Leucine-rich repeat kinase 2 induces alpha-synuclein expression via the extracellular signal-regulated kinase pathway

Abstract

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of autosomal-dominant Parkinson's disease (PD). The second known autosomal-dominant PD gene (SNCA) encodes alpha-synuclein, which is deposited in Lewy bodies, the neuropathological hallmark of PD. LRRK2 contains a kinase domain with homology to mitogen-activated protein kinase kinase kinases (MAPKKKs) and its activity has been suggested to be a key factor in LRRK2-associated PD. Here we investigated the role of LRRK2 in signal transduction pathways to identify putative PD-relevant downstream targets. Over-expression of wild-type [wt]LRRK2 in human embryonic kidney HEK293 cells selectively activated the extracellular signal-regulated kinase (ERK) module. PD-associated mutants G2019S and R1441C, but not kinase-dead LRRK2, induced ERK phosphorylation to the same extent as [wt]LRRK2, indicating that this effect is kinase-dependent. However, ERK activation by mutant R1441C and G2019S was significantly slower than that for [wt]LRRK2, despite similar levels of expression. Furthermore, induction of the ERK module by LRRK2 was associated to a small but significant induction of SNCA, which was suppressed by treatment with the selective MAPK/ERK kinase inhibitor U0126. This pathway linking the two dominant PD genes LRRK2 and SNCA may offer an interesting target for drug therapy in both familial and sporadic disease.

Fig. 1

Expression and distribution of LRRK2. (A) A LRRK2-specific rabbit polyclonal antibody was raised against the region 800–1000 amino acids (MID) between the ANK and LRR domains of human LRRK2. Location of the mutations used in the study is shown in red. R1441C in the COR domain and G2019S in the kinase domain are PD-associated mutations, whereas K1906N is a synthetic mutant with an impaired kinase activity. (B) Extracts of HEK293 cells transfected for 48 h with [wt]LRRK2 were run on a 6% polyacrylamide gel, immunoblotted with the antibody against LRRK2-MID (lower panel) and reprobed with an antibody against the HA tag (upper panel). LRRK2 protein is detected at approximately 280 kDa by both HA-specific and LRRK2-specific antibodies. The LRRK2 antibody detected also endogenous LRRK2 in extracts from mock-transfected cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Activation of ERK1/2 in response to LRRK2 expression. 30 μg of protein lysate from LRRK2-inducible or HEK293 transiently transfected with wild-type, G2019S and kinase-dead LRRK2 cells was loaded on 7.5% polyacrylamide gels and subjected to immunoblot analysis using specific antibodies against the activated dually phosphorylated forms of ERK1/2, JNK and p38^MAPK. Membranes were stripped and reprobed with antibodies against the total MAPK protein. Similar LRRK2 expression levels were assessed with the specific LRRK2 antibody. LRRK2 expression in inducible wild-type and R1441C cell lines was achieved by 48 h treatment with 1 μg/ml tetracycline. Immunoblots are representative from 3 independent experiments.

Fig. 3

Time course of ERK1/2 activation in inducible cell lines. [wt]LRRK2 and [R1441C] LRRK2 inducible HEK293 cells were treated with tetracycline for the indicated times and subjected to immunoblot analysis with antibodies against phospho- and total ERK1/2. Relative densitometry intensities for phospho-ERK1/2 immunoreactivity showed that over-expression of the PD-causative mutation R1441C led to a delayed activation of ERK1/2 compared to [wt]LRRK2. Each value is averaged from 4 independent experiments.

Fig. 4

Delayed activation of ERK1/2 by LRRK2 mutant G2019S. (A) HEK293 cells were transfected with wild-type and G2019S mutant LRRK2 for 12, 24 and 48 h and subjected to immunoblot analysis. The samples were probed for activated phospho-ERK1/2 and total ERK1/2. (B) Relative band intensities for phospho-ERK1/2 immunoreactivity in wild-type, G2019S and kinase-dead LRRK2 at the indicated time points after transfection. Each value is averaged from 4 independent experiments. Mock = cells transfected with the empty vector pCI/HA.

Fig. 5

Delayed activation of the specific ERK1/2 upstream kinases MEK1/2. (A) HEK293 cells were transfected for 12, 24 and 48 h with the indicated LRRK2/HA constructs or with MEK1, non-treated or treated with the pharmacological inhibitor of the MEK/ERK kinase pathway U0126. Cell lysates were immunoblotted and probed for phospho- and total-MEK1/2 and for phospho- and total ERK1/2. (B) Relative band densitometry for phospho-MEK1/2 in transiently transfected HEK293 cells. Image is representative from 3 independent blots. Mock = cells transfected with the empty vector pCI/HA.

Fig. 6

Up-regulation of SNCA by LRRK2. Endogenous mRNA levels of SNCA in HEK293 cells 48 h after transfection with the indicated LRRK2/HA constructs were determined by real-time qRT-PCR. Total RNA was purified on spin columns from cell lysates and quantified in the LightCycler with specific hybridization probes for SNCA. Results are given as the ratio between SNCA and the housekeeping gene h-PBGD.

Fig. 7

LRRK2-mediated induction of SNCA is repressed by the phospho-MEK1/2 inhibitor U0126. HEK293 cells transiently transfected with different LRRK2 constructs were treated with 10 μg/ml U0126 or vehicle (DMSO) for 12 h prior to harvesting. Total mRNA was extracted and SNCA-specific mRNA quantified by real-time RT-PCR to measure the induction levels of SNCA. Similar results were achieved in 3 independent experiments.