TEMPOL was acquired from Tocris Bioscience (Fisher Scientific). Animal experiments were conducted per the guidelines established by the Vanderbilt University’s Institutional Animal Care and Use Committee (IACUC) and the Division of Animal Care. The performed work was approved by Vanderbilt IACUC with an extended protocol, M1700044-01. In addition, all of the works involving live animals were compliant with the ARRIVE guidelines. In a typical imaging procedure, anesthetized mice received 2% of isoflurane via inhalation, supplied with oxygen using a precision vaporizer.
General synthesis
All commercially available reagents and solvents were used as received. L-(+)-Ergothionene was purchased from Cayman Chemicals. Reactions were montitored by a Agilent LC/MS 1260 Infinity II. Products were purified using a Telodyne Combiflash Rf automated purification instrument using normal phase or reverse phase. NMR spectra were recorded on a 400 MHz Bruker AVANCE 400 equipped with a 9.3 T Oxford Magnet or 600 MHz Bruker AVIII console equipped with a 14.1 T Bruker Magnet. 1H NMR chemical shifts were referenced to the residual solvent signal; 13C NMR chemical shifts were referenced to the deuterated solvent signal. Data are presented as follows: chemical shift d (ppm), multiplicity (s = singlet, d = doublet, dd = doublet of doublet, t = triplet, m = multiplet).
Synthesis of the PET precursor (Fig. 3)
N
τ-(((9H-Fluoren-9-yl)methoxy)carbonyl)-N
α-(tert-butoxycarbonyl)-L-histidine 2
To a stirring solution of histidine amino acid 1 (1.0 g, 3.9 mmol), FMOC-succinimide (1.45 g, 4.3 mmol), and DIPEA (0.605 g, 4.7 mmol) in methylene chloride (50 mL) and stirred overnight. Reaction was diluted with water and methylene chloride. The product was extracted 3 × with methylene chloride. The organic layers were combined, washed with 1 M HCl, saturated sodium bicarbonate, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to provide a desired product with 60% yield.
1H CDCl3 (400.13 mHz): 8.26 (d, J = 1.2 Hz, 1H); 7.79 (d, J = 7.6 Hz, 2H); 7.56 (d, J = 7.6 Hz, 2H); 7.43, (t, J = 7.2 Hz, 2H); 7.34 (t, J = 7.2 Hz, 2H); 7.24 (s, 1H); 5.51 (d, J = 6.0 Hz, 1H); 4.70 (dd, J1 = 7.2 Hz, J2 = 1.6 Hz, 2H); 4.56 (t, J = 7.6 Hz, 1H); 4.35 (t, J = 6.8 Hz, 1H); 3.32 (dd, J1 = 14.8 Hz, J2 = 2.8 Hz, 1H); 3.20 (dd, J1 = 14.4 Hz, J2 = 5.2 Hz, 1H); 1.47 (s, 9H). 13C CDCl3 (100.6 mHz): 172.9, 168.6, 155.4, 148.1, 142.7, 141.5, 137.5, 128.4, 127.6, 124.9, 120.5, 115.6, 80.0, 70.6, 53.6, 52.8, 46.6, 29.9, 28.5.
(9H-Fluoren-9-yl)methyl(S)-4-(3-(tert-butoxy)-2-((tert-butoxycarbonyl)amino)-3-oxopropyl)-1H-imidazole-1-carboxylate 3
To a stirring solution of 2 (3.90 mmol) in methylene chloride (50 mL) and tert-butanol (10 mL)at 0 C was added: EDC-HCl (744 mg, 3.9 mmol), and DMAP (95 mg, 0.78 mmol. This solution was allowed to warm up to r.t. and stirred overnight. Reaction was diluted with water and methylene chloride. The product was extracted 3 × with methylene chloride. The organic layers were combined, washed with 1 M HCl, saturated sodium bicarbonate, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to provide the desired product (12% yield).
1H CDCl3 (400.13 mHz): 7.99 (s, 1H); 7.81 (d, J = 7.6 Hz, 2H); 7.58 (d, J = 7.6 Hz, 2H); 7.48, (t, J = 7.2 Hz, 2H); 7.35 (t, J = 7.2 Hz, 2H); 7.17 (s, 1H); 5.03 (m, 1H); 4.72 (dd, J1 = 6.4 Hz, J2 = 2.0 Hz, 2H); 4.20 (m, 1H); 3.03 (m, 2H); 1.44 (s, 9H).
(9H-Fluoren-9-yl)methyl (S)-4-(2-amino-3-(tert-butoxy)-3-oxopropyl)-1H-imidazole-1-carboxylate 4
To a stirring solution of 3 (3.90 mmol) in methylene chloride at − 78 °C was added TFA (5 mL) dropwise. This solution was allowed to warm up to 0 °C and stirred until completion. The solution was then neutralized with sodium bicarbonate and diluted with water and methylene chloride. The product was extracted 3 × with methylene chloride. The organic layers were combined, washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to provide the desired product with 63% yield.
1H CDCl3 (400.13 mHz): 7.99 (s, 1H); 7.77 (d, J = 7.6 Hz, 2H); 7.57 (d, J = 6.8 Hz, 2H); 7.41, (t, J = 6.0 Hz, 2H); 7.33 (t, J = 7.6 Hz, 2H); 7.14 (s, 1H); 5.03 (m, 1H); 4.59 (m, 2H); 4.18 (m, 1H); 3.05 (m, 2H); 1.38 (s, 9H).
Tert-Butyl N
α,N
α-dimethyl-L-histidinate 5
To a stirring solution of 4 in MeOH (20 mL) was added NaBH3CN (362 mg, 5.8 mmol) and CH2O (37% in H20, 702 mg, 23.4 mmol). The reaction was capped and stirred for 2 h. The resulting solution was then concentrated via rotovap. The crude oil was purified by reverse phase chromatography. The product was verified by LC/MS and used as is in the next step with a 82% yield. The Fmoc group was removed in this step through interaction with the NaBH3CN to provide the desired product.
1H D2O (400.13 mHz): 7.47 (s, 1H); 6.83 (s, 1H); 4.79 (m, 1H); 4.29 (m, 2H); 2.58 (s, 6H); 1.24 (s, 9H).
Tert-Butyl (S)-2-(dimethylamino)-3-(2-thioxo-2,3-dihydro-1H-imidazol-4-yl)propanoate 6
To a stirring solution of 5 (31.5 mg, 0.13 mmol) in water (2 mL) and diethyl ether (2 mL) was added sodium bicarbonate (65 mg, 0.78 mmol), and phenylchloro thionoformate (24.7 mg, 0.14 mmol) and stirred overnight. Reaction was diluted with water and diethyl ether. The product was extracted 3 × with diethyl ether. The organic layers were combined, washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The resulting oil was then redissolved in MeOH (5 mL) and triethylamine (55 μL) was added. This solution was stirred overnight. The resulting solution was then concentrated under reduced pressure. Product was purified by reverse phase chromatography to provide a final product (16.0 mg) 45% yield. And the product was immediately used for the next step.
Tert-Butyl (S)-4-(3-(tert-butoxy)-2-(dimethylamino)-3-oxopropyl)-2-((tert-butoxycarbonyl)thio)-1H-imidazole-1-carboxylate 7
To a stirring solution of 6 (16.0 mg, 0.066 mmol) in methylene chloride (10 mL) was added: Boc anhydride (32 mg, 0.145 mmol), and DIPEA (19 mg, 0.145 mmol) and stirred over the course of 48 h. The resulting solution was concentrated and purified by flash chromatography (0–50% CH2Cl2/(20%MeOH/CH2Cl2)) to afford the precursor 7 (15.0 mg) with 48% yield.
1H CDCl3 (600.13 mHz): 7.16 (s, 1H); 3.40 (m, 1H); 2.86 (dd, J1 = 14.4 Hz, J2 = 8.4 Hz, 1H); 2.72 (dd, J1 = 14.4 Hz, J2 = 6.6 Hz, 1H); 2.28 (s, 6H); 1.48 (s, 9H); 1.37 (s, 9H); 1.32 (s, 9H). 13C CDCl3 (150.9 mHz): 170.9, 165.3, 146.7, 139.9, 135.1, 119.8, 86.8, 85.8, 81.2, 67.3, 41.8, 29.8, 28.6, 28.3, 28.2, 27.9, 22.3, 22.0.
MS: calculated: 471.2403, detected: 471.1845.
[11C]ERGO radiotracer synthesis
The [11C]CO2 was made by irradiating a target filled with nitrogen and 1% oxygen gas with protons. The [11C]CO2 was then trapped on nickel Shimalite with molecular sieves at room temperature. The [11C]CO2 was then converted to [11C]CH4 by heating the trapped [11C]CO2 to 400 °C in the presence of hydrogen gas. The [11C]CH4 was then released from the nickel Shimalite at 400 °C and isolated on molecular sieves at -75 °C. The [11C]CH4 was then converted to [11C]MeI via a recirculation through gaseous iodine at ~ 720 °C, with the [11C]MeI being trapped on Porapak N with each cycle. The [11C]MeI was then released from the Porapak N by heating with a gentle flow of helium that is passed through an AgOTf impregnated column at ~ 200 °C to convert the [11C]MeI to [11C]MeOTf. This [11C]MeOTf was bubbled into a solution of precursor in 250 μL acetonitrile at − 10 °C. After the activity transfer was complete, the reaction mixture was heated to 80 °C for 2 min. At this time, hydrochloric acid (6 M, 250 μL) was added, and the reaction mixture was heated at 70 °C for 5 min, cooled to room temperature, and diluted with water (1 mL). The reaction mixture was passed through an ion-retardation resin (Ag11–A8, 3 g) into the product vial, and the resin was rinsed with water (5 mL) into the product vial. The product was then transferred to the final vial, and an aliquot was removed for quality control analysis.
The radiochemical purity and the identity of the [11C]ERGO were characterized using an analytical HPLC system, equipped with a UV absorption detector (λ = 254 nm) and a radio-detector (Bioscan Flow-Count). The chromatography setup included a SeQuant ZIC-HILIC 150 × 4.6 mm with a typical mobile phase of acetonitrile (75%) in water at a flow rate of 1 mL/min. The identity of the [11C]ERGO was confirmed by comparing the retention time with co-injected and unlabeled ERGO along with the gamma peak. The molar activity of the radioligand is 690 TBq/mmol.
PET/CT data processing and analysis
The dynamic acquisition was divided into twelve 5 s frames, four 60 s frames, five 120 s frames, three 5 min frames, and six 10 min scans. The data from all possible lines of response (LOR) were saved in the list mode raw data format. The raw data were then binned into 3D sinograms with a span of 3 and ring difference of 47. The images were reconstructed into transaxial slices (128 × 128 × 159) with voxel sizes of 0.0815 × 0.0815 × 0.0796 cm3, using the MAP algorithm with 16 subsets, 4 iterations, and a beta of 0.0468. For anatomical co-registration, immediately following the PET scans, the mice received a CT scan in a NanoSPECT/CT (Mediso, Washington DC) at an X-ray beam intensity of 90 mAs and x-ray peak voltage of 45 kVp. The CT images were reconstructed into 170 × 170 × 186 voxels at a voxel size of 0.4 × 0.4 × 0.4 mm3. The PET/CT images were uploaded into Amide software (www.sourceforge.com), co-registered to an MRI template made in-house, and volumetric regions-of-interest were drawn around the cortex, hippocampus, striatum, thalamus, and cerebellum in addition to the whole brain. The PET images were normalized to the injected dose, and the time-activity-curves (TACs) of the mean activity within the ROIs were estimated for the entire duration of the scans.
Static PET scan
The mice were injected with ~ 20 MBq/0.1 mL 11C-ERGO via the tail vein and returned to their cages for 10 min. Then they were anesthetized with 2% isofluorane and imaged in an Inveon microPET (Siemens Pre-clinical, Knoxville, TN, USA) for 30 min in static mode followed by a CT scan using a NanoSPECT/CT (Mediso, Washington DC) at an X-ray beam intensity of 90 ms and X-ray voltage of 45 kVp. PET images were reconstructed using the iterative MAP reconstruction algorithm with 18 iterations and a beta smoothing value of 0.001 into 128 × 128 × 95 slices with a voxel size of 0.388 mm × 0.388 mm × 0.796 mm. The CT images were reconstructed into 170 × 170 × 126 slices at a voxel size of 0.4 × 0.4 × 0.4 mm3. The PET and CT images were co-registered using the imaging tool AMIDE.
Cardiac perfusion procedure and tissue collection
At 5 or 10 min post injection, anesthetized mice were laid on the shallow tray filled with crushed ice and the thoracic cavity was accessed using a scalpel after making 5–6 cm mid-line incision from the abdominal area. After careful separation of the liver from the diaphragm, the thoracic opening was stabilized with a retractor. Perfusion was performed as we described in the past55,56,57. Following removal of air bubbles, approximately 30 mL (pH7.4) of ice-cold PBS buffer is slowly injected in the left ventricle toward the ascending aorta using a 25-G syringe while the right atrium was snipped off using a curved point squeeze snip scissors to facilitate drainage of the systemic venous return. Then, 30 mL of 4% paraformaldehyde (PFA, pH 7.4) was perfused. When completed, tissues were harvested and weighed before counting using the automatic gamma counter (Hidex).
Animal models
5XFAD and control C57BL/6J mice were maintained at Vanderbilt University under standard conditions, in a 12-h light/dark cycle, and with free access to food and water. The 5XFAD mice over express both mutant human amyloid precursor protein (APP) and presenilin 1 (PS1), correlating with high burden and accelerated accumulation of the Aβ. A colony of 5XFAD transgenic mice obtained from Jackson Laboratories was maintained by crossing 5XFAD mice with a WT C57BL/6J strain. The 5XFAD mice were maintained as heterozygous.
The mouse model of LPS-induced neuroinflammation was developed based on past reports50,51. The LPS derived from Escherichia coli O111:B4 (Sigma Aldrich, St Louis, MO) was formulated in sterilized dd. water and given a high dose of LPS formulation (5 mg/kg) through intraperitoneal injection 24 h before PET imaging. This high dose would result in approximately 12–13% body weight loss over the course of 24 h. After PET imaging, animals were sacrificed, and the brains were collected for histology analysis.
Immunohistochemistry
Brains embedded in OCT were cut into sagittal sections (10 µm) using a Tissue-Tek cryostat and mounted onto charged glass slides. Prior to staining, slides were washed with PBS (10 min); then, they were treated with blocking buffer (5% normal goat serum, 0.2% Triton X-100, 0.5% bovine albumin in PBS) for 1 h at room temperature. The treated sections were then incubated overnight at 4 °C with primary anti-GFAP antibody (1:100 dilution, Biolegend San Diego, CA, USA). Slides were washed with PBS (3×) for 10 min each, the sections were subsequently incubated with secondary antibody goat anti-mouse Alexa Fluor 488 (1:200 dilution, Thermo Fisher Scientific, Carlsbad, CA, USA) for 30 min at room temperature. The sections were then washed with PBS twice for 10 min and once for 30 min, and cover slipped with an antifade mounting medium (Vector Laboratories, Burlingame, CA, USA) before observation under a fluorescence microscope.
Statistical analysis
Unpaired t test was used to compare the mean signal (%ID/g) difference between two independent subjects. A p value of ≤ 0.05 was considered as a statistically significant difference.
