Systematic characterization of LRRK2 monoclonal antibodies reveal novel LRRK2 localization in the mouse, rat and human brain
Monday, Oct 15, 2012, 1:00 PM - 2:00 PM
*N. N. SUKAR
, P. DAVIES
, K. HINKLE
, R. MESIAS
, B. SEPULVEDA
, G. SERRANO
, D. ALESSI
, T. G. BEACH
, D. L. BENSON
, C. L. WHITE, III
, R. M. COWELL
, S. S. DAS
, H. MELROSE
, A. B. WEST
Psychiatry, Univ. of Alabama at Birmingham, Birmingham, AL;
MRC Protein Phosphorylation Unit, Univ. of Dundee, Dundee, United Kingdom;
Neurosci., Mayo Clin. Jacksonville, Jacksonville, FL;
Neurosci., Mount Sinai Sch. of Med., New York, NY;
Civin Lab. for Neuropathology, Banner Sun Hlth. Res. Inst., Sun City, AZ;
Neuropathology, Univ. of Texas Southwestern Med. Sch., Dallas, TX;
The Michael J. Fox Fndn. for Parkinson's Res., New York, NY
Missense mutations in the leucine-rich repeat kinase 2 (LRRK2) gene represent a major cause of late-onset Parkinson’s disease (PD) in select ethnic populations. The LRRK2 gene encodes a large multi-domain protein that possesses both kinase and GTPase activity. Results of LRRK2 expression and localization studies that may provide insight into the role of LRRK2 function in neurodegeneration have varied greatly and may have been hampered by the lack of sensitive and specific reagents. In recent years, the Michael J. Fox Foundation for Parkinson’s Research sponsored the creation of eight monoclonal LRRK2 antibodies, in addition to the development of both rat and mouse LRRK2 knockout animals. We tested a total of 10 rabbit and mouse monoclonal antibodies using cell lines and brain tissue derived from mice, rats and humans. We optimized protocols for the detection of LRRK2 in a number of different assays including immunoblot, immunoprecipitation, immunocytochemistry in cultured neurons, and immunohistochemistry using frozen, PFA-fixed, and formalin-fixed paraffin embedded brain tissue. Several highly-specific and sensitive antibodies were identified, although species specific antibodies were not identified. Endogenous LRRK2 protein from brain tissue was immunoprecipitated and found to retain kinase acitivity that was highly dependent on post-mortem intervals. Differences in LRRK2 localization between rodents and humans were noted in several brain regions, most dramatically in the cerebellum, where LRRK2 is expressed in Purkinje neurons in rodents, but in Bergmann glia in humans. LRRK2 is highly expressed in some neuromelanin positive neurons in the substantia nigra in humans, with weaker expression in the striatum, whereas in rodents LRRK2 is preferentially expressed in medium-spiny neurons in the striatum, with weaker expression in the substantia nigra. Within neurons, LRRK2 concentrates in small clusters consistent with its localization to vesicular populations. LRRK2 monoclonal antibodies that are theoretically unlimited in availability together with standardized protocols should aid definition of LRRK2 function in health and disease.
Michael J. Fox Foundation for Parkinson’s Research
[Authors]. [Abstract Title]. Program No. XXX.XX. 2012 Neuroscience Meeting Planner. New Orleans, LA: Society for Neuroscience, 2012. Online.
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