Mutations in the gene encoding LRRK2 (aka tyrosine kinase leucine-rich repeat kinase 2) are the most common cause of hereditary Parkinson disease. LRRK2 was discovered in 2004, and five years later it is not yet clear what the protein does or how pathogenic mutations affect its function. PD-causing mutations are dominant, suggesting a gain of function, and most increase the kinase activity of the protein. Because of that, LRRK2 and points downstream offer attractive therapeutic targets, and two papers published this week offer some new possibilities in that regard. A report in this week’s Proceedings of the National Academy of Sciences from Valina and Ted Dawson at Johns Hopkins University School of Medicine in Baltimore, Maryland, offers evidence that the concentration of LRRK2 protein in neurons is regulated through the ubiquitin proteasomal pathway. In particular, their data indicate that LRRK2 is a substrate for the CHIP (carboxy terminus of HSC70-interacting) ubiquitin ligase, which regulates its degradation. CHIP and LRRK2 form a complex with the HSP90 (heat shock protein 90) chaperone, the scientists show; treating cells with an HSP90 inhibitor lowered LRRK2 levels and partially protected cells from the toxic effects of mutant LRRK2 proteins.

A second report suggests that mutant LRRK2 proteins can trigger cell death via the extrinsic apoptotic pathway—this is a cascade separate from the intrinsic, mitochondria-centered pathway normally set off by cell stress or abnormal proteins. The extrinsic pathway usually gears up in response to cell surface receptors such as Fas or TNF receptor, but William Dauer and colleagues at New York City’s Columbia University present new evidence that mutant LRRK2 interacts with its intracellular adaptor proteins to activate caspase-8 and trigger cell death. Inhibition of either adaptor protein or caspase-8 activation prevented LRRK2-mediated neuronal death in cultured cells. This work appears in the January 28 issue of the Journal of Neuroscience.

In the first study, Dawson and colleagues explore the role of the HSP90/CHIP quality control mechanism in regulating LRRK protein levels. The chaperone HSP90 tries to refold damaged or toxic proteins, and when that fails, CHIP tags them for degradation through the ubiquitin-proteasome pathway. LRRK2 had been shown to interact with HSP90, so first author Han Seok Ko looked for evidence that it also might be associated with CHIP. Using co-immunoprecipitation, Ko found CHIP and LRRK2 associated in cells overexpressing both proteins, and in mouse brain extracts. The interaction was not altered by PD-associated mutations in LRRK2.

Using co-expression and siRNA knockdowns, the researchers found that CHIP complex regulates levels of normal and mutated LRRK2 in cells. Higher CHIP expression led to lower LRRK2 protein, while in CHIP knockout mice, LRRK2 was elevated in the brain. CHIP also modulated LRRK2 toxicity. Transfection of mutant LRRK2 into SH-SY5Y neuronal cells decreased their viability, and this effect was partially blocked by CHIP overexpression. On the other hand, CHIP knockdown made toxicity worse.

Along with CHIP, the researchers found HSP90 in the LRRK2 complex. The HSP90 inhibitor 17AAG has been shown to increase degradation of other neurotoxic proteins that are CHIP/HSP90 substrates, including tau (see ARF related news story), and LRRK2 was no different. Treating cells with 17AAG decreased LRRK2 levels and significantly protected the cells from death, the researchers showed. “Together, these findings suggest that LRRK2 is a client of the CHIP-HSP chaperone system, and that regulation of LRRK2 levels through this system is potentially involved in LRRK2 toxicity,” the authors write.

The work from the Dauer lab looks at a different LRRK2 association, one that may lead directly to cell death. The researchers noted the similarity of LRRK2 to kinases of the extrinsic cell death pathway that signal downstream of the Fas and TNFα death receptors in association with adaptor proteins FADD (Fas-associated protein with death domain) and TRADD. First author Cherry Cheng-Ying Ho found that LRRK2 can associate with FADD and TRADD, but not with death receptors themselves. PD-linked mutations enhanced the interaction, and kinase-minus mutants lost the ability to interact. By using dominant-negative FADD proteins, the researchers showed that FADD function was required for cell death in primary mouse cortical neurons expressing LRRK2 mutant proteins.

The FADD-LRRK2 complex also included caspase-8, and RNAi knockdown of that protease inhibited LRRK2-mediated cell death in primary neurons. In contrast, decreasing levels of caspase-9, which lies in the intrinsic pathway, did not block LRRK2-induced cell death. “Our results suggest that capase-8 activation may be a pathological event in PD patients with LRRK2 mutations,” the authors write. In support of that idea, they looked at caspase-8 activation in brain lysates from PD patients with LRRK2 mutations. In four of four patients, they found evidence of caspase-8 cleavage and activation, but none in three of three controls.

Previous work has implicated the apoptotic pathway in neurodegeneration in PD, but one question has been whether the apoptotic markers indicate a primary event, or merely a downstream consequence of disease processes. The current study suggests the intrinsic pathway could happen early on. Previous work has also implicated the intrinsic pathway in LRRK2 toxicity (Iaccarino et al., 2007), so it remains to be seen how these two pathways might interact.

Though the most common cause of inherited PD, LRRK2 mutations account for less than 1 percent of total cases of the disease. However, a few percent of cases of sporadic PD show acquired LRRK2 mutations, and it is possible that alterations in the LRRK2 pathway in the absence of mutations will turn out to play a role in other sporadic cases. If so, then the identification of LRRK2’s partners in crime may lead to more widely useful therapies.—Pat McCaffrey


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News Citations

  1. Therapeutic Takedown: Hsp90 Inhibitors Tackle Tau

Paper Citations

  1. . Apoptotic mechanisms in mutant LRRK2-mediated cell death. Hum Mol Genet. 2007 Jun 1;16(11):1319-26. PubMed.

Further Reading

Primary Papers

  1. . The Parkinson disease protein leucine-rich repeat kinase 2 transduces death signals via Fas-associated protein with death domain and caspase-8 in a cellular model of neurodegeneration. J Neurosci. 2009 Jan 28;29(4):1011-6. PubMed.
  2. . CHIP regulates leucine-rich repeat kinase-2 ubiquitination, degradation, and toxicity. Proc Natl Acad Sci U S A. 2009 Feb 24;106(8):2897-902. PubMed.