Uncovering elevated tau TPP motif phosphorylation in the brain of Alzheimer's disease patients

Abstract

INTRODUCTION

A wide array of post‐translational modifications of the tau protein occurs in Alzheimer's disease (AD) and they are critical to pathogenesis and biomarker development. Several promising tau markers, pT181, pT217, and pT231, rely on increased phosphorylation within a common molecular motif threonine‐proline‐proline (TPP).

METHODS

We validated new and existing antibodies against pT217, pT231, pT175, and pT181, then combined immunohistochemistry (IHC) and immunoassays (ELISA) to broadly examine the phosphorylation of the tau TPP motif in AD brains.

RESULTS

The tau burden, as examined by IHC and ELISA, correlates to Braak stages across all TPP sites. Moreover, we observed regional variability across four TPP motif phosphorylation sites in multiple brains of sporadic AD patients.

DISCUSSION

We conclude that there is an elevation of TPP tau phosphorylation in AD brains as disease advances. The regional variability of pTPP tau suggests that examining different phosphorylation sites is essential for a comprehensive assessment of tau pathology.

1. INTRODUCTION

The diversity of pathological tau species, differential solubility and molecular weight of tau aggregates, the abundance of tau protein, and six splice variants engender substantial technical and conceptual challenges to comprehensively studying tau across individuals and disease states. ¹ The diversity of tau species is driven by various post‐translational modifications (PTMs) that are seen under physiological and pathological conditions, including truncation, acetylation, ubiquitination, and phosphorylation. ² These tau PTMs vary substantially across disease states, and quantifying tau PTMs forms the basis of several existing and emerging biomarkers for Alzheimer's disease (AD). pT181, pT217, and pT231 tau are being increasingly studied as valuable measures of AD pathology and predictors of current and future cognitive decline, highlighting the potential for tau PTM‐based biomarkers in the clinic. ³ , ⁴ , ⁵ Of note, a common molecular motif is found across the tau phosphorylation sites at amino acids (aa) threonine 181, 217, and 231, as these are all phosphorylated within a short stretch containing threonine followed by two prolines (pTPP). Within the longest tau isoform in the central nervous system (aa 1‐441), four sites are phosphorylated within the TPP motif, which are pT175, pT181, pT217, and pT231. These phospho‐threonine sites have been heavily studied in brain tissue and biofluids, and blood and CSF measures of pT181 and 217 are among the most promising biomarkers available for AD. However, despite the intense study of these tau phosphorylation sites, it remains unclear whether phosphorylation on one of these sites is more closely tied to the pathological progression of AD and whether unique information is provided with respect to disease progression by one site over others. Consideration of this question has been difficult due to the incomplete specificity of commonly used antibodies to specifically bind one phospho‐epitope over another.

Employing a set of antibodies that exhibit strong phosphorylation site specificity, we investigated the extent to which tau pT175, pT181, pT217, and pT231, all sharing a common pTPP motif, may differ across the spectrum of AD tau pathology. To do this, we examined brain sections and tissue using a combination of immunohistochemistry (IHC) and serial ELISAs, which were reported as the most sensitive. ³ , ⁴ We observed that specific antibodies for each pTPP phospho‐epitope robustly recognized tau neurofibrillary tangles (NFTs) at different stages of maturation and that, except for some regional variations, pTau175, 181, 217, and 231 all showed similar relationships to Braak stage. The findings of this study indicate that there is a widespread increase in pTPP tau levels in the human brain as AD pathology advances.

2. MATERIAL AND METHODS

2.1. Generation of human Tau‐441 expression vectors

The coding sequence of the wild type human Tau‐441 isoform was synthesized by Integrated DNA Technologies (IDT, Coralville, IA) and subcloned into a pcDNA3.1 vector, which was further used to generate vectors expressing specific alanine mutations. Polymerase chain reaction (PCR) amplified the template into two DNA fragments with an overlapping sequence containing the target locus to introduce mutations. Overlap PCR was done to generate the whole open‐reading frame containing the alanine mutation. PCR products were subcloned into pcDNA3.1 vector for expression under a human cytomegalovirus promoter. Vectors were sequenced from 5′ and 3′ ends to confirm successful mutagenesis.

RESEARCH IN CONTEXT

1. Systematic review: Extensive research has focused on tau phosphorylation as a key feature of Alzheimer's disease (AD) pathology. A shared molecular motif (threonine‐proline‐proline, TPP) is identified at disease‐related tau phosphorylation sites, threonine 175, 181, 217, and 231. The question remains as to whether the phosphorylation of one of these sites is more closely associated with the progression of AD pathology and whether any site offers distinctive insights into disease advancement.

2. Interpretation: All four pTPP tau examined in this study elevate to a similar extent in cortical regions as AD advances. With limited exceptions, both histochemical and biochemical data appear to correlate within and between the phosphorylation sites examined, suggesting that they convey similar information regarding pathological tau burden and disease progression.

3. Future directions: Given the presence of regional variations in different pTPP tau forms, a comprehensive assessment of regional tau pathology may necessitate the examination of multiple phosphorylation sites.

2.2. Tissue culture and transfection of adherent cells

Adherent human embryonic kidney 293 (HEK) cells were cultured in complete growth medium: Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), two mM L‐glutamine, 10 units/mL penicillin, and 10 μg /mL streptomycin. Adherent HEK cells were seeded in 24‐well dishes at a density of 5 × 10⁵ cells per well for transfection. Transfection was carried out with jetPrime reagent. Cells were incubated for 48 h before the lysates were harvested for ELISA and western blot (WB).

2.3. Antibodies

BT‐2 (RRID: AB_10975238), AT8 (RRID: AB_223647), and AT‐270 (pT181) (RRID: AB_223651) were from Thermo Fisher Scientific, Tau‐13 (RRID: AB_291452) was from Biolegend, pT175 (Cat: MM‐0147‐P) was from MÉDIMABS, and Cell Signaling Technology developed pT217 (E9Y4S), pT231 (E7O3C), and pTPP (D61C3).

2.4. Electrophoresis and western blot

Samples were loaded onto 4% to 12% Bis‐Tris gels (SurePAGE, Genscript) using MES‐SDS running buffer (Genscript), transferred to nitrocellulose membranes, and probed for various proteins using standard western blot (WB). Enhanced luminol‐based chemiluminescent substrate (Biolegend) detected the resultant blots, and signals were captured by film.

2.5. Immunostaining of human brain sections

The Harvard Brain and Tissue Resource Center (HBTRC) provided autopsied brain sections. Formalin‐fixed, paraffin‐embedded sections (5 μm) of the entorhinal cortex (ERc) and dorsolateral prefrontal cortex BA9 (DLPFC) from 35 subjects covering the entire span of AD neuropathology from Braak stage 0 to VI were used in this part of the study. The demographic characteristics of the patients are shown in Table 1. The paraffin sections were rehydrated, and endogenous peroxidases were inactivated by treatment with 0.3% H₂O₂ in phosphate‐buffered saline (PBS) containing 0.03% Triton X‐100. The sections were incubated with primary antibodies at 4°C overnight and then with biotinylated secondary antibodies for 1 h. For visualization, sections were treated with avidin/biotin‐HRP complex (Vector) and then with 3,3′‐Diaminobenzidine substrate (Tokyo Chemical Industry Co., Ltd. Tokyo, Japan) with intensification by nickel ammonium sulfate. Photomicrographs were taken with an ECLIPSE E600 microscope (Nikon) equipped with a Mlchrome 5 Pro camera (Tucsen), and the brightness/contrast/threshold was adjusted with ImageJ 1.54f (NIH). Auto‐stitching tile scans were taken using the Mosaic 2 (Tucsen) live stitching function. Whole‐slide photos were taken with a well‐calibrated Perfection V850 Pro Photo Scanner (Epson).

TABLE 1.

Patient demographics in current study.

Cohort	Age (mean ± SD)	Sex (% male)	Neuropathology (N, %)
HBTRC ERc and BA9 (n = 35) paraffin sections	80.8 ± 8.4	54.2	Braak Stage 0 (1, 2.9) Braak Stage I (7, 20) Braak Stage II (6, 17.1) Braak Stage III (5, 14.3) Braak Stage IV (5, 14.3) Braak Stage V (9, 25.7) Braak Stage VI (2, 5.7)
HBTRC 16 different brain regions (n = 4) frozen tissue	79.5 ± 1.7	50	Braak Stage V (4, 100)
HBTRC Insular cortex (n = 67) frozen tissue	80.6 ± 9.8	52.2	Braak Stage 0 (4, 6) Braak Stage I (12, 17.9) Braak Stage II (7, 10.4) Braak Stage III (9, 13.4) Braak Stage IV (14, 20.9) Braak Stage V (17, 25.4) Braak Stage VI (4, 6)
HBTRC BA32 cortex (n = 42) frozen tissue	80.3 ± 11.1	50	Braak Stage 0 (1, 2.3) Braak Stage I (5, 11.9) Braak Stage III (9, 21.4) Braak Stage IV (12, 28.6) Braak Stage V (12, 28.6) Braak Stage VI (3, 7.1)

2.6. Densitometric analysis of IHC

For densitometric analysis of histological sections stained with AT8, pT231, pT217, pT181, and pT175 antibodies, three digital images were taken in the cortical regions of ERc (n = 34) and BA9 (n = 35) of patients at different Braak stages. Each picture represented an area of 1324 × 1108 μm (about 1.4 mm²) and was acquired as 2448 × 2048 pixels in 16‐bit TIFF format (65,536 levels of gray) using the same exposure time. Each image was opened using ImageJ 1.54f, and the mean gray value was measured. After averaging three picture values, statistical evaluation was performed using Brown‐Forsythe for ANOVA followed by a multiple‐comparisons corrected Dunnett's T3 post hoc test to determine the statistical significance of pairwise comparisons.

2.7. Human brain tissue extracts

The insular cortex and dorsal anterior cingulate cortex (BA32) were obtained from 67 and 42 HBTRC participants, respectively. Sixteen different regions, including BA4, BA8, BA17, BA40, insular cortex, EC, hippocampus, amygdala, caudate nucleus, putamen, nucleus accumbens, thalamus, substantia nigra, pons, cerebellum, dentate nucleus, were each obtained from four HBTRC donors, respectively. The demographic characteristics of the patients are shown in Table 1. Individuals were selected based on clinical and pathological diagnoses of AD across different Braak stages as quantified during regular neuropathological examination and age‐matched control patients. The gray matter was dissected and triturated in Tris‐buffered saline (TBS) by pipetting and centrifuged at 20,000 × g for 30 min to analyze tau levels in the supernatants of the brain extracts. The supernatants were collected as TBS‐soluble fractions and measured by multiple tau ELISAs. The TBS‐insoluble pellet was solubilized with 8 M urea and centrifuged at 20,000 × g for 30 min. The resultant supernatants were collected as the UREA‐soluble fractions and assayed by multiple tau ELISAs.

2.8. Tau ELISA using MesoScale Discovery (MSD) technology

HEK cell lysates and human brain extracts were diluted with 1% bovine serum albumin (BSA) in wash buffer (TBS supplemented with 0.03% Tween). For pT175, pT181, pT217, pT231, N‐terminal tau, AT8, and pTPP assays, each well of an uncoated 96‐well multi‐array plate (MesoScale Discovery) was coated with 30 μL of a PBS solution containing 1 μg/mL of Tau‐13 capture antibody to the N‐terminus region of Tau (epitope aa 15–25) and incubated at room temperature (RT) overnight. The aforementioned detection antibody solutions were prepared to have biotinylated monoclonal antibodies explicitly recognizing the phospho‐epitopes or general epitope (for N‐terminal tau assay), plus 100 ng/mL Streptavidin Sulfo‐TAG (MSD, No. R32AD‐5) and 1% BSA diluted in wash buffer. Following overnight incubation at RT, 50 μL/well of the sample, followed by 25 μL/well of detection antibody solution were incubated for 2 h at RT with shaking at 850 rpm and washing of wells with wash buffer between incubations. The plate was read and analyzed according to the MSD manufacturer's protocol. Full‐length tau was phosphorylated by GSK‐3β through their co‐expression in BL21 (DE3) Escherichia coli cells and purified for use as a calibrator for all assays.

2.9. Quantification and statistical analysis

All statistical analyses were conducted using GraphPad Prism 9. Z‐scores were calculated using the following formula: Z = (X−μ)/σ. For the IHC analysis, X represents the individual case's IHC intensity, μ represents the mean IHC intensity of the entire cohort (34 for ERc and 35 for BA9), and σ represents the standard deviation of the IHC intensity across the entire cohort. For the tau ELISA analysis of 16 different brain regions, X represents a specific type of tau analyte from a subject's specific region, μ represents for the mean of the specific type of tau quantification from that specific subject's 16 regions, and σ represents the standard deviation of the tau quantification in the same 16 regions. The correlation was assessed using both Spearman and Pearson correlation coefficients, with the corresponding ρ/r and p values displayed on the figure or in the text. A Supplementary Table containing all raw data and detailed statistical analyses is provided, and additional statistical information is included in the text and figure legends.

3. RESULTS

3.1. Validation of antibodies recognizing phosphorylation of the TPP motif of tau at threonine 175, 181, 217, and 231

To broadly examine the phosphorylation of the TPP motif of tau, we first validated the specificity of two novel recombinant antibodies developed against pT217 and pT231. We also examined the specificity of two commercially available antibodies against pT175 and pT181 (the widely used AT270 ⁶ ). To assess specificity for a particular phosphorylation site, we tested the binding of each antibody to a series of tau species, including both wild‐type tau and mutant forms of human tau modified to prevent phosphorylation at one of the four TPP sites under study. Specifically, plasmids expressing the longest form human tau (wild‐type Tau‐441) and its mutant forms in which the four TPP phosphorylation sites were mutated to alanine (T175A, T181A, T217A, and T231A Tau‐441) were each expressed in HEK293 cells, and the recognition of phospho‐tau species was assessed using each antibody. Analysis of cell lysates from transfected 293 cells by immunoblotting showed robust and equal production of each tau construct using non‐phosphorylation‐dependent antibodies against either the mid‐region of Tau‐441 (BT2: epitope = aa 194–198) (Figure 1C, left/top) or the N‐terminus (Tau13: epitope = aa 15–25) (Figure 1C, left/bottom). Detection of phosphorylated tau in the cell lysates was assessed using each of the four antibodies under validation (Figure 1C, right). First, we found that the endogenous kinases in HEK293 cells were sufficient to phosphorylate tau at all four sites. Second, compared to the negative control (empty vector transfection), we found that antibodies directed against tau phosphorylated at residues 175, 181, 217, and 231 failed to detect T175A, T181A, T217A, and T231A tau, respectively. This approach indicates that each antibody specifically recognizes the intended phosphorylation site and does not bind tau phosphorylated at other sites that share the same TPP motif.

FIGURE 1.

pTPP antibodies are highly specific and recognize NFTs in AD brains. (A–B) Schematic of tau protein sequence with antibody epitopes and pTPP motifs. (C) Lysates of HEK293 cells transfected with different tau constructs were blotted by indicated antibodies. (D) IHC of pT175, pT181, pT217, and pT231 on AD brain sections, showing NFTs at different maturation stages, scale bar = 5 μm. AD, Alzheimer's disease; IHC, immunohistochemistry; NFT, neurofibrillary tangle.

3.2. Immunohistochemical examination using TPP motif antibodies labels neurofibrillary tangles of different maturations

We next tested these antibodies by IHC on paraffin sections of the AD brain [parahippocampal and entorhinal cortices from AD (Braak stage VI)] to see whether they could recognize tau pathology in situ. All four antibodies readily identified NFTs at differing levels of maturity ⁷ as well as dystrophic neurites within mature neuritic (amyloid) plaques (Figures 1D and 3). Qualitative levels of labeling intensity varied across antibodies, however with roughly equal labeling intensity for pT231 and pT217, lesser intensity for pT181, and lesser still for pT175 (Figures 2C and Figure S1). Of note, the IHC performed here was done without epitope retrieval and with a relatively low concentration of antibodies (< 1 μg/mL), suggesting that the antibodies validated here may be suitable for efficient, high‐throughput screening of brain tissue.

FIGURE 3.

pT175 antibodies label parahippocampus (PHc) but not ERc. IHC of AT8, pT175, pT181, pT217, pT231 and counterstaining using NR on (A) ERc and (B) PHc. Scale bar = 400 μm. IHC, immunohistochemistry; NR, neutral red.

FIGURE 2.

pT231 antibodies label ERc but not prefrontal cortex (BA9). (A) Illustration of ERc region: threshold image of AT8 staining. (B) IHC of AT8 and counterstaining using NR on ERc section of AD brain. (C) IHC of pT175, pT181, pT217, and pT231 on serial sections of ERc. (D) IHC of AT8, pT181, pT217, and counterstaining using NR on serial sections of BA9. Scale bar = 1 mm. ERc, entorhinal cortex; IHC, immunohistochemistry; NR, neutral red.

3.3. Limited regional variability across TPP motif phosphorylation sites is observed

To address the amount and regional variability of pTPP phosphorylated tau species, we first selected paraffin‐fixed tissue from 35 cases examined in the HBTRC, including 15 control cases without significant AD pathology (Braak 0 to II) and 20 AD cases (Braak Stage III–VI). Cases were age and sex‐matched between AD and non‐AD groups. For this report, we focused mainly on pTau IHC in the entorhinal cortex (ERc) and prefrontal cortex (BA9), as these two sites represent early and relatively late sites of tau cytopathology in AD, respectively. ERc is a small area functionally connected with different cortical and subcortical neurons in the brain and surrounded by the subiculum, perirhinal cortex, and other structures with distinct cytoarchitectures (Figure 2A). Sequential sections containing ERc from AD patients (Braak VI) were stained with AT8 and each of the four pTPP antibodies (pT175, pT181, pT217, and pT231). Neutral red (NR) was used for counterstaining. As shown in Figure 3A (higher magnification) and Figure S2B (lower magnification), AT8, pT181, pT217, and pT231 identified NFTs and neuronal threads (NTs) in ERc. Again, pT217 demonstrated a better resolution with a lower background among the five antibodies. pT175 had very limited labeling in the ERc (Figure 3A and Figure S2B). When we examined regions surrounding the ERc, we found that pT175 labeled most of the AT8‐positive NFTs in the parahippocampal cortex (PHc) (Figure 3B and Figure S2C), suggesting regional heterogeneity in tau in the ERc compared to PHc, two areas separated by just a few millimeters. We next examined BA9, a late site of tau accumulation in AD. Sequential sections from the same AD donors (Braak VI) were stained with the same methods as the ERc. As shown in Figure 2D, the global labeling intensities were AT8 ≈ pT217 > pT181. Notably, little or no labeling beyond background was observed when using pT175 or pT231 antibodies (as shown in Figure S2A at a higher magnification). This suggests that pT231 tau may be relatively absent in the BA9 while being highly abundant in the ERc (Figure S1). Meanwhile, all other antibodies demonstrated a similar profile in these two brain regions. Among them, pT217 showed consistency with strong labeling and low background (compared to AT8 and pT231), enabling excellent anatomical resolution (Figure 3A,B and Figure S2B,C).

In addition, we performed a densitometric assessment of our immunohistochemical data from ERc and BA9 (prefrontal cortex) regions in the same cases. As shown in Figure 4A–C, we found a high degree of correlation between Braak stage and the density of IHC staining in the ERc for pT181, pT217, pT231, and AT8. In BA9, IHC of pT217 and AT8 correlated well with Braak stages. The correlation of AT8 IHC signal in ERc has a nearly linear correlation with Braak stages (Spearman ρ = 0.83, Pearson r = 0.82). Regarding the negative correlation between pT231 IHC in BA9 and Braak stage, it resulted from a generally low level of staining in this brain region, as shown in Figure S2A (right panel).

FIGURE 4.

Quantification of IHC staining by all five antibodies on ERc and BA9. (A) IHC quantification in ERc for pT175, pT181, pT217, pT231, and AT8. (B) IHC quantification in ERc for pT175, pT181, pT217, pT231, and AT8. Brown‐Forsythe for ANOVA followed by Dunnett's T3 multiple‐comparisons test: ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001. (C) Correlation matrix of IHC semi‐quantification with Braak stages, age, and PMI. The number represents Spearman ρ, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001. ERc, entorhinal cortex; IHC, immunohistochemistry; PMI, post mortem interval.

3.4. ELISA‐based measurements of phosphorylation of TPP motif in tau further reveal regional heterogeneity in pTau abundance

To explore further the regional heterogeneity of pTPP tau abundance revealed by IHC, we developed a series of immunoassays to assess region‐by‐region quantification of each pTPP tau isoform in a high‐throughput manner. Specifically, we used the previously validated pT175, pT181, pT217, and pT231 antibodies as detector antibodies paired with Tau‐13 as the capture antibody in a series of sandwich ELISA measurements of brain tissue. We first used the same HEK293 cells transfected different tau constructs to evaluate the assay specificity. Similar to the results seen with WB (Figure 1C), each sandwich ELISA had the expected pattern of recognition of wild‐type and mutant tau species, with a failure to detect an above‐background signal of T175A, T181A, T217A, and T231A in the pTau 175, 181, 217, and 231 assays, respectively (Figure 5A).

FIGURE 5.

Specific pTPP ELISA quantifies pTau abundance in 16 different brain regions. (A) Lysates of HEK293 transfected with different tau constructs were analyzed with indicated MSD ELISA. (B) Different tau measurements from 16 brain regions were standardized into z‐scores. (C) Correlation matrix of all five tau ELISA quantification in different brain regions. The number represents Spearman ρ, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001.

To examine the possible regional variability of TPP tau phosphorylation, we chose four late Braak stage AD cases from the HBTRC with tissue available across a set of 16 brain regions, including ERc, hippocampus, amygdala, BA4, BA8, BA40, BA17, insular cortex, caudate nucleus, putamen, nucleus accumbens, thalamus, substantial nigra, pons, cerebellum, and dentate nucleus. Tissue from each area was dissected and homogenized in TBS, and the TBS‐soluble centrifugal fraction was used to quantify the phosphorylation of the pTPP motif using these site‐specific ELISAs. Data from 16 regions were standardized into z‐scores on a per case per analyte basis. As shown in Figure 5B, we found that, among the regions examined, brain tissue from the limbic system (four regions) had the most abundant phosphorylation of the TPP motif, followed by the neocortex (four regions), the basal ganglia (six regions), and the cerebellum (two regions). In addition, we observed that BA17 (primary visual cortex) had substantially lower phosphorylation of the TPP motif among neocortical regions, which is consistent with a previous report. ⁸

Phosphorylation events at the four TPP motif sites were highly inter‐correlated among the four limbic and four neocortical regions (shown as a correlation matrix in Figure 5C). Moderate correlations between the four pTPP analytes in the basal ganglia were observed. An observable correlation was found only between pT181 and pT217 in the cerebellum, while no such correlation was observed among other pTPP analytes in the cerebellum. More broadly, we observed that pT231 was more highly abundant than the next most abundant pTau isoform (pT217) in all regions examined, as shown in the Supplementary Table, and that the extent to which pTau231 was more abundant varied by brain region. Specifically, we observed 2.5, 2.0, 8.2, and 16.9 ratios of pTau231: pTau217 in the neocortex, limbic system, basal ganglia, and cerebellum.

3.5. Patterns of TPP motif phosphorylation of tau in TBS‐soluble and TBS‐insoluble fractions correlate with Braak stage

We next examined the extent to which the phosphorylation of the TPP motif in tau correlated with the staging of AD tau pathology. Using homogenized tissue (insular cortex) from 67 cases ranging from Braak stage 0 to VI, we assessed phosphorylation of the TPP motif in tau using ELISAs for pTau175, 181, 217, and 231 in the TBS‐soluble fraction (Figure 6A) and TBS‐insoluble (urea soluble) fraction (Figure 6G). All four pTPP analytes correlated well in TBS‐soluble fractions (Supplementary Table); pT175 and pT217 correlated best with the Braak stage (Figure 5F). To confirm these findings, we quantified the phosphorylation of the TPP motif using a conformational antibody against this general phosphorylated peptide motif, pTPP, together with a tau‐specific antibody (Tau‐13) to examine tau phosphorylated at any of the four TPP motif sites (pTPP motif assay, Figure 6B,C). ELISAs using the pTPP motif assay provided similar rises across increasing Braak stages as those using the four site‐specific phospho‐tau antibodies (Figure 6A). To provide further context for these findings, we also assayed each brain sample using a non‐phospho‐specific N‐terminal tau assay and an assay using the commonly employed AT8 tau antibody as a detector (Figure 6D,E). We observed no significant changes in N‐terminal tau across the Braak stages, whereas the AT8 assay performed similarly to the pTPP assays, as expected. Variations in donor age or post mortem interval did not statistically explain these associations (Figure 6F).

FIGURE 6.

The abundance of pTPP tau in brain TBS‐ and urea‐soluble fractions correlates to Braak stage. (A) Indicated concentrations of pTPP tau in the TBS‐soluble fractions of insular cortex from 67 patients. (B) Schematic illustration of the immunoassay quantifying pTPP tau among four phosphorylation sites. (C–E) Indicated concentrations of pTPP‐motif tau, N‐terminal tau, and AT8 tau in the TBS soluble fractions of insular cortex from 67 patients. (F) All tau analytes correlate to the Braak stage, age, and PMI in the TBS soluble fractions. The number represents Spearman ρ, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001. (G) Indicated concentrations of pTPP tau in urea‐soluble fractions of insular cortex from 67 patients. (H) Correlation of pT217 and pT231, respectively, in TBS‐soluble fractions to urea‐soluble fractions. (I) Correlation of Braak stages with standardized pT217 and pT231 concentrations in both TBS‐ and urea‐soluble fractions. Spearman correlation was used with the Spearman ρ and p value listed in each figure. PMI, post mortem interval; TBS, Tris‐buffered saline.

We next examined whether correlations between phospho‐tau species and the Braak stage may vary between TBS‐soluble and TBS‐insoluble fractions from the insular cortex. To examine the TBS‐insoluble fraction of each sample, we used 8 M urea to solubilize the pellets remaining from the TBS‐soluble fraction. These urea‐solubilized tissue fractions were then assayed using the same ELISAs as for the TBS‐soluble fractions. Levels of pTau175 and pTau181 in the TBS‐insoluble fractions were below the detection limit, whereas pTau217 and pTau231 were readily detectable and at > 10‐fold higher levels than those in the TBS‐soluble fraction (compare Figures 6A–G). Though pTau217 and 231 levels were higher in the TBS‐insoluble fraction, relative levels of these analytes in the TBS‐insoluble and TBS‐soluble fractions were highly correlated within each brain sample (Figure 6H). They showed very similar correlations with the Braak stage (Figure 6I and Figure S3A [with all the data plotted]).

Several additional confirmatory analyses were performed to provide context for the preceding observations. First, a parallel set of phospho‐tau ELISAs was performed in TBS‐soluble fractions of the anterior cingulate cortex BA32 (Figure S3B,C). We observed similar associations between the levels of TPP motif phosphorylation of tau and Braak stage in the anterior cingulate compared to the associations seen using insular cortex tissue (Figure S3E), and levels of each phospho‐tau analyte in the anterior cingulate were highly correlated with levels observed in the insular cortex from the same donor (see Figure S2D for correlations of pT217 as an example). Among all cases with ELISA data, IHC data of ERc and prefrontal cortex (BA9) are available for the same cases [28 for the insular cortex and 14 for anterior cingulate (BA32), respectively]. All the ELISA data correlated very well with the same individual's ERc and IHC data, with Spearman ρ value as high as 0.93 for pT181 ERc IHC and 0.96 for pT217 BA9 IHC, respectively, correlating with BA32 ELISA of pTPP and AT8 assay (Figure S4).

4. DISCUSSION

The accumulation of hyperphosphorylated tau in the form of NFTs and dystrophic neurites is a defining feature of AD neuropathology and a central aspect of AD molecular pathogenesis. Many of the most heavily studied and pathophysiologically relevant phosphorylation events on the tau protein (including phosphorylation at residues 175, 181, 217, and 231) occur on a common molecular motif of a threonine residue followed by two proline residues (the TPP motif). ² The present report broadly examined phosphorylation events at the TPP motifs within tau. We correlated the levels and patterns of this conserved phosphorylation motif with the degree of tau pathology at an individual level. To facilitate this examination, we validated several novel antibodies and immunoassays that allow for specific examination of TPP phosphorylation events at a single site or across all four TPP sites using both immunohistochemistry and biochemical quantification. These antibodies exhibit specificity for single phosphorylation sites, rendering them valuable tools in post mortem brain diagnosis and fundamental research into tauopathy in the human brain, complementing the commonly used and less specific AT8 antibody. Using immunohistochemical techniques, we observe that phosphorylation at all examined TPP sites was strongly present in NFTs across various maturation stages. The staining intensity by IHC varied somewhat across site‐specific pTPP antibodies, with the strongest signal observed using pTau231 and pTau217 in the ERc. Importantly, pT181, pT217, and pT231 staining exhibited similarly strong correlations with the Braak stage (Spearman ρ = 0.8, 0.79, and 0.72, respectively, for the ERc) across brain regions. Consistent with the formulation of Braak staging, the highest degrees of correlation between p‐tau staining and tau pathologic staging were observed in the ERc region, where IHC intensity for AT8, pT181, and pT217 shows a nearly linear correlation with Braak stages, as determined by the Pearson correlation (r = 0.82, 0.77, and 0.77, respectively, shown in Supplementary Table). In contrast, when analyzed via ELISA in the insular cortex, all pTPP analytes display strong correlations with Braak stages, though these correlations were slightly weaker and were primarily driven by data from brains above Braak IV. As insular cortex tauopathy onset typically occurs after Braak IV, this is an expected result.

Results were generally similar when using ELISA‐based quantitation of TPP phosphorylation, with all four TPP phosphorylation sites we examined showing elevations at later Braak stages. Comparing TBS‐soluble and TBS‐insoluble cortical fractions, we observed similar correlations between pTau217 and pTau231 phosphorylation and Braak stages. Protein concentrations of pTau species varied substantially across regions, with the highest phospho‐TPP levels seen in limbic regions that often harbor high levels of NFT pathology, consistent with AD neuropathology ⁹ and recent tau PET in vivo imaging. ¹⁰ In addition, variations in phosphoprotein concentration across brain regions were evident between TPP sites, with pTau231 having the highest relative concentration in nearly every brain region examined, including non‐neocortical regions, followed by relative elevations in pTau217.

A high degree of correlation was generally observed in TPP phosphorylation of tau in the limbic system and cortical regions as depicted in Figures 4C and 5C and Figure S4, suggesting that the occupancy of each of the four examined tau phosphorylation sites goes up to a similar extent with Braak stage in cortical regions. However, these correlations were weaker in subcortical regions, as shown in Figure 5C. Importantly, a modest level of regional variability in TPP phosphorylation was observed, including low levels of staining for pT175 and pTau231 in the prefrontal cortex (BA9) relative to other pTPP tau species and very low levels of pTau175 staining seen in ERc compared to PHC. At the same time, we clearly understand the limitation of the neuropathological staging, ¹¹ which is largely driven by the onset of the ERc tau burden, ⁹ suggesting that, to some extent, this regional variability is expected, as exemplified by the different levels of linear correlation with the Braak stage when using data from the ERc or insular cortex.

Collectively, the pattern of results suggests that while the absolute levels of phosphorylation on the TPP motif of tau may vary across sites, increasing levels of phosphorylation of each of the TPP motifs of tau are consistently seen with progressive “spreading” of NFT pathology in brain, as measured by Braak stage. The presence of regional variation in tau phosphorylation of the TPP motif suggests that examination across multiple phosphorylation sites may be needed to assess regional tau pathology comprehensively.

AUTHOR CONTRIBUTIONS

Jean‐Pierre Bellier, Yuqi Cai, Sarah M. Alam, Thorsten Wiederhold, Arica Aiello, and Lei Liu conducted the experiments. Jonathan S. Vogelgsang, Sabina Berretta, Jasmeer P. Chhatwal, Dennis J. Selkoe, and Lei Liu analyzed and interpreted data. Jasmeer P. Chhatwal and Lei Liu designed the experiments and wrote the paper.

CONFLICT OF INTEREST STATEMENT

D.J.S. is a director and consultant of Prothena Biosciences. L.L. is a consultant of Korro Bio, Inc. T.W. and A.A. are employees of Cell Signaling Technology, Inc. All other authors have nothing to disclose.

CONSENT STATEMENT

All human subjects provided informed consent.

Supporting information

Supporting Information

Supporting Information

Supporting Information

Supporting Information

Supporting Information

Supporting Information

Bellier J‐P, Cai Y, Alam SM, et al. Uncovering elevated tau TPP motif phosphorylation in the brain of Alzheimer's disease patients. Alzheimer's Dement. 2024;20:1573–1585. 10.1002/alz.13557