LY2444296, a κ-opioid receptor antagonist, selectively reduces alcohol drinking in male and female Wistar rats with a history of alcohol dependence.

Alcohol use disorder (AUD) remains a major public health concern. The dynorphin (DYN)/κ-opioid receptor (KOP) system is involved in actions of alcohol, particularly its withdrawal-associated negative affective states. This study tested the ability of LY2444296, a selective, short-acting, KOP antagonist, to decrease alcohol self-administration in dependent male and female Wistar rats at 8 h abstinence. Animals were trained to orally self-administer 10% alcohol (30 min/day for 21 sessions) and were made dependent via chronic intermittent alcohol vapor exposure for 6 weeks or exposed to air (nondependent). After 6 weeks, the effect of LY2444296 (0, 3, and 10 mg/kg, p.o.) was tested on alcohol self-administration at 8 h of abstinence. A separate cohort of rats was prepared in parallel, and their somatic withdrawal signs and alcohol self-administration were measured after LY2444296 administration at 8 h, 2 weeks, and 4 weeks abstinence. LY2444296 at 3 and 10 mg/kg significantly reduced physical signs of withdrawal in dependent rats at 8 h abstinence, only. Furthermore, 3 and 10 mg/kg selectively decreased alcohol self-administration in dependent rats at only 8 h abstinence. These results highlight the DYN/KOP system in actions of alcohol during acute abstinence, suggesting KOP antagonism could be beneficial for mitigating acute withdrawal signs and, in turn, significantly reduce excessive alcohol consumption associated with AUD.

Mapping the Direction of Nucleocytoplasmic Transport of Glucocorticoid Receptor (GR) in Live Cells Using Two-Foci Cross-Correlation in Massively Parallel Fluorescence Correlation Spectroscopy (mpFCS).

Nucleocytoplasmic transport of transcription factors is vital for normal cellular function, and its breakdown is a major contributing factor in many diseases. The glucocorticoid receptor (GR) is an evolutionarily conserved, ligand-dependent transcription factor that regulates homeostasis and response to stress and is an important target for therapeutics in inflammation and cancer. In unstimulated cells, the GR resides in the cytoplasm bound to other molecules in a large multiprotein complex. Upon stimulation with endogenous or synthetic ligands, GR translocation to the cell nucleus occurs, where the GR regulates the transcription of numerous genes by direct binding to glucocorticoid response elements or by physically associating with other transcription factors. While much is known about molecular mechanisms underlying GR function, the spatial organization of directionality of GR nucleocytoplasmic transport remains less well characterized, and it is not well understood how the bidirectional nucleocytoplasmic flow of GR is coordinated in stimulated cells. Here, we use two-foci cross-correlation in a massively parallel fluorescence correlation spectroscopy (mpFCS) system to map in live cells the directionality of GR translocation at different positions along the nuclear envelope. We show theoretically and experimentally that cross-correlation of signals from two nearby observation volume elements (OVEs) in an mpFCS setup presents a sharp peak when the OVEs are positioned along the trajectory of molecular motion and that the time position of the peak corresponds to the average time of flight of the molecule between the two OVEs. Hence, the direction and velocity of nucleocytoplasmic transport can be determined simultaneously at several locations along the nuclear envelope. We reveal that under ligand-induced GR translocation, nucleocytoplasmic import/export of GR proceeds simultaneously but at different locations in the cell nucleus. Our data show that mpFCS can characterize in detail the heterogeneity of directional nucleocytoplasmic transport in a live cell and may be invaluable for studies aiming to understand how the bidirectional flow of macromolecules through the nuclear pore complex (NPC) is coordinated to avoid intranuclear transcription factor accretion/abatement.

The interwoven fibril-like structure of amyloid-beta plaques in mouse brain tissue visualized using super-resolution STED microscopy.

BACKGROUND: Standard neuropathologic analysis of Alzheimer's brain relies on traditional fluorescence microscopy, which suffers from limited spatial resolution due to light diffraction. As a result, it fails to reveal intricate details of amyloid plaques. While electron microscopy (EM) offers higher resolution, its extensive sample preparation, involving fixation, dehydration, embedding, and sectioning, can introduce artifacts and distortions in the complex brain tissue. Moreover, EM lacks molecular specificity and has limited field of view and imaging depth. RESULTS: In our study, we employed super-resolution Stimulated Emission Depletion (STED) microscopy in conjunction with the anti-human APP recombinant antibody 1C3 fluorescently labelled with DyLightTM633 (1C3-DyLight633). This combination allowed us to visualize amyloidogenic aggregates in vitro and in brain sections from a 17-month-old 3×Tg-AD mouse with sub-diffraction limited spatial resolution. Remarkably, we achieved a spatial resolution of 29 nm in vitro and 62 nm in brain tissue sections, surpassing the capabilities of conventional confocal microscopy by 5-10 times. Consequently, we could discern individual fibrils within plaques, an achievement previously only possible with EM. CONCLUSIONS: The utilization of STED microscopy represents a groundbreaking advancement in the field, enabling researchers to delve into the characterization of local mechanisms that underlie Amyloid (Aß) deposition into plaques and their subsequent clearance. This unprecedented level of detail is especially crucial for comprehending the etiology of Alzheimer's disease and developing the next generation of anti-amyloid treatments. By facilitating the evaluation of drug candidates and non-pharmacological interventions aiming to reduce amyloid burden, STED microscopy emerges as an indispensable tool for driving scientific progress in Alzheimer's research.

Naltrexone blocks alcohol-induced effects on kappa-opioid receptors in the plasma membrane.

Naltrexone (NTX), a homologue of the opiate antidote naloxone, is an orally active long-acting mu-opioid receptor (MOP) antagonist used in the treatment of opiate dependence. NTX is also found to relieve craving for alcohol and is one of the few FDA-approved drugs for alcohol use disorder (AUD). Reports that NTX blocks the actions of endogenous opioids released by alcohol are not convincing, suggesting that NTX interferes with alcohol actions by affecting opioid receptors. MOP and kappa-opioid receptor (KOP) are structurally related but functionally different. MOP is mainly located in interneurons activated by enkephalins while KOP is located in longer projections activated by dynorphins. While the actions of NTX on MOP are well established, the interaction with KOP and addiction is not well understood. We used sensitive fluorescence-based methods to study the influence of alcohol on KOP and the interaction between KOP and NTX. Here we report that alcohol interacts with KOP and its environment in the plasma membrane. These interactions are affected by NTX and are exerted both on KOP directly and on the plasma membrane (lipid) structures ("off-target"). The actions of NTX are stereospecific. Selective KOP antagonists, recently in early clinical trials for major depressive disorder, block the receptor but do not show the full action profile of NTX. The therapeutic effect of NTX treatment in AUD may be due to direct actions on KOP and the receptor environment.

Preventing recurrence in Sonic Hedgehog Subgroup Medulloblastoma using the OLIG2 inhibitor CT-179.

Recurrence is the primary life-threatening complication for medulloblastoma (MB). In Sonic Hedgehog (SHH)-subgroup MB, OLIG2-expressing tumor stem cells drive recurrence. We investigated the anti-tumor potential of the small-molecule OLIG2 inhibitor CT-179, using SHH-MB patient-derived organoids, patient-derived xenograft (PDX) tumors and mice genetically-engineered to develop SHH-MB. CT-179 disrupted OLIG2 dimerization, DNA binding and phosphorylation and altered tumor cell cycle kinetics in vitro and in vivo, increasing differentiation and apoptosis. CT-179 increased survival time in GEMM and PDX models of SHH-MB, and potentiated radiotherapy in both organoid and mouse models, delaying post-radiation recurrence. Single cell transcriptomic studies (scRNA-seq) confirmed that CT-179 increased differentiation and showed that tumors up-regulated Cdk4 post-treatment. Consistent with increased CDK4 mediating CT-179 resistance, CT-179 combined with CDK4/6 inhibitor palbociclib delayed recurrence compared to either single-agent. These data show that targeting treatment-resistant MB stem cell populations by adding the OLIG2 inhibitor CT-179 to initial MB treatment can reduce recurrence.

Small Molecule Decoys of Aggregation for Elimination of Aß-Peptide Toxicity.

Several lines of evidence suggest that a characteristic of the neuropathology of Alzheimer's disease (AD) is the aggregation of the amyloid beta peptides (Aß), fragments of the human amyloid precursor protein (hAPP). The dominating species are the Aß40 and Aß42 fragments with 40 and 42 amino acids, respectively. Aß initially forms soluble oligomers that continue to expand to protofibrils, suggestively the neurotoxic intermediates, and thereafter turn into insoluble fibrils that are markers of the disease. Using the powerful tool of pharmacophore simulation, we selected small molecules not known to possess central nervous system (CNS) activity but that might interact with Aß aggregation, from the NCI Chemotherapeutic Agents Repository, Bethesda, MD. We assessed the activity of these compounds on Aß aggregation using the thioflavin T fluorescence correlation spectroscopy (ThT-FCS) assay. Förster resonance energy transfer-based fluorescence correlation spectroscopy (FRET-FCS) was used to characterize the dose-dependent activity of selected compounds at an early stage of Aß aggregation. Transmission electron microscopy (TEM) confirmed that the interfering substances block fibril formation and identified the macrostructures of Aß aggregates formed in their presence. We first found three compounds generating protofibrils with branching and budding never observed in the control. One compound generated a two-dimensional sheet structure and another generated a double-stranded filament. Importantly, these compounds generating protofibrils with altered macrostructure protected against Aß-induced toxicity in a cell model while showing no toxicity in a model of cognition in normal mice. The data suggest that the active compounds act as decoys turning the aggregation into nontoxic trajectories and pointing toward novel approaches to therapy.

Non-Peptide Opioids Differ in Effects on Mu-Opioid (MOP) and Serotonin 1A (5-HT1A) Receptors Heterodimerization and Cellular Effectors (Ca2+, ERK1/2 and p38) Activation.

The importance of the dynamic interplay between the opioid and the serotonin neuromodulatory systems in chronic pain is well recognized. In this study, we investigated whether these two signalling pathways can be integrated at the single-cell level via direct interactions between the mu-opioid (MOP) and the serotonin 1A (5-HT1A) receptors. Using fluorescence cross-correlation spectroscopy (FCCS), a quantitative method with single-molecule sensitivity, we characterized in live cells MOP and 5-HT1A interactions and the effects of prolonged (18 h) exposure to selected non-peptide opioids: morphine, codeine, oxycodone and fentanyl, on the extent of these interactions. The results indicate that in the plasma membrane, MOP and 5-HT1A receptors form heterodimers that are characterized with an apparent dissociation constant Kdapp = (440 ± 70) nM). Prolonged exposure to all non-peptide opioids tested facilitated MOP and 5-HT1A heterodimerization and stabilized the heterodimer complexes, albeit to a different extent: Kd, Fentanylapp = (80 ± 70) nM), Kd,Morphineapp = (200 ± 70) nM, Kd, Codeineapp = (100 ± 70) nM and Kd, Oxycodoneapp = (200 ± 70) nM. The non-peptide opioids differed also in the extent to which they affected the mitogen-activated protein kinases (MAPKs) p38 and the extracellular signal-regulated kinase (Erk1/2), with morphine, codeine and fentanyl activating both pathways, whereas oxycodone activated p38 but not ERK1/2. Acute stimulation with different non-peptide opioids differently affected the intracellular Ca2+ levels and signalling dynamics. Hypothetically, targeting MOP−5-HT1A heterodimer formation could become a new strategy to counteract opioid induced hyperalgesia and help to preserve the analgesic effects of opioids in chronic pain.

Serum Amyloidogenic Nanoplaques and Cytokines in Alzheimer's Disease: Pilot Study in a Small Naturalistic Memory Clinic Cohort.

BACKGROUND: Neuroinflammation is a central component of Alzheimer's disease (AD) and correlates closely with amyloid pathology. Markers of inflammation such as cytokines, and amyloidogenic aggregates, so-called nanoplaques, are both promising biomarker candidates for AD. We have previously shown that there is a relationship between the levels of nanoplaques and cytokines in cerebrospinal fluid, but it is unknown whether this association extends to serum. OBJECTIVE: Investigate in a naturalistic memory clinic cohort whether the associations between nanoplaques and cytokines in the cerebrospinal fluid extends to serum. METHODS: We collected serum from 49 patients assessed for cognitive complaints at the Oslo University Hospital Memory Clinic (15 with clinical AD). We assessed the levels of serum nanoplaques with the novel Thioflavin-T fluorescence correlation spectroscopy (ThT-FCS) assay. Serum levels of nine cytokines (eotaxin-1, granulocyte colony-stimulating factor [G-CSF], interleukin [IL]-6, IL-7, IL-8, monocyte chemoattractant protein-1 (MCP-1), gamma induced protein 10 (IP-10), macrophage inflammatory protein [MIP]-1α, and MIP-1ß) were quantified with a multiplex assay and read on a Luminex IS 200 instrument. RESULTS: Serum nanoplaques were not increased in clinical AD patients compared to non-AD memory clinic patients and nanoplaques were not associated with any cytokines. The cytokines IL-8 and G-CSF were increased in patients with clinical AD compared to non-AD patients. CONCLUSION: In this small pilot study, serum nanoplaques were not associated with serum cytokines. Nanoplaque levels could not be used to separate clinical AD patients from non-AD patients in this unselected memory clinic cohort.

Cytosolic GPR37, but not GPR37L1, multimerization and its reversal by Parkin: A live cell imaging study.

Biochemical data have shown aggregated G protein-coupled receptor 37 (GPR37) in the cytoplasm and Lewy bodies in Parkinson's disease (PD). Properly folded GPR37 at the plasma membrane appears to be neuroprotective. GPR37, and its homologue GPR37L1, are orphan G protein-coupled receptors and their homo- and hetero-dimers have not been established. We therefore examined GPR37 and GPR37L1 dimerization and extended studies of multimerization of GPR37 to live cells. In this study, we investigated GPR37 and GPR37L1 dimerization and multimerization in live cells using three quantitative imaging methods: Fluorescence Cross-Correlation Spectroscopy, Förster Resonance Energy Transfer, and Fluorescence Lifetime Imaging Microscopy. Our data show that GPR37 and GPR37L1 form homo- and heterodimers in live N2a cells. Importantly, aggregation of GPR37, but not GPR37L1, was identified in the cytoplasm, which could be counteracted by Parkin overexpression. These data provide further evidence that GPR37 participate in cytosolic aggregation processes implicated in PD pathology.

Dynamic Cellular Cartography: Mapping the Local Determinants of Oligodendrocyte Transcription Factor 2 (OLIG2) Function in Live Cells Using Massively Parallel Fluorescence Correlation Spectroscopy Integrated with Fluorescence Lifetime Imaging Microscopy (mpFCS/FLIM).

Compartmentalization and integration of molecular processes through diffusion are basic mechanisms through which cells perform biological functions. To characterize these mechanisms in live cells, quantitative and ultrasensitive analytical methods with high spatial and temporal resolution are needed. Here, we present quantitative scanning-free confocal microscopy with single-molecule sensitivity, high temporal resolution (∼10 µs/frame), and fluorescence lifetime imaging capacity, developed by integrating massively parallel fluorescence correlation spectroscopy with fluorescence lifetime imaging microscopy (mpFCS/FLIM); we validate the method, use it to map in live cell location-specific variations in the concentration, diffusion, homodimerization, DNA binding, and local environment of the oligodendrocyte transcription factor 2 fused with the enhanced Green Fluorescent Protein (OLIG2-eGFP), and characterize the effects of an allosteric inhibitor of OLIG2 dimerization on these determinants of OLIG2 function. In particular, we show that cytoplasmic OLIG2-eGFP is largely monomeric and freely diffusing, with the fraction of freely diffusing OLIG2-eGFP molecules being fD,freecyt = (0.75 ± 0.10) and the diffusion time τD,freecyt = (0.5 ± 0.3) ms. In contrast, OLIG2-eGFP homodimers are abundant in the cell nucleus, constituting ∼25% of the nuclear pool, some fD,boundnuc = (0.65 ± 0.10) of nuclear OLIG2-eGFP is bound to chromatin DNA, whereas freely moving OLIG2-eGFP molecules diffuse at the same rate as those in the cytoplasm, as evident from the lateral diffusion times τD,freenuc = τD,freecyt = (0.5 ± 0.3) ms. OLIG2-eGFP interactions with chromatin DNA, revealed through their influence on the apparent diffusion behavior of OLIG2-eGFP, τD,boundnuc (850 ± 500) ms, are characterized by an apparent dissociation constant Kd,appOLIG2-DNA = (45 ± 30) nM. The apparent dissociation constant of OLIG2-eGFP homodimers was estimated to be Kd,app(OLIG2-eGFP)2 ≈ 560 nM. The allosteric inhibitor of OLIG2 dimerization, compound NSC 50467, neither affects OLIG2-eGFP properties in the cytoplasm nor does it alter the overall cytoplasmic environment. In contrast, it significantly impedes OLIG2-eGFP homodimerization in the cell nucleus, increasing five-fold the apparent dissociation constant, Kd,app,NSC50467(OLIG2-eGFP)2 ≈ 3 µM, thus reducing homodimer levels to below 7% and effectively abolishing OLIG2-eGFP specific binding to chromatin DNA. The mpFCS/FLIM methodology has a myriad of applications in biomedical research and pharmaceutical industry. For example, it is indispensable for understanding how biological functions emerge through the dynamic integration of location-specific molecular processes and invaluable for drug development, as it allows us to quantitatively characterize the interactions of drugs with drug targets in live cells.

Associations of cerebrospinal fluid amyloidogenic nanoplaques with cytokines in Alzheimer's disease.

BACKGROUND: The aggregation of amyloid ß (Aß) is central in the pathogenesis of Alzheimer's disease (AD). Recently it has been shown that specifically, larger, Thioflavin T-binding Aß aggregates are associated with increased neuroinflammation and cytokine release. This study was aimed to quantify fibrillary amyloid aggregates, so-called nanoplaques, and investigate their relationship with cytokines in the cerebrospinal fluid (CSF). METHODS: CSF was collected from 111 patients assessed for cognitive complaints at the Oslo University Hospital Memory Clinic. The patients were grouped based on their amyloid status. The CSF nanoplaque concentration was quantified with the Thioflavin T-fluorescence correlation spectroscopy (ThT-FCS) assay. The levels of nine cytokines (eotaxin-1, granulocyte stimulating factor, interleukin [IL]-6, IL-7, IL-8, monocyte chemoattractant protein-1, gamma-induced protein 10, macrophage inflammatory protein [MIP]-1α, and MIP-1ß) were quantified with a magnetic bead-based multiplex assay and read on a Luminex IS 200 instrument. RESULTS: There were 49 amyloid-negative and 62 amyloid-positive patients in the cohort; none of the cytokines differed significantly between the amyloid groups. The increased nanoplaque levels were associated with levels of MIP-1ß below the lower limit of quantification, and with decreased levels of MIP-1α and IL-8. The associations remained significant when adjusted for age, sex, cognitive function, apolipoprotein ε4 status and CSF core biomarker levels. CONCLUSION: The cytokine levels were not associated with amyloid status in this cohort. The nanoplaque levels were negatively associated with MIP-1ß, MIP-1α and IL-8, which is in line with recent findings suggesting that the upregulation of some cytokine markers has a protective role and is negatively associated with AD progression.

Iso-α-acids in Nonalcoholic and Alcoholic Beer Stimulate Growth of Neuron-like SH-SY5Y Cells and Neuroepithelial Stem Cells.

With the increasing popularity of nonalcoholic beer, the association between beer drinking and alcohol intake is lost. In the present study, we show that nonalcoholic beer can stimulate the expansion of neuron-like cell lines and neuroepithelial stem cells in culture, yielding an effect comparable to that of alcoholic beer. One ingredient in beer is hops, which is derived from the flower of hop plants. The female flower contains humulones, which are transformed into iso-α-acids during wort boiling and give beer its bitter taste. In this study, we tested the effects of these iso-α-acids and/or alcohol on the proliferation of neuron-like cells and neuroepithelial stem cells in culture. Iso-α-acids enhanced cell expansion, showing a bimodal dose-response curve with peaks around 2-30 nM and 2-5 µM, of which nanomolar concentrations are relevant in beer drinking. The more lipophilic trans-iso-α-acids, found to a greater extent in beer foam, are even more potent. Our results indicate that iso-α-acids, acting via peroxisome proliferator-activated receptors could be responsible for the observed effects. Altogether, our results indicate that nonalcoholic beer with ingredients such as iso-α-acids stimulate the proliferation of neuroepithelial stem cells.

Amyloidogenic Nanoplaques in Cerebrospinal Fluid: Relationship to Amyloid Brain Uptake and Clinical Alzheimer's Disease in a Memory Clinic Cohort.

BACKGROUND: Aggregation of amyloid-ß (Aß) is an early pathological event in Alzheimer's disease (AD). Consequently, measures of pathogenic aggregated Aß are attractive biomarkers for AD. Here, we use a recently developed Thioflavin-T-Fluorescence Correlation Spectroscopy (ThT-FCS) assay to quantify structured ThT-responsive protein aggregates, so-called nanoplaques, in the cerebrospinal fluid (CSF). OBJECTIVE: The overall aim of this work was to assess whether ThT-FCS determined CSF nanoplaque levels could predict amyloid brain uptake as determined by 18F-Flutemetamol PET analysis. Further, we assess whether nanoplaque levels could predict clinical AD. METHODS: Nanoplaque levels in the CSF from 54 memory clinic patients were compared between sub-groups classified by 18F-Flutemetamol PET as amyloid-positive or amyloid-negative, and by clinical assessment as AD or non-AD. RESULTS: Nanoplaque levels did not differ between amyloid groups and could not predict brain amyloid uptake. However, nanoplaque levels were significantly increased in patients with clinical AD, and were significant predictors for AD when adjusting for age, sex, cognitive function, and apolipoprotein E (APOE) genotype. CONCLUSION: The concentration of nanoplaques in the CSF differentiates patients with clinical AD from non-AD patients.

Methylation of Brain Derived Neurotrophic Factor (BDNF) Val66Met CpG site is associated with early onset bipolar disorder.

BACKGROUND: The brain-derived neurotrophic factor (BDNF) rs6265 (Val66Met) Met allele is associated with early onset (≤ 19 years old) bipolar disorder (BD). Val66Met (G196A) creates a CpG site when the Val/G allele is present. We sought to study the methylation of the BDNF promoter and its interaction with Val66Met genotype in BD. METHODS: Sex/age-matched previously genotyped DNA samples from BD-Type 1 cases [N = 166: early onset (≤ 19 years old) n = 79, late onset (> 20 years old) n = 87] and controls (N = 162) were studied. Pyrosequencing of four CpGs in Promoter-I, four CpGs in promoter-IV, and two CpGs in Promoter-IX (CpG2 includes G= Val allele) was performed. Logistic regression adjusting for batch effect was used to compare cases vs. controls. Analyses also included stratification by disease onset and adjustment for Val66Met genotype. Secondary exploratory analyses for the association of life stressors, comorbid substance abuse, and psychotropic use with methylation patterns were performed. RESULTS: Comparing all BD cases vs. controls and adjusting for Val66Met genotype, BD cases had significantly higher methylation in promoter -IX/CPG-2 (p = 0.0074). This was driven by early onset cases vs. controls (p = 0.00039) and not late onset cases vs. controls (p = 0.2). LIMITATION: Relatively small sample size. CONCLUSION: Early onset BD is associated with increased methylation of CpG site created by Val=G allele of the Val66Met variance. Further studies could include larger sample size and postmortem brain samples in an attempt to replicate these findings.

A strategy for designing allosteric modulators of transcription factor dimerization.

Transcription factors (TFs) are fundamental in the regulation of gene expression in the development and differentiation of cells. They may act as oncogenes and when overexpressed in tumors become plausible targets for the design of antitumor agents. Homodimerization or heterodimerization of TFs are required for DNA binding and the association interface between subunits, for the design of allosteric modulators, appears as a privileged structure for the pharmacophore-based computational strategy. Based on this strategy, a set of compounds were earlier identified as potential suppressors of OLIG2 dimerization and found to inhibit tumor growth in a mouse glioblastoma cell line and in a whole-animal study. To investigate whether the antitumor activity is due to the predicted mechanism of action, we undertook a study of OLIG2 dimerization using fluorescence cross-correlation spectroscopy (FCCS) of live HEK cells transfected with 2 spectrally different OLIG2 clones. The selected compounds showed an effect with potency, which correlated with the earlier observed antitumor activity. The OLIG2 proteins showed change in diffusion time under compound treatment in line with dissociation from DNA. The data suggest a general approach of drug discovery based on the design of allosteric modulators of protein-protein interaction.

Comparison of Cerebrospinal Fluid Amyloidogenic Nanoplaques With Core Biomarkers of Alzheimer's Disease.

Accurate biomarkers of Alzheimer's disease (AD) are essential for early diagnosis and intervention. Available biomarkers are not sufficient to permit the monitoring of AD progression over time, and additional biomarkers are required. Measures of aggregated amyloid-ß (Aß) could be useful biomarkers for AD. Here, we investigate whether levels of Thioflavin-T (ThT) positive amyloid aggregates, i.e., nanoplaques, in cerebrospinal fluid (CSF) could serve as useful biomarkers for AD. One-hundred and eighteen memory clinic patients were AT(N) classified, and CSF nanoplaque concentrations were compared between patients on the "Alzheimer's continuum" (A+ patients) and patients with "Normal AD biomarkers" or "Non-AD pathologic change" (A- patients). CSF nanoplaque concentrations and sizes were quantified using the novel ThT-Fluorescence Correlation Spectroscopy (ThT-FCS) assay, and core biomarkers (Aß42, total tau and phosphorylated tau) were determined by enzyme-linked immunosorbent assays. We investigated the association between nanoplaque concentrations and core biomarkers, and the diagnostic value of nanoplaque levels. Nanoplaque levels were increased in A+ patients compared to A- patients. Nanoplaque concentrations were negatively associated with Aß42, but not related to total tau or phosphorylated tau measures. Quantification of nanoplaques did not improve the classification of patients on the Alzheimer's continuum compared to the core biomarkers alone. Dynamic changes in nanoplaques concentration and size throughout AD stages should be explored in longitudinal studies.

Functional Fluorescence Microscopy Imaging: Quantitative Scanning-Free Confocal Fluorescence Microscopy for the Characterization of Fast Dynamic Processes in Live Cells.

Functional fluorescence microscopy imaging (fFMI), a time-resolved (21 µs/frame) confocal fluorescence microscopy imaging technique without scanning, is developed for quantitative characterization of fast reaction-transport processes in solution and in live cells. The method is based on massively parallel fluorescence correlation spectroscopy (FCS). Simultaneous excitation of fluorescent molecules in multiple spots in the focal plane is achieved using a diffractive optical element (DOE). Fluorescence from the DOE-generated 1024 illuminated spots is detected in a confocal arrangement by a matching matrix detector comprising 32 × 32 single-photon avalanche photodiodes (SPADs). Software for data acquisition and fast auto- and cross-correlation analysis by parallel signal processing using a graphic processing unit (GPU) allows temporal autocorrelation across all pixels in the image frame in 4 s and cross-correlation between first- and second-order neighbor pixels in 45 s. We present here this quantitative, time-resolved imaging method with single-molecule sensitivity and demonstrate its usefulness for mapping in live cell location-specific differences in the concentration and translational diffusion of molecules in different subcellular compartments. In particular, we show that molecules without a specific biological function, e.g., the enhanced green fluorescent protein (eGFP), exhibit uniform diffusion. In contrast, molecules that perform specialized biological functions and bind specifically to their molecular targets show location-specific differences in their concentration and diffusion, exemplified here for two transcription factor molecules, the glucocorticoid receptor (GR) before and after nuclear translocation and the Sex combs reduced (Scr) transcription factor in the salivary gland of Drosophila ex vivo.

Amyloidogenic Nanoplaques in Blood Serum of Patients with Alzheimer's Disease Revealed by Time-Resolved Thioflavin T Fluorescence Intensity Fluctuation Analysis.

BACKGROUND: Biomarkers are central to current research on molecular mechanisms underlying Alzheimer's disease (AD). Their further development is of paramount importance for understanding pathophysiological processes that eventually lead to disease onset. Biomarkers are also crucial for early disease detection, before clinical manifestation, and for development of new disease modifying therapies. OBJECTIVE: The overall aim of this work is to develop a minimally invasive method for fast, ultra-sensitive and cost-effective detection of structurally modified peptide/protein self-assemblies in the peripheral blood and in other biological fluids. Specifically, we focus here on using this method to detect structured amyloidogenic oligomeric aggregates in the blood serum of apparently healthy individuals and patients in early AD stage, and measure their concentration and size. METHODS: Time-resolved detection of Thioflavin T (ThT) fluorescence intensity fluctuations in a sub-femtoliter observation volume element was used to identify in blood serum ThT-active structured amyloidogenic oligomeric aggregates, hereafter called nanoplaques, and measure with single-particle sensitivity their concentration and size. RESULTS: The concentration and size of structured amyloidogenic nanoplaques are significantly higher in the blood serum of individuals diagnosed with AD than in control subjects. CONCLUSION: A new method with the ultimate, single-particle sensitivity was successfully developed. The proposed approach neither relies on the use of immune-based probes, nor on the use of radiotracers, signal-amplification or protein separation techniques, and provides a minimally invasive test for fast and cost-effective early determination of structurally modified peptides/proteins in the peripheral blood, as shown here, but also in other biological fluids.

Control of Hox transcription factor concentration and cell-to-cell variability by an auto-regulatory switch.

The variability in transcription factor concentration among cells is an important developmental determinant, yet how variability is controlled remains poorly understood. Studies of variability have focused predominantly on monitoring mRNA production noise. Little information exists about transcription factor protein variability, as this requires the use of quantitative methods with single-molecule sensitivity. Using Fluorescence Correlation Spectroscopy (FCS), we have characterized the concentration and variability of 14 endogenously tagged TFs in live Drosophila imaginal discs. For the Hox TF Antennapedia, we investigated whether protein variability results from random stochastic events or is developmentally regulated. We found that Antennapedia transitioned from low concentration/high variability early, to high concentration/low variability later, in development. FCS and temporally resolved genetic studies uncovered that Antennapedia itself is necessary and sufficient to drive a developmental regulatory switch from auto-activation to auto-repression, thereby reducing variability. This switch is controlled by progressive changes in relative concentrations of preferentially activating and repressing Antennapedia isoforms, which bind chromatin with different affinities. Mathematical modeling demonstrated that the experimentally supported auto-regulatory circuit can explain the increase of Antennapedia concentration and suppression of variability over time.