Lo Cascio et al JBC 295, 14807-25, 2020

 Modulating disease-relevant tau oligomeric strains by small molecules

Filipa Lo Cascio, Stephanie Garcia, Mauro Montalbano, Nicha Puangmalai, Salome McAllen, Andrea Pace, Antonio Palumbo Piccionello, Rakez Kayed

claim curcumin derivitives bind and change the conformation of toxic tau oligomers into larger, less toxic aggregates

something to look at with our compounds

disease relevant brain derived tau oligomers from AD, DLB, and PSP by IP with T22 antibody then amplified using full length 2N4R recombinant tau

primary cortical neurons exposed to 0.5uM BDTOs alone or BDTOs pretreated with 5uM compound for 24h, measure cyttoxicity by LDH - we could try this

Preparation of recombinant tau oligomers for cell assay studies:

300 μl of tau stock (0.3 mg) was added to 700 μl of 1× PBS and incubated for 1 h on an orbital shaker at room temperature. After shaking, the resulting TauO were purified by FPLC (Superdex 200 Increase 10/300 column, Amersham Biosciences).

primary cells used C57BL/6 mice (Jackson Laboratory, stock number 000664) exposed to brainderived oligomers 0.125 to 1µM +/- 5µM compound for 24h, then MTS or LDH viability assay

disagree with statement of different proteinase patterns of AD vs DLB vs PSP in Fig 1 E and F, looks more like difference in quantity rather than species. however, the band patterns in undigested (Fig1d) certainly look different

densitometry in Fig 2 does not seem to match with blots shown, some bands are distinctly more dense than the control and yet the quantification shows no significant difference. conversely, some bands are less than half the density of the control but the quantification shows only a 10% drop

fig 3 shows a change in band pattern of BDTO as a result incubation with compound. a decrease in higher molecular weight bands without a corresponding increase in monomers is supportive that the compound causes an increase in very large aggregates (>250kDa)

fig 4 drives me crazy due to the changing scales but again supports generation of large aggregates

fig 5 shows compound partial rescue of BDTO induced toxicity in primary neurons - by IF

fig 7 is intriguing subcellular fractionation after exposure of primary neurons to BDTO +/-preincubation with compound - a decrease in monomeric tau is found in all 3 compartments when incubated with oligomers. authors claim this is evidence of seeding activity. densitometry data again makes no sense, opposite results with cmpound in AD vs PSP, very confusing, but a very interesting concept

fig 8 is using biosensor seeding assay: transfect with BDTOs in Lipofectamine 2000 6µL Lipo, incubate 24 hours, analyze by IF

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

 

Brandt, Trushina, and Bakota

Much More Than a Cytoskeletal Protein: Physiological and Pathological Functions of the Non-microtubule Binding Region of Tau Front. Neurol., 19 October 2020

Tau review article

 

Tau mostly in axons, MAP2 in somatodendritic compartment. Tau ko or inactivation has no major effect in mouse models or cultured nerve cells - likely funciton is to regulate microtubule dynamicity not stability (Qiang L, Sun X, Austin TO, Muralidharan H, Jean DC, Liu M, et al. Tau does not stabilize axonal microtubules but rather enables them to have long labile domains. Curr Biol. (2018) 28:2181–9 e4. doi: 10.1016/j.cub.2018.05.045)

Bioinformatics analysis showed a minimal interactome of 73 direct binding partners (18), and 175 potential new and known tau interacting proteins were recently identified by MALDI-TOF mass spectrometry (19, 19. Sinsky J, Majerova P, Kovac A, Kotlyar M, Jurisica I, Hanes J. Physiological tau interactome in brain and its link to tauopathies. J Proteome Res. (2020) 19:2429–42. doi: 10.1021/acs.jproteome.0c00137). Thus, tau appears to be a multifunctional protein with many interaction partners, and pathological changes in its interactome could contribute significantly to disease development in AD and other tauopathies.

Strong support for a mechanism of action that targets the pathogenic subpopulation of tau

Intrinsically disordered regions highest in N terminal region and proline rich region, increased disorder through vertebrate evolution, MAP2 has no evolutionary trend

MBD

Association of MBD positive lysines with MT negative glutamate electrostatic interactions

Most phosphorylation sites are located in two regions flanking MBD

acetylation of lysines found in MBD neutralizes positive charge

MBD also interacts with heat shock proteins, tau itself, end-binding proteins (EB2), LRP1 (possible involvement in endocytosis)

NTD

aspartate and glutamate = acidic

associated proteins involved in plasma membrane binding and function, exo/endocytosis (annexins, synapsin, synaptotagmin, cynatogyrin) interaction can be phosphorylation dependent, membrane associated tau less phosphorylated than cytosolic tau

also signaling molecules, GSK3beta, 14-3-3 proteins (also PRD, MBD)

PRD

phosphorylation in this domain decreases MT association and PM association

Pathology

move to somatodendritic compartment, enriched in membrane enriched fraction in AD samples