Skin tau-SAA can specifically differentiate AD and non-AD tauopathies from normal controls and PiD cases using 4RCF as the substrate

In contrast to PrP in PrD and α-synuclein in PD, the tau molecule in the human brain exhibits 6 isoforms, of which there are 3 isoforms with 4 microtubule-binding repeats (4R tau) and 3 with three repeats (3R tau), resulting from alternative mRNA splicing. In order to find an appropriate tau substrate for SAA of skin tau (sTau-SAA) with RT-QuIC, based on our recent finding with autopsy brain tissues from cadavers with AD and other non-AD tauopathies [20], we first examined 4 types of tau substrates including 2 full-length tau isoforms (2N3R/2N4R) and 2 truncated tau fragments consisting of 4RCF (cysteine-free, equivalent to K18CFh)/3RCF (equivalent to K19CFh) using autopsy skin samples from AD cadavers diagnosed neuropathologically. 4RCF represents the aggregation-prone core sequences of the 4R tau isoforms while 3RCF fragment is the aggregation-prone core sequences of the 3R tau isoforms [20, 21]. The only difference between 4RCF and 3RCF is that 3RCF lacks the R2 region. Compared to the full-length tau, the truncated tau substrates-based RT-QuIC revealed higher endpoint fluorescence readings (exceeding 100,000 RFU) and shorter lag phases (less than 25 h) (Fig. S1). As a result, we used the two truncated tau fragments as the substrates for the following examinations in our study.

Using RT-QuIC with 4RCF truncated tau as the substrate, we analyzed autopsy skin samples from AD (n = 46), CBD (n = 5), PSP (n = 33), PiD (n = 6), and NC (n = 43). 4RCF-based RT-QuIC of skin sTau-SA exhibited that CBD cases had the highest ThT fluorescence intensity, followed by AD, PSP, and PiD (Fig. 1A, B). We also compared lag phases of sTau-SA in each type of tauopathy, the time period between the beginning of the RT-QuIC measurement and the time point at which the curves reflecting the ThT fluorescence started to hit the half of the maximum of ThT fluorescence. Consistent with the endpoint ThT values, CBD displayed the shortest lag phase, while PiD was the longest (Fig. 1C). Our 4RCF-based RT-QuIC assay of the skin samples from AD and non-AD tauopathies yielded a sensitivity of 80.49% and a specificity of 95.35%. To quantitate the sTau-SA of each type of tauopathy, we conducted endpoint titration of RT-QuIC assay of each skin sample from different tauopathies. The x-axis represents SD50 that referred to the 50% seeding activity. PSP samples exhibited the highest sTau-SA, followed by AD samples. In contrast, PiD skin samples displayed markedly lower tau-SA, as shown in Fig. 1D. This observed variation is likely attributable to the incompatibility between seed and substrate, particularly noting that PiD-derived misfolded tau is mainly comprised of 3R tau. AUC analysis revealed an area value of 0.88 based on the comparison between AD and normal controls and 0.79 based on the comparison between tauopathies and normal controls (Fig. 1E, F).

Fig. 1

Tau-seeding activity of skin samples from patients with tauopathies using 4RCF-based RT-QuIC. A Kinetic curves displaying the mean and standard deviation (SD) of tau-SA over time of skin samples from CBD (n = 5), AD (n = 46), PSP (n = 33), PiD (n = 6) and NC (n = 43). B Scatter plot illustrating the distribution of tau-SA across different tauopathies detected in panel A. C Lag phase, defined as the initial period before a significant increase in the ThT fluorescence intensity. D The end-point dilution analysis of quantitative tau-SA of skin samples from tauopathies. The half of maximal SA (SD50) was determined by Spearman-Kärber analyses and is shown as log SD50/mg skin tissue. E Receiver operating characteristic (ROC) curve analysis comparing tau-SA of skin samples between AD and control subjects, with an area under the curve (AUC) of 0.82. F ROC curve analysis comparing tau-SA of skin samples between total tauopathy and control subjects, with an AUC of 0.79. ns: p > 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001

The sTau-SAA of AD and all other tauopathies can be specifically differentiated from that of normal controls using 3RCF as the substrate

We next used 3RCF (K19)-based RT-QuIC assays to examine the same samples detected in 4RCF studies. The sTau-SA was significantly higher in tauopathies than in non-tauopathies, of which PiD was the highest, followed by AD, PSP, and CBD (Fig. 2A, B). We also compared their lag phases, which were inversely proportional to those of 4R tau: PiD exhibited the shortest lag phase, while CBD showed the longest lag phase (Fig. 2C). PiD and PSP demonstrated the highest seeding dose while CBD was lowest (Fig. 2D). In addition, the 3RCF-based skin tau RT-QuIC assay generated a sensitivity of 75% and a specificity of 100%, which was similar to that of 4RCF-based RT-QuIC in general. The AUC values (0.85 vs. 0.79) of 3RCF-based RT-QuIC assay of skin tau were similar to those detected with 4RCF-based RT-QuIC shown above (Fig. 2E, F). Notably, in contrast to the 4RCF-based sTau-SA, 3RCF-based sTau-SA from PiD was the highest, in addition to differentiating other tauopathies from the normal controls.

Fig. 2

Tau-seeding activity of skin samples from patients with tauopathies using 3RCF-based RT-QuIC assay. A Kinetic curves displaying the mean and SD of tau-SA over time of skin samples from CBD (n = 5), AD (n = 46), PSP (n = 33), PiD (n = 6) and NC (n = 43). B Scatter plot illustrating the distribution of tau-SA across different tauopathies. C Comparison of lag phases of skin tau-SAA from different tauopathies and NC, same as above, as the initial delay before the ThT fluorescence intensity begins to rise. D The end-point dilution analysis of quantitative tau-SA of skin samples from various tauopathies. The half of maximal SA (SD50) determined by Spearman-Kärber analyses is shown as log SD50/mg skin tissue. E ROC curve analysis comparing skin tau-SA between AD and control subjects, with an AUC of 0.77. F ROC curve analysis comparing skin tau-SA between total tauopathy and control subjects, with an AUC of 0.72. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001

Then we compared the tau-SA of brain and skin samples from AD using the two truncated tau fragments as substrates. Tau-SA was significantly lower in skin than in brain tissues either with 4RCF [skin (n = 20), 182,400.8 ± 60,353.3 (mean ± SD) vs. brain (n = 12), 252,170.1 ± 11,147.6; p = 0.0034 < 0.005] or with 3RCF as the substrate [skin (n = 20), 177,442.3 ± 62,953.4 vs. brain (n = 12), 215,374.9 ± 48,246.7; p = 0.0061 < 0.01] (Fig. S2A, B).

The correlation of tau-SA in the skin and brain of 8 cadavers was analyzed, from whom we obtained both brain and skin tissues of the same individuals for RT-QuIC assays. There was a trend of a positive correlation of tau-SA between skin and brain tissues with both 4RCF and 3RCF substrates although they were not statistically significant (p > 0.05) (Fig. S2C-F).

To determine whether there is a difference in tau between brain and skin tissues, we examined the phosphorylated tau of brain and skin tissues from AD cadavers by western blotting with pS396 and pT231 antibodies against phosphorylated tau. There were significant differences in the levels and gel profiles of phosphorylated tau between the two tissues (Fig. S3A, B). We also examined the ratio of the skin 3R/4R tau isoforms by western blotting with antibodies specifically directed against 3R (RD3) and 4R (RD4) antibodies (Fig. S3C, D). By densitometry of protein bands on western blots, the ratio of 3R/4R tau in the skin was 1.3:1, which is consistent with that reported in the brain tissues of AD [22]. We performed a quantitative analysis of AD brain and skin samples from tauopathies based on the Western blot, as presented in Fig. S3E (pT231) and Fig. S3F (pS396). Our findings show that the pS396 level in the brain is approximately 160 times higher than in the skin of tauopathy patients, while the pT231 level in the brain is around 60 times higher than in the skin.

We also conducted the western blotting of skin samples from AD and other tauopathies with the anti-tau antibody HT7 (Fig. S4). Notably, HT7 detects tau bands with high molecular weights including 100 kDa and higher close to 250 kDa, greater than those of typical monomeric tau proteins. They could be the dimers or oligomers based on their molecular weights. Meanwhile, HT7 also detects small tau fragments migrating between 12 and 35 kDa, whose molecular weights are smaller than those of any individual monomers of 6 isoforms (Fig. S4). Based on their smaller molecular weights, they are most likely the truncated forms of tau isoforms.

Given that the tau-SA was not significantly different among some of tauopathies, the specificity of the amplification reaction may not be certain. To determine whether the tau-SA is indeed specifically from misfolded tau isoforms, we used tau immunodepletion (ID) by immunoprecipitation of tau with an anti-tau antibody from skin samples prior to RT-QuIC assay. We revealed that tau-SA was significantly decreased in the samples subjected to ID compared to the samples without ID [41,285 ± 20,657 (AD_ID skin, mean ± SD) vs. 149,573 ± 61,571 (AD skin), p < 0.0001] (Fig. S5), confirming the specificity of tau-SA by RT-QuIC.

The sTau-SA is significantly elevated over an increase in the Braak staging

Based on Aβ/tau-pathology and Aβ/tau-positron emission tomography (PET) scan, the accumulation of Aβ/tau leading to clinical AD is a continuum process. To determine whether the skin tau-SA can reflect the severity or Braak staging in the AD brain, next we associated skin tau-SA with the Braak staging in autopsy brain tissues examined. Notably, when sTau-SA with both 4RCF and 3RCF served as a function of Braak staging, there was a clear association observed: the sTau-SA was significantly elevated upon an increase in the Braak staging (Fig. 3). Specifically, the overall ThT intensity for 4RCF-based skin tau-SA was increased following advancing of the Braak stages, although no significant difference was noted between stages IV and V (Fig. 3A). A similar trend was observed for 3RCF, where the overall ThT intensity increased with the progression of Braak stage (Fig. 3B), yet no significant difference was found between stages V and VI.

Fig. 3

Correlation of tau-SA of skin samples from AD with their different Braak stages. Scatter plot of ThT fluorescence readings at end-points detected by RT-QuIC assays with the substrate 4RCF (A) or 3RCF (B) as a function of different Braak stages [IV (n = 20), V (n = 6) and VI (n = 20)]. Scatter plot of Braak stages with LogSD50/mg of skin tau-SA detected by RT-QuIC with the substrate 4RCF (C) or 3RCF (D), ns: p > 0.05; ** p < 0.01; ****p < 0.0001

We also explored whether variables such as age, gender, and post-mortem interval (PMI) could influence Tau-SA. We observed no significant differences between males and females in both 4RCF (Fig. S6A, B) and 3RCF (Fig. S6C, D) assays. In the correlation analysis between ThT fluorescence intensity and age/PMI of the autopsied skin tissues, no correlation was found for 4RCF with either age (Fig. S6E) (r = 0.07247, p = 0.5047 > 0.05) or PMI (Fig. S6F) (r = -0.02187, p = 0.8406 > 0.05). For 3RCF, a slight positive correlation was observed between age and Tau-SA end-point ThT fluorescence, though this was not statistically significant (Fig. S6G) (r = 0.1042, p = 0.3366 > 0.05); similarly, a slight negative correlation was noted between PMI and ThT fluorescence (r = -0.1225, p = 0.2583 > 0.05) (Fig. S6H).

The sTau-SA is significantly higher in PD and dementia with Lewy bodies than in multiple system atrophy and normal controls but it is still lower than that in AD

Accumulation of tau aggregates in the brain has been observed in some of cases with synucleinopathies including PD, dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) [23,24,25,26,27]. Next, we further explored whether tau-SA can be detected in the skin of synucleinopathies by our sTau-SAA. Autopsy skin samples from AD (n = 21), PD (n = 10), MSA (n = 6), DLB (n = 6), and NC (n = 17) were examined by 4RCF-based tau-SAA. The ThT endpoint fluorescence intensity of sTau-SAA was dramatically higher in AD than in synucleinopathies, whereas the skin-tau fluorescence intensity was also significantly increased in synucleinopathies except for MSA than in the control group (Fig. 4), consistent with the previous observations that some of cases with synucleinopathies can have tau-pathology [23, 25,26,27]. Notably, 4 out of 6 DLB samples also had coexisting AD pathology as indicated in Table 1. In MSA samples, none of these cases had co-morbidities with confirmed tauopathies. However, tau tangles and Aβ plaques were detectable in their brain tissues (Table 1). Ten PD cadavers showed no AD pathology (Table 1) but they had significantly higher skin tau-SA compared to the normal controls (Fig. 4).

Fig. 4

Tau-SAA of skin samples from participants with AD, synucleinopathies, and NC using 4RCF-based RT-QuIC assay. Scatter plot illustrating the distribution of tau-SA at the endpoint fluorescence readings across skin samples from 21 cases with AD, different synucleinopathies including 6 cases with DLB, 6 with MSA and 10 with PD as well as 17 NCs. *: p < 0.05, **: p < 0.01, ***: p < 0.001; ****: p < 0.0001

Table 1 Demographic and neuropathological features of autopsied cases in different groupsBiopsy sTau-SA is significantly higher in tauopathies than in normal controls

We then used the above 4RCF- or 3RCF-based SAA (4RCF- or 3RCF-SAA) to examine biopsied skin samples from AD (n = 16), PSP (n = 8) and NCs (n = 10). Skin 4RCF-SA was significantly higher in AD than in normal controls [83,091 ± 57,912 (mean ± SD) vs. 20,490 ± 9,307, p = 0.0026 < 0.005]; skin 4RCF-SA was also significantly greater in PSP than in normal controls (73,360 ± 49,625 vs. 20,490 ± 9,307, p = 0.0043 < 0.005) (Fig. 5A). Similar to skin 4RCF-SA, 3RCF-SA was significantly higher in AD than in NCs (97,511 ± 54,115 vs. 30,090 ± 13,657, p = 0.0008 < 0.001) and greater in PSP than in NCs (60,449 ± 20,492 vs. 30,090 ± 13,657, p = 0.0017 < 0.005) (Fig. 5B). There were no significant differences in sTau-SA between AD and PSP cases with both substrates (Fig. 5). We also conducted a correlation analysis between tau seeding amplification assay (SAA) results and cognitive decline, as measured by the Mini-Mental State Examination (MMSE). Our findings indicate an inverse correlation between both 4R and 3R tau SAA and MMSE scores. Specifically, 4R tau-SA demonstrated an inverse correlation with MMSE (r = -0.7026, p = 0.002; Fig. 5C), while 3R tau-SA showed a milder, yet statistically significant, inverse correlation with MMSE (r = -0.5073, p = 0.0135; Fig. 5D). This observation implied the potential for sTau-SA to serve as an antemortem diagnostic biomarker to differentiate tauopathies from normal controls. The individual clinical data are listed in Tables 2 and 3.

Fig. 5

Examination of tau-SA in biopsied skin samples from patients with AD and PSP. The scatter plot displays the endpoint ThT fluorescence percentage of skin tau-SA from AD (n = 16), PSP (n = 8), and NC (n = 10) detected by RT-QuIC using 4RCF (A) and 3RCF (B) as the substrates. Correlation analysis between MMSE and skin tau RT-QuIC seeding activity is shown in C (4RCF) and D (3RCF) **: p < 0.01; ***: p < 0.001

Table 2 Demographic and clinical features of biopsied cases in different groups Table 3 Demographic and clinical features of individual biopsied cases in different groupsThT fluorescence levels of sTau-SAA end-point correlate with dot-blot intensity of captured tau aggregates of the RT-QuIC end products

To determine whether ThT fluorescence levels reflecting sTau-SA represent the formation of skin tau-seeded aggregates, we correlated the end-point ThT fluorescence levels with the dot-blot intensity of tau aggregates captured by a filter-trap assay (FTA) (Fig. 6). After obtaining the end point ThT fluorescence levels of sTau-SA of 4 cases each from AD, PSP, CBD, PiD and NC with 4RCF or 3RCF as the substrate (Fig. 6A, B), we then ran FTA with their corresponding end products, followed by probing the dot-blots with anti-4R tau antibody (RD4) (Fig. 6C) and anti-3R antibody (RD3) (Fig. 6D). The semiquantitative densitometric scanning of protein dot intensity on the dot-blots revealed that similar to ThT fluorescence levels, the intensity of tau aggregates captured by FTA on the blots was significantly higher in tauopathies than in normal controls by both RD4 (Fig. 6E) and RD3 antibodies (Fig. 6F). Correlation analyses demonstrated that the intensity of the trapped aggregates from the end products correlated positively with the ThT fluorescence levels (r = 0.87 for 4R, p < 0.001; r = 0.68 for 3R, p < 0.009) (Fig. 6G, H).

Fig. 6

Characterization of RT-QuIC end products of skin tau from tauopathies using filter-trap assay probed with anti-3R (RD3) and anti-4R (RD4) tau antibodies. Scatter plots of ThT fluorescence values skin-tau RT-QuIC end products of selected samples from PSP (n = 4), AD (n = 4), CBD (n = 4), PiD (n = 4) and NC (n = 4), with 4RCF (A) and 3RCF(B) as the substrates. The filter-trap assay (FTA) of 3RCF-/4RCF-based RT-QuIC end products of skin samples from different tauopathies including PSP, AD, CBD, PiD and NC (4 cases/group) probed with RD4 (C) or RD3 (D) antibodies. Quantitative densitometry of FTA-dot blotting with 4RCF- (E) and 3RCF (F) -based RT-QuIC end products from panels (C) and (D). Correlation analysis between FTA-trapped protein dot intensity and skin tau-SA of 4RCF- (G)/3RCF(H) -based RT-QuIC end products

Transmission electron microscopy of 4RCF- or 3RCF-based RT-QuIC end products displays the protofibril-like structures

While our FTA apparently was able to detect captured tau aggregates, to further determine the morphology of the amplified skin tau aggregates, we performed transmission electron microscopy (TEM) of the RT-QuIC end-products of skin misfolded tau from 3 cases each of AD, PSP, CBD, PiD and NCs with either 4RCF or 3RCF as the substrate (Fig. 7). For 4RCF, TEM revealed that except for PiD and NCs (Fig. 7A, E), other end-products had a small number of protofibril-like structures. For 3RCF, while NCs showed only oligomer-like structure (Fig. 7F), protofibrils were detectable in the end-products of tau RT-QuIC of cases with all tauopathies by TEM (Fig. 7G and J).

Fig. 7

Transmission electron microscopy of SAA end products of skin misfolded tau from AD, other tauopathies and normal controls. Panels (A) through (E) show the representative images of tau 4RCF-based RT-QuIC end-products with normal controls (NC, A), AD (B), PSP (C), CBD (D) and PiD (E). Panels (F) through (J) exhibit the representative images of tau 3RCF-based RT-QuIC end-products with NC (F), AD (G), PSP (H), CBD (I) and PiD (J). Scale bars: 200 nm

The end-products of 4RCF- and 3RCF-SAA exhibit different patterns of resistance to proteinase K digestion

The pattern of protein aggregates from the skin tau RT-QuIC end-products to proteinase K (PK) digestion has been widely believed to reflect the conformational properties of misfolded proteins examined. Since the small fragments of 3RCF tau lower than 7 kDa were more difficult to digest by PK, we next performed the titration of varied PK concentrations ranging from 0, 1.25 µg/mL, 2.5 µg/mL, 3.75 µg/mL, 5 µg/mL, 7.5 µg/mL, to 10 µg/mL for the 4R (Fig. 8A) and 0, 1.25 µg/mL, 5 µg/mL, 10 µg/mL, 12.5 µg/mL and 25 µg/ml for the 3R tau (Fig. 8B) SAA end-products of skin tau from AD and non-AD subjects. Without PK treatment, the end-products of 4RCF-based RT-QuIC of skin tau from non-AD exhibited 3 protein bands by the RD4 tau antibody, migrating at approximately 25–26 kDa, 12–14 kDa, and 7–10 kDa (Fig. 8A). The short exposure of our blots showed that the 7–10 kDa bands actually consisted of 7 kDa and 10 kDa proteins. As a result, the above four bands could represent the trimer and dimer of a 7 kDa band as well as the monomers of a full-length 4RCF (~ 12 kDa) and a truncated 4RCF (~ 7–10 kDa), respectively. Of them, the two lower monomeric bands were predominant, whereas the top dimeric and trimeric tau bands were underrepresented, accounting for less than 1–2% of total tau (Fig. 8A, C). In contrast, the end-product from AD skin samples without PK-treatment exhibited an additional band migrating at approximately 48–50 kDa in addition to the four bands found in the end-product of non-AD skin described above. This band could be oligomers of full-length or truncated tau molecules. In addition, the intensity of truncated trimers and dimer of tau migrating at 24–27 kDa was significantly increased compared to that of non-AD end-products.

Fig. 8

PK-resistance and conformational stability assay of the end products of 4RCF-/3RCF-based tau RT-QuIC assay of skin samples from AD and controls. A-C Western blotting of PK titration of 4RCF-based SAA end-products of AD and non-AD skin samples and quantitative analysis of intensity of PK-resistant tau fragments with different molecular weights by densitometry. D-F: Western blotting of PK titration of 3RCF-based SAA end products of AD and non-AD skin samples and quantitative analysis of intensity of PK-resistant tau fragments with different molecular weights by densitometry. Probed with RD3 and RD4 antibodies against 3R or 4R tau isoforms, respectively. The 4RCF-(G)/3RCF(J)-based RT-QuIC end products of AD and non-AD skin samples were treated with different concentrations of GdnHCl and followed by PK digestion prior to western blotting probed with anti-tau antibodies RD4 against 4R (G) and RD3 against 3R (J) tau fragments. Quantitative analysis of GdnHCl/PK-resistant protein band intensity with different molecular weights by densitometry on blots (G and J). H and I for 4R tau; K and L: for 3R tau

Upon PK-treatment, for the non-AD end-products, the intensity of the two lower monomeric tau bands was significantly decreased while they were still detected at PK of 10 µg/mL (Fig. 8A, B). The intensity of the tau band migrating at ~ 25–27 kDa was increased first up to PK of 2.5 µg/mL and then decreased, until became undetectable at PK of 10 µg/mL. The band migrating at ~ 12 kDa seemed to be completely PK-sensitive and no band was detectable even at the lowest PK concentration at 1.25 µg/mL. In contrast, after PK-treatment the end-products of the RT-QuIC with AD skin samples showed the decreased intensity of the tau band migrating at ~ 25–27 kDa but generated additional smaller band migrating at about 24 kDa (Fig. 8A, C, red arrow). This band was most likely derived from the truncation of the 25–27 kDa band since it was generated and enhanced over the increase in the PK concentration. In contrast with the non-AD end-products, AD end-products also exhibited an additional band between 7 kDa and 10 kDa migrating at about 8 kDa in the PK-treated AD skin en- products (marked with the red arrow in Fig. 8A). The intensity of this band was similar to that of 7 kDa and 10 kDa bands, and showed no changes upon the increase in the PK concentration (Fig. 8A, C).

Regarding the end-products of sTau-SAA using 3RCF as the substrate, without PK-treatment, the AD and non-AD samples all mainly exhibited 3 bands migrating at about 7 kDa, 10–11 kDa, and 22–23 kDa on the gels (Fig. 8D). According to the sequence of the 3RCF molecule, the molecular weight of the monomeric 3RCF should be 10.5 kDa. Therefore, the 7 kDa band could be a truncated fragment of 3RCF while 22–23 kDa band could be a dimer of 3RCF. Since we got high intensity of the low molecular weight band (~ 7 kDa), we increased the PK concentration to 25 µg/mL for 3R and decreased the loading amounts of samples (Fig. 8D, E). The intensity of the 3 bands from non-AD end-products all decreased while there was a faint band emerging, migrating at approximately 5 kDa over the increase in PK concentrations. The intensity of the 3 bands from AD skin tau RT-QuIC end-products was also all decreased while there were two additional bands emerging, migrating at approximately 16–18 kDa and 5–6 kDa over the increase in PK concentrations (Fig. 8D, F, red arrows). The monomers of truncated 4RCF (at ~ 12 kDa) and 3RCF (at ~ 10–11 kDa) exhibited no resistance to PK treatment (Fig. 8A, D). However, the dimers at higher molecular weights demonstrated increased resistance to PK, with the exception of the 4R negative end-product dimers, which were digested at PK concentrations exceeding 7.5 µg/mL. The low molecular weight bands below the monomers were more resistant to being digested within the chosen PK concentration range, especially with 4RCF.

Conformational-stability assay of skin tau aggregates amplified by tau-SAA

We treated 4RCF (Fig. 8G) and 3RCF (Fig. 8J) tau-SAA end-products with GdnHCl ranging from 0 to 3.2 M, followed by PK digestion at 10 µg/mL and quantitative analyses of GdnHCl/PK-resistant protein intensity of each treated sample. This approach is grounded on the principle that subtle differences in protein structure can be ascertained by assessing conformational stability when the protein is exposed to a denaturant such as GdnHCl at appropriate concentration ranges [28]. In the absence of GdnHCl and PK, 3 tau bands migrating at 48 kDa, 25 kDa and 7–12 kDa were observed for 4RCF-based RT-QuIC end-products, while 2 bands migrating at 22–23 kDa and 5–10 kDa were detected in 3RCF-based RT-QuIC end-products (Fig. 8G, J). In contrast, both AD skin 4RCF-/3RCF-based tau RT-QuIC end-products were found to have multiple or smear bands above 30 kDa (Fig. 8G, J). After GdnHCl and PK-treatment, there were virtually no PK-resistant tau bands detectable from both non-AD 4RCF/3RCF-based RT-QuIC end products. In contrast, positive skin tau RT-QuIC from AD participants with either 4RCF or 3RCF as the substrate showed PK-resistant tau fragments, especially for bands migrating at 25 kDa or lower for low concentration of GdnHCl (Fig. 8G through L). Notably, there was an additional partially PK-resistant tau fragment migrating between 7 kDa and 5 kDa bands for 3RCF-based RT-QuIC end-products (Fig. 8J, L), which was not detectable in the 4RCF-based skin tau RT-QuIC end-products (Fig. 8G, I). The GdnHCl concentrations required to make half of the tau end-product sensitive to PK, referred to as GdnHCl1/2, were 2.6 M for positive 4R tau and 2.3 M for positive 3R tau for bands migrating at 22–25 kDa and 10 kDa, indicating that the positive 4R tau end-products were approximately 1.13-fold more stable than the 3R tau end product. But, the 3RCF-based RT-QuIC end products from AD cases generated stable 7 kDa band (Fig. 8J, L).