Fig 2: Pharmacological inhibition of Rab7 abolished the neuroprotective effect of NRBF2 against SAH at 24 h. A The lysates from cortex tissue were immunoprecipitated with anti-NRBF2. Then, immunoprecipitates were analyzed by western blotting with anti-NRBF2 and anti-Rab7. n = 6. B Immunostaining for NRBF2 and Rab7 in left cortex area. n = 3. C, D Quantification of neurological function with two different scoring systems at 24 after SAH. n = 17. E Quantification of brain water content. n = 6. F Representative microphotographs of immunofluorescence double staining showing the localization of LC3B (red) and Lamp2 (green). Scale bar = 50 μm. n = 5. (G-O) Representative western blotting images and quantitative analyses of LC3 II/I, Lamp2, CHOP, Romo-1, TXNIP, NLRP3, TNF-α, and IL-1β expression at 24 h after SAH. n = 6. Data are represented as mean ± SD (E, H–O) or median (interquartile range) (C, D). *P < 0.05 versus sham group. #P < 0.05 versus SAH + NC-AAV group. &P < 0.05 versus SAH+NRBF2-AAV + vehicle group

Fig 3: NRBF2 interacted with Rab7 and the MIT domain was indispensable for the interaction between NRBF2 and Rab7. A The lysates from HT22 cells were immunoprecipitated with anti-NRBF2. Then, immunoprecipitates were analyzed by western blotting with anti-NRBF2 and anti-Rab7. n = 5. B Immunostaining for NRBF2 and Rab7 in HT22 cells. n = 3. C Schematic diagram of NRBF2. D Cell lysates were immunoprecipitated with an anti-Rab7 antibody and subjected to western blotting with the indicated antibodies, including anti-NRBF2, -CCZ1, -MON1A, and -Rab7. n = 5

Fig 4: Activity and cellular localization of LRRK1 and LRRK1 variants in vitro.A Western blot showing phosphorylation of Rab7A, and relative LRRK1 expression level of the indicated LRRK1 mutants. B Quantification of membranes shown in (A), i) quantification of total LRRK1, normalized to a-tubulin, ii) quantification of pRab7, normalized to total Rab7. Data are mean ± SEM (n = 3 independent experiments). C Representative confocal images of U2OS cells transfected with the indicated LRRK1 variant, stained for total LRRK1 (flag) and GFP-Rab7A. D Quantification of LRRK1/Rab7A colocalization. Data are mean ± SEM (n = 22–28 cells per group over one independent experiment). One-way ANOVA with Dunnett’s was used for multiple comparisons analysis. Effect size was measure as R2 = 0.4322. **** means p value < 0.0001. Source data is provided as a source data file.

Fig 5: The Roc-COR-kinase interface.A Effect of mutations in this interface on Rab7A phosphorylation by recombinant LRRK1. Results presented are the quantification of multiple kinase assays from at least three independent protein preparations (see “Methods”, Supplementary Fig. 7D for representative membrane images and Supplementary Fig. 8D for complete membrane images, values are mean ± SEM, n = 6–36 independent assays from 9 independent protein preparations (WT, K1270M) or 3 independent protein preparations (all other mutants)). B Related to C–E, structure of LRRK1, with the position of the aC helix, COR-B DK and a1 helices and K746 indicated in the structure. C Cryo-EM density supporting the Roc/COR interface, additionally showing that the aC helix is positioned away from the interface in the inactive state. D Structural changes between the inactive-state cryo-EM model, and the active-state Alphafold model, highlighting a change in the position of K746 relative to the DK helix, and in the position of R1034. E Overlay of the DK helix/activation loop interface between the cryo-EM and Alphafold models, highlighting the movement of the DK helix towards the activation loop. In C the map shown is the composite cryo-EM density map. Source data is provided as a source data file.