Back
 APD  Vol.6 No.4 , November 2017
Lack of Evidence for Decreased Protein Stability in the 2397 (Met) Haplotype of the Leucine Rich Repeat Kinase 2 Protein Implicated in Parkinson’s Disease
Abstract:
Missense mutations in the leucine rich repeat kinase 2 (LRRK2) gene are the leading genetic cause of autosomal dominant familial Parkinson’s disease. We previously reported that two mutations within the ROC domain, namely R1441C and A1442P, exhibit increased protein degradation leading to lowered steady state LRRK2 protein levels in HEK293 cells. More recently, the common WD40 domain LRRK2 haplotype, Met2397, which is a risk factor for Crohn’s disease, has been shown to lower steady state protein levels in HEK293 cells. In view of recent evidence implicating LRRK2 and inflamemation in PD, we investigated the effects of Met2397 on LRRK2 expression, and compared them to the Thr2397 variant and other LRRK2 mutants. In this study, we transfected HEK293 cells with plasmid constructs encoding the different LRRK2 variants, and analyzed the resulting protein levels by Western blot and flow cytometry. Here we found that both the Met2397 and Thr2397 haplotypes yield similar levels of LRRK2 protein expression and do not appear to impact cell viability in HEK293 cells, compared to other LRRK mutants. Thus, we have concluded that the Met2397 haplotype is unlikely to play a role in LRRK2 mediated or idiopathic PD.
Cite this paper: Anderton, R. , Hill, L. , Morris, R. , Mastaglia, F. , Greene, W. and Boulos, S. (2017) Lack of Evidence for Decreased Protein Stability in the 2397 (Met) Haplotype of the Leucine Rich Repeat Kinase 2 Protein Implicated in Parkinson’s Disease. Advances in Parkinson's Disease, 6, 113-123. doi: 10.4236/apd.2017.64012.
References

[1]   Schapira, A.H. and Jenner, P. (2011) Etiology and Pathogenesis of Parkinson’s Disease. Movement Disorders, 26, 1049-1055. https://doi.org/10.1002/mds.23732

[2]   Corti, O., Lesage, S. and Brice, A. (2011) What Genetics Tells Us about the Causes and Mechanisms of Parkinson’s Disease. Physiological Reviews, 91, 1161-1218.

[3]   Smith, W.W., et al. (2005) Leucine-Rich Repeat Kinase 2 (LRRK2) Interacts with Parkin, and Mutant LRRK2 Induces Neuronal Degeneration. Proceedings of the National Academy of Sciences of the United States of America, 102, 18676-18681. https://doi.org/10.1073/pnas.0508052102

[4]   Mills, R.D., et al. (2014) Prediction of the Repeat Domain Structures and Impact of Parkinsonism-Associated Variations on Structure and Function of all Functional Domains of Leucine-Rich Repeat Kinase 2 (LRRK2). Human Mutation, 35, 395-412.
https://doi.org/10.1002/humu.22515

[5]   Greene, I.D., et al. (2014) Evidence that the LRRK2 ROC Domain Parkinson’s Disease-Associated Mutants A1442P and R1441C Exhibit Increased Intracellular Degradation. Journal of Neuroscience Research, 92, 506-516. https://doi.org/10.1002/jnr.23331

[6]   West, A.B., et al. (2005) Parkinson’s Disease-Associated Mutations in Leucine-Rich Repeat Kinase 2 Augment Kinase Activity. Proceedings of the National Academy of Sciences of the United States of America, 102, 16842-16847. https://doi.org/10.1073/pnas.0507360102

[7]   Greggio, E., et al. (2006) Kinase Activity Is Required for the Toxic Effects of Mutant LRRK2/Dardarin. Neurobiology of Disease, 23, 329-341.
https://doi.org/10.1016/j.nbd.2006.04.001

[8]   Gilsbach, B.K. and Kortholt, A. (2014) Structural Biology of the LRRK2 GTPase and Kinase Domains: Implications for Regulation. Frontiers in Molecular Neuroscience, 7.

[9]   Sheng, D., et al. (2010) Deletion of the WD40 Domain of LRRK2 in Zebrafish Causes Parkinsonism-Like Loss of Neurons and Locomotive Defect. PLoS Genet, 6, e1000914.

[10]   Jorgensen, N.D., et al. (2009) The WD40 Domain Is Required for LRRK2 Neurotoxicity. PLoS One, 4, e8463.

[11]   Liu, Z., et al. (2011) The Kinase LRRK2 Is a Regulator of the Transcription Factor NFAT That Modulates the Severity of Inflammatory Bowel Disease. Nat Immunol, 12, 1063-1070.
https://doi.org/10.1038/ni.2113

[12]   Anderton, R.S., et al. (2014) Investigation of a Recombinant SMN Protein Delivery System to Treat Spinal Muscular Atrophy. Translational Neuroscience, 5, 8-16.
https://doi.org/10.2478/s13380-014-0201-2

[13]   Anderton, R.S., et al. (2011) Survival of Motor Neuron Protein Over-Expression Prevents Calpain-Mediated Cleavage and Activation of Procaspase-3 in Differentiated Human SH-SY5Y Cells. Neuroscience, 181, 226-233. https://doi.org/10.1016/j.neuroscience.2011.02.032

[14]   Ohta, E., Kubo, M. and Obata, F. (2010) Prevention of Intracellular Degradation of I2020T Mutant LRRK2 Restores Its Protectivity against Apoptosis. Biochemical and Biophysical Research Communications, 391, 242-247. https://doi.org/10.1016/j.bbrc.2009.11.043

[15]   Barrett, J.C., et al. (2008) Genome-Wide Association Defines More than 30 Distinct Susceptibility Loci for Crohn’s Disease. Nature Genetics, 40, 955-962. https://doi.org/10.1038/ng.175

[16]   Tan, E.K., et al. (2007) The LRRK2 Gly2385Arg Variant Is Associated with Parkinson’s Disease: Genetic and Functional Evidence. Hum Genet, 120, 857-863. https://doi.org/10.1007/s00439-006-0268-0

[17]   Carlessi, R., et al. (2011) GTP Binding to the ROC Domain of DAP-Kinase Regulates Its Function through Intramolecular Signalling. EMBO Reports, 12, 917-923.
https://doi.org/10.1038/embor.2011.126

[18]   Smith, W.W., et al. (2006) Kinase Activity of Mutant LRRK2 Mediates Neuronal Toxicity. Nature Neuroscience, 9, 1231-1233. https://doi.org/10.1038/nn1776

[19]   MacLeod, D., et al. (2006) The Familial Parkinsonism Gene LRRK2 Regulates Neurite Process Morphology. Neuron, 52, 587-593. https://doi.org/10.1016/j.neuron.2006.10.008

[20]   Dzamko, N. and Halliday, G.M. (2012) An Emerging Role for LRRK2 in the Immune System. Biochemical Society Transactions, 40, 1134-1139. https://doi.org/10.1042/BST20120119

[21]   Gillardon, F., Schmid, R. and Draheim, H. (2012) Parkinson’s Disease-Linked Leucine-Rich Repeat Kinase 2(R1441G) Mutation Increases Proinflammatory Cytokine Release from Activated Primary Microglial Cells and Resultant Neurotoxicity. Neuroscience, 208, 41-48.
https://doi.org/10.1016/j.neuroscience.2012.02.001

[22]   Nagatsu, T., et al. (2000) Changes in Cytokines and Neurotrophins in Parkinson’s Disease. Journal of Neural Transmission. Supplementa, 60, 277-290.

[23]   Sawada, M., Imamura, K. and Nagatsu, T. (2006) Role of Cytokines in Inflammatory Process in Parkinson’s Disease. Journal of Neural Transmission. Supplementa, 70, 373-381.

[24]   Long-Smith, C.M., Sullivan, A.M. and Nolan, Y.M. (2009) The Influence of Microglia on the Pathogenesis of Parkinson’s Disease. Progress in Neurobiology, 89, 277-287.
https://doi.org/10.1016/j.pneurobio.2009.08.001

 
 
Top