Hiroyuki Hakozaki

Fibrinogen-induced perivascular microglial clustering is required for the development of axonal damage in neuroinflammation

Blood-brain barrier disruption, microglial activation and neurodegeneration are hallmarks of multiple sclerosis. However, the initial triggers that activate innate immune responses and their role in axonal damage remain unknown. Here we show that the blood protein fibrinogen induces rapid microglial responses toward the vasculature and is required for axonal damage in neuroinflammation. Using in vivo two-photon microscopy, we demonstrate that microglia form perivascular clusters before myelin loss or paralysis onset and that, of the plasma proteins, fibrinogen specifically induces rapid and sustained microglial responses in vivo. Fibrinogen leakage correlates with areas of axonal damage and induces reactive oxygen species release in microglia. Blocking fibrin formation with anticoagulant treatment or genetically eliminating the fibrinogen binding motif recognized by the microglial integrin receptor CD11b/CD18 inhibits perivascular microglial clustering and axonal damage. Thus, early and progressive perivascular microglial clustering triggered by fibrinogen leakage upon blood-brain barrier disruption contributes to axonal damage in neuroinflammatory disease.

Automated microscopy system for mosaic acquisition and processing.

An automatic mosaic acquisition and processing system for a multiphoton microscope is described for imaging large expanses of biological specimens at or near the resolution limit of light microscopy. In a mosaic, a larger image is created from a series of smaller images individually acquired systematically across a specimen. Mosaics allow wide‐field views of biological specimens to be acquired without sacrificing resolution, providing detailed views of biological specimens within context. The system is composed of a fast‐scanning, multiphoton, confocal microscope fitted with a motorized, high‐precision stage and custom‐developed software programs for automatic image acquisition, image normalization, image alignment and stitching. Our current capabilities allow us to acquire data sets comprised of thousands to tens of thousands of individual images per mosaic. The large number of individual images involved in creating a single mosaic necessitated software development to automate both the mosaic acquisition and processing steps. In this report, we describe the methods and challenges involved in the routine creation of very large scale mosaics from brain tissue labelled with multiple fluorescent probes.

Protein Kinase Cδ-mediated Phosphorylation of Connexin43 Gap Junction Channels Causes Movement within Gap Junctions followed by Vesicle Internalization and Protein Degradation

Background: Connexin43, a ubiquitous gap junction protein, is phosphorylated by protein kinase C on serine 368. Results: After PKCδ activation, phospho-Ser-368 Connexin43 channels segregated into the gap junction center and were subsequently internalized and degraded. Conclusion: PKCδ phosphorylation triggered internalization and degradation of Connexin43 channels without dephosphorylation. Significance: Differential phosphorylation events are used to sort and traffic Connexin43 channels within gap junctions and into the cytoplasm. Phosphorylation of gap junction proteins, connexins, plays a role in global signaling events involving kinases. Connexin43 (Cx43), a ubiquitous and important connexin, has several phosphorylation sites for specific kinases. We appended an imaging reporter tag for the activity of the δ isoform of protein kinase C (PKCδ) to the carboxyl terminus of Cx43. The FRET signal of this reporter is inversely related to the phosphorylation of serine 368 of Cx43. By activating PKC with the phorbol ester phorbol 12,13-dibutyrate (PDBu) or a natural stimulant, UTP, time lapse live cell imaging movies indicated phosphorylated Ser-368 Cx43 separated into discrete domains within gap junctions and was internalized in small vesicles, after which it was degraded by lysosomes and proteasomes. Mutation of Ser-368 to an Ala eliminated the response to PDBu and changes in phosphorylation of the reporter. A phosphatase inhibitor, calyculin A, does not change this pattern, indicating PKC phosphorylation causes degradation of Cx43 without dephosphorylation, which is in accordance with current hypotheses that cells control their intercellular communication by a fast and constant turnover of connexins, using phosphorylation as part of this mechanism.

High-resolution large-scale mosaic imaging using multiphoton microscopy to characterize transgenic mouse models of human neurological disorders

The thorough characterization of transgenic mouse models of human central nervous system diseases is a necessary step in realizing the full benefit of using animal models to investigate disease processes and potential therapeutics. Because of the labor- and resource-intensive nature of high-resolution imaging, detailed investigation of possible structural or biochemical alterations in brain sections has typically focused on specific regions of interest as determined by the researcher a priori. For example, Parkinsons disease researchers often focus imaging on regions of the brain expected to exhibit pathology such as the substantia nigra and striatum. Because of limitations in acquiring and storing high-resolution imaging data, additional data contained in the specimen is not usually acquired or disseminated/reported to the research community. Here we present a method of imaging large regions of brain at close to the resolution limit of light microscopy using a mosaic imaging technique in conjunction with multiphoton microscopy. These maps are being used to characterize several genetically modified animal models of neurological disease by filling the information “gap” among techniques such as magnetic resonance imaging and electron microscopic analysis.

Prototype of Kepler Processing Workflows For Microscopy And Neuroinformatics

We report on progress of employing the Kepler workflow engine to prototype “end-to-end” application integration workflows that concern data coming from microscopes deployed at the National Center for Microscopy Imaging Research (NCMIR). This system is built upon the mature code base of the Cell Centered Database (CCDB) and integrated rule-oriented data system (IRODS) for distributed storage. It provides integration with external projects such as the Whole Brain Catalog (WBC) and Neuroscience Information Framework (NIF), which benefit from NCMIR data. We also report on specific workflows which spawn from main workflows and perform data fusion and orchestration of Web services specific for the NIF project. This “Brain data flow” presents a user with categorized information about sources that have information on various brain regions.

Petabyte Data Management and Automated Data Workflow in Neuroscience: Delivering Data from the Instruments to the Researcher's Fingertips

The advent of new large area detectors and high-speed data acquisition technologies in light and electron microscopy are now producing data sets on the order of multiple terabytes. With the development of detectors such as the 8k x 8k lens-coupled CCD camera system developed at the National Center for Microscopy and Imaging Research (NCMIR), it is clear that it is no longer possible to store, view, and visualize these large data on a single desktop machine. With instrument automation, it is now possible to collect million by million pixel images with a data size of just over 2 terabytes. Additionally, with automated montaged tomograms up to 24k x 24k pixels per image, 120 tilts per rotation axis, and up to six rotation axis, we can produce just under 1 terabyte of raw data. The final computed volumes are approaching 10 terabytes and could exceed 100 terabytes for serial section tomography. With data this size, large memory, multi-node computation clusters must be used for processing, presentation, and visualization of this data. The results can then be displayed over the Internet, using dynamic binning to match the native screen resolution. With these large data sets, transfer of a single completed data set from the instrument where the data is acquired to networked storage units can bring the network and disk access to a standstill. With multiple users on various instruments and processing workstations all trying to access the network disks simultaneously, the technical challenges only multiply. It vital that these data be stored in databases and shared with the community as a whole, whether to use reuse the data for an alternate analysis or for educational purposes. We report on a new petabyte data management, workflow, and database system that allows us to overcome these challenges. This system is built upon the mature code base of the Cell Centered Database (CCDB), an integrated rule-oriented data system (IRODS) for distributed storage, scientific workflow engine “Kepler”, a set of advance hardware, newly developed code to manage the data and metadata from each microscope, and a NCMIR developed Web Image Browser (WIB). This system also utilizes service oriented architecture (SOA) to provide integration with external projects such as the Whole Brain Catalog (WBC), the International Neuroinformatics Coordinating Center (INCF), and Neuroscience Information Framework (NIF), which are benefiting from NCMIR data. A key objective for NCMIR has been to develop our current information technology infrastructure for managing distributed microscopy imaging data to serve the requirements of the mesoscale technological research and development and associated collaborative activities. Through the Cell Centered Database (CCDB), Telescience, and the Neuroscience Information Framework (NIF), NCMIR has developed a robust infrastructure for storing, sharing, searching, and disseminating microscopy image information via the CCDB and a secure web portal. CCDB has made available to the scientific community light and electron microscopic data sets contributed by NCMIR, NCMIR collaborators, and outside contributors. Over 50 data sets were released to the public in 2010, including live cell imaging, electron tomography, serial tomography, and correlated LM and EM. 276 doi:10.1017/S143192761100225X Microsc. Microanal. 17 (Suppl 2), 2011

Fibrin-targeting immunotherapy protects against neuroinflammation and neurodegeneration

Activation of innate immunity and deposition of blood-derived fibrin in the central nervous system (CNS) occur in autoimmune and neurodegenerative diseases, including multiple sclerosis (MS) and Alzheimer’s disease (AD). However, the mechanisms that link disruption of the blood–brain barrier (BBB) to neurodegeneration are poorly understood, and exploration of fibrin as a therapeutic target has been limited by its beneficial clotting functions. Here we report the generation of monoclonal antibody 5B8, targeted against the cryptic fibrin epitope γ377–395, to selectively inhibit fibrin-induced inflammation and oxidative stress without interfering with clotting. 5B8 suppressed fibrin-induced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and the expression of proinflammatory genes. In animal models of MS and AD, 5B8 entered the CNS and bound to parenchymal fibrin, and its therapeutic administration reduced the activation of innate immunity and neurodegeneration. Thus, fibrin-targeting immunotherapy inhibited autoimmunity- and amyloid-driven neurotoxicity and might have clinical benefit without globally suppressing innate immunity or interfering with coagulation in diverse neurological diseases.Fibrin deposition occurs after the blood–brain barrier is breached. Akassoglou and colleagues generate a therapeutic monoclonal antibody that targets a cryptic fibrin epitope to suppress activation of innate immune responses in the CNS and diminish neuroinflammation.

Quantification and Representation of Structural Heterogeneities in a Continuum Model of the Mouse Heart

With the availability of genetically engineered mice models of heart disease, there is a need for more structurally detailed computational models of murine ventricular anatomy suitable for predictive simulations of physiological function. In the heart, gap junctions (GJ) provide low resistance pathways, enabling coordinated propagation of action potential between cells. A decrease of GJ coupling causes a slowing of conduction, which is a key ingredient of reentrant arrhythmias [2]. In order to study the regional variations in structure and cell connectivity in detail, the purpose of the study was to measure and quantify the heterogeneities of GJ distribution and statistically represent them in a finite element (FE) model of the mouse heart.

Probability Map Viewer: near real-time probability map generator of serial block electron microscopy collections

Summary: To expedite the review of semi‐automated probability maps of organelles and other features from 3D electron microscopy data we have developed Probability Map Viewer, a Java‐based web application that enables the computation and visualization of probability map generation results in near real‐time as the data are being collected from the microscope. Probability Map Viewer allows the user to select one or more voxel classifiers, apply them on a sub‐region of an active collection, and visualize the results as overlays on the raw data via any web browser using a personal computer or mobile device. Thus, Probability Map Viewer accelerates and informs the image analysis workflow by providing a tool for experimenting with and optimizing dataset‐specific segmentation strategies during imaging. Availability and implementation: https://github.com/crbs/probabilitymapviewer. Contact: mellisman@ucsd.edu Supplementary information: Supplementary data are available at Bioinformatics online.