Abstract
Missense mutations in LRRK2 (leucine-rich repeat kinase 2) are a major cause of PD (Parkinson's disease). Several antibodies against LRRK2 have been developed, but results using these polyclonal antibodies have varied widely leading to conflicting conclusions. To address this challenge, the Michael J. Fox Foundation for Parkinson's Research generated a number of monoclonal antibodies targeting epitopes across the LRRK2 protein. In the present paper, we report optimized protocols and results for ten monoclonal antibodies for immunoblotting, immunohistochemistry, immunoprecipitation and kinase activity assays, in rat, mouse and human brain tissue. Several efficacious antibodies were identified, but results demonstrate that the mouse monoclonal N241A/34 is suitable for most applications, with the best overall rabbit monoclonal antibody being c41-2. These antibodies produced a dominant band of the expected size via immunoblotting and a lack of labelling in tissue derived from LRRK2-knockout animals under optimized conditions. A significant proportion of LRRK2 protein localizes to insoluble fractions and no evidence of truncated LRRK2 protein was detected in any fraction from rodent or human tissues. An assay was developed for the robust detection of LRRK2 kinase activity directly from frozen mouse and human brain tissue, but precipitous declines in activity were observed that corresponded to increasing post-mortem intervals and processing times. Finally, we demonstrate the highest levels of brain-localized LRRK2 in the striatum, but note differential expression patterns between rat and mouse in both striatum and cortex. Anti-LRRK2 monoclonal antibodies that are unlimited in availability together with the proposed standardized protocols should aid in the definition of LRRK2 function in both health and disease.
Highlights
Rare missense mutations and more common genetic variability in the leucine-rich repeat kinase 2 (LRRK2) gene are known to be important susceptibility factors to late-onset typical PD (Parkinson’s disease) [1]
Antigens used for the production of anti-LRRK2 monoclonal antibodies include recombinant N-terminal fragments of LRRK2, readily soluble from Escherichia coli expression systems, as well as kinase-active fragments of LRRK2 lacking the N-terminus, generated in insect cells, and a short peptide synthesized in vitro c The Authors Journal compilation c 2013 Biochemical Society
LRRK2 has become a focus of intense research because of strong genetic associations with PD
Summary
Rare missense mutations and more common genetic variability in the LRRK2 (leucine-rich repeat kinase 2) gene are known to be important susceptibility factors to late-onset typical PD (Parkinson’s disease) [1]. The LRRK2 protein is a large multidomain enzyme with both kinase activity and GTPase activity. The physiological cellular function of LRRK2 is not clear despite strong evolutionary conservation of this class of protein. The pathogenic mechanisms and pathways underlying LRRK2 function in disease are not fully understood. In vitro data evaluating LRRK2 pathogenic mutations generally point towards a gain of function caused by enhanced activity [4,5]. Further investigation into LRRK2 may provide insight into pathways and mechanisms that are important in late-onset PD-related neurodegeneration
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