MAP1B rescues LRRK2 mutant-mediated cytotoxicity

Molecular Brain MAP1B rescues LRRK2 mutant-mediated cytotoxicity Sharon L Chan 0 Ling-Ling Chua 0 Dario C Angeles 3 Eng-King Tan 1 0 National Neuroscience Institute, SGH Campus , Singapore, Singapore 1 National Neuroscience Institute (SGH Campus) and Duke-NUS Graduate Medical School Singapore, Singhealth Research Facilities , Blk A 2 02-02 , Singapore 169611 , Singapore 3 Department of Neurology, Singapore General Hospital, Singapore , Singapore 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. LRRK2; MAP1B; LC1; Phosphorylation; Apoptosis - Introduction Parkinsons 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 microtubuleassociated protein 1B (MAP1B), at the pre-synapse [7]. The MAP1B complex comprises of the heavy chain (HC) and light chain (LC1) subunits [8]. LC1 has been reported to dimerize or oligomerize [9] and its overexpression can lead to endoplasmic reticulum stress-induced apoptosis [10]. 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 Immunocytochemistry SKNSH cells were fixed in 4% paraformaldehyde and probed with LRRK2 (Novus) and LC1 (Santa Cruz) primary antibodies (1:200) at 4C 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. Kinase assays LRRK2 kinase assay was carried out using the ADP Hunter assay (BMG labtech, Offenburg, Germany), truncated LRRK2 protein (Invitrogen), 10 mM ATP (SigmaAldrich) 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. Western blotting 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 [11] 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 -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 [12] and tabulated in a bar graph. Functional assays Cell viability assays utilised Methylthiazolyldiphenyltetrazolium bromide (MTT, Sigma-Aldrich); MTT (0.5 mg/mL) was added to transfected cells and incubated at 37C 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 manufacturers instructions. Statistical analysis was carried out using the Students t-test. Results Yeast-two-hybrid screening was performed as previously mentioned [13]. Briefly, a human brain cDNA library w (...truncated)