24 Oct 2012

Polymorphisms in the gene for leucine-rich repeat kinase 2 (LRRK2) confer risk for both familial and sporadic forms of Parkinson’s disease (PD). LRRK2 substrates have been hard to pin down, and the kinase's role in pathogenesis remains unclear. At this year’s Society for Neuroscience annual meeting, held 13-17 October in New Orleans, Louisiana, several groups proposed myriad functions for LRRK2. If all those functions prove true, the kinase is one busy protein.

"We are at this early stage of research where we are just getting a handle on what LRRK2 is doing,” said Darren Moore of the Swiss Federal Institute of Technology in Lausanne (EPFL). “After we do that, we can work out which pathway is pathologically the most relevant."

Rachel Bailey, from Jada Lewis’ lab at the University of Florida, Gainesville, put forth the idea that LRRK2 phosphorylates tau. Hyperphosphorylated and aggregated forms of the microtubule-binding protein tend to afflict a subset of PD patients who have LRRK2 mutations. One common LRRK2 variant, with a glycine-to-serine substitution at position 2019 (G2019S), boosts kinase activity. Could that extra oomph be responsible for the increased tau seen in PD?

The researchers incubated LRRK2 with recombinant human tau in vitro and subsequently detected multiple phosphorylated sites on tau by mass spectrometry. The researchers then created a mouse model expressing both human wild-type LRRK2 and human mutant tau by crossing the BAC-LRRK2 mouse (Melrose et al., 2007) with the rTg4510 strain (Santacruz et al., 2005). Mice expressing both human genes accumulated more tau in their brains than did the tau transgenics, demonstrating that LRRK2 enhances tau pathology. Bailey presented no data on how these changes alter tau neurotoxicity, pathology, or behavior, but plans to address those points in future research.

A different presentation tied LRRK2 to a protein involved in Wnt signaling. Wnt is crucial in signal transduction, synaptic plasticity, and is increasingly implicated in neurodegenerative disease, said Daniel Berwick and Kirsten Harvey from the University College London School of Pharmacy, U.K. Previous results from Harvey’s lab pointed to LRRK2’s interaction with the disheveled family of phosphoproteins, important regulators of Wnt signaling (see Sancho et al., 2009). Berwick presented new yeast-two-hybrid screen, co-immunoprecipitation, and confocal microscopy data suggesting that LRRK2 directly interacts with another Wnt signaling component, i.e., low-density lipoprotein receptor-related protein 6 (LRP6). This transmembrane protein binds a cytoplasmic complex that destroys β-catenin while Wnt pathways are dormant.

Under basal conditions, LRRK2 resides in the cytoplasmic β-catenin destruction complex. Once the Wnt pathway is stimulated, however, LRRK2 binds to LRP6 in the membrane, and the two, together with the destruction complex, are taken up by endocytosis. With destruction complex components tied up in endosomes, β-catenin accumulates in the cytoplasm and eventually translocates into the nucleus, where it affects gene transcription.

Together, these observations suggest that LRRK2 bridges membrane and cytosolic components of Wnt signaling, said Berwick. Both a LRRK2 inhibitor and PD-associated LRRK2 mutations weakened the interaction with LRP6 and reduced Wnt signaling. Since those pathways are crucial for basic neuronal function and neurogenesis, their deregulation could lead to neurodegeneration, he and Harvey suggested in a related paper (see Berwick and Harvey, 2012). However, some scientists believe overactivation of the kinase contributes to pathology and are pursuing LRRK2 inhibitors as potential therapeutics. According to Berwick and Harvey’s data, inhibitors aimed at reducing LRRK2 activity may not be therapeutic if they also deregulate Wnt signaling, Berwick said.

Alexandra Beilina from Mark Cookson’s lab at the National Institutes of Health in Bethesda, Maryland, favors yet a different LRRK2 interactor. By protein microarray and co-immunoprecipitation, these researchers found that LRRK2 binds BCL2-associated athanogene 5 (BAG5) in both mammalian cells and in mouse brain. This protein acts as a co-chaperone for heat shock proteins, which are involved in protein folding activities in the cell, and has previously been implicated in Parkinson's (see ARF related news story). It is not clear what happens when LRRK2 binds BAG5. In Beilina’s hands, the interaction did not depend on kinase activity and LRRK2 did not phosphorylate the co-chaperone. Both proteins independently reduced neurite length when expressed in primary cortical neurons. BAG5 might act downstream of LRRK2, Beilina suggested.

A related presentation detailed a LRRK2 interaction that seems to clear the kinase from the cytoplasm. Frederick Nucifora in collaboration with Christopher Ross at Johns Hopkins University School of Medicine, Baltimore, Maryland, found with a yeast-two-hybrid screen and co-immunoprecipitation that both wild-type and mutant G2019S LRRK2 bound to an E3 ubiquitin ligase called WSB1. WSB1 and LRRK2 co-localized in lysosomes of cultured neuroblastoma cells. Evidence suggested that WSB1 tagged LRRK2 with ubiquitin, marking it for lysosomal destruction. Since boosting WSB1 prevented LRRK2 toxicity in cultured primary neurons, Nucifora proposed that activating the ligase to boost LRRK2 degradation may be a therapeutic strategy for PD.

In addition, Lewy bodies and Lewy neurites of postmortem human brains from sporadic PD patients contained WSB1, leading the team to hypothesize that the ligase encourages the formation of Lewy bodies to protect neurons in humans.

Still other evidence links LRRK2 function to nuclear organization, according to an October 17 Nature paper from Juan Carlos Izpisua Belmonte, Salk Institute for Biological Studies, La Jolla, California, and colleagues. Since aging correlates with nuclear envelope abnormalities, joint first authors Guang-Hui Liu, Jing Qu, and Keiichiro Suzuki wondered if diseases associated with aging also came with wrinkles in the nuclear architecture. They zeroed in on Parkinson’s and the G2019S mutation of LRRK2.

The researchers aged cultured human neural stem cells and found that those that express the mutant form of LRRK2 developed enlarged and convoluted (rather than spherical) nuclei with unusual folds in their nuclear membranes. The membranes lacked the filament proteins lamin B1 and lamin B2, which regulate nuclear structure. It turns out that these proteins were hyperphosphorylated relative to those in wild-type cells, suggesting that too much LRRK2 activation boosts phosphorylation of lamin B proteins.

Epigenetic changes to DNA accompanied the misshapen nuclear membranes, particularly affecting genes involved in neurogenesis and neural function. Furthermore, aged LRRK2 mutant cells divided and differentiated less well. These problems disappeared when the researchers introduced wild-type LRRK2 into the neural stem cells with a lentivirus. The results seemed to relate to disease pathology, since similarly deformed nuclei turned up in hippocampal cells from postmortem PD brains either with or without the G2019S mutation. Taken together, the results suggest involvement of the nucleus in PD pathology.

These proposed doings of LRRK2 add to previous evidence implicating it in synaptic vesicle endocytosis (see ARF related news story) and perturbation of the Golgi network (see ARF related news story). Unfortunately, no one has yet been able to finger the true pathogenic role of LRRK2. Several groups recently put together a consortium to study some 2,000 people with LRRK2 mutations worldwide (see ARF related news story).

"It could be that LRRK2 has myriad functions and activities, and only one or a few of them are pathological,” said Moore. "It’s going to be difficult to work out which one is most relevant to the disease."—Gwyneth Dickey Zakaib

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References

News Citations

1. Parkin Adversary Is BAGged 23 Dec 2004
2. New Substrate for Parkinson’s Protein Is Picky About Phosphate 5 Oct 2012
3. α-Synuclein Conspires With LRRK2 to Corrupt Neurons 28 Dec 2009

Paper Citations

1. Melrose HL, Kent CB, Taylor JP, Dachsel JC, Hinkle KM, Lincoln SJ, Mok SS, Culvenor JG, Masters CL, Tyndall GM, Bass DI, Ahmed Z, Andorfer CA, Ross OA, Wszolek ZK, Delldonne A, Dickson DW, Farrer MJ. A comparative analysis of leucine-rich repeat kinase 2 (Lrrk2) expression in mouse brain and Lewy body disease. Neuroscience. 2007 Jul 29;147(4):1047-58. PubMed.
2. Santacruz K, Lewis J, Spires T, Paulson J, Kotilinek L, Ingelsson M, Guimaraes A, DeTure M, Ramsden M, McGowan E, Forster C, Yue M, Orne J, Janus C, Mariash A, Kuskowski M, Hyman B, Hutton M, Ashe KH. Tau suppression in a neurodegenerative mouse model improves memory function. Science. 2005 Jul 15;309(5733):476-81. PubMed.
3. Sancho RM, Law BM, Harvey K. Mutations in the LRRK2 Roc-COR tandem domain link Parkinson's disease to Wnt signalling pathways. Hum Mol Genet. 2009 Oct 15;18(20):3955-68. PubMed.
4. Berwick DC, Harvey K. LRRK2 functions as a Wnt signaling scaffold, bridging cytosolic proteins and membrane-localized LRP6. Hum Mol Genet. 2012 Nov 15;21(22):4966-79. PubMed.

Other Citations

1. ARF related news story

Further Reading

Papers

1. Ray S, Liu M. Current understanding of LRRK2 in Parkinson's disease: biochemical and structural features and inhibitor design. Future Med Chem. 2012 Sep;4(13):1701-13. PubMed.
2. Lee S, Imai Y, Gehrke S, Liu S, Lu B. The synaptic function of LRRK2. Biochem Soc Trans. 2012 Oct;40(5):1047-51. PubMed.
3. Sai Y, Zou Z, Peng K, Dong Z. The Parkinson's disease-related genes act in mitochondrial homeostasis. Neurosci Biobehav Rev. 2012 Oct;36(9):2034-43. PubMed.
4. Heutink P, Verhage M. Neurodegeneration: new road leads back to the synapse. Neuron. 2012 Sep 20;75(6):935-8. PubMed.

News

1. α-Synuclein Conspires With LRRK2 to Corrupt Neurons 28 Dec 2009
2. The Human Connectome Project: Deciphering the Brain’s Wiring 23 Oct 2012
3. Evidence Piles Up for Lysosomal Dysfunction in Parkinson’s 1 Jun 2012
4. More Than a LRRK: PD Field Thinks Big With Genetic Cohort 8 Feb 2012
5. Come and Get ’em—Foundation Develops Antibodies for PD Research 25 Mar 2010
6. New Substrate for Parkinson’s Protein Is Picky About Phosphate 5 Oct 2012
7. Parkin Adversary Is BAGged 23 Dec 2004