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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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Related in: MedlinePlus

Follicle cells undergo premature caspase-dependent cell death in lrrk NS flies. (A) Schematic depicting a stage-12 egg chamber, with anterior to the left and posterior to the right. Most of the exterior of the egg chamber is composed of an epithelial layer of somatic follicle cells that surrounds the developing oocyte, whereas the anterior of the egg chamber contains the germline nurse cells, which are clonally related to the oocyte. (B–E) Note that, whereas B and C depict whole egg chambers, D and E are higher-magnification views of follicle cells, similar to what is outlined by the box in A. (B,C) Single stage-12 egg chambers from wild-type (B) and lrrk1/4 (C) females labeled by TUNEL to highlight apoptotic nuclei, show a dramatic increase in follicle cell death in lrrk NS egg chambers. The egg chamber is outlined with a dashed line for ease of visualization. Note that the area to the anterior (right) of the lrrk NS egg chamber (C) is devoid of TUNEL-positive signal because this area is occupied predominantly by nurse cells rather than follicle cells. (D,E) Higher-magnification images of follicle cells co-stained for activated caspase-3 (called Drice in Drosophila) (D,E) and TUNEL (D′,E′) demonstrates increased caspase activation in apoptotic follicle cells from stage-12 lrrk1/2 egg chambers (E) relative to wild type (D). Merged images show that, in lrrk NS flies, follicle cells that accumulate activated Drice are also TUNEL-positive (E″), suggesting that caspase activation occurs specifically in cells undergoing apoptosis. (F) Quantification of average number of TUNEL-positive follicle cells per egg chamber of the indicated genotype. Whereas lrrk NS alleles show a dramatic increase in follicle cell apoptosis relative to wild type, this is significantly rescued by a myc-tagged lrrk genomic rescue transgene, and by follicle-cell-specific expression of either Drosophila lrrk or human LRRK2. Apoptosis of lrrk NS follicle cells is also suppressed by expression of the caspase inhibitor diap1, suggesting that this process is caspase-dependent. n=56, 41, 58, 26, 107 and 61 egg chambers each, respectively, for the genotypes in panel F. *P<0.0001. **P=0.002. Scale bar in D: 10 μm.
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f2-0071351: Follicle cells undergo premature caspase-dependent cell death in lrrk NS flies. (A) Schematic depicting a stage-12 egg chamber, with anterior to the left and posterior to the right. Most of the exterior of the egg chamber is composed of an epithelial layer of somatic follicle cells that surrounds the developing oocyte, whereas the anterior of the egg chamber contains the germline nurse cells, which are clonally related to the oocyte. (B–E) Note that, whereas B and C depict whole egg chambers, D and E are higher-magnification views of follicle cells, similar to what is outlined by the box in A. (B,C) Single stage-12 egg chambers from wild-type (B) and lrrk1/4 (C) females labeled by TUNEL to highlight apoptotic nuclei, show a dramatic increase in follicle cell death in lrrk NS egg chambers. The egg chamber is outlined with a dashed line for ease of visualization. Note that the area to the anterior (right) of the lrrk NS egg chamber (C) is devoid of TUNEL-positive signal because this area is occupied predominantly by nurse cells rather than follicle cells. (D,E) Higher-magnification images of follicle cells co-stained for activated caspase-3 (called Drice in Drosophila) (D,E) and TUNEL (D′,E′) demonstrates increased caspase activation in apoptotic follicle cells from stage-12 lrrk1/2 egg chambers (E) relative to wild type (D). Merged images show that, in lrrk NS flies, follicle cells that accumulate activated Drice are also TUNEL-positive (E″), suggesting that caspase activation occurs specifically in cells undergoing apoptosis. (F) Quantification of average number of TUNEL-positive follicle cells per egg chamber of the indicated genotype. Whereas lrrk NS alleles show a dramatic increase in follicle cell apoptosis relative to wild type, this is significantly rescued by a myc-tagged lrrk genomic rescue transgene, and by follicle-cell-specific expression of either Drosophila lrrk or human LRRK2. Apoptosis of lrrk NS follicle cells is also suppressed by expression of the caspase inhibitor diap1, suggesting that this process is caspase-dependent. n=56, 41, 58, 26, 107 and 61 egg chambers each, respectively, for the genotypes in panel F. *P<0.0001. **P=0.002. Scale bar in D: 10 μm.

Mentions: Previously, we and others have reported that lrrke03680 homozygous females show a dramatic reduction in female fertility (Dodson et al., 2012; Imai et al., 2008; Lee et al., 2007), which can be rescued by restoring wild-type lrrk function specifically in follicle cells (Dodson et al., 2012). Follicle cells are a somatic epithelial cell monolayer that surrounds the developing oocyte during oogenesis (Fig. 2A) (King, 1970; Spradling, 1993). Follicle cells have a number of advantages that make them an attractive cell biological system, including their large size, ease of accessibility and squamous morphology that aids in visualization of subcellular structures. As expected, all trans-heterozygous combinations of lrrk NS alleles showed reduced female fertility that could be rescued by follicle-cell-specific expression of wild-type lrrk (Fig. 1C). Expression of lrrkGS, which is analogous to the most common PD-causing mutation in human LRRK2 (G2019S) (Bonifati, 2007; Taylor et al., 2006), also suppressed female infertility in lrrk NS flies (Fig. 1C). This suggests that lrrkGS retains at least some of the functions of wild-type lrrk, consistent with what we have previously reported (Dodson et al., 2012). Moreover, follicle-cell-specific expression of human LRRK2 also restored fertility to lrrk NS flies (Fig. 1C), suggesting that the human and Drosophila proteins are functionally conserved.


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)

Follicle cells undergo premature caspase-dependent cell death in lrrk NS flies. (A) Schematic depicting a stage-12 egg chamber, with anterior to the left and posterior to the right. Most of the exterior of the egg chamber is composed of an epithelial layer of somatic follicle cells that surrounds the developing oocyte, whereas the anterior of the egg chamber contains the germline nurse cells, which are clonally related to the oocyte. (B–E) Note that, whereas B and C depict whole egg chambers, D and E are higher-magnification views of follicle cells, similar to what is outlined by the box in A. (B,C) Single stage-12 egg chambers from wild-type (B) and lrrk1/4 (C) females labeled by TUNEL to highlight apoptotic nuclei, show a dramatic increase in follicle cell death in lrrk NS egg chambers. The egg chamber is outlined with a dashed line for ease of visualization. Note that the area to the anterior (right) of the lrrk NS egg chamber (C) is devoid of TUNEL-positive signal because this area is occupied predominantly by nurse cells rather than follicle cells. (D,E) Higher-magnification images of follicle cells co-stained for activated caspase-3 (called Drice in Drosophila) (D,E) and TUNEL (D′,E′) demonstrates increased caspase activation in apoptotic follicle cells from stage-12 lrrk1/2 egg chambers (E) relative to wild type (D). Merged images show that, in lrrk NS flies, follicle cells that accumulate activated Drice are also TUNEL-positive (E″), suggesting that caspase activation occurs specifically in cells undergoing apoptosis. (F) Quantification of average number of TUNEL-positive follicle cells per egg chamber of the indicated genotype. Whereas lrrk NS alleles show a dramatic increase in follicle cell apoptosis relative to wild type, this is significantly rescued by a myc-tagged lrrk genomic rescue transgene, and by follicle-cell-specific expression of either Drosophila lrrk or human LRRK2. Apoptosis of lrrk NS follicle cells is also suppressed by expression of the caspase inhibitor diap1, suggesting that this process is caspase-dependent. n=56, 41, 58, 26, 107 and 61 egg chambers each, respectively, for the genotypes in panel F. *P<0.0001. **P=0.002. Scale bar in D: 10 μm.
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f2-0071351: Follicle cells undergo premature caspase-dependent cell death in lrrk NS flies. (A) Schematic depicting a stage-12 egg chamber, with anterior to the left and posterior to the right. Most of the exterior of the egg chamber is composed of an epithelial layer of somatic follicle cells that surrounds the developing oocyte, whereas the anterior of the egg chamber contains the germline nurse cells, which are clonally related to the oocyte. (B–E) Note that, whereas B and C depict whole egg chambers, D and E are higher-magnification views of follicle cells, similar to what is outlined by the box in A. (B,C) Single stage-12 egg chambers from wild-type (B) and lrrk1/4 (C) females labeled by TUNEL to highlight apoptotic nuclei, show a dramatic increase in follicle cell death in lrrk NS egg chambers. The egg chamber is outlined with a dashed line for ease of visualization. Note that the area to the anterior (right) of the lrrk NS egg chamber (C) is devoid of TUNEL-positive signal because this area is occupied predominantly by nurse cells rather than follicle cells. (D,E) Higher-magnification images of follicle cells co-stained for activated caspase-3 (called Drice in Drosophila) (D,E) and TUNEL (D′,E′) demonstrates increased caspase activation in apoptotic follicle cells from stage-12 lrrk1/2 egg chambers (E) relative to wild type (D). Merged images show that, in lrrk NS flies, follicle cells that accumulate activated Drice are also TUNEL-positive (E″), suggesting that caspase activation occurs specifically in cells undergoing apoptosis. (F) Quantification of average number of TUNEL-positive follicle cells per egg chamber of the indicated genotype. Whereas lrrk NS alleles show a dramatic increase in follicle cell apoptosis relative to wild type, this is significantly rescued by a myc-tagged lrrk genomic rescue transgene, and by follicle-cell-specific expression of either Drosophila lrrk or human LRRK2. Apoptosis of lrrk NS follicle cells is also suppressed by expression of the caspase inhibitor diap1, suggesting that this process is caspase-dependent. n=56, 41, 58, 26, 107 and 61 egg chambers each, respectively, for the genotypes in panel F. *P<0.0001. **P=0.002. Scale bar in D: 10 μm.
Mentions: Previously, we and others have reported that lrrke03680 homozygous females show a dramatic reduction in female fertility (Dodson et al., 2012; Imai et al., 2008; Lee et al., 2007), which can be rescued by restoring wild-type lrrk function specifically in follicle cells (Dodson et al., 2012). Follicle cells are a somatic epithelial cell monolayer that surrounds the developing oocyte during oogenesis (Fig. 2A) (King, 1970; Spradling, 1993). Follicle cells have a number of advantages that make them an attractive cell biological system, including their large size, ease of accessibility and squamous morphology that aids in visualization of subcellular structures. As expected, all trans-heterozygous combinations of lrrk NS alleles showed reduced female fertility that could be rescued by follicle-cell-specific expression of wild-type lrrk (Fig. 1C). Expression of lrrkGS, which is analogous to the most common PD-causing mutation in human LRRK2 (G2019S) (Bonifati, 2007; Taylor et al., 2006), also suppressed female infertility in lrrk NS flies (Fig. 1C). This suggests that lrrkGS retains at least some of the functions of wild-type lrrk, consistent with what we have previously reported (Dodson et al., 2012). Moreover, follicle-cell-specific expression of human LRRK2 also restored fertility to lrrk NS flies (Fig. 1C), suggesting that the human and Drosophila proteins are functionally conserved.

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