An increasing age is the greatest risk factor for dementia and related disorders. Therefore, much attention has been focus on researches to understand mechanisms of disease-related brain aging. Neurodegenerative diseases including Alzheimer’s disease (AD), dementia with lewy bodies, and frontotemporal lobar degeneration are mostly diagnosed by neuropathological features with protein inclusions such as Aβ, tau, α-synuclein, TDP-43, and FUS. These proteins are expected to lose physiological functions and mutual interaction with functional molecule with aging. Consecutively, acquired pathogenicities of aged proteins are accumulated and propagated in neural cells. The research for “Brain protein aging” is developed for understanding the mechanisms of initiation and pathogenicity of aging. Tau protein is one of major components of neurofibrillary tangles, which are closely associated with the severity of brain function loss of AD. To investigate tau protein’s Brain protein aging, we have currently developed the in vivo multimodal imaging techniques for visualizing the progression of tau pathology. In this review, we will introduce such a novel imaging-based diagnostic procedures on a mouse model of tauopathy.
Sepsis is the leading cause of death in critically ill patients, and its incidence continues to rise. Sepsis is now defined as life-threatening organ dysfunction due to a dysregulated host response to infection. Histamine assumes a critical role as a major mediator of many pathologic disorders with inflammation and immune reactions. However, direct evidence has not been provided showing the involvement of histamine in the development of multiple organ dysfunction or failure in sepsis. We have found that sepsis-induced major end-organ (lung, liver, and kidney) injury is attenuated in histidine decarboxylase (HDC) gene knockout mice. H1/H2-receptor gene-double knockout mice apparently behave similar to HDC knockout mice in reducing sepsis-related pathologic changes. Here we provide an overview on the role of endogenous histamine as an aggregating mediator that could contribute to the development of major end-organ injury in sepsis.
Histamine acts as a neurotransmitter to regulate various physiological functions in CNS. Recent reports showed the involvement of histaminergic dysfunction in neurological disorders. Neurotransmitter clearance is essential to determine brain neurotransmitter concentration. However, molecular mechanism of brain histamine clearance remains largely unknown. First, we examined the molecular mechanism of histamine clearance in primary human astrocytes. We demonstrated that extracellular histamine was transported through organic cation transporter (OCT) 3 and plasma membrane monoamine transporter (PMAT), and subsequently intracellular histamine was inactivated by histamine N-methyltransferase (HNMT) in cytosol. Next, we generated HNMT knockout (HNMT KO) mice to investigate the role of HNMT in vivo. HNMT deficiency dramatically enhanced brain histamine concentration, indicating the important role of HNMT in histamine inactivation. HNMT KO mice showed high aggression via abnormal histamine H2 receptor (H2R) activation and the disrupted sleep-wake cycle via excessive H1R activation. These observations show that HNMT plays a pivotal role in regulating brain histamine concentration, and modulates aggression as well as the sleep-wake cycle. Although importance of OCT3 and PMAT in histaminergic nervous system remains still unknown, our preliminary data show the contribution of PMAT to brain histamine concentration. We also try to find novel inhibitors targeting brain histamine clearance. We hope our study could lead a better understanding of neuropsychiatric disorders and the development of new drugs inhibiting HNMT, OCT3 and PMAT activity.
Inositol 1,4,5-trisphosphate (IP3) is an important intracellular messenger produced by phospholipase C via the activation of G-protein-coupled receptor- or receptor-tyrosine-kinase-mediated pathways, and is involved in numerous responses to hormones, neurotransmitters, and growth factors through the releases of Ca2+ from intracellular stores via IP3 receptors. IP3-mediated Ca2+ signals often exhibit complex spatial and temporal organizations, such as Ca2+ oscillations. Recently, new methods have become available to measure IP3 concentration ([IP3]) using AlphaScreen technology, fluorescence polarization, and competitive ligand binding assay (CFLA). These methods are useful for the high throughput screening in drug discovery. Calcium ions generate versatile intracellular signals such as Ca2+ oscillations and waves. Fluorescent sensors molecules to monitor changes in [IP3] in single living cells are crucial to study the mechanism for the spatially and temporally regulated Ca2+ signals. In particular, FRET-based IP3 sensors are useful for the quantitative monitoring intracellular [IP3], and allowed to uncovered the oscillatory IP3 dynamics in association with Ca2+ oscillations. A mathematical model of coupled Ca2+ and IP3 oscillations predicts that Ca2+ oscillations are the result of modulation of the IP3 receptor by intracellular Ca2+, and that the period is modulated by the accompanying IP3 oscillations. These model predictions have also been confirmed experimentally. At present, however, usefulness of FRET-based IP3 sensors are limited by their relatively small change in fluorescence. Development of novel IP3 sensors with improve dynamic range would be important for understanding the regulatory mechanism of Ca2+ signaling and for in vivo IP3 imaging.
Electrophysiological methods are commonly used in neuroscience and pharmacology to reveal the mechanisms of drug action. In vivo analysis of the mechanisms of drug action is a particularly important method in neuropharmacology. Here, we show the juxtacellular recording method to characterize the electrophysiological and neurochemical properties of neurons. Using juxtacellular recording, researchers can record the membrane potential from single neurons, and examine action potential parameters, such as the width and coefficient variance of inter-spike intervals. Additionally, recorded neurons can be labeled using neurobiotin, and neurochemical properties can be revealed by a combination of immunohistochemical staining and in situ hybridization. We introduce an experiment testing the effects of a phosphodiesterase 4 (PDE4) inhibitor on the fronto-striatal circuit using juxtacellular recording. The cerebral cortex-nucleus accumbens (NAcc)-external segment of globus pallidus (GPe)-subthalamic nucleus (STN)-substantia nigra pars reticulata (SNr) pathway is the neurobiological basis of many neuropsychiatric disorders. Several components of this pathway are particularly important for the regulation of motor action and cognitive function: 1) STN-SNr pathway (hyperdirect pathway), 2) NAcc-SNr pathway (direct pathway), and 3) GPe-STN-SNr pathway (indirect pathway). Researchers can record tri-phasic responses reflecting these pathways using electro-stimulation in cerebral cortex. A PDE4 inhibitor, roflumilast, affected the 2) direct pathway as well as the 3) indirect pathway, but not the 1) hyperdirect pathway. The current findings suggest that PDE4 inhibition could be considered as a possible treatment for cognitive deficits related to fronto-striatal disorders such as attention deficit/hyperactivity disorder, and Parkinson’s disease.
Three-dimensional (3D) cardiac myoblast tissues derived from iPS cells were constructed by cell coating technology with nanometer-sized extracellular matrix. Vascularized 3D-cardiac tissues were also fabricated by employing cardiac endothelial cells. These 3D-cardiac tissues are expected to apply to pharmaceutical assays.
Clostridium difficile (C. difficile), an enterobacteria, flourishes and produces potent toxins, toxin A (TcdA) and toxin B (TcdB), after the disruption of the normal colonic microbiota by antibiotic therapy. C. difficile infection (CDI) may induce life-threatening complications such as fulminant colitis through damage of the intestinal wall by the toxins, therefore the prevention of CDI recurrence is the most important in CDI treatment. Bezlotoxumab is a human monoclonal antibody that neutralizes the activity of TcdB directly. The antibody inhibited cytotoxicity by TcdB derived from various ribotypes of C. difficile at a concentration (EC50) of 1/150 or less of the serum concentration (Cmax: 169 μg/mL) in CDI patients at the clinical dose. Moreover the anti-cytotoxicity effects of the antibody were also observed against 81 clinically isolated C. difficile strains (incl. 018 [smz] and 369 [trf]: Japanese prevalent ribotypes; 027: hypervirulent ribotype) obtained in Japan and western countries. The antibody prolonged survival time of hamster and rat CDI models in a dose-dependent manner. In clinical phase III studies (MODIFY I and II), the recurrence rate of CDI up to 12 weeks after administration of the bezlotoxumab group was significantly lower (P<0.0001) than the placebo group. Bezlotoxumab is the world’s first drug with an indication for reduce recurrence of CDI. In Japan, bezlotoxumab was approved for marketing in September, and launched in December in 2017. Bezlotoxumab is effective for broad ribotypes of C. difficile, therefore it expects to contribute to CDI treatment through the reduce recurrence of the CDI.