Role of Neuroinflammation in the Trajectory of Alzheimer’s Disease and in vivo Quantification Using PET

Abstract

Recent evidence suggests that neuroinflammation and immunity play a significant role in Alzheimer’s disease and other neurodegenerative diseases. It has also been observed that, independent of the presence of aggregated proteins, neuroinflammation could be present in different neurodegenerative diseases. It has also been suggested that neuroinflammation could occur well ahead of amyloid deposition in AD. Recent genetic studies and other preclinical studies specifically point to a role of neuroinflammation and, in this review, we evaluate the evidence of neuroinflammation in the Alzheimer’s disease trajectory and the different imaging modalities by which we could monitor neuroinflammation in vivo in humans.

Keywords

Alzheimer’s disease astrocytes microglia neurodegeneration neuroinflammation

INTRODUCTION

Neuroinflammation is an innate response in the central nervous system (CNS) against harmful changes in brain milieu such as formation of abnormal protein aggregates, invasion of pathogens, traumatic and vascular lesions, and autoimmune responses to brain material such as myelin. It has been proposed that intrinsic neuroinflammation in the form of glial activation is a component of neurodegenerative diseases such as Alzheimer’s disease (AD) and other dementias, Parkinsonian disorders, and Huntington’s disease. While there are several mediators of inflammation which lead to neuronal damage, the pro-inflammatory cytokines and interleukin-1 β, IL-1, IL-8, and IL-33 play a significant role.

While neuroinflammation is a response that involves all cells present within affected region of the CNS, including neurons, microglia, and other inflammatory cells, there are several factors which influence how neuroinflammation affects the neurodegenerative process. These include environmental factors, previous immune sensitization, genetic factors, epigenetic factors, and several intrinsic and extrinsic factors. Preclinical models have shown that lipopolysaccharide (LPS) can induce

toll-like receptor protein (TLR) signaling, which activates several signal transduction pathways including protein kinase B, mitogen protein activated kinase, and mammalian target of rapamycin in turn activating NF-κb. NF-κb mediates production of cytokines, chemokines, inducible nitric oxide synthase and cox2 promoting neurodegenerative processes.

There are several players involved in intrinsic neuroinflammatory process: these include microglia, astrocytes, oligodendrocytes, and inflammatory mediators such as cytokines, chemokines, and LPS.

MICROGLIA

At rest, microglia regulate the homeostasis of the brain but, if activated, become the resident macrophages of the CNS involved in the immune defense mechanism. They account for 10–15% of the non-neuronal brain cells. There is significant controversy regarding the precise nature of microglial progenitors. It has been suggested that microglia could arise from intrinsic brain embryonic progenitor cells. It has also been proposed that they could originate from meningeal macrophages penetrating the brain during embryonic development. There is still uncertainty about what proportion of the microglia are derived from blood monocytes and it is possible that monocytes may be recruited to the neonatal and adult brain when there is an injury and then differentiate into microglial cells. While there is controversy regarding the origin of microglial cells, the consensus is that microglial differentiation occurred primarily in the CNS [1]. While it has been shown that microglial progenitors invade the brain in the early stages, it is now established that microglia arise from the yolk sac [2]. Microglia serve as a part of the innate immune system which is constantly scanning and surveying signals for any danger to the brain cells. As a primary response to injury, microglia become activated in order to protect the CNS from tissue damage and facilitate tissue repair and clearance. Microglia also contribute to the control of neuronal proliferation and differentiation and influence synaptic connections. It has been shown that there is an interaction between the microglia and synaptic connections in the healthy brain. Microglia help regulate the wiring of the brain circuits allowing adaptive recovery processes to occur and control the growth of dopaminergic axons and neocortical neurons [3, 4].

While the origin of microglia has been debated, it seems clear that microglia are of monocyte lineage and present in the brain from birth. They are the resident macrophages of the CNS which constantly survey the brain to maintain normal homeostasis. During normal homeostasis, they maintain the plasticity of the neuronal circuit and contribute to the protection and remodeling of the synapses. It has been suggested this protective effect of microglia is mediated by the release of trophic factors such as brain-derived neurotrophic factors which are implicated in memory formation. Resting microglia exhibit a highly ramified morphology, which can exceed 50μm in length, suitable for monitoring the environment. In response to an activating signal, they begin to withdraw the ramified branches (the withdrawal stage). When these processes are withdrawn, new protrusions may appear (the transitional stage) and then move on to a motile stage where the newly generated protrusions can grow and shrink at a rate exceeding 4μm per minute. These motile cells begin to contact the neighboring cells and, during the motile stage, microglia can move through the tissue at the rate of 110μm per hour and engulf other cells [5].

It has also been established that, while microglia engulf dead cells and cellular debris, they can also transiently ensheath a cell sized object and then move on without ingesting the object. These transient ensheathing events indicate their dynamic nature and possible role in tissue surveillance. It is proposed that this transient ensheathing (frisking), where the frisked object may very well be neurons or other cells which maintain the normal milieu of the brain plays a protective role as does the microglial responsibility for clearance of Aβ and other toxic proteins from the brain.

Amyloid-β (Aβ) clearance is an important process of microglial function. In AD, microglia can bind to soluble Aβ oligomers and Aβ fibrils via cell-surface receptors. The cell surface receptors include CD36, CD14, α6 β1 integrin, CD47 and TLR (TLR2, TLR4, TLR6, and TLR9). It has been shown that CD36, TLR4, and TLR6 trigger a pro-inflammatory response while binding Aβ. Further experiments have demonstrated that deletion of CD36, TLR4, and TLR6 reduces Aβ induced cytokine production and amyloid accumulation.

Microglia engulf Aβ fibrils by phagocytosis whilst soluble Aβ is degraded by various extracellular proteases [6]. Microglia contribute to CNS homeostasis and neuroprotection during development by synaptic pruning and phagocytosis of redundant neurons. They are involved in cortical laminar formation and axon bundle fasciculation. It has been shown that peripherally delivered lipopolysaccharide (LPS) can activate TLR receptors on the luminal surface of brain endothelial cells, which then secrete cytokines and activate microglia. It has been demonstrated that activated microglia can strip axosomatic inhibitory synapses from neuronal soma which induces neuroprotection by upregulation of BCL1, FGF2, or MCL1, which are anti-apoptotic molecules. These microglia can assume an M2-AP phenotype able to secrete ceruloplasmin, CD163, SAA3, YM-1, and MSR1 during the initial phase of neuronal injury [7].

It is generally accepted that phenotypes of microglia fall into two main classes: 1) A pro-inflammatory or M1 phenotype which is activated the classical complement pathway and changes in brain milieu; and 2) An anti-inflammatory or M2 phenotype which is activated by the alternative complement pathway. The M1 phenotype responds to LPS in combination with interferon gamma (IFN-γ), leading to a massive inflammatory response producing cytokines including interleukin-1β, IL-12, TNF-α, and inducible nitric oxide. The M2 phenotype has three sub-phenotypes, M2a, which usually responds to IL-4 and IL-13, while M2b is stimulated by TLR or IL-1β activation. M2c represents the deactivated macrophages and contributes to the suppression of pro-inflammatory cytokines [8, 9]. It has been suggested that these phenotypes can interconvert depending on the stimuli and so models based purely on inducing an M1 or M2 phenotype is over simplistic. Despite this, a simplified model where pro-inflammatory phenotypes are regarded to be predominantly detrimental while anti-inflammatory phenotypes are regarded as predominantly involved in the repair process of the neurons has proved useful.

While there is evidence to suggest microglial activation can be deleterious, the beneficial effect is highlighted in circumstances where repair is happening, as in after stroke, during myelin repair, removal of toxic aggregated proteins and cell debris from the CNS, as well as secretion of neurotrophic factors to prevent neuronal injury [10 –12].

While it is agreed that the M1/M2 microglial classification is an over simplified model, these two phenotypes have been studied extensively in cell culture and it has been demonstrated that the relative populations have differential influences over pathological outcome in CNS human diseases.

ASTROCYTES

Astrocytes are glial cells characterized by star-shaped cell bodies with a number of processes. There are two types of astrocytes: 1) Protoplasmic astrocytes, which are found in the grey matter and their processes end in sheet like appendages; and 2) fibrous astrocytes, which are found in the white matter and have long fine processes. While the function of astrocytes is still debated, it is generally thought that they provide nutrition for neurons and insulate nerves and synaptic connections from each other. They help regulate the potassium concentration in the space between the neurons [13]. More importantly, they perform the housekeeping chores that promote efficient signaling between neurons and they maintain surrounding neurons by releasing growth factors.

Astrocytes enfold all the blood vessels of the brain and ensheath synapses. As their physical association with synapses is closer than 1μm, astrocytes can regulate local extracellular concentration of ions, neurotransmitters, and other molecules. The pathological response of astrocytes is reactive astrogliosis forming scars whereas remodeling of astrocytes is generally aimed at neuroprotection and recovery of injured neuronal tissue [14, 15]. Reactive astrocytes are characterized by increased expression of glial fibrillary acidic protein (GFAP). However, many healthy astrocytes do not express detectable levels of GFAP and the expression of GFAP can depend on the anatomical location of the astrocytes as well as the species in which GFAP expression is being examined. Aging is the leading risk factor for the common dementias, and astrocytes in the aging brain show features of senescence and expression of a senescence associated secretory phenotype.

Initially it was thought that astrocytes appeared activated in AD brain as a secondary or non-specific response to the disease process [16]. However, it is now understood that astrocytes are central to the pathogenic mechanism in neurodegeneration. This could be due to their production of cytokines and chemokines or loss of physiological functions such as neuronal support and spatial buffering. It has been suggested that disruption of normal glioneuronal interaction can lead to synaptic dysfunction and contribute to cognitive impairment [15, 17]. Wyss-Corey et al. first demonstrated in vitro that astrocytes are able to take up and degrade Aβ using cultured mouse astrocytes. Histopathological studies of AD brain have shown the presence of astrocytes which contain Aβ suggesting they are involved in the clearance of this peptide [18 –21]. Engulfment of Aβ by astrocytes, however, can lead to their death and give rise to secondary plaques [21].

The mechanisms governing the receptor-mediated uptake of Aβ and its consequences are not fully understood. For instance, does uptake of Aβ induce a change in astrocyte phenotype altering their usual neurosupportive function? Low density lipoprotein receptor-related protein 1 is involved in the uptake and clearance of Aβ and is also a receptor for the uptake of ApoE4 and complexes of ApoE-Aβ highlighting the importance of this receptor in the astrocytic clearance of Aβ [22 –24].

Neuroinflammation is a prominent and early feature of AD which plays a key role in modulating the progression of disease via inflammatory mediators and neurotoxic compounds. It has also been suggested that an astrocyte mediated inflammatory response can contribute to the neurodegenerative process through expression of pro-inflammatory cytokines and chemokines, activation of complement cascade as well as reactive oxygen and nitrogen species [25 –27]. Studies also show that astrocytes can suppress innate immunity through αB-crystallin suggesting that they have a more deleterious influence on neuroinflammation. In animal models of AD, it has also been shown that the astrocyte contribution to neuroinflammation is significant and an important therapeutic target [28, 29].

Apart from microglia and astrocytes, other cells such as blood derived monocytes may also play a significant role in AD. However, the precise role of these cells in human studies is unclear although there are animal studies demonstrating infiltration of these peripheral mononuclear cells associated with amyloid deposition. Ablation of CD11b-positive cells in APP/PS1 models of AD have suggested that peripheral monocytes do play an important role in clearing amyloid plaques [11, 30].

P2X7 RECEPTOR

The purinergic P2X7 receptor (P2X7R) plays an important role in the CNS binding ATP. The P2XR is expressed by activated microglia and, following brain injury, ATP can be released in large quantities leading to stimulation of low affinity P2X7Rs resulting in glial necrosis/apoptosis or proliferation, demonstrating two opposing effects of neuroinflammation [31, 32].

P2X7Rs or ATP-gated non-selective cation channels are made up of 595 amino acid subunits. The common structural motifs of P2X7R are the two transmembrane domains and a large glycosylated cysteine-rich extracellular loop as short intracellular and terminal domain and intracellular C-terminal domain [33 –35]. Activation of P2X7R results in the opening of the channel pore, allowing the passage of small cations (Na⁺, Ca⁺, and K⁺). Additionally, P2X7 is characterized by opening of a non-selective pore in response to repeated or prolonged activation, allowing permeation of larger molecular weight organic cations up to 600–800 Da. Patency of the large pore eventually results in membrane blebbing and cell death [36 –38].

Cytokines are the major mediators of neuroinflammation, including pro-inflammatory and anti-inflammatory processes, chemo-attraction and Aβ deposition in response to microglial activation. It has also been suggested that, as Aβ concentration increases in aging transgenic mouse models, it is associated with increased levels of the cytokines TNF-α, IL-6, interleukin 1-α, and GM-CSF, suggesting pathological accumulation of Aβ could drive a neuroinflammatory response [39 –41].

Chemokines regulate microglial migration to areas of neuroinflammation and enhance local inflammation in AD. There is upregulation of CCL2, CCR3, and CCR5 expression by microglia, whereas CCL4 is expressed by reactive astrocytes. It has been shown that Aβ deposition leads to generation of interleukin-8, CCL2, and CCL3. It has also been suggested that CX3CR1/CX3CL1 is involved in neuronal survival, plaque load, and cognition [42 –44].

The complement system is a major constituent of the immune system in the defense against pathogens. Activation of the proteolytic complement cascade results in opsonization. Major sources of complement system proteins include microglia and, to a lesser extent, astrocytes [45, 46]. It has also been shown that Aβ can activate the complement pathway. The protein clusterin is involved in the processing and clearance of immune complexes and is also a regulator of C3 convertase activity. Raised clusterin levels are associated with an increased risk of AD.

PET IMAGING OF NEUROINFLAMMATION

Studies have shown microglial activation to be a component of many CNS disorders including multiple sclerosis, focal epilepsy, stroke, and brain tumors. It is clear that all neurodegenerative diseases are associated with significant levels of neuroinflammation but that this inflammatory process is different from the autoimmune diseases of brain. While in relapsing remitting multiple sclerosis, it has been shown that the inflammatory process is accompanied by T-cell activation with specificity for CNS antigen infiltrates, the inflammatory reaction in AD is associated with activation of microglia in close proximity to Aβ plaques [47, 48]. During the activation process, microglia express the translocator protein (TSPO)3/4previously known as the peripheral benzodiazepine receptor (PBR)3/4on the outer surface of their mitochondria. This protein binds isoquinolines such as PK11195, and diazepam, and is present in peripheral tissues such as kidney, liver, and lungs. It was later demonstrated that TSPO/PBR is different from the central benzodiazepine receptor which is a component of the GABA_A complex found in the CNS [49]. TSPO forms a multimeric complex with a 32 kDa voltage dependent anion channel called mitochondrial porin and 30 kDa adenine nucleotide carrier in the outer mitochondrial membrane [49, 50]. Recent studies have shown that TSPO transports cholesterol, anions, and other substrates across the mitochondria and helps maintain the membrane potential [51, 52].

The enzyme monoamine oxidase B (MAO-B) is expressed by astrocytes and hydrolyses trace amines, phenylethyl amine, and dopamine. It binds deprenyl and D2-deprenyl. MAO-B expression increases with age and is thought to contribute to age-related neurodegeneration. Astrocytes upregulate expression of MAO-B under physiological and pathological conditions and so levels of brain MAO-B reflect astrocytosis.

Arachidonic acid is a polyunsaturated omega-6 fatty acid present in the phospholipid bilayer membranes in the brain. It serves as a second messenger and is involved in the upregulation of several signaling enzymes. It is now considered that arachidonic acid plays an important role in the inflammatory process. It has been suggested that binding of cytokines derived from microglia to calcium channel receptors on astrocytes activates phospholipase enzyme that releases arachidonic acid from membrane lipoproteins. Owing to these properties, arachidonic acid has been suggested to be a useful marker of neuroinflammation. Additionally, cyclooxygenase catalyzes the breakdown of arachidonic acid into prostaglandins. There are two isoforms of cyclooxygenase, Cox-1 and Cox-2, where Cox-1 is predominantly found in microglia whereas Cox-2 is expressed post-synaptically in neurons in the cortex, amygdala, and hippocampus [53]. Cox is also involved in the inflammatory cascade and is thus considered as a biomarker for neuroinflammation.

IMAGING TRANSLOCATOR PROTEIN

There are several TSPO radioligands which have been used to detect microglial activation in vivo in humans.

The TSPO radiotracer that has been most widely used is [¹¹C]-R-PK11195, an isoquinoline [1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide]. It is a selective antagonist for TSPO and, during the development of this radiotracer, it was shown that the R-enantiomer has two-fold higher affinity for TSPO compared to the S-enantiomer. Initial studies with PK11195 were conducted more than two decades ago, following which many papers have been published demonstrating neuroinflammation can be detected in a variety of neurodegenerative diseases and in neuroinflammatory conditions [54 –57].

Cagnin et al. reported the first AD study with ¹¹C-PK11195 PET and reported up to 40% increases in temporal lobe binding using a region of interest approach [58]. Increased microglial activation with aging was also seen. Subsequent studies generally confirmed the increased ¹¹C-PK11195 uptake in AD brain, though some studies failed to detect this [59 –61].

Studies have also evaluated the relationship between amyloid load and neuroinflammation. It has been reported that neuroinflammation correlates with amyloid load in AD and mild cognitive impairment (MCI) cases with raised amyloid deposition. While clusters of significant correlation between amyloid deposition and neuroinflammation have been demonstrated, these studies have also shown regional discrepancy between these two pathological processes, suggesting that neuroinflammation could also be triggered by other pathologies such as tau tangles and alpha synuclein aggregation. It has also been pointed out that, while we are able to image amyloid deposition using imaging ligands for beta sheeted protein, we are still unable to detect oligomeric Aβ which is most likely the toxic species contributing to the microglial activation [62, 63]. The trajectories of amyloid aggregation and microglial activation are likely to be different as the first precedes the second and rises to a stable level while inflammation rises and then may fall. Whether correlations are seen between amyloid and PK11195 uptake in brain regions may depend on the time point of the disease. While positive correlations have been detected between PK11195 and PIB uptake, one group could not demonstrate any such correlation, while another group found a negative correlation between amyloid and neuroinflammation [60, 64]. Longitudinal studies on preclinical and prodromal AD cases are really needed to sort this inter-relationship out. As other pathologies could also contribute to neuroinflammation, the advent of tau imaging agents is now allowing groups to evaluate the inter-relationships between amyloid, tau, and neuroinflammation.

While there have been discrepancies between the results across centers, it is now generally accepted that there is increased cortical microglial activation in AD most closely tracking an amyloid pattern. Several recent studies have also shown that there is increased microglial activation in amyloid positive MCI subjects and that this microglial activation can be seen before the conversion to dementia. Measurement of microglial activation using PK11195 PET shows increased regional tracer binding in the entorhinal, temporoparietal, and cingulate cortex in AD and MCI subjects. It has also been shown that microglial activation is increased in Parkinson’s disease, Parkinson’s disease with dementia, Lewy body dementia, schizophrenia, traumatic brain injury, multiple sclerosis, stroke, and several neuroinflammatory diseases. Despite the demonstration of microglial activation in these conditions using PK11195 PET tracer, there has been considerable controversy over its utility because of its relatively low signal-to-noise ratio. This has led to the development of several novel second-generation TSPO PET radiotracers with higher affinity and lower background signals. These include ¹¹C-PBR28, ¹¹C-DAA1106, ¹¹C-DPA713, ¹⁸F-FEDAA1106, ¹⁸F-PBR06, ¹⁸F-FEPPA, ¹⁸F-DPA-714, and ¹⁸F-GE180. [54 , 65–70]

These second-generation TSPO PET tracers were developed to overcome the shortcomings of ¹¹C-PK11195. However, one of the main limitations of the second-generation TSPO tracers is that their binding is influenced by the TSPO polymorphism expressed by subjects leading to differential binding across the general population due to variations in TSPO binding affinity. A polymorphism on the TSPO gene consisting of one amino acid substitution (Ala147Thr) results in the population having a high affinity binding (HAB) phenotype, mixed affinity binding (MAB) phenotype, or low affinity binding (LAB) phenotype for TSPO ligands other than PK11195. The Ala/Ala TSPO genotype (wild-type) results in HAB, while the Ala/Thr results in MAB, and Thr/Thr results in LAB. It has been shown that roughly 50% of the general population are high affinity binders while around 40% are mixed affinity binders and 10% of the population are low affinity binders. Hence, for a homogeneous population, one should select high and/or mixed affinity but not low affinity binders for study with 2^nd generation TSPO tracers [71]. While concerns have been raised regarding the utility of the studies conducted in subgroups of the population, it has been demonstrated that, in AD and MCI subjects, studies performed in apoE4 or apoE3 genetic subgroups could be generalized to the entire AD/MCI population, at least in observational studies. However, one could speculate that this could hold true even in intervention studies, as long as the treatments were not influencing cholesterol metabolism.

Studies using ¹¹C-PBR28 have shown a very high specific signal for microglial activation with an increased 80-fold affinity in animal models. Studies in AD subjects demonstrated that there is increased microglial activation specifically in the inferior parietal lobule, precuneus, occipital cortex, hippocampus, and entorhinal cortex [68, 72]. However, surprisingly these workers were unable to detect inflammation in amyloid positive MCI cases.

Despite the significant interest in the second generation tracers, results using [¹¹C]PBR28 have been inconsistent. While some groups have been able to demonstrate a significant difference between the AD and healthy control subjects, other groups were unable to show consistent differences. The average percentage increase in AD subjects compared to the control subjects was similar to that seen with [¹¹C](R)PK11195 PET (around 30% ). While no head-to-head study has compared [¹¹C]PBR28 and [¹¹C](R)PK11195 PET in AD, there is no convincing evidence to suggest that one tracer is more sensitive than the other. As there is no typical reference devoid of microglial activation in the brain in neurodegenerative diseases, TSPO ligands are also affected by the quantification issues. While supervised cluster analysis has been used to define a reference tissue cluster representing normal grey matter uptake kinetics for [¹¹C](R)PK11195, such an approach has not been feasible for [¹¹C]PBR28. Hence, a cerebellar reference has been used to reflect non-specific uptake approach for [¹¹C]PBR28 which is likely to overestimate this component. There is also considerable variability in the plasma protein binding of TSPO ligands across subjects and disease states [73-75]. This makes using an arterial plasma input reference function difficult due to the variability in time activity curves.

Initial studies with ¹¹C-DPA-713 demonstrated that it provided better sensitivity than ¹¹C-PK11195 and showed more TSPO density in widespread regions of ageing subjects and also AD subjects [76, 77].

While [¹¹C]DPA-713 was being evaluated, a newer, higher affinity, higher specific to non-specific binding tracer with a longer half-life, [¹⁸F]DPA-714 was developed and evaluated [66, 78]. [¹⁸F]DPA-714 has demonstrated significant increases in the frontal, temporal, and parietal cortex of AD cases, again suggesting that microglial activation could be detected with both first and second generation TSPO tracers. Interestingly, highest tracer binding was seen in prodromal AD suggesting that inflammation may reduce as MCI progresses to AD.

Other second-generation tracers include [¹⁸F]FEPPA, where PET has shown that there is significant uptake in the grey matter of the hippocampus, prefrontal cortex, temporal, parietal, occipital cortex, posterior limb of internal capsule, and cingulum of AD cases [79, 80]. [¹⁸F]FEMPA PET detected significant uptake in the medial and lateral temporal cortex, posterior cingulate, caudate, putamen, and thalamus in AD. [¹¹C]DAA-1106, [¹⁸F]FEDAA-1106, and [¹¹C]vinpocetine PET have also demonstrated significant microglial activation in AD and other neurodegenerative diseases [60 , 81].

In the early stages of AD (amyloid-positive MCI subjects), [¹¹C]DAA-1106 and [¹⁸F]DPA-714 PET have demonstrated high increases in binding in the frontal, temporal and parietal cortex. This was consistent with previous observations using [¹¹C](R)PK11195 PET [82]. While initial studies with ¹¹C-PBR28 failed to demonstrate increased microglial activation in MCI subjects, recent studies have shown that increased microglial activation in MCI subjects can be seen on a single subject analysis.

Microglial activation has been reported in AD variants such as posterior cortical atrophy, where PBR28 has demonstrated significantly increased binding in occipital, posterior parietal, and temporal regions. While there have been some reports concerning correlation with age, later reports using [¹⁸F]DPA-714 in a larger cohort have not shown an age effect. There has been significant negative correlation between TSPO binding and cognitive performance using ¹¹C-PBR28, [¹¹C](R)PK11195 and [¹⁸F]FEPPA. Interestingly, a recent study in a large number of AD and MCI subjects demonstrated that MMSE was positively correlated with microglial activation [70]. Studies have already demonstrated that, in AD subjects, there is a negative correlation between microglial activation and atrophy while, using [¹⁸F]DPA-714, microglial activation was positively correlated with the grey matter volume in MCI and AD patients. Studies have already demonstrated correlations between amyloid load using [¹¹C]PIB and [¹¹C](R)PK11195, [¹¹C]PIB and [¹¹C]PBR28 and [¹⁸F]DPA714 in different cortices, precuneus, hippocampus, and parahippocampal gyrus.

LONGITUDINAL EVALUATION OF MICROGLIAL ACTIVATION

There are only a handful of studies which have evaluated the longitudinal relationship of microglial activation and disease progression. Fan et al. demonstrated that there is increased microglial activation as the disease progresses in established AD, while in MCI subjects there was a longitudinal reduction. Please see Fig. 1. The authors argued that the microglial activation detected by TSPO tracers in the early and late stages of the disease could be phenotypically different, and in the early stage of the disease it may be detecting the anti-inflammatory phenotype while during the later stages of the disease it may be detecting the pro-inflammatory phenotype. It has also been suggested that, while the anti-inflammatory phenotype becomes ineffective in clearing amyloid and toxic debris, there is progressive amyloid deposition and neuronal damage. In contrast, as the disease progresses there is persistent activation of the pro-inflammatory phenotype which is also detected by the microglial tracer as a persistent elevation of microglial activation. This later phase of microglial activation is also detected by the TSPO tracer and continues to rise as the disease progresses and correlates with the cognitive impairment [83].

Fig.1

Hypothetical model of dual peak of microglial activation in the Alzheimer’s disease (AD) trajectory. The upper panel demonstrates the hypothetical model of morphological changes in microglia in AD trajectory, where ramified microglia transform to anti-inflammatory (protective) microglial phenotype and pro-inflammatory (toxic) microglial phenotypes. The lower panel shows the microglial activation in relation to other biomarkers detectable using positron emission tomography where two peaks of microglial activation are present in AD trajectory (Reprinted from Brain [83]).

Kreisl et al. demonstrated that in AD subjects there is increased binding of [¹¹C]-PBR28 in the inferior parietal lobule, precuneus occipital cortex, hippocampus, entorhinal cortex, middle and inferior temporal cortex. Longitudinally there was an annual increase of 3.9 to 6.3% in patients with AD. It is also proposed that the annual rate of increased TSPO binding in the tempoparietal region was about five-fold higher in patients with clinical progression compared to those who did not progress [84].

Hamelin et al. evaluated 64 patients with AD and 32 controls. They demonstrated that higher microglial activation was present in slow decliners compared with fast decliners. They also demonstrated that microglial activation is present in prodromal and possibly at the preclinical stage of AD and was found to play a protective role in in the clinical progression of the disease. This study further substantiates the concept that microglial activation could be protective in early stages of the disease [70].

IMAGING ASTROCYTE ACTIVATION

L-deprenyl is an irreversible monoamine oxidase-B (MAO-B) inhibitor, which exists on the outer mitochondrial membrane of astrocytes [85, 86]. The radiotracer [¹¹C]deuterium-L-deprenyl ([¹¹C]DED) has high affinity and specificity for MAO-B increases in most brain regions in healthy older adults. Activity of MAO-B increases in AD patients’ brains where the enzyme is over expressed by reactive astrocytes. Autoradiographic studies have demonstrated that [¹¹C]DED can be used as an in vivo PET ligand for assessing MAO-B in AD brains. In a study of eight MCI subjects, seven AD subjects, and 14 healthy controls it has been shown that there is increased astrocyte activation in the left temporal, left insular cortex, bilateral anterior cingulate, right parahippocampal cortex, right hippocampus, right caudate, and left putamen [87]. It was also shown that increased [¹¹C]DED binding to MAO-B was more evident in the amyloid-positive MCI subjects compared to the amyloid-negative subjects and AD subjects.

Novel astrocyte markers are being tested which

includes markers of imidazoline binding. Preliminary data using the tracer [¹¹C]BU99008 have demonstrated significant uptake in different cortical regions in healthy control subjects. While the results of further studies are awaited, the results from the healthy control subjects are promising.

While it is recognized that neuroinflammation is a prominent and early feature of AD which plays a key role in modulating disease progression, the role of astrocyte activation is still being debated. Several studies indicate that astrocyte-mediated inflammatory processes also contribute to neurodegeneration in AD through increased astrocytic expression of pro-inflammatory cytokines and chemokines, activation of the complement cascade as well as reactive oxygen and nitrogen species. To understand the role of astrocytes, further studies are necessary using in vivo imaging agents which would allow us to track the progression of astrocyte activation longitudinally.

NOVEL TARGETS OF NEUROINFLAMMATION

Apart from targeting TSPO, further work is necessary to develop new targets to detect the migratory capacity of microglia or their ability to phagocytose toxic products. While such targets could be of very significant interest, new approaches such as cell type specific transcriptional profiling and identification of numerous cell specific changes may provide a challenge and is being still pursued as a novel strategy to identify microglial activation.

The cannabinoid type 2 receptor (CB2R) is part of the endogenous cannabinoid system which is an alternative membrane marker of microglial activation. PET tracers showing high affinity for CB2R have been developed, one of which is [¹¹C]NE40. However, this tracer showed lower uptake in AD patients compared to the control subjects. It was suggested this could be due to low level of CB2R expression and insufficient selectivity for CB2R. Several other high affinity agonists are also being evaluated as CB2R tracers, such as [¹¹C]MA2, [¹⁸F]MA3, [¹⁸F]RS126 [88, 89].

It has been shown that [¹¹C]KTP-Me is a pro-radiotracer for ketoprofen (KTP) and animal studies have suggested that [¹¹C]KTP is retained in inflammatory lesions due to the expression of Cox-1. While a first human study in healthy volunteers showed that [¹¹C]KTP-Me could be a potential PET tracer with good penetration in human brain, subsequent studies did not find a difference between controls and AD subjects [90]. Nicotinic acetylcholine receptors (nAChR) are upregulated in neuroinflammation. The ligand targeting α4β2 nAChR has been demonstrated to have similar patterns of uptake as [¹¹C]-PK11195. However, despite the initial enthusiasm, several nicotinic acetylcholine receptor tracers have not been successful. New compounds such as [¹⁸F]ASEM and [¹⁸F]DBT-10 are now being evaluated [91].

Recent studies have shown that the P2X7 receptor is widely present in neuroinflammation. Studies have shown that deletion and pharmacological blockade of P2X7Rs alter responsiveness in animal models of neurological disorders. P2X7 receptors are expressed in the cell-surface membrane of hematopoietic cells such as macrophages and microglia. Novel PET tracers targeting P2X7 receptors include [¹¹C]GSK1482160, [¹¹C]A740003, and [¹¹C]JNJ-54173717.

Other targets of interest include phospholipase A2 (PLA2) activity. It has been shown that inflammatory cytokines released from microglia can bind to astrocyte receptors which are coupled to PLA2. When this enzyme is activated it hydrolyses arachidonic acid (AA) from the membrane. Hence, by measuring the brain uptake of [¹¹C]arachidonic acid, one could determine the metabolic loss of arachidonic acid in the brain. It was proposed that increased incorporation of [¹¹C]AA could represent upregulated AA metabolism due to neuroinflammation.

Another target is adenosine A2A receptors (A2AR). The binding of adenosine to A2AR tends to attenuate inflammation by endogenously limiting the inflammatory response and leads to upregulation of these receptors at the sites of inflammation. While these mechanisms have been proposed, to date no definite tracer which could replace the TSPO tracer has been developed.

CONCLUSION

It is now clear that neuroinflammation plays a significant role in AD and neurodegenerative diseases. Microglia and astrocytes play a significant role in neuroinflammation; however, activation of microglia and astrocytes can vary depending on the stage of the disease and the trajectories are still uncertain. While we are now able to image activation of microglia and astrocytes, further research is necessary to evaluate whether initially they have a protective and later a cidal influence on neurodegeneration as some series have suggested. There have been significant advances in imaging microglia, with further recent advances in imaging astrocytes. More evidence is emerging regarding the differential role of microglial and astrocyte activation in different stages of neurodegenerative disease, which will form the basis of future research in neuroinflammation in the coming decades. As there are many other processes involved in neuroinflammation, future research will need to develop biomarkers to evaluate new markers such as chemokine receptor function to differentiate the pro-inflammatory and anti-inflammatory molecules involved in neuroinflammation. Current evidence suggests that not all the neuroinflammatory processes happening in the brain are detrimental, and further research is necessary to separate and understand them.

DISCLOSURE STATEMENT

Authors’ disclosures available online (https://www.j-alz.com/manuscript-disclosures/17-9929).

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