Figure 1.
Expression of LRRK2 domain fragments reduces the viability of yeast.
(A) LRRK2 domain fragments reduce yeast viability. Yeast cells (BY4741 MATa) were transformed with galactose-inducible high copy expression constructs containing the following human LRRK2 domain fragments: GTPase domain (GTP, residues 1300–1514), kinase domain (Kin, residues 1843–2163), kinase domain plus the C-terminal region (Kin-CT, residues 1843–2527), GTPase-COR-kinase domains (GTP-COR-Kin, residues 1300–2163), GTPase-COR-kinase domains plus the C-terminal region (GTP-COR-Kin-CT, residues 1300–2527), and a LRRK2 fragment lacking the N-terminal LRRK2-specific repeat region (ΔN-LRRK2, residues 570–2527) and low copy expression construct containing the GTP-COR-Kin region. Empty vectors (pYES/CT, p416GAL) are used as controls. Cells were spotted onto media containing glucose (LRRK2 Off, repressed, left panel) or galactose (LRRK2 On, induced, right panel) and incubated at 30°C for 2–3 days. Shown are five-fold serial dilutions (from left to right, as indicated by graded open box) starting with equal numbers of cells. Protein domain structure of each LRRK2 fragment relative to the full-length protein is also indicated. (B) Expression of LRRK2 domain fragments in yeast cells following galactose induction was detected by Western blot analysis with anti-V5 antibody, with anti-PGK antibody as a protein loading control. (C) Growth curve analysis in liquid media containing galactose was used to monitor the growth rate of yeast cells expressing each LRRK2 domain fragment or with empty vector as a control. Data are taken from three independent experiments with each data point representing the mean ± SEM (n = 3).
Figure 2.
GTPase activity modulates LRRK2-induced toxicity in yeast.
(A) Table of LRRK2 sequence variants employed in this study and their predicted functional effects. (B) GTPase mutations, K1347A and T1348N, markedly enhance LRRK2-induced toxicity in yeast compared to WT or other GTPase mutations. Yeast cells were transformed with galactose-inducible expression constructs containing the central GTP-COR-Kin fragment of LRRK2 harboring various functional GTPase variants (WT, R1441C, R1398L, R1398Q/T1343G, K1347A, and T1348N) or empty vector as a control. Spotting experiments were conducted to examine the viability of yeast cells due to the expression of each truncated LRRK2 GTPase variant. Shown are five-fold serial dilutions (from left to right, as indicated by graded open box) starting with equal numbers of cells grown on media containing glucose (LRRK2 Off, left panel) or galactose (LRRK2 On, right panel). (C) Expression of LRRK2 GTPase variants in the GTP-COR-Kin fragment in yeast following galactose induction was detected by Western blot analysis with anti-V5 antibody, with anti-PGK antibody as control for protein loading. (D) Growth curve analysis in liquid media containing galactose was used to measure the growth rate of yeast cells expressing each truncated LRRK2 GTPase variant relative to an empty vector control. Data are taken from three independent experiments with each data point representing the mean ± SEM (n = 3). (E) GTP-binding activity was determined for each LRRK2 GTPase variant (in the GTP-COR-Kin fragment) derived from yeast cell lysates following galactose induction by measuring the relative levels of GTP-bound LRRK2 with normalization to input levels of total LRRK2. LRRK2 levels were determined from western blot images by densitometric analysis. Data are expressed as GTP-binding as a percent of WT LRRK2 levels with each bar representing the mean ± SEM from three independent experiments. An example Western blot probed with anti-V5 antibody is shown indicating the levels of GTP-bound GTP-COR-Kin LRRK2 fragment and input levels. (F) GTP hydrolysis activity was determined in yeast by measuring the concentration of free Pi released from GTP for each truncated LRRK2 GTPase variant and normalized to LRRK2 input levels. Input levels of immunoprecipitated GTP-COR-Kin LRRK2 derived from yeast total lysates were detected by Western blot analysis with anti-V5 antibody, as shown, with densitometric analysis. GTP hydrolysis activity for each LRRK2 variant is expressed as Pi release as a percent of WT LRRK2 activity with each bar representing the mean ± SEM from three independent experiments. (G) GTP hydrolysis activity was measured for disease-associated mutations in full-length human LRRK2. Input levels of immunoprecipitated myc-tagged LRRK2 derived from HEK-293T cell lysates were detected by Western blot analysis with anti-MYC antibody, as shown, with densitometric analysis. GTP hydrolysis activity for each LRRK2 variant is expressed as Pi release as a percent of WT LRRK2 activity with each bar representing the mean ± SEM from five independent experiments. Data were analyzed for statistical significance by two-tailed unpaired Student's t-test compared to WT-LRRK2 (*P<0.01 and **P<0.001). n.s., non-significant.
Figure 3.
LRRK2 GTPase variants induce defects in endocytic vesicular trafficking.
(A) Endocytosis of the lipophilic fluorescent dye FM4–64 (red) was employed to monitor the effects of LRRK2 GTPase variants (in the GTP-COR-Kin fragment) on vesicular trafficking to the vacuole in yeast. Cells carrying empty vector display normal ring-like vacuolar membrane staining (asterix). Expression of the GTPase mutants, K1347A or T1348N, markedly disrupts FM4–64 vacuole localization with the appearance of multiple large punctate structures (arrows). (B) Quantification of endocytic trafficking defect showing the average number of FM4–64-positive punctate structures per cell. A total of 100 cells were analyzed in one experiment and data are representative of at least two independent experiments. Bars represent the mean ± SEM. Data were analyzed for statistical significance by two-tailed unpaired Student's t-test relative to WT LRRK2, or by pair-wise comparisons with vector controls where indicated by horizontal lines (*P<0.01). n.s., non-significant versus WT. (C) Frequency distribution showing the percent (%) of cells with different numbers of FM4–64-positive punctate structures as a measure of endocytic trafficking defects between truncated LRRK2 GTPase variants. Data are taken from one experiment (n = 100 cells) and are representative of at least two independent experiments. (D) Yeast cells carrying LRRK2 GTPase variants have normal vacuolar morphology. DIC images are employed to visualize the morphology of vacuoles for each LRRK2 construct following galactose induction and FM4–64 staining.
Figure 4.
LRRK2 GTPase variants induce defects in autophagy.
(A) Transmission electron micrographs demonstrating increased autophagic vacuoles (asterix) within the vacuole of yeast cells expressing the toxic GTPase variants, K1347A or T1348N, in the LRRK2 GTP-COR-Kin fragment. V, vacuoles; N, nucleus; Scale bars, 2 µm. (B) Quantification of autophagic defects showing the percentage of cells with autophagic vacuoles. A total of 100 cells were analyzed in one experiment and data are representative of at least two independent experiments. Bars represent the mean ± SEM. Data were analyzed for statistical significance by two-tailed unpaired Student's t-test relative to WT LRRK2, or by pair-wise comparisons with vector controls where indicated by horizontal lines (*P<0.01 and **P<0.001). n.s., non-significant versus WT.
Figure 5.
GTPase activity modulates LRRK2-induced neuronal toxicity.
(A) Human LRRK2 domain fragments containing the GTPase domain (GTP and GTP-COR-Kin) but not the kinase domain (Kin) alone induce neuronal toxicity similar to full-length WT LRRK2. Representative fluorescent images (eGFP) showing mouse primary cortical neurons co-transfected with LRRK2 constructs and eGFP in a 10∶1 molar ratio or transduced with HSV-WT-LRRK2/CMV-eGFP virus expressing full-length WT LRRK2. Neuronal viability was analyzed at 48 hrs post-transfection (DIV 12) with non-viable neurons exhibiting obvious neurite process and/or nuclear fragmentation (arrows). (B) Quantification of neuronal viability induced by LRRK2 expression. Bars indicate the viability of eGFP-positive neurons (n = 200) for each transfection condition expressed as a percent (%) of control neurons (eGFP only). Data represent the mean ± SEM from three independent experiments. Data were analyzed for statistical significance by two-tailed unpaired Student's t-test compared to control neurons (*P<0.01 and **P<0.001). n.s., non-significant. (C) LRRK2 GTPase variants (in GTP-COR-Kin fragment) induce neuronal toxicity. Representative fluorescent images (eGFP) of neurons at 48 hrs post-transfection containing truncated LRRK2 GTPase variants and eGFP. Arrows indicate non-viable neurons. (D) Quantification of neuronal viability induced by truncated LRRK2 GTPase variants. Bars indicate the viability of eGFP–positive neurons (n = 200) for each transfection condition expressed as a percent (%) of control neurons (eGFP only). Data represent the mean ± SEM from three independent experiments. Data were analyzed for statistical significance by two-tailed unpaired Student's t-test compared to control neurons, or by pair-wise comparisons where indicated by horizontal lines (*P<0.05 and **P<0.005). n.s., non-significant versus control.
Figure 6.
Expression of LRRK2 causes vesicular trafficking defects in neurons.
(A) Hippocampal neuronal synapses transduced with HSV-WT-LRRK2/CMV-eGFP or HSV-PrPUC/CMV-eGFP as a control following FM4–64 dye loading by synaptic vesicle endocytosis and unloading by synaptic vesicle exocytosis. Arrows indicate synaptic boutons. (B) Dynamic real-time quantification of FM4–64 fluorescence intensity in synaptic boutons following dye loading and unloading over a 10 min period. Notice reduced initial loading and delayed release of the FM dye due to WT LRRK2 expression (C) Quantification of FM4–64 fluorescence intensity in synaptic boutons following dye loading at time point 0 sec and unloading at time point 480 sec. Data represent the mean ± SEM from three independent experiments (15 boutons for each experiment). Data were analyzed for statistical significance by two-tailed unpaired Student's t-test between HSV-FL-LRRK2 neurons and HSV-GFP control neurons (*P<0.01).
Figure 7.
A genome-wide genetic screen identifies modifiers of LRRK2 toxicity in yeast.
(A) Yeast gene deletion strains ahc2Δ and rmd8Δ markedly enhance LRRK2-induced toxicity in yeast compared to the WT strain. Yeast WT strain BY4741 and gene deletion strains ahc2Δ and rmd8Δ cells were transformed with galactose-inducible expression constructs containing the central GTP-COR-Kin fragment of WT LRRK2 or empty vector as a control. (B) Yeast gene deletion strains gcn4Δ, ade16Δ, yhl005cΔ, slt2Δ, gfd2Δ, cce1Δ, and gcs1Δ suppress LRRK2-induced toxicity in yeast compared to the WT strain. Yeast WT strain BY4741 and gene deletion strains gcn4Δ, ade16Δ, yhl005cΔ, slt2Δ, gfd2Δ, cce1Δ, and gcs1Δ cells were transformed with galactose-inducible expression constructs containing the central GTP-COR-Kin fragment of WT LRRK2 or empty vector as a control. (C) Yeast gene deletion strains gcn4Δ, ade16Δ, yhl005cΔ, slt2Δ, gfd2Δ, cce1Δ, and gcs1Δ markedly suppress LRRK2 mutant K1347A and T1348N-induced toxicity in yeast compared to the WT strain. Yeast WT strain BY4741 and gene deletion strains gcn4Δ, ade16Δ, yhl005cΔ, slt2Δ, gfd2Δ, cce1Δ, and gcs1Δ cells were transformed with galactose-inducible expression constructs containing K1347A and T1348N mutations in the central GTP-COR-Kin fragment of LRRK2 or empty vector as a control. Spotting experiments were conducted to examine the viability of yeast cells due to the expression of the LRRK2 fragment (A–C). Shown are five-fold serial dilutions (from left to right, as indicated by graded open box) starting with equal numbers of cells grown on media containing glucose (LRRK2 Off, left panel) or galactose (LRRK2 On, right panel). (D) Endocytosis of FM4–64 (red) was employed to monitor vesicular trafficking in yeast gene deletion strains ahc2Δ and rmd8Δ carrying the WT LRRK2 GTP-COR-Kin fragment. WT yeast cells expressing the GTP-COR-Kin fragment display normal ring-like vacuolar membrane staining (asterix) in addition to some punctate structures (arrow). Yeast gene deletion strains ahc2Δ and rmd8Δ expressing the GTP-COR-Kin fragment markedly disrupts FM4–64 vacuole localization with the appearance of multiple large punctate structures (arrows) whereas the ahc2Δ and rmd8Δ strains alone show normal vacuole staining. (E) Endocytosis of FM4–64 (red) was employed to monitor vesicular trafficking in yeast gene deletion strains gcn4Δ, ade16Δ, yhl005cΔ, slt2Δ, gfd2Δ, cce1Δ, and gcs1Δ carrying the LRRK2 GTP-COR-Kin mutant T1348N. WT yeast cells expressing the T1348N mutant display multiple large punctate structures (arrows). Yeast gene deletion strains gcn4Δ, ade16Δ, yhl005cΔ, slt2Δ, gfd2Δ, cce1Δ, and gcs1Δ carrying the mutant T1348N generally exhibit normal ring-like vacuolar membrane staining and a decrease in punctate structures.
Table 1.
Yeast deletion strains synthetically sick with truncated LRRK2 or suppressors of the toxicity induced by truncated LRRK2.
Figure 8.
GTPase activity plays a key role in the pathobiology of LRRK2.
GTP hydrolysis activity but not GTP binding or kinase activity is sufficient to modify toxicity induced by truncated human LRRK2 variants. Truncated GTPase variants with impaired GTP hydrolysis induce marked defects in the endocytic vesicular trafficking and autophagy pathways, which may underlie LRRK2-induced toxicity. Truncated GTPase variants with enhanced GTPase activity show reduced LRRK2-induced toxicity. Lines with arrows indicate promoting or activating effects while lines with blunt ends indicate inhibitory effects.