Figure S1. (A) SDS-PAGE separation of GST-fusion proteins purified from E.coli BL21 strain is shown. An equal amount of GST-tag control, LRRK2 LRR

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1 Figure S1. (A) SDS-PAGE separation of GST-fusion proteins purified from E.coli BL21 strain is shown. An equal amount of GST-tag control, LRRK2 LRR and LRRK2 WD40 GST fusion proteins (5 µg) were loaded onto a 1 0% SDS-gel and stained with colloidal Coomassie. GST, LRRK2 LRR and LRRK2 WD40 fusion proteins were used as baits to pull-down putative LRRK2 interactors from mouse total brain lysate. Eluted samples were analyzed by silver-stain (B) or western-blotting after SDS-PAGE (C).

2 Figure S2. (A) SDS-PAGE separation of GST-fusion proteins purified from E.coli BL21 strain is shown. Equal amounts of GST-tag control, LRRK2 WD40 wild type, LRRK2 WD40 G2385R and RACK1 GST fusion proteins (5 µg) were loaded on a 1 0% SDS-gel and stained with colloidal Coomassie. (B) RACK1 failed to pull-down LRRK2, NSF, syntaxin1 A, actin and MAP2. (C) AntiGST antibody stains multiple bands generated by partial degradation of the fusion proteins.

3 Figure S3. The list of putative interactors of LRRK2 WD40 was elaborated on STRING (accessible on and further processed by Cytoscape. The network reports putative interactors of LRRK2 WD40 and their reciprocal connections as reported in STRING. Network nodes are grouped according to their main GO terms. Solid lines depict interactions annotated on STRING while dashed ones highlight the interactions described in this manuscript.

4 Figure S4. Subcellular distribution of ectopic DsRed, RFP LRRK2 WD40, RFP LRRK2 WD40 G2385R, RFP RACK1 and FLAG LRRK proteins was assayed via co-localization with the synaptic protein SNAP-25. Images show signals acquired for DsRed or RFP or FLAG (channel 1, CH1 ), superimposition of CH1 and SNAP-25 (CH1 +CH2) and the relative merge with GFP (merge). Cortical neurons were transfected at DIV1 0 and fixed at DIV1 6. Panel size shown is 200 x 200 µm.

5 Figure S5. (A) Sequence alignment analysis of the WD40 domains of LRRK2 and RACK1. In this alignment, the conserved hydrophobic residues are shown in orange, negatively charged residues in red, positively charged residues in blue and the rest of the highly conserved residues in green. For comparison, the secondary structure feature of the RACK1 WD40 domain (PDB ID:

6 3DM0) is also included. The G2385 residue is labeled and highlighted with a red star and the residues that are predicted to conflict with the G2385R mutant are labeled and highlighted with black asterisks. (B) Representative TEM micrographs of negatively stained (1 % uranyl actetate) GST-tag control and LRRK2 WD40, RACK1 or LRRK2 WD40 G2385R GST fusion proteins. Scale bars are as indicated. (C) Electron microscopy of negatively stained (2% ammonium molybdate) 6xHIS LRRK2 WD40 reveals the typical WD40 ring-like fold independent of the solubility-enhancing GST-tag or a specific staining solution. Upper panel: representative micrograph, scale as indicated. Lower panel: four representative averaged single particle 2D projections (scale bar represents 5 nm). (D) Increasing nanomolar amounts of GST-tag protein were incubated with unstripped (US) or salt-stripped SV (SSV) before high-speed sedimentation. A representative Western blot show initial amount of GST-tag protein (total) and the yield of GST-tag protein precipitated by US or SSV (bound). GST-tag proteins were incubated with equal amount of SV (monitored by anti synaptophysin staining).

7 Figure S6. Expression of the LRRK2 WD40 G2385R domain is sufficient to induce neurotoxicity. (A) Cortical neurons were transfected at DIV1 0 with GFP and either DsRed alone, RFP LRRK2 WD40 (WD40 wild type) or RFP LRRK2 WD40 G2385R (WD40 G2385R) constructs and fixed at DIV1 6. The over-expression of LRRK2 WD40 or LRRK2 WD40 G2385R significantly reduced the number of processes and increase the amount of swollen or fragmented neurites compared to DsRed transfected neurons. Images show signals acquired for GFP (channel 1, CH1 ), DsRed or RFP (channel 2, CH2), the relative merge and tracing. Graphs report the number of total

8 neurites (B) and number of fragmented processes (C); n=1 5, ** p<0.01 vs DsRed condition, ANOVA, Tuckey s post hoc test. The exo-endo assay was performed at DIV1 2 on cortical neurons transfected with GFP and either DsRed, RFP LRRK2 WD40 or RFP LRRK2 WD40 G2385R at DIV1 0. (D) Cycling SV appear as synaptotagmin (s-tagmin) positive clusters along neuron processes while total number of SV was determined by staining with anti SV2A antibodies upon permeabilization. Images show signals acquired in RFP LRRK2 WD40 G2385R transfected neurons for s-tagmin or SV2A (channel 1, CH1 ) and DsRed or RFP (channel 2, CH2) and the relative merge plus GFP. Panels are 1 0 µm long. (E) SV cycling is severely reduced upon either LRRK2 WD40 or LRRK2 WD40 G2385R over-expression. The graph reports the number of s-tagmin positive clusters per 1 0 µm of GFP-positive process. (F) Total SV pool, monitored as SV2A-positive dots along 1 0 µm of neuronal processes, was not modified by LRRK2 WD40 or LRRK2 WD40 G2385R domains over-expression. (G) Cycling SV distribution is confined near the soma in neurons over-expressing LRRK2 WD40 or LRRK2 WD40 G2385R. The graph reports the percentage of s-tagmin positive clusters distributed within the proximal half of the process. (H) Distribution of total SV pools is not affected by either LRRK2 WD40 or LRRK2 WD40 G2385R domains expression. The graph reports the percentage of SV2A-positive clusters distributed within the proximal half of the process. Data are expressed as mean±se; * p<0.05 vs DsRed condition, ANOVA, Tukey s post hoc test, n=4, 7 neurons were analyzed for each experimental case.