Hung Lo et al. Alz Dem 15(11):1489-1502, 2019

 I was looking at protein misfolding conferences, and saw a Gordon conference that took place in Texas earlier this year, pre-Covid. One talk jumped out at me by Elizabeth Rhoades. This led me to her publications for which this paper seemed interesting. It is cited by a paper with Rudi Tanzi published on October, which will be my next review. Liz Rhoades website is hilarious and she seems like a pretty cool researcher. Will see if a collaboration is possible.

tldr: Sachs lab has developed full length tau biosensor and can detect oligomer signal using fluorescence lifetime measurements, Rhoades lab developed single molecule FRET to measure conformational changes of monomeric tau

Targeting the ensemble of heterogeneous tau oligomers in cells: A novel small molecule screening platform for tauopathies

Chih Hung Lo1, Colin Kin-Wye Lim1, Zhipeng Ding1, Sanjula P. Wickramasinghe2, Anthony R. Braun1, Karen H. Ashe3,4,5,6, Elizabeth Rhoades2,7, David D. Thomas8,9, Jonathan N. Sachs1,*

compound stabilizes on pathway oligomer based on shorter lag phase in ThT assay

time resolved FRET biosensor, cell based

"no specific toxic tau oligomer species ha been isolated or identified to date" [29-31]

in vitro assays lack chaperone proteins that may be reuired to produce the ensemble oif tau oligomers, and post translational modifications

building upon diamond biosensor

FRET biosensors express full length 2N4R WT tau and fluorescent protein fusion constructs GFP or RFP, optimal after 48 hours of expression

2N4R WT tau does not fibrillize without seeding [49-52]

mk886 has ec50 of 1.06uM in SH-SY5Y cells, rescues tau induced cell cytotoxicity iwth IC50 of 0.523uM oe of P301L tau results in 37% cel death compared to vector control, 23%

spr Kd=0.178 WT tau, 0.375 p301L tau

single molecule FRET - two different doubly fluorescent labeled tau (labeled at mid and N=-terminal dmain) shows perturbation in prolin/MT domains more so than Nterminal domaisn, pretty sure this is the part that Liz Rhoades lab contributed:

Discussion about recent advances of NMR in cells [75-77]

Methods

Molecular biology To generate tau-GFP and tau-RFP, cDNA encoding full-length 2N4R tau (441 amino acids) was fused to the N-terminus of EGFP and TagRFP vectors. The P301L mutation was introduced by QuikChange mutagenesis (Agilent Technologies, Santa Clara, CA) and sequenced for confirmation. The GFP-tau-RFP was generated by fusing the N-terminal of tau to the C-terminus of GFP and the C-terminus of tau to the N-terminus of RFP. All constructs contain the monomeric mutation A206K to prevent constitutive fluorophore clustering [88].

Transient transfection of HEK293 using lipofectamine 3000

P301L was ransiently expressed in SH-SY5Ycells also with lipfectamine 3000

Stable cell lines of single vectors only

cells were transfected and incubated 2 days before harvesting, washing, diluted to 1 million cells/ml, 50ul per well were dispensed into 384 well plate contining compounds and allowed to incubate at room temperature for 2 hours before readings were taken by the fluorescence liftime plate reader (Fluorescence Innovations) [46, 47]

SPR - BIAcore S200 - recombinant proteins imobilized via amine coupling, compounds tested at 8 concentrations from 1nM to 5uM prepared in HEPES-EP and 2% DMSO