MAP1B rescues LRRK2 mutant-mediated cytotoxicity
© Chan et al.; licensee BioMed Central Ltd. 2014
Received: 7 March 2014
Accepted: 15 April 2014
Published: 22 April 2014
Leucine-rich repeat kinase 2 (LRRK2) mutations are the most common cause of dominant and sporadic Parkinson’s disease (PD), a common neurodegenerative disorder. Yeast-two-hybrid screening using human LRRK2 kinase domain as bait identified microtubule associated protein 1B (MAP1B) as a LRRK2 interactor. The interacting domains were LRRK2 kinase and the light chain portion of MAP1B (LC1). LRRK2 + LC1 interaction resulted in LRRK2 kinase inhibition. LRRK2 mutants (R1441C, G2019S and I2020T) exhibited decreased endogenous LC1 expression and its co-expression with LC1 rescued LRRK2 mutant-mediated toxicity. This study presented the first data on the effects of LRRK2 + LC1 interaction and also suggested that LCI possibly rescued LRRK2 mutant-induced cytotoxicity by inhibiting LRRK2 kinase activity. Compounds that upregulate LC1 expression may therefore hold therapeutic potential for LRRK2-linked diseases.
Parkinson’s disease (PD), a neurodegenerative disorder, has been estimated to afflict six million people worldwide [1, 2] and mutations in the leucine-rich repeat kinase 2 (LRRK2) gene is the most common cause of dominant and sporadic PD [3, 4]. Common pathogenic LRRK2 mutations like R1441C/G, Y1699C, G2019S and I2020T reside within the Roc-COR-kinase domains and are associated with increased kinase activity, which is in turn linked to increased neurotoxicity [5, 6]. Though the enzymatic functions of LRRK2 have been extensively studied, its physiological mechanism remains unknown. Hence, recent LRRK2 research focused on the identification of LRRK2 interactors/substrates as they will provide vital pathophysiologic clues.
Recently, LRRK2 was reported to phosphorylate and negatively regulate Futsch, the fly homolog of microtubule-associated protein 1B (MAP1B), at the pre-synapse . The MAP1B complex comprises of the heavy chain (HC) and light chain (LC1) subunits . LC1 has been reported to dimerize or oligomerize  and its overexpression can lead to endoplasmic reticulum stress-induced apoptosis .
Utilising yeast-two-hybrid screening with human LRRK2 kinase domain as bait, LC1 was identified as a LRRK2 interactor. This study presented the first data on the effects of LRRK2 and LC1 interaction and also suggested that LCI possibly rescued LRRK2 mutant-induced cytotoxicity by inhibiting LRRK2 kinase activity.
Materials and methods
SKNSH cells were fixed in 4% paraformaldehyde and probed with LRRK2 (Novus) and LC1 (Santa Cruz) primary antibodies (1:200) at 4°C overnight. Subsequently, secondary antibodies (Invitrogen), Alexa488 (LRRK2) and Alexa546 (LC1), were added to cells and incubated at room temperature for one hour. Cells were mounted with DAPI and immunofluorescence was visualised.
LRRK2 kinase assay was carried out using the ADP Hunter assay (BMG labtech, Offenburg, Germany), truncated LRRK2 protein (Invitrogen), 10 mM ATP (Sigma-Aldrich) and purified LC1-GST. Reagents were mixed and incubated at room temperature for two hours; reaction was stopped by adding sample loading dye and boiling for five minutes. Primary antibodies, phospho-threonine (Cell Signaling) and phospho-serine (Millipore) were used at the concentration 1:1000.
Human LRRK2 WT, G2019S and I2020T were cloned into pEGFP-N1 vectors and LC1 was cloned into pGEX5X1 vector. R1441C plasmid was cloned by Mark Cookson  and obtained from Addgene (plasmid 25046). Human neuroblastoma cells (SKNSH; ATCC) was transfected using Turbofect (Thermo Scientific) and incubated for 48 hours before cell lysates were collected and resolved by SDS-PAGE. Primary antibodies, LC1 (Santa Cruz) and β-actin (Sigma-Aldrich), were used at the concentration 1:1000 and all secondary antibodies (Santa Cruz) were used at the concentration 1:2000. Resultant western blot bands were quantified using the NIH ImageJ software  and tabulated in a bar graph.
Cell viability assays utilised Methylthiazolyldiphenyl-tetrazolium bromide (MTT, Sigma-Aldrich); MTT (0.5 mg/mL) was added to transfected cells and incubated at 37°C for three hours before resultant formazan was solubilised in DMSO and quantified. Caspase-Glo 3/7 luminescent apoptosis assay (Promega, Wisconsin, USA) was carried out according to manufacturer’s instructions. Statistical analysis was carried out using the Student’s t-test.
MAP1B was identified as a LRRK2 interactor through yeast-two-hybrid screening using human LRRK2 kinase domain as bait; the interacting domains were narrowed down to the LRRK2 kinase and LC1. The MAP1B complex comprises of the HC and LC1 subunits. LC1 has microtubule stabilizing activity and the HC has been reported to act as the regulatory subunit of the MAP1B complex to control LC1 activity . LC1 appeared to inhibit LRRK2 kinase activity and a decrease in endogenous LC1 expression was observed in LRRK2 mutants that exhibited increased toxicity. Subsequent functional assays showed that transient LRRK2 mutant expression caused acute toxicity through apoptosis, which could be rescued by the co-expression of LC1. LRRK2 kinase activity is central to its pathogenicity as LRRK2 kinase-dead mutants are not toxic . These observations suggest that LCI is an important mediator of LRRK2 mutant-related toxicity and this is possibly linked to LRRK2 kinase inhibition.
The interaction between LRRK2 and LC1 is supported by an earlier report which showed that LRRK2 phosphorylates Futsch, a fly homolog of MAP1B . LRRK2 was reported to negatively regulate the presynaptic function of Futsch in controlling microtubule dynamics  and this study showed that LRRK2 kinase phosphorylated LC1 at serine residues while concurrently inhibiting LRRK2 autophosphorylation at threonine residues. Similarly, as the co-expression of Futsch was able to rescue synaptic morphology defects induced by human LRRK2 overexpression , this report demonstrated that the co-expression of LC1 was able to neutralise or rescue the observed apoptotic effect with wild-typed LRRK2 and mutant LRRK2 respectively.
In conclusion, this study validated MAP1B as a LRRK2 interactor and specified LC1 as its interacting domain. It showed concurrent LRRK2 kinase inhibition while phosphorylating LC1 and the effect of this interaction possibly neutralised LRRK2 mutant-mediated cytotoxicity by inhibiting its kinase activity. This provided additional insight into LRRK2 mutant-mediated pathogenicity and the regulation of LRRK2+ LC1 interaction is a potential avenue for the development of LRRK2 therapeutics.
We thank National Medical Research Council and Singapore Millennium Foundation for their support.
- Dauer W, Przedborski S: Parkinson’s disease: mechanisms and models. Neuron. 2003, 39: 889-909. 10.1016/S0896-6273(03)00568-3.PubMedView ArticleGoogle Scholar
- Braak H, Del Tredici K, Rub U, de Vos RA, Jansen Steur EN, Braak E: Staging of brain pathology related to sporadic Parkinson’s disease. Neurobiol Aging. 2003, 24: 197-211. 10.1016/S0197-4580(02)00065-9.PubMedView ArticleGoogle Scholar
- Kumari U, Tan EK: LRRK2 in Parkinson’s disease: genetic and clinical studies from patients. FEBS J. 2009, 276: 6455-6463. 10.1111/j.1742-4658.2009.07344.x.PubMedView ArticleGoogle Scholar
- Wider C, Dickson DW, Wszolek ZK: Leucine-rich repeat kinase 2 gene-associated disease: redefining genotype-phenotype correlation. Neurodegener Dis. 2010, 7: 175-179. 10.1159/000289232.PubMedPubMed CentralView ArticleGoogle Scholar
- West AB, Moore DJ, Choi C, Andrabi SA, Li X, Dikeman D, Biskup S, Zhang Z, Lim KL, Dawson VL, Dawson TM: Parkinson’s disease-associated mutations in LRRK2 link enhanced GTP-binding and kinase activities to neuronal toxicity. Hum Mol Genet. 2007, 16: 223-232.PubMedView ArticleGoogle Scholar
- Berwick DC, Harvey K: LRRK2 signaling pathways: the key to unlocking neurodegeneration?. Trends Cell Biol. 2011, 21: 257-265. 10.1016/j.tcb.2011.01.001.PubMedView ArticleGoogle Scholar
- Lee S, Liu HP, Lin WY, Guo H, Lu B: LRRK2 kinase regulates synaptic morphology through distinct substrates at the presynaptic and postsynaptic compartments of the Drosophila neuromuscular junction. J Neurosci. 2010, 30: 16959-16969. 10.1523/JNEUROSCI.1807-10.2010.PubMedPubMed CentralView ArticleGoogle Scholar
- Hammarback JA, Obar RA, Hughes SM, Vallee RB: MAP1B is encoded as a polyprotein that is processed to form a complex N-terminal microtubule-binding domain. Neuron. 1991, 7: 129-139. 10.1016/0896-6273(91)90081-A.PubMedView ArticleGoogle Scholar
- Togel M, Wiche G, Propst F: Novel features of the light chain of microtubule-associated protein MAP1B: microtubule stabilization, self interaction, actin filament binding, and regulation by the heavy chain. J Cell Biol. 1998, 143: 695-707. 10.1083/jcb.143.3.695.PubMedPubMed CentralView ArticleGoogle Scholar
- Wang Z, Zhang Y, Zhang S, Guo Q, Tan Y, Wang X, Xiong R, Ding J, Chen S: DJ-1 can inhibit microtubule associated protein 1 B formed aggregates. Mol Neurodegener. 2011, 6: 38-10.1186/1750-1326-6-38.PubMedPubMed CentralView ArticleGoogle Scholar
- Greggio E, Jain S, Kingsbury A, Bandopadhyay R, Lewis P, Kaganovich A, van der Brug MP, Beilina A, Blackinton J, Thomas KJ, Ahmad R, Miller DW, Kesavapany S, Singleton A, Lees A, Harvey RJ, Harvey K, Cookson MR: Kinase activity is required for the toxic effects of mutant LRRK2/dardarin. Neurobiol Dis. 2006, 23: 329-341. 10.1016/j.nbd.2006.04.001.PubMedView ArticleGoogle Scholar
- Schneider CA, Rasband WS, Eliceiri KW: NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012, 9: 671-675. 10.1038/nmeth.2089.PubMedView ArticleGoogle Scholar
- Angeles DC, Gan BH, Onstead L, Zhao Y, Lim KL, Dachsel J, Melrose H, Farrer M, Wszolek ZK, Dickson DW, Tan EK: Mutations in LRRK2 increase phosphorylation of peroxiredoxin 3 exacerbating oxidative stress-induced neuronal death. Hum Mutat. 2011, 32: 1390-1397. 10.1002/humu.21582.PubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.