Positron emission tomography in animal models of Alzheimer’s disease amyloidosis

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 are essential. Recent advances in positron emission tomography have allowed the non-invasive visualization of the alterations in the brain of animal models as well as in patients with Alzheimer’s disease, These tools have facilitated our understanding of disease mechanisms, and provided longitudinal monitoring of treatment effect 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 on-chip characterization of imaging biomarkers towards clinical translation. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 15 October 2021 © 2021 by the author(s). Distributed under a Creative Commons CC BY license.


Introduction
Alzheimer's disease (AD) is the most common cause of dementia, afflicting 50 million people worldwide [1]. AD is pathologically featured by amyloid-beta(A) plaques and neurofibrillary tangles formed by hyperphosphorylated tau, gliosis, neurotransmitter deficits, and neuronal loss leading to cognitive impairment [2,3]. Ais produced from through sequential cleavages of amyloid precursor protein (APP) by -secretase and -secretase. An imbalance in the production and clearance of A leads to its abnormal cerebral accumulation of A in different forms (oligomers, protofibrils, fibrils and amyloid plaques) [4]. The abnormal accumulation of Aβ deposits, especially the neurotoxic oligomeric A plays a crucial role in the disease pathogenesis in animal models and in patients with AD [5][6][7][8]. Recent advances in positron emission tomography (PET) using [ 18 F]fluorodeoxyglucose (FDG), tracers for A pathology and tauopathy, structural magnetic resonance imaging and cerebrospinal fluid biomarkers have provided valuable insights into the time course of pathophysiology of AD continuum, assisted the early and differential diagnosis, and facilitated the development of therapeutics for AD [9][10][11][12][13]. A range of molecular imaging tracers for neuroinflammation, synaptic density and neurotransmitter receptor deficits have been developed and provided comprehensive picture of AD [13][14][15][16]. Disease animal models recapitulating AD amyloidosis have been developed including transgenic APP/PS1, APP23, APPswe, J20, PS2APP, arcA, 5×FAD, 3×Tg mice, TgF344 and McGill-R-Thy1-APP rats [17][18][19][20][21][22][23][24], 2 nd generation App NL-G-F , App hu/hu knock-in mice [25,26], 3 rd generation mouse models [27,28] as well as non-human primate model [29]. The animal models accumulate cerebral A pathology, develop gliosis, metabolic and synaptic deficits and cognitive impairment assessed by behavior tests, facilitated the understanding of disease mechanisms and the development of treatment strategies. In this review, we focus on the recent development in PET imaging for A, alterations in cerebral glucose metabolism, synaptic neurotransmitter receptors, bloodbrain barrier as and neuroinflammation in rodent models of AD amyloidosis.

Amyloid imaging
Ex vivo immunohistochemistry in brain tissues from amyloidosis mouse or rat models has revealed that Apathology initiates first in the cortical region, and spreads to the limbic region and finally to the cerebellum [30], in a animal line-dependent manner. More pronounced load of A deposits was observed in 5×FAD mice compared to that in APPswe mice [30][31][32]. In addition to the parenchymal A plaques, cerebral amyloid angiopathy (CAA) is also observed in different amyloidosis animal models especially in the APPDutch mice, Tg-SwDI, APP/London, APP23, arcAβ and APPswe mice [33,34].
Several A imaging tracers have been developed and applied in animal models of amyloidosis, including benzothiazole derivatives [ 11    imaging study [61] (Fig. 1d). Moreover, comparative studies of amyloid imaging tracers have been performed in a head-to-head manner in animal models, such as comparing [

Cerebral glucose metabolism imaging
Brain glucose dysregulation plays an important role in AD [75]. Post-mortem studies reported higher levels of brain tissue glucose concentration, lower levels of glucose transporter 3, and glycolytic flux in brain from patients with AD compared to controls, associating with the severity of AD pathology [75]. Accumulating evidence also indicates a link between diabetes and AD [76]. [ 18 F]FDG PET 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 [11,77] bound specifically to SV2A in mouse brain, and that the radioligand binding can be quantified by kinetic modeling using an image-derived input function [103]. Toyonaga showed that in vivo [ 11 C]UCB-J detected reduced levels of SV2A in APP/PS1 mice, and the treatment effects of tyrosine kinase Fyn inhibitor Saracatinib in mitigating the [ 11 C]UCB-J reduction [104]. Xiong  standard uptake value (SUV) across the whole brain of APP/PS1 mice compared to non-transgenic mice [107]. The results from a static (30-60 min post-injection) [ 18 F]SynVesT-1 PET scan was found comparable to kinetic modeling results [107].

Glutamate receptors
The glutamate receptors are classified into the N-methyl-D-aspartate receptor (NMDAR), α-amino-3hydroxy-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 [108,109]. Glutamate excitotoxicity, and disruption of the glutamate receptor mediated normal signaling are implicated in AD [110,111]. Aβ reduces glutamatergic transmission and inhibits synaptic plasticity [112,113]. Direct interaction between Aβ oligomers and glutamate receptors including NMDAR [114], mGluR subunit mGluR5 [115], AMPA receptor subunit GluA3 [116] and GluA1 [117] have been demonstrated, leading to impaired synaptic plasticity in the animal models [118]. Chronic pharmacological inhibition of mGluR5 has been shown to prevent the cognitive impairment and reduce pathological development in APP/PS1 mice [119]. Thus glutamate receptors have been important target for AD therapeutics. Several imaging tracers for glutamate receptors have been developed including [ 11 C]K-2 [120] and [ 11 C]HMS011 [121] for AMPA receptor, [ 18 F]GE-179 [122] and [ 18 F]PK-209 for NMDAR [123], [124] for NMDAR GluN1/GluN2B subunits [125], as well as [ 18 [126][127][128]. In patients with AD, PET using [ 18 F]FPEB [129] and [130] revealed consistant reductions in regional mGluR5 binding in the hippocampus and amygdala compared to non-demented controls. Sofar only mGluR5 imaging have been reported in amyloidosis animal models and showed conflicting results probably due to different animal models utilized ( measures of mGluR5 in the cortical and subcortical brain areas in 5×FAD mice at 9 month compared to 3 month-of-age, validated by ex vivo assessment of mGluR5 protein expression level [131]. However Varlow et al. showed that [ 18 F]FPEB uptake increased in the brain of 10 month-old APP/PS1 mice compared with controls [132]. Fang et al. reported similar levels of [ 18 F]FPEB uptake in the brain of Tg-ArcSwe mice compared to control mice at different ages [133]. However immunoblotting results indicated that the level of mGluR5 in Tg-ArcSwe mouse brain lysate was higher compared to control mice, at 12 month-of-age, not at 8 and 16 month-of age [133]. Further studies are needed to elucidate the dynamic alteration in glutamate receptors in AD animal models.

Blood-brain barrier
Blood-brain barrier (BBB) is impaired at an early disease stage in AD [155,156]. Whether the BBB dysfunction is secondary to A 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 arcA, APP/PS1, but not prevalent in certain mouse line such as PS2APP line [157,158]. Several receptors presented in the BBB have been explored as PET imaging targets, such as adenosine triphosphatebinding 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 [159]. The levels of Pgp expression and activity were found decreased in the brains of AD patients compared to that in control cases, as well as in APP mouse model compared to wild-type mice [160]. Several P-gp tracers  [161][162][163][164][165][166][167][168] (Table 2).
Zoufal et al. demonstrated an age-dependent reduction in the cerebral P-gp function in APP/PS1 mice compared to wild-type mice assessed by PET using (R)-[ 11 C]verapamil [161] (Figs. 2d-g) and by using [162]. 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 increased level of A pathology in the brain of APP/PS1 mice compared to wild-type mice [165]. The increase in the ABCC1 level has been assumed relating to upregulation of its expression in astrocytes as a protective mechanism. Imaging of ABCG2 by PET using [ 11 C]erlotinib have been reported in APP/PS1 mice: no alteration in the level of ABCG2 compared to wild-type mice was observed [166].
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 [169,170]. The expression level of RAGE was found increased in post-mortem AD brains compared to that in control cases [169]. RAGE tracers such as [ 11 C]FPS-ZM1 [171], [ 18 F]RAGER [172], [173], and [ 64 Cu]Rho-G4-CML nanoparticle (multimodal) have been developed [174].
The only imaging study conducted in AD animal model by Luzi et al. showed that [ 11 C]FPS-ZM1 uptake in the brain of APPswe was similar compared to that of wild-type mice [175]. Further development and studies are needed to evaluate RAGE imaging tracers in AD animal models and in patients with AD.

Neuroinflammation imaging
Several recent articles have provided thorough reviews on neuroinflammation PET imaging in AD patients and AD animal models [16,[176][177][178][179][180]. Thus here we discuss briefly the recent development in neuroinflammation imaging in AD amyloidosis animal models. Neuroinflammation plays an important role in the pathogenesis of AD and appears early in the disease development [181][182][183]. Microglia are the resident macrophages in the central nervous system, engulf Aβ plaques and are important for maintaining the brain homeostasis [183,184]. Recent single cell sequencing and transcriptomics have demonstrated a transcriptionally-distinct and neurodegeneration-specific profile of microglia termed disease-associated-microglia (DAM) [185][186][187]. The 18 kDa translocator protein (TSPO) that located on the outer mitochrondria membrane of microglia has been the most investigated target for microgliosis PET imaging. Three generations of TSPO tracers have been developed with improved properties, from the 1 st generation (R)-[ 11 C]PK11195 [188]; 2 nd generation [ 11 C]PBR28 [189] [191]. PET using various 18 kDa translocator protein (TSPO) tracers have demonstrated an early microgliosis preceding the Aβ deposition in several animal models of amyloidosis including APP23, hAPP-J20, APPSL70, App NL-G-F and PS2APP mice [184,[192][193][194][195][196][197]. Due to the diverse cellular location of TSPO expression on astrocytes and endothelial cells in addition to that on microglia, tracers specific for microglial expression and of disease-associated profile are of high interest [198][199][200].

Discussion
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, such as 1) metal dysregulation and copper trafficking e.g. using [ 64 Cu]GTSM [208]; 2) reactive oxygen species [209] and pH alterations [210]; 3) microtubule using [ 11  Astrocytes are essential for maintaining the homeostasis, synaptic plasticity and inflammatory response in the central nervous system [220]. Astrocytes play key roles in the onset and progression of AD.
Reactive astrocytes show disease-associated profiles and exert dynamic functions (neuroprotection and neurotoxicity) in AD [221][222][223][224][225]. Few studies have been reported on PET imaging of astrocytosis in AD animal models. PET using irreversible mono-amine oxidase B (MAO-B) inhibitors [ 11 C]deuterium-Ldeprenyl (DED) showed an early astrocytosis preceding the A accumulation assessed by using [ 11 C]AZD2184 in the brain of APPswe at 6 months-of-age compared to wild-type mice (Figs. 3a, b).
In vivo longitudinal imaging in animal models of AD amyloidosis has provided valuable insights on the spatiotemporal links between different pathophysiology. The challenges in bridging the translational gaps of PET imaging in rodent models and in patients with AD may include: -Animal model: Different rodent models of AD demonstrated divergent time courses and patterns of pathophysiological development [32,233,234]. Thus rational selection of optimal animal model and age for investigation are thus critical in PET imaging studies in tracer evaluation [235]. In addition, species difference in cell types, protein expression level, available binding sites, post-translational modification of the target added to the complexity [236]. For example, the A deposits formed in the APP mouse models and in aged primates are structurally different from that in the brain from patients with AD [237]. Thus, models that better recapitulate the human AD pathology will greatly boost the AD research, such as the recent Aβ-KI mouse model of late-onset AD [28], 3 rd generation mouse model [27]; Moreover, databases of comprehensive deep phenotyping in disease animal models such as "MODEL-AD" by the Alzheimer consortium think tank [238,239] (www.model-ad.org/) are instrumental in facilitating the translational research. Systems biology approaches including single cell sequencing, transcriptomics, biochemical characterization, and behavioural assessments along with in vivo imaging data will provide accurate interpretation of the readouts [240][241][242][243].

Conclusions
We provide an overview of PET imaging in animal models of AD amyloidosis, highlighting recent development in visualizing A, 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.

Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.