Antibody-Based In Vivo PET Imaging Detects Amyloid-β Reduction in Alzheimer Transgenic Mice After BACE-1 Inhibition

Animals

tg-ArcSwe mice (20) were scanned with PET to study disease progression at the age of 7, 10, 13, and 16 mo. The effect of NB-360 treatment was investigated in mice aged 10 mo at the start of the treatment, that is, at an age characterized by moderate Aβ pathology (23) but no detectable ¹¹C-PiB PET signal (15). The number of mice in each age group is displayed in Supplemental Table 1 (supplemental materials are available at http://jnm.snmjournals.org).

Throughout the experiment, the animals were kept with free access to food and water in rooms with controlled temperature and humidity in an animal facility at Uppsala University. All experiments were approved by the Uppsala County Animal Ethics board (approval C17/14) following the rules and regulations of the Swedish Animal Welfare Agency and complied with the European Communities Council Directive of September 22, 2010.

Radioligand

The recently described radioligand ¹²⁴I-RmAb158-scFv8D3 was used for antibody-based PET imaging. Its expression and pharmacokinetics were described by Hultqvist et al. in 2017 (17). The radioligand is based on the well-studied antibody mAb158, which displays a selective binding to Aβ protofibrils (6,14,15). To increase the transport of mAb158 across the blood–brain barrier into the brain, 2 single-chain variable fragments of the transferrin receptor antibody 8D3 (16) were attached using short linkers to the C termini of the light chains of mAb158. This step enables monovalent transferrin receptor binding, which leads to efficient transcytosis over the blood–brain barrier. RmAb158-scFv8D3 was recombinantly expressed in Expi293 cells and purified as described previously (17).

Radiochemistry

RmAb158-scFv8D3 was labeled with ¹²⁴I using direct radioiodination (24). Radiolabeling was done in 6 batches; a 119 ± 31.3 MBq ¹²⁴I solution (PerkinElmer Inc.) was preincubated for 15 min with NaI at a concentration of 10 μM. RmAb158-scFv8D3 was mixed with phosphate-buffered saline (PBS) and added to the iodine solution at 0.6 μg/MBq to a final volume of 380 μL. The reaction was activated by addition of 40 μL of chloramine-T solution (1 mg/mL) and quenched after 120 s by addition of 80 μL of sodium metabisulfite solution (1 mg/mL). The radiolabeled protein was purified of free iodine and low-molecular-weight components with a disposable NAP-5 size-exclusion column with a molecular-weight cutoff of 5 kDa (GE Healthcare). The high-molecular-weight fraction containing the labeled protein was eluted with 1 mL of PBS.

PET Imaging

PET imaging was performed with ¹²⁴I-RmAb158-scFv8D3 using a Triumph Trimodality System (TriFoil Imaging, Inc.). To reduce thyroidal uptake of ¹²⁴I, the mice were given 0.5% NaI in the drinking water 1 d before radioligand injection and then 0.2% NaI until the PET scan. The mice in the disease-progression investigation were intravenously injected with 14.8 ± 2.6 MBq of ¹²⁴I-RmAb158-scFv8D3 with a specific activity of 234 MBq/nmol and scanned 4 d afterward. The mice in the NB-360 treatment experiment were intravenously injected with 7.4 ± 1.3 MBq of ¹²⁴I-RmAb158-scFv8D3 with a specific activity of 161 ± 12.7 MBq/nmol and scanned 6 d afterward. Before undergoing PET, the animals were anesthetized with 2.5% isoflurane, moved to the heated scanner bed, and kept anesthetized with 1.5%–2% isoflurane during the scan. The duration of the scan was 60 min, followed by a CT examination of 3 min (field of view, 8.0 cm).

Directly after scanning, the mice underwent a 2-min intracardiac perfusion with 0.9% NaCl. Brain and blood samples were collected, and ex vivo radioactivity was measured with a well counter (GE Healthcare).

PET data were reconstructed using 3-dimensional ordered-subsets expectation maximization (20 iterations). CT raw files were reconstructed using filtered backprojection. All subsequent processing of the PET and CT images was performed using the imaging software Amide, version 1.0.4 (25). The CT scans were manually aligned with a T2-weighted, MRI-based mouse brain atlas (26) containing outlined regions of interest for whole brain, cortex, and hippocampus. The PET images were then aligned with the CT images, and the atlas and PET data could be quantified in regions of interest.

Ex Vivo Autoradiography

Frozen right hemispheres of ¹²⁵I-RmAb158-scFv8D3–injected mice were cryosectioned (20 μm) with a Leica CM 1850 at −25°C. Sections were stored at −20°C until use. Fresh tissue sections were placed in an x-ray cassette and exposed to positron-sensitive phosphor screens (MultiSensitive; PerkinElmer) for 6 d. The plates were then scanned with a Cyclone Plus Imager system (Perkin Elmer) at a resolution of 600 dpi. Images were analyzed by ImageJ software, version 1.51.

NB-360 Treatment

Three groups of mice were investigated (Fig. 1; Supplemental Table 1). A NB-360–treated group was provided food containing the BACE-1 inhibitor NB-360 (Novartis) at a concentration of 0.5 g/kg for 3 mo, whereas a vehicle group was maintained on control food until the age of 13 mo. Mice from 4 litters were randomly allocated to the NB-360– or vehicle-treated groups, and all mice were studied during the same period. A baseline group, included to provide an estimate of the Aβ pathology level before treatment, had free access to control food until the age of 10 mo, when PET scans and analyses were conducted. All groups were scanned on the same occasion.

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FIGURE 1.

Overview of treatment and PET imaging. At age of 10 mo, animals were given either food containing BACE-1 inhibitor NB-360 or control food. At age of 13 mo, animals were injected with radioligand and were scanned with PET 6 d later. A third group was scanned with PET and analyzed at baseline age of 10 mo for comparison at starting point.

Brain Sample Preparation

After perfusion, brains were removed and dissected in half. The right hemisphere was either fixed in 4% PFA and embedded in paraffin or frozen on dry ice for later autoradiography analysis. The left hemisphere was immediately frozen on dry ice for further extraction and measurement of Aβ concentrations, as previously described (15,26). In short, brain tissue was homogenized with a tissue grinder (Teflon [The Chemours Co.] pestle), 2 × 10 strokes on ice, at a 1:5 weight-to-volume ratio in Tris-buffered saline (TBS) with cOmplete Protease Inhibitor Cocktail Tablets (Roche). A 250-μL volume of the homogenate was mixed with another 250 μL of TBS and centrifuged for 1 h at 16,000g. The supernatants were stored at −80°C until analysis. For extraction of TBS insoluble proteins, 27 μL of the original extract and 73 μL of 96% formic acid (FA) for an FA concentration of 70% were mixed for 30 s with a Kimble Pestle pellet grinder (Sigma-Aldrich) followed by 1 h of centrifugation at 16,000g. Supernatants were stored at −80°C until analysis.

Biochemical Aβ Analyses

To break Aβ aggregates for better detection (27), TBS extracts from all transgenic mice were supplemented with 1% sodium dodecyl sulfate and kept for 5 min at 95°C in a compact dry heat block (ThermoFisher) and then further diluted to 0.05% sodium dodecyl sulfate.

Prepared samples were analyzed with a V-PLEX Aβ Peptide Panel 1 Kit (Meso Scale Diagnostics) for Aβ38, Aβ40, and Aβ42 using neoepitope-specific antibodies to the different Aβ C termini in combination with 6E10, which binds near the Aβ N terminus. The assay was conducted according to the user’s manual, and the plates were read using a Meso Scale Diagnostics imager.

Aβ protofibrils and oligomers were measured with a homogeneous ELISA, using the Aβ N terminus–specific 82E1 (IBL International/Tecan Trading AG) as both capture and detection antibody and a calibration curve of synthetic Aβ protofibrils for quantification (18). A 96-well half-area plate was coated overnight with 12.5 ng of 82E1 per well, followed by blocking with 1% bovine serum albumin in PBS. TBS brain extracts were diluted 1:25 and incubated overnight at 4°C. Soluble Aβ aggregates with a size of at least a dimer were then detected with biotinylated 82E1 (0.25 μg/mL) and streptavidin–horseradish peroxidase (Mabtech AB), diluted 1:4,000. Signals were developed with K blue aqueous tetramethylbenzidine substrate (Neogen Corp.) and read with a spectrophotometer at 450 nm.

For ELISA measurement of total Aβx-40 and Aβx-42, 96-well plates were coated overnight with 100 ng per well of polyclonal rabbit anti-Aβ40 or anti-Aβ42 (Agrisera) and blocked with 1% bovine serum albumin in PBS. FA-soluble brain extracts were neutralized with 2 M Tris and diluted 1:10,000 for Aβ40 and 1:750 for Aβ42 analysis and then were incubated overnight at 4°C. After incubation with biotinylated 6E10 (Nordic BioSite) (0.5 μg/mL) and streptavidin–horseradish peroxidase (Mabtech AB), diluted 1:5,000, signals were developed and read as above. All sample and secondary antibody dilutions were made in ELISA incubation buffer (0.1% bovine serum albumin, 0.05% polysorbate 20 in PBS).

Immunohistochemistry

Five-micrometer sections from fixed, paraffin-embedded right-brain hemispheres were processed with an electronic rotary microtome (Hm340E; Thermofisher). Sections were deparaffinized and incubated in 3% H₂O₂ and 10% methanol in water for 15 min. Nonspecific binding was blocked with 3% bovine serum albumin in PBS-polysorbate 20 (0.1%) for 1 h and incubated overnight with a polyclonal rabbit-anti-Aβ40 antibody (Agrisera). Sections were incubated with biotinylated goat anti-rabbit antibody (Vector Laboratories Inc.) for 2 h at room temperature, followed by PBS washes and a 45-min incubation with avidin/biotin complex (Vector Laboratories). The staining was visualized by 3-min 3,3′-diaminobenzidine development and mounted with DPX mounting medium (a mixture of distyrene, tricresyl phosphate and xylene; Sigma-Aldrich). Pictures were captured with a DXM1200F microscope (Nikon Instruments Inc.) at a magnification of ×5 and further processed with Photoshop CC photomerge (Adobe) to a whole-brain panorama.

For Aβ and glial fibrillary acidic protein (GFAP) immunofluorescence double staining, sections were deparaffinized, underwent antigen retrieval in citrate buffer at 86°C for 20 min, and were further permeabilized and blocked for 1 h with 5% normal goat serum and 0.3% Triton X-100 (The Dow Chemical Co.) in PBS. Aβ and GFAP were stained with 6E10 (Nordic BioSite) and anti-GFAP (Dako), respectively. Alexa Fluor 488 anti-mouse and Alexa Fluor 555 anti-rabbit (Life Technologies) were used as secondary antibodies. Pictures were captured with an LSM700 confocal laser scanning microscope (Zeiss), and the software Zen 2012 was used for image processing.

Statistics

All statistical analyses and graphs were done in Prism, version 6 (GraphPad Software, Inc.). Group results are reported as mean ± SD. Data were analyzed with 1-way ANOVA followed by the Bonferroni post hoc test.