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, 60 180 mins. MLi-2 was used at 500 nM for 90 mins 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, 60 mins. (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, 60 mins. MLi-2 was used at 500 nM for 90 mins 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, 60 mins. MLi-2 was used at 500 nM for 90 mins 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’).

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 2 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 2 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 an 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 mins) 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 assessing Lrrk2 Ser935 and Rab8 (pan Rabs) phosphorylation Differences between the two genotypes have been evaluated using Student’s t-test (significance *P<0.05 pSer935), n=3 animals per genotype per age.

Capacitance and access resistance of LRRK2 KO and WT cortical neurons at 50, 70 and 90 DIV with or without BDNF stimulation.

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 was 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 proteins 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 5 most significant terms from the Cellular Component (CC) and Biological Process (BP) domains are shown. (G) Sunburst plot showing enriched SynGO cellular component (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.

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, 180 mins). Anti-TrkB antibodies were used to confirmed 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 show 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, 60 mins) 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 show 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 hours. 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 represents individual segments (ný20 neurites from n=5-6 neurons per replicate) and colors define neuronal cultures prepared in different days from pulled pups (ný8 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.

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=0.2489. 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 form n=6 Lrrk2 WT and n=6 Lrrk2 KO one-month old mice. Statistical significance has been calculated with Student’s t-test. (G) Western blot analysis of 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.

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 hrs. (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).

(A) Western blot analysis of undifferentiated versus differentiated naïve 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 mins or with 500 nM MLi-2 for 90 mins. (C) On the left, schematic representation of experimental setting. On the right, BDNF stimulation of GFP-LRRK2 SH-SY5Y cells prior to MLi-2 (500 nM) treatment for 90 minutes. Western blot were performed using anti LRRK2 (phospho-Ser935 and total), anti-RAB10 (phospho Thr73 and total) and anti-RAB8 (pan-phopho-RABs and total RAB8).

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 naïve 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 naïve 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; aminoacid 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.

(A) Functional enrichment analysis; the analysis was performed using g:Profiler g:GOSt, proteins related to actin cytoskeleton were manually identified and blue-colored, and the first ten 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.

Analysis of the predicted aminoacid pathogenicity in drebrin using AlphaMissense pathogenicity score.

Aminoacid change of S339 or its neighbor S337 is predicted to be benign, suggesting that phosphorylation at this site may play a regulatory function.

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.

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.

No significant change in the frequency of mEPSC between WT and KO in (A) DIV50 and (B) DIV90 due to the addition of BDNF (50 mg/ml for 24 hrs). One-way ANOVA with Tukey’s post-doc test. P>0.05 for all comparisons (N=2 independent cultures). (C) 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). (D) 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.

(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.