PMC

(A) Double fluorescence staining of AD NFTs and Pick bodies (PBs) in Pick’s disease with PBBs, other tau ligands and anti-phospho-tau antibody (AT8). FSB and PBBs sensitively captured AD NFTs and PBs. AD NFTs were labeled with THK523. Meanwhile, PBs were not visualized by these compounds. NFTs and PBs were barely recognizable by using FDDNP. (B) Double fluorescence staining of neuronal tau inclusions (arrows) in PSP and CBD and putative astrocytic plaques (arrowheads) in CBD. A substantial portion of tau fibrils in neurons were captured by PBB3 and PBB5, but a much smaller subset of phosphorylated tau aggregates in astrocytic plaques were labeled with these compounds. Scale bar: 20 μm (A, B).

(A) Double fluorescence staining of intraneuronal tau aggregates in postmortem brain stem slices of a 12-month-old PS19 mouse with PBB, other amyloid ligands and anti-phospho-tau antibody (AT8). (B) Binding of intravenously administered PBBs (0.1 mg/kg PBB5 and 1 mg/kg PBB1 and PBB3) to NFTs in PS19 mice at 10–12 months of age. The tissues were sampled at 60 min after tracer administration. The brain stem (top row) and spinal cord (second and third rows from the top) sections abundantly contained neurons showing strong fluorescence (left), and subsequent staining with FSB or AT8 (right) indicated that these cells were laden with tau amyloid fibrils (right). Putative intraneuronal tau inclusions in unsectioned spinal cords (arrowheads in the bottom row) removed from PS19 mice at 60 min after intravenous injection of PBB2 and PBB4 were also clearly visible by using a two-photon (2P) fluorescence microscopic system. Arrow in the bottom row indicates a cluster of autofluorescence signals from blood cells. Scale bars: 25 μm (A); 30 μm (top to third rows in B); 20 μm (bottom row in B).

(A–H) A maximum projection of fluorescence in a 3-D volume of the spinal cord of a living PS19 mouse at 12 months of age before (A) and at various time points after (B–H) intravenous administration of PBB3 (1 mg/kg). Blood vessels were labeled with Sulforhodamine 101 (red) intraperitoneally injected at 15 min before PBB3 administration. Green fluorescence indicates a rapid transfer of PBB3 from the plasma to tissue parenchyma (B–E) and subsequent washout from the tissue (F). Background PBB3 signals were further attenuated beyond 300 sec, while somatodendritic labeling by this compound was observed in a subset of neurons (arrowheads in G, H). (I) Fluorescence image of WT spinal cord at 300 sec after PBB3 injection, demonstrating no overt retention of the tracer in the tissue. (J, K) Ex vivo microscopy for a brain stem section of the same Tg mouse. Tissues were obtained at 60 min after PBB3 injection. Signals of intravenously administered PBB3 (J) overlapped with AT8 immunoreactivity (K). Scale bars: 50 μm (A–F); 25 μm (G–I), 25 μm (J, K).

(A) Time course of unmetabolized [¹¹C]PBB3 fraction in plasma following intravenous radiotracer injection. The plot was generated by averaging data from 6 individuals. (B) Time-radioactivity curves in different brain regions of cognitively normal control subjects over 70 min after intravenous injection of [¹¹C]PBB3. Data were generated by averaging values in two individuals, and are presented as standard uptake values (SUVs). (C, D) Comparisons of time-radioactivity curves in the medial temporal region (C) and precuneus (D) of normal controls (black symbols and lines; n = 3) and AD patients (red symbols and lines; n = 3). (E–H) Scatterplots illustrating correlation of SUVRs with MMSE scores in the medial temporal region (E), precuneus (F) and lateral temporal (G) and frontal (H) cortices. Numbers beside symbols denote subject ID as indicated in . Coefficients of determination (r²) and p values by t-test are displayed in graphs. (I) [¹¹C]PBB3- and [¹¹C]PIB-PET images in a subject with clinical diagnosis of corticobasal syndrome. Images were generated as in and . Accumulation of [¹¹C]PBB3 was noticeable in the basal ganglia (red arrowheads) with right-side dominance and an area containing the thalamus and midbrain (yellow arrowhead).

(A) Confocal fluorescence images of frontal cortex sections from an AD patient. Following fluorescence labeling (pseudocolors are converted to green) with PIB (top row) and FSB (middle row), the samples were immunostained with an antibody against AβN3(pE) (red in the right column). PIB intensely labeled Aβ plaques (white arrowheads), but did not clearly label NFTs (arrows). By contrast, NFTs and neuropil threads were intensely labeled by FSB, while the staining of diffuse plaques was negligible. A section was also doubly immunolabeled (bottom row) with AT8 (green) and anti-AβN3(pE) antibodies (red in the right panel), to demonstrate the abundance of tau and Aβ amyloids in this area. Yellow arrowheads indicate tau-positive dystrophic neurites associated with senile plaques. (B) Structures of PBBs. Neutral benzothiazoles (PBB1–4) are newly synthesized chemicals, and a charged benzothiazolium, PBB5, is identical to a commercially available near-infrared laser dye. (C) Confocal fluorescence images of PBBs (pseudocolors are converted to green) and AβN3(pE) (red in the right column) staining in sections adjacent to those displayed in A. The intensity of plaque staining (arrowheads) relative to that of NFTs (arrows) was positively associated with the lipophilicity of PBBs. As compared with PBB1 (top row) staining, labeling of diffuse plaques with PBB3 (middle row) was substantially attenuated. PBB5 was nearly unreactive with diffuse plaques (bottom row), and subsequent double immunofluorescence staining of the same section (bottom row in C) illustrated good agreement of PBB5 labeling with the distribution of AT8-positive NFTs. Scale bar: 50 μm (A, C).

(A) Autoradiographic labeling of adjacent brain sections from an AD patient with 10 nM of [¹¹C]PBB3 (left) and [¹¹C]PIB (middle). The slices contain the hippocampus (Hi), parahippocampal gyrus (PH), fusiform gyrus (FF) and white matter (asterisks). Total binding (top) of [¹¹C]PBB3 and [¹¹C]PIB was markedly abolished (bottom) by addition of nonradioactive PBB5 (100 μM) and thioflavin-S (10 μM), respectively, except for the nonspecific (NS) labeling of white matter with [¹¹C]PIB. The hippocampal CA1 sector and subiculum displayed intense [¹¹C]PBB3 signals without noticeable binding of [¹¹C]PIB, and binding of [¹¹C]PBB3 in cortical areas flanking the collateral sulcus (identified by a red dot) and hippocampal CA2 sector (arrows) was also abundant relative to that of [¹¹C]PIB. FSB staining of amyloid fibrils in the sections used for autoradiography indicated the predominance of NFTs and diffuse plaques in the hippocampal subiculum (Sub) and fusiform gyrus (FF), respectively (right panels), supporting the strong reactivity of [¹¹C]PBB3 with AD NFTs. (B) MRI (left) and PET imaging with [¹¹C]PBB3 (middle) and [¹¹C]PIB (right) performed in the same AD (top) and normal control (NC; bottom) subjects. Coronal images containing the hippocampal formation (arrowheads) are displayed. [¹¹C]PBB3- and [¹¹C]PIB-PET images were generated by estimating SUVRs at 30–70 min and 50–70 min after radiotracer injection, respectively, and were superimposed on individual MRI data. In the hippocampal formation, prominently increased retention of [¹¹C]PBB3 in the AD patient was in sharp contrast to the modest or negligible changes in [¹¹C]PIB binding as compared with NC. Scale ranges for SUVRs were 0.75–1.50 ([¹¹C]PBB3) and 0.75–3.00 ([¹¹C]PIB).

Data are displayed as parametric maps for SUVR. The [¹¹C]PBB3 binding to the hippocampal formation (arrowheads) was increased consistently in AD patients in contrast to minimum radiotracer retention in normal control (NC) subjects with MMSE score of 29–30 points (Subjects 1 and 2). Another NC subject with MMSE score of 27 points (Subject 3) was negative for [¹¹C]PIB-PET, but exhibited slight accumulation of radiotracer signals primarily around the hippocampus, resembling fibrillar tau deposition at Braak stage III–IV or earlier. Sagittal slices around the midline illustrate that radioligand signals were the most intense in the limbic system but began to expand to the neocortex in a patient with the mildest AD (Subject 4), in agreement with the tau pathology at Braak stage V–VI, and was further intensified in most neocortical areas, corresponding to Braak stage VI, apparently as a function of the disease severity assessed by MMSE (Subjects 5 and 6). The AD patient with the lowest MMSE score (Subject 6) displayed less profound increase of [¹¹C]PBB3 retention in the lateral temporal and parietal cortices than did the other two AD cases, and this is attributable to marked cortical atrophy in this individual and/or toxic loss of tau-bearing neurons in these brain areas at an advanced pathological stage. In contrast to the spatial profiles of [¹¹C]PBB3 binding, the distribution of [¹¹C]PIB signals appeared unchanged among AD subjects.

(A) Baseline autofluorescence signals (middle) are overlaid on the visible background image of a shaven non-Tg WT mouse head (left). Ellipsoidal ROIs are defined above the frontal cortex (FC), brain stem (BS) and cervical spinal cord (SC) guided by a relatively intense emission from the FC region (right). (B) Fluorescence intensity maps in 12-month-old WT (top) and PS19 (Tg; bottom) mice before and at 30 and 240 min after the intravenous administration of PBB5 (0.1 mg/kg). The intensity maps (A, B) are normalized by the FC ROI value at 30 min after tracer injection. Long-lasting retention of the tracer was noted in the BS and SC ROIs of the Tg mouse. C, D, Target-to-FC ratios of fluorescence intensity in the BS (C) and SC (D) ROIs over the image acquisition time in the WT (open circles; n = 7) and PS19 (closed circles; n = 7) mice. There were significant main effects of time, region and genotype in 2-way, repeated-measures ANOVA (time, F_{(11, 132)} = 17.6, p < 0.001; region, F_{(1, 12)} = 29.9, p < 0.001; genotype, F_{(1, 12)} = 23.6, p < 0.001). (E) Target-to-FC ratios in the BS and SC ROIs of the WT (open columns) and tau Tg (closed columns) mice at 240 min after tracer injection. *, p < 0.05; **, p < 0.01; 2-way repeated-measures ANOVA with Bonferroni’s post hoc analysis. (F) Scatterplots of target-to-FC ratios at 240 min versus the number of FSB-positive NFTsper unit area of postmortem 20 -μm tissue slices in BS (blue symbols) and SC (red symbols) ROIs of tau Tg mice. Solid lines represent regressions; p values were determined by t-test. Vertical bars in the graphs represent SEs.

(A) In vitro autoradiograms of PS19 and non-Tg WT hindbrains (coronal sections) and AD frontal cortex. Fibrillar aggregates in the mouse brain stem and AD gray matter produced intense radiolabeling with both tracers, but nonspecific background signals were also observed at a considerably high level with the use of [¹¹C]PBB2. Binding of [¹¹C]PBB3 was profoundly abolished by the addition of nonradioactive PBB3 (10 μM). (B) Autoradiographic labeling with intravenously injected [¹¹C]PBB2 and [¹¹C]PBB3 in PS19 (Tg) and WT mice. The brains were removed at 45 min after injection, and were cut into sagittal slices. The autoradiographic section of PS19 brain was also stained with FSB. Arrows indicate the brain stem containing numerous tau inclusions displayed at intermediate and high magnifications. (C) Sagittal and coronal PET images generated by averaging dynamic scan data at 60–90 min after intravenous administration of [¹¹C]PBB3. The images are overlaid on the MRI template (images of the template alone are presented at the bottom). Arrows and asterisks indicate the brain stem and striatum, respectively, and arrowhead denotes intense radiolabeling in the medial brain stem of the PS19 mouse. (D) FSB staining of PS19 mouse brain shown in c. Sagittal (left) and coronal (middle) images and a high-power view of fibrillar inclusions (right) are displayed. Corresponding to high-level retention of [¹¹C]PBB3 in PET scans, abundant FSB-positive lesions were found in the medial brain stem (arrow and arrowhead). (E) Time-radioactivity curves (left) in the striatum (ST) and brain stem (BS) and BS-to-ST ratio of radioactivity (right) over the imaging time in PS19 (Tg; red symbols) and WT (black symbols) mice (n = 5 each). Vertical bars in the graphs denote SEs. Scale bars: 1 cm (A, top, middle and bottom-left panels in B); 1 cm (C, left and middle panels in D); 100 μm (bottom-middle panel in B); 100 μm (bottom-right panel in B, right panel in D).