Ca2+-activated K+ channels modulate membrane potential in the human parathyroid cell: Possible role in exocytosis.

The relationships between parathyroid hormone (PTH) secretion and parathyroid cell membrane potential, including the identities and roles of K+ channels that regulate and/or modulate membrane potential are not well defined. Here we have used Western blot/immunohistochemistry as well as patch-clamp and perifusion techniques to identify and localize specific K+ channels in parathyroid cells and to investigate their roles in the control of membrane potential and PTH secretion. We also re-investigated the relationship between membrane potential and exocytosis. We showed that in single human parathyroid cells K+ current is dependent on at least two types of Ca2+-activated K+ channels: a small-conductance Ca2+-activated K+ channel (KSK) and a large-conductance voltage and Ca2+-activated K+ channel (KBK). These channels were sensitive to specific peptide blocking toxins including apamin, charybdotoxin, and iberiotoxin. These channels confer sensitivity of the membrane potential in single cells to high extracellular K+, TEA, and peptide toxins. Blocking of KBK potently inhibited K+ channel current, and KBK was shown to be expressed in the plasma membrane of parathyroid cells. In addition, when using the capacitance technique as an indicator of exocytosis, clamping the parathyroid cell at -60 mV prevented exocytosis, whereas holding the membrane potential at 0 mV facilitated it. Taken together, the results show that human parathyroid cells have functional KBK and KSK channels but the data presented herein suggest that KBK/KSK channels likely contribute to the maintenance of the membrane potential, and that membrane potential, per se, modulates exocytosis independently of [Ca2+]i.

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.

Increased intracellular crowding during hyperosmotic stress.

Hyperosmotic stress activates in live cells numerous processes and also promotes intracellular protein/RNA aggregation and phase separation. However, the time course and the extent of these changes remain largely uncharacterized. To investigate dynamic changes in intracellular macromolecular crowding (MMC) induced by hyperosmotic stress in live cells, we used fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy (FCS) to quantify changes in the local environment by measuring the fluorescence lifetime and the diffusion of the monomeric enhanced green fluorescent protein (eGFP), respectively. Real-time monitoring of eGFP fluorescence lifetime showed that a faster response to environmental changes due to MMC is observed than when measuring the acceptor/donor emission ratio using the MMC-sensitive Förster resonance energy transfer sensor (GimRET). This suggests that eGFP molecular electronic states and/or collision frequency are affected by changes in the immediate surroundings due to MMC without requiring conformational changes as is the case for the GimRET sensor. Furthermore, eGFP diffusion assessed by FCS indicated higher intracellular viscosity due to increased MMC during hyperosmotic stress. Our findings reveal that changes in eGFP fluorescence lifetime and diffusion are early indicators of elevated intracellular MMC. Our approach can therefore be used to reveal in live cells short-lived transient states through which MMC builds over time, which could not be observed when measuring changes in other physical properties that occur at slower time scales.

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.

Interactions of ultrashort laser pulses with hemoglobin: Photophysical aspects and potential applications.

Hemoglobin (Hb), a life-sustaining and highly abundant erythrocyte protein, is not readily fluorescent. A few studies have already reported Two-Photon Excited Fluorescence (TPEF) of Hb, however, the mechanisms through which Hb becomes fluorescent upon interaction with ultrashort laser pulses are not completely understood. Here, we characterized photophysically this interaction on Hb thin film and erythrocytes using fluorescence spectroscopy upon single-photon/two-photon absorption, and UV-VIS single-photon absorption spectroscopy. A gradual increase of the fluorescence intensity, ending up with saturation, is observed upon prolonged exposure of Hb thin layer and erythrocytes to ultrashort laser pulses at 730 nm. When compared to protoporphyrin IX (PpIX) and oxidized Hb by H2O2, TPEF spectra from a thin Hb film and erythrocytes showed good mutual agreement, broad peaking at 550 nm, supporting hemoglobin undergoes degradation and that same fluorescent specie(s) originating from the heme moiety are generated. The uniform square shaped patterns of the fluorescent photoproduct exhibited the same level of the fluorescence intensity even after 12 weeks from the formation, indicating high photoproduct stability. We finally demonstrated the full potential of the formed Hb photoproduct with TPEF scanning microscopy towards spatiotemporally controlled micropatterning in HTF and single human erythrocyte labelling and tracking in the whole blood.

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.

Plaque Evaluation by Ultrasound and Transcriptomics Reveals BCLAF1 as a Regulator of Smooth Muscle Cell Lipid Transdifferentiation in Atherosclerosis.

BACKGROUND: Understanding the processes behind carotid plaque instability is necessary to develop methods for identification of patients and lesions with stroke risk. Here, we investigated molecular signatures in human plaques stratified by echogenicity as assessed by duplex ultrasound. METHODS: Lesion echogenicity was correlated to microarray gene expression profiles from carotid endarterectomies (n=96). The findings were extended into studies of human and mouse atherosclerotic lesions in situ, followed by functional investigations in vitro in human carotid smooth muscle cells (SMCs). RESULTS: Pathway analyses highlighted muscle differentiation, iron homeostasis, calcification, matrix organization, cell survival balance, and BCLAF1 (BCL2 [B-cell lymphoma 2]-associated transcription factor 1) as the most significant signatures. BCLAF1 was downregulated in echolucent plaques, positively correlated to proliferation and negatively to apoptosis. By immunohistochemistry, BCLAF1 was found in normal medial SMCs. It was repressed early during atherogenesis but reappeared in CD68+ cells in advanced plaques and interacted with BCL2 by proximity ligation assay. In cultured SMCs, BCLAF1 was induced by differentiation factors and mitogens and suppressed by macrophage-conditioned medium. BCLAF1 silencing led to downregulation of BCL2 and SMC markers, reduced proliferation, and increased apoptosis. Transdifferentiation of SMCs by oxLDL (oxidized low-denisty lipoprotein) was accompanied by upregulation of BCLAF1, CD36, and CD68, while oxLDL exposure with BCLAF1 silencing preserved MYH (myosin heavy chain) 11 expression and prevented transdifferentiation. BCLAF1 was associated with expression of cell differentiation, contractility, viability, and inflammatory genes, as well as the scavenger receptors CD36 and CD68. BCLAF1 expression in CD68+/BCL2+ cells of SMC origin was verified in plaques from MYH11 lineage-tracing atherosclerotic mice. Moreover, BCLAF1 downregulation associated with vulnerability parameters and cardiovascular risk in patients with carotid atherosclerosis. CONCLUSIONS: Plaque echogenicity correlated with enrichment of distinct molecular pathways and identified BCLAF1, previously not described in atherosclerosis, as the most significant gene. Functionally, BCLAF1 seems necessary for survival and transdifferentiation of SMCs into a macrophage-like phenotype. The role of BCLAF1 in plaque vulnerability should be further evaluated.

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.

Smallest Secondary Nucleation Competent Aß Aggregates Probed by an ATP-Independent Molecular Chaperone Domain.

Protein oligomerization is a commonly encountered strategy by which the functional repertoire of proteins is increased. This, however, is a double-edged sword strategy because protein oligomerization is notoriously difficult to control. Living organisms have therefore developed a number of chaperones that prevent protein aggregation. The small ATP-independent molecular chaperone domain proSP-C BRICHOS, which is mainly trimeric, specifically inhibits fibril surface-catalyzed nucleation reactions that give rise to toxic oligomers during the aggregation of the Alzheimer's disease-related amyloid-ß peptide (Aß42). Here, we have created a stable proSP-C BRICHOS monomer mutant and show that it does not bind to monomeric Aß42 but has a high affinity for Aß42 fibrils, using surface plasmon resonance. Kinetic analysis of Aß42 aggregation profiles, measured by thioflavin T fluorescence, reveals that the proSP-C BRICHOS monomer mutant strongly inhibits secondary nucleation reactions and thereby reduces the level of catalytic formation of toxic Aß42 oligomers. To study binding between the proSP-C BRICHOS monomer mutant and small soluble Aß42 aggregates, we analyzed fluorescence cross-correlation spectroscopy measurements with the maximum entropy method for fluorescence correlation spectroscopy. We found that the proSP-C BRICHOS monomer mutant binds to the smallest emerging Aß42 aggregates that are comprised of eight or fewer Aß42 molecules, which are already secondary nucleation competent. Our approach can be used to provide molecular-level insights into the mechanisms of action of substances that interfere with protein aggregation.

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.

Alpha-Gal on the Protein Surface Hampers Transcytosis through the Caco-2 Monolayer.

Transepithelial transport of proteins is an important step in the immune response to food allergens. Mammalian meat allergy is characterized by an IgE response against the carbohydrate moiety galactosyl-α-1,3-galactose (α-Gal) present on mammalian glycoproteins and glycolipids, which causes severe allergic reactions several hours after red meat consumption. The delayed reaction may be related to the processing of α-Gal carrying proteins in the gastrointestinal tract. The aim of this study was to investigate how protein glycosylation by α-Gal affects the susceptibility to gastric digestion and transport through the Caco-2 cell monolayer. We found that α-Gal glycosylation altered protein susceptibility to gastric digestion, where large protein fragments bearing the α-Gal epitope remained for up to 2 h of digestion. Furthermore, α-Gal glycosylation of the protein hampered transcytosis of the protein through the Caco-2 monolayer. α-Gal epitope on the intact protein could be detected in the endosomal fraction obtained by differential centrifugation of Caco-2 cell lysates. Furthermore, the level of galectin-3 in Caco-2 cells was not affected by the presence of α-Gal glycosylated BSA (bovine serum albumin) (BSA-α-Gal). Taken together, our data add new knowledge and shed light on the digestion and transport of α-Gal glycosylated proteins.

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.