Fig 1: Interferon-? signaling synergizes with LRRK2 in human neurons and microglia.IFN-? response is conserved in DA neurons, and IFN-? induces LRRK2 expression in DA neurons and microglia (a). The PD-associated mutation LRRK2 G2019S sensitizes DA neurons to IFN-? by decreasing AKT phosphorylation (b) and suppressing NFAT nuclear shuttling (c) that, in turn, leads to defects in neurite outgrowth (d). LRRK2-dependent defects of NFAT translocation are linked to defects in calcium buffering capacity (e) and possibly to an increased complexity of the microtubule network (f). Defects in NFAT shuttling are also observed in LRRK2 G2019S microglia (g), which display a more activated phenotype upon stimulation. LRRK2 G2019S modulates microglia activation by interfering with the metabolic switch toward glycolysis that normally occurs upon mononuclear phagocyte activation (h) and impairing NF-?B p65 nuclear translocation upon stimulation with IFN-? or LPS (i).
Fig 2: Aberrant synaptic PKA activities underlie the dopamine-dependent RC impaired motor learning.a Western blot analysis of striatal extracts from WT and RC mice injected with AAV-sh-control and AAV-sh-PKARIIß viral constructs probed for p-PKA and ß-actin. b Quantification of p-PKA bands normalized to ß-actin. Summary graphs represent the mean, while error bars SEM, *p < 0.05, ***p < 0.001, Tukey post hoc following one-way ANOVA (n = 3). c, d The latency to fall of RC mice treated with the dopamine receptors antagonists’ cocktail and injected either with control AAV-sh-control and AAV-sh-PKARIIß viruses was assessed in the rotarod paradigm described in Fig. 3a. A subset of mice was administered saline and used as a reference for the effect of D1 and D2 antagonism (saline treated group nRCsaline = 10, D1 + D2 antagonists treated groups nRCsh-control = 13, and nRC-shPKARIIß = 10). The average latency in the drug-free recovery phase of the three late sessions (16–18) are summarized in e. Asterisks show statistical significance for Tukey’s multiple comparison tests after two-way ANOVA. d Dopamine receptor antagonists treated WT mice were injected with shPKARIIß virus, and their latency to fall was assessed in the accelerating test. WT mice administered saline and cocktail are used as reference, saline treated group nWT = 10, D1 + D2 receptor antagonist treated groups nRC = 12, and nRC-shPKARIIß = 11. The average latency in the three late sessions (16–18) of the rotarod test is shown in f. Asterisks show statistical significance for Tukey’s multiple comparison tests after two-way ANOVA. g Working model of aberrant PKA synaptic activities at a blowup of a spine of an SPN (left). Presynaptic dysregulation of LRRK2 activity in RC and GS mutants has a common disruption of the synaptic vesicle cycle, which leads to decreased dopamine release. In addition, in RC mutants, there is a postsynaptic translocation of PKA into the dendritic spines with aberrant increased PKA activity, which disrupts synaptic signaling and interferes with motor learning and performance (measured by decreased latency to fall off rotarod).
Fig 3: Schematic representation of the generation of clonal LUHMES cells expressing WT and G2019S LRRK2 using AMAXA nucleofection. Proliferating naïve LUHMES cells (gray) were transfected with plasmids expressing either WT or G2019S LRRK2 proteins (blue) using the AMAXA nucleofection technology. After transfection, a mixture of untransfected (gray) and transfected cells (blue) was obtained. Antibiotic selection removed untransfected (gray) cells, and a pool of transfected cells (LUHMES pools) expressing different levels of transgene (indicated by different shades of blue) was obtained. Serial dilution of transfected pool generated single clones that subsequently formed clonal colonies. LUHMES cell pools and clonal colonies were differentiated into mature neurons and further characterized as described in the main text.
Fig 4: Characterisation of Rab GTPase behaviour after sterile damage RAW264.7 macrophages were electroporated with EGFP-Rab3A, EGFP-Rab8A, EGFP-Rab10 and EGFP-Rab35. Cells were treated with 1 mM of LLOMe for 30 min, and Rab recruitment to LAMP1 + compartments was monitored by high-content immunofluorescence imaging. Scale bar = 10 µm.WT and LRRK2 KO RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min. Cells were separated into cytosolic (C) and membrane (M) fractions and analysed for Rab8A pT72, Rab8A and Rab10 by Western blot.WT and Rab8A KO RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min, and Rab8A and Rab8A pT72 levels were analysed by Western blot.RAW264.7 macrophages were electroporated with EGFP-Rab8A-WT, EGFP-Rab8A-Q67L, EGFP-Rab8A-T22N and EGFP-Rab8A-T72A. Cells were treated with 1 mM of LLOMe for 30 min, and CHMP4B recruitment was assessed by confocal microscopy. Scale bar = 5 µm.CHMP4B integrated fluorescence density was analysed per cell. Data show values from single cells and mean. Source data are available online for this figure.
Fig 5: Microglial motility is retarded and accelerated, respectively, by the LRRK2 GS mutation and LRRK2 deficiency.(a) Time-lapse images of non-Tg and GS-Tg microglia were obtained every 5 s for 20 min after addition of 100 µM ADP. Black and white arrowheads represent lamellipodia. (b,c) Membrane dynamics of microglia (n=25) were quantified using SACED as described in the Methods section. Two-tailed Student's t-test, *P<0.05, **P<0.01. (d) Motilities of non-Tg and GS-Tg microglia were measured using transwells (upper panel). Number of cells migrated to the bottom of the transwells were counted at 12 h after plating (lower panel). Values are means±s.e.m. of three independent experiments. One-way ANOVA with Newman–Keuls post hoc test, **P<0.01. (e) Unlike NT microglia, LRRK2-KD microglia showed a morphological polarity with a leading edge corresponding to lamellipodia (white arrowheads) and a tail (black arrowheads) even in the absence of ADP. (f) NT and LRRK2-KD cells were stained with Alexa-488 phalloidin, and Z-stack scanned images were obtained at 2-µm intervals from the bottom. Lamellipodia attached to the bottom were enriched with F-actin in LRRK2-KD cells (arrowheads). (g) Time-lapse images of NT and LRRK2-KD cells were taken at 2-min intervals for 72 min. Asterisks and arrow heads chased two different cells that moved in the images. (h) Spontaneous migration paths of NT and LRRK2-KD cells (15 cells each) were tracked for 6 h. The location of each cell was determined every 10 min and connected to depict its migration route. Scale bar, 50 µm (a,d); 10 µm (e–g). Data are representative of at least three independent experiments unless indicated. ANOVA, analysis of variance.
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