Heterodimerization of Lrrk1-Lrrk2: Implications for LRRK2-associated Parkinson disease

Abstract

LRRK2 mutations are recognized as the most frequent genetic cause of both familial and sporadic parkinsonism identified to date. A remarkable feature of this form of parkinsonism is the variable penetrance of symptom manifestation resulting in a wide range of age-at-onset in patients. Herein we use a functional approach to identify the Lrrk1 protein as a potential disease modifier demonstrating an interaction and heterodimer formation with Lrrk2. In addition, evaluation of LRRK1 variants in our large Lrrk2 p.G2019S-parkinsonism series from a Tunisian (n=145) identified a missense mutation (p.L416M) resulting in an average 6.2 years younger age at disease onset. In conclusion we show that the interaction of Lrrk1-Lrrk2 can form protein dimers and this interaction may influence the age of symptomatic manifestation in Lrrk2-parkinsonism patients.

Figure 1

Schematic of Lrrk1 and Lrrk2 domain structures: ARM= armadillo, ANK= ankyrin, LRR= leucine-rich, ROC= Ras of complex, COR= C-terminus of ROC.

Figure 2

Absolute LRRK1 and LRRK2 mRNA expression levels in human control brain. Copy numbers for LRRK1 and LRRK2 were calculated by comparison to a standard curve of co-amplified plasmids encoding full length Lrrk1 or Lrrk2 proteins.

Figure 3

Lrrk1 and Lrrk2 proteins interact in vitro: A. Co-immunoprecipitation of full length Lrrk1-V5 with FLAG-Lrrk2 in HEK293T cells. The left panel shows the presence of Lrrk1-V5 but not LacZ-V5 in the FLAG-Lrrk2 immunoprecipitate (IP). The inputs on the right panel represent the amounts of transfected proteins introduced in the immunoprecipitation assay.B. Co-precipitation of Lrrk2-V5 with FLAG-Lrrk1. Successful co-pull-down of Lrrk2-V5 occurs only in the presence of FLAG-Lrrk1 (IP left; Inputs right). A stronger detection signal was observed when Lrrk2 was used to pull-down Lrrk1 than vice versa, which may indicate a higher affinity of Lrrk2 for Lrrk1, whereas Lrrk1 might preferentially engage in interactions with other proteins. However as co-immunoprecipitation assays do not provide a reliable quantitative assessment of binding affinities this warrants further investigation.

Figure 3

Lrrk1 and Lrrk2 proteins interact in vitro: A. Co-immunoprecipitation of full length Lrrk1-V5 with FLAG-Lrrk2 in HEK293T cells. The left panel shows the presence of Lrrk1-V5 but not LacZ-V5 in the FLAG-Lrrk2 immunoprecipitate (IP). The inputs on the right panel represent the amounts of transfected proteins introduced in the immunoprecipitation assay.B. Co-precipitation of Lrrk2-V5 with FLAG-Lrrk1. Successful co-pull-down of Lrrk2-V5 occurs only in the presence of FLAG-Lrrk1 (IP left; Inputs right). A stronger detection signal was observed when Lrrk2 was used to pull-down Lrrk1 than vice versa, which may indicate a higher affinity of Lrrk2 for Lrrk1, whereas Lrrk1 might preferentially engage in interactions with other proteins. However as co-immunoprecipitation assays do not provide a reliable quantitative assessment of binding affinities this warrants further investigation.