Synthesis and Assessment of Novel Probes for Imaging Tau Pathology in Transgenic Mouse and Rat Models

Article abstract of DOI:10.1021/acschemneuro.0c00790

Aggregated tau protein is a core pathology present in several neurodegenerative diseases. Therefore, the development and application of positron emission tomography (PET) imaging radiotracers that selectively bind to aggregated tau in fibril form is of importance in furthering the understanding of these disorders. While radiotracers used in human PET studies offer invaluable insight, radiotracers that are also capable of visualizing tau fibrils in animal models are important tools for translational research into these diseases. Herein, we report the synthesis and characterization of a novel library of compounds based on the phenyl/pyridinylbutadienylbenzothiazoles/benzothiazolium (PBB3) backbone developed for this application. From this library, we selected the compound LM229, which binds to recombinant tau fibrils with high affinity (Kd = 3.6 nM) and detects with high specificity (a) pathological 4R tau aggregates in living cultured neurons and mouse brain sections from transgenic human P301S tau mice, (b) truncated human 151-351 3R (SHR24) and 4R (SHR72) tau aggregates in transgenic rat brain sections, and (c) tau neurofibrillary tangles in brain sections from Alzheimer's disease (3R/4R tau) and progressive supranuclear palsy (4R tau). With LM229 also shown to cross the blood-brain barrier in vivo and its effective radiolabeling with the radioisotope carbon-11, we have established a novel platform for PET translational studies using rodent transgenic tau models.

Full text of DOI:10.1021/acschemneuro.0c00790

pubs.acs.org/chemneuro
Research Article
Synthesis and Assessment of Novel Probes for Imaging Tau
Pathology in Transgenic Mouse and Rat Models
Lindsay McMurray, Jennifer A. Macdonald, Nisha Kuzhuppilly Ramakrishnan, Yanyan Zhao,
̌
David W. Williamson, Ole Tietz, Xiaoyun Zhou, Steven Kealey, Steven G. Fagan, Tomás Smolek,
Veronika Cubinkova, Norbert Zilka, Maria Grazia Spillantini, Aviva M. Tolkovsky, Michel Goedert,
̌
and Franklin I. Aigbirhio*
Cite This: https://dx.doi.org/10.1021/acschemneuro.0c00790
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: Aggregated tau protein is a core pathology present in
several neurodegenerative diseases. Therefore, the development and
application of positron emission tomography (PET) imaging radio-
tracers that selectively bind to aggregated tau in fibril form is of
importance in furthering the understanding of these disorders. While
radiotracers used in human PET studies offer invaluable insight,
radiotracers that are also capable of visualizing tau fibrils in animal
models are important tools for translational research into these
diseases. Herein, we report the synthesis and characterization of a
novel library of compounds based on the phenyl/pyridinylbutadienyl-
benzothiazoles/benzothiazolium (PBB3) backbone developed for this
application. From this library, we selected the compound LM229,
which binds to recombinant tau fibrils with high affinity (Kd = 3.6 nM) and detects with high specificity (a) pathological 4R tau
aggregates in living cultured neurons and mouse brain sections from transgenic human P301S tau mice, (b) truncated human 151-
351 3R (SHR24) and 4R (SHR72) tau aggregates in transgenic rat brain sections, and (c) tau neurofibrillary tangles in brain sections
from Alzheimer’s disease (3R/4R tau) and progressive supranuclear palsy (4R tau). With LM229 also shown to cross the blood−
brain barrier in vivo and its effective radiolabeling with the radioisotope carbon-11, we have established a novel platform for PET
translational studies using rodent transgenic tau models.
KEYWORDS: PET, imaging, tau, neurodegeneration, mouse, rat
While the final aim is to employ tau PET probes for human
diagnostics and investigation of disease mechanisms, there is a
need to underpin their application by studying experimental
animal models of neurodegeneration. These provide a critical
translational link between laboratory research from cellular and
animal models to human disease and, conversely, allow human
pathophysiological data to be used to design more appropriate
experimental models and studies. In particular, transgenic
animal models of dementia are important for understanding
the involvement of Aβ and NFTs in disease, especially given
their use in longitudinal and behavioral studies.13 Therefore,
the development of PET radiotracers that are capable of
visualizing tau aggregates within these animal models is of
INTRODUCTION
■
Imaging protein aggregates, characteristic features of a variety
of neurodegenerative diseases,1 has become a powerful means
of investigating their pathology. In particular, positron
emission tomography (PET) is the method of choice to
study their pathophysiology in vivo using imaging probes
selective for the various aggregated protein forms. PET probes
were initially developed for imaging β-amyloid (Aβ)
aggregated protein, present as plaque deposits in Alzheimer’s
disease (AD),2 then probes selective for protein tau aggregates,
which form intracellular neurofibrillary tangles (NFT) in
AD.3,4 In addition to AD, pathological tau aggregates are a
constituent feature of several other neurodegenerative
disorders, collectively termed tauopathies;5−7 hence tau PET
imaging is a key biomarker technology. Since the first reports
of [18F]T807 ([18F]AV1451)8 as a suitable PET radiotracer for
selective imaging of NFTs, several radiotracers capable of
binding to and visualizing tau have now been used for clinical
PET studies,4,9 including [11C]PBB3,10 [18F]PI-2620,11 and
[18F]MK6240.12
Received: December 11, 2020
Accepted: February 9, 2021
© XXXX The Authors. Published by
American Chemical Society
https://dx.doi.org/10.1021/acschemneuro.0c00790
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
A

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
Scheme 1. (A) Development of Novel Compounds for Imaging Tau Aggregation in Animal Models of Tau and (B)
Streamlined Convergent Synthetic Route for a Compound Library
great importance in advancing both the understanding of the
disease and the development of new therapeutics alongside
clinical studies.
aggregation. Notably, imaging tau pathology in a rat model of
tauopathy has yet to be reported. With the rat brain being
approximately six times larger than the mouse brain, this would
bring distinct advantages for research involving small animal
PET imaging. For example, it would enable more accurate
quantification of radioactivity concentration in brain regions
and blood sampling for gold standard blood-based kinetic
analysis.
Therefore, to improve the development of PET imaging
probes for visualizing tau pathology in transgenic models of
neurodegeneration, herein, we report the synthesis and
characterization of a novel series of compounds based on the
phenyl/pyridinylbutadienylbenzothiazoles/benzothiazolium
(PBB3) structure (Scheme 1A), which has been shown to bind
to tau aggregates in both mouse models and humans.10
Binding affinities to heparin-induced synthetic recombinant tau
fibrils were determined as well as binding to aggregated tau in a
transgenic human P301S tau mouse model.18 The most
promising compound from this series, LM229, was then
While the present range of tau PET radiotracers has shown
excellent affinities for tau fibrils, they are still limited in their
ability to visualize tau NFTs in animal models as well as in
humans. For example, [18F]T807 (AV1451) showed no
specific binding to tau tangles in transgenic mice expressing
mutant human P301L tau as assessed by in vitro14 and
microPET in vivo15 imaging studies. Studies with P301S tau
mice indicated a difference in retention of [18F]AV1451 in
their brainstem of no more than 23% compared to control
wild-type (WT) mice.16 More successful have been the
radiotracers [11C]PBB3, which has been shown to bind to
tau pathology in PS19 tau transgenic mice10 and [18F]-
THK5117, which bound to tau aggregates in two transgenic
mice mouse models, P301S tau and GSK3-β X P301L tau
(biGT).17 Even though these can detect tau fibrils, higher
specificity is needed to analyze subtle early changes in tau
B
https://dx.doi.org/10.1021/acschemneuro.0c00790
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
assessed for its binding to pathological tau in transgenic mouse
and rat brain tissues in vitro and in vivo and in two human
tauopathies. The successful radiolabeling of LM229 with the
carbon-11 (t1/2 = 20 min) PET radioisotope is described.
P301S tau mice, which express the shortest isoform (0N4R) of
human 4-repeat (4R) tau,18 using their autofluorescence
properties to rapidly analyze their binding. Although some of
the compounds recognized tau aggregates, they also showed
significant fluorescence in WT brain sections, giving a high
background and low specificity, therefore unlikely to be useful
as PET radiotracers. However, compound LM229, showed
intense, specific binding that correlated with hyperphosphory-
lated pathological tau (AT8 antibody, pS202/pS205) and β-
sheet-rich filamentous tau (AT100 antibody, pT212/pS214/
pT21718,22), consistent with its high binding affinity to
recombinant tau fibrils (Figure 1).
RESULTS AND DISCUSSION
■
Synthesis of a Compound Library. At the outset of our
investigation, a library of unlabeled analogues based on the
core structure of PBB319 was synthesized. To rapidly access
compounds of interest, we developed a streamlined convergent
route centered on a Horner−Wadsworth−Emmons coupling
of two key fragments: aldehyde C and phosphonate D
(Scheme 1B). Synthesis of aldehyde C commenced with
palladium(II)-catalyzed Heck reaction of a range of commer-
cially available bromopyridines (A) with methyl acrylate.
DIBAL reduction followed by immediate benzylic oxidation
with manganese dioxide furnished the desired aldehyde C. The
desired benzothiazoles (B) were either available from
commercial sources or synthesized from simple starting
materials. Deprotonation with lithium diisopropylamide
(LDA) followed by addition of diethylchlorophosphate
afforded the desired phosphonates D. Coupling of the
aldehyde C and phosphonate D proceeded smoothly for all
derivatives to furnish the desired library of PBB3 analogues.
Assessment of Binding Affinities. All compounds were
assessed for their ability to bind to synthetic recombinant tau
fibrils using a fluorescence quenching assay20 (Table 1), for
Table 1. Kd (nM) of Compound Binding to Heparin-
Induced Synthetic Tau Fibrils Determined by Fluorimetry
Figure 1. LM229 binding to mouse brain sections is specific to
neurons with pathological tau aggregates. (Top) No specific binding
to brainstem sections from WT animals but coincident LM229
binding (green) and (middle) AT8 or (bottom) AT100 antibody
staining (red) in phenotypic P301S tau mice; AT100 binding
correlates with tau fibrils. Scale bar = 20 μm.
Kd
(nM)
name
R1
R2
R3
X
Y
PBB3
148
152
H
H
H
F
OH
OH
OH
OH
OMe
H
H
H
H
H
NHMe
F
F
NHMe
NHMe
F
F
F
C(H)
C(H)
N
C(H)
C(H)
C(H)
N
C(H)
N
C(H)
C(H)
C(H)
N
N
C(H)
N
N
N
C(H)
N
C(H)
N
N
N
1.4
56
353
3.6
1.1
229
225
F
To further confirm that binding of LM229 was specific to
aggregated tau rather than to soluble tau, binding to brain
sections from P301S tau mice at 2 and 6 months of age was
compared, based on the fact that a significant amount of tau
aggregates is detected in older mice (>3 months).18,23 Rare
binding was detected in brain sections from 2 months old
mice, but significant binding to large populations of neurons
was evident at 6 months of age (Supporting Information,
Figure S1).
a
171
OH
OH
H
a
166
169
a
a
164
H
F
236
OCH2CH2F
H
F
NHMe
NHMe
NHMe
454
a
207
F
H
a
141
a
Due to negligible change of fluorescence intensity, binding affinities
could not be determined.
We next looked at the ability of LM229 to cross the blood−
brain barrier (BBB), a key property of any viable neuro-
radiotracer. LM229 was injected intravenously (i.v.) (1 mg/kg
weight), and after 1 h, WT and P301S tau mice were perfused-
fixed and brain sections were imaged under a fluorescence
microscope. Figure 2 shows binding of LM229 to some nerve
cells from P301S tau transgenic mice, with little binding to the
brains of non-transgenic control mice., confirming that the
compound crosses the BBB. Moreover, when compared with
PBB3 (Figure 3), and based on the compounds having similar
extinction coefficients, LM229 showed comparably less non-
specific binding and more specific binding to tau aggregates by
fluorescence microscopy, suggesting that LM229 was highly
suitable as a ligand for tau aggregate tracing within the
transgenic mouse model. However, we await a comparison of
PBB3 and LM229 by PET imaging to determine their relativity
properties as in vivo imaging probes.
which we first established a Kd value for PBB3 of 1.4 nM, in
good agreement with the previously reported value of 1.3
nM.21 Binding affinities for some compounds (141, 164, 166,
169, 171, and 207) could not be assessed with this assay
because of negligible changes in fluorescence intensity. While
several compounds showed affinities below the micromolar
range, LM3-229 (abbreviated LM229 or 229) showed a Kd of
3.8 nM, in the nanomolar range expected of a PET ligand,
combined with having chemical moieties with the potential to
be radiolabeled by either carbon-11 or fluorine-18 for
development as a PET imaging probe. This compound was
then selected for further characterization and development.
Binding to Transgenic P301S Tau Mouse Brain
Sections. We then screened all of the compounds for their
binding to tau inclusions in brain sections from transgenic
C
https://dx.doi.org/10.1021/acschemneuro.0c00790
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
Figure 2. Fluorescence image of brain section from WT (A) and
P301S tau mice (B) fixed 1 h after i.v. injection of LM229 showing
entry into brain from periphery. Scale bar = 100 μm.
Figure 3. P301S tau brain tissue from (E) ex vivo experiments with
PBB3 (i.v. injection 1 h prior to injection) colocalized with AT8
showing binding of PBB3 with significant nonspecific binding; (F) ex
vivo experiments with LM229 showing significant specific binding to
tau aggregates as confirmed with colocalization with AT8.
Binding of LM229 to Cultured Neurons from P301S
Tau Transgenic Mice. P301S tau mice with aggregated tau in
the brain also express aggregated tau in DRG neurons.24−26
Unlike neurons from the brain, which do not survive in culture
when extracted beyond neonatal ages, DRG neurons with tau
filaments can be cultured from adult mice when filamentous
tau pathology is well-developed. To estimate the apparent
affinity of LM229 to tau aggregates directly in living neurons,
we imaged live neurons after 20 min incubation with various
concentrations of LM229 (Figure 4A). Analysis of fluorescence
intensity by nonlinear curve fitting to a Michaelis−Menten
equation yielded an IC50 of 2.7 0.7 μM (R2 = 0.97) (Figure
4B). A dose−response conducted after fixation and permeabi-
lization of the neurons (Figure 4C) gave a similar affinity (IC50
Figure 4. (A) Fluorescence images of LM229 binding to live DRG
neurons (green). Phase contrast images show total live neurons. (B)
Fluorescence intensity per neuron as a function of LM229
concentration (mean SD, 5−15 neurons per concentration, three
independent cultures; black line, result of nonlinear curve fitting,
green and blue dashed lines, 95% confidence intervals; live neurons:
IC50 = 2.97 0.73; R2 = 0.968; fixed neurons: IC50 = 1.67 0.28; R2
= 0.990). (C) Dose−response of LM229 binding to fixed neurons.
(D) Co-staining of LM229 (10 μM, green) and antiphospho-tau
antibody AT100 (red). Blue in merged image shows cell nuclei in the
culture.
= 1.7
0.3 μM, R2 = 0.99, Figure 4B), showing that the
plasma membrane does not form a barrier to LM229 entry/
binding. LM229 binding was specific to neurons with
aggregated tau, as detected with the AT100 antibody18,22,23,26
(Figure 4D). Notably, inspection of the neurons 3 days after
LM229 exposure and washes showed that the LM229
fluorescence was still retained albeit to a lower intensity.
Although PBB3 also bound to DRG neurons with tau
aggregates at 30−100 μM, it was not possible to derive an
IC50 value because of its low affinity in this assay (Supporting
Information, Figure S2). Thus, LM229 is highly cell
permeable, and binding follows a single saturable binding
algorithm. The shift from nanomolar to micromolar affinity
between binding to heparin tau fibrils and tau aggregates in
P301S tau neurons may reflect the different environment and
assay conditions or the different conformations that tau
aggregates assume in neurons compared to heparin−tau
complexes.27
Binding to Tau Transgenic Rat Brain Tissue. Since
future PET studies for development of these compounds
would require kinetic modeling for their assessment in vivo, we
tested their performance in tau transgenic rats. Rat brains and
blood volumes are about 6- and 10-fold larger, respectively,
than those of mice and thus enable higher resolution imaging
and ample volume for repeated blood sampling. We used two
transgenic rat lines expressing human-truncated tau comprising
the proline-rich region and either three microtubule binding
domains (3R tau151-391, SH24)28 or four microtubule
binding domains (4R tau151-391, SHR72).29 Both LM229
(Figure 5A) and PBB3 (Figure 6A) bound with similar
properties to pathological aggregated tau in brain sections from
SHR24 rats, which develop tangle pathology in the cortex, as
confirmed by colocalization with AT8 immunofluorescence
(Figures 5B and 6B). Likewise, with similar properties, both
LM229 (Figure 5C) and PBB3 (Figure 6C) bound to tau in
D
https://dx.doi.org/10.1021/acschemneuro.0c00790
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
Figure 7. (A) Post-mortem PSP tissue stained with LM229; (B)
Double staining of PSP tissue with LM229 and AT8 (red) to confirm
colocalization of LM229 with tau aggregates.
Figure 5. LM229 (green) binding to (A) 3R tau in cortical sections
from SHR24 rat brains and (B) 4R tau in brainstem sections from
SHR72 rats. (C, D) Colocalization with AT8 (red).
Figure 8. Post mortem AD tissue stained with LM229 and (A,C) and
double staining with AT8 (B) and beta-amyloid antibodies (D),
suggesting binding to tau rather than amyloid aggregates.
In slices from frontal cortex of AD brains, LM229 gave rise
to strong signal (Figure 8A), which colocalized with AT8
immunofluorescence (Figure 8B). In contrast, LM229
fluorescence intensity that colocalized with Aβ (Figure 8C)
was lower (Figure 8D),suggesting selectivity for tau versus β-
amyloid protein.
Radiosynthesis of [11C]LM229. Having characterized the
in vitro properties of unlabeled LM229, we next synthesized a
[11C]radiolabeled version for application as a PET imaging
probe. An analogous route to that reported by Wang et al.19 for
the synthesis of [11C]PBB3 was developed. [11C]methyl iodide
was trapped in a mixture of tert-butyldimethylsilyl (TBS)-
protected E and potassium hydroxide in dimethylsulfoxide.
The resulting mixture was heated to 125 °C for 5 min to access
intermediate F. Addition of water, followed by heating to 60
°C for 2 min, enabled the deprotection of the TBS group to
furnish [11C]LM229 (Scheme 2). It was found that a loading
of potassium hydroxide higher than that reported was required
to prevent formation of undesired side products. Due to the
heterogeneity of the reaction, mixing of the suspension of E
and KOH via vortex prior to the reaction also proved to be
crucial in the formation of [11C]LM229. Purification of the
crude reaction mixture by semipreparative HPLC yielded
[11C]LM229 (Supporting Information, Figure S1), which was
reformulated into ethanolic saline using a C18-light Sep-Pak
cartridge to afford [11C]LM229 1.0−1.4 GBq at end-of-
synthesis (10% DCY, 96.6% RCP, 250 GBq/μmol molar
activity).
Figure 6. Binding of PBB3 (green) to (A) 3R tau in the cortex of
SHR24 brain tissue and (B) to 4R tau in the brainstem of SHR72 and
(C, D) colocalization with staining AT8 (red).
brain sections from SHR72 rats, which develop tangles mainly
in the brainstem, as confirmed by colocalization with AT8
immunofluorescence (Figures 5D and Figure 6D). However,
compared to the SHR24 rat brain sections, there were less
inclusions detected, which seems to indicate the compounds
have preferential binding toward the three repeat forms of tau
in these rat models. To our knowledge, this is the first time
that tau PET imaging compounds have been shown to bind to
pathological tau in a rat transgenic model. These results
establish the basis to perform in vivo PET imaging studies,
including further investigations into the selectivity of LM229
toward the three and four repeat forms of tau.
Binding to Human Tau Pathology. To further validate
LM229 as a potential radiotracer for translational research, we
investigated its binding to tau aggregates in human globus
pallidus/putamen from progressive supranuclear palsy (PSP)
subjects (Figure 7) and its selectivity for tau over β-amyloid
pathology in AD frontal cortex sections (Figure 8). An intense
signal was emitted by LM229 binding to PSP sections (Figure
6A), which was extensively colocalized with AT8 binding.
PET Studies with [11C]LM229. We then performed
preliminary in vivo PET studies with [11C]L229 in both WT
and transgenic P310S tau mice. These studies confirmed
E
https://dx.doi.org/10.1021/acschemneuro.0c00790
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
Scheme 2. Two-Step Radiosynthesis of [11C]LM229
inclusions. To our knowledge, this is the first time any tau
PET imaging compounds have been shown to bind to tau
inclusions in a transgenic rat model. Finally, we have
established that LM229 can be used for in vivo translational
PET research, by developing a method for radiolabeling with
the carbon-11 radioisotope and confirmation of BBB entry in
mice.
METHODS
■
General Methods and Materials. All reagents and solvents were
used as supplied or purified using standard procedures as necessary.
Analytical separation was conducted on an Ultimate 3000 HPLC
system (Thermo Scientific). [11C]CO2 was produced via the 14N(p,α)
nuclear reaction using GE PETtrace cyclotron applying 16.5 MeV
protons onto 0.5% oxygen in nitrogen gas. [11C]Methyl iodide was
synthesized on a GE Medical Systems MeI MicroLab. A F-4500 FL
spectrophotometer (Chemical Engineering department, University of
Cambridge) was used to do fluorescence scan of all of the compounds
mixed with tau fibrils. Excitation wavelength was 365 nm, and
emission was scanned from 400 to 600 nm. With parameters, setting
excitation slit was 5.0 nm, emission slit was 20.0 nm, PMT voltage was
950 V, and response was 0.5 s.
All mouse studies were carried out in the Laboratory of Molecular
Imaging or the Clifford Allbutt Building, University of Cambridge,
and the MRC Laboratory of Molecular Biology. P301S tau transgenic
mice18 and WT mice were on a pure C57Bl/6 Jax background. The
SHR24, SHR72, and SHR wild-type rat brain slices were developed
and obtained from AXON Neuroscience.28 PSP brain tissue was
obtained from the Cambridge Brain Bank, with the PSP cases being
part of the cohort created by Prof. James Rowe. AD brain tissue was
supplied by the London Neurodegenerative Diseases Brain Bank and
Brains for Dementia Research.
blood−barrier brain penetration by the radiotracer with
maximum brain uptake (%ID/g max; WT = 1.56, P301S =
2.38) within the first minute followed by washout within the
90 min scan (Figure 9). The brain exposure was higher in the
Preparation of tau fibrils. Monomeric tau (tau-441 human)
lyophilized powder was obtained from Eurogentec. Tau monomers
(100 μM) were prepared in 20 mM BES buffer (pH 7.4 with 25 mM
NaCl and 2 mM dithiothreitol and incubated at 56 °C for 10 min.
After being cooled to room temperature in a water bath, heparin (25
μM) and a protease inhibitor mix were added and incubated at 37 °C
for 10 days.
In Vitro and Ex Vivo Fluorescence Microscopy. Mice were
perfused with PBS followed by 4% PFA. Following 24 h in 4% PFA,
the brains were transferred to 20% sucrose prior to being embedded
in OCT for cryo-sectioning. Sections (14 μm) were incubated with 50
μM LM229 or PBB3 for 1 h, washed in PBS + 0.1% Triton X-100,
and cover-slipped with Vectashield mounting medium. Sections were
imaged using a Leica DM6000 microscope. Mouse monoclonal AT8
(MN1020) or AT100 (MN1060) were from ThermoFisher and used
at 1:1000. Anti-mouse secondary antibody was conjugated to
AlexaFluor647.
Figure 9. Time−activity curves for [11C]LM229 from in vivo PET
studies in wild-type and P301S mice.
P301S mouse (area under the curve, AUC = 60) compared to
the wild-type mouse (AUC = 34). Further PET studies are
required to fully characterize the in vivo pharmacokinetics of
the radiotracer in both mice and rats.
Ex Vivo Mice Studies. Mice were injected intravenously (i.v.)
with 50 μM LM229 or PBB3 (DMSO) diluted in 50:50 ethanol/PBS.
Animals were then perfused (4% PFA) 1 h post-i.v.
Rat Brain. Fresh frozen rat brain coronal sections (10 μm) were
stored on slides at −80 °C until use. Sections were fixed in ice cold
acetone for 10 min and air-dried and then rehydrated twice for 5 min
with buffer (10 mM PBS with 0.1% Triton X-100) before application
of 5% bovine serum albumin for 1 h and two more 5 min buffer
washes. Primary monoclonal antibody AT8 (Thermo MN1020,
1:1000) was added overnight at 4 °C, followed by 1 h incubation with
a mixture of secondary AlexaFluor555 goat anti-mouse antibody
(1:1000) and 50 μM PBB3 or LM229 in 50:50 ethanol/buffer.
Following two 5 min washes in buffer, the slides were air-dried and
cover-slipped using Fluorsave before analysis. The green autofluor-
escence of PBB3 and LM229 was visualized using a 488 nm filter and
fluorescence of AT8 using a 568 nm filter on a Leica DM6000
microscope.
CONCLUSION
■
In order to establish transgenic tau rodent models as a
translational platform for PET imaging research in neuro-
degenerative disorders, we have developed a range of novel
compounds based on the PBB3 structure. One of these
compounds, LM229, is shown to have high binding affinity to
recombinant tau fibrils. Further evaluation of LM229 showed it
to bind to pathological tau inclusions in transgenic human
P301S tau mouse brains, with selectivity for tau inclusions in
PSP and AD, dementias with different fibrillar tau isoform
compositions. In addition, LM229 and PBB3 PET compounds
were both shown to bind to tau pathology in brain tissue from
two transgenic tau rat models with either 3R and 4R tau
inclusions with preferential binding toward the 3R tau
DRG Neurons. Neurons were prepared from phenotypic end stage
5−7 month old P301S tau mice and cultured as described
previously.24 For live staining, neurons cultured for 2 days were
F
https://dx.doi.org/10.1021/acschemneuro.0c00790
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
washed in PBS and incubated with various concentrations of LM229
for 20 min at room temperature. After three washes in PBS, neurons
were imaged on a Leica DMI 4000B microscope using a DFC3000 G
camera and application suite 4.0.0.11706. The dead cell stain
Propidium iodide (1 μg/mL) was added to ensure that only live
cells were imaged. Phase contrast images were taken to show viable
neurons. For fixed cell staining, neurons were fixed for 20 min at room
temperature in 4% paraformaldehyde, washed in PBS, and
permeabilized in PBS containing 0.3% Triton X-100. LM299 was
added as described for live cells, and cells were probed with AT100
(1:1000, MN1060) followed by anti-mouse AlexaFluor568 secondary
antibody. Cultures were co-stained with the nuclear dye DAPI (blue)
to reveal total cells.
Synthetic and analytical data of compounds, immuno-
histochemistry, and binding affinity methods (PDF)
AUTHOR INFORMATION
Corresponding Author
■
Franklin I. Aigbirhio − Molecular Imaging Chemistry
Laboratory, Wolfson Brain Imaging Centre and Department
of Clinical Neurosciences, University of Cambridge,
Cambridge CB2 0QQ, United Kingdom; orcid.org/0000-
0001-9453-5257; Email: fia20@wbic.cam.ac.uk
Radiosynthesis of [11C]LM229. TBS-protected precursor E (1.5
mg) in DMSO (150 μL) was added to a suspension of powdered
KOH (15 mg) in DMSO (300 μL), and the suspension mixed via
vortex for 1 min. [11C]Methyl iodide was trapped in the suspension
and heated to 125 °C for 5 min. Water (200 μL) was then added and
the mixture heated to 60 °C for 2 min. The mixture was diluted with
water (0.8 mL), and the radioactive material was loaded into a
preparative HPLC system for purification (42% acetonitrile/50 mM
ammonium formate, 6 mL/min, Luna 5μ, 10.00 mm internal diameter
× 250 mm (Phenomenex)). The fraction corresponding to
[11C]LM229 was collected in water (100 mL) and reformulated
into saline (6 mL) containing ethanol (400 μL) and 25% ascorbic
acid (200 μL) using a C18-light sep-pak cartridge to afford
[11C]LM229 (1.0−1.4 GBq, 10% DCY). Analytical HPLC (Luna
5μ, 4.60 mm internal diameter × 250 mm (Phenomenex), 50%
acetonitrile/50 mM ammonium formate, 1 mL/min) confirmed the
formulated product was radiochemically pure (>95%). The specific
activity of [11C]LM229 was >200 GBq/μmol.
Authors
Lindsay McMurray − Molecular Imaging Chemistry
Laboratory, Wolfson Brain Imaging Centre, University of
Cambridge, Cambridge CB2 0QQ, United Kingdom
Jennifer A. Macdonald − MRC Laboratory of Molecular
Biology, Cambridge CB2 0QH, United Kingdom
Nisha Kuzhuppilly Ramakrishnan − Molecular Imaging
Chemistry Laboratory, Wolfson Brain Imaging Centre,
University of Cambridge, Cambridge CB2 0QQ, United
Kingdom
Yanyan Zhao − Molecular Imaging Chemistry Laboratory,
Wolfson Brain Imaging Centre, University of Cambridge,
Cambridge CB2 0QQ, United Kingdom
David W. Williamson − Molecular Imaging Chemistry
Laboratory, Wolfson Brain Imaging Centre, University of
Cambridge, Cambridge CB2 0QQ, United Kingdom
Ole Tietz − Molecular Imaging Chemistry Laboratory,
Wolfson Brain Imaging Centre, University of Cambridge,
Cambridge CB2 0QQ, United Kingdom
Xiaoyun Zhou − Molecular Imaging Chemistry Laboratory,
Wolfson Brain Imaging Centre, University of Cambridge,
Cambridge CB2 0QQ, United Kingdom
Steven Kealey − Molecular Imaging Chemistry Laboratory,
Wolfson Brain Imaging Centre, University of Cambridge,
Cambridge CB2 0QQ, United Kingdom
Steven G. Fagan − Department of Clinical Neurosciences,
University of Cambridge, Cambridge CB2 0QQ, United
Kingdom
PET Studies with [11C]LM229. These studies were regulated
under the Animals (Scientific Procedures) Act 1986 Amendment
Regulations 2012 following ethical review by the University of
Cambridge Animal Welfare and Ethical Review Body (AWERB). The
mice were anesthetized with 5% isoflurane, and general anesthesia was
maintained using 1.5% isoflurane. The anesthetized mice were placed
prone on the bed of a microPET Focus-220 scanner (Concorde
Microsystems, Knoxville, TN). Body temperature was maintained at
37 °C using a heating blanket connected to a rectal thermistor probe.
Blood oxygen saturation as well as heart rate and breathing rate were
monitored and maintained within normal limits using a noninvasive
mouseOX pulse-oximeter sensor (Starr Life Science Corp., Oakmont,
PA) attached to the thigh. Before the injection of a tracer, a
transmission scan was performed with a 68Ge point source for
attenuation and scatter correction of 511 keV photons. [11C]LM229
was injected via the tail vein over 30 s, followed by a 15 s heparin−
saline flush. Dynamic data were acquired in list mode for 90 min. Data
were subsequently Fourier rebinned in following time frames: 12 × 5
s, 6 × 10 s, 3 × 20 s, 4 × 30 s, 5 × 60 s, 10 × 120 s, 12 × 5 min.
Corrections were applied for randoms, dead time, normalization,
attenuation, and decay. Fourier rebinning was used to compress the
4D sinograms to 3D before reconstruction with a 2D-filtered back
projection with a Hann window cutoff at the Nyquist frequency. The
image voxel size was 0.95 × 0.95 × 0.80 mm, with an array size of 128
× 128 × 95.
̌
Tomás Smolek − Axon Neuroscience R&D Services SE,
Bratislava, Slovak Republic 811 02
Veronika Cubinkova − Axon Neuroscience R&D Services SE,
Bratislava, Slovak Republic 811 02
̌
Norbert Zilka − Axon Neuroscience R&D Services SE,
Bratislava, Slovak Republic 811 02
Maria Grazia Spillantini − Department of Clinical
Neurosciences, University of Cambridge, Cambridge CB2
0QQ, United Kingdom
Aviva M. Tolkovsky − Department of Clinical Neurosciences,
University of Cambridge, Cambridge CB2 0QQ, United
Kingdom
Three-dimensional volumes of interest (VOIs) for mice brain MRI
template available on PMOD software (version 3.8; PMOD
technologies, Zurich, Switzerland) were modified to obtain a whole
brain VOI. Individual PET images were then co-registered with this
MRI template, and the VOI transferred from MRI to PET. Whole-
brain time−activity curves were obtained for each of the animals. The
results were expressed as % injected dose per gram, assuming a
specific gravity of 1 g·mL−1 for brain tissue.
Michel Goedert − MRC Laboratory of Molecular Biology,
Cambridge CB2 0QH, United Kingdom
Complete contact information is available at:
https://pubs.acs.org/10.1021/acschemneuro.0c00790
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acschemneuro.0c00790.
Notes
The authors declare no competing financial interest.
G
https://dx.doi.org/10.1021/acschemneuro.0c00790
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
PET Tracer for In Vivo Quantification of Human Neurofibrillary
Tangles. J. Nucl. Med. 57 (10), 1599−1606.
ACKNOWLEDGMENTS
■
Funding was provided by UK Medical Research Council grants
(MR/K02308X/1 and a Proximity to Discovery award), a UK
Engineering and Physical Science Council grant (EP/
P008224/1) and EU/EFPIA/Innovative Medicines Initiative
2 Joint Undertaking (IMPRiND Grant No. 116060). We
gratefully acknowledge the London Neurodegenerative Dis-
eases Brain, The Cambridge Brain Bank, and Brains for
Dementia Research for human AD and PSP tissue. PSP brain
tissue was part of the cohort created by Professor James Rowe.
The Cambridge Brain Bank is supported by the National
Institute for Health Research (NIHR) Cambridge Biomedical
Research Centre. Mass spectrometry data were acquired at the
EPSRC UK National Mass Spectrometry Facility at Swansea
University.
(13) Götz, J., Bodea, L. G., and Goedert, M. (2018) Rodent models
for Alzheimer disease. Nat. Rev. Neurosci. 19 (10), 583−598.
(14) Declercq, L., Celen, S., Lecina, J., Ahamed, M., Tousseyn, T.,
Moechars, D., Alcazar, J., Ariza, M., Fierens, K., and Bottelbergs, A.
(2016) Comparison of New Tau PET-Tracer Candidates With
[18F]T808 and [18F]T807. Mol. Imaging 15, 1−15.
(15) Xia, C. F., Arteaga, J., Chen, G., Gangadharmath, U., Gomez, L.
F., Kasi, D., Lam, C., Liang, Q., Liu, C., Mocharla, V. P., et al. (2013)
[(18)F]T807, a novel tau positron emission tomography imaging
agent for Alzheimer’s disease. Alzheimer's Dementia 9 (6), 666−76.
(16) Brendel, M., Yousefi, B. H., Blume, T., Herz, M., Focke, C.,
Deussing, M., Peters, F., Lindner, S., von Ungern-Sternberg, B.,
Drzezga, A., et al. (2018) Comparison of (18)F-T807 and (18)F-
THK5117 PET in a Mouse Model of Tau Pathology. Front. Aging
Neurosci. 10, 174.
(17) Brendel, M., Jaworska, A., Probst, F., Overhoff, F., Korzhova,
V., Lindner, S., Carlsen, J., Bartenstein, P., Harada, R., Kudo, Y., et al.
(2016) Small-Animal PET Imaging of Tau Pathology with 18F-
THK5117 in 2 Transgenic Mouse Models. J. Nucl. Med. 57 (5), 792−
8.
(18) Allen, B., Ingram, E., Takao, M., Smith, M. J., Jakes, R., Virdee,
K., Yoshida, H., Holzer, M., Craxton, M., Emson, P. C., et al. (2002)
Abundant tau filaments and nonapoptotic neurodegeneration in
transgenic mice expressing human P301S tau protein. J. Neurosci. 22
(21), 9340−51.
(19) Wang, M., Gao, M., Xu, Z., and Zheng, Q. H. (2015) Synthesis
of a PET tau tracer [(11)C]PBB3 for imaging of Alzheimer’s disease.
Bioorg. Med. Chem. Lett. 25 (20), 4587−92.
(20) Bragina, M. E., Stergiopulos, N., and Fraga-Silva, R. A. (2017)
Fluorescence-Based Binding Assay for Screening Ligands of
Angiotensin Receptors. Methods Mol. Biol. 1614, 165−174.
(21) Ono, M., Sahara, N., Kumata, K., Ji, B., Ni, R., Koga, S.,
Dickson, D. W., Trojanowski, J. Q., Lee, V. M., Yoshida, M., et al.
(2017) Distinct binding of PET ligands PBB3 and AV-1451 to tau
fibril strains in neurodegenerative tauopathies. Brain 140 (3), 764−
780.
(22) Delobel, P., Lavenir, I., Fraser, G., Ingram, E., Holzer, M.,
Ghetti, B., Spillantini, M. G., Crowther, R. A., and Goedert, M. (2008)
Analysis of tau phosphorylation and truncation in a mouse model of
human tauopathy. Am. J. Pathol. 172 (1), 123−31.
ABBREVIATIONS
■
PET, positron emission tomography; Aβ, β-amyloid; AD,
Alzheimer’s disease; PSP, progressive supranuclear palsy; PiB,
Pittsburgh Compound B; NFT, neurofibrillary tangle; DTT,
dithiothreitol; 4R, four repeat tau; 3R, three repeat tau
REFERENCES
■
(1) Chiti, F., and Dobson, C. M. (2017) Protein Misfolding,
Amyloid Formation, and Human Disease: A Summary of Progress
Over the Last Decade. Annu. Rev. Biochem. 86, 27−68.
(2) Cistaro, A., Alongi, P., Caobelli, F., and Cassalia, L. (2018)
Radiotracers for Amyloid Imaging in Neurodegenerative Disease:
State-of-the-Art and Novel Concepts. Curr. Med. Chem. 25 (26),
3131−3140.
(3) Goedert, M., Yamaguchi, Y., Mishra, S. K., Higuchi, M., and
Sahara, N. (2018) Tau Filaments and the Development of Positron
Emission Tomography Tracers. Front Neurol 9, 70.
(4) Saint-Aubert, L., Lemoine, L., Chiotis, K., Leuzy, A., Rodriguez-
Vieitez, E., and Nordberg, A. (2017) Tau PET imaging: present and
future directions. Mol. Neurodegener 12 (1), 19.
(5) Kovacs, G. G. (2018) Tauopathies. Handb Clin Neurol 145,
355−368.
(23) Macdonald, J. A., Bronner, I. F., Drynan, L., Fan, J., Curry, A.,
Fraser, G., Lavenir, I., and Goedert, M. (2019) Assembly of transgenic
human P301S Tau is necessary for neurodegeneration in murine
spinal cord. Acta Neuropathol Commun. 7 (1), 44.
(6) Arendt, T., Stieler, J. T., and Holzer, M. (2016) Tau and
tauopathies. Brain Res. Bull. 126, 238−292.
(7) Goedert, M. (2018) Tau filaments in neurodegenerative
diseases. FEBS Lett. 592 (14), 2383−2391.
(24) Mellone, M., Kestoras, D., Andrews, M. R., Dassie, E.,
Crowther, R. A., Stokin, G. B., Tinsley, J., Horne, G., Goedert, M.,
Tolkovsky, A. M., et al. (2013) Tau pathology is present in vivo and
develops in vitro in sensory neurons from human P301S tau
transgenic mice: a system for screening drugs against tauopathies. J.
Neurosci. 33 (46), 18175−89.
(25) Brelstaff, J., Spillantini, M. G., and Tolkovsky, A. M. (2015)
pFTAA: a high affinity oligothiophene probe that detects filamentous
tau in vivo and in cultured neurons. Neural Regener. Res. 10 (11),
1746−7.
(8) Liang, S. H., Yokell, D. L., Normandin, M. D., Rice, P. A.,
Jackson, R. N., Shoup, T. M., Brady, T. J., Fakhri, G. E., Collier, T. L.,
and Vasdev, N. (2014) First human use of a radiopharmaceutical
prepared by continuous-flow microfluidic radiofluorination: proof of
concept with the tau imaging agent [18F]T807. Mol. Imaging 13,
7290.2014.00025.
(9) Leuzy, A., Chiotis, K., Lemoine, L., Gillberg, P. G., Almkvist, O.,
Rodriguez-Vieitez, E., and Nordberg, A. (2019) Tau PET imaging in
neurodegenerative tauopathies-still a challenge. Mol. Psychiatry 24 (8),
1112−1134.
(26) Brelstaff, J., Ossola, B., Neher, J. J., Klingstedt, T., Nilsson, K.
P., Goedert, M., Spillantini, M. G., and Tolkovsky, A. M. (2015) The
fluorescent pentameric oligothiophene pFTAA identifies filamentous
tau in live neurons cultured from adult P301S tau mice. Front.
Neurosci. 9, 184.
(10) Maruyama, M., Shimada, H., Suhara, T., Shinotoh, H., Ji, B.,
Maeda, J., Zhang, M. R., Trojanowski, J. Q., Lee, V. M., Ono, M., et al.
(2013) Imaging of tau pathology in a tauopathy mouse model and in
Alzheimer patients compared to normal controls. Neuron 79 (6),
1094−108.
(27) Zhang, W., Falcon, B., Murzin, A. G., Fan, J., Crowther, R. A.,
Goedert, M., and Scheres, S. H. (2019) Heparin-induced tau filaments
are polymorphic and differ from those in Alzheimer’s and Pick’s
diseases. eLife 8, e43584.
(28) Filipcik, P., Zilka, N., Bugos, O., Kucerak, J., Koson, P., Novak,
P., and Novak, M. (2012) First transgenic rat model developing
progressive cortical neurofibrillary tangles. Neurobiol. Aging 33 (7),
1448−56.
(11) Mueller, A., Bullich, S., Barret, O., Madonia, J., Berndt, M.,
Papin, C., Perrotin, A., Koglin, N., Kroth, H., Pfeifer, A., et al. (2020)
Tau PET imaging with (18)F-PI-2620 in patients with Alzheimer’s
disease and healthy controls: a first-in-human study. J. Nucl. Med. 61,
911.
(12) Hostetler, E. D., Walji, A. M., Zeng, Z., Miller, P., Bennacef, I.,
Salinas, C., Connolly, B., Gantert, L., Haley, H., Holahan, M., et al.
(2016) Preclinical Characterization of 18F-MK-6240, a Promising
H
https://dx.doi.org/10.1021/acschemneuro.0c00790
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
(29) Koson, P., Zilka, N., Kovac, A., Kovacech, B., Korenova, M.,
Filipcik, P., and Novak, M. (2008) Truncated tau expression levels
determine life span of a rat model of tauopathy without causing
neuronal loss or correlating with terminal neurofibrillary tangle load.
Eur. J. Neurosci 28 (2), 239−46.
I
https://dx.doi.org/10.1021/acschemneuro.0c00790
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX