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Novel ethyl methanesulfonate (EMS)-induced alleles of the Drosophila homolog of LRRK2 reveal a crucial role in endolysosomal functions and autophagy in vivo.

Dodson MW, Leung LK, Lone M, Lizzio MA, Guo M - Dis Model Mech (2014)

Bottom Line: Using these alleles, we show that lrrk loss-of-function causes striking defects in the endolysosomal and autophagy pathways, including the accumulation of markedly enlarged lysosomes that are laden with undigested contents, consistent with a defect in lysosomal degradation. lrrk loss-of-function also results in the accumulation of autophagosomes, as well as the presence of enlarged early endosomes laden with mono-ubiquitylated cargo proteins, suggesting an additional defect in lysosomal substrate delivery.Interestingly, the lysosomal abnormalities in these lrrk mutants can be suppressed by a constitutively active form of the small GTPase rab9, which promotes retromer-dependent recycling from late endosomes to the Golgi.Collectively, our data provides compelling evidence of a vital role for lrrk in lysosomal function and endolysosomal membrane transport in vivo, and suggests a link between lrrk and retromer-mediated endosomal recycling.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University of California, Los Angeles, CA 90095, USA. Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA.

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Lrrk physically binds to Rab9, and expression of activated rab9 rescues lrrk NS phenotypes. (A) In lysates from transiently transfected cultured Drosophila S2 cells, Lrrk-Myc co-immunoprecipitates with YFP-tagged Rab5 and more strongly with Rab9, but not with Rab11 or YFP alone. (B–D) Lysotracker staining of stage-12 follicle cells from wild-type flies (B), lrrk NS flies (C) and lrrk NS flies expressing constitutively active rab9 (rab9CA) specifically in follicle cells (D) shows dramatic suppression of the abnormal lysosome morphology seen in lrrk NS flies when rab9 is expressed. (E) Quantification of average lysosome size in Lysotracker-stained follicle cells shows suppression of lysosome enlargement by rab9CA in lrrk NS flies. n=16, 16, 34 and 33 egg chambers each, respectively, for the genotypes in E. **P<0.0001. (F) Similarly, premature follicle cell death in lrrk NS flies, as assessed by TUNEL staining, is suppressed by rab9CA. n=61, 68, 99 and 102 egg chambers each, respectively, for the genotypes in F. *P<0.05. (G–J) Staining of the presynaptic motor neuron at the neuromuscular junction (NMJ) at abdominal segment 4, muscle 4 in late third instar larvae with anti-cysteine string protein (CSP; red), which labels synaptic vesicles. Compared with wild type (G), lrrk NS larvae (H) show an overgrowth phenotype in which numerous small satellite boutons (marked with arrowheads) form adjacent to the larger normal boutons. This overgrowth phenotype is significantly suppressed by expression of either wild-type lrrk (I) or rab9CA (J) using the motor neuron-specific OK6-Gal4 driver. (K) Quantification of the numbers of boutons per NMJ in the indicated genotypes shows significant suppression of lrrk NS synaptic overgrowth with expression of both wild-type lrrk and rab9CA. n=27 NMJs analyzed for each genotype in K. ***P=0.0003, *P<0.05. lrrk NS is lrrk1/2. Scale bars: 5 μm in B–D, 10 μm in G–J.
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f7-0071351: Lrrk physically binds to Rab9, and expression of activated rab9 rescues lrrk NS phenotypes. (A) In lysates from transiently transfected cultured Drosophila S2 cells, Lrrk-Myc co-immunoprecipitates with YFP-tagged Rab5 and more strongly with Rab9, but not with Rab11 or YFP alone. (B–D) Lysotracker staining of stage-12 follicle cells from wild-type flies (B), lrrk NS flies (C) and lrrk NS flies expressing constitutively active rab9 (rab9CA) specifically in follicle cells (D) shows dramatic suppression of the abnormal lysosome morphology seen in lrrk NS flies when rab9 is expressed. (E) Quantification of average lysosome size in Lysotracker-stained follicle cells shows suppression of lysosome enlargement by rab9CA in lrrk NS flies. n=16, 16, 34 and 33 egg chambers each, respectively, for the genotypes in E. **P<0.0001. (F) Similarly, premature follicle cell death in lrrk NS flies, as assessed by TUNEL staining, is suppressed by rab9CA. n=61, 68, 99 and 102 egg chambers each, respectively, for the genotypes in F. *P<0.05. (G–J) Staining of the presynaptic motor neuron at the neuromuscular junction (NMJ) at abdominal segment 4, muscle 4 in late third instar larvae with anti-cysteine string protein (CSP; red), which labels synaptic vesicles. Compared with wild type (G), lrrk NS larvae (H) show an overgrowth phenotype in which numerous small satellite boutons (marked with arrowheads) form adjacent to the larger normal boutons. This overgrowth phenotype is significantly suppressed by expression of either wild-type lrrk (I) or rab9CA (J) using the motor neuron-specific OK6-Gal4 driver. (K) Quantification of the numbers of boutons per NMJ in the indicated genotypes shows significant suppression of lrrk NS synaptic overgrowth with expression of both wild-type lrrk and rab9CA. n=27 NMJs analyzed for each genotype in K. ***P=0.0003, *P<0.05. lrrk NS is lrrk1/2. Scale bars: 5 μm in B–D, 10 μm in G–J.

Mentions: As we have previously reported, Drosophila Lrrk colocalizes with Rab9 at the late-endosome membrane (Dodson et al., 2012). Interestingly, we found that Lrrk-myc co-immunoprecipitated with Rab9-YFP, but did not show significant binding with either YFP alone or with Rab11-YFP (Fig. 7A), which served as controls. Expression of a constitutively active form of rab9 (rab9CA) in follicle cells resulted in marked suppression of the lysosome-enlargement phenotype observed in lrrk NS cells (Fig. 7D vs 7C; Fig. 7E). Similarly, rab9CA significantly, although incompletely, suppressed premature follicle cell death in lrrk NS cells (Fig. 7F). One mechanism by which lrrk could be required for rab9-dependent processes is through regulation of the membrane recruitment of Rab9; however, we did not detect any significant difference in the subcellular localization of Rab9 between the wild-type and lrrk-NS-mutant backgrounds (supplementary material Fig. S6). Collectively, these data suggest that augmenting Rab9 function can, at least in part, bypass the need for lrrk.


Novel ethyl methanesulfonate (EMS)-induced alleles of the Drosophila homolog of LRRK2 reveal a crucial role in endolysosomal functions and autophagy in vivo.

Dodson MW, Leung LK, Lone M, Lizzio MA, Guo M - Dis Model Mech (2014)

Lrrk physically binds to Rab9, and expression of activated rab9 rescues lrrk NS phenotypes. (A) In lysates from transiently transfected cultured Drosophila S2 cells, Lrrk-Myc co-immunoprecipitates with YFP-tagged Rab5 and more strongly with Rab9, but not with Rab11 or YFP alone. (B–D) Lysotracker staining of stage-12 follicle cells from wild-type flies (B), lrrk NS flies (C) and lrrk NS flies expressing constitutively active rab9 (rab9CA) specifically in follicle cells (D) shows dramatic suppression of the abnormal lysosome morphology seen in lrrk NS flies when rab9 is expressed. (E) Quantification of average lysosome size in Lysotracker-stained follicle cells shows suppression of lysosome enlargement by rab9CA in lrrk NS flies. n=16, 16, 34 and 33 egg chambers each, respectively, for the genotypes in E. **P<0.0001. (F) Similarly, premature follicle cell death in lrrk NS flies, as assessed by TUNEL staining, is suppressed by rab9CA. n=61, 68, 99 and 102 egg chambers each, respectively, for the genotypes in F. *P<0.05. (G–J) Staining of the presynaptic motor neuron at the neuromuscular junction (NMJ) at abdominal segment 4, muscle 4 in late third instar larvae with anti-cysteine string protein (CSP; red), which labels synaptic vesicles. Compared with wild type (G), lrrk NS larvae (H) show an overgrowth phenotype in which numerous small satellite boutons (marked with arrowheads) form adjacent to the larger normal boutons. This overgrowth phenotype is significantly suppressed by expression of either wild-type lrrk (I) or rab9CA (J) using the motor neuron-specific OK6-Gal4 driver. (K) Quantification of the numbers of boutons per NMJ in the indicated genotypes shows significant suppression of lrrk NS synaptic overgrowth with expression of both wild-type lrrk and rab9CA. n=27 NMJs analyzed for each genotype in K. ***P=0.0003, *P<0.05. lrrk NS is lrrk1/2. Scale bars: 5 μm in B–D, 10 μm in G–J.
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f7-0071351: Lrrk physically binds to Rab9, and expression of activated rab9 rescues lrrk NS phenotypes. (A) In lysates from transiently transfected cultured Drosophila S2 cells, Lrrk-Myc co-immunoprecipitates with YFP-tagged Rab5 and more strongly with Rab9, but not with Rab11 or YFP alone. (B–D) Lysotracker staining of stage-12 follicle cells from wild-type flies (B), lrrk NS flies (C) and lrrk NS flies expressing constitutively active rab9 (rab9CA) specifically in follicle cells (D) shows dramatic suppression of the abnormal lysosome morphology seen in lrrk NS flies when rab9 is expressed. (E) Quantification of average lysosome size in Lysotracker-stained follicle cells shows suppression of lysosome enlargement by rab9CA in lrrk NS flies. n=16, 16, 34 and 33 egg chambers each, respectively, for the genotypes in E. **P<0.0001. (F) Similarly, premature follicle cell death in lrrk NS flies, as assessed by TUNEL staining, is suppressed by rab9CA. n=61, 68, 99 and 102 egg chambers each, respectively, for the genotypes in F. *P<0.05. (G–J) Staining of the presynaptic motor neuron at the neuromuscular junction (NMJ) at abdominal segment 4, muscle 4 in late third instar larvae with anti-cysteine string protein (CSP; red), which labels synaptic vesicles. Compared with wild type (G), lrrk NS larvae (H) show an overgrowth phenotype in which numerous small satellite boutons (marked with arrowheads) form adjacent to the larger normal boutons. This overgrowth phenotype is significantly suppressed by expression of either wild-type lrrk (I) or rab9CA (J) using the motor neuron-specific OK6-Gal4 driver. (K) Quantification of the numbers of boutons per NMJ in the indicated genotypes shows significant suppression of lrrk NS synaptic overgrowth with expression of both wild-type lrrk and rab9CA. n=27 NMJs analyzed for each genotype in K. ***P=0.0003, *P<0.05. lrrk NS is lrrk1/2. Scale bars: 5 μm in B–D, 10 μm in G–J.
Mentions: As we have previously reported, Drosophila Lrrk colocalizes with Rab9 at the late-endosome membrane (Dodson et al., 2012). Interestingly, we found that Lrrk-myc co-immunoprecipitated with Rab9-YFP, but did not show significant binding with either YFP alone or with Rab11-YFP (Fig. 7A), which served as controls. Expression of a constitutively active form of rab9 (rab9CA) in follicle cells resulted in marked suppression of the lysosome-enlargement phenotype observed in lrrk NS cells (Fig. 7D vs 7C; Fig. 7E). Similarly, rab9CA significantly, although incompletely, suppressed premature follicle cell death in lrrk NS cells (Fig. 7F). One mechanism by which lrrk could be required for rab9-dependent processes is through regulation of the membrane recruitment of Rab9; however, we did not detect any significant difference in the subcellular localization of Rab9 between the wild-type and lrrk-NS-mutant backgrounds (supplementary material Fig. S6). Collectively, these data suggest that augmenting Rab9 function can, at least in part, bypass the need for lrrk.

Bottom Line: Using these alleles, we show that lrrk loss-of-function causes striking defects in the endolysosomal and autophagy pathways, including the accumulation of markedly enlarged lysosomes that are laden with undigested contents, consistent with a defect in lysosomal degradation. lrrk loss-of-function also results in the accumulation of autophagosomes, as well as the presence of enlarged early endosomes laden with mono-ubiquitylated cargo proteins, suggesting an additional defect in lysosomal substrate delivery.Interestingly, the lysosomal abnormalities in these lrrk mutants can be suppressed by a constitutively active form of the small GTPase rab9, which promotes retromer-dependent recycling from late endosomes to the Golgi.Collectively, our data provides compelling evidence of a vital role for lrrk in lysosomal function and endolysosomal membrane transport in vivo, and suggests a link between lrrk and retromer-mediated endosomal recycling.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University of California, Los Angeles, CA 90095, USA. Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA.

Show MeSH
Related in: MedlinePlus