Glial Nrf2 signaling mediates the neuroprotection exerted by Gastrodia elata Blume in Lrrk2-G2019S Parkinson’s disease

  1. Yu-En Lin
  2. Chin-Hsien Lin
  3. En-Peng Ho
  4. Yi-Ci Ke
  5. Stavroula Petridi
  6. Christopher JH Elliott
  7. Lee-Yan Sheen  Is a corresponding author
  8. Cheng-Ting Chien  Is a corresponding author
  1. Institute of Molecular Biology, Academia Sinica, Taiwan
  2. Institute of Food Science and Technology, National Taiwan University, Taiwan
  3. Department of Neurology, National Taiwan University Hospital, Taiwan
  4. Department of Clinical Neurosciences and MRC Mitochondrial Biology Unit, University of Cambridge, United Kingdom
  5. Department of Biology and York Biomedical Research Institute, University of York, United Kingdom
  6. Neuroscience Program of Academia Sinica, Academia Sinica, Taiwan
11 figures, 1 table and 3 additional files

Figures

Figure 1 with 2 supplements
Water extract of Gastrodia elata Blume (WGE) treatment rescues the diminished locomotion of Ddc>G2019S flies.

(AC) Climbing activities of Ddc>G2019S flies fed on food supplemented with 0.1, 0.5, or 1.0% WGE (A), 0.1 or 1.0 mM gastrodin (B), and 0.1 or 1.0 mM 4-hydroxybenzyl alcohol (4-HBA) (C). Controls …

Figure 1—figure supplement 1
Climbing activity assay of Ddc>mCD8-GFP and Ddc>Lrrk2 flies.

(A) Comparable climbing activities were detected for Ddc>mCD8-GFP and Ddc>Lrrk2 flies. Both exhibited better climbing activities than Ddc>G2019S flies in the climbing assay from weeks 1 to 6. Bar …

Figure 1—figure supplement 2
Locomotion improvement of Ddc>G2019S flies starting water extract of Gastrodia elata Blume (WGE) feeding at week 4.

The climbing activities of Ddc>Lrrk2, Ddc>G2019S, and Ddc>G2019S with 0.1% WGE feeding at week 4 were assessed at weeks 3–6. Bar graphs show success rates (mean ± SEM, N = 6) of flies climbing over …

Figure 2 with 1 supplement
Water extract of Gastrodia elata Blume (WGE) prevents loss of dopaminergic neurons in Ddc>G2019S flies.

(A) Representative adult brain images showing TH-positive dopaminergic neurons in the PPL1 cluster of 2-, 4-, and 6-week-old flies of the Ddc>Lrrk2, Ddc>G2019S, and 0.1% WGE-fed Ddc>G2019S groups. …

Figure 2—figure supplement 1
Water extract of Gastrodia elata Blume (WGE) treatment prevents dopaminergic neuron loss in Ddc>G2019S flies.

(A–C) Representative adult whole-brain images for TH staining to reveal dopaminergic neurons in the PPL1, PPL2, PPM1/2, and PPM3 clusters of 2-, 4-, and 6-week-old flies. Scale bar: 40 μm. The …

Figure 3 with 1 supplement
Water extract of Gastrodia elata Blume (WGE) modulates Lrrk2 accumulation and hyperactivation in elav>G2019S flies.

(A) Representative immunoblots of 3-day-old adult brain lysates showing expression levels of Lrrk2, pLrrk2 (phosphorylated at Ser1292), Akt, and pAkt (phosphorylated at Ser505) in elav>Lrrk2 and elav…

Figure 3—figure supplement 1
Water extract of Gastrodia elata Blume (WGE) specifically modulates Lrrk2 accumulation and hyperactivation in elav>G2019S but not elav> Lrrk2 flies.

(A) Representative immunoblots of 2-week-old adult brain lysates showing levels of Lrrk2 and pLrrk2 (Ser1292) in elav>Lrrk2, WGE-fed elav>Lrrk2, elav>G2019S, and WGE-fed elav>G2019S flies. (B, C) …

Water extract of Gastrodia elata Blume (WGE) activates the Akt-Nrf2 pathway in elav>G2019S flies.

(A) Representative immunoblots of 2- and 4-week-old adult brain lysates showing levels of Akt and pAkt in brain extracts of elav>Lrrk2, elav>G2019S, and WGE-fed elav>G2019S flies. (B) Quantification …

Figure 5 with 2 supplements
Activation of Nrf2 in glia rescues locomotion defects in Ddc>G2019S flies.

(A, B) Requirement of Nrf2 in glia but not neurons for water extract of Gastrodia elata Blume (WGE)-improved Ddc>G2019S climbing activity. (A) Climbing success rates of flies in which Ddc-GAL4

Figure 5—figure supplement 1
Water extract of Gastrodia elata Blume (WGE) treatment specifically activates glial Nrf2 signals in the ARE-GFP reporter flies.

(A, B) WGE treatment activates glial Nrf2 signals in 1-week-old ARE-GFP reporter flies. ARE-GFP (green) and glial Repo (red) in whole-mount adult brains without (A) or with 0.1% WGE treatment (B) …

Figure 5—figure supplement 2
Water extract of Gastrodia elata Blume (WGE) rescues the locomotion defect displayed by Ddc-LexA>G2019S flies.

WGE treatment improves the climbing ability of Ddc-LexA>G2019S flies. Bar graph shows percentage (mean ± SEM, N = 6) of flies that successfully climbed above 8 cm within 10 s. One-way ANOVA and …

Figure 6 with 2 supplements
Nrf2 in astrocyte-like and ensheathing glia mediates the effect of water extract of Gastrodia elata Blume (WGE) treatment in Ddc>G2019S flies.

(A) Nrf2 knockdown in astrocyte-like and ensheathing glia abolishes the improved locomotion elicited by WGE treatment in Ddc>G2019S flies. Composite bar graph shows climbing success rates for …

Figure 6—figure supplement 1
Lrrk2 and pLrrk2 levels are maintained upon glial Nrf2 overexpression.

(A) Representative immunoblots of 2-week-old adult brain lysates showing protein expression levels of Lrrk2 and pLrrk2 (Ser1292) in Ddc-LexA>G2019S; repo-GAL4 and Ddc-LexA>G2019S; repo>cncC-FL2

Figure 6—figure supplement 2
Water extract of Gastrodia elata Blume (WGE) treatment induces a mild Nrf2 activation in dopaminergic neurons in Ddc>G2019S flies.

(A, B) Quantifications (mean ± SEM, n > 20 for each genotype) of GFP intensities in TH-positive dopaminergic neurons nearby the astrocyte-like glia (A) or ensheathing glia (B). GFP intensities were …

Figure 7 with 1 supplement
Water extract of Gastrodia elata Blume (WGE) downregulates G2019S-induced BMP/Mad signaling.

(A) Representative images of glial pMad staining in the adult PPL1 clusters of Ddc>Lrrk2, Ddc>G2019S, and WGE-fed Ddc>G2019S flies. White arrows indicate pMad signals co-localized with Repo, with …

Figure 7—figure supplement 1
Mad heterozygosity rescues the impaired locomotion of Ddc>G2019S flies.

Removing one copy of Mad improves Ddc>G2019S climbing activity. Bar graphs show climbing success rates (mean ± SEM, N = 6) of 6-week-old Ddc>Lrrk2, Ddc>G2019S, and Ddc>G2019S, Mad+/− flies. One-way …

Nrf2 antagonizes BMP/Mad signaling in glia.

(A) Heterozygosity of Mad suppresses G2019S mutation-induced locomotion impairment in a negative geotaxis assay. Bar graph shows percentages (mean ± SEM, N = 6) of 6-week-old flies that climbed …

Water extract of Gastrodia elata Blume (WGE) treatment rescues impaired locomotion of LRRK2-G2019S mice.

(A) Video-tracked paths for nTg, LRRK2-G2019S, and WGE-fed LRRK2-G2019S mice (8.5 and 11.5 months old) during the open-field test. (B) Quantification of total distance (m), average velocity (cm/s), …

Figure 10 with 1 supplement
Water extract of Gastrodia elata Blume (WGE) prevents dopaminergic neuron loss, microglial activation, and phosphorylation of LRRK2, Smad2, and Smad3.

(A, C) Representative images showing TH-positive dopaminergic neurons (A) or Iba-1-positive microglia (C) in the substantia nigra of 11.5-month-old nTg, LRRK2-G2019S, and WGE-fed LRRK2-G2019S mice. …

Figure 10—figure supplement 1
Water extract of Gastrodia elata Blume (WGE) suppresses microglia activation in LRRK2-G2019S mice.

Single-channel images of Figure 10C show Iba-1-positive microglia in the substantia nigra of 11.5-month-old nTg, LRRK2-G2019S, and WGE-fed LRRK2-G2019S mice. Bars: 50 μm.

The proposed model of water extract of Gastrodia elata Blume (WGE) in the G2019S-induced neurodegeneration.

Accumulation of the hyperactivated G2019S mutant protein enhances the BMP ligand (Gbb) maturation via upregulation of Furin 1 translation in dopaminergic neurons. Secreted Gbb binds to the BMP …

Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Genetic reagent (Drosophila melanogaster)UAS-mCD8-GFPBloomington Drosophila Stock CenterBDSC Cat# 5137; RRID:BDSC_5137
Genetic reagent (D. melanogaster)UAS-cncTRiPBloomington Drosophila Stock CenterBDSC Cat# 25984; RRID:BDSC_25984
Genetic reagent (D. melanogaster)UAS-tkvQ253DBloomington Drosophila Stock CenterBDSC Cat# 36536; RRID:BDSC_36536
Genetic reagent (D. melanogaster)UAS-Flag-
LRRK2-WT
Lin et al., 2010N/A
Genetic reagent (D. melanogaster)UAS-Flag-
LRRK2-G2019S
Lin et al., 2010N/A
Genetic reagent (D. melanogaster)UAS-cncC-FL2Sykiotis and Bohmann, 2008N/A
Genetic reagent (D. melanogaster)UAS-MadTakaesu et al., 2006N/A
Genetic reagent (D. melanogaster)elav-GAL4Bloomington Drosophila Stock CenterBDSC Cat# 8760; RRID:BDSC_8760
Genetic reagent (D. melanogaster)repo-GAL4Bloomington Drosophila Stock CenterBDSC Cat# 7215; RRID:BDSC_7215
Genetic reagent (D. melanogaster)alrm-GAL4Bloomington Drosophila Stock CenterBDSC Cat# 67032; RRID:BDSC_67032
Genetic reagent (D. melanogaster)R56F03-GAL4Bloomington Drosophila Stock CenterBDSC Cat# 39157; RRID:BDSC_39157
Genetic reagent (D. melanogaster)NP2222-GAL4Bloomington Drosophila Stock CenterBDSC Cat# 112830; RRID:BDSC_112830
Genetic reagent (D. melanogaster)NP6293-GAL4Kyoto Stock
Center
DGRC Cat# 105188;RRID:Kyoto Stock Center_105188
Genetic reagent (D. melanogaster)Ddc-GAL4Sang et al., 2007N/A
Genetic reagent (D. melanogaster)moody-GAL4Bainton et al., 2005N/A
Genetic reagent (D. melanogaster)Tub-GAL80tsBloomington Drosophila Stock CenterBDSC Cat# 7108; RRID:BDSC_7108
Genetic reagent (D. melanogaster)Ddc-LexABloomington Drosophila Stock CenterBDSC Cat# 54218; RRID:BDSC_54218
Genetic reagent (D. melanogaster)MadK00237Bloomington Drosophila Stock CenterBDSC Cat# 10474; RRID:BDSC_10474
Genetic reagent (D. melanogaster)ARE-GFPSykiotis and Bohmann, 2008N/A
Genetic reagent (D. melanogaster)LexAop-LRRK2-WTThis paperN/ASee Materials and methods, ‘Drosophila stocks and maintenance’
Genetic reagent (D. melanogaster)LexAop-LRRK2-
G2019S
This paperN/ASee Materials and methods, ‘Drosophila stocks and maintenance’
Genetic reagent (Mus musculus)FVB/NJThe Jackson LaboratoryJAX stock #001800
Genetic reagent (M. musculus)FVB/N-Tg(LRRK2*
G2019S)1Cjli/J
The Jackson LaboratoryJAX stock #009609
AntibodyAnti-TH(mouse monoclonal)ImmunostarCat# 22941; RRID:AB_572268IF (1:1000)
AntibodyAnti-TH(rabbit polyclonal)MilliporeCat# AB152;RRID:AB_390204Mouse-IHC (1:200)
AntibodyAnti-Repo(mouse monoclonal)Hybridoma Bank DSHBCat# 8D12; RRID:AB_528448IF (1:500)
AntibodyAnti-GFP(chicken polyclonal)AbcamCat# ab13970; RRID:AB_300798IF (1:10000)
AntibodyAnti-LRRK2(rabbit monoclonal)AbcamCat# ab133474;RRID:AB_2713963Fly-WB (1:1000) mouse-WB(1:5000)
AntibodyAnti-LRRK2 (phospho
Ser1292)(rabbit monoclonal)
AbcamCat# ab203181Fly-WB (1:500) mouse-WB(1:1000)
AntibodyAnti-Akt(rabbit monoclonal)Cell SignalingCat# 4691;RRID:AB_915783WB (1:1000)
AntibodyAnti-phospho-
Drosophila
Akt
(Ser505)(rabbit polyclonal)
Cell SignalingCat# 4054;RRID:AB_331414WB (1:500)
AntibodyAnti-Nrf2(rabbit polyclonal)ThermoFisherScientificCat# 710574;RRID:AB_2532742WB (1:1000)
AntibodyAnti-phospho-
Nrf2 (Ser40)(rabbit polyclonal)
ThermoFisherScientificCat# PA5-67520;RRID:AB_2691678WB (1:1000)
AntibodyAnti-HO-1-1(mouse monoclonal)ThermoFisherScientificCat# MA1-112;RRID:AB_2536823WB (1:1000)
AntibodyAnti-GAPDH(rabbit polyclonal)GeneTexCat# GTX100118;RRID:AB_1080976WB (1:5000)
AntibodyAnti-alpha tubulin(rabbit polyclonal)Cell SignalingCat# 2144;RRID:AB_2210548WB (1:10,000)
AntibodyAnti-Smad2(rabbit monoclonal)Cell SignalingCat# 5339;RRID:AB_10626777Mouse-WB (1:1000)
AntibodyAnti-phospho-Smad2 (Ser465/467)(rabbit monoclonal)Cell SignalingCat# 3108;RRID:AB_490941Mouse-WB (1:1000)
AntibodyAnti-Smad3(rabbit monoclonal)Cell SignalingCat# 9523;RRID:AB_2193182Mouse-WB (1:1000)
AntibodyAnti-Smad3 (phospho
S423 + S425)(rabbit monoclonal)
AbcamCat# ab52903; RRID:AB_882596IF (1:250) mouse-WB (1:1000)
AntibodyAnti-beta actin(mouse monoclonal)Sigma-AldrichCat# A5441;RRID:AB_476744Mouse-WB (1:5000)
AntibodyAnti-Iba-1(rabbit polyclonal)GeneTexCat# GTX100042;RRID:AB_1240434Mouse-IHC (1:200)
AntibodyAnti-mouse Alexa 488(goat polyclonal)InvitrogenCat# A28175;RRID:AB_2536161IF (1:500)
AntibodyAnti-rabbit Alexa 488(goat polyclonal)InvitrogenCat# A27034;RRID:AB_2536097IF (1:500)
AntibodyAnti-rabbit DyLight 488(goat polyclonal)ThermoFisherScientificCat# 35552;RRID:AB_844398Mouse-IHC (1:300)
AntibodyAnti-rabbit Alexa 546(goat polyclonal)ThermoFisherScientificCat# A-11035;RRID:AB_2534093Mouse-IHC (1:200)
AntibodyAnti-mouse FITC(goat polyclonal)Jackson ImmunoResearchCat#115-095-003;RRID:AB_2338589IF (1:500)
AntibodyAnti-mouse Cy3(goat polyclonal)Jackson ImmunoResearchCat# 115-165-003;RRID:AB_2338680IF (1:500)
AntibodyAnti-rat IgG Cy5(goat polyclonal)InvitrogenCat# A10525;RRID:AB_2534034IF (1:500)
AntibodyAnti-rabbit peroxidase(goat polyclonal)Jackson ImmunoResearchCat# 111-035-144;RRID:AB_2307391WB (1:7500)
AntibodyAnti-mouse
peroxidase
(goat polyclonal)
Jackson ImmunoResearchCat# 115-035-003;RRID:AB_10015289WB (1:7500)
AntibodyAnti-mouse
peroxidase(goat polyclonal)
GeneTexCat# GTX213111-01;RRID:AB_10618076Mouse-WB (1:5000)
AntibodyAnti-rabbit peroxidase(goat polyclonal)GeneTexCat# GTX213110-01;RRID:AB_10618573Mouse-WB (1:5000)
Chemical compound, drugPhalloidin-TRITCSigma-AldrichCat# P1951;RRID:AB_2315148IF (1:5000)
Chemical compound, drugWGELin et al., 2016a;Lin et al., 2018N/AKO DA Pharmaceutical Co. Ltd.
Chemical compound, drugGastrodinLin et al., 2016bN/AWuhan YC Fine Chemical Co.
Chemical compound, drug4-HBASigma-AldrichCat# H20806-10G
Recombinant DNA reagent(Homo sapiens)pDEST53-LRRK2-WT (plasmid)AddgeneAddgene plasmid # 25044;RRID:Addgene_25044
Recombinant DNA reagent(H. sapiens)pDEST53-LRRK2-G2019S (plasmid)AddgeneAddgene plasmid # 25045; RRID:Addgene_25045
Recombinant DNA reagent(D. melanogaster)pJFRC19-13XLexAop2-IVS- myr::GFP (plasmid)AddgeneAddgene plasmid # 26224; RRID:Addgene_26224
Software, algorithmImageJPMID:22930834https://imagej.nih.gov/ij;RRID:SCR_003070
Software, algorithmCtraxPMID:19412169N/A
Software, algorithmCatWalk XTLin et al., 2020N/ANoldus Information Technology
Software, algorithmPrism 6GraphPadRRID:SCR_002798
OtherDAPIGeneTexCat# GTX30920

Additional files

Transparent reporting form
https://cdn.elifesciences.org/articles/73753/elife-73753-transrepform1-v2.pdf
Source data 1

Statistical information.

The file includes all data and the statistical analyses in this article.

https://cdn.elifesciences.org/articles/73753/elife-73753-supp1-v2.xlsx
Source data 2

Data—western blotting.

The file includes the uncropped images of the western blotting in this article.

https://cdn.elifesciences.org/articles/73753/elife-73753-supp2-v2.pdf

Download links