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Efficacy of chronic BACE1 inhibition in PS2APP mice depends on the regional Aβ deposition rate and plaque burden at treatment initiation

Beta secretase (BACE) inhibitors are promising therapeutic compounds currently in clinical phase II/III trials. Preclinical [18F]-florbetaben (FBB) amyloid PET imaging facilitates longitudinal monitoring of amyloidosis in Alzheimer's disease (AD) mouse models. Therefore, we applied this theranostic concept to investigate, by serial FBB PET, the efficacy of a novel BACE1 inhibitor in the PS2APP mouse, which is characterized by early and massive amyloid deposition. Methods: PS2APP and C57BL/6 (WT) mice were assigned to treatment (PS2APP: N=13; WT: N=11) and vehicle control (PS2APP: N=13; WT: N=11) groups at the age of 9.5 months. All animals had a baseline PET scan and follow-up scans at two months and after completion of the four-month treatment period. In addition to this longitudinal analysis of cerebral amyloidosis by PET, we undertook biochemical amyloid peptide quantification and histological amyloid plaque analyses after the final PET session. Results: BACE1 inhibitor-treated transgenic mice revealed a progression of the frontal cortical amyloid signal by 8.4 ± 2.2% during the whole treatment period, which was distinctly lower when compared to vehicle-treated mice (15.3 ± 4.4%, p<0.001). A full inhibition of progression was evident in regions with <3.7% of the increase in controls, whereas regions with >10% of the increase in controls showed only 40% attenuation with BACE1 inhibition. BACE1 inhibition in mice with lower amyloidosis at treatment initiation showed a higher efficacy in attenuating progression to PET. A predominant reduction of small plaques in treated mice indicated a main effect of BACE1 on inhibition of de novo amyloidogenesis. Conclusions: This theranostic study with BACE1 treatment in a transgenic AD model together with amyloid PET monitoring indicated that progression of amyloidosis is more effectively reduced in regions with low initial plaque development and revealed the need of an early treatment initiation during amyloidogenesis.


Aβ-PET
Mice were anesthetized with isoflurane (1.5%, delivered at 3.5 L/min) and received an injection of 10.6 ± 2.4 MBq [ 18 F]-florbetaben to a tail vein. After placement in the aperture of the Inveon DPET (Preclinical Solutions, Siemens Healthcare Molecular Imaging), a single frame emission recording was obtained in the interval 30-60 min p.i., followed by a 15-min transmission scan using a rotating [ 57 Co] point source. The image reconstruction procedure consisted of a 3D ordered subset expectation maximization (OSEM, 4 iterations) and a 3D maximum a posteriori (MAP,32 iterations) at a zoom factor of 1.0, and with scatter, attenuation, and decay corrections, resulting in a final voxel dimension of 0.78 x 0.78 x 0.8 mm. The origin of the Aβ-PET images was concealed from the operator for further analyses.
Static 30-60 min datasets were co-registered to an MRI mouse atlas (Dorr, Sled et al. 2007) by a manual rigid-body transformation (TXrigid) using the PMOD fusion tool (V3.5, PMOD Technologies Ltd.). Templates were generated by averaging all age specific PET scans of TG and WT groups. In the second step, a reader-independent fine co-registration to specific templates was performed (Overhoff, Brendel et al. 2016). Here, the initial manual µPET-to-MRI atlas fusion images were normalized by non-linear brain normalization (TXBN) to the age-specific templates using the PMOD brain normalization tool (equal modality; smoothing by 0.6 mm; nonlinear warping; 16 iterations; frequency cutoff 3; regularization 1.0; no thresholding). The concatenation of TXrigid and TXBN was then applied to µPET frames in the native space, so as to obtain optimal resampling with a minimum of interpolation. For VOI-based analyses, a hindbrain white matter VOI comprising 67 mm³ served for normalization and two bilateral frontal cortex VOIs comprising 12 mm³ each, were employed for c alculation of Aβ-PET cortex-to-cerebellum standardized-uptake-value-ratios (SUVRCTX/CBL), as described earlier (Overhoff, Brendel et al. 2016).

Histochemical analyses
Cerebral hemispheres randomly selected for histochemistry were fixed by immersion in 4% paraformaldehyde at 4 °C for two days. A mean of five representative 50 µm thick slices per animal were then cut in the sagittal plane about 1.5 mm from the midline using a vibratome (VT 1000 S, Leica, Wetzlar, Germany). The slices were permeabilized overnight in 2% Triton X-100 in phosphate buffered saline (PBS; pH 7.4) at room temperature. The unbound dye was removed by three washing steps with PBS, and the slices were then mounted on microscope slides with fluorescent mounting medium (Dako, Germany). 3D image stacks for each hemisphere were acquired on an anepi-fluorescence microscope (Axio Imager.M2 with ApoTome.2, Jena, Zeiss, Germany). Imaging of the whole slice was performed in tile scan mode, which allows automatic stitching of an array of fields of view. For this imaging, th e methoxy-X04 was excited at 405 nm, and the emitted light was collected from 410 to software (Imaris 7. 6.5; Bitplane, Zurich) in a region (0.27 ± 0.04 mm³) defined to match the frontal cortex VOI as applied for Aβ-PET image analysis.

Supplemental Figure S2
Supplemental Figure S2: Isobaric lines (2% steps) depict amyloid progression in TG-VEH mice between baseline and terminal PET scans. Axial slices through the cranial half of the mouse brain are illustrated upon a T1w MRI template.

Supplemental Figure S3
Supplemental Figure S3: Individual longitudinal Aβ-PET imaging of BACE1 inhibition versus vehicle in high (A) and low (B) Aβ accumulating brain regions.