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, Dunnett’s multiple comparison test (*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 (G) (n=4 independent experiments). One-way ANOVA, Dunnett’s multiple comparison test (*P < 0.05 t=0 vs. t=5’).

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 | FC < −0.5 or (ii) FC > 1 | FC < −1. 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 enriched categories of BDNF-increased interactors (top, blue bar graph) versus BDNF-decreased interactors (bottom, orange bar graph). (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) 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. (H) Validation of increased drebrin:LRRK2 interaction upon BDNF treatment (15 mins). Samples are the same analyzed in the three rounds of MS (A). (I) Quantification of (H).

An actin-synaptic cluster from the LRRK2 protein-protein interaction network.

(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) Network graph as in (A) highlighting actin cytoskeletal proteins in blue and the proportion over the total (pie chart). (D) Network graph as in (A) highlighting synaptic proteins in red and the proportion over the total (pie chart).

BDNF-induced spinogenesis 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 expeiments. 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 (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.

Striatal spine maturation is delayed in 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) Morphological classification of protrusions into four classes (filopodia, thin, mushroom, branched) and quantified as % of the total number. Each dot represents one segment (n ζ 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. (E) Representative transmission electron microscopy (TEM) micrographs of striatal synapses from 1-month and 18-month old WT vs KO mouse brain slices (n=3-4 mice per genotype). Scale bar: 200 nm. (F) 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). (G) Quantitative PCR of Bdnf, TrkB, Psd95 and Shank3 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. (H) 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 animals per genotype per age.

Effect of BDNF exposure on spontaneous electrical activity recorded in LRRK2 WT and KO cortical neurons.

(A) Schematic representation of the experimental setup. (B) Representative spontaneous traces of hiPSC-derived cortical neurons WT in the presence or absence of 50 ng/ml BDNF for 24 hrs (upper). Left plot: frequency of mini postsynaptic excitatory currents (mEPSC) in LRRK2 WT (vehicle: 0.16 ± 0.03 Hz (n=15, N=3); BDNF: 1.36 ± 0.38 Hz (n=11, N=3). Statistical significance was determined using Student’s paired t-test (LRRK2 WT: vehicle vs. BDNF **P < 0.01; LRRK2 KO: vehicle vs. BDNF P > 0.05). Right plot: cumulative probability curves of interevent interval (IEIs) in WT -/+ BDNF treatment. (C) Representative spontaneous traces of hiPSC-derived cortical neurons KO in the presence or absence of 50 ng/ml BDNF for 24 hrs (upper). Left plot: frequency of mini postsynaptic excitatory currents (mEPSC) in LRRK2 KO (vehicle: 1.09 ± 0.21 Hz (n=11, N=3); BDNF: 1.11 ± 0.25 Hz (n=12, N=3). Statistical significance was determined using Student’s paired t-test (LRRK2 KO: vehicle vs. BDNF P > 0.05). Right plot: cumulative probability curves of interevent interval (IEIs) in KO -/+ BDNF treatment. (D) Left plot: amplitude of mini postsynaptic excitatory currents (mEPSC) in LRRK2 WT. BDNF treatment has no effect on the average peak amplitude (vehicle: 11.00 ± 1.85 pA (n=14, N=3); BDNF: 7.4 ± 0.77 pA (n=10, N=3)). Statistical significance was determined using Student’s paired t-test (vehicle vs. BDNF P > 0.05). Right plot: cumulative probability curves of peak amplitude in WT -/+ BDNF treatment. (E) Left plot: amplitude of mini postsynaptic excitatory currents (mEPSC) in LRRK2 KO. BDNF treatment has no effect on the average peak amplitude (vehicle 10.42 ± 1.53 pA (n=11, N=3), BDNF 11.12 ± 1.76 pA (n=12, N=3)). Statistical significance was determined using Student’s paired t-test (vehicle vs. BDNF P > 0.05). Right plot: cumulative probability curves of peak amplitude in WT -/+ BDNF treatment.

Phosphoproteome of Lrrk2G2019S synaptosomes is enriched in postsynaptic structure organization SynGO terms.

(A) Overview of experimental design. (B) Volcano plot of 2957 differentially phosphopeptides common between WT and Lrrk2G2019S striatal synaptosomes (labeled with gene name) isolated from Lrrk2 WT and Lrrk2G2019S KI mice. Common genes between this analysis and the AP-MS analysis are orange labelled for peptides with decreased phosphorylation and blue labelled for peptides with increased phosphorylation. (C) Sunburst plot showing enriched SynGO biological processes (BP) categories (267 proteins, at least one significant peptide, P < 0.05 and fold change > 0.5). (D) Sunburst plot showing enriched SynGO cellular component (CC) categories (267 proteins, at least one significant peptide, P < 0.05 and fold change > 0.5).