PET molecular imaging for pathophysiological visualization in Alzheimer’s disease

¹¹C-Pittsburgh compound B (¹¹C-PIB) is the first and one of the most successful selective Aβ tracers after a non-specific radiotracer named ¹⁸F-FDDNP, which greatly drives AD researches forward. As a derivative of Thioflavin T, a dye widely used to visualize amyloid in vitro, ¹¹C-PIB could bind to beta-sheet structures, especially fibrillar Aβ in plaques with high affinity (Ki = 20.2 nM) [24]. To some extent, ¹¹C-PIB also binds to Aβ oligomers, a neurotoxic form of Aβ gaining increasing attention [112]. Though ¹¹C-PIB was found bind to tau [113] and α-synuclein [114] as well, in the concentrations clinically used, neurofibrillary tangles [115] and Lewy bodies [114] do not contribute to the retention of PIB. These characteristics lead to a sensitive and specific quantification of Aβ burden in vivo.

A robust inverse correlation was observed between ¹¹C-PIB retention and Aβ₄₂ level in CSF [116]. And in comparison with CSF biomarkers, PET techniques greatly improved the accuracy of clinical diagnosis [25]. As postmortem studies suggested, the retention of ¹¹C-PIB in PET images is highly correlated to the distribution areas of Aβ at autopsy [117]. Similar to studies conducted by Braak, AD patients mostly show increased uptake in frontal, cingulate, precuneus, striatum, parietal, and lateral temporal cortices. And regions of occipital, sensorimotor, and mesial temporal lobe are usually spared [7]. The striking similarity in the lobes affected and cortices supporting memory function further suggests the importance of amyloid PET in indicating early AD stages [118]. Besides, as amyloid distribution is different in various dementias [119, 120] and AD subtypes [7], the spatial patterns of amyloid deposition measured by PET may help to differentiate AD from other neurodegenerative diseases and determine AD phenotypes.

Additionally, ¹¹C-PIB positive is a sensitive predictor of disease conversion from MCI to AD [26]. With the faster converters commonly showing higher PIB uptake in brain [108], PET presents a better accuracy than CSF biomarker to classify stable MCI and progressed MCI [121]. And the specificity of conversion predicting would constantly increase as the follow-up period elongates [122]. Thus, ¹¹C-PIB PET has been applied to help researchers select subjects of preclinical AD, of which the group is extensively focused on as the target of Aβ-related therapies. ¹¹C-PIB also possesses great potential to assess treatment response, providing much crucial information about the reduction of amyloid burden and its effects on cognitive decline [123].

Though ¹¹C-PIB possesses plenty of virtues, the short half-life of carbon-11 (half-life of 20 min) limits its access only in PET centers with an on-site cyclotron and experienced carbon-11 radiochemistry technicists. To solve this dilemma, multiple fluorine-18 (half-life of 110 min) labeled radiotracers were developed, including ¹⁸F-florbetapir (a.k.a. Amyvid) [27], ¹⁸F-flutemetamol (a.k.a. Vizamyl) [28], and ¹⁸F-florbetaben (a.k.a. Neuraseq) [29]. They were known as the second generation of amyloid imaging radiotracers. And to date, these compounds have been approved by the US Food and Drug Administration (FDA). The longer half-life of fluorine-18 enables these tracers to be centrally produced and regionally distributed like ¹⁸F-FDG, which may greatly lower the cost and allow AD researches be extensively conducted.

Research using these three tracers have confirmed their high sensitivity and specificity [19]. And mounting evidence has suggested their potential role in amyloidosis early detecting, AD subjects discriminating, and disease progression prediction [7]. In general, results derived from ¹¹C-PIB could mostly be replicated. Although different binding characteristics can be observed both in white matter and grey matter, cortical retention of these tracers was highly correlated with that of ¹¹C-PIB. The strong link between them may allow PET data be transformed and compared in multicenter studies that use different tracers [124].

These tracers have been extensively adopted by researchers around the world, and few drawbacks were reported. But in a minority of subjects, appreciable white matter retention can be observed, which may hamper the visual assessment of clinicians [30]. And according to a study evaluating the effect of concurrent pathologies on the uptake of florbetapir, while other diseases do not affect the tracer retention, Lewy bodies significantly lower the ratio of standard uptake value (SUV), the underlying mechanisms worth further exploring [125].

¹⁸F-NAV4694, formerly known as ¹⁸F-AZD4694, is a more novel radiotracer being considered to outperform ligands currently available. In vitro studies of AZD4694 have shown its high affinity for Aβ-fibrils (K (d) = 2.3 + / − 0.3 nM). And in brain tissues of AD patients, selective binding to Aβ deposits of NAV4694 can be observed in grey matter, while the non-specific signal in white matter is low [31]. Besides, according to results of clinical validation, ¹⁸F-NAV4694 possesses a favorable kinetic profile and a relatively low test–retest variability [32]. When comparing ¹⁸F-NAV4694 with ¹¹C-PIB, nearly identical imaging characteristics were reported (Fig. 1) [33]. All these results suggest the tremendous potential of ¹⁸F-NAV4694 to be widely applied both in clinical practice and scientific research.

It is worth noting that most radioligands available now are developed to bind to Aβ in insoluble fibrils. However, increasing evidence has suggested that soluble Aβ aggregates are the neurotoxic form of Aβ causing neural dysfunction. Moreover, diffuse plaque pathology in AD patients usually presents low PIB retention in brain due to the relatively low level of fibrillar Aβ [126]. Soluble Aβ usually better correlated to the severity of AD than amyloid plaques [127]. These results call for agents to visualize soluble oligomers and further investigate their role in AD pathology. In 2003, an antibody-based radioactive ligand, ¹²⁵I-mAb158, was reported to show high affinity for soluble protofibrils [34]. And recently, some derivatives of this radioligand were described in both diagnostic and therapeutic research. ¹²⁴I-RmAb158-scFv8D3 was used to evaluate the distribution of an antibody RmAb158-scFv8D3 in a protofibril-clearing immunotherapy research [36]. And ¹²⁴I-Di-scFv-3D6-8D3 was developed with the ability to cross the blood–brain barrier and bind to both fibrillary Aβ and soluble aggregates [35].

Although amyloid PET has contributed much to a better understanding of AD pathophysiology, some problems remain unsolved. Histologically determined Aβ deposition was reported to poorly correlate to regional uptake in some PIB negative patients, calling for further postmortem research to evaluate the binding characteristics [128]. As there are some other mechanisms involved in disease progression [129], the dynamic alternation of amyloid pathology and its correlation with other biomarkers should be further explored. Besides, according to some studies comparing amyloid PET and other measures, PET is a sensitive technique to predict disease progression with limited specificity. So other biomarkers such as ¹⁸F-FDG metabolism or brain atrophy should be taken into account to improve the prediction specificity [130, 131]. Additionally, different PET centers might apply various protocols to complete Aβ imaging acquisition. Meanwhile, the interpretation of PET images relied on the clinical experience of physicians. Therefore, more standardized approaches involved in image acquisition and interpretation could be helpful to reduce the variation. A guideline or widely accepted protocol could assist physicians to perform a standardized Aβ imaging acquisition and minimize errors produced by operators or PET machines. The combination of visual interpretation and SUVR in Aβ imaging interpretation could establish a semiquantitative analysis system which decreased the variation developed by visual interpretation to some extent [132]. And as studies of prefibrillar Aβ imaging are rather few so far, more research is warranted to develop better prefibrillar Aβ tracers in the future.

¹⁸F-AV-1451 (a.k.a. ¹⁸F-T807 or ¹⁸F-flortaucipir) was the first promising PET tracer for visualization and quantification of NFT pathology in AD patients. And to date, it has been the most widely used tau-binding radiotracer [37]. According to results of autoradiography and immunohistochemistry staining in human cortical sections, the selectivity of ¹⁸F-AV-1451 for tau is 29-fold over Aβ. It also presents high affinity to PHF-tau in the nanomolar range (Kd = 14.6 nM), very low non-specific binding in white matter, and favorable tracer kinetics [37]. Moreover, good reproducibility was reported in a test–retest study, making ¹⁸F-AV-1451 PET a powerful means in longitudinal researches [38, 39].

Studies comparing tau PET imaging and CSF biomarkers revealed a significant correlation between tau in CSF and ¹⁸F-AV-1451 uptake, especially in some specific cortical regions [147]. And in patients with memorial dysfunction, ¹⁸F-AV-1451 is able to distinguish AD from other non-AD neurodegenerative disorders with high accuracy [40]. Abnormally higher cortical retention can be observed in MCI and AD patients compared with cognitive normal subjects, and the elevated binding of ¹⁸F-AV-1451 is better correlated to the severity of cognitive impairment than ¹¹C-PIB [148]. Besides, it is suggested that the distribution of tracer retention can be classified into patterns as Braak prescribed (Fig. 2) [149], enabling tau PET to monitor progression of tau pathology and evaluate the cause of clinical symptoms. ¹⁸F-AV-1451 positive results can also be observed in preclinical AD patients, with faster increase of tracer retention being observed in the group of high amyloid burden [150], which may further suggest a close relationship between tau and amyloid-beta, making ¹⁸F-AV-1451 PET a valuable method to screen and determine subjects in disease-modifying clinical trials. And more recently, international consensus suggested to consider the application of ¹⁸F-AV-1451 PET imaging for definitive diagnosis, differential diagnosis, and severity determination of AD [151].

However, this tracer has some limitations. As a study has suggested, the kinetics of this tracer in brain varies in different disease stages, and the uptake curve does not plateau during the typical imaging duration; further dynamic researches are warranted to determine the best timing of scanning for accurately diagnosing [41]. Additionally, in some cortical areas with no tau pathology, off-target binding of ¹⁸F-AV-1451 was reported, especially in basal ganglia, substantia nigra, and choroid plexus [42, 43], so more imaging-pathological studies on postmortem materials should be conducted for a better interpretation of PET imaging.

¹⁸F-AV-680 is another benzimidazole-pyrimidines derivative showing promise for the tau imaging in AD patients. It also displays high selectivity and affinity to PHF-tau, little white matter retention, and minimal off-target binding. In contrast to ¹⁸F-AV-1451, ¹⁸F-AV-680 demonstrates a substantial defluorination in rodents, and a faster kinetics in human brains [44]. But studies are warranted to further verify these findings.

The first ¹⁸F-labelling quinoline derivatives created for tau imaging were ¹⁸F-THK523, but its clinical application was hampered due to several limitations, including the high white matter retention and relatively low selectivity of tau over Aβ [152]. Then, two new derivatives: ¹⁸F-THK-5105 and ¹⁸F-THK-5117, were developed to improve the tau affinity and selectivity. These two compounds showed better imaging capability both in vitro and in vivo [45], but nonspecific binding in white matter still existed [46]. After that, ¹⁸F-THK-5351 was further developed. Encouragingly, ¹⁸F-THK-5351 showed favorable pharmacokinetics, higher affinity for tau protein fibrils, and lower retention in white matter, allowing for better tau visualization [47].

Using ¹⁸F-THK-5351, the spatial distribution of retention in AD patients is corresponded to the pattern reported by Braak previously [153]. In AD patients, significant higher retention is observed in association cortices and limbic areas than healthy subjects, and the level of tracer uptake was clearly associated with the cognitive function [154]. And by combining the topology information of tau pathology, various neurodegenerative diseases can be differentiated [155‐157]. However, while ¹⁸F-THK-5351 seems to target similar binding site with ¹⁸F-AV-1451, it is less sensitive and specific to tau of AD and shows higher off-target binding than ¹⁸F-AV-1451 [48]. Additionally, the availability of monoamine oxidase B (MAO-B) was reported to affect the uptake of ¹⁸F-THK-5351 in brain [49]. So the retention in areas of commonly off-targeted and tissues containing MAO-B should be interpreted with caution.

Another series of probes, phenyl/pyridinyl-butadienyl-benzothiazoles/benzothiazoliums (PBBs), were also developed as potential tau-targeted PET tracers. And among these derivatives, ¹¹C-PBB3 was the most promising candidate, demonstrating a good binding affinity in the nanomolar range and excellent selectivity for tau over amyloid (40–50-fold) [50]. And compared to other classes of tracers, ¹¹C-PBB3 is the first compound reported to detect a broad range of tau aggregates, binding other tau fibril types as well as AD PHFs [51]. Additionally, ¹¹C-PBB3 has shown the potential to differentiate patients in the AD continuum group from biomarker-negative individuals and suspected non-AD pathophysiology group [158].

Nevertheless, some shortcomings of this tracer should be overcome in the future. While nonspecific signals of ¹¹C-PBB3 are generally low, retention in venous sinuses is noticeable [50]. And ¹¹C-PBB3 is vulnerable to light due to its structure [52]; the instability will add complexity to its synthesis, purification, and clinical usage. The short half-life of ¹¹C also limits its availability. Besides, a radiometabolite of this tracer could enter the brain and confound the imaging results [159].

Now several kinds of ligands have been developed for tau imaging. But as tau proteins are heterogeneous in isoforms and conformations, and the roles of various tau pathologies are elusive, further researches are warranted to better classify these tracers and interpret the binding pattern of each tracer in longitudinal studies. And as quite a few studies have reported off-target retention of these tracers, more imaging-to-postmortem researches should be conducted to validate their binding characteristics. Since tau PET imaging is bound to be applied to monitor disease progression and therapeutic efficacy, reproducibility assessment of these compounds is critical for accurately comparing results. Besides, studies comparing the dynamic change of tau tracer retention and other biomarkers are necessary for a better exploration of the interactions of different pathology, as well as helping determining the best means used for disease evaluation at the right time.

Neuroinflammation is regarded as an important participant in AD pathophysiology, though the exact role remains elusive. Some studies have suggested that the inflammation is involved in promoting tau hyperphosphorylation and driving neurodegenerative processes [173], while there is evidence showing its protective role at early AD stages [174]. Currently, numerous PET ligands are available to evaluate neuroinflammation by targeting underlying biological processes [175]. For example, microglial activation could be visualized by quantifying the 18-kDa translocator protein using radiotracers such as ¹¹C-PK11195 [59], ¹¹C-DAA1106 [60], ¹⁸F-FEDAA1106 [61], etc. ¹¹C-DED can be utilized to bind monoamine oxidase B (MAO-B) and then measure reactive astrocytosis [62]. And the level of ¹¹C-AA (arachidonic acid) uptake in brain is a reliable biomarker for detecting the up-regulation of phospholipase activity [63].

High content of metal ions like Zn²⁺ and Cu²⁺ was observed in and around the formation site of amyloid plaques [176]. And these metals are able to mediate the protein aggregation and lead to the formation of amyloid plaques [177]. According to the data, a “Metal Hypothesis of AD” was proposed, and many ionophores and metal-chelators were developed as disease-modifying drugs [178]. The first PET radiotracer discovered to bind metal ions in amyloid plaques is ¹⁸F-CABS13, showing promising preliminary results in a mouse model [64]. And in recent years, ¹¹C-L2-b and ¹⁸F-FL2-b were synthesized and evaluated to investigate the metallobiology of AD [65], but further researches on the metal-Aβ radioligands are warranted.

Glycogen synthase kinase 3 (GSK3) is a wide expressed serine/threonine kinase involved in a range of cellular processes, including apoptosis, glucose regulation, and cell signaling [179‐181]. It has been shown that GSK3 activity is associated with the formation of both amyloid plaques and NFTs [182, 183]. And inhibitors of GSK3 were reported to reduce Aβ production and tau aggregation [184]. These evidence encourage the “GSK3 hypothesis of AD” [185]. Some radiolabeled compounds are now available for GSK3 monitoring [186]. ¹¹C-A1070722 and several ¹¹C-labelling isonicotinamides were developed recently showing better binding characteristics than that synthesized previously [66, 67]. And more recently, glycogen synthase kinase 3β could be visualized by using ¹⁸F-labelling maleimide-based and isonicotinamide-based ligands which showed moderate brain uptake [68, 69].

Glutamate is the most abundant excitatory neurotransmitter in brain, playing a vital role in learning and memory [187, 188]. Glutamate mediates ionotropic and metabotropic receptors (iGluRs and mGluRs, respectively), both of which are implicated in a spectrum of neurodegenerative diseases including AD [189, 190]. During the past decades, a number of radioligands have been developed for imaging iGluRs, but few PET tracers presented favorable in vivo results [70]. In terms of probes of mGluRs, no promising ligands are now available for imaging group III mGluRs (including mGluRs 4, 6, 7, and 8) and mGluR3. However, mGluR2 can be evaluated by a prospective tracer ¹¹C-JNJ42491293. For visualizing mGluR1, ¹⁸F-FITM, ¹¹C-ITMM, ¹¹C-ITDM, and ¹⁸F-FIMX are clinically useful. And the most widely studied receptor, mGluR5, can be measured in vivo using ¹⁸F-FPEB, ¹⁸F-SP203, and ¹¹C-ABP688 [71].

As a few alternations of the endogenous cannabinoid system (ECS) were reported in post-mortem studies [191], the ECS gets increasing attention as a novel target in some AD-therapeutic researches. Though the exact role of ECS in AD remains elusive, accumulating evidence suggests that the activation of cannabinoid signaling could alleviate AD symptoms by reducing excitotoxicity, neuroinflammation, and oxidative stress [192]. Currently, a series of compounds have been developed for the imaging of ECS. ¹⁸F-MK-9470 [72],¹⁸F-FMPEP-d2 [73], ¹⁸F-FPATPP [74], and ¹¹C-SD5024 [75] are promising imaging tools for ECS researches in terms of type 1 cannabinoid receptor (CB1R). And the availability of type 2 cannabinoid receptor (CB2R) could be measured by several radioligands like ¹⁸F-labelling 1,3,5-triazine derivatives or ¹¹C-labelling 4-oxoquinoline derivatives [76]. Fatty acid amide hydrolase (FAAH), the principal catabolic enzyme metabolizing endogenous cannabinoid, could also be detected using tracers like ¹¹C-CURB [77], ¹¹C-MK-3168 [78], and ¹¹C-FAAH-1906 [79].

The cholinergic dysfunction has also been found to be crucially involved in the pathophysiology of AD. A few post-mortem studies have suggested the cholinergic alternation in the course of dementia [193, 194]. And by using PET imaging techniques with selective tracers, diverse components of the cholinergic system could be measured in vivo [195]. Specifically, cerebral acetylcholinesterase (AChE) activity could be measured by using ¹¹C-PMP [80] and ¹¹C-MP4A [81]. ¹¹C-MK-6884 [82] and ¹⁸F-ASEM [83] can be used to quantify the binding potential of the muscarinic and nicotinic acetylcholine receptors (mAChR and nAChR), respectively. And the imaging of vesicular acetylcholine transporter (VAChT) can be achieved by a series of compounds, such as ¹¹C-TZ659 [84], ¹⁸F-VAT [85], and radiolabeled benzovesamicol VAChT analogs [86].

Although the cholinergic system is primarily affected in AD pathology, neurotransmitters of monoaminergic system are also implicated, including serotonin (5-HT), dopamine (DA), and norepinephrine (NE) [196, 197]. And the disruption of the dynamic balance of monoaminergic system may impair the functional neural networks of AD patients involved in affective regulation and executive function [198]. Serotonin 5-HT_1A receptor, which is particularly relevant to AD, could be selectively bound by ¹¹C-WAY100635 [87] and ¹⁸F-MPPF [88]. And tracers targeting dopaminergic system have been extensively utilized in Parkinsonian disorders [199]. In terms of norepinephrine transporter imaging, ¹¹C-MRB was considered as the most promising ligand [89].