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Pathway-specific dysregulation of striatal excitatory synapses by LRRK2 mutations

  1. Chuyu Chen
  2. Giulia Soto
  3. Vasin Dumrongprechachan
  4. Nicholas Bannon
  5. Shuo Kang
  6. Yevgenia Kozorovitskiy  Is a corresponding author
  7. Loukia Parisiadou  Is a corresponding author
  1. Department of Pharmacology, Feinberg School of Medicine, Northwestern University, United States
  2. Department of Neurobiology, Northwestern University, United States
Research Article
Cite this article as: eLife 2020;9:e58997 doi: 10.7554/eLife.58997
6 figures, 4 tables and 1 additional file

Figures

Figure 1 with 1 supplement
LRRK2 RC mutation increases synaptic glutamate receptor content in the striatum.

(A) Schematic diagram of LRRK2 protein highlighting the armadillo repeats (ARM), ankyrin (ANK) repeats, Ras of complex (ROC), C-terminal of ROC (COR), kin (KIN), and WD40 domains. Knock- in mice expressing the R1441C and G2019S mutations found in the ROC and kinase domains respectively, crossed with either Drd1-Tomato or Drd2-eGFP mouse lines. (B) Workflow schematic for subcellular fractionation of striatal homogenate for the enrichment of postsynaptic density fraction (PSD). (C) Representative western blot analysis of the subcellular fractionation results, showing supernatant (S1), crude synaptosomal preparation (P2), PSD, and Triton soluble fractions (TSF). (D) Western blot analysis of +/+, +/RC, and +/GS P2 striatal fractions probed for p-PKA substrates, pS845 GluA1, total GluA1, pT72Rab8A, total Rab8A, pT73Rab10, total Rab10, and PSD95. (E) Western blot analysis of +/+, +/RC, and +/GS mice probed for GluA1, p-PKA, and PSD95. S1 and PSD fractions are shown. (F-G) Quantification of GluA1 and p-PKA proteins in PSD fractions normalized to PSD95. Summary graphs reflect the mean, error bars reflect SEM. *p<0.05, Tukey post-hoc test following one-way ANOVA.

Figure 1—figure supplement 1
LRRk2 mutations do not alter PSD95 levels.

(A–B) Supernatant (S1), crude synaptosomal preparation (P2), PSD, and Triton soluble fractions (TSF) of wild type, +/RC and +/GS striata were run on the same blot. Ponceau staining shows similar loading across genotypes at a given fraction. Western blot analysis of the fractions described in A, probed for PSD95 and Homer-1. (C) Quantification of PSD95 band intensities normalized to Homer-1. Summary graphs represent the mean, while error bars SEM. (D) Quantification of PSD95 band intensities expressed as a fraction of the intensity of PSD95 wild type in each one of the S1, P2, and PSD fractions.

Figure 2 with 2 supplements
LRRK2 RC mutation restructures the nanoscale synaptic organization of dSPNs.

(A) Schematic depicting experimental design. (B) Structured Illumination super-resolution microscopy (SIM) image of dendritic spines on +/+, +/RC, and +/GS Drd1-Tomato expressing SPNs, labeled with antibodies to GluA1 (purple), and PSD95 (green). Open arrowheads, GluA1 nanodomains; arrowheads, PSD95 nanodomains. (C) Schematic diagram and object masks depicting GluA1, PSD95, and overlap nanodomains within a dendritic spine. Minimum distance between GluA1-PSD95 is measured from the closest edge of the two nanodomains, as shown. (D, E) Summary graphs and cumulative distribution of the minimum distance between GluA1 and PSD95 nanodomains. (F, G) Summary data and cumulative frequency for the overlap area of GluA1 and PSD95 nanodomains within dendritic spines. Asterisk in D and F reflect statistical significance for Tukey’s multiple comparison tests after one-way ANOVA, whereas asterisks in E and G show statistical significance for Bonferroni post-hoc comparisons after Kolmogorov-Smirnov tests. (H) Bar graphs showing the ratio of GluA1-PSD95 overlap area in E relative to PSD95 area, across genotypes. Data are represented as mean ± SEM. (I-K) Correlation plots of overlap in GluA1-PSD95 area versus PSD95 area for +/+, +/RC, and +/GS Drd1-Tomato expressing SPNs. *p<0.05, **p<0.01, ***p<0.001.

Figure 2—figure supplement 1
LRRK2 mutations alter postsynaptic density area of SPNs.

(A) Summary graph, PSD95 area across genotypes of dSPNs. Error bars reflect SEM. (B) Cumulative percentage of PSD95 area per dendritic spine across dSPN genotypes. (C) Summary graph, PSD95 area across genotypes of iSPNs. Error bars reflect SEM; asterisks, Tukey post-hoc comparisons after one-way ANOVA, +/+ vs. +/GS p<0.001 (D) Cumulative percentage of PSD95 areas per dendritic spine across iSPN genotypes.

Figure 2—video 1
3D Reconstruction of dendritic spine of an RC dSPN.

GluA1 (purple), and PSD95 (green), overlap area (white), as in Figure 2B. A surface mask is added for a fraction of the video, enabling the visualization of GluA1 and PSD95 in spine head.

LRRK2 mutations alter the nanoscale synaptic organization of iSPNs.

(A) SIM image of +/+, +/RC, and +/GS Drd2-eGFP expressing SPNs immunostained with GluA1 (purple), and PSD95 (orange). Open arrowheads, GluA1 nanodomains; arrowheads, PSD95 nanodomains. GFP antibody was used to amplify the Drd2-eGFP signal. (B) Surface intensity through a dendritic spine head in an RC iSPN. (C) Schematic diagram and object masks depicting GluA1, PSD95, overlap nanodomains, and minimum distance between nanodomains within a dendritic spine head. (D) Bar graphs showing the minimum distance between GluA1 and PSD95 nanodomains. (E) Cumulative distribution of data shown in D. (F, G) Summary data and cumulative distribution of the overlap area between GluA1 and PSD95 nanodomains in dendritic spine heads across genotypes. Asterisks in F show statistical significance for Tukey’s multiple comparison tests after one-way ANOVA; asterisks in G reflect statistical significance for Bonferroni post-hoc comparisons after Kolmogorov-Smirnov tests. (H) The ratio of overlap area between GluA1 and PSD95 to the PSD95 area for +/+, +/RC, and +/GS iSPNs. (I-K) Correlation plots of overlap areas versus PSD95 area for iSPNs across genotypes. *p<0.05, **p<0.01, ***p<0.001.

Figure 4 with 1 supplement
Pathway-specific functional alterations of SPN synapses in LRRK2 mutants.

(A) Example miniature excitatory postsynaptic current (mEPSC) traces from individual neurons of six genotype-pathway combinations. GFP-, dSPNs; GFP+, iSPNs; color, as defined in the figure. Scale bars, 10 pA and 2.5 s. (B) Summary graph showing the frequency of pharmacologically isolated mEPSCs, in GFP- and GFP+ SPNs in controls, compared to both RC and GS mutations. Asterisks reflect statistical significance for Bonferroni post hoc comparisons after two-way ANOVA. (C) Same as B, but for mEPSC amplitude. (D) Left, cumulative distribution of inter-event intervals (IEI) for mEPSCs across genotypes for GFP- SPNs. Right, same as left, but for GFP+ SPNs. (E) Binned histograms and cumulative distribution of mEPSC amplitude data. X axis starts at 5 pA, reflecting the amplitude threshold for mEPSC identification. *p<0.05, **p<0.01.

Figure 4—figure supplement 1
Analyses of mEPSC amplitude distributions.

Left, gamma curve fits to density histogram of mEPSC amplitudes by genotype and cell type (bin width, 1 pA). Gamma AIC for GFP- controls +/+, +/RC, and +/GS was 11813, 7721, and 4920. Gamma AIC for GFP+ controls +/+, +/RC, and +/GS was 9477, 6825, and 4881. Right, overlaid gamma curve fits by genotype and cell type. (B) Amplitudes estimates from GLM using the gamma family of curves, 95% confidence intervals in blue. Bonferroni corrected post-hoc pairwise comparisons, GPF- neurons, ***p<0.0001 for all comparisons; GFP+ neurons, p>0.7.

Figure 5 with 1 supplement
Pathway-specific analysis of dendritic spine morphology in LRRK2 mutants.

(A) Example confocal maximum projection image of a +/RC;Adora2a-Cre iSPN expressing AAV8/Flex-GFP. Representative dendritic fragment with dendritic spines and the corresponding 3D Imaris generated filament. (B) Summary graph showing the dendritic spine density in pathway-identified SPNs across genotypes. Quantification of dendritic spine head width (C), and dendritic spine length (D) in d- and iSPNs. (E) Confocal maximum projection image and the corresponding 3D Imaris generated filament with classified dendritic spines. Red, stubby; green, mushroom; purple, filopodia. (F) Left, summary data showing the density of each dendritic spine category in dSPNs. Right, same as left, but for iSPNs.

Figure 5—video 1
3D Reconstruction of an identified +/RC;Adora2a-Cre (Drdr1-, iSPN) neuron transduced with a Cre-dependent AAV eGFP virus (AAV8/Flex-GFP) in neonatal pups.
LRRK2 RC mutation increases glutamate uncaging-evoked currents in dSPNs.

(A) Schematic illustrating experimental design. (B) Example projection of a two-photon laser scanning microscopy stack showing an +/RC; GFP+ SPN (iSPN). Scale bar, 20 µm. (C) Close up images of representative dendrites from SPNs in the six genotype-pathway combinations, shown using inverse greyscale LUT. (D) Example single synapse AMPA-receptor-mediated currents, evoked by focal uncaging of MNI-glutamate. Scale bar, 20 pA and 100 ms. (E) Summary graph showing uncaging-evoked EPSCs (uEPSCs) for GFP-SPNs in controls, RC, and GS mutants. Asterisks reflect statistical significance for Tukey post-hoc comparisons after one-way ANOVA. (F) Same as E, but for GFP+ SPNs. (G) Summary schematic for the study, illustrating changes in synaptic content of glutamate receptors in LRRK2 mutations.

Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
AntibodyPhospho-PKA Substrate
(RRXS*/T*) (100G7E) rabbit monoclonal
Cell Signaling TechnologyRRID:AB_331817WB (1:1000)
AntibodyAMPA Receptor 1 (GluA1) (D4N9V) rabbit monoclonalCell Signaling TechnologyRRID:AB_2732897WB (1:1000)
AntibodyPhospho-AMPA Receptor 1 (GluA1)
(Ser845) (D10G5) rabbit monoclonal
Cell Signaling TechnologyRRID:AB_10860773WB (1:1000)
AntibodySynaptophysin rabbit polyclonalCell Signaling TechnologyRRID:AB_1904154WB (1:1000)
AntibodyAnti-RAB8A antibody [EPR14873] rabbit monoclonalAbcamRRID:AB_2814989WB (1:1000)
AntibodyRAB8A (phospho T72) [MJF-R20] rabbit monoclonalAbcamRRID:AB_2814988WB (1:1000)
AntibodyRab10 (D36C4) XP Rabbit mAb rabbit monoclonalCell signaling technologyRRID:AB_10828219WB (1:1000)
AntibodyAnti-RAB10 (phospho T73) antibody [MJF-R21] rabbit monoclonalAbcamRRID:AB_2811274WB (1:1000)
AntibodyHOMER1 polyclonal antibody rabbit polyclonalProteintechRRID:AB_2295573WB (1:1000)
AntibodyGluA1/GluR1 glutamate receptor clone N355/1
mouse monoclonal
UC Davis/NIH NeuroMab FacilityRRID:AB_2315840IF (1:300)
AntibodyPSD-95 monoclonal (6G6-1C9) mouse monoclonalInvitrogenRRID:AB_325399WB (1:1000)
IF (1:300)
AntibodyPSD-95 polyclonal rabbit polyclonalInvitrogenRRID:AB_87705IF (1:300)
AntibodyGFP chicken polyclonalInvitrogenRRID:AB_2534023IF (1:1000)
AntibodymCherry (16D7) rat monoclonalInvitrogenRRID:AB_2536611IF (1:1000)
AntibodyGoat anti-Mouse IgG (H+L) Secondary Antibody, HRPInvitrogenRRID:AB_2533947WB (1:5000)
AntibodyGoat anti-Rabbit IgG (H+L) Secondary Antibody, HRPInvitrogenRRID:AB_2533967WB (1:5000)
AntibodyDonkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647InvitrogenRRID:AB_162542IF (1:300)
AntibodyDonkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 568InvitrogenRRID:AB_2534017IF (1:300)
AntibodyGoat anti-Rat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568InvitrogenRRID:AB_2534121IF (1:300)
AntibodyGoat anti-Chicken IgY (H+L) Secondary Antibody, Alexa Fluor 488InvitrogenRRID:AB_2534096IF (1:300)
AntibodyDonkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488InvitrogenRRID:AB_2535792IF (1:300)
OtherFetal Bovine SerumSigma-AldrichF09265%
OtherHorse Serum, heat inactivated, New Zealand originGibco260500885%
Peptide, recombinant proteinLaminin Mouse Protein, NaturalGibco2301701510 μg/ml
Chemical compound, drugPoly-D-lysine hydrobromideSigmaP089950 μg/ml
Chemical compound, drugGlutaMAX Supplement (100x)Gibco35050061
Chemical compound, drugB-27 Supplement (50X), serum freeGibco17504044
Chemical compound, drugN-2 Supplement (100X)Gibco17502001
Chemical compound, drugAntibiotic Antimycotic Solution (100×), StabilizedSigma-AldrichA5955
Chemical compound, drugBasal Medium EagleSigma-AldrichB1522
Chemical compound, drugCytosine β-D-arabinofuranosideSigma-AldrichC17682.5 μM
Chemical compound, drugPDS Kit, Papain VialWorthington Biochemical CorporationLK003178
Chemical compound, drugDNase Vial (D2)Worthington Biochemical CorporationLK003172
Chemical compound, drugSucroseSigma-AldrichS7903
Chemical compound, drugHEPES solutionSigma-AldrichH0887
Chemical compound, drugUltraPure 1M Tris-HCI, pH 8.0Invitrogen15568025
Chemical compound, drugMgCl2Sigma-AldrichM8266
Chemical compound, drugCalcium chloride solutionSigma-Aldrich21115
Chemical compound, drugTriton X-100Sigma-AldrichX100
Chemical compound, drugEthylenediaminetetraacetic acid disodium salt solutionSigma-AldrichE7889
Chemical compound, drugHalt Protease and Phosphatase Inhibitor Cocktail, EDTA-free (100X)Thermo Scientific78441
Chemical compound, drugTris Buffered Saline (10x)Sigma-AldrichT5912
Chemical compound, drugTWEEN 20Sigma-AldrichP1379
Chemical compound, drugMNI-L-glutamateTocris1490
Chemical compound, drugAlexa Fluor 594Thermo Fisher ScientificA10438
Chemical compound, drugSodium chlorideSigma-AldrichS3014
Chemical compound, drugPotassium chlorideSigma-AldrichP9541
Chemical compound, drugSodium bicarbonateSigma-AldrichS5761
Chemical compound, drugSodium phosphate monobasicSigma-AldrichS3139
Chemical compound, drugCalcium chlorideSigma-AldrichC5670
Chemical compound, drugD-(+)-GlucoseSigma-AldrichG7021
Chemical compound, drugCesium methanesulfonateSigma-AldrichC1426
Chemical compound, drugCesium chlorideSigma-AldrichC3032
Chemical compound, drugHEPESSigma-Aldrich54457
Chemical compound, drugEthylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acidSigma-AldrichE3889
Chemical compound, drugGabazine/SR 95531 hydrobromideTocris1262
Chemical compound, drugScopolamine hydrobromideTocris1414
Chemical compound, drugPhosphocreatine disodium salt hydrateSigma-AldrichP7936
Chemical compound, drugQX-314 ChlorideTocris2313
Chemical compound, drugAdenosine 5’-triphosphate magnesium saltSigma-AldrichA9187
Chemical compound, drugGuanosine 5’-triphosphate sodium salt hydrateSigma-Aldrich51120
Chemical compound, drug(R)-CPPTocris0247
Chemical compound, drugTetrodotoxinTocris1078
Chemical compound, drugPotassium gluconateSigma-Aldrich1550001
Commercial assay or kitiBlot Transfer Stack, nitrocellulose, regular sizeInvitrogenIB301031
Commercial assay or kitNuPAGE 4–12% Bis-Tris Protein Gels, 1.5 mm, 15-wellInvitrogenNP0336BOX
Commercial assay or kitNuPAGE MES SDS Running Buffer (20X)InvitrogenNP0002
Commercial assay or kitNuPAGE AntioxidantInvitrogenNP0005
Commercial assay or kitNuPAGE Transfer Buffer (20X)InvitrogenNP0006
Commercial assay or kitNuPAGE LDS Sample Buffer (4X)InvitrogenNP0008
Commercial assay or kitNuPAGE Sample Reducing Agent (10X)InvitrogenNP0009
Commercial assay or kitPierce BCA Protein Assay KitThermo Scientific23225
Commercial assay or kitRestore Western Blot Stripping BufferThermo Scientific21059
OtherProLong Diamond Antifade Mountant with DAPIInvitrogenP36971
OtherBLUeye Prestained Protein LadderSigma-Aldrich94964
Commercial assay or kitImmobilon ECL Ultra Western HRP SubstrateMilliporeWBULS0500
Software, algorithmGraphPad Prism 7GraphPad Software IncRRID:SCR_002798
Software, algorithmNIS-elements 5.10Nikon Instruments IncRRID:SCR_014329
Software, algorithmImaris 9.21Bitplane IncRRID:SCR_007370
Software, algorithmiBright Analysis SoftwareThermo ScientificRRID:SCR_017632
Software, algorithmMATLABMathWorksRRID:SCR_001622
Software, algorithmFIJISchindelin et al., 2012http://fiji.sc/; RRID:SCR_002285
Strain, strain backgroundrAAV8/Flex-GFPUNC GTC vector coreLot, AV4910B
Author response table 1
Genotype/
Cell-type
Gaussian/
Normal AIC
Gamma AIC
BAC, GFP-13166.7511812.54
RC, GFP-8252.9107721.240
GS, GFP-5373.3604919.803
BAC, GFP+10312.099476.535
RC.GFP+7324.1176825.080
GS, GFP+5305.2984880.877
Author response table 2
Cell typeGenotypeMean amplitude (pA)SELCL*UCL*
GFP-+/+ (BAC)9.330.1069.089.59
+/GS8.260.1387.958.61
+/RC10.110.1439.7810.47
GFP++/+ (BAC)8.910.1108.669.19
+/GS8.690.1488.359.06
+/RC8.890.1288.609.21
Author response table 3
Cell typeComparisonBonferroni corrected p-values
GFP-+/+ vs GS< 0.0001
+/+ vs RC< 0.0001
GS vs RC< 0.0001
GFP++/+ vs GS0.7070
+/+ vs RC1.0000
GS vs RC0.9278

Data availability

All data generated during this study are included in the manuscript and supporting files.

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