Dynamic and redundant regulation of LRRK2 and LRRK1 expression - PubMed (original) (raw)
Comparative Study
Dynamic and redundant regulation of LRRK2 and LRRK1 expression
Saskia Biskup et al. BMC Neurosci. 2007.
Abstract
Background: Mutations within the leucine-rich repeat kinase 2 (LRRK2) gene account for a significant proportion of autosomal-dominant and some late-onset sporadic Parkinson's disease. Elucidation of LRRK2 protein function in health and disease provides an opportunity for deciphering molecular pathways important in neurodegeneration. In mammals, LRRK1 and LRRK2 protein comprise a unique family encoding a GTPase domain that controls intrinsic kinase activity. The expression profiles of the murine LRRK proteins have not been fully described and insufficiently characterized antibodies have produced conflicting results in the literature.
Results: Herein, we comprehensively evaluate twenty-one commercially available antibodies to the LRRK2 protein using mouse LRRK2 and human LRRK2 expression vectors, wild-type and LRRK2-null mouse brain lysates and human brain lysates. Eleven antibodies detect over-expressed human LRRK2 while four antibodies detect endogenous human LRRK2. In contrast, two antibodies recognize over-expressed mouse LRRK2 and one antibody detected endogenous mouse LRRK2. LRRK2 protein resides in both soluble and detergent soluble protein fractions. LRRK2 and the related LRRK1 genes encode low levels of expressed mRNA species corresponding to low levels of protein both during development and in adulthood with largely redundant expression profiles.
Conclusion: Despite previously published results, commercially available antibodies generally fail to recognize endogenous mouse LRRK2 protein; however, several antibodies retain the ability to detect over-expressed mouse LRRK2 protein. Over half of the commercially available antibodies tested detect over-expressed human LRRK2 protein and some have sufficient specificity to detect endogenous LRRK2 in human brain. The mammalian LRRK proteins are developmentally regulated in several tissues and coordinated expression suggest possible redundancy in the function between LRRK1 and LRRK2.
Figures
Figure 1
Biochemical characterization of LRRK2 and testing of LRRK2 antibodies on mouse tissue. A) Over-expressed mouse LRRK2 detected by JH5514 versus empty vector transfected HEK-293T cells. Whole mouse brain was mechanically homogenized in either PBS (low salt buffer), high salt buffer (PBS supplemented to 600 mM NaCl), 1% Triton X-100 (in PBS), 1% SDS (in PBS) or RIPA (1% SDS, 1% sodium deoxycholate, 1% NP-40). All buffers contained complete protease inhibitors (Roche). A knockout mouse brain is homogenized in PBS (low salt) alone and shown as control. B) Lysates derived from HEK-293T cells transfected with mLRRK2 plasmids were incubated in 1 × Laemmli sample buffer at the indicated temperatures for 10 minutes and then analyzed by SDS-PAGE. Plasticware used to process the protein samples were either siliconized or left untreated. Indicated samples were supplemented with 5% Formic acid (FA) or 4M urea after 10 minute incubation at the indicated temperature. C) LRRK2 antibodies Novus 267 and 268, Abgent AP7099b, and Chemicon (AB9682) in addition to Alexis (AT106) and Cell Signaling (2567) were tested on over-expressed mouse LRRK2 protein and wildtype and _LRRK2_-deficient mouse brain homogenized in PBS alone. All antibodies recognize cross-reactive bands near the expected size of LRRK2. An arrow denotes the position of LRRK2 in the Alexis (AT106) blot. All antibodies were tested on at least two independent membranes and lysates using optimized exposure conditions with similar results.
Figure 2
Testing of LRRK2 antibodies on human tissue. A) Antibodies able to detect over-expressed human LRRK2 protein. B) Fresh frozen tissue samples from the human temporal lobe (Htl) is subjected to different extraction buffers as in figure legend 1A. Novus 267, Novus 268, Abgent AP7099e and JH5514 have sufficient specificity to detect endogenous human LRRK2. All antibodies were tested on at least two independent membranes and lysates using optimized exposure conditions with similar results.
Figure 3
LRRK1 and LRRK2 mRNA levels in development and adulthood. A) Neonatal LRRK1 and LRRK2 mRNA levels. Relative LRRK expression was calculated by normalization to TBP (TATA binding protein) within each tissue sample using the delta delta CT method. Similar results were obtained with internal normalization to GAPDH. Error bars represent standard error mean derived from two each independently analyzed male and female 14 day old CD-1 mice B) LRRK1 and LRRK2 mRNA levels in adult mouse tissues as normalized to TBP. Similar results were obtained with normalization to GAPDH. Error bars represent standard error mean independently derived from two each female and male 3 month old CD-1 mice.
Figure 4
LRRK1 and LRRK2 mRNA levels in development and adulthood. A) Northern blot analysis of different embryonic stages (whole mouse embryo) using a LRRK2 specific riboprobe. B) LRRK2 protein can be detected by JH5514 in whole brain lysates on E17 and throughout post-natal development C) Relative LRRK1 and LRRK2 mRNA levels in direct comparison calculated from whole brain, lung, heart and liver during embryonic stages E11.5 to E19.5, with internal normalization to TBP transcript. Error bars represent standard error mean independently derived from a total of four CD-1 embryos at the indicated age.
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