Positron Emission Tomography in Animal Models of Alzheimer’s Disease Amyloidosis: Translational Implications

Animal models of Alzheimer’s disease amyloidosis that recapitulate cerebral amyloid-beta pathology have been widely used in preclinical research and have greatly enabled the mechanistic understanding of Alzheimer’s disease and the development of therapeutics. Comprehensive deep phenotyping of the pathophysiological and biochemical features in these animal models is essential. Recent advances in positron emission tomography have allowed the non-invasive visualization of the alterations in the brain of animal models and in patients with Alzheimer’s disease. These tools have facilitated our understanding of disease mechanisms and provided longitudinal monitoring of treatment effects in animal models of Alzheimer’s disease amyloidosis. In this review, we focus on recent positron emission tomography studies of cerebral amyloid-beta accumulation, hypoglucose metabolism, synaptic and neurotransmitter receptor deficits (cholinergic and glutamatergic system), blood–brain barrier impairment, and neuroinflammation (microgliosis and astrocytosis) in animal models of Alzheimer’s disease amyloidosis. We further propose the emerging targets and tracers for reflecting the pathophysiological changes and discuss outstanding challenges in disease animal models and future outlook in the on-chip characterization of imaging biomarkers towards clinical translation.


Cerebral Glucose Metabolism Imaging
Brain glucose dysregulation plays an important role in AD [89]. Post-mortem studies reported higher levels of brain tissue glucose concentration, lower levels of glucose transporter 3, and glycolytic flux in the brain from patients with AD, compared with controls, associating with the severity of AD pathology [89]. [ 18 F]FDG PETs have been routinely used for detecting the reduced cerebral glucose metabolism (CMRglc) in disease-specific brain regions in patients with AD, Frontotemporal dementia, and Parkinson's disease to improve the diagnostic accuracy [9,90]. In lab settings, [  mental conditions such as anesthesia and handling, as well as genotype, age, and gender of the animal models [96]. Most of the studies in rodent amyloidosis models reported a global reduction in CMRglc, although few exceptions of increased CMRglc (associating with gliosis) were also reported [61]. A recent study by Xiang et al. further showed that microglial activation states drive glucose uptake and [ 18 F]FDG-PET alterations [97]. Table 2. PET imaging in of neurotransmitter receptors, blood-brain barriers, enzymes, metabolism, and synaptic density in animal models of Alzheimer's disease amyloidosis.

Synaptic Vesicle Glycoprotein 2A
Synapse loss is reported in the post-mortem frontal cortex of patients with AD, correlating with cognitive severity [135]. Synaptic vesicle glycoprotein 2A (SV2A) is located at the synapses across the entire brain and is the binding site for the antiepileptic drug levetiracetam [136]. SV2A involves in vesicle trafficking exocytosis and is crucial for neurotransmission and postnatal brain development [137]. Mendoza-Torreblanca et al. suggested that SV2A either regulates the presynaptic Ca 2+ levels during repetitive activity or is a target for residual Ca 2+ . Higher loads of cerebral Aβ deposits have been reported in the brain of SV2A knock-out mice, compared with control littermates [138]. A 40% reduction in SV2A signal by PET using [ 11 C]UCB-J was observed in the hippocampus in patients with AD, compared with cognitively normal control cases [139,140]. Kong et al. showed that SV2A over-expression was associated with the downregulation of β-site APP-cleaving enzyme 1 and apolipoprotein E genes, indicating that SV2A impacts Aβ production. However, Nowack et al. showed that overexpression of SV2A increased synaptic levels of the calcium-sensor protein synaptotagmin, resulting in a neurotransmission deficit [141]. Thus, modulation of SV2A as a potential treatment requires careful dosing and close monitoring of the SV2A levels. Several SV2A PET imaging tracers have been developed including [ Table 2). PET measures of Aβ deposition were found associated with regional synaptic density measured by [ 11 C]UCB-J in patients with early AD [139,146]. Few studies have reported on SV2A imaging in AD animal models. Bertoglio et al. demonstrated that [ 11 C]UCB-J is bound specifically to SV2A in mouse brain and that the radioligand binding can be quantified by kinetic modeling using an image-derived input function [147]. Toyonaga [115]. The results from a static (30-60 min post-injection) [ 18 F]SynVesT-1 PET scan were found comparable to kinetic modeling results [115].

Glutamate Receptors
The glutamate receptors are classified into the N-methyl-D-aspartate receptor (NM-DAR), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-kainate receptor, and metabotropic glutamate receptors (mGluRs). The glutamate receptors mediate excitatory neurotransmission, involve in multiple second messenger systems, and are essential in learning and memory [149,150]. Glutamate excitotoxicity and disruption of the glutamate receptor-mediated normal signaling are implicated in AD [151,152]. Aβ reduces glutamatergic transmission and inhibits synaptic plasticity [153,154]. Direct interaction between Aβ oligomers and glutamate receptors including NMDAR [155], mGluR subunit mGluR5 [156], AMPA receptor subunit GluA3 [157], and GluA1 [158] have been demonstrated, leading to impaired synaptic plasticity in the animal models [159]. Chronic pharmacological inhibition of mGluR5 has been shown to prevent cognitive impairment and reduce pathological development in APP/PS1 mice [160]. Thus, glutamate receptors have been important targets for AD therapeutics. Several imaging tracers for glutamate receptors have been developed, including [ 11 C]K-2 [161] and [ 11 C]HMS011 [162] for AMPA receptor, [ 18 F]GE-179 [163] and [ 18 F]PK-209 for NMDAR [164], [ 11 C]Me-NB1 [165] for NMDAR GluN1/GluN2B subunits [166], as well as [ 18 F]FPEB, [ 11 C]ABP688, and [ 18 F]PSS232 for mGluR5 [167][168][169]. In patients with AD, PET using [ 18 F]FPEB [170] and [ 11 C]ABP688 [171] revealed consistent reductions in regional mGluR5 binding in the hippocampus and amygdala, compared with non-demented controls. Sofar only mGluR5 imaging has been reported in amyloidosis animal models and showed conflicting results probably due to different animal models utilized (Table 2). Lee et al. demonstrated an age-dependent 35% decrease in the level of [ 18 F]FPEB measures of mGluR5 in the cortical and subcortical brain areas in 5 × FAD mice at 9 months of age, compared with 3 months of age, validated by ex vivo assessment of mGluR5 protein expression levels [116]. However, Varlow et al. showed that [ 18 F]FPEB uptake increased in the brain of 10-month-old APP/PS1 mice, compared with controls [118]. Fang et al. reported similar levels of [ 18 F]FPEB uptake in the brain of Tg-ArcSwe mice, compared with control mice at different ages [119]. However, immunoblotting results indicated that the level of mGluR5 in Tg-ArcSwe mouse brain lysate was higher, compared with control mice, at 12 months of age, not at 8 and 16 months of age [119]. Further studies are needed to elucidate the dynamic alteration in glutamate receptors in AD animal models.

Cholinergic System
The cholinergic system is essential for learning, memory formation, attention, and regulating inflammation [172]. The cholinergic system includes nicotinic acetylcholine receptors (nAChR), muscarinic acetylcholine receptors (mAChR), acetylcholinesterase (AChE), and butyrylcholinesterase (BChE). α7 nAChR and α4β2 nAChR are the most abundant nAChR subtypes in the brain. The cholinergic system is impaired early in AD associated with the cognitive, behavioral, and global functioning decline [172][173][174]. Reduced basal forebrain cholinergic neurons, increased levels of α7 nAChR [175,176], and reduced levels of M1 mAChR [177] were reported in the cortical regions of post-mortem brain from AD patients, compared with control. Interaction between α7 and α4β2 nAChR and different forms of Aβ aggregates have also been reported [178][179][180][181] F]florbetaben, respectively, in brain of 5 × FAD mice at 4-12 months of age, compared with wild-type mice [121] (Figure 2h-j). In comparison, comparable levels of AChE were observed in APP23, compared with wild-type mice at 10-13 months of age, assessed by PET using [ 11 C]MP4A [120].

Blood-Brain Barrier
Blood-brain barrier (BBB) is impaired at an early disease stage in AD [189,190]. Whether the BBB dysfunction is secondary to Aβ pathology or a causal factor has not been fully elucidated. In amyloidosis animal models of AD, BBB disruption is observed in mouse models such as arcAβ and APP/PS1 but not prevalent in certain mouse lines such as the PS2APP line [191,192]. Several receptors presented in the BBB have been explored as PET imaging targets, such as adenosine triphosphate-binding cassette (ABC) transporter ABCC1, ABCG2, ABCB1 (P-glycoprotein, P-gp), and receptor for advanced glycation endproducts (RAGE). P-gp plays an important role in the clearance and efflux of Aβ from the brain into the blood across the brain endothelial luminal membrane [193]. The levels of P-gp expression and activity were found to be decreased in the brains of AD patients, compared with that in control cases, as well as in the APP mouse model, compared with wild-type mice [194]. Several P-gp tracers such as (R)-O-[ 18 [130][131][132][133][195][196][197][198] (Table 2). Zoufal et al. demonstrated an age-dependent reduction in the cerebral P-gp function in APP/PS1 mice, compared with wild-type mice assessed by PET using (R)-[ 11 C]verapamil [133] (Figure 3a-d) and by using [ 11 C]metoclopramide [132].
However, (R)-[ 11 C]verapamil showed suboptimal brain uptake, and further improvement and evaluation of P-gp function using novel tracers with improved properties are needed. In addition, PET using 6-bromo-7-[ 11 C]methylpurine ([ 11 C]BMP) showed an increased level of ABCC1 along with [ 11 C]PiB detection of an increased level of Aβ pathology in the brain of APP/PS1 mice, compared with wild-type mice [130]. The increase in the ABCC1 level has been assumed to be related to the upregulation of its expression in astrocytes as a protective mechanism. Imaging of ABCG2 by PET using [ 11 C]erlotinib has been reported in APP/PS1 mice: no alteration in the level of ABCG2, compared with wild-type mice, was observed [131].
Receptor for advanced glycation end products (RAGE) is a BBB transporter and a binding site for advanced glycation end products and mediates Aβ transportation across the BBB into the brain [199,200]. The expression level of RAGE was found increased in post-mortem AD brains, compared with that in control cases [199]. RAGE tracers such as [ 11 C]FPS-ZM1 [201], [ 18 F]RAGER [202], [ 18 F]InRAGER [203], and [ 64 Cu]Rho-G4-CML nanoparticle (multimodal) have been developed [204]. The only imaging study conducted in the AD animal model by Luzi et al. showed that [ 11 C]FPS-ZM1 uptake in the brain of APPswe was similar, compared with that of wild-type mice [129]. Further development and studies are needed to evaluate RAGE imaging tracers in AD animal models and in patients with AD.

Discussion
In vivo longitudinal imaging in animal models of AD amyloidosis has provided valuable insights on the spatiotemporal links between different pathophysiology. A range of molecular imaging tracers for neuroinflammation, synaptic density, and neurotransmitter receptor deficits have been developed and provided a comprehensive picture of AD [11,210,236,237]. In addition to the aforementioned targets, many emerging targets show potential as indicators for pathological alterations in AD and are yet to be further investigated in amyloidosis animal models. These include (1) microgliosis; (2) astrocytosis; (3) metal dysregulation and copper trafficking, e.g., using [ 64 Cu]GTSM [125]; (4) reactive oxygen species [238] and pH alterations [239]; (5) [128,245].
Among the aforementioned emerging microgliosis tracers, the tracers for purinergic P2X7 receptor [230,231], P2Y12 receptor [232] are of high interest due to their specific cellular location on microglia. In addition, astrocytes are essential for maintaining the homeostasis, synaptic plasticity, and inflammatory response in the central nervous system [246] and play key roles in the onset and progression of AD. Reactive astrocytes show diseaseassociated profiles and exert dynamic functions (neuroprotection and neurotoxicity) in AD [247][248][249][250][251]. Few studies have been reported on PET imaging of astrocytosis in AD animal models. PET using irreversible monoamine oxidase B (MAO-B) inhibitors [ 11 C]deuterium-L-deprenyl (DED) showed an early astrocytosis preceding the Aβ accumulation assessed by using [ 11 C]AZD2184 in the brain of APPswe at 6 months of age, compared with wild-type mice (Figure 3e,f). A similar finding of an early increase in [ 11 C]DED binding was reported in Tg-ArcSwe mice, compared with wild-type littermates [252]. Several novel MAO-B tracers have been developed including [ 11 C]SMBT-1 [253] based on (S)-[ 18 F]THK5117 structure [254] and [ 18 F]6 [255]. In addition, a novel astrocytic tracer [ 11 C]BU99008, which targets imidazoline-2 binding sites (I2BS), has shown specific and high-affinity binding properties in post-mortem characterization [256] and demonstrated promising results in the recent in vivo PET studies in patients with AD [257,258].
Several earlier studies have reported the complicated temporal and spatial association between [ 134], and increased microglial activation using [ 18 F]GE-180 [134] (Figure 2a-d), and [ 18 F]DPA-714 has been reported in animal models [105]. Tsukada  F]BCPP-EF for mitochondrial complex 1 in the brain of aged monkeys [70]. Given the recent finding of microglial [ 18 F]FDG-PET uptake [97], further studies may potentially use [ 18 F]FDG-PET for monitoring the microglial status in treatment targeting at microglia. In addition, markers that can specifically reflect synaptic and neuronal function are needed. Amyloidosis animal models show cortical, hippocampal atrophy, and enlargement of ventricle assessed by using structural magnetic resonance imaging, although to a less extent, compared with that in tauopathy animal models [259,260]. Multi-modal imaging [261] or multi-tracer imaging studies combining microgliosis, [ 18 F]FDG, and SV2A imaging to provide more comprehensive functional and molecular readouts are thus highly desired [262].
The challenges in bridging the translational gaps of PET imaging in rodent models and in patients with AD may include (1) different rodent models of AD demonstrated divergent time courses and patterns of pathophysiological development. Thus, rational selection of optimal animal models and age for investigation is thus critical in PET imaging studies in tracer evaluation [263]; (2) in addition, species difference in cell types, protein expression level, available binding sites, and post-translational modification of the target added to the complexity [264]. For example, the Aβ deposits formed in the APP mouse models and in aged primates are structurally different from that in the brain from patients with AD [265]. Thus, models that better recapitulate the human AD pathology will greatly boost the AD research, such as the Aβ-KI mouse modeling late-onset AD [23] and the thirdgeneration mouse model [22]; databases of comprehensive deep phenotyping in disease animal models such as "MODEL-AD" by the Alzheimer Consortium Think Tank [266,267] (www.model-ad.org/, accessed on 15 October 2021) are instrumental in facilitating the translational research. Systems biology approaches, including single-cell sequencing, transcriptomics, biochemical characterization, and behavioral assessments, along with in vivo imaging data, will provide accurate interpretation of the readouts [268].

Conclusions
We provided an overview of PET imaging in animal models of AD amyloidosis, highlighting recent development in visualizing Aβ, cerebral glucose metabolism, synaptic and neurotransmitter receptor deficits, BBB impairment, and neuroinflammation, and proposed outstanding challenges for future development to increase the translational power of preclinical PET in AD.