Amyloid-beta impairs TOM1-mediated IL-1R1 signaling.

Defects in interleukin-1ß (IL-1ß)-mediated cellular responses contribute to Alzheimer's disease (AD). To decipher the mechanism associated with its pathogenesis, we investigated the molecular events associated with the termination of IL-1ß inflammatory responses by focusing on the role played by the target of Myb1 (TOM1), a negative regulator of the interleukin-1ß receptor-1 (IL-1R1). We first show that TOM1 steady-state levels are reduced in human AD hippocampi and in the brain of an AD mouse model versus respective controls. Experimentally reducing TOM1 affected microglia activity, substantially increased amyloid-beta levels, and impaired cognition, whereas enhancing its levels was therapeutic. These data show that reparation of the TOM1-signaling pathway represents a therapeutic target for brain inflammatory disorders such as AD. A better understanding of the age-related changes in the immune system will allow us to craft therapies to limit detrimental aspects of inflammation, with the broader purpose of sharply reducing the number of people afflicted by AD.

Transgenic Mouse Models of Alzheimer's Disease: An Integrative Analysis.

Alzheimer's disease (AD) constitutes the most prominent form of dementia among elderly individuals worldwide. Disease modeling using murine transgenic mice was first initiated thanks to the discovery of heritable mutations in amyloid precursor protein (APP) and presenilins (PS) genes. However, due to the repeated failure of translational applications from animal models to human patients, along with the recent advances in genetic susceptibility and our current understanding on disease biology, these models have evolved over time in an attempt to better reproduce the complexity of this devastating disease and improve their applicability. In this review, we provide a comprehensive overview about the major pathological elements of human AD (plaques, tauopathy, synaptic damage, neuronal death, neuroinflammation and glial dysfunction), discussing the knowledge that available mouse models have provided about the mechanisms underlying human disease. Moreover, we highlight the pros and cons of current models, and the revolution offered by the concomitant use of transgenic mice and omics technologies that may lead to a more rapid improvement of the present modeling battery.

Direct Measurement of Two-Photon Action Cross Section.

We present a simple and fast methodology for measuring the two-photon (2P) action cross section of phototriggers. The method uses a standard 2P microscopy setup for both uncaging and detection and a set of lithographically made microcuvettes in order to reduce the total excitation volume and, thus, the photolysis time. The procedure does not need a standard and can be used for any caged compounds that present different emission properties before and after uncaging. We tested the method with 2P active ruthenium-based caged serotonin and compared the obtained value with a standard measure involving fluorescein as reference.

Prescribed fire and partial overstory removal alter an acorn-rodent conditional mutualism.

In eastern North America, oak (Quercus) regeneration failure has spurred management using silvicultural approaches better aligned with the autecology of oaks. In particular, shelterwood harvests can create favorable intermediate light conditions for oak establishment and prescribed fire is predicted (by the oak-fire hypothesis) to favor oak regeneration. These approaches substantially modify forest structure and may affect crucial trophic interactions including the conditional mutualism between oaks and granivorous rodents that scatterhoard acorns, which shifts along a continuum from antagonistic to mutualistic depending on external factors. We investigated how overwinter survival and dispersal of northern red oak (Quercus rubra) acorns were influenced by location within or outside of group shelterwood harvests (small canopy gaps created throughout an intact forest stand) with and without prescribed fire. We conducted two concurrent experiments to test (1) dispersal and survival of acorns presented on the forest floor and (2) acorn pilferage rates from caches that mimic squirrel scatterhoards in shelterwood gap/group interiors, edges, and the uncut forest matrix in burned and unburned forest stands. In both experiments, acorn survival was generally higher in burned than unburned stands. Acorn survival from forest floor presentations was higher in the unharvested forest matrix than harvest gap interiors; however, there was no effect of proximity to harvest gaps on survival of cached acorns. Survival of cached acorns was associated with understory vegetative cover (-), coarse woody debris cover (-), and distance to nearest tree (+), but uncorrelated with canopy cover above the cache. Our results suggest that reduced understory cover following prescribed fire may increase perceived habitat riskiness for granivores resulting in higher acorn survival up to 2 yr post-fire. These findings unify the oak-fire and oak-granivore conditional mutualism hypotheses, and suggest that the environmental conditions following prescribed fire and group shelterwood harvests may shift the oak-granivore conditional mutualism in a direction beneficial for oak regeneration.

Phagocytic clearance of presynaptic dystrophies by reactive astrocytes in Alzheimer's disease.

Reactive astrogliosis, a complex process characterized by cell hypertrophy and upregulation of components of intermediate filaments, is a common feature in brains of Alzheimer's patients. Reactive astrocytes are found in close association with neuritic plaques; however, the precise role of these glial cells in disease pathogenesis is unknown. In this study, using immunohistochemical techniques and light and electron microscopy, we report that plaque-associated reactive astrocytes enwrap, engulf and may digest presynaptic dystrophies in the hippocampus of amyloid precursor protein/presenilin-1 (APP/PS1) mice. Microglia, the brain phagocytic population, was apparently not engaged in this clearance. Phagocytic reactive astrocytes were present in 35% and 67% of amyloid plaques at 6 and 12 months of age, respectively. The proportion of engulfed dystrophic neurites was low, around 7% of total dystrophies around plaques at both ages. This fact, along with the accumulation of dystrophic neurites during disease course, suggests that the efficiency of the astrocyte phagocytic process might be limited or impaired. Reactive astrocytes surrounding and engulfing dystrophic neurites were also detected in the hippocampus of Alzheimer's patients by confocal and ultrastructural analysis. We posit that the phagocytic activity of reactive astrocytes might contribute to clear dysfunctional synapses or synaptic debris, thereby restoring impaired neural circuits and reducing the inflammatory impact of damaged neuronal parts and/or limiting the amyloid pathology. Therefore, potentiation of the phagocytic properties of reactive astrocytes may represent a potential therapy in Alzheimer's disease.

Relationship between virulence factors, resistance to antibiotics and phylogenetic groups of uropathogenic Escherichia coli in two locations in Mexico. / Relación entre factores de virulencia, resistencia a antibióticos y los grupos filogenéticos de Escherichia coli uropatógena en dos localidades de México.

INTRODUCTION: Escherichia coli is the major causative agent of urinary tract infections (UTI), and virulence factors are responsible for the severity of these emerging infections. The aim of this study was to evaluate the relationship between virulence determinants and antibiotic susceptibility with phylogenetic groups of E.coli isolates of UTI in two locations in Mexico. METHODS: An analysis was performed on 50 isolates of E.coli from the centre of the country and 57 from a town in the southwest. The isolates were characterized by phenotype (serotyping assays, in vitro adhesion, biofilm formation, production of haemolysin, and antibiotic susceptibility) and genotype (phylogenetic groups and virulence genes). RESULTS: In the centre of the country location the phylogenetic group B2 (60%) and F (12%) were significantly more prevalent and had a higher frequency of genes, fimH (96%), iutA (66%), sat (36%), compared to the southwest location, where the group A (35%) and B1 (21%) were significantly predominant and had fewer virulence genes. About one-fifth (21.5%) of all isolates belonged to the O25-ST131 group. Haemolysin and biofilm producing strains were significantly higher in the southwest location. Resistance to ampicillin (92.5%), tetracycline (76.6%), and trimethoprim/sulfamethoxazole (70.1%) were the most common in both groups. CONCLUSION: The phylogenetic group, virulence factors, and antibiotic susceptibility of the E.coli that causes UTI in the community, varies significantly among the Mexican populations studied. Phylogenetic groups A and B1 may be multidrug resistant and have the ability to produce UTI.

Soluble phospho-tau from Alzheimer's disease hippocampus drives microglial degeneration.

The role of microglial cells in the development and progression of Alzheimer's disease (AD) has not been elucidated. Here, we demonstrated the existence of a weak microglial response in human AD hippocampus which is in contrast to the massive microglial activation observed in APP-based models. Most importantly, microglial cells displayed a prominent degenerative profile (dentate gyrus > CA3 > CA1 > parahippocampal gyrus), including fragmented and dystrophic processes with spheroids, a reduced numerical density, and a significant decrease in the area of surveillance ("microglial domain"). Consequently, there was a substantial decline in the area covered by microglia which may compromise immune protection and, therefore, neuronal survival. In vitro experiments demonstrated that soluble fractions (extracellular/cytosolic) from AD hippocampi were toxic for microglial cells. This toxicity was abolished by AT8 and/or AT100 immunodepletion, validating that soluble phospho-tau was the toxic agent. These results were reproduced using soluble fractions from phospho-tau-positive Thy-tau22 hippocampi. Cultured microglial cells were not viable following phagocytosis of SH-SY5Y cells expressing soluble intracellular phospho-tau. Because the phagocytic capacity of microglial cells is highly induced by apoptotic signals in the affected neurons, we postulate that accumulation of intraneuronal soluble phospho-tau might trigger microglial degeneration in the AD hippocampus. This microglial vulnerability in AD pathology provides new insights into the immunological mechanisms underlying the disease progression and highlights the need to improve or develop new animal models, as the current models do not mimic the microglial pathology observed in the hippocampus of AD patients.

Improving image contrast in fluorescence microscopy with nanostructured substrates.

Metallic and dielectric nanostructures can show sharp contrasted resonances, sensitive to the environment, and high field enhancement in sub-wavelength volumes. For this reason, these structures are commonly used as molecular sensors. Only few works have focused on their application in optical microscopy, in particular in superresolution. In this work we have designed, fabricated and optically tested a nanostructured TiO(2) substrate, fabricated by direct embossing of TiO(2) derived film, as a substrate for fluorescence microscopy. Moreover, using numerical simulations, we have compared the signal to background noise with respect to other metallo-dielectric structures. We show that the TiO(2) structure is a good candidate for reducing the thickness of the fluorescence excitation down to ~100 nm. Therefore, this substrate can be used to obtain Total Internal Reflection (TIRF) axial resolution without a TIRF-Microscopy system.

Adipose tissue as a therapeutic target for vascular damage in Alzheimer's disease.

Adipose tissue has recently been recognized as an important endocrine organ that plays a crucial role in energy metabolism and in the immune response in many metabolic tissues. With this regard, emerging evidence indicates that an important crosstalk exists between the adipose tissue and the brain. However, the contribution of adipose tissue to the development of age-related diseases, including Alzheimer's disease, remains poorly defined. New studies suggest that the adipose tissue modulates brain function through a range of endogenous biologically active factors known as adipokines, which can cross the blood-brain barrier to reach the target areas in the brain or to regulate the function of the blood-brain barrier. In this review, we discuss the effects of several adipokines on the physiology of the blood-brain barrier, their contribution to the development of Alzheimer's disease and their therapeutic potential. LINKED ARTICLES: This article is part of a themed issue From Alzheimer's Disease to Vascular Dementia: Different Roads Leading to Cognitive Decline. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.6/issuetoc.

Comparison of MSD analysis from single particle tracking with MSD from images. Getting the best of both worlds.

Fluorescence microscopy can provide valuable information about cell interior dynamics. Particularly, mean squared displacement (MSD) analysis is widely used to characterize proteins and sub-cellular structures' mobility providing the laws of molecular diffusion. The MSD curve is traditionally extracted from individual trajectories recorded by single-particle tracking-based techniques. More recently, image correlation methods like iMSD have been shown capable of providing averaged dynamic information directly from images, without the need for isolation and localization of individual particles. iMSD is a powerful technique that has been successfully applied to many different biological problems, over a wide spatial and temporal scales. The aim of this work is to review and compare these two well-established methodologies and their performance in different situations, to give an insight on how to make the most out of their unique characteristics. We show the analysis of the same datasets by the two methods. Regardless of the experimental differences in the input data for MSD or iMSD analysis, our results show that the two approaches can address equivalent questions for free diffusing systems. We focused on studying a range of diffusion coefficients between D = 0.001µm2s-1and D = 0.1µm2s-1, where we verified that the equivalence is maintained even for the case of isolated particles. This opens new opportunities for studying intracellular dynamics using equipment commonly available in any biophysical laboratory.

Monocyte-derived cells invade brain parenchyma and amyloid plaques in human Alzheimer's disease hippocampus.

Microglia are brain-resident myeloid cells and play a major role in the innate immune responses of the CNS and the pathogenesis of Alzheimer's disease (AD). However, the contribution of nonparenchymal or brain-infiltrated myeloid cells to disease progression remains to be demonstrated. Here, we show that monocyte-derived cells (MDC) invade brain parenchyma in advanced stages of AD continuum using transcriptional analysis and immunohistochemical characterization in post-mortem human hippocampus. Our findings demonstrated that a high proportion (60%) of demented Braak V-VI individuals was associated with up-regulation of genes rarely expressed by microglial cells and abundant in monocytes, among which stands the membrane-bound scavenger receptor for haptoglobin/hemoglobin complexes or Cd163. These Cd163-positive MDC invaded the hippocampal parenchyma, acquired a microglial-like morphology, and were located in close proximity to blood vessels. Moreover, and most interesting, these invading monocytes infiltrated the nearby amyloid plaques contributing to plaque-associated myeloid cell heterogeneity. However, in aged-matched control individuals with hippocampal amyloid pathology, no signs of MDC brain infiltration or plaque invasion were found. The previously reported microglial degeneration/dysfunction in AD hippocampus could be a key pathological factor inducing MDC recruitment. Our data suggest a clear association between MDC infiltration and endothelial activation which in turn may contribute to damage of the blood brain barrier integrity. The recruitment of monocytes could be a consequence rather than the cause of the severity of the disease. Whether monocyte infiltration is beneficial or detrimental to AD pathology remains to be fully elucidated. These findings open the opportunity to design targeted therapies, not only for microglia but also for the peripheral immune cell population to modulate amyloid pathology and provide a better understanding of the immunological mechanisms underlying the progression of AD.

Age-dependent accumulation of soluble amyloid beta (Abeta) oligomers reverses the neuroprotective effect of soluble amyloid precursor protein-alpha (sAPP(alpha)) by modulating phosphatidylinositol 3-kinase (PI3K)/Akt-GSK-3beta pathway in Alzheimer mouse model.

Neurotrophins, activating the PI3K/Akt signaling pathway, control neuronal survival and plasticity. Alterations in NGF, BDNF, IGF-1, or insulin signaling are implicated in the pathogenesis of Alzheimer disease. We have previously characterized a bigenic PS1×APP transgenic mouse displaying early hippocampal Aß deposition (3 to 4 months) but late (17 to 18 months) neurodegeneration of pyramidal cells, paralleled to the accumulation of soluble Aß oligomers. We hypothesized that PI3K/Akt/GSK-3ß signaling pathway could be involved in this apparent age-dependent neuroprotective/neurodegenerative status. In fact, our data demonstrated that, as compared with age-matched nontransgenic controls, the Ser-9 phosphorylation of GSK-3ß was increased in the 6-month PS1×APP hippocampus, whereas in aged PS1×APP animals (18 months), GSK-3ß phosphorylation levels displayed a marked decrease. Using N2a and primary neuronal cell cultures, we demonstrated that soluble amyloid precursor protein-α (sAPPα), the predominant APP-derived fragment in young PS1×APP mice, acting through IGF-1 and/or insulin receptors, activated the PI3K/Akt pathway, phosphorylated the GSK-3ß activity, and in consequence, exerted a neuroprotective action. On the contrary, several oligomeric Aß forms, present in the soluble fractions of aged PS1×APP mice, inhibited the induced phosphorylation of Akt/GSK-3ß and decreased the neuronal survival. Furthermore, synthetic Aß oligomers blocked the effect mediated by different neurotrophins (NGF, BDNF, insulin, and IGF-1) and sAPPα, displaying high selectivity for NGF. In conclusion, the age-dependent appearance of APP-derived soluble factors modulated the PI3K/Akt/GSK-3ß signaling pathway through the major neurotrophin receptors. sAPPα stimulated and Aß oligomers blocked the prosurvival signaling. Our data might provide insights into the selective vulnerability of specific neuronal groups in Alzheimer disease.

Abnormal accumulation of autophagic vesicles correlates with axonal and synaptic pathology in young Alzheimer's mice hippocampus.

Dystrophic neurites associated with amyloid plaques precede neuronal death and manifest early in Alzheimer's disease (AD). In this work we have characterized the plaque-associated neuritic pathology in the hippocampus of young (4- to 6-month-old) PS1(M146L)/APP(751SL) mice model, as the initial degenerative process underlying functional disturbance prior to neuronal loss. Neuritic plaques accounted for almost all fibrillar deposits and an axonal origin of the dystrophies was demonstrated. The early induction of autophagy pathology was evidenced by increased protein levels of the autophagosome marker LC3 that was localized in the axonal dystrophies, and by electron microscopic identification of numerous autophagic vesicles filling and causing the axonal swellings. Early neuritic cytoskeletal defects determined by the presence of phosphorylated tau (AT8-positive) and actin-cofilin rods along with decreased levels of kinesin-1 and dynein motor proteins could be responsible for this extensive vesicle accumulation within dystrophic neurites. Although microsomal Aß oligomers were identified, the presence of A11-immunopositive Aß plaques also suggested a direct role of plaque-associated Aß oligomers in defective axonal transport and disease progression. Most importantly, presynaptic terminals morphologically disrupted by abnormal autophagic vesicle buildup were identified ultrastructurally and further supported by synaptosome isolation. Finally, these early abnormalities in axonal and presynaptic structures might represent the morphological substrate of hippocampal dysfunction preceding synaptic and neuronal loss and could significantly contribute to AD pathology in the preclinical stages.

Spectroscopic properties of gold nanoparticles at the single-particle level in biological environments.

Labeling cells and tissues with fluorescent probes, such as organic dyes and quantum dots (Qdots) is a widespread and successful technique for studying molecular dynamics both in vitro and in vivo. However, those probes usually suffer from undesirable photophysical/photochemical processes, such as blinking and photobleaching, limiting their utilization. The main challenges in fluorescent probe design are to improve their absorption/emission properties, and to provide higher stability against photobleaching. In the last few years, metallic nanoparticles (NPs) of various sizes, shapes, and compositions have been used as a new alternative for cellular microscopy. This is in part because-unlike common organic dyes and Qdots-metallic NPs do not bleach or blink upon continuous illumination, are extremely stable, very bright, and their luminescence spans over the visible spectrum. These characteristics make them attractive contrast agents for cell imaging both in vitro and in vivo. For these reasons, the emission of metallic NPs in bulk solutions has already been extensively characterized. In contrast with bulk experiments, where billions of molecules are measured simultaneously, single-particle techniques allow the observation of characteristics and dynamical processes otherwise hidden in the measured average. A full understanding of the photophysical properties of the NPs is critical when they are used for single-molecule applications. Photophysical processes can be a source of artifacts if they are not interpreted accordingly, and thus a careful characterization of these labels at the single-particle level became crucial for the correct interpretation of the experimental results. Herein, we study some of their unique optical properties at the single-particle level and show examples that illustrate their intrinsic heterogeneity when used in biological environments.

Intracellular trafficking of cationic liposome-DNA complexes in living cells.

Three-dimensional single-particle tracking (SPT) was used to calculate the mean square displacement (MSD) and the diffusion coefficients of multicomponent cationic liposome-DNA complexes (lipoplexes) in CHO-K1 living cells. In untreated (NT) control cells, we found that the intracellular lipoplex motion was either directed or Brownian with active transportation being definitely more frequent (more than 70%) than Brownian diffusion. The MSD analysis was supported by the calculation of the three-dimensional asphericity, A3, which was close to unity, denoting the preponderant occurrence of movement along a direction. To elucidate the role of the cytoskeleton structure in the lipoplex trafficking, cells were treated with cytoskeleton (actin microfilaments and microtubules) polymerization inhibitors (latrunculin B and nocodazole, respectively). When cells were treated with inhibitors, the lipoplex movement tended towards a random walk at the expense of directed motion. The disassembly of microtubules had a stronger effect on the reduction of directional movement than that of actin microfilaments. Relevance of the results for enhanced gene delivery is discussed.

3D nanometer images of biological fibers by directed motion of gold nanoparticles.

Using near-infrared femtosecond pulses, we move single gold nanoparticles (AuNPs) along biological fibers, such as collagen and actin filaments. While the AuNP is sliding on the fiber, its trajectory is measured in three dimensions (3D) with nanometer resolution providing a high-resolution image of the fiber. Here, we systematically moved a single AuNP along nanometer-size collagen fibers and actin filament inside chinese hamster ovary K1 living cells, mapping their 3D topography with high fidelity.

Correlation analyses reveal differential diffusion behavior of eisosomal proteins between mother and daughter cells.

Eisosomes are nanoscale plasma membrane domains shaped as furrow-like invaginations. InSaccharomyces cerevisiaethese relatively immobile and uniform structures are mainly composed of two cytoplasmic proteins Pil1 and Lsp1. The present work uses fluctuation of fluorescence signals and analytical methods to determine Pil1 and Lsp1 dynamics at different subcellular locations. Using scanning techniques and autocorrelation analysis we determine that the cytoplasmic pools of Pil1 and Lsp1 behave mainly by passive diffusion. Single-point FCS experiments performed at several subcellular locations reveal that Pil1 mobility is faster in daughter cells. Furthermore, pair correlation function analysis indicates a rapid dynamic of Pil1 near the plasma membrane of growing yeast buds, where the membrane is expected to be actively assembling eisosomes.

Dengue Virus Capsid-Protein Dynamics in Live Infected Cells Studied by Pair Correlation Analysis.

It has become increasingly evident that unveiling the mechanisms of virus entry, assembly, and virion release is fundamental for identifying means for preventing viral spread and controlling viral disease. Due to virus mobility and structural and/or functional heterogeneity among viral particles, high spatiotemporal resolution single-virus/single-particle techniques are required to capture the behavior of viral particles inside infected cells.In this chapter, we present fluorescence imaging analysis methods for studying the mobility of fluorescently labeled dengue virus (DENV) proteins in live infected cells. Some of the most recent Fluorescence Fluctuation Spectroscopy (FFS) methods will be presented and, in particular, the pair Correlation Functions (pCF) approach will be discussed. The pCF method does not require individual molecule isolation, as in a particle-tracking experiment, to capture single viral protein behavior. In this regard, image acquisition is followed by the spatiotemporal cross-correlation function at increasing time delays, yielding a quantitative view of single-particle mobility in intact live infected cells.We provide a general overview and a practical guidance for the implementation of advanced FFS techniques, and the pair Correlation Functions analysis, as quantitative tools to reveal insights into previously unreported DENV mechanisms. We expect this protocol report will serve as an incentive for further applying correlation imaging studies in virology research.

Animal and Cellular Models of Alzheimer's Disease: Progress, Promise, and Future Approaches.

Alzheimer's disease (AD) is an incurable neurodegenerative disease affecting over 45 million people worldwide. Transgenic mouse models have made remarkable contributions toward clarifying the pathophysiological mechanisms behind the clinical manifestations of AD. However, the limited ability of these in vivo models to accurately replicate the biology of the human disease have precluded the translation of promising preclinical therapies to the clinic. In this review, we highlight several major pathogenic mechanisms of AD that were discovered using transgenic mouse models. Moreover, we discuss the shortcomings of current animal models and the need to develop reliable models for the sporadic form of the disease, which accounts for the majority of AD cases, as well as human cellular models to improve success in translating results into human treatments.

In vivo pair correlation microscopy reveals dengue virus capsid protein nucleocytoplasmic bidirectional movement in mammalian infected cells.

Flaviviruses are major human disease-causing pathogens, including dengue virus (DENV), Zika virus, yellow fever virus and others. DENV infects hundreds of millions of people per year around the world, causing a tremendous social and economic burden. DENV capsid (C) protein plays an essential role during genome encapsidation and viral particle formation. It has been previously shown that DENV C enters the nucleus in infected cells. However, whether DENV C protein exhibits nuclear export remains unclear. By spatially cross-correlating different regions of the cell, we investigated DENV C movement across the nuclear envelope during the infection cycle. We observed that transport takes place in both directions and with similar translocation times (in the ms time scale) suggesting a bidirectional movement of both C protein import and export.Furthermore, from the pair cross-correlation functions in cytoplasmic or nuclear regions we found two populations of C molecules in each compartment with fast and slow mobilities. While in the cytoplasm the correlation times were in the 2-6 and 40-110 ms range for the fast and slow mobility populations respectively, in the cell nucleus they were 1-10 and 25-140 ms range, respectively. The fast mobility of DENV C in cytoplasmic and nuclear regions agreed with the diffusion coefficients from Brownian motion previously reported from correlation analysis. These studies provide the first evidence of DENV C shuttling from and to the nucleus in infected cells, opening new venues for antiviral interventions.