Under Heparin-Free Conditions Unsaturated Phospholipids Inhibit the Aggregation of 1N4R and 2N4R Tau

A progressive aggregation of Tau proteins in the brain is linked to both Alzheimer’s disease (AD) and various Tauopathies. This pathological process can be enhanced by several substances, including heparin. However, very little if anything is known about molecules that can inhibit the aggregation of Tau isoforms. In this study, we examined the effect of phosphatidylserines (PSs) with various lengths and saturations of fatty acids (FAs) on the aggregation properties of Tau isoforms with one (1N4R) and two (2N4R) N-terminal inserts that enhance binding of Tau to tubulin. We found that PS with unsaturated and short-length FAs inhibited Tau aggregation and drastically lowered the toxicity of Tau oligomers that were formed in the presence of such phospholipids. Such an effect was not observed for PS with fully saturated long-chain FAs. These results suggest that a short-chain irreversible disbalance between saturated and unsaturated lipids in the brain could be the trigger of Tau aggregation.

2N4R and 1N4R Tau aggregation: In a lipid-free environment, 30 μM of protein was in PBS, and the solution pH was adjusted to pH 7.4.For the aggregation study of 2N4R and 1N4R Tau in the presence of phosphatidylserine (PS), 30 μM of protein was mixed with a (1:5) ratio of the concentration of the corresponding large unilamellar vesicles (LUVs).The pH of the final solution was adjusted to pH 7.4 using concentrated HCl.Subsequently, the samples were dispensed into a 96-well plate, which was placed in a plate reader (Tecan, Männedorf, Switzerland) at 37°C for 150 h under 510 rpm agitation..

Kinetic measurements:
Rates of protein aggregation were measured using thioflavin T (ThT) fluorescence assay.For this, samples were mixed with 2 mM of ThT solution and placed into 96 well-plate that was kept in the plate reader (Tecan, Männedorf, Switzerland) at 37 °C for 150 h under 510 rpm agitation.Fluorescence measurements were taken every 10 min (excitation 450 nm; emission 488 nm).Each kinetic curve is an average of four independent measurements.

Atomic force microscopy (AFM) imaging:
We utilized an AIST-NT-HORIBA system (Edison, NJ) AFM setup for the morphological analysis of protein aggregates.Silicon tapping-mode AFM probes from Appnano (Mountain View, CA, USA) were employed, featuring a force constant of 2.7 N/m and a resonance frequency of 50-80 kHz.The probe's diameter was 10 nm.To prepare for AFM imaging, each sample aliquot was diluted with DI water and deposited onto pre-cleaned glass coverslips.After exposure for 20-30 minutes, excess solution was removed from the glass surface, and the coverslips were dried using a flow of dried nitrogen.Approximately 20-30 individual aggregates were measured for each sample.Postacquisition processing of the AFM images was performed using AIST-NT software (Edison, NJ, USA).We report their heights only because height measurements are absolute (artifact-free) in AFM, whereas widths have tip-convolution error and, therefore, should not be reported because they do not provide accurate information about the width of aggregates.

Attenuated total reflectance Fourier-transform Infrared (ATR-FTIR) spectroscopy:
After 150 h of incubation at 37 °C, 2N4R and 1N4R samples were placed onto ATR crystal of 100 FTIR spectrometer (Perkin-Elmer, Waltham, MA, USA) and dried at room temperature.Three spectra were collected from each sample.

Atomic force microscopy-infrared spectroscopy (AFM-IR):
Imaging and spectral analysis were obtained using a nanoIR3 system (Bruker, Santa Barbara, CA, USA), equipped with a QCL laser.ContGB-G AFM probes specific to contact mode were utilized with probe parameters: frequency of 13 kHZ, spring constant of 0.2 N/m, and a length of 450 µm.The tip was optimized using a polymethyl acrylate standard for the wavenumbers: 1400-1800 cm -1 .Laser parameters include a power of 25.49%, polarization at 90 degrees, IR focus of 70456, and a pulse rate around 828 kHz.Image dimensions of 1-10 µm were acquired at a scan rate of 0.5-0.8Hz, an I and P gain ranging from 1/2 to 5/10, and a resolution of 256 for both the X and Y.A total of 30 spectra per sample were obtained with a co-average of 3 for each spectrum at a spectral resolution of 2 cm -1 /pt.In each sample, ~10 individual aggregates were analyzed.Protein samples were first deposited onto a 70 nm gold-coated silicon wafer at a volume of 2.5 µL, left to dry at room temperature for roughly 15 minutes or until a visible coffee ring under the drop is present, then rinsed with distilled water and dried using a N 2 air flow.The spectra were zapped at the 1648-1652 points to remove an artifact caused by the chip-to-chip transition of the laser at this region.Spectral processing was conducted using MATLAB, equipped with a PLS Toolbox version 9.0 (Eigenvector Research, Inc., Manson, WA).Spectra are first applied a smoothing processing of Savitzky-Golay at a polynomial order of 0, area normalized, and baselined with automated weighted least squares.After processing, spectra are then peak fitted using GRAMS/AI Spectroscopy Software to assign peaks respective to the secondary structure of the protein.Estimated area for each secondary structure is then run under ANOVA by first applying an Anderson-Darling Test to determine normal distribution, if so, a one-way ANOVA with Tukey HSD posthoc test is run to determine statistical significance.
Cell toxicity assays: Rat midbrain dopaminergic N27 cells were cultured in RPMI 1640 Medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Waltham, MA, USA) in 96-well plates at a seeding density of 10,000 cells per well.The cells were then incubated at 37°C in a humidified atmosphere with 5% CO 2 .After 24 hours, the cells had adhered fully to the culture surface.
Subsequently, 100 μL of the cell culture medium was replaced with 100 μL of RPMI 1640 Medium containing 5% FBS and supplemented with 10 μL of 2N4R and 1N4R protein aggregates.Following 24 hours of incubation with the protein aggregates, the toxicity of the aggregates was assessed using a lactate dehydrogenase (LDH) assay kit (G1781, Promega, Madison, WI, USA).Absorbance measurements were taken at 490 nm using a plate reader (Tecan, Männedorf, Switzerland).

Figure S1 .
Figure S1.CD spectra acquired from 1N4R Tau (A) and 2N4R Tau (B) fibrils grown in the lipid-free environment and in the presence of lipids.
Figure S2.Representative AFM images of 1N4R aggregates (left panels) AFM images with sites of AFM-IR spectral acquisition (blue dots) (right panels).