Maiko Ono

Imaging of tau pathology in a tauopathy mouse model and in Alzheimer patients compared to normal controls

Accumulation of intracellular tau fibrils has been the focus of research on the mechanisms of neurodegeneration in Alzheimers disease (AD) and related tauopathies. Here, we have developed a class of tau ligands, phenyl/pyridinyl-butadienyl-benzothiazoles/benzothiazoliums (PBBs), for visualizing diverse tau inclusions in brains of living patients with AD or non-AD tauopathies and animal models of these disorders. In vivo optical and positron emission tomographic (PET) imaging of a transgenic mouse model demonstrated sensitive detection of tau inclusions by PBBs. A pyridinated PBB, [(11)C]PBB3, was next applied in a clinical PET study, and its robust signal in the AD hippocampus wherein tau pathology is enriched contrasted strikingly with that of a senile plaque radioligand, [(11)C]Pittsburgh Compound-B ([(11)C]PIB). [(11)C]PBB3-PET data were also consistent with the spreading of tau pathology with AD progression. Furthermore, increased [(11)C]PBB3 signals were found in a corticobasal syndrome patient negative for [(11)C]PIB-PET.

Longitudinal, Quantitative Assessment of Amyloid, Neuroinflammation, and Anti-Amyloid Treatment in a Living Mouse Model of Alzheimer's Disease Enabled by Positron Emission Tomography

We provide the first evidence for the capability of a high-resolution positron emission tomographic (PET) imaging system in quantitatively mapping amyloid accumulation in living amyloid precursor protein transgenic (Tg) mice. After the intravenous administration of N-[11C]methyl-2-(4′-methylaminophenyl)-6-hydroxybenzothiazole (or [11C]PIB for “Pittsburgh Compound-B”) with high-specific radioactivity, the Tg mice exhibited high-level retention of radioactivity in amyloid-rich regions. PET investigation for Tg mice over an extended range of ages, including longitudinal assessments, demonstrated age-dependent increase in radioligand binding consistent with progressive amyloid accumulation. Reduction in amyloid levels in the hippocampus of Tg mice was also successfully monitored by multiple PET scans along the time course of anti-amyloid treatment using an antibody against amyloid β peptide (Aβ). Moreover, PET scans with [18F]fluoroethyl-DAA1106, a radiotracer for activated glia, were conducted for these individuals parallel to amyloid imaging, revealing treatment-induced neuroinflammatory responses, the magnitude of which intimately correlated with the levels of pre-existing amyloid estimated by [11C]PIB. It is also noteworthy that the localization and abundance of [11C]PIB autoradiographic signals were closely associated with those of N-terminally truncated and modified Aβ, AβN3-pyroglutamate, in Alzheimers disease (AD) and Tg mouse brains, implying that the detectability of amyloid by [11C]PIB positron emission tomography is dependent on the accumulation of specific Aβ subtypes. Our results support the usefulness of the small animal-dedicated PET system in conjunction with high-specific radioactivity probes and appropriate Tg models not only for clarifying the mechanistic properties of amyloidogenesis in mouse models but also for preclinical tests of emerging diagnostic and therapeutic approaches to AD.

Imaging of Peripheral Benzodiazepine Receptor Expression as Biomarkers of Detrimental versus Beneficial Glial Responses in Mouse Models of Alzheimer's and Other CNS Pathologies

We demonstrate the significance of peripheral benzodiazepine receptor (PBR) imaging in living mouse models of Alzheimers disease (AD) as biomarkers and functional signatures of glial activation. By radiochemically and immunohistochemically analyzing murine models of the two pathological hallmarks of AD, we found that AD-like Aβ deposition is concurrent with astrocyte-dominant PBR expression, in striking contrast with nonastroglial PBR upregulation in accumulations of AD-like phosphorylated tau. Because tau-induced massive neuronal loss was distinct from the marginal neurodegeneration associated with Aβ plaques in these models, cellular localization of PBR reflected deleterious and beneficial glial reactions to tau versus Aβ pathologies, respectively. This notion was subsequently examined in models of various non-AD neuropathologies, revealing the following reactive glial dynamics underlying differential PBR upregulation: (1) PBR(−) astrogliosis uncoupled with microgliosis or coupled with PBR(+) microgliosis associated with irreversible neuronal insults; and (2) PBR(+) astrogliosis coupled with PBR(− or ±) microgliosis associated with minimal or reversible neuronal toxicity. Intracranial transplantation of microglia also indicated that nontoxic microglia drives astroglial PBR expression. Moreover, levels of glial cell line-derived neurotrophic factor (GDNF) in astrocytes were correlated with astroglial PBR, except for increased GDNF in PBR(-) astrocytes in the model of AD-like tau pathology, thereby suggesting that PBR upregulation in astrocytes is an indicator of neurotrophic support. Together, PBR expressions in astrocytes and microglia reflect beneficial and deleterious glial reactions, respectively, in diverse neurodegenerative disorders including AD, pointing to new applications of PBR imaging for monitoring the impact of gliosis on the pathogenesis and treatment of AD.

In vivo positron emission tomographic imaging of glial responses to amyloid-beta and tau pathologies in mouse models of Alzheimer's disease and related disorders.

Core pathologies of Alzheimers disease (AD) are aggregated amyloid-β peptides (Aβ) and tau, and the latter is also characteristic of diverse neurodegenerative tauopathies. These amyloid lesions provoke microglial activation, and recent neuroimaging technologies have enabled visualization of this response in living brains using radioligands for the peripheral benzodiazepine receptor also known as the 18 kDa translocator protein (TSPO). Here, we elucidated contributions of Aβ and tau deposits to in vivo TSPO signals in pursuit of mechanistic and diagnostic significance of TSPO imaging in AD and other tauopathies. A new antibody to human TSPO revealed induction of TSPO-positive microgliosis by tau fibrils in tauopathy brains. Emergence of TSPO signals before occurrence of brain atrophy and thioflavin-S-positive tau amyloidosis was also demonstrated in living mice transgenic for mutant tau by positron emission tomography (PET) with two classes of TSPO radioligands, [11C]AC-5216 and [18F]fluoroethoxy-DAA1106. Meanwhile, only modest TSPO elevation was observed in aged mice modeling Aβ plaque deposition, despite the notably enhanced in vivo binding of amyloid radiotracer, [11C]Pittsburgh Compound-B, to plaques. In these animals, [11C]AC-5216 yielded better TSPO contrasts than [18F]fluoroethoxy-DAA1106, supporting the possibility of capturing early neurotoxicity with high-performance TSPO probes. Furthermore, an additional line of mice modeling intraneuronal Aβ accumulation displayed elevated TSPO signals following noticeable neuronal loss, unlike TSPO upregulation heralding massive neuronal death in tauopathy model mice. Our data corroborate the utility of TSPO-PET imaging as a biomarker for tau-triggered toxicity, and as a complement to amyloid scans for diagnostic assessment of tauopathies with and without Aβ pathologies.

Mechanistic involvement of the calpain-calpastatin system in Alzheimer neuropathology

The mechanism by which amyloid‐β peptide (Aβ) accumulation causes neurodegeneration in Alzheimers disease (AD) remains unresolved. Given that Aβ perturbs calcium homeostasis in neurons, we investigated the possible involvement of calpain, a calcium‐activated neutral protease. We first demonstrated close postsynaptic association of calpain activation with Aβ plaque formation in brains from both patients with AD and transgenic (Tg) mice overexpressing amyloid precursor protein (APP). Using a viral vector‐based tracer, we then showed that axonal termini were dynamically misdirected to calpain activation‐positive Aβ plaques. Consistently, cerebrospinal fluid from patients with AD contained a higher level of calpain‐cleaved spectrin than that of controls. Genetic deficiency of calpastatin (CS), a calpain‐specific inhibitor protein, augmented Aβ amyloidosis, tau phosphorylation, microgliosis, and somatodendritic dystrophy, and increased mortality in APP‐Tg mice. In contrast, brain‐specific CS overexpression had the opposite effect. These findings implicate that calpain activation plays a pivotal role in the Aβ‐triggered pathological cascade, highlighting a target for pharmacological intervention in the treatment of AD.—Higuchi, M., Iwata, N., Matsuba, Y., Takano, J., Suemoto, T., Maeda, J., Ji, B., Ono, M., Staufenbiel, M., Suhara, T., Saido, T. C. Mechanistic involvement of the calpain‐calpastatin system in Alzheimer neuropathology. FASEB J. 26, 1204‐1217 (2012). www.fasebj.org

Radiosynthesis, Photoisomerization, Biodistribution, and Metabolite Analysis of 11C-PBB3 as a Clinically Useful PET Probe for Imaging of Tau Pathology

2-((1E,3E)-4-(6-(11C-methylamino)pyridin-3-yl)buta-1,3-dienyl)benzo[d]thiazol-6-ol (11C-PBB3) is a clinically useful PET probe that we developed for in vivo imaging of tau pathology in the human brain. To ensure the availability of this probe among multiple PET facilities, in the present study we established protocols for the radiosynthesis and quality control of 11C-PBB3 and for the characterization of its photoisomerization, biodistribution, and metabolism. Methods: 11C-PBB3 was synthesized by reaction of the tert-butyldimethylsilyl desmethyl precursor (1) with 11C-methyl iodide using potassium hydroxide as a base, followed by deprotection. Photoisomerization of 11C-PBB3 under fluorescent light was determined. The biodistribution and metabolite analysis of 11C-PBB3 was determined in mice using the dissection method. Results: 11C-PBB3 was synthesized with 15.4% ± 2.8% radiochemical yield (decay-corrected, n = 50) based on the cyclotron-produced 11C-CO2 and showed an averaged synthesis time of 35 min from the end of bombardment. The radiochemical purity and specific activity of 11C-PBB3 were 98.0% ± 2.3% and 180.2 ± 44.3 GBq/μmol, respectively, at the end of synthesis (n = 50). 11C-PBB3 showed rapid photoisomerization, and its radiochemical purity decreased to approximately 50% at 10 min after exposure to fluorescent light. After the fluorescent light was switched off, 11C-PBB3 retained more than 95% radiochemical purity over 60 min. A suitable brain uptake (1.92% injected dose/g tissue) of radioactivity was observed at 1 min after the probe injection, which was followed by rapid washout from the brain tissue. More than 70% of total radioactivity in the mouse brain homogenate at 5 min after injection represented the unchanged 11C-PBB3, despite its rapid metabolism in the plasma. Conclusion: 11C-PBB3 was produced with sufficient radioactivity and high quality, demonstrating its clinical utility. The present results of radiosynthesis, photoisomerization, biodistribution, and metabolite analysis could be helpful for the reliable production and application of 11C-PBB3 in diverse PET facilities.

Distinct binding of PET ligands PBB3 and AV-1451 to tau fibril strains in neurodegenerative tauopathies

Diverse neurodegenerative disorders are characterized by deposition of tau fibrils composed of conformers (i.e. strains) unique to each illness. The development of tau imaging agents has enabled visualization of tau lesions in tauopathy patients, but the modes of their binding to different tau strains remain elusive. Here we compared binding of tau positron emission tomography ligands, PBB3 and AV-1451, by fluorescence, autoradiography and homogenate binding assays with homologous and heterologous blockades using tauopathy brain samples. Fluorescence microscopy demonstrated intense labelling of non-ghost and ghost tangles with PBB3 and AV-1451, while dystrophic neurites were more clearly detected by PBB3 in brains of Alzheimers disease and diffuse neurofibrillary tangles with calcification, characterized by accumulation of all six tau isoforms. Correspondingly, partially distinct distributions of autoradiographic labelling of Alzheimers disease slices with 11C-PBB3 and 18F-AV-1451 were noted. Neuronal and glial tau lesions comprised of 4-repeat isoforms in brains of progressive supranuclear palsy, corticobasal degeneration and familial tauopathy due to N279K tau mutation and 3-repeat isoforms in brains of Picks disease and familial tauopathy due to G272V tau mutation were sensitively detected by PBB3 fluorescence in contrast to very weak AV-1451 signals. This was in line with moderate 11C-PBB3 versus faint 18F-AV-1451 autoradiographic labelling of these tissues. Radioligand binding to brain homogenates revealed multiple binding components with differential affinities for 11C-PBB3 and 18F-AV-1451, and higher availability of binding sites on progressive supranuclear palsy tau deposits for 11C-PBB3 than 18F-AV-1451. Our data indicate distinct selectivity of PBB3 compared to AV-1451 for diverse tau fibril strains. This highlights the more robust ability of PBB3 to capture wide-range tau pathologies.

In-vivo visualization of key molecular processes involved in Alzheimer's disease pathogenesis: Insights from neuroimaging research in humans and rodent models.

Diverse age-associated neurodegenerative disorders are featured at a molecular level by depositions of self-aggregating molecules, as represented by amyloid beta peptides (Abeta) and tau proteins in Alzheimers disease, and cascade-type chain reactions are supposedly commenced with biochemical aberrancies of these amyloidogenic components. Mutagenesis and multiplication of the genes encoding Abeta, tau and other pathogenic initiators may accelerate the incipient process at the cascade top, rationalizing generations of transgenic and knock-in animal models of these illnesses. Meanwhile, these genetic manipulations do not necessarily compress the timelines of crucial intermediate events linking amyloidogenesis and neuronal lethality, resulting in an incomplete recapitulation of the diseases. Requirements for modeling the entire cascade can be illustrated by a side-by-side comparison of humans and animal models with the aid of imaging-based biomarkers commonly applicable to different species. Notably, key components in a highly reactive state are assayable by probe-assisted neuroimaging techniques exemplified by positron emission tomography (PET), providing critical information on the in-vivo accessibility of these target molecules. In fact, multispecies PET studies in conjunction with biochemical, electrophysiological and neuropathological tests have revealed putative neurotoxic subspecies of Abeta assemblies, translocator proteins accumulating in aggressive but not neuroprotective microglia, and functionally active neuroreceptors available to endogenous neurotransmitters and exogenous agonistic ligands. Bidirectional translational studies between human cases and model strains based on this experimental paradigm are presently aimed at clarifying the tau pathogenesis, and would be expanded to analyses of disrupted calcium homeostasis and mitochondrial impairments. Since reciprocal causalities among the key processes have indicated an architectural interchangeability between cascade and network connections as an etiological representation, longitudinal imaging assays with manifold probes covering the cascade from top to bottom virtually delineate the network dynamics continuously altering in the course of the disease and its treatment, and therefore expedite the evaluation and optimization of therapeutic strategies intended for suppressing the neurodegenerative pathway over its full length.

Fluorescence and autoradiographic evaluation of tau PET ligand PBB3 to α-synuclein pathology

Background: The tau PET ligand 2‐((1E,3E)‐4‐(6‐([11C]methylamino)pyridin‐3‐yl)buta‐1,3‐dienyl)benzo[d]thiazol‐6‐ol ([11C]PBB3) binds to a wide range of tau pathology; however, binding property of PBB3 to non‐tau inclusions remains unknown. To clarify whether [11C]PBB3 binds to α‐synuclein pathology, reactivity of PBB3 was assessed by in vitro fluorescence and autoradiographic labeling of brain sections from α‐synucleinopathies patients.

In Vivo SPECT Imaging of Amyloid-β Deposition with Radioiodinated Imidazo[1,2-a]Pyridine Derivative DRM106 in a Mouse Model of Alzheimer's Disease

Noninvasive determination of amyloid-β peptide (Aβ) deposition has important significance for early diagnosis and medical intervention for Alzheimers disease (AD). In the present study, we investigated the availability of radiolabeled DRM106 (123/125I-DRM106 [6-iodo-2-[4-(1H-3-pyrazolyl)phenyl]imidazo[1,2-a]pyridine]), a compound with sufficient affinity for the synthesis of human Aβ fibrils and satisfactory metabolic stability, as a SPECT ligand in living brains. Method: The sensitivity of 125I-DRM106 for detecting Aβ deposition was compared with that of 125I-IMPY (2-(4′-dimethylaminophenyl)-6-iodo-imidazo[1,2-a]pyridine), a well-known amyloid SPECT ligand, by ex vivo autoradiographic analyses in 18-mo-old amyloid precursor protein transgenic mice. To verify the sensitivity and quantitation of radiolabeled DRM106 for in vivo imaging, we compared the detectability of Aβ plaques with 123I-DRM106 and a well-known amyloid PET agent, 11C-labeled Pittsburgh compound B (11C-PiB), in 29-mo-old transgenic mice and age-matched nontransgenic littermates. Additionally, we compared the binding characteristics of 125I-DRM106 with those of 11C-PiB and 11C-PBB3, which selectively bind to Aβ plaques and preferentially to tau aggregates, respectively, in postmortem AD brain sections. Results: Ex vivo autoradiographic analysis showed that measurement with 125I-DRM106 has a higher sensitivity for detecting Aβ accumulation than with 125I-IMPY in transgenic mice. SPECT imaging with 123I-DRM106 also successfully detected Aβ deposition in living aged transgenic mice and showed strong correlation (R = 0.95, P < 0.01) in quantitative analysis for Aβ plaque detection by PET imaging with 11C-PiB, implying that sensitivity and quantitation of SPECT imaging with 123I-DRM106 are almost as good as 11C-PiB PET for the detectability of Aβ deposition. Further, the addition of nonradiolabeled DRM106 fully blocked the binding of 125I-DRM106 and 11C-PiB, but not 11C-PBB3, to AD brain sections, and 125I-DRM106 showed a lower binding ratio of the diffuse plaque–rich lateral temporal cortex to the dense-cored/neuritic plaque–rich hippocampal CA1 area, compared with 11C-PiB. Conclusion: All of these data demonstrated the high potential of 123I-DRM106 for amyloid imaging in preclinical and clinical application, and it might more preferentially detect dense-cored/neuritic amyloid deposition, which is expected to be closely associated with neuropathologic changes of AD.