[18F]GE-180 PET Detects Reduced Microglia Activation After LM11A-31 Therapy in a Mouse Model of Alzheimer's Disease

Abstract

Microglial activation is a key pathological feature of Alzheimer's disease (AD). PET imaging of translocator protein 18 kDa (TSPO) is a strategy to detect microglial activation in vivo. Here we assessed flutriciclamide ([¹⁸F]GE-180), a new second-generation TSPO-PET radiotracer, for its ability to monitor response to LM11A-31, a novel AD therapeutic in clinical trials. AD mice displaying pathology were treated orally with LM11A-31 for 3 months. Subsequent [¹⁸F]GE-180-PET imaging revealed significantly lower signal in cortex and hippocampus of LM11A-31-treated AD mice compared to those treated with vehicle, corresponding with decreased levels of TSPO immunostaining and microglial Iba1 immunostaining. In addition to detecting decreased microglial activation following LM11A-31 treatment, [¹⁸F]GE-180 identified activated microglia in AD mice with greater sensitivity than another second-generation TSPO radiotracer, [¹⁸F]PBR06. Together, these data demonstrate the promise of [¹⁸F]GE-180 as a potentially sensitive tool for tracking neuroinflammation in AD mice and for monitoring therapeutic modulation of microglial activation.

Keywords: Alzheimer's disease; LM11A-31.; PET; TSPO; [18F]GE-180; flutriciclamide; neuroinflammation.

Figure 1

Schematic representation of experimental approach. (A) Head-to-head comparison of two second generation PET radiotracers targeting the translocator protein 18 kDa (TSPO) - i.e., [¹⁸F]PBR06 and [¹⁸F]GE-180 - in a mouse model of Alzheimer's disease (AD). The same 8.5-10 month old APP^L/S mice and wild-type (wild-type) littermates were imaged with [¹⁸F]PBR06 and [¹⁸F]GE-180, 2 days apart. (B) Timeline and study design for evaluating TSPO-PET as an imaging biomarker strategy to monitor response to a novel AD therapeutic (i.e., LM11A-31) currently in clinical trials. Based on findings from the head-to-head comparison study, the most sensitive TSPO-PET ligand was selected for therapy monitoring studies. Baseline PET imaging of APP^L/S mice with pathology and wild-type littermates (5.5-7 months of age) was performed just prior to commencing treatment with LM11A-31, and again at the conclusion of the study after mice had been treated daily with LM11A-31 for 3 months. Ex vivo autoradiography and histology were conducted immediately following the final PET imaging time-point. TSPO and Iba1 immunostaining were performed to investigate the relationship between TSPO-PET signal and levels of target (i.e., TSPO and activated microglia).

Figure 2

[¹⁸F]GE-180 is more sensitive than [¹⁸F]PBR06 for detecting elevated levels of microglial activation in APP^L/S mice. (A) Time activity curves show the accumulation of [¹⁸F]GE-180 in cortex and hippocampus of 8.5-10 month old APP^L/S (n = 6) and wild-type mice (n = 6), and in APP^L/S mice pre-treated with PK11195 (1 mg/kg) (n = 4). (B) Graphs from head-to-head comparison studies depict uptake (% injected dose per gram, % ID/g) of either [¹⁸F]PBR06 or [¹⁸F]GE-180 in the same 8.5-10 month old APP^L/S (n = 7) versus wild-type mice (n = 6). *p-value <0.05.

Figure 3

Identification of the rostral thalamus as a suitable pseudo-reference region for [¹⁸F]GE-180 in APP^L/S and wild-type mice. (A) [¹⁸F]GE-180 uptake in cerebellum, hypothalamus, medulla, midbrain, olfactory, pons, striatum, and thalamus of wild-type (n = 6) and APP^L/S mice (n = 7) aged 8.5-10 months. Uptake values are shown as percent injected dose per gram (% ID/g). (B) Brain regions of interest (ROIs) defined using a segmented 3D mouse brain atlas and VivoQuant image analysis software after co-registering brain atlas to PET/CT data. ROI for the rostral thalamus (rThal) is shown in dark blue in four consecutive coronal brain slices used for analysis. Cortex (grey), corpus callosum (apricot), ventricles (light blue), white matter (red), striatum (pink), pallidum (yellow), and hypothalamus (green), are also shown. (C) Representative 20x pictures of TSPO immunostaining in rThal and caudal thalamus (cThal), (D) [¹⁸F]GE-180 uptake (% ID/g) in rThal and cThal, and (E) PET uptake ratio values determined by dividing % ID/g in cortex or hippocampus by % ID/g in rThal. Standard error of mean (SEM) is shown. *p<0.05, **p<0.01.

Figure 4

[¹⁸F]GE-180-PET detects a significant drug effect in AD mice treated with LM11A-31 (C31). (A) Representative coronal brain PET/MR images of APP^L/S and wild-type mice that have been treated with either C31 or vehicle (veh). Images visually depict increased PET signal in cortex and hippocampus of APP^L/S-veh mice (n = 9) compared to age-matched wild-type littermates (n = 10) and APP^L/S mice treated with C31 (n = 8). White and red arrows point to cortex and hippocampus respectively. (B) Quantitation of [¹⁸F]GE-180 uptake in cortex and hippocampus using the rostral thalamus (rThal) as a reference region. Error bars are standard error of mean (SEM). **p-value <0.01, ***p-value <0.005.

Figure 5

[¹⁸F]GE-180 ex vivo autoradiography of 8.5-10 month old APP^L/S and wild-type mice following 3 months treatment with LM11A-31 (C31) or vehicle. (A) Representative autoradiography images and Nissl staining of the same brain sections show a similar pattern of uptake to that observed in [¹⁸F]GE-180-PET/MR images. White and red arrows point to cortex and hippocampus respectively. (B) Mean signal intensity for specific brain regions normalized to rostral thalamus (rThal) for all four groups of mice (n = 7 per group). *p-value <0.05, **p-value <0.01, ***p-value <0.005.

Figure 6

LM11A-31 (C31) attenuates microglial activation in cortex and hippocampus of APP^L/S mice. Representative 10x images of TSPO and Iba1 staining in cortex and hippocampus from 8.5-10 month old APP^L/S and wild-type mice, treated daily via oral gavage for 3 months with LM11A-31 (C31) or vehicle (veh). Scale bar = 250 μm. Quantitation of mean pixel intensity or percent cortical/hippocampal area occupied for TSPO and Iba1 staining respectively (n = 7-8 per group). *p-value <0.05, **p-value <0.01, ***p-value <0.005.

Figure 7

Correlation between the levels of microglial activation, TSPO, and [¹⁸F]GE-180 brain-PET signal in 8.5-10 month old APP^L/S and wild-type mice after treatment. Correlation between Iba1 and TSPO immunostaining in (A) cortex, and (B) hippocampus (n = 7-8 per group). Correlation between Iba1 immunostaining and PET signal in (C) cortex, and (D) hippocampus (n = 6-7 per group). Correlation between TSPO immunostaining and PET signal in (E) cortex, and (F) hippocampus (n = 6-7 per group).