ACS Chemical Neuroscience
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
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Franklin I. Aigbirhio − Molecular Imaging Chemistry
Laboratory, Wolfson Brain Imaging Centre and Department
of Clinical Neurosciences, University of Cambridge,
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.
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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:
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
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* Supporting Information
The Supporting Information is available free of charge at
Notes
The authors declare no competing financial interest.
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ACS Chem. Neurosci. XXXX, XXX, XXX−XXX