LRRK2 regulates synaptic function through modulation of actin cytoskeletal dynamics
- Department of Biology, University of Padova, Italy
- UK Dementia Research Institute, Cardiff University, Hadyn Ellis Building, United Kingdom
- School of Medicine, Cardiff University, Hadyn Ellis Building, United Kingdom
- Northwestern University, United States
- Institute for Biochemistry, Charité Universitatsmedizin Berlin, Germany
- Freie Universität Berlin, Germany
- School of Pharmacy, University College London, United Kingdom
- CIBIO, University of Trento, Italy
- Department of Experimental Medicine, University of Genova, Italy
- IRCCS, Azienda Ospedaliera Metropolitana, Italy
- Department of Biomedical Sciences, University of Padova, Italy
- IRCCS San Camillo Hospital, Italy
Figures
Figure 1 with 1 supplement
LRRK2 phosphorylation at Ser935 rapidly and transiently increases upon BDNF stimulation in primary mouse cortical neurons and differentiated SH-SY5Y cells.
(A) Schematic representation of the experimental setting of (B). (B) Phospho-Ser935 and total LRRK2 protein levels of primary cortical neurons at DIV14 treated with 100 ng/mL BDNF for 0, 5, 30, 60, and 180 min. MLi-2 was used at 500 nM for 90 min to inhibit LRRK2 kinase activity. (C) Quantification of n=6 independent experiments of (B). One-way ANOVA (***p<0.001), Dunnett’s multiple comparison test (t=0 compared with different time points, *p<0.05 t=0 vs. t=5’). (D) Schematic representation of the experimental setting of (F) and (G). (E) TrkB protein levels in undifferentiated or retinoic acid-differentiated SH-SY5Y cells. (F) Phospho Thr202/185 ERK1/2, total ERK1/2, and (G) phospho Ser473 AKT, total AKT protein levels of retinoic-acid-differentiated SH-SY5Y cells stimulated with 100 ng/mL BDNF for 0, 5, 30, and 60 min. (H) Phospho-Ser935 and total LRRK2 protein levels of retinoic-acid-differentiated SH-SY5Y cells stimulated with 100 ng/mL BDNF for 0, 5, 30, and 60 min. MLi-2 was used at 500 nM for 90 min to inhibit LRRK2 kinase activity. (I) Quantification of (H) (n=4 independent experiments). One-way ANOVA (**p<0.01), Dunnett’s multiple comparison test (*p<0.05 t=0 vs. t=5’). (J) Phospho-T73 and total RAB10 protein levels of retinoic-acid-differentiated SH-SY5Y cells stimulated with 100 ng/mL BDNF for 0, 5, 30, and 60 min. MLi-2 was used at 500 nM for 90 min to inhibit LRRK2 kinase activity. (K) Quantification of (J) (n=4 independent experiments). One-way ANOVA (p>0.5), Dunnett’s multiple comparison test (*p<0.05 t=0 vs. t=30’).
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Figure 1—source data 1
Original western blot files for Figure 1.
- https://cdn.elifesciences.org/articles/95987/elife-95987-fig1-data1-v1.zip
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Figure 1—source data 2
PDF files containing original western blots for Figure 1, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/95987/elife-95987-fig1-data2-v1.zip
Figure 1—figure supplement 1
LRRK2 expression in naïve SH-SY5Y and characterization of SH-SY5Y stably expressing GFP-LRRK2.
(A) Western blot analysis of undifferentiated versus differentiated naive SH-SY5Y and western blot with anti-phospho-Ser935 LRRK2 and total LRRK2 antibodies. (B) Cells stably overexpressing GFP-LRRK2 treated with 100 ng/mL BDNF for 5 min or with 500 nM MLi-2 for 90 min. (C) On the left, schematic representation of the experimental setting. On the right, BDNF stimulation of GFP-LRRK2 SH-SY5Y cells prior to MLi-2 (500 nM) treatment for 90 min (MLi-2 for 90’, washout, BDNF stimulation for 1’, 5’, 15’, 60’, and 180’). The timepoint 0’ corresponds to non-BDNF treated. Western blots were performed using anti-LRRK2 (phospho-Ser935 and total), anti-RAB10 (phospho Thr73 and total), and anti-RAB8 (pan-phospho-RABs and total RAB8).
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Figure 1—figure supplement 1—source data 1
Original western blot files for Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/95987/elife-95987-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
PDF files containing original western blots for Figure 1—figure supplement 1, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/95987/elife-95987-fig1-figsupp1-data2-v1.zip
Figure 2 with 1 supplement
BDNF promotes LRRK2 interaction with post-synaptic actin cytoskeleton components.
(A) Volcano plot of GFP-LRRK2 versus GFP enriched interactors from differentiated SH-SY5Y cells (n=6 replicates: n=3 independent experiments each with two technical replicates). Interactors that were considered for SynGO analysis have adjusted p-value <0.05 and FC >2 (red dots). (B) Donut charts with % of SynGO genes among the total 207 LRRK2 interactors (A) and with % of presynaptic and postsynaptic proteins among SynGO genes. SynGO-CC terms visualized with a sunburst plot showing significant categories (below). (C) Volcano plot of GFP-LRRK2+/-BDNF enriched interactors from differentiated SH-SY5Y cells (n=6 replicates: n=3 independent experiments each with two technical replicates). Interactors selected for pathway enrichment analysis fall into two categories: (i) adjusted p-value <0.05 and FC >0.5 or FC <–0.5, or (ii) |FC|>1 regardless of the p value. Proteins with increased interaction upon BDNF stimulation are blue-labeled, proteins with decreased interaction are orange-labeled. Donut chart showing the number of SynGO annotated genes versus non-synaptic proteins in BDNF-up versus BDNF-down interactors (top right). (D) G:profiler g:GOSt pathway enrichment analysis showing the top 5 enriched biological processes (BP) categories of BDNF-increased interactors (top, blue bar graph) versus BDNF-decreased interactors (bottom, orange bar graph). GO analysis was performed with g:Profiler on 04/14/2025 (version e112_eg59_p19_25aa4782, database updated on 03/02/2025); enriched terms determined using g:SCS correction and size terms set to <500 to increase specificity. (E) Protein-Protein Interaction Networks built with STRING (https://string-db.org/) of all BDNF(+) interactors and SynGO BDNF-increased interactors (inset) and (F) of SynGO BDNF-decreased interactors. (G) Validation of increased drebrin:LRRK2 interaction upon BDNF treatment (15 min) and quantification (one-sample t-test, p<0.01). Samples are the same analyzed in the three rounds of MS (A). (H) Western blot analysis of brain samples from WT and Dbn1 (drebrin) KO mice (2-month-old) assessing Lrrk2 Ser935. Differences between the two genotypes have been evaluated using Student's t-test (significance *p<0.05 pSer935), n=10 animals per genotype.
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Figure 2—source data 1
Original western blots files for Figure 2.
- https://cdn.elifesciences.org/articles/95987/elife-95987-fig2-data1-v1.zip
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Figure 2—source data 2
PDF files containing original western blots for Figure 2, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/95987/elife-95987-fig2-data2-v1.zip
Figure 2—figure supplement 1
Western blot analysis validating the specificity of LRRK2-Drebrin interaction.
(A) Confocal immunofluorescence of DIV14 primary neurons co-stained with MAP2 (neuronal marker), PSD95 (postsynaptic marker), and drebrin. Scale bar: 100 μm; magnifications scale bar: 10 μm. (B) To rule out that drebrin does not bind the resin but exclusively GFP-LRRK2 bound to anti-GFP nanobodies, GFP-LRRK2 was purified from cell lysates containing a mix of GFP-LRRK2 OE SH-SY5Y cells and naive SH-SY5Y cells at different ratios: 4:0, 3:1, 2:2, 1:3, 0:4 (OE:naïve). The levels of drebrin bound to GFP-LRRK2 diminish proportionally with the reduction of LRRK2 purified from the lysate, with a complete lack of signal in the eluate from the resin incubated with naive cells only (0:4 condition), confirming the specificity of drebrin binding to GFP-LRRK2. (C) Co-immunoprecipitation of 3xFlag-LRRK2 with YFP-drebrin domains (full-length, N-terminus, and C-terminus; amino acid boundaries are indicated in the figure). Drebrin constructs were immunoprecipitated with GFP-trap resin and co-precipitated LRRK2 visualized by western blot using anti-Flag antibodies.
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Figure 2—figure supplement 1—source data 1
Original western blots files for Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/95987/elife-95987-fig2-figsupp1-data1-v1.zip
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Figure 2—figure supplement 1—source data 2
PDF files containing original western blots for Figure 2—figure supplement 1, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/95987/elife-95987-fig2-figsupp1-data2-v1.zip
Figure 3 with 2 supplements
Convergence on actin and synaptic processes in literature-curated LRRK2 interactors and the LRRK2 G2019S striatal phospho-proteome.
(A) Network graph showing one of the topological clusters extracted from the LRRK2 protein-protein interaction network. Proteins are represented as nodes while protein-protein interactions are represented as edges. Node size is proportional to the degree centrality: the larger the node, the higher the degree, the more interactions the node has in the network. (B) Gene Ontology biological-processes (GO-BPs) enrichment; the analysis was performed using g:Profiler, GO-BPs with term size >500 were discarded as general terms, and the remaining terms were clustered based on semantics using the keywords: Postsynaptic (Actin) Cytoskeleton, Lamellipodium, Synaptic Vesicle, Actin Polymerization/Nucleation, DNA Recombination/Repair, Cell/Neuron Projection. (C) Upper network graph as in (A) highlighting actin cytoskeletal proteins in blue and the proportion over the total (pie chart) and lower network graph as in (A) highlighting synaptic proteins in red and the proportion over the total (pie chart). (D) Experimental design of phospho-proteomic analysis comparing WT and G2019S knockin mouse striata. Phosphopeptide abundances were normalized to total protein levels. (E) Volcano plot of differentially enriched phosphopeptides, expressed as Log2 fold change (FC) between G2019S-KI versus WT. Highlighted peptides correspond to LRRK2 interactors discovered by MS in Figure 2. (F) GO enrichment analysis performed with g:Profiler (accessed on 01/23/2025; version e94_eg41_p11), filtering for GO terms with size <200 to avoid overly general categories. The top five most significant terms from the Cellular Component (CC) and Biological Process (BP) domains are shown. (G) Sunburst plot showing enriched SynGO CC categories and the percentage of SynGO genes among the differentially phosphorylated proteins. (H) Venn diagrams showing common and unique hits across the three datasets: GFP-LRRK2 interactomics, synaptic cluster, and phospho-MS.
Figure 3—figure supplement 1
Actin Cytoskeleton and Synaptic Pathway Enrichment Analysis in literature-curated LRRK2 interactors.
(A) Functional enrichment analysis; the analysis was performed using g:Profiler g:GOSt, proteins related to the actin cytoskeleton were manually identified and blue-colored, and the first 10 GO-BPs categories with term size <500 were graphed. (B) SynGO of enriched biological-processes and cellular components; synaptic proteins were red-colored, and significant terms in SynGO-BP and SynGO-CC categories were graphed.
Figure 3—figure supplement 2
Analysis of the predicted amino acid pathogenicity in drebrin using AlphaMissense pathogenicity score.
Amino acid change of S339 or its neighbor S337 is predicted to be benign, suggesting that phosphorylation at this site may play a regulatory function.
Figure 4 with 1 supplement
BDNF signaling is impaired in Lrrk2 knockout neurons.
(A) Six-day RA-differentiated SH-SY5Y naïve and CRISPR-LRRK2 KO cells stimulated with BDNF at different time points (0, 1, 5, 15, 60, and 180 min). Anti-TrkB antibodies were used to confirm expression of BDNF receptor. To compare BDNF-induced signaling in naïve vs LRRK2-KO cells, phosphorylated Akt (S473) and Erk1/2 (T185/T202) were evaluated. (B) Quantification of phosphorylated proteins shows from n=3 independent differentiation experiments. Two-way ANOVA; phospho-Akt: interaction p=0.7079, F (5, 24)=0.5896; genotype: **p=0.0014, F (1, 24)=13.07; time: p=0.5610, F (5, 24)=0.7994. Phospho-Erk1: interaction p=0.0783, F (5, 24)=2.284; genotype: ***p=0.0003, F (1, 24)=17.58; time: *p=0.0244, F (5, 24)=3.174. Phospho-Erk2: interaction: p=0.3725, F (5, 24)=1.128; genotype: ***p=0.0008 F (1, 24)=14.59; time: p=0.1356, F (5, 24)=1.879. (C) DIV14 primary cortical neurons from WT vs KO mice stimulated with BDNF at different time points (0, 5, 15, 30, and 60 min) in the presence or absence of LRRK2 inhibitor MLi-2 (90 min, 500 nM). To compare BDNF-induced signaling in WT vs KO neurons, phosphorylated Akt (S473) and Erk1/2 (T185/T202) were evaluated. Phosphorylated LRRK2 was assessed with pS935 antibodies. (D) Quantification of phosphorylated proteins shows from n=9 independent cultures. Two-way ANOVA; phospho-Akt: interaction p=0.1186, F (3, 63)=2.031; genotype: **p=0.0037, F (1, 63)=9.101; time: p=0.0342, F (3, 63)=3.069. Phospho-Erk1: interaction p=0.3256, F (3, 64)=1.177; genotype: *p=0.0201, F (1, 64)=5.680; time: ****p<0.0001, F (3, 64)=10.04. Phospho-Erk2: interaction P=0.8524, F (3, 62)=0.2622; genotype: p=0.7528, F (1, 62)=0.1001; time: ***p=0.0003, F (3, 62)=7.428. (E) Overview of the experimental workflow to induce spine formation/maturation. (F) Representative confocal images of primary cortical neurons stimulated with 100 ng/mL of BDNF or vehicle control for 24 hr. GFP has been transfected at DIV4 to fill the neuroplasm and visualize individual dendrites, MAP2 is a neuronal marker and PSD95 is a marker of mature spines. (G) Quantification of the number of PSD-positive puncta per unit of length (100 µm). Dots represent individual segments (n≅20 neurites from n=5–6 neurons per replicate), and colors define neuronal cultures prepared in different days from pulled pups (n≅8 pups per culture per genotype, n=3 independent cultures). Two-way ANOVA; interaction **p=0.0038, F (1, 191)=8.584; genotype: p=0.5833, F (1, 191)=0.3020; treatment: *p=0.0126, F (1, 191)=6.346. Šídák’s multiple comparisons test: vehicle vs. BDNF (WT) ***p=0.0005; vehicle vs. BDNF (KO) p=0.9440; Vehicle (WT) vs. vehicle (KO) p=0.3756.
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Figure 4—source data 1
Original western blot files for Figure 4.
- https://cdn.elifesciences.org/articles/95987/elife-95987-fig4-data1-v1.zip
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Figure 4—source data 2
PDF files containing original western blots for Figure 4, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/95987/elife-95987-fig4-data2-v1.zip
Figure 4—figure supplement 1
Western blot of different monoclonal populations of SH-SY5Y cells transfected with pSpCas9(BB)–2A-Puro (PX459) V2.0 vector (Addgene, Watertown, MA, US) for co-expression of Cas9 and gRNAs (gRNA1, gRNA2, and gRNAB) and subsequent single clone isolation and expansion.
The band intensities corresponding to the total amount of endogenous LRRK2 were normalized to GAPDH, used as loading control.
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Figure 4—figure supplement 1—source data 1
Original western blot files for Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/95987/elife-95987-fig4-figsupp1-data1-v1.zip
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Figure 4—figure supplement 1—source data 2
PDF files containing original western blots for Figure 4—figure supplement 1, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/95987/elife-95987-fig4-figsupp1-data2-v1.zip
Figure 5 with 1 supplement
Postsynaptic structural changes in young Lrrk2 knockout mice.
(A) Overview of experimental design. (B) Representative images of neurite segments from Golgi-Cox stained neurons of dorsal striatum. Scale bar: 3 µm. (C) Quantification of average spine number (top), width (middle), and length (bottom) of n=3 animals per group (same segments analyzed in B). Statistical significance was determined by two-way ANOVA with Šídák’s multiple comparisons test. Number of protrusions: interaction p=0.3820, F (2, 12)=1.044; age: ****p<0.0001, F (2, 12)=27.12; genotype: p=0.0840, F (1, 12)=3.550; WT vs. KO (1 month) p=0.2013; WT vs. KO (4 months) p=0.5080; WT vs. KO (18 months) p>0.9999. Width: interaction p=0.0815, F (2, 12)=3.112; age: ***p=0.0004, F (2, 12)=16.14; genotype: ***p=0.0007, F (1, 12)=20.77; WT vs. KO (1 month) **p=0.0017; WT vs. KO (4 months) p=0.4602; WT vs. KO (18 months) p=0.p2489. Length: interaction *p=0.0345, F (2, 12)=4.514; age: *p=0.0203, F (2, 12)=5.488; genotype: ***p=0.0006, F (1, 12)=21.53; WT vs. KO (1 month), ***p=0.0008; WT vs. KO (4 months), p=0.2875; WT vs. KO (18 months), p=0.5948. (D) Representative transmission electron microscopy (TEM) micrographs of striatal synapses from 1-month and 18-month-old WT vs KO mouse brain slices. Scale bar: 200 nm. (E) Quantification of post-synaptic density (PSD) length. Graphs show the length of individual synapses (grey dots) and the average PSD length per animal (colored dots). Statistical significance has been calculated with Student’s t-test: 1-month-old mice (n=4 mice, 95 synapses WT, 118 synapses KO, ***p=0.0003); 18-month-old mice (n=3 mice, 108 synapses WT, 119 synapses KO, p=0.8502). (F) Quantitative PCR of Bdnf, TrkB, Psd95, Shank3, and Dbn1 mRNA expression in striatum, cortex, and midbrain from n=6 Lrrk2 WT and n=6 Lrrk2 KO 1-month-old mice. Statistical significance has been calculated with Student’s t-test. (G) Western blot analysis of whole brain samples from the same Lrrk2 WT and KO mice where Golgi-Cox staining has been performed. The reduction in drebrin content is significant in Lrrk2 KO mice at 1 month of age. Differences between the two genotypes have been evaluated using Student’s t-test (significance **p<0.01), n=3–4 animals per genotype per age.
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Figure 5—source data 1
Original western blot files for Figure 5.
- https://cdn.elifesciences.org/articles/95987/elife-95987-fig5-data1-v1.zip
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Figure 5—source data 2
PDF files containing original western blots for Figure 5, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/95987/elife-95987-fig5-data2-v1.zip
Figure 5—figure supplement 1
Morphological classification of protrusions into four classes (filopodia, thin, mushroom, branched) and quantified as % of the total number.
Each dot represents one segment (n≥20 segments analyzed per animal; genotype: wild-type vs. Lrrk2 KO; age: 1, 4, 18-month-old; n=3 mice per group) and error bars represent the mean ± SD of n=3 mice (color coded). Statistical significance was determined by two-way ANOVA with Šídák’s multiple comparisons test on mean values. One-month: interaction: **p=0.0017, F (3, 16)=8.018; genotype: p=0.5731, F (1, 16)=0.3309; class of protrusion: ****p<0.0001, F (3, 16)=158.4; filopodia WT vs. KO p=0.0201; thin WT vs. KO *p=0.0102; mushroom WT vs. KO p=0.9585; branched WT vs. KO p=0.8726. Four-months: interaction: p=0.3178, F (3, 16)=1.271; genotype: p=0.7300, F (1, 16)=0.1233; class of protrusion: ****p<0.0001, F (3, 16)=438.9; filopodia WT vs. KO p=0.8478; thin WT vs. KO p=0.4921; mushroom WT vs. KO p=0.8882; branched WT vs. KO p=0.9858. Eighteen-months: interaction: p=0.3237, F (3, 16)=1.253; genotype: p=0.6880, F (1, 16)=0.1672; class of protrusion: ****p<0.0001, F (3, 16)=69.67; filopodia WT vs. KO p=0.9756; thin WT vs. KO p=0.6413; mushroom WT vs. KO p>0.9999; branched WT vs. KO p=0.5290.
Figure 6 with 2 supplements
Effect of BDNF exposure on spontaneous electrical activity recorded in DIV70 LRRK2 WT and KO cortical neurons.
(A) Schematic representation of the experimental setup. (B) Representative spontaneous traces of hiPSC-derived WT and KO neurons in the presence or absence of 50 ng/mL BDNF for 24 hr. (C) Frequency of miniature postsynaptic excitatory currents (mEPSC) in LRRK2 WT and KO cultures (WT vehicle: 0.16±0.03 Hz [n=15, N=3]; WT BDNF: 1.36±0.38 Hz [n=11, N=3]; KO vehicle: 1.09±0.21 Hz [n=11, N=3]; KO BDNF: 1.11±0.25 Hz [n=12, N=3]). Statistical significance was determined using one-way ANOVA (WT vehicle vs. WT BDNF **p<0.01; WT BDNF vs. KO BDNF *p<0.05; WT vehicle vs. KO BDNF *p<0.05; p>0.05 for all the other comparisons). (D) Cumulative probability curves of interevent interval (IEIs) in DIV70 WT and KO -/+ BDNF-treated cultures. (E) Amplitude of miniature postsynaptic excitatory currents (mEPSC) in DIV70 LRRK2 WT and KO cultures. BDNF treatment has no effect on the average peak amplitude (WT vehicle: 11.00±1.85 pA [n=14, N=3]; WT BDNF: 7.9±0.84 pA [n=11, N=3]; KO vehicle 10.42±1.53 pA [n=11, N=3]; KO BDNF 11.12±1.76 pA [n=12, N=3]). Statistical significance was determined using one-way ANOVA (p>0.05 for all comparisons). (F) Cumulative probability curves of peak interval in DIV70 WT and KO -/+ BDNF-treated cultures. (G) mEPSC frequency in WT and LRRK2 KO neurons at DIV50, DIV70 (data shown in C), and DIV90 under vehicle (circles) or BDNF (triangles) conditions. WT: DIV50 vehicle, 0.38±0.14 Hz (n=12, N=3); DIV50 BDNF, 0.54±0.25 Hz (n=10, N=3); DIV70 vehicle, 0.16±0.03 Hz (n=15, N=3); DIV70 BDNF, 1.36±0.38 Hz (n=11, N=3); DIV90 vehicle, 1.35±0.46 Hz (n=9, N=3); DIV90 BDNF, 1.63±0.69 Hz (n=8, N=3). KO: DIV50 vehicle, 0.48±0.16 Hz (n=9, N=3); DIV50 BDNF, 0.67±0.30 Hz (n=10, N=3); DIV70 vehicle, 1.09±0.21 Hz (n=11, N=3); DIV70 BDNF, 1.11±0.25 Hz (n=12, N=3); DIV90 vehicle, 0.26±0.13 Hz (n=8, N=3); DIV90 BDNF, 1.56±0.76 Hz (n=7, N=3). Two-way ANOVA (DIV × treatment) showed significant effects of DIV (F(2,59)=4.61, P=0.0138) and BDNF (F(1,59)=4.07, P=0.0482) in WT neurons, with no interaction (F(2,59)=1.69, P=0.1936); no significant effects were detected in KO neurons (DIV: F(2,51)=1.51, P=0.2299; BDNF: F(1,51)=3.70, P=0.0600; interaction: F(2,51)=2.10, P=0.1325). Bars represent mean ± SEM; n, cells; N, independent cultures.
Figure 6—figure supplement 1
LRRK2 Deletion and BDNF Treatment Do Not Alter Bassoon–Homer Colocalization in Cortical Neurons.
(A) Immunocytochemistry of WT and LRRK2 KO cortical neurons at DIV70 using Bassoon (pre-synaptic marker), Homer (post-synaptic marker), MAP2 (neuronal marker), and DAPI (scale bars 20 µm; zoom 5 µm). (B) Quantification of the % of Homer puncta colocalizing with total Bassoon (on MAP2-positive processes). BDNF was applied at 50 mg/mL for 24. One-way ANOVA with Tukey’s post-doc test. p>0.05 for all comparisons. N=4 cultures with 5–25 neurons imaged per condition per culture.
Figure 6—figure supplement 2
Neuronal and Synaptic Enrichment of Genes Associated with Significant GWAS Loci.
(A) Functional enrichment analysis of the nearest genes to each of the 134 significant loci (obtained from table S3, column F from https://doi.org/10.1101/2025.03.14.24319455) was performed using g:Profiler g:GOSt with term size <500 to increase specificity. (B) SynGO analysis of 32 genes mapped from the input list (from GWAS) revealed significant enrichment of synaptic Cellular Component (5 terms) and Biological Process (6 terms) annotations at 1% FDR. Annotations were based on 31 genes for Cellular Components and 27 for Biological Processes, using a brain-expressed background of 18,035 genes. Experimental-evidence filtering was not applied. SynGO version 20231201 was used.
Author response image 1
Western blot analysis of N=7 WT and N=7 drebrin KO whole brains.
Author response image 2
Western blot (same experiments as in figure 1) was performed using antibodies against phosphoThr202/185 ERK1/2, total ERK1/2 and phospho-Ser473 AKT, total AKT protein levels.
Retinoic acid-differentiated SH-SY5Y cells stimulated with 100 ng/mL BDNF for 0, 5, 30, 60 mins. MLi-2 was used at 500 nM for 90 mins to inhibit LRRK2 kinase activity.
Tables
Table 1
Capacitance and access resistance of LRRK2 KO and WT cortical neurons at 50, 70 and 90 DIV with or without BDNF stimulation.
| Capacitance 50 DIV | Access resistance 50 DIV | ||||||
|---|---|---|---|---|---|---|---|
| Mean (pF) | SEM | n | Mean (MΩ) | SEM | n | ||
| WT - BDNF | 14.14 | 1.858 | 12 | WT - BDNF | 18.54 | 1.312 | 12 |
| WT +BDNF | 15.1 | 1.331 | 12 | WT +BDNF | 18.27 | 1.053 | 12 |
| KO - BDNF | 16.21 | 2.299 | 9 | KO - BDNF | 20.11 | 1.467 | 9 |
| KO +BDNF | 14.66 | 1.295 | 11 | KO +BDNF | 18.71 | 1.884 | 11 |
| 70 DIV | 70 DIV | ||||||
| Mean (pF) | SEM | n | Mean (MΩ) | SEM | n | ||
| WT - BDNF | 23 | 2.177 | 16 | WT - BDNF | 21.18 | 1.372 | 16 |
| WT +BDNF | 22.4 | 2.579 | 12 | WT +BDNF | 21.6 | 1.273 | 12 |
| KO - BDNF | 24.64 | 2.555 | 13 | KO - BDNF | 22.36 | 1.835 | 13 |
| KO +BDNF | 20.03 | 2.232 | 13 | KO +BDNF | 15.1 | 1.122 | 13 |
| 90 DIV | 90 DIV | ||||||
| Mean (pF) | SEM | n | Mean (MΩ) | SEM | n | ||
| WT - BDNF | 12.51 | 1.994 | 8 | WT - BDNF | 17.6 | 2.221 | 8 |
| WT +BDNF | 20.9 | 6.365 | 7 | WT +BDNF | 18.64 | 1.68 | 7 |
| KO - BDNF | 24.23 | 3.062 | 7 | KO - BDNF | 16.66 | 1.609 | 9 |
| KO +BDNF | 24.96 | 6.49 | 8 | KO +BDNF | 14.64 | 1.591 | 8 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Mus musculus) | Lrrk2 knock-out (KO) mice | Mayo Clinics | JAX:016121; RRID:IMSR_JAX:016121 | Provided by Dr. Heather Melrose |
| Genetic reagent (M. musculus) | G2019S-Lrrk2 knock-in (KI) mice | Novartis | MGI:5295368; registered Novartis Lrrk2tm1.1Npa allele | Provided by Prof. Michele Morari and Dr. Derya R Shimshek |
| Genetic reagent (M. musculus) | Dbn1 knock-out (KO) mice | Previously described | MGI:5501061; PMID:28198431; the relevant allele is Dbn1tm1b(KOMP)Wtsi. | See Willmes et al., 2017 |
| Genetic reagent (Homo sapiens) | LRRK2 KO SH-SY5Y monoclonal cell line | Generated in this study | Not available | Generated by CRISPR/Cas9 |
| Genetic reagent (H. sapiens) | GFP-LRRK2 wild type expressing SH-SY5Y cells | Generated in this study | Not available | |
| Biological sample (M. musculus) | Primary cortical neurons | This study | Not available | Isolated from P0 C57BL/6 J mice and Lrrk2 knock-out (KO) mice |
| Cell line (H. sapiens) | SH-SY5Y human neuroblastoma cell line | Interlab Cell Line Collection (ICLC) | HTL95013, RRID:CVCL_0019 | Originally from ECACC; authenticated by STR profiling; mycoplasma negative |
| Cell line (H. sapiens) | HEK293T | ATCC | ATCC CRL-3216; RRID:CVCL_0063 | Human embryonic kidney cell line |
| Cell line (H. sapiens) | Isogenic WT and LRRK2 KO hiPSC lines | Mark R. Cookson laboratory | RRID:CVCL_RM92; RRID:CVCL_A8ZU; RRID:CVCL_A8ZV | Provided by Mark R. Cookson’s laboratory |
| Antibody | α-LRRK2 (rabbit monoclonal) | Abcam | Cat#: ab133474 RRID:AB_2713963 | WB (1:300) |
| Antibody | α-Phospho-Ser935 (rabbit monoclonal) | Abcam | Cat# ab133450, RRID:AB_2732035 | WB (1:300) |
| Antibody | α–21Phospho-Ser473-AKT (mouse monoclonal) | Santa Cruz Biotechnology | Cat# sc-293125, RRID:AB_2847909 | WB (1:500) |
| Antibody | α-AKT (rabbit polyclonal) | Cell Signaling Technology | Cat# 9272, RRID:AB_329827 | WB (1:1000) |
| Antibody | α-p44/42 MAPK (Erk1/2) (rabbit monoclonal) | Cell Signaling Technology | Cat# 4695, RRID:AB_390779 | WB (1:1000) |
| Antibody | Α-phospho Erk1/2 (Thr202 /Tyr204, Thr185/Tyr187) (rabbit monoclonal) | Millipore | Cat# 05–797 R RRID:AB_1587016 | WB (1:1500) |
| Antibody | α-βIII tubulin (mouse monoclonal) | Sigma-Aldrich | Cat# T8578, RRID:AB_1841228 | WB (1:40,000) |
| Antibody | α-Drebrin (mouse monoclonal) | Thermo Fisher Scientific | Cat# MA1-20377, RRID:AB_2089494 | WB (1:500) IF (1:400) |
| Antibody | α-Flag M2-HRP (mouse monoclonal) | Sigma-Aldrich | Cat# A8592, RRID:AB_439702 | WB (1:5000) |
| Antibody | α-Mouse IgG-HRP (rabbit polyclonal) | Sigma-Aldrich | Cat# A9044, RRID:AB_258431 | WB (1:80,000) |
| Antibody | α-Rabbit IgG-HRP (goat polyclonal) | Sigma-Aldrich | Cat# A9169, RRID:AB_258434 | WB (1:16,000) |
| Antibody | α-PSD95 (mouse monoclonal) | Abcam | Cat# ab2723, RRID:AB_303248 | IF (1:200) |
| Antibody | α-MAP2 (rabbit polyclonal) | Santa Cruz Biotechnology | Cat# sc-20172, RRID:AB_2250101 | IF (1:200) |
| Antibody | α-MAP2 (chicken polyclonal) | Antibodies | Cat# A85363, RRID:AB_2748940 | IF (1:1000) |
| Antibody | α-Homer 1 (rabbit polyclonal) | Synaptic Systems | Cat# 160 003, RRID:AB_887730 | IF (1:500) |
| Antibody | α-Bassoon (guinea pig polyclonal) | Synaptic Systems | Cat# 141 004, RRID:AB_2290619 | IF (1:500) |
| Antibody | α-Mouse-Alexa Fluor 568 (goat polyclonal) | Molecular Probes | Cat# A-11004, RRID:AB_2534072 | IF (1:200) |
| Antibody | α-Rabbit-Alexa Fluor 488 (goat polyclonal) | Molecular Probes | Cat# A-11034, RRID:AB_2576217 | IF (1:200) |
| Antibody | α-Rabbit-Alexa Fluor 568 (goat polyclonal) | Molecular Probes | Cat# A-11036, RRID:AB_10563566 | IF (1:200) |
| Antibody | α-Rabbit-Alexa Fluor 405 (goat polyclonal) | Molecular Probes | Cat# A-31556, RRID:AB_221605 | IF (1:200) |
| Antibody | α-Guinea pig-Alexa-488 (goat polyclonal) | Molecular Probes | Cat# A-11073, RRID:AB_2534117 | IF (1:200) |
| Antibody | α-Chicken-Alexa 647 (goat polyclonal) | Molecular Probes | Cat# A-21449, RRID:AB_2535866 | IF (1:200) |
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/95987/elife-95987-mdarchecklist1-v1.pdf
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Supplementary file 1
Quantitative proteomic analysis of LRRK2-associated proteins under basal and BDNF-stimulated conditions.
- https://cdn.elifesciences.org/articles/95987/elife-95987-supp1-v1.xlsx
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Supplementary file 2
Functional enrichment and semantic classification of LRRK2-associated proteins involved in actin-cytoskeleton and synaptic processes.
- https://cdn.elifesciences.org/articles/95987/elife-95987-supp2-v1.xlsx
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Supplementary file 3
Comparative phosphoproteomic analysis of saline-treated LRRK2 G2019S and C57BL/6 mouse samples.
- https://cdn.elifesciences.org/articles/95987/elife-95987-supp3-v1.xlsx
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LRRK2 regulates synaptic function through modulation of actin cytoskeletal dynamics
eLife 13:RP95987.
https://doi.org/10.7554/eLife.95987.4
