In vivo Imaging of Glial Activation in Alzheimer's Disease

Alzheimer's disease (AD) is characterized by memory loss and decline of cognitive function, associated with progressive neurodegeneration. While neuropathological processes like amyloid plaques and tau neurofibrillary tangles have been linked to neuronal death in AD, the precise role of glial activation on disease progression is still debated. It was suggested that neuroinflammation could occur well ahead of amyloid deposition and may be responsible for clearing amyloid, having a neuroprotective effect; however, later in the disease, glial activation could become deleterious, contributing to neuronal toxicity. Recent genetic and preclinical studies suggest that the different activation states of microglia and astrocytes are complex, not as polarized as previously thought, and that the heterogeneity in their phenotype can switch during disease progression. In the last few years, novel imaging techniques e.g., new radiotracers for assessing glia activation using positron emission tomography and advanced magnetic resonance imaging technologies have emerged, allowing the correlation of neuro-inflammatory markers with cognitive decline, brain function and brain pathology in vivo. Here we review all new imaging technology in AD patients and animal models that has the potential to serve for early diagnosis of the disease, to monitor disease progression and to test the efficacy and the most effective time window for potential anti-inflammatory treatments.


INTRODUCTION
Neuroinflammation is the term used to denote the response of the central nervous system to harmful stimuli such as protein aggregation, pathogens, and any other insult to the brain. While timely initiated and resolved, the inflammatory response is necessary reaction to noxious stimuli and hence protective, sustained and/or disproportionate (neuro)inflammation will likely contribute, exacerbate or induce tissue damage and thereby aggravate disease pathology (1). The nature of the inflammatory process is therefore complex and dynamic and changes along different stages of the disease, involving phenotypic alterations in all cells present within the CNS including neurons, microglia, astrocytes, and other inflammatory cells.
Microglia, act as part of the innate immune system, are constantly scanning and surveying the local microenvironment for signals of infection and injury [for a review see (2)]. Amyloid-β has been reported to "prime" or activate microglial cells (3). Different activation states were described in the past, so called "M1" or classically activated microglia or "M2" alternatively activated (2). The classically activated or pro-inflammatory phenotype has been associated with disease aggravation. However, this classification has been recently challenged by single-cells transcriptomics, which suggests that the gene expression profile progressively switches with the disease and that this may even depend on how close they are to the amyloid plaques (4).
Astrocytes, are key mediators of many essential processes in the CNS. As for microglia, astrocytes have been classified into two distinct reactive states, A1 (inflammatory) and A2 (ischemic) states (5), although this classification seems to be over-simplistic. Accumulation of hypertrophic reactive astrocytes around senile plaques has been observed in post-mortem human tissue from AD patients [Reviewed by (6)] and in animal models of the disease (7). It is worth noting that astrocytes and microglia communicate with each other and this cross talk is important in promoting glial activation (8,9).
In order to follow-up changes in microglial and astrocytic activation in vivo, radioactive tracers for positron emission and single-photon emission computed tomography (PET and SPECT) have been developed in the past decades. In this review, we will analyze different in vivo imaging techniques that allow the visualization of changes in neuroinflammation in animals and humans.

IN VIVO IMAGING OF MICROGLIA ACTIVATION PET Imaging With TSPO Ligands in AD Patients
Following activation, microglia proliferate, and express a series of genes for pro-inflammatory cytokines and certain receptors on their surface, including the 18 kDa translocator protein (TSPO). TSPO, primarily but not exclusively expressed in the mitochondrial membrane of microglia, was previously identified as the peripheral-type benzodiazepine receptor (PBR) (10). Besides microglia, TSPO has been detected in other types of gliosis as well, such as in astrocytoma (11), and is generally expressed in highly proliferating cells. While the exact function of TSPO still remains to be elucidated, initially its role was associated with the transport of cholesterol, with the TSPO complex being a rate-limiting step in the synthesis of steroid hormones (12). Initial attempts to create TSPO knockout mice reported a non-viable phenotype [described in (13)]. However, the successful development of conditional TSPO knockout mice suggested that TSPO might not be a crucial part of steroid hormone synthesis, e.g., testosterone production (14). In addition, the mitochondrial expression-paradigm was challenged by reports of TSPO expression in other subcellular locations (15), e.g., nuclear/perinuclear-located TSPO, where is believed to play a part in cell proliferation (16). Moreover, plasma membrane bound TSPO has been observed as well, for instance in mature human red blood cells, lacking mitochondria (17,18).
In the mammalian brain, the expression of TSPO turned out to be very low, compared to other tissues (19). Only the olfactory bulb and non-parenchymal regions, such as the ependymal and choroid plexus, showed higher TSPO densities in comparison with most gray and white matter structures (20,21). However, under conditions of local inflammatory responses, e.g., caused by a multitude of brain injuries, neoplasms and infections, TSPO appears to be upregulated. This effect was quickly recognized and made TSPO a potentially ideal and sensitive biomarker of brain injury (10,11,(22)(23)(24)(25). Therefore, PET tracers for TSPO were developed in the past decade as markers for microglia activation and neuroinflammation in AD (10, 26) (see Table 1).
Cagnin et al. published the first PET study using [ 11 C]PK11195 in 2001, demonstrating an increase of tracer uptake in AD cases (28). While subsequent reports generally have shown increased tracer binding in AD brains, some publications found no differences between Alzheimer's subjects and healthy individuals (29,33,35). In spite of these conflicting results, it is generally accepted that there is increased microglial activation in AD. Recent studies have extended this to patients with Mild cognitive impairment (MCI), showing that glia activation can precede clinical AD (70).
Recent reports have evaluated the relationship between amyloid load and neuroinflammation, suggesting that microglial activation is associated with amyloid load. Interestingly, using [ 11 C]PK11195 and the amyloid tracer [ 11 C]PIB, one study did not show any correlation between the binding of these tracers, while another one suggested a negative correlation between amyloid-β and TSPO density (31,33). The reason behind these different outcomes could be related to the limitations of the current amyloid ligands; while we are able to image amyloid plaque deposition using amyloid imaging agents, other forms of amyloid, such as β-amyloid oligomers, which may be contributing to the microglial activation, are not currently detectable by PET (36,71).
However, while affinity and nonspecific binding properties were usually found to be improved as compared to PK11195, it quickly became apparent that these new TSPO tracers are affected by genetic variability of TSPO binding site induced by the rs6971 single-nucleotide polymorphism (80), resulting in high-, mixed

PET Imaging in Animal Models of AD
The first studies carried out in animal models of amyloidosis demonstrated a significant age-dependent increase in the specific binding of [ 3 H]PK11195 in the TASTPM model (APPswxPS1M146V) by autoradiography (8), in agreement with age-dependent increases in CD68 immunoreactivity colocalized with Aβ deposits. However, reports on [ 11 C]PK11195 PET imaging in mouse models of amyloidosis have exposed conflicting results, depending on the model used and age of the animals. A higher [ 11 C]PK11195 uptake was shown in the brains of older APP/PS1 mice when compared with age-matched controls (68,86). Surprisingly, [ 11 C]PK11195 binding in younger transgenic APP/PS1 mice was not different from their controls, even though immunostaining revealed activated microglia in close proximity of amyloid deposits. Similar to human data, it is likely that different modeling approaches and reference regions may contribute to the seemingly conflicting in vivo findings.
Our own recent autoradiographic and PET data provided evidence of an increased binding of [ 3 H]PBR28 in the brain of the aggressive 5XFAD mouse model, compared with wildtype controls, which coincided with the strongly increased immunoreactivity of the microglial marker Iba1 in the same brain areas (87). These results provided support for the suitability of PBR28 as a tool for monitoring of (micro)-glial activation.
[ 3 H]PBR28 binding was significantly higher in female animals and positively correlated between Aβ plaque load and tracer binding. In addition, using [ 11 C]PBR28 in healthy rats, in vitro brain autoradiography showed a 19% increase of binding in aged (19.6 months) as compared in young rats (4 months) (88).
Besides PBR28, other new generation tracers have exhibited similar patterns in animal models of AD. Increases in TSPO density were reported from 10 months old Thy1-hAPPLond/Swe (APPL/S) mice compared with wild-type controls, using ex vivo autoradiography with [ 18 F]PBR06, but this increase was only observed in older mice, at 16 months of age by PET (89). Similar findings were published by Liu et al. (90), who performed [ 18 F]GE180 PET in young and old wild-types (WTs) and APP/PS1dE9 transgenic mice, showing higher uptake in transgenic and WT mice at 24 months of age but not in young 4 months old transgenics (90). In a different study, [ 18 F]GE180 uptake was slightly increased in PS2APP mice at 5 mo and markedly elevated at 16 mo. Over this age range, there was a highly positive correlation between TSPO PET uptake, amyloid load and likewise with tracers for brain metabolism (91). However, a recent study in APP23 mice showed that the increased rate of (micro)glia activation detected with [ 18 F]GE-180 appears to be of less magnitude than the elevation in amyloidosis detected with [ 11 C]PIB over time. In fact, [ 18 F]GE-180 binding seems to plateau at an earlier stage of pathogenesis, whereas amyloidosis continues to increase. These results suggest that TSPO might be a good marker for early pathogenesis detection, but not for tracking long-term disease progression (92).
These tracers have also served to assess and monitor the efficacy of anti-inflammatory treatments. LM11A-31 is a p75 neurotrophin receptor ligand that was shown to reduce the hyperphosphorylation and misfolding of tau, decrease neurite degeneration, and attenuate microglial activation. LM11A-31treated APPL/S mice displayed significantly lower [ 18 F]GE-180 binding in cortex and hippocampus of as compared to vehicletreated animals, corresponding to decreased TSPO and Iba1 staining (93).
As AD is characterized by substantial aggregation of hyperphosphorylated tau, second-generation TSPO ligands have also been employed in transgenic models of tau pathology, such as the PS19 mice. Here, uptake of [ 11 C]AC-5216 was found linearly proportional to the phospho-tau immunolabelling (94).
While TSPO is the most widely recognized biomarker of neuroinflammations, other targets have been explored in recent years. Radiolabeled ketoprofen methyl ester, [ 11 C]-KTP-Me is a highly selective tracer for the cyclooxygenase-1 (COX-1). In APP transgenic mice, [ 11 C]-KTP-Me uptake was significantly increased in the brain of 16 to 24 mo old mice in comparison to their age-matched controls, coinciding with the histopathologic appearance of abundant Aβ plaques and activated microglia. Furthermore, [ 11 C]-KTP-Me accumulation was observed in the frontal cortex and hippocampus, whereby COX-1-expressing activated microglia appeared surrounding Aβ plaques, indicating neuroinflammation that originated with Aβ deposition (95). Another currently investigated alternative to TSPO ligands are tracers for the cannabinoid 2 receptor, such as [ 11 C]Sch225336 and [ 11 C]A-836339 (96)(97)(98) and tracers for the purinergic receptors P2Y12 and P2X7 (99).

Magnetic Resonance Spectroscopy (MRS)
Magnetic resonance spectroscopy (MRS) is a new technique that can provide information about several relevant metabolites for neuroinflammation and neurodegeneration. Recently, chemical exchange saturation transfer (CEST), as a novel molecular MR imaging approach, has been developed, which uses proton exchange as a means of enhancing the contrast of specific molecules in the body (100). Endogenous CEST compounds include hydroxyl (OH), amine (NH2), and amide groups (NH). In the last few years, several studies have explored the possibility of imaging neuroinflammatory and neurodegeneration biomarkers in vivo with CEST, such as CEST imaging of myo-inositol (101), glutamate (102) and glucose (103) in AD mouse models.

IN VIVO IMAGING OF ASTROCYTES PET Imaging of Astrocytes in Humans
The best-known tracer for astrocytes so far is [ 11 C]deuterium-L-deprenyl [[ 11 C]DED], which is an irreversible monoamine oxidase B (MAO-B) inhibitor. This is based on previous findings showing that astrocytes express elevated levels of MAO-B during their activation. The ligand has been therefore employed as biomarker of astrocytosis in pathologies such as AD (59) and amyotrophic lateral sclerosis (ALS) (104). Increased [ 11 C]DED binding throughout the brain was detected in MCI [ 11 C]PIBpositive patients compared with controls and MCI [ 11 C]PIBnegative and AD patients (59). In autosomal dominant AD carriers, astrocytosis measured by [ 11 C]DED was found initially high and then declining, contrasting with the increasing amyloidβ plaque load during disease progression, suggesting astrocyte activation is implicated in the early stages of AD pathology (61).
In the last years, new ligands for the potential imaging of astrocytes have been developed, including those for type-2 imidazoline receptors (I2Rs), which were found to be expressed primarily in astrocytes. These receptors were described for the first time in the 90's and the first studies performing in vitro binding with [ 3 H]idazoxan in postmortem cortical membranes showed increased density in AD patients (105). Later on, work carried out with the I2R PET tracer [ 11 C]FTIMD reported specific binding to these receptors in rat and monkey brains, but exhibiting a relative low binding specificity (106,107). More recently, [ 11 C]BU99008 (2-(4,5-Dihydro-1Himidazol-2-yl)-1-methyl-1H-indole) was developed as a more potent PET ligand for I2Rs imaging (108,109), displaying relatively high binding specificity and brain penetration in the porcine and rhesus monkey brain (108,110). There are ongoing human PET imaging trials in Alzheimer's and healthy control patients at the moment using [ 11 C]BU99008 and the preliminary results have shown good brain delivery, reversible kinetics, heterogeneous distribution specific binding signal consistent with I2BS distribution and good test-retest reliability (111).

PET Imaging for Astrocytes
Imaging studies with [ 11 C]DED carried out in transgenic APP Swedish (APPswe) mice and wild-type animals at different ages, have demonstrated that tracer uptake was significantly higher at 6 months than at 18-24 months in APPswe mice, preceding Aβ deposition (112). However, no differences in [ 3 H]-L-deprenyl obtained by autoradiography were observed between WT and APPswe mice across different ages. Furthermore, staining of the astrocyte marker GFAP was increased in older transgenic APPSwe mice as compared to younger mice (112), raising questions about the specificity of this ligand as marker for astrocytes.

In vivo Bioluminescence Imaging (BPI)
Bioluminescence describes the light produced by the enzymatic reaction of a luciferase with its substrate (luciferin) and the emitted light is detected with a camera. The technique allows for fast acquisition times so that subjects can be imaged quickly, serially over time and with minimal distress. The Prusiner's lab developed in vivo bioluminescence imaging and quantitative determination of inflammation in a model of prion related neurodegenerative disease (113). Additionally, bigenic mice overexpressing APP and GFAP-Luc were reported to show an age-dependent increase in signal that was corresponded to major areas of Aβ deposition. Bioluminescence signals began to increase in 7-mo-old Tg(CRND8:Gfap-luc) mice and at 14-mo-old in Tg(APP23:Gfap-luc) mice (114).
Gfap-luciferase reporter mice have also been crossed-bred with hTau40AT/C57BL/6N mice. In vivo bioluminescence imaging (BLI) showed activation of astrocytes in response to aggregation of Tau, from 5 months of age compared with wildtype animals (115).

CONCLUSIONS AND FUTURE PERSPECTIVES
The development of new neuroinflammation tracers in the last decade has allowed characterizing the pattern of glia activation in AD patients, showing that it occurs ahead of amyloid deposition, correlates in many cases with amyloid plaque density and allows limited predictions of disease progression. The longitudinal studies have shown that this glial activation, as detected by PET, fluctuates during disease progression. Although reports in animal models of AD have helped confirming the specificity of TSPO tracers for microglia, the situation is not the same for tracers for astrocytes and more research needs to be done regarding this aspect. In addition, new tracers able to differentiate between the potential M1 and M2 microglial phenotypes would be advantageous in identifying their function in vivo (116).
Future studies should include imaging in patients after intervention with anti-inflammatory drugs; however so far, there are no reports in that aspect in AD cases. Therefore, imaging studies are key to test the efficacy and the most effective time window for potential anti-inflammatory treatments.

AUTHOR CONTRIBUTIONS
PE and MS designed this review outline, PE performed most of the literature review on TSPO imaging in humans, CD edited and the manuscript, performed the literature review for the table and MS wrote the rest of the manuscript.