PD patients with LRRK2 mutations have pleiomorphic pathology, where α
-synuclein and/or tau and/or ubiquitin have been detected in intraneuronal aggregates.3
Furthermore, the phenotype of LRRK2 PD is very similar to the idiopathic PD.3, 5
Accordingly, it is believed that LRRK2 acts far upstream in the unknown pathway leading to the development of the disease, and that deciphering the roles of LRRK2 will provide significant insight into PD pathogenesis of PD.
The abnormal accumulation of cytoplasmic inclusions with ubiquitin is a common feature in PD and other neurodegenerative diseases. This could arise if an interference with protein degradation was one of the principal mechanisms leading to the development of the diseases. Our data demonstrate the involvement of LRRK2 in protein homoeostasis. We have provided a mechanism for the accumulation of multiple proteins due to LRRK2 overexpression by showing that this results in impaired flux through the UPS. This would result in the accumulation α
-synuclein, tau and ubiquitin in PD patients with LRRK2 mutations, if the disease is due to excess LRRK2 function, as has been proposed.7, 8
Our data are further supported by the observation that LRRK2 overexpression enhances ubiquitin accumulation in mice.7
Our data suggest that UPS malfunction is the major cause behind the accumulation of proteins in this context, as LRRK2 overexpression resulted in no additional protein reporter protein accumulation in proteasome-inhibited cells (). Furthermore, LRRK2 overexpression led to protein accumulation in autophagy-incompetent cells (), suggesting that the effects are autophagy-independent. The idea of UPS impairment by LRRK2 is further supported by the accumulation of a specific proteasome substrate in both LRRK2-expressing cells and zebrafish ().
We have shown that LRRK2 did not affect the catalytic activity of the proteasome or expression levels of proteasomal core subcomplexes (). Moreover, co-expression of HSP 70 decreased the tendency of LRRK2 to form aggregates and at the same time enhanced LRRK2-mediated protein accumulation (). Together, these findings suggest that accumulation of proteins after LRRK2 expression is not caused by sequestration of these proteins or proteasomal subunits into LRRK2 aggregates, but rather by mono- or oligomeric forms of LRRK2 acting on the UPS. It is not clear at which level the UPS is perturbed by LRRK2. The cascade of reactions acting upstream of the proteasome catalytic activity is complex and finely tuned. It is possible that ubiquitination or de-ubiquitination may be perturbed or that recognition or transport of ubiquitinated proteins to the proteasome may be altered.
Disease-causing LRRK2 mutations are found throughout the protein. Although increased kinase activity was consistently shown for the most prominent G2019S mutation, data for other mutations are contradictory.11, 26
Thus, mutant LRRK2 may confer pathogenic effects through other functions. In our experimental conditions, the wild-type form of LRRK2 had a significant effect on reporter protein levels and ubiquitin levels that were similar to G2019S-mutated LRRK2. Also, chemical or genetic inhibition of LRRK2 kinase activity did not modulate the accumulation of proteins, suggesting that LRRK2 kinase activity is not a major determinant of its UPS-inhibitory phenotype. This is supported by in vivo
Mutations in LRRK2 cause PD through a gain-of-function mechanism, and we therefore overexpressed the protein in order to mimic a gain-of-function. Although the kinase activity does not seem to be critical for the impairment of the UPS system, it is surprising that the G2019S mutation does not confer additional toxicity compared with WTLRRK2. It is feasible that a kinase-independent toxic mechanism depends on exceeding a threshold level of activity, which may be lowered by pathogenic mutations. This kind of mechanism is supported by the finding that homo- and heterozygous carriers of LRRK2 mutations are clinically indistinguishable.28
If we assume that LRRK2 function needs to exceed a threshold level in order to become toxic, then this may explain why we could not observe a difference in the effect of WTLRRK2 and GSLRRK2 on the impairment of the UPS in an overexpression study.
Alternatively, we may have missed a small but biologically significant difference in effect. The overexpression of LRRK2 may result in a saturated environment where target proteins or auxiliary factors become rate limiting and therefore reduce the true effect size. Even if these other proteins are in abundance, overexpression might over-represent the impact of the protein to such an extent that small differences are not detectable. However, even a small difference may have deleterious consequences over the course of a lifetime, as in PD.
Lastly, we cannot exclude the possibility that UPS inhibition is a wild-type function of LRRK2 unrelated to PD. Although we do not consider this a likely scenario, we believe the involvement of LRRK2 with the UPS as relevant, as it provides some insights into LRRK2's function, which is poorly understood. It is also critical to understand the consequences of LRRK2 overexpression for understanding models of LRRK2 PD, as all Drosophila
,29, 30, 31
mouse7, 32, 33
and cell-based9, 12, 13, 21, 22, 34, 35, 36
models published to date have relied on overexpression.
Altogether our data support a role for LRRK2 in the UPS pathway. It is possible that the accumulation of UPS substrates in LRRK2-mediated PD may be caused by a similar mechanism to LRRK2 overexpression, consistent with a gain-of-function model. A better knowledge of the function of LRRK2 will assist further generation of cellular and animal models and allow for the development of rational therapeutic strategies.