Phenotypic Insights Into Anti-IgLON5 Disease in IgLON5-Deficient Mice.

BACKGROUND AND OBJECTIVES: Anti-IgLON5 disease is an autoimmune neurodegenerative disorder characterized by various phenotypes, notably sleep and movement disorders and tau pathology. Although the disease is known to be associated with the neuronal cell adhesion protein IgLON5, the physiologic function of IgLON5 remains elusive. There are conflicting views on whether autoantibodies cause loss of function, activation of IgLON5, or inflammation-associated neuronal damage, ultimately leading to the disease. We generated IgLON5 knockout (-/-) mice to investigate the functions of IgLON5 and elucidate the pathomechanism of anti-IgLON5 disease. METHODS: IgLON5 knockout (-/-) mice underwent behavioral tests investigating motor function, psychiatric function (notably anxiety and depression), social and exploratory behaviors, spatial learning and memory, and sensory perception. Histologic analysis was conducted to investigate tau aggregation in mice with tauopathy. RESULTS: IgLON5-/- mice had poorer performance in the wire hang and rotarod tests (which are tests for motor function) than wild-type mice. Moreover, IgLON5-/- mice exhibited decreased anxiety-like behavior and/or hyperactivity in behavior tests, including light/dark transition test and open field test. IgLON5-/- mice also exhibited poorer remote memory in the contextual fear conditioning test. However, neither sleeping disabilities assessed by EEG nor tau aggregation was detected in the knockout mice. DISCUSSION: These results suggest that IgLON5 is associated with activity, anxiety, motor ability, and contextual fear memory. Comparing the various phenotypes of anti-IgLON5 disease, anti-IgLON5 disease might partially be associated with loss of function of IgLON5; however, other phenotypes, such as sleep disorders and tau aggregation, can be caused by gain of function of IgLON5 and/or neuronal damage due to inflammation. Further studies are needed to elucidate the role of IgLON5 in the pathogenesis of anti-IgLON5 diseases.

Proteomic characterization of aging-driven changes in the mouse brain by co-expression network analysis.

Brain aging causes a progressive decline in functional capacity and is a strong risk factor for dementias such as Alzheimer's disease. To characterize age-related proteomic changes in the brain, we used quantitative proteomics to examine brain tissues, cortex and hippocampus, of mice at three age points (3, 15, and 24 months old), and quantified more than 7000 proteins in total with high reproducibility. We found that many of the proteins upregulated with age were extracellular proteins, such as extracellular matrix proteins and secreted proteins, associated with glial cells. On the other hand, many of the significantly downregulated proteins were associated with synapses, particularly postsynaptic density, specifically in the cortex but not in the hippocampus. Our datasets will be helpful as resources for understanding the molecular basis of brain aging.

A next-generation iPSC-derived forebrain organoid model of tauopathy with tau fibrils by AAV-mediated gene transfer.

It is known that the human cellular models of Alzheimer's disease (AD) and tauopathy can only recapitulate the very early stage of the disease. To overcome these limitations, we developed a technology to make forebrain organoids (FBOs) from feeder-free induced pluripotent stem cells (iPSC)s by regulating a FGF2 concentration and applied this method to generate FBOs from patients with familial AD (fAD FBOs). The obtained fAD FBOs recapitulated the amyloid-ß pathology and increased tau phosphorylation but not tau aggregates. To fully induce the tau pathology, FBOs were injected with adeno-associated virus (AAV)-expressing P301L mutant tau. In these Tau-P301L FBOs, tau fibrils were observed in the neuronal cell body and neurites with immunoelectron microscopy, in addition to the sarkosyl-insoluble and thioflavin S-positive phospho-tau aggregates. Collectively, this model can be used as a platform for investigating pathogenetic mechanisms and evaluation of target molecules for drug discovery for tauopathy.

Co-expression network analysis of human tau-transgenic mice reveals protein modules associated with tau-induced pathologies.

Abnormally accumulated tau protein aggregates are one of the hallmarks of neurodegenerative diseases, including Alzheimer's disease (AD). In order to investigate proteomic alteration driven by tau aggregates, we implemented quantitative proteomics to analyze disease model mice expressing human MAPT P301S transgene (hTau-Tg) and quantified more than 9,000 proteins in total. We applied the weighted gene co-expression analysis (WGCNA) algorithm to the datasets and explored protein co-expression modules that were associated with the accumulation of tau aggregates and were preserved in proteomes of AD brains. This led us to identify four modules with functions related to neuroinflammatory responses, mitochondrial energy production processes (including the tricarboxylic acid cycle and oxidative phosphorylation), cholesterol biosynthesis, and postsynaptic density. Furthermore, a phosphoproteomics study uncovered phosphorylation sites that were highly correlated with these modules. Our datasets represent resources for understanding the molecular basis of tau-induced neurodegeneration, including AD.

Lyn Kinase Suppresses the Transcriptional Activity of IRF5 in the TLR-MyD88 Pathway to Restrain the Development of Autoimmunity.

Interferon regulatory factor-5 (IRF5), a transcription factor critical for the induction of innate immune responses, contributes to the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE) in humans and mice. Lyn, a Src family kinase, is also implicated in human SLE, and Lyn-deficient mice develop an SLE-like disease. Here, we found that Lyn physically interacted with IRF5 to inhibit ubiquitination and phosphorylation of IRF5 in the TLR-MyD88 pathway, thereby suppressing the transcriptional activity of IRF5 in a manner independent of Lyn's kinase activity. Conversely, Lyn did not inhibit NF-κB signaling, another major branch downstream of MyD88. Monoallelic deletion of Irf5 alleviated the hyperproduction of cytokines in TLR-stimulated Lyn(-/-) dendritic cells and the development of SLE-like symptoms in Lyn(-/-) mice. Our results reveal a role for Lyn as a specific suppressor of the TLR-MyD88-IRF5 pathway and illustrate the importance of fine-tuning IRF5 activity for the maintenance of immune homeostasis.

Prediction of relaxin-3-induced downstream pathway resulting in anxiolytic-like behaviors in rats based on a microarray and peptidome analysis.

The effect of the intracerebroventricular (i.c.v.) injection of relaxin-3 (RLX3) was evaluated using anxiety-related behavioral tests in rats. RLX3-injected animals showed normal locomotion activity in a habituated environment and declined anxiety cognition in the elevated plus maze test and the shock probe-burying test. The measurement of spontaneous locomotor activity in a novel environment also suggested that RLX3 reduced the stress response. To elucidate the regulatory mechanisms of the downstream signaling pathways underlying RLX3 activity and its relation to anxiolytic and hyperphagic behavior phenotypes, RLX3-i.c.v.-injected rat hypothalamic responses were examined using a microarray analysis. Ingenuity Pathway Analysis software listed the phenotype-relating genes and they showed characteristic expression patterns in the rat hypothalamus. When peptidome data sets for the same listed genes was analyzed using a semi-quantitative approach, the expressions of two neuropeptides were found to have increased. One of these neuropeptides, oxytocin (Oxt), exhibited increased expression in both the microarray and the peptidomic analysis, and a Western blot analysis validated the mass spectrometry results. A cross-omics data analysis is useful for predicting downstream signaling pathways, and the anxiolytic-like behavior of RLX3 may be mediated by an oxytocin signaling pathway in rats. These results suggest that RLX3 acts as an anxiolytic peptide and that the downstream pathways mediated by its receptors may be potential candidates for the treatment of anxieties in the future.

Capture molecules preconditioned for kinetic analysis of high-affinity antigen-antibody complex in Biacore A100.

Surface plasmon resonance (SPR) is routinely applied on determining association or dissociation constant rates of antigen-antibody complexes. In a SPR system such as Biacore, the capture method is a widely accepted procedure in kinetic analysis for association or dissociation of soluble antigen analytes with antibody ligands initially captured by anti-Fc molecules immobilized on the sensor chip. Appropriate preparations of anti-immunoglobulin G (IgG)-Fc molecules on sensor chips have not been examined yet for stable kinetic analysis of antibodies with several affinities to soluble antigens. Here, we constructed murine monoclonal antibodies (MoAbs) with various affinities to hen egg lysozyme (HEL) and performed kinetic analysis of these MoAbs captured by rat MoAbs against mouse IgG-Fc immobilized on the sensor chip. When capture molecules maximally immobilized on the sensor chip, we observed no apparent dissociation of MoAbs with extremely high affinity to soluble HEL antigens. In contrast, on the limited amount (1000-2000 response units) of capture molecule immobilized on the sensor chip, we could perform stable kinetic analysis of MoAbs with highest affinities to the antigen as well as those with lower or moderate binding affinities. Thus, in some cases, accurate kinetic analysis of high-affinity antibodies can be performed by minimization of capture molecule densities on the sensor chip in SPR.

N-type calcium channel in the pathogenesis of experimental autoimmune encephalomyelitis.

One of the family of voltage-gated calcium channels (VGCC), the N-type Ca(2+) channel, is located predominantly in neurons and is associated with a variety of neuronal responses, including neurodegeneration. A precise mechanism for how the N-type Ca(2+) channel plays a role in neurodegenerative disease, however, is unknown. In this study, we immunized N-type Ca(2+) channel α(1B)-deficient (α(1B)(-/-)) mice and their wild type (WT) littermates with myelin oligodendrocyte glycoprotein 35-55 and analyzed the progression of experimental autoimmune encephalomyelitis (EAE). The neurological symptoms of EAE in the α(1B)(-/-) mice were less severe than in the WT mice. In conjunction with these results, sections of the spinal cord (SC) from α(1B)(-/-) mice revealed a reduction in both leukocytic infiltration and demyelination compared with WT mice. No differences were observed in the delayed-type hypersensitivity response, spleen cell proliferation, or cytokine production from splenocytes between the two genotypes. On the other hand, Western blot array analysis and RT-PCR revealed that a typical increase in the expression of MCP-1 in the SC showed a good correlation with the infiltration of leukocytes into the SC. Likewise, immunohistochemical analysis showed that the predominant source of MCP-1 was activated microglia. The cytokine-induced production of MCP-1 in primary cultured microglia from WT mice was significantly higher than that from α(1B)(-/-) mice and was significantly inhibited by a selective N-type Ca(2+) channel antagonist, ω-conotoxin GVIA or a withdrawal of extracellular Ca(2+). These results suggest that the N-type Ca(2+) channel is involved in the pathogenesis of EAE at least in part by regulating MCP-1 production by microglia.

MPR1 as a novel selection marker in Saccharomyces cerevisiae.

L-Azetidine-2-carboxylic acid (AZC) is a toxic four-membered ring analogue of L-proline that is transported into cells by proline transporters. AZC and L-proline in the cells are competitively incorporated into nascent proteins. When AZC is present in a minimum medium, misfolded proteins are synthesized in the cells, thereby inhibiting cell growth. The MPR1 gene has been isolated from the budding yeast Saccharomyces cerevisiae Sigma1278b as a multicopy suppressor of AZC-induced growth inhibition. MPR1 encodes a novel acetyltransferase that detoxifies AZC via N-acetylation. Since MPR1 is absent in the laboratory strain of S. cerevisiae S288C, it could be a positive selection marker that confers AZC resistance in the S288C background strains. To examine the usefulness of MPR1, we constructed some plasmid vectors that harboured MPR1 under the control of various promoters and introduced them into the S288C-derived strains. The expression of MPR1 conferred AZC resistance that was largely dependent on the expression level of MPR1. In an additional experiment, the galactose-inducible MPR1 and ppr1(+), the fission yeast Schizosaccharomyces pombe homologue of MPR1, were used for gene disruption by homologous recombination, and here AZC-resistant colonies were also successfully selected. We concluded that our MPR1-AZC system provides a powerful tool for yeast transformation.

The utilization of gene targeting models during in preclinical study of drug discovery process--example of phenotypic and functional analysis of Cacna1beta gene product.

Using gene knockout mice of particular genes is one of the most effective methods in conducting successful study on the mode of action of target gene products in targeted organs. So called the knockout technology is now a powerful tool that can lead us to find clear understanding on difficult questions such as the effects of full antagonist against target molecules. Cacna1b (alpha(1B)) gene knockout mouse was generated to study mechanisms of N-type calcium (Ca(2+)) channel. The model was able to overcome physiological obstacles in studies of N-type Ca(2+) channel selective blockers, such as unspecific binding to structurally similar molecules, and failed distribution to targeted organs. In the case of N-type Ca(2+) channel studies, knockout technology was successfully applied to various cardiovascular, sympathetic, nociceptive, sleep-awake cycles, metabolic and neurodegenerative experiments using homozygous mutants of the alpha(1B) gene that turned out to be viable. These studies were able to confirm not only the predicted phenotypes, but were able to present completely unexpected phenotypes that are great interest for future study. Thus the outputs from the knockout mouse studies lead to gain the proof of concept as a drug for specific inhibitors of the gene products and enabled us to make further prediction of side-effects of these inhibitors in the drug discovery and development process.

Evaluation of comprehensive multidimensional separations using reversed-phase, reversed-phase liquid chromatography/mass spectrometry for shotgun proteomics.

Two-dimensional liquid-chromatographic (LC) separation followed by mass spectrometric (MS) analysis was examined for the identification of peptides in complex mixtures as an alternative to widely used two-dimensional gel electrophoresis followed by MS analysis for use in proteomics. The present method involves the off-line coupling of a narrow-bore, polymer-based, reversed-phase column using an acetonitrile gradient in an alkaline mobile phase in the first dimension with octadecylsilanized silica (ODS)-based nano-LC/MS in the second dimension. After the first separation, successive fractions were acidified and dried off-line, then loaded on the second dimension column. Both columns separate peptides according to hydrophobicity under different pH conditions, but more peptides were identified than with the conventional technique for shotgun proteomics, that is, the combination of a strong cation exchange column with an ODS column, and the system was robust because no salts were included in the mobile phases. The suitability of the method for proteomics measurements was evaluated.

Pseudo internal standard approach for label-free quantitative proteomics.

Quantitative proteome analysis has become a versatile tool to understand biological functions. Although stable isotope labeling is the most reliable method for quantitative mass spectrometry, preparation of isotope-labeled compounds is time-consuming and expensive. Simple label-free approaches have been introduced, but intensity-based quantitation without standards is not generally accepted as reliable, especially for small molecules. We have developed a novel label-free quantitative proteome analysis using pseudo internal standards (PISs). This idea was derived from northern blotting analysis, in which housekeeping genes are used as standards to normalize and compare target gene expression levels in different samples. In many proteomics studies, most proteins do not change their expression levels under different conditions, and therefore, these proteins can be employed as pseudo internal standards. This new approach is simple and does not require additional standards or labeling reagents. The PIS method represents a novel approach for mass spectrometry-based comprehensive quantitatitation and may also be applicable to quantitative metabolome analysis.

Enhancement of the efficiency of phosphoproteomic identification by removing phosphates after phosphopeptide enrichment.

Immobilized metal affinity chromatography (IMAC) and titanium oxide (TiO2) chromatography are simple, widely used, and cost-effective methods to enrich phosphopeptides, but the sample loading buffer composition, desalting procedure, and control of loading amount are critical to avoid nonspecific interactions and to achieve efficient phosphopeptide enrichment. Although the combination of MS3 analysis and high-resolution mass spectrometry (MS) is helpful to identify phosphopeptides, the quality of many MS/MS spectra having a neutral loss peak of phosphate is still too poor to allow sequence identification, and this results in many false-negative as well as false-positive identifications. Here, we present a novel strategy, which is based on the use of alkaline phosphatase to remove phosphates and analysis of phospho/dephosphopeptide retention times to increase the reliability of identification. The use of phospho/dephosphopeptide retention time ratios allows the identification of phosphopeptides with high confidence with the aid of a focused database of dephosphopeptides. This approach was very effective to identify multiple phophorylations in tryptic peptides. A 'true' phosphorylation data set should contain about 90% phospho-Ser and a few percent phospho-Tyr, and this ratio can be used as a quality criterion for evaluation of data sets. By applying this efficient approach, we were able to identify more than one thousand phosphopeptides.

Characterization of mRNA species that are associated with postsynaptic density fraction by gene chip microarray analysis.

We previously reported the partial identification by random sequencing of mRNA species that are associated with the postsynaptic density (PSD) fraction prepared from the rat forebrain [Tian et al., 1999. Mol. Brain Res. 72, 147-157]. We report here further characterization by gene chip analysis of the PSD fraction-associated mRNAs, which were prepared in the presence of RNase inhibitor. We found that mRNAs encoding various postsynaptic proteins, such as channels, receptors for neurotransmitters and neuromodulators, proteins involved in signaling, scaffold and adaptor proteins and cytoskeletal proteins, were highly concentrated in the PSD fraction, whereas those encoding housekeeping proteins, such as enzymes in the glycolytic pathway, were not. We extracted approximately 1900 mRNA species that were highly concentrated in the PSD fraction. mRNAs related to certain neuronal diseases were also enriched in the PSD fraction. We also constructed a cDNA library using the PSD fraction-associated mRNAs as templates, and identified 1152 randomly selected clones by sequencing. Our data suggested that the PSD fraction-associated mRNAs are a very useful resource, in which a number of as yet uncharacterized mRNAs are concentrated. Identification and functional characterization of them are essential for complete understanding of synaptic function.

Multiplexed two-dimensional liquid chromatography for MALDI and nanoelectrospray ionization mass spectrometry in proteomics.

We developed a multiplexed two-dimensional separation system based on reversed phase (RP)--strong cation exchange (SCX) chromatography as a front-end device for matrix-assisted laser desorption ionization (MALDI) or nanoelectrospray ionization (nanoESI) mass spectrometry. Tryptic peptide mixtures were fractionated on a reversed-phase HPLC column, and each fraction was loaded onto multiplexed SCX microcolumns. Because this second chromatography was carried out in parallel, the analysis time is independent of the fraction number in the first RP-HPLC separation. The resultant samples were desalted/concentrated and eluted onto a MALDI plate with matrix-containing elution solutions in parallel, or eluted with optimized solutions for nanoESI and loaded onto nanoESI sprayers by an automated instrument. The soluble portion of HCT116 lysate was digested and fractionated using a 48-plexed chromatography system. Approximately 1000 unique peaks were detected in MALDI-MS with 3000 MS/MS spectra, while 724 peptides with ultrahigh peptide mass accuracy (sub-ppm error) were identified in nanoESI-FTICR mass spectrometry with five integrated selected ion monitoring scans. Since MS measurement with this off-line LC-LC approach is not restricted by continuous LC elution, it is expected to be useful especially in cases where repeated analysis with different scan modes or long-term data acquisition is required.

Chronic intracerebroventricular administration of relaxin-3 increases body weight in rats.

Bolus-administered intracerebroventricular (ICV) relaxin-3 has been reported to increase feeding. In this study, to examine the role of relaxin-3 signaling in energy homeostasis, we studied the effects of chronically administered ICV relaxin-3 on body weight gain and locomotor activity in rats. Two groups of animals received vehicle or relaxin-3 at 600 pmol/head/day, delivered with Alzet osmotic minipumps. In animals receiving relaxin-3, food consumption and weight gain were statistically significantly higher than those in the vehicle group during the 14-day infusion. During the light phase on days 2 and 7 and the dark phase on days 3 and 8, there was no difference in locomotor activity between the two groups. Plasma concentrations of leptin and insulin in rats chronically injected with relaxin-3 were significantly higher than in the vehicle-injected controls. These results indicate that relaxin-3 up-regulates food intake, leading to an increase of body weight and that relaxin-3 antagonists might be candidate antiobesity agents.

Altered nociceptive response in ADAM11-deficient mice.

Mice that lack A Disintegrin And Metalloprotease 11 (ADAM11) protein showed normal responses to stimuli in the von Frey test and the hot plate test, but showed reduced responses in the formalin paw test and acetic acid writhing test. Our results indicate that the cell adhesion-related molecule ADAM11 may play a role in pain transmission and in inflammatory regulation mechanisms underlying changes in the threshold for pain perception.

Deficits in spatial learning and motor coordination in ADAM11-deficient mice.

BACKGROUND: ADAM11 is a member of the ADAM gene family and is mainly expressed in the nervous system. It is thought to be an adhesion molecule, since it has a disintegrin-like domain related to cell-cell or cell-matrix interactions. To elucidate the physiological functions of ADAM11, we generated ADAM11-deficient mice by means of gene targeting. RESULTS: ADAM11-deficient mice were apparently normal, and survived more than one year with no major histological abnormalities in the brain or spinal cord. Because ADAM11 is highly expressed in the hippocampus and cerebellum, we have examined ADAM11 mutant mice for learning using visual and hidden water maze tasks, and their motor coordination using a rotating rod task. Our results showed that their visual water maze task results are normal, but the hidden water maze and rotating rod task skills are impaired in ADAM11-deficient mice. CONCLUSION: Our results indicate that ADAM11 mutation does not affect cell migration and differentiation during development, but affects learning and motor coordination. Thus, ADAM11 might play an important signalling or structural role as a cell adhesion molecule at the synapse, and may thus participate in synaptic regulation underlying behavioural changes.

Quantitative chemical proteomics for identifying candidate drug targets.

We have developed a systematic strategy for drug target identification. This consists of the following sequential steps: (1) enrichment of total binding proteins using two differential affinity matrixes upon which are immobilized positive and negative chemical structures for drug activity, respectively; (2) covalent labeling of the proteins with a new cleavable isotope-coded affinity tag (ICAT) reagent, followed by proteolysis of the combined proteins; (3) isolation, identification, and relative quantification of the tagged peptides by liquid chromatography-mass spectrometry; (4) array-based transcription profiling to select candidate proteins; and (5) confirmation of direct interaction between the activity-associated structure and the selected proteins by using surface plasmon resonance. We present a typical application to identify the primary binding protein of a novel class of anticancer agents exemplified by E7070. Our results suggest that this approach provides a new aspect of quantitative proteomics to find specific binding proteins from protein mixture and should be applicable to a wide variety of biologically active small molecules with unidentified target proteins.

Medicinal genetics approach towards identifying the molecular target of a novel inhibitor of fungal cell wall assembly.

Glycosylphosphatidylinositol (GPI)-anchored cell wall mannoproteins are required for the adhesion of pathogenic fungi, such as Candida albicans, to human epithelium. Small molecular inhibitors of the cell surface presentation of GPI-anchored mannoproteins would be promising candidate drugs to block the establishment of fungal infections. Here, we describe a medicinal genetics approach to identifying the gene encoding a novel target protein that is required for the localization of GPI-anchored cell wall mannoproteins. By means of a yeast cell-based screening procedure, we discovered a compound, 1-[4-butylbenzyl]isoquinoline (BIQ), that inhibits cell wall localization of GPI-anchored mannoproteins in Saccharomyces cerevisiae. Treatment of C. albicans cells with this compound resulted in reduced adherence to a rat intestine epithelial cell monolayer. A previously uncharacterized gene YJL091c, named GWT1, was cloned as a dosage-dependent suppressor of the BIQ-induced phenotypes. GWT1 knock-out cells showed similar phenotypes to BIQ-treated wild-type cells in terms of cell wall structure and transcriptional profiles. Two different mutants resistant to BIQ each contained a single missense mutation in the coding region of the GWT1 gene. These results all suggest that the GWT1 gene product is the primary target of the compound.