Compact Luminol Chemiluminophores for In Vivo Detection and Imaging of ß-Sheet Protein Aggregates.

Protein aggregation has been found in a wide range of neurodegenerative protein-misfolding diseases. The demand for in vivo technologies to identify protein aggregation is at the leading edge for the pathogenic study, diagnostic development, and therapeutic intervention of these devastating disorders. Herein, we report a series of luminol analogues to construct a facile chemiluminescence (CL)-based approach for in vivo detection and imaging of ß-sheet protein aggregates. The synthesized compounds exhibited a distinct chemiluminescent response with long emission wavelengths toward reactive oxygen species under physiological conditions and displayed signal amplification in the presence of ß-sheet protein aggregates, including α-synuclein, ß-amyloid, and tau. Among them, CyLumi-3 was further evaluated as a chemiluminescent probe in preclinical models. By intravenous administration into the model mice via the tail vein, in vivo CL imaging noninvasively detected the specific CL of the probe targeting the α-synuclein aggregates in the brains of living mice. Based on its structural characteristics, CyLumi-3 can readily interact with α-synuclein aggregates with significantly enhanced fluorescence and can identify α-synuclein aggregates in vivo via distinctive CL amplification, which could pave the way for a more comprehensive understanding of protein aggregation in preclinical studies and would provide new hints for developing small-molecule chemiluminophores for protein aggregates.

Synthesis and Evaluation of Fluorine-18 Labeled 2-Phenylquinoxaline Derivatives as Potential Tau Imaging Agents.

In this study, three pairs of optically pure 18F-labeled 2-phenylquinoxaline derivatives were evaluated as Tau imaging agents for the diagnosis of Alzheimer's disease (AD). The chiral 2-fluoromethyl-1,2-ethylenediol side chain was attached to the 2-phenylquinoxaline backbone to increase hydrophilicity, thereby improving the binding affinity of the probe to tangles and their selectivity toward Tau tangles over ß-amyloid plaques (Aß). These probes displayed excellent fluorescent properties and high selectivity for tangles on brain sections from transgenic mice (rTg4510) and AD patients. Quantitative binding assays with AD homogenates showed that the probes (R)-5 and (S)-16 have a high affinity (Ki = 4.1 and 10.3 nM, respectively) and high selectivity (30.5-fold and 34.6-fold, respectively) for tangles over Aß. The high affinity and selectivity of (R)-[18F]5 and (S)-[18F]16 for tangles were further confirmed with autoradiography on AD brain tissue in vitro. In addition, they displayed sufficient blood-brain barrier penetration (7.06% and 10.95% ID/g, respectively) and suitable brain kinetics (brain2 min/brain60 min = 10.1, 6.5 respectively) in normal mice. Ex vivo metabolism studies and micro-positron emission computed tomography (PET) revealed high brain biostability, good brain kinetic properties, and low nonspecific binding for (S)-[18F]16. Together, these results demonstrate that (R)-[18F]5 and (S)-[18F]16 are promising PET probes for Tau tangles imaging.

Quantum energy levels of glutamate modulate neural biophotonic signals.

Glutamate is the most abundant excitatory neurotransmitter in the brain, and it plays an essential and important role in neural functions. Current studies have shown that glutamate can induce neural biophotonic activity and transmission, which may involve the mechanism of photon quantum brain; however, it is unclear whether such a mechanism follows the principle of quantum mechanics. Here we show that the action of glutamate on its receptors leads to a decrease in its quantum energy levels, and glutamate then partially or completely loses its function to further induce the biophotonic activity in mouse brain slices. The reduced quantum energy levels of glutamate can be restored by direct-current electrical discharges and the use of energy transfer of chloroplast photosynthesis; hence, the quantum energy recovered glutamate can again induce significant biophotonic activity. Furthermore, the changes in quantum energy levels of glutamate are related to the exchange and transfer of electron energy on its active hydrogen atom. These findings suggest that the glutamate-induced neural biophotonic signals may be involved in the transfer of the quantum energy levels of glutamate, which implies a quantum mechanism of neurotransmitter action.

Discovery of Diphenoxy Derivatives with Flexible Linkers as Ligands for ß-Amyloid Plaques.

The highly rigid and planar scaffolds with π-conjugated systems have been widely considered to be indispensable for ß-amyloid (Aß) binding ligands. In this study, a library of diphenoxy compounds with different types of more flexible linkers as Aß ligands were synthesized and evaluated. Most of them displayed good affinity (Ki < 100 nM) for Aß1-42 aggregates, and some ligands even showed values of Ki less than 10 nM. Structure-activity relationship analysis revealed that modification on the linkers or substituents tolerated great flexibility, which challenged the long-held belief that rigid and planar structures are exclusively favored for Aß binding. Three ligands were labeled by iodine-125, and they exhibited good properties in vitro and in vivo, which further supported that this flexible scaffold was potential and promising for the development of Aß imaging agents.

Folium Sennae and emodin reverse airway smooth muscle contraction.

The objective of this project was to find a bronchodilatory compound from herbs and clarify the mechanism. We found that the ethanol extract of Folium Sennae (EEFS) can relax airway smooth muscle (ASM). EEFS inhibited ASM contraction, induced by acetylcholine, in mouse tracheal rings and lung slices. High-performance liquid chromatography assay showed that EEFS contained emodin. Emodin had a similar reversal action. Acetylcholine-evoked contraction was also partially reduced by nifedipine (a selective inhibitor of L-type voltage-dependent Ca2+ channels, LVDCCs), YM-58483 (a selective inhibitor of store-operated Ca2+ entry, SOCE), as well as Y-27632 (an inhibitor of Rho-associated protein kinase). In addition, LVDCC- and SOCE-mediated currents and cytosolic Ca2+ elevations were inhibited by emodin. Emodin reversed acetylcholine-caused increases in phosphorylation of myosin phosphatase target subunit 1. Furthermore, emodin, in vivo, inhibited acetylcholine-induced respiratory system resistance in mice. These results indicate that EEFS-induced relaxation results from emodin inhibiting LVDCC, SOCE, and Ca2+ sensitization. These findings suggest that Folium Sennae and emodin may be new sources of bronchodilators.

Paracetamol inhibits Ca2+ permeant ion channels and Ca2+ sensitization resulting in relaxation of precontracted airway smooth muscle.

The purpose of this study was to screen a bronchodilator from old drugs and elucidate the underlying mechanism. Paracetamol (acetaminophen) is a widely used analgesic and antipyretic drug. It has been reported that it inhibits the generation of prostaglandin and histamine, which play roles in asthma. These findings led us to explore whether paracetamol could be a potential bronchodilator. Paracetamol inhibited high K+- and acetylcholine (ACH)-induced precontraction of mouse tracheal and bronchial smooth muscles. Moreover, the ACH-induced contraction was partially inhibited by nifedipine (selective blocker of LVDCCs), YM-58483 (selective inhibitor of store-operated Ca2+ entry (SOCE), canonical transient receptor potential 3 (TRPC3) and TRPC5 channels) and Y-27632 (selective blocker of ROCK, a linker of the Ca2+ sensitization pathway). In single airway smooth muscle cells, paracetamol blocked the currents sensitive to nifedipine and YM-58483, and inhibited intracellular Ca2+ increases. In addition, paracetamol inhibited ACH-induced phosphorylation of myosin phosphatase target subunit 1 (MYPT1, another linker of the Ca2+ sensitization pathway). Finally, in vivo paracetamol inhibited ACH-induced increases of mouse respirator system resistance. Collectively, we conclude that paracetamol inhibits ASM contraction through blocking LVDCCs, SOCE and/or TRPC3 and/or TRPC5 channels, and Ca2+ sensitization. These results suggest that paracetamol might be a new bronchodilator.

Transcriptomic Landscape of von Economo Neurons in Human Anterior Cingulate Cortex Revealed by Microdissected-Cell RNA Sequencing.

The von Economo neurons (VENs) are specialized large bipolar projection neurons with restricted distribution in the human brain, and they are far more abundant in humans than in non-human primates. However, VEN functions remain elusive due to the difficulty of isolating VENs and dissecting their connections in the brain. Here, we combined laser-capture-microdissection with RNA sequencing to describe the transcriptomic profile of VENs from human anterior cingulate cortex (ACC). Using pyramidal neurons as reference cells, we identified 344 genes with VEN-associated expression differences, including 215 higher and 129 lower expression genes. Functional enrichment and protein-protein interaction network analyses showed that these genes with VEN-associated expression differences are involved in VEN morphogenesis and functions, such as dendrite branching and axon myelination, and many of them are associated with human social-emotional disorders. With the use of in situ hybridization and immunohistochemistry assays, we validated four novel VEN markers (VAT1L, CHST8, LYPD1, and SULF2). Collectively, we generated a full-spectrum expression profile of VENs from human ACC, greatly enlarging the pool of genes with VEN-associated expression differences that can help researchers to understand the role of VENs in normal and disordered human brains.

Azithromycin inhibits muscarinic 2 receptor-activated and voltage-activated Ca2+ permeant ion channels and Ca2+ sensitization, relaxing airway smooth muscle contraction.

Azithromycin (AZM) has been used for the treatment of asthma and chronic obstructive pulmonary disease (COPD); however, the effects and underlying mechanisms of AZM remain largely unknown. The effects of AZM on airway smooth muscles (ASMs) and the underlying mechanisms were studied using isometric muscle force measurements, the examination of lung slices, imaging, and patch-clamp techniques. AZM completely inhibited acetylcholine (ACH)-induced precontraction of ASMs in animals (mice, guinea pigs, and rabbits) and humans. Two other macrolide antibiotics, roxithromycin and Klaricid, displayed a decreased inhibitory activity, and the aminoglycoside antibiotics penicillin and streptomycin did not have an inhibitory effect. Precontractions were partially inhibited by nifedipine (selective inhibitor of L-type voltage-dependent Ca2+ channels (LVDCCs)), Pyr3 (selective inhibitor of TRPC3 and/or STIM/Orai channels, which are nonselective cation channels (NSCCs)), and Y-27632 (selective inhibitor of Rho-associated kinase (ROCK)). Moreover, LVDCC- and NSCC-mediated currents were inhibited by AZM, and the latter were suppressed by the muscarinic (M) 2 receptor inhibitor methoctramine. AZM inhibited LVDCC Ca2+ permeant ion channels, M2 receptors, and TRPC3 and/or STIM/Orai, which decreased cytosolic Ca2+ concentrations and led to muscle relaxation. This relaxation was also enhanced by the inhibition of Ca2+ sensitization. Therefore, AZM has potential as a novel and potent bronchodilator. The findings of this study improve the understanding of the effects of AZM on asthma and COPD.

Human high intelligence is involved in spectral redshift of biophotonic activities in the brain.

Human beings hold higher intelligence than other animals on Earth; however, it is still unclear which brain properties might explain the underlying mechanisms. The brain is a major energy-consuming organ compared with other organs. Neural signal communications and information processing in neural circuits play an important role in the realization of various neural functions, whereas improvement in cognitive function is driven by the need for more effective communication that requires less energy. Combining the ultraweak biophoton imaging system (UBIS) with the biophoton spectral analysis device (BSAD), we found that glutamate-induced biophotonic activities and transmission in the brain, which has recently been demonstrated as a novel neural signal communication mechanism, present a spectral redshift from animals (in order of bullfrog, mouse, chicken, pig, and monkey) to humans, even up to a near-infrared wavelength (∼865 nm) in the human brain. This brain property may be a key biophysical basis for explaining high intelligence in humans because biophoton spectral redshift could be a more economical and effective measure of biophotonic signal communications and information processing in the human brain.

Structure-Property Relationships of Polyethylene Glycol Modified Fluorophore as Near-Infrared Aß Imaging Probes.

To optimize the lipophilicity and improve in vivo pharmacokinetics of near-infrared probes targeted Aß plaques, we designed, synthesized, and evaluated a series of polyethylene glycol modified probes with hydroxyl and methoxyl terminals. The relationships between chemical structure and optical, biological properties were systemically elucidated. The results indicated that a desired Aß probe should keep a balance among molecular rigidity, size, and lipophilicity. Probe 12d displayed improved properties including intense and selective response to Aß1-42 aggregates ( Kd = 7.3 nM, 22-fold fluorescence enhancement and emission maxima at 715 nm upon interaction with Aß1-42 aggregates), sufficient blood-brain barrier penetration (3.04% ID/g), and fast wash out from the brain (brain2 min/brain60 min = 10.1). Clear fluorescence signals retention in transgenic mice than control mice in in vivo near-infrared imaging. Hence, polyethylene glycol modified probes retained favorable optical properties but displayed great improvement of biological properties for Aß detection.

Smart D-π-A Type Near-Infrared Aß Probes: Effects of a Marked π Bridge on Optical and Biological Properties.

To expand the scope of D-π-A based near-infrared (NIR) probes for detecting ß-amyloid (Aß) plaques and to systematically explore the relationship among their structural characteristics, optical properties, and biological properties, three series of smart NIR probes with different aromatic rings and up to seven trans double bonds were synthesized and evaluated. Marked correlations between the conjugated π system and properties of these probes, such as optical data, binding ability, and brain uptake, were observed. One probe, PHC-4, displayed improved properties as a NIR probe for the in vivo detection of Aß plaques.

Amyloid-ß Deposits Target Efficient Near-Infrared Fluorescent Probes: Synthesis, in Vitro Evaluation, and in Vivo Imaging.

The formation of extracellular amyloid-ß (Aß) plaques is a common molecular change that underlies several debilitating human conditions, including Alzheimer's disease (AD); however, the existing near-infrared (NIR) fluorescent probes for the in vivo detection of Aß plaques are limited by undesirable fluorescent properties and poor brain kinetics. In this work, we designed, synthesized, and evaluated a new family of efficient NIR probes that target Aß plaques by incorporating hydroxyethyl groups into the ligand structure. Among these probes, DANIR 8c showed excellent fluorescent properties with an emission maximum above 670 nm upon binding to Aß aggregates and also displayed a high sensitivity (a 629-fold increase in fluorescence intensity) and affinity (Kd = 14.5 nM). Because of the improved hydrophilicity that was induced by hydroxyls, 8c displayed increased initial brain uptake and a fast washout from the brain, as well as an acceptable biostability in the brain. In vivo NIR fluorescent imaging revealed that 8c could efficiently distinguish between AD transgenic model mice and normal controls. Overall, 8c is an efficient and veritable NIR fluorescent probe for the in vivo detection of Aß plaques in the brain.

99mTc-Labeled 2-Arylbenzothiazoles: Aß Imaging Probes with Favorable Brain Pharmacokinetics for Single-Photon Emission Computed Tomography.

A series of 2-arylbenzothiazole derivatives conjugated with bis(aminoethanethiol) (BAT) chelating groups were designed and synthesized. A competitive binding assay-based screening was used to select seven rhenium complexes with potent binding affinity toward Aß1-42 aggregates (Ki < 50 nM) for 99mTc labeling and further evaluation. The 99mTc-labeled probes showed good affinity and specificity to Aß plaques in Tg mouse brain tissue in in vitro autoradiography studies. Moreover, [99mTc]14b exhibited favorable brain pharmacokinetics in normal mice (2.11% ID/g at 2 min and 0.62% ID/g at 60 min). Ex vivo autoradiography revealed extensive labeling of Aß plaques by [99mTc]14b in the brain of Tg mice. Furthermore, we performed the first single-photon emission computed tomography (SPECT) imaging study in nonhuman primates with 99mTc-labeled Aß probes. The semiquantitative data showed that [99mTc]14b penetrated the brains of rhesus monkeys. These results indicate that [99mTc]14b could be utilized as a SPECT imaging probe for Aß plaques.

Synthesis and Monkey-PET Study of (R)- and (S)-18F-Labeled 2-Arylbenzoheterocyclic Derivatives as Amyloid Probes with Distinctive in Vivo Kinetics.

This study describes an effective strategy to improve pharmacokinetics of Aß imaging agents, offering a novel class of (R)- and (S)-18F-labeled 2-arylbenzoheterocyclic derivatives which bear an additional chiral hydroxyl group on the side chain. These ligands displayed binding abilities toward Aß aggregates with Ki values ranging from 3.2 to 195.6 nM. Chirality-related discrepancy was observed in biodistribution, and (S)-2-phenylbenzoxazole enantiomers exhibited vastly improved brain clearance with washout ratios higher than 20. Notably, (S)-[18F]28 possessed high binding potency (Ki = 7.6 nM) and exceptional brain kinetics (9.46% ID/g at 2 min, brain2min/brain60min = 27.8) that is superior to well-established [18F]AV45. The excellent pharmacokinetics and low nonspecific binding of (S)-[18F]28 were testified by dynamic PET/CT scans in monkey brains. In addition, (S)-[18F]28 clearly labeled Aß plaques both in vitro and ex vivo. These results might qualify (S)-[18F]28 to detect Aß plaques with high signal-to-noise ratio.

99mTc(CO)3-Labeled Benzothiazole Derivatives Preferentially Bind Cerebrovascular Amyloid: Potential Use as Imaging Agents for Cerebral Amyloid Angiopathy.

Cerebral amyloid angiopathy (CAA) is a disorder affecting the elderly that is characterized by amyloid-ß (Aß) deposition in blood vessel walls of the brain. A series of 99mTc(CO)3-labeled benzothiazole derivatives as potential SPECT imaging probes for cerebrovascular Aß deposition is reported. Rhenium surrogate displayed high affinities to Aß aggregates with Ki values ranging from 106 to 42 nM, and they strongly stained Aß deposits in transgenic mice (Tg) and Alzheimer's disease (AD) patients. In vitro autoradiography on brain sections of Tg and AD patients confirmed that [99mTc]24 possessed sufficient affinity for Aß plaques, and [99mTc]24 could only label Aß deposition in blood vessels but not Aß plaques in the parenchyma of the brain of AD patients. Moreover, [99mTc]24 possessed favorable initial uptake (1.21% ID/g) and fast blood washout (blood2 min/blood60 min=23) in normal mice. These preliminary results suggest that [99mTc]24 may be used as an Aß imaging probe for the detection of CAA.

Discovery and Evaluation of Imidazo[2,1-b][1,3,4]thiadiazole Derivatives as New Candidates for α-Synuclein PET Imaging.

α-synuclein (α-syn) pathologies are central to the development of synucleinopathies including Parkinson's disease (PD). Positron emission tomography (PET) imaging of α-syn pathologies is one strategy to facilitate the diagnosis, understanding, and treatment of synucleinopathies, but has been restricted by the lack of specific α-syn PET probes. In this work, we identified 2,6-disubstituted imidazo[2,1-b][1,3,4]thiadiazole (ITA) as a new α-syn-binding scaffold. Through autoradiography studies, we discovered an iodinated lead compound [125I]ITA-3, with moderate binding affinity (IC50 = 55 nM) to α-syn pathologies in human PD brain sections. Modified from [125I]ITA-3, we developed a potential PET tracer, [18F]FITA-2 (radiochemical yield >25%, molar activity >110 GBq/µmol), which demonstrated clear signals in α-syn-rich regions in human PD brain tissues (IC50 = 245 nM), good brain uptake (SUVpeak = 2.80 ± 0.45), and fast clearance rate in rats. Overall, [18F]FITA-2 appears to be a promising candidate for α-syn PET imaging and merits further development.

Brain eQTLs of European, African American, and Asian ancestry improve interpretation of schizophrenia GWAS.

Research on brain expression quantitative trait loci (eQTLs) has illuminated the genetic underpinnings of schizophrenia (SCZ). Yet, the majority of these studies have been centered on European populations, leading to a constrained understanding of population diversities and disease risks. To address this gap, we examined genotype and RNA-seq data from African Americans (AA, n=158), Europeans (EUR, n=408), and East Asians (EAS, n=217). When comparing eQTLs between EUR and non-EUR populations, we observed concordant patterns of genetic regulatory effect, particularly in terms of the effect sizes of the eQTLs. However, 343,737 cis-eQTLs (representing ∼17% of all eQTLs pairs) linked to 1,276 genes (about 10% of all eGenes) and 198,769 SNPs (approximately 16% of all eSNPs) were identified only in the non-EUR populations. Over 90% of observed population differences in eQTLs could be traced back to differences in allele frequency. Furthermore, 35% of these eQTLs were notably rare (MAF < 0.05) in the EUR population. Integrating brain eQTLs with SCZ signals from diverse populations, we observed a higher disease heritability enrichment of brain eQTLs in matched populations compared to mismatched ones. Prioritization analysis identified seven new risk genes ( SFXN2 , RP11-282018.3 , CYP17A1 , VPS37B , DENR , FTCDNL1 , and NT5DC2 ), and three potential novel regulatory variants in known risk genes ( CNNM2 , C12orf65 , and MPHOSPH9 ) that were missed in the EUR dataset. Our findings underscore that increasing genetic ancestral diversity is more efficient for power improvement than merely increasing the sample size within single-ancestry eQTLs datasets. Such a strategy will not only improve our understanding of the biological underpinnings of population structures but also pave the way for the identification of novel risk genes in SCZ.

Temporal profiles of axonal injury following impact acceleration traumatic brain injury in rats--a comparative study with diffusion tensor imaging and morphological analysis.

Traumatic axonal injury (TAI) plays a major role in the development of neurological impairments after traumatic brain injury (TBI), but it is commonly difficult to evaluate it precisely and early with conventional histological biomarkers, especially when the patients experience short-term survival after TBI. Diffusion tensor imaging (DTI) has shown some promise in detecting TAI, but longitudinal studies on the compromised white matter with DTI at early time points (≤72 h) following impact acceleration TBI are still absent. In the present study, rats were subjected to the Marmarou model and imaged with DTI at 3, 12, 24, and 72 h (n = 5 each) post-injury. Using a region-of-interest-based approach, the regions of interest including the corpus callosum, bilateral external capsule, internal capsule, and pyramidal tract were studied. Two DTI parameters, fraction anisotropy and axial diffusivity, were significantly reduced from 3 to 72 h in each region after trauma, corresponding to the gradient of axonal damage demonstrated by immunohistochemical staining of ß-amyloid precursor protein and neurofilament light chain. Remarkably, DTI changes predicted the approximate time in the acute phase following TBI. These results indicate that the temporal profiles of diffusion parameters in DTI may be able to provide a tool for early diagnosis of TAI following impact acceleration TBI.

Discovery and evaluation of aza-fused tricyclic derivatives for detection of Tau pathology in Alzheimer's disease.

For various neurodegenerative diseases, including Alzheimer's disease (AD), the abnormal aggregation of Tau is not only the predominant contributing factor but also a major biomarker for disease diagnosis. In this study, a series of aza-fused tricyclic derivatives were designed and synthesized. By changing the position and number of nitrogen atoms on the fused tricyclic core, the imidazonaphthyridine scaffold was screened and reported for the first time which could potentially detect Tau aggregates. Through a series of in vitro and in vivo biological evaluations, probe [125I]5 possessed exceptional binding affinity (IC50 = 1.63 nM) to neurofibrillary tangles in the AD brain, high selectivity over Aß plaques (23.4-fold), clean off-target profile to monoamine oxidase A/B (MAO-A/B), and suitable pharmacokinetics (initial brain uptake = 3.22% ID/g).