Leucine-Rich Repeat Kinase-2 Controls the Differentiation and Maturation of Oligodendrocytes in Mice and Zebrafish

Abstract

Leucine-rich repeat kinase-2 (LRRK2), a gene mutated in familial and sporadic Parkinson's disease (PD), controls multiple cellular processes important for GLIA physiology. Interestingly, emerging studies report that LRRK2 is highly expressed in oligodendrocyte precursor cells (OPCs) compared to the pathophysiology of other brain cells and oligodendrocytes (OLs) in PD. Altogether, these observations suggest crucial function(s) of LRRK2 in OPCs/Ols, which would be interesting to explore. In this study, we investigated the role of LRRK2 in OLs. We showed that LRRK2 knock-out (KO) OPC cultures displayed defects in the transition of OPCs into OLs, suggesting a role of LRRK2 in OL differentiation. Consistently, we found an alteration of myelin basic protein (MBP) striosomes in LRRK2 KO mouse brains and reduced levels of oligodendrocyte transcription factor 2 (Olig2) and Mbp in olig2:EGFP and mbp:RFP transgenic zebrafish embryos injected with lrrk2 morpholino (MO). Moreover, lrrk2 knock-down zebrafish exhibited a lower amount of nerve growth factor (Ngf) compared to control embryos, which represents a potent regulator of oligodendrogenesis and myelination. Overall, our findings indicate that LRRK2 controls OL differentiation, affecting the number of mature OLs.

Keywords: LRRK2; Parkinson’s disease; oligodendrocytes; zebrafish.

Figure 1

LRRK2 KO OPCs exhibited a reduced number of primary cellular processes. (a) Representative contrast phase microscopy images of LRRK2 WT and KO OPCs at DIV3 of culture. (b) Cumulative frequency distributions of the number of primary processes per cell in LRRK2 WT and KO cultures (LRRK2 WT = 181 cells and LRRK2 KO = 174 cells; *** p ≤ 0.001; Kolmogorov–Smirnov test). Data are representative of three independent experiments (~60 cells for each experiment). Scale bar 25 µm.

Figure 2

LRRK2 controls the transition of OPCs into mature OLs. (a) Representative image of the immunofluorescence staining for NG2 and MBP markers in LRRK2 WT and LRRK2 KO OPCs culture. Scale bars 50 µm. (b) Quantification of MBP⁺ cells in LRRK2 KO compared to WT cultures. Data are representative of three experiments performed on three different cultures and are expressed as % MPB⁺ cells/total cells analyzed. At least 60 cells were analyzed for each experiment. Data were analyzed using unpaired t-test, ** p = 0.003. (c) Quantification of NG2⁺ cells in LRRK2 KO compared to WT cultures. Data are representative of six experiments performed on two different cultures and are expressed as % NG2⁺ cells/total cells analyzed. At least 60 cells were analyzed for each experiment. Data were analyzed using unpaired t-test, ** p = 0.007.

Figure 3

LRRK2 KO mouse brains displayed alterations of MBP+ striosomes. (a) Maximum intensity Z-projection confocal images of 13-month-old LRRK2 WT and KO mice immunostained for MBP. LRRK2 WT: (a’) Z = 3 mm, (a’’) Z = 4 mm; LRRK2 KO: (a’) Z = 3 mm, (a’’) Z = 2.33 mm. Scale bars 20 µm. (b) Cumulative frequency distributions of MBP negative signal in LRRK2 WT and LRRK2 KO mice (LRRK2 WT = 249 striosomes and LRRK2 KO = 242 striosomes; ** p < 0.01, Kolmogorov–Smirnov test, n = 3 animals per group).

Figure 4

Effects of lrrk2 MO on zebrafish embryo development. (a) Analysis of morphological phenotypes of zebrafish embryos injected with lrrk2 MO (0.1 mM–0.001 mM), 0.1 mM std MO, lrrk2 MO (0.1 mM–0.001 mM) + 0.1 mM p53 MO, lrrk2 MO (0.1 and 0.01 mM) + human LRRK2 mRNA (25, 50, and 100 ng/μL corresponding to 100, 200, and 400 pg). Quantification of the absolute percentage of embryos showing the wild-type-like (white closed bars), mild (gray closed bars), and severe (black closed bars) morphological phenotypes out of the total embryos analyzed. (b) Representative images of the phenotypes observed after the injection with std MO and lrrk2 MO. Number of observed embryos: not injected (n = 350); 0.1 mM std MO (n = 65); 0.1 mM std MO + p53 MO (n = 60); 0.1 mM lrrk2 MO (n = 230); 0.01 mM lrrk2 MO (n = 196); 0.001 mM lrrk2 MO (n = 112); 0.1 mM lrrk2 MO + 0.1 mM p53 MO (n = 131); 0.01 mM lrrk2 MO + 0.1 mM p53 MO (n = 110); 0.1 mM lrrk2 MO + 25 ng/μL hLRRK2 mRNA (n = 44); 0.1 mM lrrk2 MO + 50 ng/μL hLRRK2 mRNA (n = 57); 0.1 mM lrrk2 MO + 100 ng/μL hLRRK2 mRNA (n = 47); 00.1 mM lrrk2 MO + 50 ng/μL hLRRK2 mRNA (n = 56); 0.01 mM lrrk2 MO + 100 ng/μL hLRRK2 mRNA (n = 53). (c) A pool of fifty embryos per condition (not injected, lrrk2 MO, lrrk2 MO + 100 pg hLRRK2 mRNA, lrrk2 MO + 200 pg hLRRK2 mRNA, lrrk2 MO + 400 pg hLRRK2 mRNA, std MO) were subjected to immunoblotting using Lrrk2 and Gapdh antibodies at 48 hpf.

Figure 5

tg(olig2:EGFP) transgenic zebrafish reporter line injected with lrrk2 MO exhibited a reduction of Olig2 signal. (a) A pool of fifty embryos at 48 hpf injected with std or lrrk2 MO were subjected to immunoblotting using Lrrk2 and Gapdh antibodies. (b) Representative images of tg(olig2:EGFP) injected with std or lrrk2 MO were acquired by using an AXIOZOOM V16 ZEISS fluorescence microscope. (c) Quantification of fluorescence intensity of tg(olig2:EGFP) embryos at 48 hpf injected with std or lrrk2 MO calculated as the average of fluorescence intensity pixels ± SEM from three independent experiments (total embryos analyzed: std MO = 34 and lrrk2 MO = 31). Data were analyzed by using an unpaired t-test, **** p < 0.0001.

Figure 6

tg(mbp:RFP) zebrafish reporter line injected with lrrk2 MO for 120 h exhibited a reduction of Mbp signal and Ngf levels. (a) 3D reconstruction of Z-stack confocal acquisition of tg(mbp:RFP) reporter line injected with std or lrrk2 MO for 120 h. std MO: (a’) Z = 90 mm, (a’’) Z = 80 mm and (a’’’) Z = 80 mm; Lrrk2 MO: (a’) Z = 160 mm, (a’’) Z = 60 mm and (a’’’) Z = 90 mm. Scale bar 10 µm. The asterisk indicates the eye of the zebrafish embryo. (b) Zebrafish embryos injected with std or lrrk2 MO for 120 h were subjected to immunoblotting using Lrrk2, Ngf and Gapdh antibodies. (c) Quantification of Ngf is normalized on Gapdh expression. Data are representative of three independent pools of fifty embryos. Data were analyzed using unpaired t-test, * p = 0.0111.

Figure 7

Effects of lrrk2 MO on zebrafish motor behaviors. (a) Spontaneous coiling events (tail flips) performed by wild-type zebrafish embryos injected with 0.01 mM of both control (std MO) and lrrk2 MO have been recorded at 24 hpf. Bar Graph represents the media of tail flips performed by each embryo in 30 s ± SEM (a, left panel). Touch-evoked escape response has been measured at 48 hpf on the same injected embryos. A value of 0 was attributed to completely paralyzed embryos, 1 to embryos performing only spontaneous coiling events, 2 to embryos moving short distances, and 3 to embryos swimming normally. Bar Graph reports the media of motor value for each condition ± SEM (a, right panel) Values represent the media from four independent experiments. Number of embryos analyzed for tail flip: std MO-injected (n = 90), lrrk2 MO-injected (n = 80). Number of embryos analyzed for touch-evoked escape response: std MO-injected (n = 69), lrrk2 MO-injected (n = 66). Data were analyzed by Mann–Whitney test, ** p < 0.01 and **** p < 0.0001. (b,c) Analysis of distance moved by std MO and lrrk2 MO-injected embryos under light on/light off visual stimuli has been performed at 120 hpf by using Noldus Chamber equipped with a camera recording traces of zebrafish movement and Ethovision XT software Version XT 13.0.1220 for the data analysis. Three 10 min light on/10 min light off cycles have been performed after 30 min of light habituation. (b) Traces in the graph represents the media of distance moved during time (in mm) by embryos of each condition ± SEM. (c) Bar graph reports the media of the total distance moved (in mm) by embryos of each condition ± SEM. Number of embryos analyzed: std MO-injected (n = 33); lrrk2 MO-injected (n = 29). Data were analyzed by one-way ANOVA followed by Bonferroni correction, **** p < 0.0001.