-2 (Fig. 1A). The dSH-SY5Y cells exhibited 50~60% ciliogenesis. However, neither serum starvation nor MLi-2 treatment produced a distinct difference. In detail, serum starvation showed no increase in ciliogenesis, contrasting to previous study results using undifferentiated SH-SY5Y cells . MLi-2 treatment showed an increase as reported , but the increase was very weak at ~1.1-fold (Fig. 1A), compared to the 2~3-fold increase previously reported in LRRK2 R1441C/G MEF cells [14, 16].
Because the result was different from the previous reports showing that MLi-2 treatment increased ciliogenesis in various cell types [11, 14], we tested rat cortical or hippocampal neurons with LRRK2 kinase inhibitors, GSK or MLi-2, respectively, under normal neuronal culture conditions. The primary neurons showed 22~31% ciliogenesis, but neither inhibitor treatment showed a significant difference compared to the untreated controls (Figs. 1B, 1C). The activity of MLi-2 as an LRRK2 kinase inhibitor was confirmed based on decreased levels of pT73-Rab10 in the corresponding cell lysates (Fig. 1D). We then tested rat cortical neurons with MLi-2 in the same culture conditions, but again, there was no significant difference (data not shown).
Because we could not observe the effect of the LRRK2 kinase inhibitor on ciliogenesis in neuronal cultures, we used primary cortical neurons prepared from heterozygous G2019S+/- and their littermate WT mice, and directly measured the effect of LRRK2 kinase activity on ciliogenesis without kinase inhibitor. Primary neurons were prepared, cultured, and immunostained for both Arl13B and βIII tubulin, and the Arl13B+ and βIII tubulin+ cells were counted as neurons with cilia. There were ~70~80% ciliated cells, similar to the previous reports [28, 29], but there was little difference in ciliogenesis between the G2019S+/- and WT littermate cells (Fig. 2A). Both serum starvation and autophagy have been reported as major inducers of ciliogenesis [9, 20, 30]. Although primary neuronal culture medium does not contain serum, the medium includes B27, a serum substitute [31, 32], for primary neuronal culture. To mimic serum-starvation excluding nutrients, we omitted the B27 supplement during the culture of these cortical neurons . Our B27 deprivation was toxic to the neurons derived from WT mice, but not to those from G2019S+/- mice (data bot shown). The B27 deprivation exhibited no significant effect on the ciliogenesis of G2019S+/- primary neurons (Fig. 2A). We reconfirmed the G2019S+/- genotype by polymerase chain reaction (PCR) analysis of tail DNA isolated from each mouse (Fig. 2B).
Taken together, the results we observed in primary neurons and dSH-SY5Y dopaminergic neuronal cells were different from the previous reports showing that LRRK2 kinase activity decreased ciliogenesis [11, 14, 15, 17].
To further test the effect of LRRK2 kinase activity on ciliogenesis, we used human fibroblasts originating from a PD patient with the G2019S mutation (ND38262) and WT MRC5 cells, a pair that has been used to produce neuroectodermal spheres by reprogramming technology . Under serum-fed conditions, the G2019S fibroblasts unexpectedly showed a significant ~13-fold increase in ciliated cells compared to the WT cells (Fig. 3A). Most previous studies were carried out under serum starvation, and reported that a decrease in LRRK2 kinase activity increased ciliogenesis in MEF and other cells [11, 14, 16]. When we cultured the cells under serum starvation without MLi-2, the G2019S fibroblasts again showed a ~5-fold (from 13 to 67%) increase in ciliated cells compared to the WT and the difference was maintained even after MLi-2 treatment, although the fold-difference was smaller (Fig. 3B). In contrast, MLi-2 treatment alone produced a nonsignificant slight increase within the same genotype by ANOVA, although the difference was significant in WT by the
Serum starvation and a decrease of LRRK2 kinase activity have been reported to increase ciliogenesis in various cell types [11, 14]. Therefore, we assumed that serum starvation would decrease LRRK2 kinase activity and tested it in the MRC5 cell line. WT fibroblasts were exposed to serum starvation and MLi-2 treatment for 24 h, and the cell lysates were subjected to Western blot analysis using the pRab10 antibody. The results showed that LRRK2 kinase activity was significantly decreased to ~60% after serum starvation, and to 17~19% after treatment with MLi-2, the positive control (Fig. 3C), confirming that serum starvation indeed decreased LRRK2 kinase activity. Because our ciliogenesis result was strikingly different from the previous reports, we reconfirmed each genotype by DNA sequencing of PCR products spanning a region including the G2019S mutation (Fig. 3D).
In addition, iPSCs that originated from the same WT/G2019S fibroblast pair were differentiated into neurons via NSCs , and tested their ciliogenesis with or without MLi-2 treatment. The percentages of ciliated cells in the differentiated neurons were too low (less than 2%) to conduct meaningful analysis regardless of the genotypes or MLi-2 treatment for reasons that were not clear (data not shown). We then counted the ciliated NSC cells and observed a result similar to the one in the parental fibroblasts. The ciliogenesis in G2019S cells significantly increased from 7 to 48 % and from 17 to 46 % compared to the WT cells without and with MLi-2, respectively, whereas MLi-2 treatment alone resulted in no significant difference in either genotype (Fig. 3E).
Dhekne et al.  reported that G2019S+/- iPSCs originating from the fibroblasts of a patient with a heterozygous G2019S mutation exhibited a significant decrease in ciliogenesis, from 33% to 15%, compared to its gene-corrected WT iPSCs, and that the MLi-2 treatment of the G2019S+/- cells rescued the decrease. In contrast, the genetic backgrounds of our WT/G2019S+/- fibroblast pair were different from each other (Fig. 3). To test whether the difference between our study and their study was due to the difference in the cellular genetic backgrounds, we utilized the previously reported iPSCs originating from G2019S+/- B lymphocytes and its gene-corrected WT iPSCs . The higher kinase activity of the G2019S iPSCs compared to the corrected WT counterparts was previously proven by increased levels of pRab10 in the G2019S cells . The corrected pair of iPSCs showed 16~22% of ciliogenesis with no significant difference regardless of the genotype or MLi-2 treatment (Fig. 4A). We also tested the effect of B27 deprivation in differentiated NSCs and obtained no significant difference regardless of the genotype and B27 deprivation (Fig. 4B). Unlike the previous WT/G2019S pair (Fig. 3), the isogenic WT/G2019S+/- pair exhibited no significant difference regardless of the genotype or B27 deprivation, maintaining 26~40 % ciliated cells although a slight nonsignificant increase was observed in the WT cells after B27 deprivation (Fig. 4B).
Rab10, an LRRK2 kinase substrate, has been reported to be a ciliogenesis suppressor because its expression decreased ciliogenesis in HEK 293 and A549 cells [14, 17]. We tested whether the pattern was also observed in neuronal cells. We transfected SN4741 murine dopaminergic neuronal model cell line with green fluorescent protein (GFP) or GFP-Rab10 WT and tested their ciliogenesis under serum starvation. We used only Rab10 WT and not phospho-mimic (T73D) or -dead (T73A) mutants because these mutants have been reported to be non-functional . The transfection of each cell was confirmed by its fluorescence, and the ciliated cells were immunostained using the Arl13B antibody. GFP+/Arl13B+ cells and GFP+/Arl13B- cells were counted separately. As a control, GFP-/Arl13B+ cells were also counted as untransfected ciliated cells (none in Fig. 5A). The expression of both GFP and GFP-Rab10 was also confirmed by Western blots (Fig. 5B). The results showed that ciliogenesis was similar among the samples transfected or non-transfected and the overexpression of GFP-Rab10 or control GFP caused no change in ciliogenesis, which was about 38%.
Our study started from a simple question on how LRRRK2 kinase activity affected on ciliogenesis in neurons. To answer it, we used various types of cells such as rat or mouse primary neurons and differentiated SH-SY5Y cells, and compared WT and G2019S genotype pairs of human fibroblasts as well as iPSCs and NSCs, and an isogenic WT/G2019S pair. In addition, various conditions such as serum starvation, B27 deprivation or MLi-2 treatment were applied.
Previous studies have repeatedly and clearly reported that LRRK2 pathogenic mutations such as G2019S or R1441C/G reduced ciliogenesis and LRRK2 kinase inhibitor treatment increased ciliogenesis, suggesting that LRRK2 kinase activity negatively regulates ciliogenesis [11, 14, 15, 17]. However, our results suggest that the relationship between LRRK2 kinase activity and ciliogenesis is not as simple as previously reported. We could detect little difference in ciliogenesis between WT and G2019S genotypes (Fig. 2, 4). The only significant difference was observed in WT and G2019S+/- fibroblasts and NSCs (Fig. 3), which showed an increase in ciliogenesis in G2019S+/- cells, opposite the results in previous reports. We reproducibly observed more than a~6-fold increase in ciliogenesis in G2019S+/- fibroblasts compared to WT (Fig. 3A) and similar results were obtained in a separate laboratory when the same cell pair was used. Decreases and increases in LRRK2 kinase activity by MLi-2 treatment and in the G2019S genotype, respectively, were confirmed by pT73-Rab10 levels in Western blot analysis of the corresponding cell lysates (Fig. 3Bii). In contrast to the fibroblast and NSC results in Figure 3, little difference in ciliogenesis was observed between primary neurons prepared from G2019S+/- and WT littermate mice (Fig. 2) and in another human WT/G2019S pair under an isogenic background (Fig. 4). Our data suggested that the cell type, as well as the genetic or species background, is more critical to determining ciliogenesis than the LRRK2 kinase activity.
Another difference was the small effect of LRRK2 kinase inhibitor treatment on ciliogenesis, suggesting again weak correlation between LRRK2 kinase activity and ciliogenesis. In most cases, MLi-2 treatment resulted in little difference in both WT and the pathogenic G2019S genotype (Figs. 1, 3, 4), although a weak increase was observed in ciliogenesis in dSH-SY5Y cells (Fig. 1A) and WT fibroblasts (Fig. 3B). It is worth noting that Lara Ordónez et al.  observed an increasing effect on ciliogenesis by MLi-2 treatment only in HEK 293T cells expressing LRRK2 pathogenic mutations, but not WT, under serum-fed conditions.
A few points can be considered the reasons for the difference in WT vs. G2019S results between our study and previous studies [14, 17]. The most probable explanation is that the regulation of ciliogenesis might be primarily genetic- and/or cell type-specific . Ciliogenesis induced by serum starvation was observed in several cell types . However, one study reported that HEK293T cells exhibited no stimulation of ciliogenesis by serum starvation and there was no difference between WT and G2019S knock-in mice astrocytes under serum starvation condition regardless of MLi-2 treatment . In addition, a deficit in ciliogenesis in R1441C mice was reported only in ChAT+ but not in ChAT- neurons of the striatum . We also observed an increase of ciliogenesis in one type of G2019S+/- NSC cells (Fig. 3E), but no difference in another type of G2019S+/- NSC cells, compared to the WT NSCs (Fig. 4B). This was probably due to the difference in LRRK2 expression levels among the tissues we used. The LRRK2 expression level is the highest in B-lympocytes that was used to prepare the isogenic WT/G2019S pair used in Fig. 4, and was similar between the cortex, hippocampus and skin when the levels were compared based on the Protein Atlas (https://www.genecards.org/cgi-bin/carddisp.pl?gene=LRRK2). Taken together, these results suggest that genetic backgrounds and the cellular expression patterns of proteins affecting basal LRRK2 kinase activity such as phosphatase PPM1H  are the critical determinants of ciliogenesis rather than the LRRK2 expression level. A recent study reported that the genetic backgrounds of rats resulted in different pT73-Rab10 levels despite similar LRRK2 expression levels . Rab8 and Rab10 might be such proteins affecting ciliogenesis because they function in the opposite ways in ciliogenesis although both Rab proteins are LRRK2 kinase substrates . In addition, the genetic background may differ by single base due to single nucleotide polymorphisms present in the LRRK2 gene itself used in each study. Although many amino acid variations in LRRK2 had been reported [37, 38] and their physiological roles are not clear yet, most studies only confirmed the pathogenic mutations themselves by DNA sequencing.
We found that a considerable number of cells contained cilia in dSH-SY5Y, murine primary neurons and G2019S fibroblasts under serum-starved conditions (Figs. 1A, 2, 3B). The lack of effect by serum starvation, B27 deprivation or MLi-2 treatment might be due to the high rate of ciliogenesis in neurons. However, it is worth noting that -20~-30% ciliogenesis in rat primary neurons and iPSCs of the G2019S/corrected WT isogenic pair was not increased after MLi-2 treatment (Figs. 1B, 4A).
To easily compare the previous results with our results, a summary is presented in Table 1.
Taken together, we observed no negative effect of LRRK2 kinase activity on ciliogenesis in neurons but an increase in ciliogenesis in the G2019S fibroblast cell line, opposite to many previous reports [11, 14-18, 39]. Our data recommend a careful approach to investigating the relationship between LRRK2 kinase activity and ciliogenesis and suggest that the genetic background must be considered to explain the relationship between LRRK2 kinase activity and ciliogenesis. To clarify the discrepant effect of LRRK2 kinase activity on ciliogenesis, it may be necessary to investigate ciliogenesis in several independent LRRK2 WT/mutant cells or G2019S/WT isogenic pairs.
We thank Dr. Ho, DH for his suggestion and help. This study was supported by the Basic Science Research Program through the National Research Foundation (NRF) funded by the Ministry of Education, Science and Technology, Republic of Korea (WS:2018R1D1A1B07041153; SKC:NRF-2013M3A9B4076487), the grant (JK:18721MFDS182) from the Ministry of Food and Drug Safety, and the KRIBB research initiative program (JK:KGM5362111) funded by the Ministry of Science and ICT.
The authors declare no conflict of interest.
Summary of studies on the relationship between LRRK2 and ciliogenesis
|Study||Tissue or cell line||Targets investigated||Cilia marker||Ciliogenesis change by the treatments||Reference|
|Previous||A549, RPE||Rab10KO||Arl13B||↑ by Rab10KO*||14|
|MEF-LRRK2-R1441G||MLi-2 treatment and Rab10KD||↑ by MLi-2* and by Rab10KD*|
|IPS cells||LRRK2WT/WT vs LRRK2G2019S/WT||↓ in LRRK2G2019S/WT|
|LRRK2G2019S/WT IPS cells||MLi-2 treatment||↑ by MLi-2*|
|Neurons in the cortex||WT/WT vs R1441C/R1441C||SSTR||↓ in LRRK2R1441C/R1441C*|
|Cholinergic neurons in the striatum||AC3||↓ in LRRK2R1441C/R1441C|
|Non-cholinergic neurons in the striatum||– by MLi-2|
|HEK 293 cells transfected with vector or LRRK2 WT||MLi-2 treatment||PT||– by MLi-2||17|
|HEK 293 cells expressing LRRK2 G2019S, R1441C, Y1699C||↑ by MLi-2#|
|Human fibroblast||LRRK2 WT vs G2019S||↓ in LRRK2G2019S/WT*|
|WT & G2019S human fibroblast||MLi-2 treatment||PT||– by MLi-2|
|WT primary astrocytes||Arl13B||– by MLi-2|
|G2019S KI primary astrocytes||↑ by MLi-2#|
|SH-SY5Y||Differentiation||AT||↑ by differentiation||39|
|SH-SY5Y (both differentiated and undifferentiated)||MG132 treatment||↓ by MG132*|
|Undifferentiated SH-SY5Y||α-synuclein WT, A30P, A53T expression||↓ by α-synuclein expression*|
|SH-SY5Y||Rotenone, MPP+ treatment||Arl13B||↑ by treatments#||20|
|MEF||Scrambled shRNA||Arl13B||– by Mli-2**||15|
|MEF-LRRK2-R1441C||MLi-2 treatment||SSTR3-GFP||↑ by MLi-2*||16|
|MEF-LRRK2-R1441G KI||MLi-2 treatment||Arl13B||↑ by MLi-2*||11|
|NIH3T3 transfected with LRRK2 G2019S|
|Our study||Rat cortical/ hippocampal WT primary neurons||GSK/MLi-2 treatment||Arl13B||– by treatment||This study|
|Fibroblasts (LRRK2G2019S/WT)||MLi-2 treatment||– by MLi-2|
|Fibroblasts (LRRK2WT/WT vs LRRK2G2019S/WT)||LRRK2WT/WT vs LRRK2G2019S/WT||↑ in LRRK2G2019S/WT##|
|NSCs (LRRK2WT/WT vs LRRK2G2019S/WT)||↑ in LRRK2G2019S/WT##|
|Murine cortical primary neurons||– between genotypes|
|Isogenic pairs of iPSCs and NSCs (LRRK2WT/WT vs LRRK2G2019S/WT)||LRRK2WT/WT vs LRRK2G2019S/WT||– between genotypes|
|MLi-2 treatment||– by MLi-2|
|Differentiated SH-SY5Y||MLi-2 treatment||Slightly ↑ by MLi-2##|
|Rab10 WT expression||– by expression|
–, little change; KI, Knock-in; AC3, adenylate cyclase 3; AT, acetylated tubulin; PT, polyglutamylated tubulin; SSTR, somatostatin receptor.
*Statistically significant change.
**The result was observed in the control sample (scrambled shRNA) to test effect of PPM1H knockdown on ciliogenesis.
#Statistically significant change was observed only under normal serum-fed condition, but not in the serum starved condition.
##Significant change regardless of serum condition.