Assay for Inhibitors of Tau-Mediated Microtubule Assembly
The microtubule-associated protein tau is an abundant protein in the axons of neurons that stabilizes microtubules. With its ability to modulate microtubule dynamics, tau contributes directly or indirectly, to key structural and regulatory cellular functions. Particularly important is the influence tau exerts on axonal transport, which allows signaling molecules, trophic factors and other more ..
NIH Molecular Libraries Probe Production Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLPCN Grant: X01 MH083262-01
Assay Provider: Carlo Ballatore, University of Pennsylvania
NCGC Assay Overview:
The microtubule-associated protein tau is an abundant protein in the axons of neurons that stabilizes microtubules. With its ability to modulate microtubule dynamics, tau contributes directly or indirectly, to key structural and regulatory cellular functions. Particularly important is the influence tau exerts on axonal transport, which allows signaling molecules, trophic factors and other essential cellular constituents to travel along the axons. Under pathological conditions, tau becomes sequestered into insoluble aggregates called neurofibrillary tangles. This phenomenon is believed to have pathological consequences by promoting axonal transport deficits that ultimately lead to synaptic dysfunction and neuronal loss. Because tau binds and stabilizes microtubules, tau fibrillization inhibitors were tested whether they interfere with tau-mediated tubulin polymerization. An in vitro assay was used where purified brain tubulin monomers were assembled into microtubules after the addition of GTP and tau. Microtubule assembly was monitored by the change in absorbance at 340 nm.
Hong, M., Zhukareva, V., Vogelsberg-Ragaglia, V., Wszolek, Z., Reed, L., Miller, B. I., Geschwind, D. H., Bird, T. D., McKeel, D., Goate, A., Morris, J. C., Wilhelmsen, K. C., Schellenberg, G. D., Trojanowski, J. Q., and Lee, V. M. Y. (1998) Science 282, 1914-1917.
NCGC Assay Protocol Summary:
Lyophilized bovine brain tubulin was reconstituted in RAB (100 mM MES, pH 6.9; 1 mM EDTA; 0.5 mM MgSO4) at a concentration of 10 mg/ml. Compounds (50 uM) were added to K18 tau (40 uM) in RAB and pre-incubated at room temperature for 60 minutes. To initiate the MT assembly reaction, 8.25 ul per well of 10 mg/ml tubulin was dispensed on a UV-clear 384-well plate, followed by 1.0 ul of 100 mM GTP in RAB and 41.25 ul of the compound:tau mixture. This resulted in a final reaction mixture of 30 uM tubulin, 40 uM K18 tau, 50 uM compound and 2 mM GTP. The plate was incubated in a Spectramax M5 plate reader at 37 C and the absorbance at 340 nm was read every minute for 45 minutes.
This is a kinetics assay. The kinetic traces were fit to a sigmoidal curve and the upper limit of the absorbance at equilibrium (max turbidity) and the time at which 1/2 of the maximum signal is reached were calculated. Compounds that do not achieve the max signal are considered to inhibit MT assembly. None of the compounds tested was active, i.e. inhibited MT assembly.
Data Table (Concise)