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With variants that boost risk for both familial and sporadic Parkinson’s disease, the LRRK2 gene beckons as a prime target for therapy development. But there’s a hitch: No one knows yet how leucine-rich repeat kinase 2, as the gene is formally called, promotes disease. Basic research has rustled up a plethora of functions (see ARF related news story; ARF news story), but thus far failed to pin down a clear link to disease. A few ideas seem to be taking flight, however. At the 11th International Conference on Alzheimer’s and Parkinson’s Diseases, held 6-10 March 2013 in Florence, Italy, researchers intrigued the crowd with compelling evidence for an inflammatory role, strengthened evidence that LRRK2 affects endocytosis and autophagy, and pointed to kinase activity as a plausible target for intervention.

Something New and Inflammatory
Most research on LRRK2 has focused on what it does in neurons, but Michael Schlossmacher at Ottawa Hospital Research Institute, Ontario, Canada, turned his eyes to the immune system. He pointed out that variants of the LRRK2 gene also associate with the inflammatory bowel disease Crohn’s and with leprosy, which is caused by susceptibility to the bacteria Mycobacterium leprae and lepromatosis. This suggested to Schlossmacher that the gene might function in inflammation. His group found rampant LRRK2 expression in circulating monocytes, which become macrophages in tissue, in neutrophils, B cells, and T cells (see Hakimi et al., 2011). Moreover, LRRK2 expression in these cells takes off after exposure to bacterial or viral particles, or when the cells are stimulated by the cytokine interferon, which rallies the immune system to fight infection. LRRK2 expression also spikes in white blood cells seen in capillaries of brains that are infected with rabies or HIV, or are afflicted with various forms of PD, Schlossmacher reported. Likewise, the protein burgeons in other types of inflamed tissue, such as leprosy skin biopsies, lymph nodes, and spleen.

How might LRRK2 promote PD from inside immune cells? Not by regulating cytokine release, as cells with mutant LRRK2 performed as well as wild-type cells in this regard, Schlossmacher said. He speculated that the gene could be involved in phagocytosis, the process by which macrophages gobble up harmful substances. He plans to look at this next. A phagocytic role would dovetail with findings that LRRK2 helps regulate neuronal endocytosis, a similar process, he said. Overall, the data suggest that pathogenic LRRK2 variants might increase a person’s susceptibility to Parkinson’s by weakening the immune system. The penetrance of dominantly inherited PD-associated LRRK2 alleles is only about 25 percent, and it varies with ethnicity and geography, Schlossmacher noted. This suggests that an environmental trigger, such as an infection, is needed for LRRK2 to cause PD (see Kitada et al., 2012).

Bart De Strooper at KU Leuven, Belgium, told Alzforum he was impressed by the data showing immune cells loaded with LRRK2, and said that these cells deserve further study. For his part, De Strooper recently reported that in flies, LRRK2 phosphorylates endophilin A, a protein involved in synaptic vesicle endocytosis (see ARF related news story). Endocytosis is disrupted both when there is too much and too little LRRK2 activity. This means that researchers targeting hyperactive LRRK2 with inhibitors should be careful not to overdo it, he suggested. In Florence, De Strooper extended these results to mice. He is collaborating with researchers at Janssen Pharmaceuticals, Beerse, Belgium, who showed data confirming his endocytosis findings in a poster presentation.

Something Old––Role in Autophagy
Several Parkinson’s risk genes, including LRRK2, have been previously linked to autophagy, a waste disposal system inside cells (see ARF related news story). The autophagy narrative has become a hot topic in PD research, since a blockage in this process may help explain why cells accumulate α-synuclein deposits. In Florence, researchers elaborated on the story. For example, Schlossmacher reported that levels of a key autophagy marker drop in immune cells containing mutant LRRK2, suggesting this pathway is impaired.

Ben Wolozin at Boston University, Massachusetts, found that numerous autophagy genes are co-regulated with LRRK2 (see Ferree et al., 2012). In a worm model, these genes interacted with pathogenic LRRK2 variants to damage dopaminergic neurons. In mammalian cells, Wolozin showed that one such gene, histone deacetylase 6 (HDAC6), directly binds LRRK2 and mediates the ability of the G2019S LRRK2 mutant to dial down autophagy. G2019S is the most common PD-associated variant, and causes hyperactivation of the kinase domain.

Wolozin then wove α-synuclein and the effects of aging into this picture. He crossed worms carrying either wild-type or G2019S LRRK2 to animals marked with a reporter molecule for autophagic flux. In young worms, the presence of wild-type LRRK2 pumped up autophagy, while the mutant form had no effect. However, in middle-aged and older worms, mutant LRRK2 dampened autophagy. Then Wolozin added human α-synuclein to the animals, which lack an endogenous version of this protein. Again, in young worms, the protein caused no problems, regardless of which LRRK2 variant they expressed. In old worms it was a different story. Animals expressing α-synuclein with either form of LRRK2 lost dopaminergic neurons. The G2019S variant led to slightly more cell death and much less autophagic flux than did the wild-type version, but both were harmful. Wolozin and colleagues previously found that LRRK2 jacks up α-synuclein expression (see Carballo-Carbajal et al., 2010). With age, this may lead to too much α-synuclein, resulting in aggregation and toxicity, he speculated. It remains to be seen whether these findings will translate to people, though LRRK2 has previously been found to exacerbate α-synuclein aggregation in transgenic mice (see ARF related news story). The finding is controversial, since two other mouse studies did not turn up synergy between these proteins (see Daher et al., 2012; Herzig et al., 2012).

What About the GTPase?
Besides its kinase, LRRK2 contains a second functional domain, a GTPase. Darren Moore at the Swiss Federal Institute of Technology in Lausanne (EPFL) pointed out that some PD-associated LRRK2 variants lower activity of this enzyme, suggesting this domain could be involved in disease. In Florence, Moore described a yeast screen he used to find genes that affected toxicity due to LRRK2 overexpression. Out of nine hits, only one had a human orthologue. This turned out to be ADP-ribosylation factor GTPase-activating protein 1 (ArfGAP1). LRRK2 physically interacts with ArfGAP1, and the two proteins are found together on Golgi membranes, in synaptosomes, and in the cytoplasm, Moore reported. Not only does ArfGAP1 activate LRRK2’s GTPase function, but LRRK2 also phosphorylates ArfGAP1, suggesting the two proteins may regulate each other. Silencing ArfGAP1 rescues a LRRK2-mediated shortening of neurites in primary cortical neurons. This shows that ArfGAP1 is required for LRRK2-induced toxicity, Moore concluded (see Stafa et al., 2012). He is looking for the mechanism now, and will also see if these cell culture results hold in vivo.

Moore also investigated how the protein’s kinase and GTPase activities might interact. He made synthetic mutations in LRRK2’s GTPase domain, and found that they diminished the protein’s kinase activity (see Biosa et al., 2013). To look in vivo, he injected virally encoded G2019S LRRK2 into rat striatum. About one-third of nigral neurons took up the gene, leading to the loss of dopaminergic neurons in the substantia nigra, together with axonal degeneration and ubiquitin pathology in the striatum (see Dusonchet et al., 2011). Injecting a kinase-dead version of G2019S LRRK2, or one with an activated GTPase, on the other hand, produced much less striatal pathology. Both of these approaches might be useful for tackling the G2019S mutation, he suggested.

Will one therapy work for all LRRK2 mutations, or will treatments need to be targeted to the specific variant? Many LRRK2 mutations do not affect kinase activity, and might require different strategies, such as hitting downstream targets of the protein, Moore predicted.

Keep It Simple: It’s a Kinase
Meanwhile, pharmaceutical companies are encouraged by the fact that the most common pathogenic mutation of LRRK2 leads to a hyperactive kinase. Drug developers have extensive experience in designing kinase inhibitors. Strengthening the case for this approach, biochemical and functional studies show that too much LRRK2 kinase activity poisons neurons (see Greggio et al., 2006; Lee et al., 2010; Ramsden et al., 2011). Moreover, the mechanism could extend to non-genetic forms of the disease. Warren Hirst at Pfizer, Cambridge, Massachusetts, pointed out that LRRK2 protein levels are elevated in sporadic PD (see Cho et al., 2013). This finding suggests that overactivation of LRRK2 could be a common feature in PD, Hirst said. He is looking now to see if kinase activity is, in fact, up in sporadic brains. This will be a key issue for pharmaceutical companies, as it would greatly increase the number of people who might take a kinase inhibitor drug.

The Pfizer program aims to develop selective, brain-penetrant kinase inhibitors for LRRK2, Hirst said. One challenge is that few good tools exist for studying LRRK2. For example, researchers lack a validated animal model. Hirst noted that the rat model described by Moore looks promising. Scientists are also hampered by the lack of a validated physiological target for LRRK2, which would be helpful for measuring whether a drug is having the desired effect. Another pressing need is for pharmacological inhibitors and probes for LRRK2. Hirst described an inhibitor Pfizer has developed as a research tool. Called LRRK2-IN-1, it inhibits the kinase with 20 nM potency, but does not cross the blood-brain barrier. Pfizer has also designed a radioligand that binds LRRK2, which is enabling them to perform tissue binding studies in transgenic mouse brain and kidney. These kinds of studies provide a knowledge base for developing drugs, Hirst said. In addition, Pfizer is now testing an inhibitor that gets into mouse brain when given at 10 mg/kg. Hirst noted that LRRK2 inhibition results in conformational changes that can be detected with antibodies (see Gillardon et al., 2013; Sheng et al., 2012), which will provide another way to measure the effectiveness of inhibitors.

Only time will tell whether this pharmaceutical approach will pan out. Hirst told Alzforum that the immunological data shown by Schlossmacher looked intriguing, as they link the LRRK2 mutation to other diseases. That may provide an alternative route to the clinic, Hirst suggested.—Madolyn Bowman Rogers.

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References

News Citations

  1. LRRK2 Pathway Offers Up New Targets in Parkinson’s
  2. The Many Faces of LRRK2
  3. New Substrate for Parkinson’s Protein Is Picky About Phosphate
  4. Evidence Piles Up for Lysosomal Dysfunction in Parkinson’s
  5. α-Synuclein Conspires With LRRK2 to Corrupt Neurons

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  11. . Inhibitors of leucine-rich repeat kinase-2 protect against models of Parkinson's disease. Nat Med. 2010 Sep;16(9):998-1000. PubMed.
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  13. . MicroRNA-205 regulates the expression of Parkinson's disease-related leucine-rich repeat kinase 2 protein. Hum Mol Genet. 2013 Feb 1;22(3):608-20. PubMed.
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Other Citations

  1. Read a PDF of the entire series.

External Citations

  1. LRRK2

Further Reading