Alzheimer's Disease Brain Homogenates Research Articles

A closer look at amyloid ligands, and what they tell us about protein aggregates.

The accumulation of amyloid fibrils is characteristic of neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease. Detecting these fibrils with fluorescent or radiolabelled ligands is one strategy for diagnosing and better understanding these diseases. A vast number of amyloid-binding ligands have been reported in the literature as a result. To obtain a better understanding of how amyloid ligands bind, we have compiled a database of 3457 experimental dissociation constants for 2076 unique amyloid-binding ligands. These ligands target Aβ, tau, or αSyn fibrils, as well as relevant biological samples including AD brain homogenates. From this database significant variation in the reported dissociation constants of ligands was found, possibly due to differences in the morphology of the fibrils being studied. Ligands were also found to bind to Aβ(1-40) and Aβ(1-42) fibrils with similar affinities, whereas a greater difference was found for binding to Aβ and tau or αSyn fibrils. Next, the binding of ligands to fibrils was shown to be largely limited by the hydrophobic effect. Some Aβ ligands do not fit into this hydrophobicity-limited model, suggesting that polar interactions can play an important role when binding to this target. Finally several binding site models were outlined for amyloid fibrils that describe what ligands target what binding sites. These models provide a foundation for interpreting and designing site-specific binding assays.

Evaluation of the α-synuclein PET radiotracer (d3)-[11C]MODAG-001 in pigs

BackgroundA positron emission tomography (PET) radiotracer to neuroimage α-synuclein aggregates would be a crucial addition for early diagnosis and treatment development in disorders such as Parkinson's disease, where elevated aggregate levels are a histopathological hallmark. The radiotracer (d3)-[11C]MODAG-001 has recently shown promise for visualization of α-synuclein pre-formed fibrils (α-PFF) in rodents. We here test the radiotracer in a pig model where proteins are intracerebrally injected immediately before scanning. Four pigs were injected in one hemisphere with 150 μg α-PFF, and in the other hemisphere, either 75 μg α-PFF or human brain homogenate from either dementia with Lewy bodies (DLB) or Alzheimer's disease (AD) was injected. All pigs underwent one or two (d3)-[11C]MODAG-001 PET scans, quantified with the non-invasive Logan graphical analysis using the occipital cortex as a reference region. ResultsThe α-PFF and AD homogenate injected brain regions had high uptake of (d3)-[11C]MODAG-001 compared to the occipital cortex or cerebellum. BPND values in 150 μg α-PFF injected regions was 0.78, and in the AD homogenate injected regions was 0.73. By contrast, the DLB homogenate injected region did not differ in uptake and clearance compared to the reference regions. The time-activity curves and BPND values in the 150 μg and 75 μg injected regions of α-PFFs show a dose-dependent effect, and the PET signal could be blocked by pretreatment with unlabeled MODAG-001. ConclusionWe find that both α-PFF and AD brain homogenates give rise to increased binding of (d3)-[11C]MODAG-001 when injected into the pig brain. Despite its limited specificity for cerebral α-synuclein pathology, (d3)-[11C]MODAG-001 shows promise as a lead tracer for future radiotracer development.

PI-2620 Lead Optimization Highlights the Importance of Off-Target Assays to Develop a PET Tracer for the Detection of Pathological Aggregated Tau in Alzheimer's Disease and Other Tauopathies.

The first candidate PI-2014 was tested in healthy controls and subjects with Alzheimer's disease (AD). As PI-2014 displayed off-target binding to monoamine oxidase A (MAO-A), a new lead with improved binding to Tau and decreased MAO-A binding was required. For compound optimization, Tau binding assays based on both human AD brain homogenate and Tau-paired helical filaments were employed. Furthermore, two MAO-A screening assays based on (1) human-recombinant MAO-A and (2) displacement of 2-fluoro-ethyl-harmine from mouse brain homogenate were employed. Removing the N-methyl group from the tricyclic core resulted in compounds displaying improved Tau binding. For the final round of optimization, the cyclic amine substituents were replaced by pyridine derivatives. PI-2620 (2-(2-fluoropyridin-4-yl)-9H-pyrrolo[2,3-b:4,5-c']dipyridine) emerged as a best candidate displaying high Tau binding, low MAO-A binding, high brain uptake, and fast and complete brain washout. Furthermore, PI-2620 showed Tau binding on brain sections from corticobasal degeneration, progressive supranuclear palsy, and Pick's disease.

VISUALIZING BRAIN ASTROCYTES - CHARACTERIZATION OF 3H-BU99008, AN IMIDAZOLINE 2 RECEPTOR LIGAND, IN THE HUMAN AD BRAIN

Since glia activation and neuroinflammation are recognized as early events in Alzheimer's disease (AD), glia seems to be a promising therapeutic target in AD. Specifically, astrocytosis has been shown to be an early phenomenon in AD, even at a presymptomatic stage (Rodriguez-Vieitez et al., Brain 2016;139(Pt 3):922-36). However, few PET tracers are available for investigating in vivo astrocyte activation, therefore there is a growing interest in developing new astrocytic tracers. Presently, the only PET tracer used for the imaging of astrocyte reactivity in AD is [11C]-deuterium-L-deprenyl ([11C]-DED). [11C]-DED binds to the enzyme monoamine oxidase-B (MAO-B), which has shown to be overexpressed in reactive astrocytes. The aim of our study is to characterize a tentative astrocytic PET tracer, BU99008, in the assessment of reactive astrocytes and neuroinflammation in AD, and to compare its characteristics with the astrocytic PET tracer [11C]-DED. BU99008 has shown to bind astrocytic mitochondrial imidazoline2 receptors (I2R), which have been linked to several central nervous system disorders, including AD. Most interestingly, the density of I2R was shown to be elevated in postmortem AD brain with approximately 63%. For the in vitro characterization of [3H]-BU99008, both competition and saturation binding assays are performed in postmortem brain tissue from both AD patients and cognitive normal control (CN). In addition, autoradiography is performed to visualize regional distribution of [3H]-BU99008. The results are compared with results for [3H]-DED binding and competition studies. Preliminary results from [3H]-BU99008 in vitro competition binding studies shows specific binding in brain homogenates for both AD and CN, however with some differences in the nanomolar range. [3H]-DED showed specific binding in the nanomolar range in the same AD brain homogenates. To be able to detect astrocytosis early in AD, specific astrocyte biomarkers are needed. Our preliminary data shows that BU99008, a tentative astrocytic PET tracer, shows binding in CN and AD brain tissue in the nanomolar range, however more characterization is needed to understand its binding properties and future use in comparison to existing astrocytic biomarkers.

Proteomic analysis of the poly‐ubiquitin profiles in Alzheimer's Disease

Several neurodegenerative diseases including Alzheimer's Disease (AD), have characteristic ubiquitin positive pathological protein aggregates. Currently, the specific ubiquitylation protein substrates associated with these aggregates in AD brain is poorly understood. We analyzed control and AD postmortem brain tissues to examine the changes in the proteome and ubiquitylation substrates associated with AD pathology. Affinity capture using tandem ubiquitin binding entities (TUBEs) was utilized to enrich poly‐ubiquitylation substrates from AD and age‐matched control postmortem brain tissues (n=3 each group). Label‐free mass spectrometry (MS) based proteomic analysis identified a total of 2112 unique proteins across all samples and bead controls. After controlling for non‐specific binding, we identified 197 proteins that were at least two‐fold enriched in AD compared to control cases. Some of these targets include ubiquitin, Tau, p62 sequestosome and 14‐3‐3 proteins, which are known to associate with AD neuropathology. A cross comparison with previous proteomic studies revealed that 108 of those 197 proteins have increased site‐specific levels of ubiquitylation in AD brain. Tau poly‐ubiquitylation from AD brain homogenates was confirmed by affinity capture using either pan or linkage‐specific (Lys48 and Lys63) poly‐ubiquitin TUBEs and immunoblotting. These findings show the utility of MS to map ubiquitylated substrates in the human brain and provide a better understanding of the mechanisms underlying post‐translational modifications of pathological proteins in AD.Support or Funding InformationNational Institute on Aging (U01AG046161‐02, R21AG054206, 5R01AG053960, RF1AG057470, and RF1AG057471) and the NINDS Emory Neuroscience Core (P30NS055077).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Quantitative Analysis of the Brain Ubiquitylome in Alzheimer's Disease.

Several neurodegenerative diseases including Alzheimer's Disease (AD) are characterized by ubiquitin-positive pathological protein aggregates. Here, an immunoaffinity approach is utilized to enrich ubiquitylated isopeptides after trypsin digestion from five AD and five age-matched control postmortem brain tissues. Label-free MS-based proteomic analysis identifies 4291 unique ubiquitylation sites mapping to 1682 unique proteins. Differential enrichment analysis shows that over 800 ubiquitylation sites are significantly altered between AD and control cases. Of these, ≈80% are increased in AD, including seven poly ubiquitin linkages, which is consistent with proteolytic stress and high burden of ubiquitylated pathological aggregates in AD. The microtubule associated protein Tau, the core component of neurofibrillary tangles, has the highest number of increased sites of ubiquitylation per any protein in AD. Tau poly ubiquitylation from AD brain homogenates is confirmed by reciprocal co-immunoprecipitation and by affinity capture using tandem ubiquitin binding entities. Co-modified peptides, with both ubiquitylation and phosphorylation sites, are also enriched in AD. Notably, many of the co-modified peptides mapped to Tau within KXGS motifs in the microtubule binding region suggesting that crosstalk between phosphorylation and ubiquitylation occurs on Tau in AD. Overall, these findings highlight the utility of MS to map ubiquitylated substrates in human brain and provides insight into mechanisms underlying pathological protein posttranslational modification in AD.

The Bri2 and Bri3 BRICHOS Domains Interact Differently with Aβ42 and Alzheimer Amyloid Plaques.

Alzheimer’s disease (AD) is the most common form of dementia and there is no successful treatment available. Evidence suggests that fibril formation of the amyloid β-peptide (Aβ) is a major underlying cause of AD, and treatment strategies that reduce the toxic effects of Aβ amyloid are sought for. The BRICHOS domain is found in several proteins, including Bri2 (also called integral membrane protein 2B (ITM2B)), mutants of which are associated with amyloid and neurodegeneration, and Bri3 (ITM2C). We have used mouse hippocampal neurons and brain tissues from mice and humans and show Bri3 deposits dispersed on AD plaques. In contrast to what has been shown for Bri2, Bri3 immunoreactivity is decreased in AD brain homogenates compared to controls. Both Bri2 and Bri3 BRICHOS domains interact with Aβ40 and Aβ42 present in neurons and reduce Aβ42 amyloid fibril formation in vitro, but Bri3 BRICHOS is less efficient. These results indicate that Bri2 and Bri3 BRICHOS have different roles in relation to Aβ aggregation.

Generation of Clickable Pittsburgh Compound B for the Detection and Capture of β-Amyloid in Alzheimer's Disease Brain.

The benzothiazole-aniline derivative Pittsburgh Compound B (PiB) is the prototypical amyloid affinity probe developed for the in vivo positron emission tomography (PET) detection of amyloid beta (Aβ) deposits in Alzheimer's disease (AD). Specific high-affinity binding sites for PiB have been found to vary among AD cases with comparable Aβ load, and they are virtually absent on human-sequence Aβ deposits in animal models, none of which develop the full phenotype of AD. PiB thus could be an informative probe for studying the pathobiology of Aβ, but little is known about the localization of PiB binding at the molecular or structural level. By functionalizing the 6-hydroxy position of PiB with a PEG3 spacer and a terminal alkyne (propargyl) moiety, we have developed a clickable PiB compound that was derivatized with commercially available azide-labeled fluorophores or affinity-tags using copper-catalyzed azide-alkyne cycloaddition reactions, commonly referred to as "click" chemistry. We have determined that both the clickable PiB derivative and its fluorescently labeled conjugate have low nanomolar binding affinities for synthetic Aβ aggregates. Furthermore, the fluorescent-PiB conjugate can effectively bind Aβ aggregates in human AD brain homogenates and tissue sections. By covalently coupling PiB to magnetic beads, Aβ aggregates were also affinity-captured from AD brain extracts. Thus, the clickable PiB derivative described herein can be used to generate a wide variety of covalent conjugates, with applications including the fluorescence detection of Aβ, the ultrastructural localization of PiB binding, and the affinity capture and structural characterization of Aβ and other cofactors from AD brains.

Preclinical Characterization of 18F-MK-6240, a Promising PET Tracer for In Vivo Quantification of Human Neurofibrillary Tangles

In vitro binding studies were conducted with 3H-MK-6240 in tissue slices and homogenates from cognitively normal and AD human brain donors to evaluate tracer affinity and selectivity for NFTs. Immunohistochemistry for phosphorylated tau was performed on human brain slices for comparison with 3H-MK-6240 binding patterns on adjacent brain slices. PET studies were performed with 18F-MK-6240 in monkeys to evaluate tracer kinetics and distribution in the brain. 18F-MK-6240 monkey PET studies were conducted after dosing with unlabeled MK-6240 to evaluate tracer binding selectivity in vivo. The 3H-MK-6240 binding pattern was consistent with the distribution of phosphorylated tau in human AD brain slices. 3H-MK-6240 bound with high affinity to human AD brain cortex homogenates containing abundant NFTs but bound poorly to amyloid plaque-rich, NFT-poor AD brain homogenates. 3H-MK-6240 showed no displaceable binding in the subcortical regions of human AD brain slices and in the hippocampus/entorhinal cortex of non-AD human brain homogenates. In monkey PET studies, 18F-MK-6240 displayed rapid and homogeneous distribution in the brain. The 18F-MK-6240 volume of distribution stabilized rapidly, indicating favorable tracer kinetics. No displaceable binding was observed in self-block studies in rhesus monkeys, which do not natively express NFTs. Moderate defluorination was observed as skull uptake. 18F-MK-6240 is a promising PET tracer for the in vivo quantification of NFTs in AD patients.

Structure-Activity Relationship of 2-Arylquinolines as PET Imaging Tracers for Tau Pathology in Alzheimer Disease.

Abnormal deposition of amyloid-β and hyperphosphorylated tau protein in the brain are the pathologic hallmark of Alzheimer disease (AD). Noninvasive detection of the lesions is considered an effective tool for early diagnosis and staging of AD. In the past decade, we developed 2-arylquinoline (2-AQ) derivatives as PET tau tracers. In this study, we synthesized new derivatives and evaluated their properties. Fifteen 2-AQ derivatives were labeled with (18)F, and their binding to tau lesions was evaluated by autoradiography using AD brain sections. The binding affinity for the AD brain homogenates was assessed by an in vitro competitive binding assay with (18)F-THK-5105. (18)F-labeled derivatives were injected into mice via the tail vein, and their pharmacokinetics over the first 120 min after injection were evaluated by an ex vivo biodistribution study. Tracer metabolism analysis was also assessed in mice. The average logP value was 2.80. This study revealed that 2-AQ derivatives having (18)F-labeled side chains on benzene or position 7 of the quinoline showed significantly lower binding affinity for tau than 6-substituted quinoline derivatives. The 2-AQ derivatives labeled with (18)F-fluoroethoxy, (18)F-fluoropropoxy, and (18)F-fluoro-polyethyleneglycol groups displayed slow clearance from blood or a high accumulation in bone, whereas derivatives labeled with the (18)F-(3-fluoro-2-hydroxy)propoxyl group did not. (18)F-THK-5151 had outstanding tau binding properties and pharmacokinetics. Furthermore, the properties of its optically pure (S)-enantiomer (THK-5351) were superior to those of the (R)-enantiomer (THK-5451), particularly in terms of its clearance from the brain and its resistance to defluorination in mice. The structure-activity relationship study of 2-AQ derivatives revealed the optimal structural features for tau imaging agents. On the basis of these results, (18)F-THK-5351 ((S)-(18)F-THK-5151) was selected as a potential agent for tau imaging.

Evidence for human transmission of amyloid-β pathology and cerebral amyloid angiopathy.

More than two hundred individuals developed Creutzfeldt-Jakob disease (CJD) worldwide as a result of treatment, typically in childhood, with human cadaveric pituitary-derived growth hormone contaminated with prions. Although such treatment ceased in 1985, iatrogenic CJD (iCJD) continues to emerge because of the prolonged incubation periods seen in human prion infections. Unexpectedly, in an autopsy study of eight individuals with iCJD, aged 36-51 years, in four we found moderate to severe grey matter and vascular amyloid-β (Aβ) pathology. The Aβ deposition in the grey matter was typical of that seen in Alzheimer's disease and Aβ in the blood vessel walls was characteristic of cerebral amyloid angiopathy and did not co-localize with prion protein deposition. None of these patients had pathogenic mutations, APOE ε4 or other high-risk alleles associated with early-onset Alzheimer's disease. Examination of a series of 116 patients with other prion diseases from a prospective observational cohort study showed minimal or no Aβ pathology in cases of similar age range, or a decade older, without APOE ε4 risk alleles. We also analysed pituitary glands from individuals with Aβ pathology and found marked Aβ deposition in multiple cases. Experimental seeding of Aβ pathology has been previously demonstrated in primates and transgenic mice by central nervous system or peripheral inoculation with Alzheimer's disease brain homogenate. The marked deposition of parenchymal and vascular Aβ in these relatively young patients with iCJD, in contrast with other prion disease patients and population controls, is consistent with iatrogenic transmission of Aβ pathology in addition to CJD and suggests that healthy exposed individuals may also be at risk of iatrogenic Alzheimer's disease and cerebral amyloid angiopathy. These findings should also prompt investigation of whether other known iatrogenic routes of prion transmission may also be relevant to Aβ and other proteopathic seeds associated with neurodegenerative and other human diseases.

Preclinical Evaluation of [(18)F]THK-5105 Enantiomers: Effects of Chirality on Its Effectiveness as a Tau Imaging Radiotracer.

Noninvasive imaging of tau and amyloid-β pathologies would facilitate diagnosis of Alzheimer's disease (AD). Recently, we have developed [(18)F]THK-5105 for selective detection of tau pathology by positron emission tomography (PET). The purpose of this study was to clarify biological properties of optically pure [(18)F]THK-5105 enantiomers. Binding for tau aggregates in AD brain section was evaluated by autoradiography (ARG). In vitro binding assays were performed to evaluate the binding properties of enantiomers for AD brain homogenates. The pharmacokinetics in the normal mouse brains was assessed by ex vivo biodistribution assay The ARG of enantiomers showed the high accumulation of radioactivity corresponding to the distribution of tau deposits. In vitro binding assays revealed that (S)-[(18)F]THK-5105 has slower dissociation from tau than (R)-[(18)F]THK-5105. Biodistribution assays indicated that (S)-[(18)F]THK-5105 eliminated faster from the mouse brains and blood compared with (R)-[(18)F]THK-5105. (S)-[(18)F]THK-5105 could be more suitable than (R)-enantiomer for a tau imaging agent.

An Intrinsically Disordered Low Complexity Domain is Required for U1‐70K Self‐association

The RNA binding protein U1‐70K aggregates via an unknown mechanism in Alzheimer's disease (AD). Interestingly, AD brain homogenates can induce the aggregation of soluble U1‐70K from control brain or recombinant U1‐70K (rU1‐70K), rendering it detergent‐insoluble. The intrinsically disordered C‐terminus of U1‐70K is necessary for aggregation and harbors two low‐complexity (LC) domains, LC1 and LC2. Similar LC domains have been shown to polymerize into amyloid‐like aggregates in vitro, and the RNA binding proteins TDP‐43 and FUS, which also harbor LC domains, form detergent‐insoluble amyloid‐like aggregates in neurodegenerative disease. Our objective is to determine if the LC domains of U1‐70K are necessary for self‐association. Addressing this question may provide mechanistic insight into the aggregation of U1‐70K in AD. To investigate this question we performed co‐immunoprecipitations of full‐length rU1‐70K and various mutants lacking either the LC1, LC2, or both LC domains. These assays revealed that the LC1 domain (amino acids 231‐308) of rU1‐70K is necessary and sufficient for association with native U1‐70K. Similarly, we utilized bio‐layer interferometry to measure the binding affinity of the LC1 domain with a synthetic peptide corresponding to amino acids 252‐270 of U1‐70K. These data provide evidence for a direct self‐association between regions of the LC1 domain. Finally, by immunocytochemistry we show that rU1‐70K mutants lacking the LC1 domain have an impaired ability to form nuclear granule structures. Together these data indicate that the disordered LC1 domain is necessary for U1‐70K self‐association and subnuclear compartmentalization.Sources of Research Support:NIRG‐12‐242297 (NTS) and T32‐NS007480 (IB)

Characterization and first human investigation of FIBT, a novel fluorinated Aβ plaque neuroimaging PET radioligand.

Imidazo[2,1-b]benzothiazoles (IBTs) are a promising novel class of amyloid positron emission tomography (PET) radiopharmaceuticals for diagnosis of neurodegenerative disorders like Alzheimer's disease (AD). Their good in vivo imaging properties have previously been shown in preclinical studies. Among IBTs, fluorinated [(18)F]FIBT was selected for further characterization and advancement toward use in humans. [(18)F]FIBT characteristics were analyzed in relation to Pittsburgh compound B (PiB) as reference ligand. [(18)F]FIBT and [(3)H]PiB were coinjected to an APP/PS1 mouse for ex vivo dual-label autoradiographic correlation. Acute dose toxicity of FIBT was examined in two groups of healthy mice. Preexisting in vivo stability and biodistribution studies in mice were complemented with analogous studies in rats. [(18)F]FIBT was titrated against postmortem human AD brain homogenate in a saturation binding assay previously performed with [(3)H]PiB. Binding of [(18)F]FIBT to human AD brain was further analyzed by in vitro incubation of human AD brain sections in comparison to [(11)C]PiB in relation to standard immunohistochemistry. Finally, [(18)F]FIBT was administered to two human subjects for a dynamic 90 min PET/MR brain investigation. Ex vivo autoradiography confirmed good uptake of [(18)F]FIBT to mouse brain and its excellent correlation to [(3)H]PiB binding. No toxicity of FIBT could be found in mice at a concentration of 33.3 nmol/kg. As in mice, [(18)F]FIBT was showing high in vivo stability in rats and comparable regional brain biodistribution dynamics to [(3)H]PiB. Radioligand saturation binding confirmed at least one high-affinity binding component of [(18)F]FIBT around 1 nM. Good binding of FIBT relative to PiB was further confirmed in binding assays and autoradiographies using post-mortem AD brain. First use of [(18)F]FIBT in humans successfully yielded clinical [(18)F]FIBT PET/MR images with very good contrast. In summary, [(18)F]FIBT has been characterized to be a new lead compound with improved binding characteristics and pharmacokinetics on its own as well as in comparison to PiB. A pilot human PET investigation provided high-quality images with a plausible tracer distribution pattern. Detailed clinical investigations are needed to confirm these first results and to explore the specific qualities of [(18)F]FIBT PET for dementia imaging in relation to established ligands.

Novel 18F-Labeled Arylquinoline Derivatives for Noninvasive Imaging of Tau Pathology in Alzheimer Disease

Neurofibrillary tangles in Alzheimer disease (AD) brains are composed of the microtubule-associated protein tau. Noninvasive monitoring of tau protein aggregates in the living brain will provide useful information regarding tau pathophysiology in AD. However, no PET probes are currently available for selective detection of tau pathology in AD. We have previously reported (18)F-labeled THK-523 ((18)F-6-(2-fluoroethoxy)-2-(4-aminophenyl)quinoline) as a tau imaging radiotracer candidate for PET. After compound optimization, we developed novel (18)F-labeled arylquinoline derivatives, (18)F-THK-5105 and (18)F-THK-5117, for use as tau imaging PET tracers. (18)F-labeled compounds were prepared from the corresponding tosylated precursors. The binding affinity of compounds to synthetic tau aggregates and tau-rich AD brain homogenates was determined by saturation and competition binding assays. The binding selectivity of compounds to tau pathology was evaluated by autoradiography of AD brain sections. The pharmacokinetics of compounds were assessed in biodistribution studies in normal mice. A 14-d toxicity study with intravenous administration of compounds was performed using rats and mice. In vitro binding assays demonstrated higher binding affinity of THK-5105 and THK-5117 than THK-523 to tau protein aggregates and tau-rich AD brain homogenates. Autoradiographic analyses of AD brain sections showed that these radiotracers preferentially bound to neurofibrillary tangles and neuropil threads, which colocalized with Gallyas-positive and immunoreactive tau protein deposits. The distribution of this radiotracer binding in AD brain sections was completely different from that of (11)C-Pittsburgh compound B, showing preferential binding to amyloid plaques. Furthermore, these derivatives demonstrated abundant initial brain uptake and faster clearance in normal mice than (18)F-THK-523 and other reported (18)F-labeled radiotracers. THK-5105 and THK-5117 showed no toxic effects related to the administration of these compounds in mice and rats and no significant binding for various neuroreceptors, ion channels, and transporters at 1-μM concentrations. (18)F-labeled THK-5105 and THK-5117 are promising candidates as PET tau imaging radiotracers.

Tissue transglutaminase is not a biochemical marker for Alzheimer's disease

Typical hallmarks of Alzheimer's disease (AD) are pathologic deposits in cortical and subcortical regions consisting of self-aggregated proteins such as amyloid-beta (Aβ) or tau. Tissue transglutaminase (tTG) catalyses calcium-dependent cross-linking between proteins (transamidation) resulting in protease-resistant isopeptide bonds. Because of this ability, tTG was suspected to participate in AD pathogenesis. Aβ and tau can be cross-linked by tTG in vitro. In AD neocortex, messenger RNA expression of tTG is increased. However, data on transamidation in AD specimens—activity of not only tTG but also other transglutaminases—are contradictory. The aim of our study was to investigate if tTG is involved in AD development and may be useful as biomarker for AD. We studied human brain samples for tTG concentration, tTG localization, and transamidation activity and cerebrospinal fluid (CSF) for tTG content by novel sensitive and highly specific methods. Neither tTG concentration nor transamidation was increased in AD brain homogenates. Immunohistologically, we found no colocalization of tTG in neocortex sections with tau or Aβ deposits but with blood vessels. Only in rare cases, tTG was detectable in CSF samples. This could be attributed to liberation from erythrocytes. Our data contradict the view that tTG is a potential biochemical marker for AD.

Preclinical Characterization of a Novel Class of18F-Labeled PET Tracers for Amyloid-β

Imaging of amyloid-β (Aβ) plaques by PET is more and more integrated into concepts for Alzheimer disease (AD) diagnosis and drug development. The objective of this study was to find novel chemical entities that can be transformed into (18)F-labeled Aβ tracers with favorable brain washout kinetics and low background signal. High-throughput screening of a large chemical library was used to identify new ligands for fibrillar aggregates of Aβ(1-42) peptide. Thirty-two fluorinated derivatives were synthesized and tested for their affinity toward AD brain homogenate. Twelve ligands have been radiolabeled with (18)F. The pharmacokinetic properties of the radioligands were investigated in mouse and monkey biodistribution studies. Binding characteristics were determined by autoradiography of AD brain sections in vitro and using amyloid precursor protein transgenic mice in vivo. The systematic search for Aβ imaging agents revealed several fluorinated derivatives with nanomolar affinity for Aβ. The fluoropyridyl derivative BAY 1008472 showed a high initial brain uptake (6.45 percentage injected dose per gram at 2 min) and rapid brain washout (ratio of percentage of injected dose per gram of tissue at 2 and 30 min after injection, 9.2) in mice. PET studies of healthy rhesus monkeys confirmed the high initial brain uptake of BAY 1008472 (2.52 standardized uptake value at peak) and a fast elimination of total radioactivity from gray and white matter areas (ratio of standardized uptake value at peak uptake and 60 min 11.0). In autoradiographic analysis, BAY 1008472 selectively detected Aβ deposits in human AD brain sections with high contrast and did not bind to τ- or α-synuclein pathologies. Finally, ex vivo autoradiography of brain sections from amyloid precursor protein-transgenic mice confirmed that BAY 1008472 is indeed suitable for the in vivo detection of Aβ plaques. A new chemical class of Aβ tracers has been identified by high-throughput screening. The fluoropyridyl derivative BAY 1008472 shows a favorable preclinical profile including low background binding in gray and white matter. These properties might qualify this new tracer, in particular, to detect subtle amounts or changes of Aβ burden in presymptomatic AD and during therapy.

Novel Indanone Derivatives as Potential Imaging Probes for β‐Amyloid Plaques in the Brain

Molecular imaging probes to detect senile plaques (SPs) might help the early diagnosis of Alzheimer's disease (AD). In this study, a novel series of indanone derivatives were synthesized and characterized. In in vitro binding studies, compound 2e exhibited a K(i) value of 16 nM with a human AD brain homogenate. Although they displayed relatively low affinities for 2i and 2j--with K(i) values of 99 and 237 nM, respectively--the SPs in AD brain sections were positively stained by 2j. A method for in situ micro-autoradiography of AD brain was developed in this study and showed clear labeling of SPs by [(125)I]2i and [(125)I]2j. Both [(125)I]2i and [(125)I]2j had suitable lipophilicities and displayed high initial uptake and rapid clearance from the mouse brains. Furthermore, [(125)I]2i and [(125)I]2j were more stable in human brain homogenates than in mouse brain homogenates. These data suggest that such indanone derivatives might represent potential amyloid imaging agents for the detection of SPs in AD.

Predicting Regional Neurodegeneration from the Healthy Brain Functional Connectome

Neurodegenerative diseases target large-scale neural networks. Four competing mechanistic hypotheses have been proposed to explain network-based disease patterning: nodal stress, transneuronal spread, trophic failure, and shared vulnerability. Here, we used task-free fMRI to derive the healthy intrinsic connectivity patterns seeded by brain regions vulnerable to any of five distinct neurodegenerative diseases. These data enabled us to investigate how intrinsic connectivity in health predicts region-by-region vulnerability to disease. For each illness, specific regions emerged as critical network "epicenters" whose normal connectivity profiles most resembled the disease-associated atrophy pattern. Graph theoretical analyses in healthy subjects revealed that regions with higher total connectional flow and, more consistently, shorter functional paths to the epicenters, showed greater disease-related vulnerability. These findings best fit a transneuronal spread model of network-based vulnerability. Molecular pathological approaches may help clarify what makes each epicenter vulnerable to its targeting disease and how toxic protein species travel between networked brain structures.

Oligomeric Aβ in Alzheimer's Disease: Relationship to Plaque and Tangle Pathology, APOE Genotype and Cerebral Amyloid Angiopathy

Despite accumulating evidence of a central role for oligomeric amyloid beta (Abeta) in the pathogenesis of Alzheimer's Disease (AD), there is scant information on the relationship between the levels and distribution of oligomeric Abeta and those of other neurodegenerative abnormalities in AD. In the present study, we have found oligomeric Abeta to be associated with both diffuse and neuritic plaques (mostly co-localized with Abeta(1-42)) and with cerebrovascular deposits of Abeta in paraffin sections of formalin-fixed human brain tissue. The amount of oligomeric Abeta that was labeled in the sections correlated with total Abeta plaque load, but not phospho-tau load, cerebral amyloid angiopathy (CAA) severity or APOE genotype. Although soluble, oligomeric and insoluble Abeta levels were all significantly increased in AD brain homogenates, case-to-case variation and overlap between AD and controls were considerable. Over the age-range studied (43-98 years), the levels of soluble Abeta, oligomeric Abeta(42), oligomeric Abeta(40) and insoluble Abeta did not vary significantly with age. Oligomeric Abeta(1-42) and insoluble Abeta levels were significantly higher in women. Overall, the level of insoluble Abeta, but neither oligomeric nor soluble Abeta, was associated with Braak stage, CAA severity and APOEepsilon4 frequency, raising questions as to the role of soluble and oligomeric Abeta in the progression of AD.