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Mutations in the LRRK2 Roc-COR tandem domain link Parkinson's disease to Wnt signalling pathways.

Sancho RM, Law BM, Harvey K - Hum. Mol. Genet. (2009)

Bottom Line: Co-expression of DVL1 increased LRRK2 steady-state protein levels, an effect that was dependent on the DEP domain.Co-expression of DVL1 with LRRK2 in mammalian cells resulted in the redistribution of LRRK2 to typical cytoplasmic DVL1 aggregates in HEK293 and SH-SY5Y cells and co-localization in neurites and growth cones of differentiated dopaminergic SH-SY5Y cells.Since the DVL1 DEP domain is known to be involved in the regulation of small GTPases, we propose that: (i) DVLs may influence LRRK2 GTPase activity, and (ii) Roc-COR domain mutations modulating LRRK2-DVL interactions indirectly influence kinase activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, The School of Pharmacy, Brunswick Square, London, UK.

ABSTRACT
Mutations in PARK8, encoding LRRK2, are the most common known cause of Parkinson's disease. The LRRK2 Roc-COR tandem domain exhibits GTPase activity controlling LRRK2 kinase activity via an intramolecular process. We report the interaction of LRRK2 with the dishevelled family of phosphoproteins (DVL1-3), key regulators of Wnt (Wingless/Int) signalling pathways important for axon guidance, synapse formation and neuronal maintenance. Interestingly, DVLs can interact with and mediate the activation of small GTPases with structural similarity to the LRRK2 Roc domain. The LRRK2 Roc-COR domain and the DVL1 DEP domain were necessary and sufficient for LRRK2-DVL1 interaction. Co-expression of DVL1 increased LRRK2 steady-state protein levels, an effect that was dependent on the DEP domain. Strikingly, LRRK2-DVL1-3 interactions were disrupted by the familial PARK8 mutation Y1699C, whereas pathogenic mutations at residues R1441 and R1728 strengthened LRRK2-DVL1 interactions. Co-expression of DVL1 with LRRK2 in mammalian cells resulted in the redistribution of LRRK2 to typical cytoplasmic DVL1 aggregates in HEK293 and SH-SY5Y cells and co-localization in neurites and growth cones of differentiated dopaminergic SH-SY5Y cells. This is the first report of the modulation of a key LRRK2-accessory protein interaction by PARK8 Roc-COR domain mutations segregating with Parkinson's disease. Since the DVL1 DEP domain is known to be involved in the regulation of small GTPases, we propose that: (i) DVLs may influence LRRK2 GTPase activity, and (ii) Roc-COR domain mutations modulating LRRK2-DVL interactions indirectly influence kinase activity. Our findings also link LRRK2 to Wnt signalling pathways, suggesting novel pathogenic mechanisms and new targets for genetic analysis in Parkinson's disease.

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Co-localization of LRRK2 and DVL1 in the cytoplasm, neurites and growth cones in differentiated dopaminergic cells. (A–F) Confocal microscopy showing expression of myc-tagged LRRK2 in SH-SY5Y cells 5 days after treatment with retinoic acid. Note that LRRK2 co-localizes with tubulin staining in neurites (A–C) and GAP43 immunostaining in growth cones (D–F). Co-expression of FLAG-tagged DVL1 with myc-tagged LRRK2 in differentiated SH-SY5Y cells shows co-localization of both proteins in multiple cytoplasmic aggregates (G–J). (J) Magnification of the process shown in (G–I). Co-localization of LRRK2 and DVL1 in an extended neuronal terminal, suggestive of a growth cone (K–N) and in growth cones co-stained for GAP43 (O–R). Scale bars: 10 µm.
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DDP337F8: Co-localization of LRRK2 and DVL1 in the cytoplasm, neurites and growth cones in differentiated dopaminergic cells. (A–F) Confocal microscopy showing expression of myc-tagged LRRK2 in SH-SY5Y cells 5 days after treatment with retinoic acid. Note that LRRK2 co-localizes with tubulin staining in neurites (A–C) and GAP43 immunostaining in growth cones (D–F). Co-expression of FLAG-tagged DVL1 with myc-tagged LRRK2 in differentiated SH-SY5Y cells shows co-localization of both proteins in multiple cytoplasmic aggregates (G–J). (J) Magnification of the process shown in (G–I). Co-localization of LRRK2 and DVL1 in an extended neuronal terminal, suggestive of a growth cone (K–N) and in growth cones co-stained for GAP43 (O–R). Scale bars: 10 µm.

Mentions: Since DVLs were previously shown to stabilize microtubules in nocodazole-treated differentiated neurones (22), we examined whether (i) a similar effect could be observed upon transfection of DVL1 into differentiated SH-SY5Y cells and (ii) whether co-expression of LRRK2 enhances or opposes DVL-induced microtubule stabilization. The formation of a stable microtubule network was assessed by staining for acetylated tubulin (22). As predicted, differentiating SH-SY5Y cells expressing FLAG-DVL1 showed an abundance of stable microtubules and neurite projections (Fig. 7A–C) that were resistant to nocodazole treatment (Fig. 7D–F). In contrast, SH-SY5Y cells expressing myc-tagged full-length LRRK2 contained acetylated tubulin (Fig. 7G–I), but this was not resistant to nocodazole treatment (Fig. 7J–L). Nocodazole-resistant acetylated tubulin was also detected in cells co-expressing FLAG-DVL1 and myc-LRRK2 (Fig. 7M–T). Because of the low co-transfection efficiency in these experiments, we could not assess whether LRRK2-DVL1 co-expression affected the length or number of neuronal processes. However, DVL1 still appeared to be able to stabilize microtubules against nocodazole treatment in the presence of LRRK2. After differentiation with retinoic acid treatment, myc-LRRK2 is observed in the cytoplasm and neuronal processes (visualized by labelling for tubulin) and appears to be enriched in growth cones as shown by double-labelling with antibodies directed against GAP43 (Fig. 8A–F), a neuronal growth cone protein thought to be involved in pathfinding. Co-transfection of myc-LRRK2 and FLAG-DVL1 resulted in co-localization of both proteins in puncta within neurites (Fig. 8G–J) and growth cones (Fig. 8K–R).


Mutations in the LRRK2 Roc-COR tandem domain link Parkinson's disease to Wnt signalling pathways.

Sancho RM, Law BM, Harvey K - Hum. Mol. Genet. (2009)

Co-localization of LRRK2 and DVL1 in the cytoplasm, neurites and growth cones in differentiated dopaminergic cells. (A–F) Confocal microscopy showing expression of myc-tagged LRRK2 in SH-SY5Y cells 5 days after treatment with retinoic acid. Note that LRRK2 co-localizes with tubulin staining in neurites (A–C) and GAP43 immunostaining in growth cones (D–F). Co-expression of FLAG-tagged DVL1 with myc-tagged LRRK2 in differentiated SH-SY5Y cells shows co-localization of both proteins in multiple cytoplasmic aggregates (G–J). (J) Magnification of the process shown in (G–I). Co-localization of LRRK2 and DVL1 in an extended neuronal terminal, suggestive of a growth cone (K–N) and in growth cones co-stained for GAP43 (O–R). Scale bars: 10 µm.
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DDP337F8: Co-localization of LRRK2 and DVL1 in the cytoplasm, neurites and growth cones in differentiated dopaminergic cells. (A–F) Confocal microscopy showing expression of myc-tagged LRRK2 in SH-SY5Y cells 5 days after treatment with retinoic acid. Note that LRRK2 co-localizes with tubulin staining in neurites (A–C) and GAP43 immunostaining in growth cones (D–F). Co-expression of FLAG-tagged DVL1 with myc-tagged LRRK2 in differentiated SH-SY5Y cells shows co-localization of both proteins in multiple cytoplasmic aggregates (G–J). (J) Magnification of the process shown in (G–I). Co-localization of LRRK2 and DVL1 in an extended neuronal terminal, suggestive of a growth cone (K–N) and in growth cones co-stained for GAP43 (O–R). Scale bars: 10 µm.
Mentions: Since DVLs were previously shown to stabilize microtubules in nocodazole-treated differentiated neurones (22), we examined whether (i) a similar effect could be observed upon transfection of DVL1 into differentiated SH-SY5Y cells and (ii) whether co-expression of LRRK2 enhances or opposes DVL-induced microtubule stabilization. The formation of a stable microtubule network was assessed by staining for acetylated tubulin (22). As predicted, differentiating SH-SY5Y cells expressing FLAG-DVL1 showed an abundance of stable microtubules and neurite projections (Fig. 7A–C) that were resistant to nocodazole treatment (Fig. 7D–F). In contrast, SH-SY5Y cells expressing myc-tagged full-length LRRK2 contained acetylated tubulin (Fig. 7G–I), but this was not resistant to nocodazole treatment (Fig. 7J–L). Nocodazole-resistant acetylated tubulin was also detected in cells co-expressing FLAG-DVL1 and myc-LRRK2 (Fig. 7M–T). Because of the low co-transfection efficiency in these experiments, we could not assess whether LRRK2-DVL1 co-expression affected the length or number of neuronal processes. However, DVL1 still appeared to be able to stabilize microtubules against nocodazole treatment in the presence of LRRK2. After differentiation with retinoic acid treatment, myc-LRRK2 is observed in the cytoplasm and neuronal processes (visualized by labelling for tubulin) and appears to be enriched in growth cones as shown by double-labelling with antibodies directed against GAP43 (Fig. 8A–F), a neuronal growth cone protein thought to be involved in pathfinding. Co-transfection of myc-LRRK2 and FLAG-DVL1 resulted in co-localization of both proteins in puncta within neurites (Fig. 8G–J) and growth cones (Fig. 8K–R).

Bottom Line: Co-expression of DVL1 increased LRRK2 steady-state protein levels, an effect that was dependent on the DEP domain.Co-expression of DVL1 with LRRK2 in mammalian cells resulted in the redistribution of LRRK2 to typical cytoplasmic DVL1 aggregates in HEK293 and SH-SY5Y cells and co-localization in neurites and growth cones of differentiated dopaminergic SH-SY5Y cells.Since the DVL1 DEP domain is known to be involved in the regulation of small GTPases, we propose that: (i) DVLs may influence LRRK2 GTPase activity, and (ii) Roc-COR domain mutations modulating LRRK2-DVL interactions indirectly influence kinase activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, The School of Pharmacy, Brunswick Square, London, UK.

ABSTRACT
Mutations in PARK8, encoding LRRK2, are the most common known cause of Parkinson's disease. The LRRK2 Roc-COR tandem domain exhibits GTPase activity controlling LRRK2 kinase activity via an intramolecular process. We report the interaction of LRRK2 with the dishevelled family of phosphoproteins (DVL1-3), key regulators of Wnt (Wingless/Int) signalling pathways important for axon guidance, synapse formation and neuronal maintenance. Interestingly, DVLs can interact with and mediate the activation of small GTPases with structural similarity to the LRRK2 Roc domain. The LRRK2 Roc-COR domain and the DVL1 DEP domain were necessary and sufficient for LRRK2-DVL1 interaction. Co-expression of DVL1 increased LRRK2 steady-state protein levels, an effect that was dependent on the DEP domain. Strikingly, LRRK2-DVL1-3 interactions were disrupted by the familial PARK8 mutation Y1699C, whereas pathogenic mutations at residues R1441 and R1728 strengthened LRRK2-DVL1 interactions. Co-expression of DVL1 with LRRK2 in mammalian cells resulted in the redistribution of LRRK2 to typical cytoplasmic DVL1 aggregates in HEK293 and SH-SY5Y cells and co-localization in neurites and growth cones of differentiated dopaminergic SH-SY5Y cells. This is the first report of the modulation of a key LRRK2-accessory protein interaction by PARK8 Roc-COR domain mutations segregating with Parkinson's disease. Since the DVL1 DEP domain is known to be involved in the regulation of small GTPases, we propose that: (i) DVLs may influence LRRK2 GTPase activity, and (ii) Roc-COR domain mutations modulating LRRK2-DVL interactions indirectly influence kinase activity. Our findings also link LRRK2 to Wnt signalling pathways, suggesting novel pathogenic mechanisms and new targets for genetic analysis in Parkinson's disease.

Show MeSH
Related in: MedlinePlus