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Graphical abstract, Highlights • Brain response to systemic inflammation is initiated by IL-1β from choroid plexus macrophages. • Choroid plexus stromal cells bear IL-1 receptors and participate in the immediate reaction to systemic inflammation. • This reaction is followed by elevated gene expression of various cytokines in the choroid plexus stroma and epithelium. • The choroid plexus immediate responses are relevant to understanding how sepsis-associated encephalopathy is initiated., Sepsis-associated encephalopathy (SAE) is characterized as diffuse brain dysfunction in patients with excessive systemic inflammatory reaction to an infection. In our previous studies using a mouse model of SAE with intraperitoneal injection of lipopolysaccharide (LPS), tissue concentrations of various cytokines were elevated in the entire brain parenchyma 4 and 24 h following LPS administration. Cytokines elevated at 4 h were produced by the choroid plexus, leptomeninges and vascular endothelium, while those at 24 h were produced by astrocytes. Interleukin (IL)-1β did not increase in the concentration in the brain parenchyma during the period from 1 to 24 h following LPS. In the present study, we hypothesized that the intracranial cells that initially respond to systemic inflammation are situated in the choroid plexus and produce IL-1β to initiate cytokine-mediated reactions. We quantified the transcript levels of related cytokines within the choroid plexus and specified the choroid plexus cells that are involved in the immediate cytokine-mediated responses. Mice received LPS or saline by intraperitoneal injection. Four hours after treatments, the choroid plexuses were isolated and subjected to cytokine gene expression analyses using real-time reverse transcription-polymerase chain reaction. Another group of mice was fixed at 1, 4 and 24 h after treatments and the expression of cytokines and receptors was studied with double immunohistofluorescence staining. The transcript levels of IL-1β, CC-motif ligand (CCL)2, CXC-motif ligand (CXCL)1, CXCL2 and IL-6 in the choroid plexus were significantly increased in mice treated with LPS compared to saline control. The IL-1β expression was remarkable in choroid plexus macrophages at 1 and 4 h but not in the brain parenchyma. Choroid plexus stromal cells expressed IL-1 receptor type 1 (IL-1R1). The IL-1R1-bearing stromal cells produced CCL2, CXCL1, CXCL2 and IL-6 at 4 h. Choroid plexus epithelial cells expressed CXCR2, a common receptor for CXCL1 and CXCL2. Choroid plexus epithelial cells also expressed CCL2, CXCL1 and CXCL2 at 4 h, and IL-1R1-bearing stromal cells expressed CXCR2. Therefore, in response to systemic LPS injection, one of the intracranial reactions was initiated within the choroid plexus using IL-1β derived from macrophages. The choroid plexus stromal cells subsequently had elevated expression of CCL2, CXCL1, CXCL2 and IL-6. The choroid plexus epithelial cells also had elevated expression of CCL2, CXCL1 and CXCL2. The presence of receptors for these cytokines on both epithelial and stromal cells raised the possibility of reciprocal interactions between these cells. The results suggested that the immediate early responses exerted by the choroid plexus are relevant to understanding how SAE is initiated in clinical settings.

The Psychoneuroimmunology Research Society (PNIRS) created an official Chinese regional affiliate in 2012, designated PNIRSChina. Now, just eight years later, the program has been so successful in advancing the science of psychoneuroimmunology that it has expanded to the whole of Asia-Oceania. In 2017, PNIRSChina became PNIRSAsia-Pacific. Between 2012 and 2019, this outreach affiliate of PNIRS organized seven symposia at major scientific meetings in China as well as nine others in Taiwan, Japan, South Korea, Australia and New Zealand. This paper summarizes the remarkable growth of PNIRSAsia-Pacific. Here, regional experts who have been instrumental in organizing these PNIRSAsia-Pacific symposia briefly review and share their views about the past, present and future state of psychoneuroimmunology research in China, Taiwan, Australia and Japan. The newest initiative of PNIRSAsia-Pacific is connecting Asia-Pacific laboratories with those in Western countries through a simple web-based registration system. These efforts not only contribute to the efforts of PNIRS to serve a truly global scientific society but also to answer the imperative call of increasing diversity in our science.

Chronic nasal inflammation induces robust olfactory bulb (OB) atrophy in mice. Here we examined initial events that occur in the OB after bilateral intranasal administration of lipopolysaccharide, focusing on the olfactory nerve fibers and meninges. We analyzed the time course of OB and meninges inflammation using histological and biochemical approaches. Within 12 h, we observed increased chemokine expression and transient infiltration of peripheral immune cells into the OB, resulting in the development of pro-inflammatory status in the OB. Meningeal immunity was activated. Resident microglia produced anti-inflammatory cytokines within 24 h. These could be the initial events that lead to OB atrophy.

Models de ratolí; Glucòlisi; Diabetis mellitus Modelos de ratón; Glucólisis; Diabetes mellitus Mouse models; Glycolysis; Diabetes mellitus Glycolytic metabolism is closely involved in physiological homeostasis and pathophysiological states. Among glycolytic enzymes, phosphoglycerate mutase (PGAM) has been reported to exert certain physiological role in vitro, whereas its impact on glucose metabolism in vivo remains unclear. Here, we report the characterization of Pgam1 knockout mice. We observed that homozygous knockout mice of Pgam1 were embryonic lethal. Although we previously reported that both PGAM-1 and -2 affect global glycolytic profile of cancers in vitro, in vivo glucose parameters were less affected both in the heterozygous knockout of Pgam1 and in Pgam2 transgenic mice. Thus, the impact of PGAM on in vivo glucose metabolism is rather complex than expected before. This work was supported in part by grants from the Japan Society for the Promotion of Science (Grants No. 26310103 to HK and No. 15K19283 to HK), and by the Japan Agency for Medical Research and Development (AMED), Core Research for Evolutional Science and Technology (CREST JP17gm0610002h0306 to HK). HK; Hiroshi Kondoh.

A growing body of evidence suggests a relationship between olfactory dysfunction and the pathogenesis of mental disorders. Our previous studies indicated that chronic nasal inflammation caused loss of olfactory sensory neurons and gross atrophy of the olfactory bulb, which may lead to olfactory dysfunction. Simultaneously, increasing numbers of reports have elucidated the importance of gut microbiota to maintain brain function and that dysbiosis may be associated with neuropsychiatric disorders. Here we examined whether chronic nasal inflammation perturbed gut microbiota and whether there were sex differences in this pattern. Eight-week-old C57BL/6 mice repeatedly received bilateral nasal administration of lipopolysaccharide (LPS) 3 times/week to cause chronic nasal inflammation or saline as a control. At 9 weeks, cecal feces were used for 16S metagenomic analysis and whole blood and fresh tissue of spleen were used for ELISA analyses. Microbiome analysis demonstrated a remarkable change of the gut microbiota in male mice with chronic nasal inflammation which was different from that in female mice. In both mice, systemic inflammation did not occur. This has shown for the first time that chronic nasal inflammation correlates with sex-dependent changes in the gut microbiota. The detailed mechanism and potential alteration to brain functions await further studies.

By comprehensively measuring changes in metabolites in the hippocampus of stress-loaded mice, we investigated the reasons for stress vulnerability and the effect of theanine, i.e., an abundant amino acid in tea leaves, on the metabolism. Stress sensitivity was higher in senescence-accelerated mouse prone 10 (SAMP10) mice than in normal ddY mice when these mice were loaded with stress on the basis of territorial consciousness in males. Group housing was used as the low-stress condition reference. Among the statistically altered metabolites, depression-related kynurenine and excitability-related histamine were significantly higher in SAMP10 mice than in ddY mice. In contrast, carnosine, which has antidepressant-like activity, and ornithine, which has antistress effects, were significantly lower in SAMP10 mice than in ddY mice. The ingestion of theanine, an excellent antistress amino acid, modulated the levels of kynurenine, histamine, and carnosine only in the stress-loaded SAMP10 mice and not in the group-housing mice. Depression-like behavior was suppressed in mice that had ingested theanine only under stress loading. Taken together, changes in these metabolites, such as kynurenine, histamine, carnosine, and ornithine, were suggested to be associated with the stress vulnerability and depression-like behavior of stressed SAMP10 mice. It was also shown that theanine action appears in the metabolism of mice only under stress loading.

Senescence-accelerated mouse prone 10 (SAMP10) exhibits cerebral atrophy and depression-like behavior. A line of SAMP10 with spontaneous mutation in the Slc5a2 gene encoding the sodium-glucose cotransporter (SGLT) 2 was named SAMP10/TaSlc-Slc5a2slc (SAMP10-&Delta, Sglt2) and was identified as a renal diabetes model. In contrast, a line of SAMP10 with no mutation in SGLT2 (SAMP10/TaIdrSlc, SAMP10(+)) was recently established under a specific pathogen-free condition. Here, we examined the mutation effect in SGLT2 on brain function and longevity. No differences were found in the survival curve, depression-like behavior, and age-related brain atrophy between SAMP10-&Delta, Sglt2 and SAMP10(+). However, memory retention was lower in SAMP10-&Delta, Sglt2 mice than SAMP10(+). Amyloid beta (A4) precursor-like protein 1 (Aplp1) expression was significantly lower in the hippocampus of SAMP10-&Delta, SGLT2 than in SAMP10(+) at 2 months of age, but was similar at 12 months of age. CaM kinase-like vesicle association (Camkv) expression was remarkably lower in SAMP10(+). These genes have been reported to be involved in dendrite function. Amyloid precursor proteins have been reported to involve in maintaining homeostasis of glucose and insulin. These results suggest that mutation in SGLT2 results in down-regulation of Aplp1 in young age, which can lead to poor memory retention in old age.

Chronic stress can impair the health of human brains. An important strategy that may prevent the accumulation of stress may be the consumption of functional foods. When senescence-accelerated mice prone 10 (SAMP10), a stress-sensitive strain, were loaded with stress using imposed male mouse territoriality, brain volume decreased. However, in mice that ingested theanine (6 mg/kg), the main amino acid in tea leaves, brain atrophy was suppressed, even under stress. On the other hand, brain atrophy was not clearly observed in a mouse strain that aged normally (Slc:ddY). The expression level of the transcription factor Npas4 (neuronal PAS domain protein 4), which regulates the formation and maintenance of inhibitory synapses in response to excitatory synaptic activity, decreased in the hippocampus and prefrontal cortex of stressed SAMP10 mice, but increased in mice that ingested theanine. Lipocalin 2 (Lcn2), the expression of which increased in response to stress, was significantly high in the hippocampus and prefrontal cortex of stressed SAMP10 mice, but not in mice that ingested theanine. These data suggest that Npas4 and Lcn2 are involved in the brain atrophy and stress vulnerability of SAMP10 mice, which are prevented by the consumption of theanine, causing changes in the expression of these genes.

The olfactory mucosa (OM) is exposed to environmental agents and therefore vulnerable to inflammation. To examine the effects of environmental toxin-initiated OM inflammation on the olfactory bulb (OB), we induced persistent rhinitis in mice and analyzed the spatial and temporal patterns of histopathological changes in the OM and OB. Mice received unilateral intranasal administration of lipopolysaccharide (LPS) or saline three times per week, and were immunohistologically analyzed at 1, 3, 7, 14 and 21 days after the first administration. LPS administration induced an inflammatory response in the OM, including the infiltration of Ly-6G-, CD11b-, Iba-1- and CD3-positive cells, the production of interleukin-1β by CD11b- and Iba-1-positive cells, and loss of olfactory sensory neurons (OSNs). In the OB, we observed activation of microglia and astrocytes and decreased expression of tyrosine hydroxylase in periglomerular cells, vesicular glutamate transporter 1, a presynaptic protein, in mitral and tufted projection neurons, and 5T4 in granule cells. Thus, the OM inflammation exerted a detrimental effect, not only on OSNs, but also on OB neurons, which might lead to neurodegeneration.

The c-RET proto-oncogene encodes a receptor-tyrosine kinase. Loss-of-function mutations of RET have been shown to be associated with Hirschsprung disease and Down's syndrome (HSCR-DS) in humans. DS is known to involve cerebellar hypoplasia, which is characterized by reduced cerebellar size. Despite the fact that c-Ret has been shown to be associated with HSCR-DS in humans and to be expressed in Purkinje cells (PCs) in experimental animals, there is limited information about the role of activity of c-Ret/c-RET kinase in cerebellar hypoplasia. We found that a loss-of-function mutation of c-Ret Y1062 in PCs causes cerebellar hypoplasia in c-Ret mutant mice. Wild-type mice had increased phosphorylation of c-Ret in PCs during postnatal development, while c-Ret mutant mice had postnatal hypoplasia of the cerebellum with immature neurite outgrowth in PCs and granule cells (GCs). c-Ret mutant mice also showed decreased numbers of glial fibers and mitogenic sonic hedgehog (Shh)-positive vesicles in the external germinal layer of PCs. c-Ret-mediated cerebellar hypoplasia was rescued by subcutaneous injection of a smoothened agonist (SAG) as well as by reduced expression of Patched1, a negative regulator for Shh. Our results suggest that the loss-of-function mutation of c-Ret Y1062 results in the development of cerebellar hypoplasia via impairment of the Shh-mediated development of GCs and glial fibers in mice with HSCR-DS.

Background Rhinitis and rhinosinusitis are olfactory disorders caused by inflammation of the nasal passage and paranasal sinuses. Although patients with chronic rhinosinusitis have smaller olfactory bulbs (OBs), there is limited knowledge regarding the influence of chronic nasal inflammation on OB neurons. Objective Repeated intranasal administration of LPS that induced persistent nasal inflammation in mice caused a loss of olfactory sensory neurons (OSNs) and gliosis and synaptic loss in the OBs within 3 weeks. The present study aimed to clarify the effects of long-term LPS treatment on the OB neurocircuit. Methods LPS was repeatedly administered into a mouse nostril for up to 24 weeks. For the recovery analyses, the mice received LPS for 10 weeks and were subsequently maintained without additional treatment for another 10 weeks. The effects of these treatments on the OBs were examined histologically. Three or more mice were analyzed per group. Results Long-term repeated LPS administration caused OB atrophy, particularly in the layers along which OSN axons travel and in the superficial external plexiform layer, in which tufted cells form synapses with interneurons. Interestingly, the OBs recovered from atrophy after cessation of LPS administration: OB volume and superficial external plexiform layer thickness returned to pretreatment levels after the nontreatment period. In contrast, OSN regeneration was incomplete. Conclusion These results suggest that chronic nasal inflammation induces structural changes in a specific OB circuit related to tufted cells, whereas tufted cells retain a high degree of plasticity that enables recovery from structural damages after inflammation subsides.

Although the brain is now known to actively interact with the immune system under non-inflammatory conditions, the site of cell–cell interactions between brain parenchymal cells and immune cells has been an open question until recently. Studies by our and other groups have indicated that brain structures such as the leptomeninges, choroid plexus stroma and epithelium, attachments of choroid plexus, vascular endothelial cells, cells of the perivascular space, circumventricular organs, and astrocytic endfeet construct the histological architecture that provides a location for intercellular interactions between bone marrow-derived myeloid lineage cells and brain parenchymal cells under non-inflammatory conditions. This architecture also functions as the interface between the brain and the immune system, through which systemic inflammation-induced molecular events can be relayed to the brain parenchyma at early stages of systemic inflammation during which the blood–brain barrier is relatively preserved. Although brain microglia are well known to be activated by systemic inflammation, the mechanism by which systemic inflammatory challenge and microglial activation are connected has not been well documented. Perturbed brain–immune interaction underlies a wide variety of neurological and psychiatric disorders including ischemic brain injury, status epilepticus, repeated social defeat, and neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease. Proinflammatory status associated with cytokine imbalance is involved in autism spectrum disorders, schizophrenia, and depression. In this article, we propose a mechanism connecting systemic inflammation, brain–immune interface cells, and brain parenchymal cells and discuss the relevance of basic studies of the mechanism to neurological disorders with a special emphasis on sepsis-associated encephalopathy and preterm brain injury.

Systemic inflammation shifts the brain microenvironment towards a proinflammatory state. However, how peripheral inflammation mediates changes in the brain remains to be clarified. We aimed to identify hippocampal cells and cytokines that respond to endotoxemia. Mice were intraperitoneally injected with lipopolysaccharide (LPS) or saline, and examined 1, 4, and 24 h after injection. Tissue cytokine concentrations in the spleens and hippocampi were determined by multiplex assays. Another group of mice were studied immunohistologically. Fourteen cytokines showed an increased concentration in the spleen, and 10 showed an increase in the hippocampus after LPS injection. Cytokines increased at 4 h (CCL2, CXCL1, CXCL2, and interleukin-6) were expressed by leptomeningeal stromal cells, choroid plexus stromal cells, choroid plexus epithelial cells, and hippocampal vascular endothelial cells, all of which were located at the brain–immune interface. Receptors for these cytokines were expressed by astrocytic endfeet. Cytokines increased at 24 h (CCL11, CXCL10, and granulocyte-colony stimulating factor) were expressed by astrocytes. Cells of the brain–immune interface therefore respond to endotoxemia with cytokine signals earlier than hippocampal parenchymal cells. In the parenchyma, astrocytes play a key role in responding to signals by using endfeet located in close apposition to the interface cells via cytokine receptors.

The senescence-accelerated mouse is a model for senescence acceleration, a higher oxidative stress status, and age-associated disorders. We studied whether fibroblasts cultured from accelerated senescence-prone SAMP11 mice could be used as in vitro models for oxidative stress in senescence. Dichlorofluorescein and hydroethidine assays demonstrated that cells from SAMP11 mice produced more reactive oxygen species than did cells from accelerated senescence-resistant SAMR1 mice. These differences were not due to the defective induction of antioxidants. Double labeling with hydroethidine and MitoTracker Green revealed that most of the reactive oxygen species were generated within the mitochondria. Nonyl acridine orange and JC-1 assays showed an increase in the mass of the mitochondria, especially those with low membrane potential, in SAMP11 cells. Ultrastructurally, mitochondria with degenerative morphology were increased in SAMP11 cells with longer culture periods. These results suggest that cells from SAMP11 mice are useful models for spontaneous higher oxidative stress in vitro due to dysfunctional mitochondria.

Experimental allergic encephalomyelitis (EAE) serves as a model for multiple sclerosis and is considered to be a CD4+ Th1 cell–mediated autoimmune disease. To investigate the role of platelet-activating factor (PAF) in this disease, PAF receptor (PAFR) KO (PAFR-KO) and wild-type (WT) mice, on a C57BL/6 genetic background, were immunized with myelin oligodendrocyte glycoprotein 35–55. The levels of PAF production and PAFR mRNA expression in the spinal cord (SC) correlated with the EAE symptoms. PAFR-KO mice showed lower incidence and less severe symptoms in the chronic phase of EAE than WT mice. However, no difference was observed in T cell proliferation, Th1-cytokine production, or titer of IgG2a between both genotypes. Before onset, as revealed by microarray analysis, mRNAs of inflammatory mediators and their receptors—including IL-6 and CC chemokine receptor 2—were down-regulated in the SC of PAFR-KO mice compared with WT mice. Moreover, in the chronic phase, the severity of inflammation and demyelination in the SC was substantially reduced in PAFR-KO mice. PAFR-KO macrophages reduced phagocytic activity and subsequent production of TNF-α. These results suggest that PAF plays a dual role in EAE pathology in the induction and chronic phases through the T cell–independent pathways.

Background: Senescence-accelerated mice (SAM) are a series of mouse strains originally derived from unexpected crosses between AKR/J and unknown mice, from which phenotypically distinct senescence-prone (SAMP) and -resistant (SAMR) inbred strains were subsequently established. Although SAMP strains have been widely used for aging research focusing on their short life spans and various age-related phenotypes, such as immune dysfunction, osteoporosis, and brain atrophy, the responsible gene mutations have not yet been fully elucidated. Results: To identify mutations specific to SAMP strains, we performed whole exome sequencing of 6 SAMP and 3 SAMR strains. This analysis revealed 32,019 to 38,925 single-nucleotide variants in the coding region of each SAM strain. We detected Ogg1 p.R304W and Mbd4 p.D129N deleterious mutations in all 6 of the SAMP strains but not in the SAMR or AKR/J strains. Moreover, we extracted 31 SAMP-specific novel deleterious mutations. In all SAMP strains except SAMP8, we detected a p.R473W missense mutation in the Ldb3 gene, which has been associated with myofibrillar myopathy. In 3 SAMP strains (SAMP3, SAMP10, and SAMP11), we identified a p.R167C missense mutation in the Prx gene, in which mutations causing hereditary motor and sensory neuropathy (Dejerine-Sottas syndrome) have been identified. In SAMP6 we detected a p.S540fs frame-shift mutation in the Il4ra gene, a mutation potentially causative of ulcerative colitis and osteoporosis. Conclusions: Our data indicate that different combinations of mutations in disease-causing genes may be responsible for the various phenotypes of SAMP strains., Article, BMC GENOMICS. 14:248 (2013)

The Senescence-Accelerated Mouse (SAM) represents a group of inbred mouse strains developed as a model for the study of human aging and age-related diseases. Senescence-prone (SAMP) strains exhibit an early onset of age-related decline in the peripheral immunity such as thymic involution, loss of CD4(+) T cells, impaired helper T cell function, decreased antibody-forming capacity, dysfunction of antigen-presenting cells, decreased natural killer activity, increased auto-antibodies, and susceptibility to virus infection. Senescence-prone SAMP10 mice undergo age-related changes in the brain such as brain atrophy, shrinkage and loss of cortical neurons, retraction of cortical neuronal dendrites, loss of dendritic spines, loss of synapses, impaired learning and memory, depressive behavior, accumulation of neuronal DNA damage, neuronal ubiquitinated inclusions, reduced hippocampal cholinergic receptors, decreased neurotrophic factors, decreased hippocampal zinc and zinc transporters, increased sphyngomyelinase, and elevated oxidative-nitrative stress. Recent data indicating increased pro-inflammatory cytokines in the brain of SAMP10 mice are directing investigators toward an integration of immune and neural abnormalities to enhance understanding of the principles of brain aging. We highlight how mouse brain cells adopt cytokine-mediated responses and how SAMP10 mice are defective in these responses. SAMP10 model would be useful to study how age-related disturbances in peripheral immunity have an impact on dysregulation of brain tissue homeostasis, resulting in age-related neurodegeneration.

Excitotoxicity is involved in seizure-induced acute neuronal death, hypoxic–ischemic encephalopathy, and chronic neurodegenerative conditions such as Alzheimer's disease. Although oxidative stress has been implicated in excitotoxicity, the target proteins of oxidative damage during the course of excitotoxic cell death are still unclear. In the present study, we performed 2D-oxyblot analysis and mass spectrometric amino acid sequencing to identify proteins that were vulnerable to oxidative damage in the rat hippocampus during kainic acid (KA)-induced status epilepticus. We first investigated the time course in which oxidative protein damage occurred using immunohistochemistry. Carbonylated proteins, a manifestation of protein oxidation, were detected in hippocampal neurons as early as 3 h after KA administration. Immunoreactivity for 8-hydroxy-2′-deoxyguanosine (8-OHdG) was also elevated at the same time point. The increase in oxidative damage to proteins and DNA occurred concomitantly with the early morphological changes in KA-treated rat hippocampus, i.e., changes in chromatin distribution and swelling of rough endoplasmic reticulum and mitochondria, which preceded the appearance of morphological features of neuronal death such as pyknotic nuclei and hypereosinophilic cytoplasm. Proteomic analysis revealed that several hippocampal proteins were consistently carbonylated at this time point, including heat shock 70 kDa protein 4, valosin-containing protein, mitochondrial inner membrane protein (mitofilin), α-internexin, and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (14-3-3 protein). We propose that oxidative damage to these proteins may be one of the upstream events in the molecular pathway leading to excitotoxic cell death in KA-treated rat hippocampus, and these proteins may be targets of therapeutic intervention for seizure-induced neuronal death.

A significantly increased risk for dominant sensorineural deafness in patients who have Hirschsprung disease (HSCR) caused by endothelin receptor type B and SOX10 has been reported. Despite the fact that c- RET is the most frequent causal gene of HSCR, it has not been determined whether impairments of c- Ret and c- RET cause congenital deafness in mice and humans. Here, we show that impaired phosphorylation of c-Ret at tyrosine 1062 causes HSCR-linked syndromic congenital deafness in c- Ret knockin (KI) mice. The deafness involves neurodegeneration of spiral ganglion neurons (SGNs) with not only impaired phosphorylation of Akt and NF-κB but decreased expression of calbindin D28k in inner ears. The congenital deafness involving neurodegeneration of SGNs in c- Ret KI mice was rescued by introducing constitutively activated RET. Taken together with our results for three patients with congenital deafness with c- RET –mediated severe HSCR, our results indicate that c- Ret and c- RET are a deafness-related molecule in mice and humans.

Oxidative stress, an imbalance between endogenous levels of oxygen radicals and antioxidative defense, increases with aging. However, it is not clear which of these two factors is the more critical. To clarify the production of oxygen radicals increases with age, we examined oxygen radical-dependent chemiluminescent signals in ex vivo brain slices using a novel photonic imaging method. The chemiluminescent intensity was significantly decreased by the membrane permeable superoxide dismutase (SOD)/catalase mimic, but not by Cu,Zn-SOD. Inhibitors for complex I, III, and IV of the mitochondrial electron transport chain transiently enhanced the chemiluminescent signal. The superoxide-dependent chemiluminescent intensity in senescence accelerated mouse (SAM) brain tissues increases with age. Moreover, the slope of the age-dependent increase was steeper in SAMP10, a strain characterized by a short lifespan and atrophy in the frontal cerebral cortex, than the senescence-resistant strain SAMR1, which has a longer lifespan. An increase in chemiluminescence with age was also observed in C57/BL6 mice, Wistar rats, and pigeons, although levels of chemiluminescence were lower in the pigeons than murines. The rate of age-related increases of superoxide-dependent chemiluminescence was inversely related to the maximum lifespan of the animals. The activity of superoxide dismutase was unchanged during the aging process in the brain. This suggested that superoxide production itself may increase with age. We speculated that reactive oxygen may be a signal to determine the aging process.

Endoplasmic reticulum (ER)p61, ERp72, and protein disulfide isomerase (PDI), which are members of the PDI family protein, are ubiquitously present in mammalian cells and are thought to participate in disulfide bond formation and isomerization. However, why the 3 different members need to be colocalized in the ER remains an enigma. We hypothesized that each PDI family protein might have different modes of enzymatic activity in disulfide bond formation and isomerization. We purified PDI, ERp61, and ERp72 proteins from rat liver microsomes and compared the effects of each protein on the folding of bovine pancreatic trypsin inhibitor (BPTI). ERp61 and ERp72 accelerated the initial steps more efficiently than did PDI. ERp61 and ERp72, however, accelerated the rate-limiting step less efficiently than did PDI. PDI or ERp72 did not impede the folding of BPTI by each other but rather catalyzed the folding reaction cooperatively with each other. These data suggest that differential enzymatic activities of ERp proteins and PDI represent a complementary contribution of these enzymes to protein folding in the ER.

Folding of secretory proteins is associated with the formation and isomerization of disulfide bonds. ERp72, a protein disulfide isomerase (PDI) family member, possesses 3 thioredoxin homology domains, but the participation of each domain in disulfide-bond formation and isomerization remains to be determined. We analyzed the function of individual domains in the insulin reduction assay system by site-directed mutagenesis with cysteine-to-serine replacement. All domains contributed to apparent steady-state binding (Km) and catalysis at saturating substrate concentrations (kcat) but in different manners. A mutant ERp72 with mutations in domains 1 and 2 (ERp72-mut-1+2) exhibited reductions in kcat of 73.9% when compared with wild type, whereas ERp72-mut-1+3 (mutations in domains 1 and 3) and ERp72-mut-2+3 (mutations in domains 2 and 3) exhibited less substantial reductions in kcat. ERp72-mut-1+3 and ERp72-mut-2+3 showed elevations in Km of 89.9% and 96.2%, respectively, when compared with wild type, whereas ERp72-mut-1+2 exhibited smaller elevations in Km. These results suggest that domains 1 and 2 make greater contributions to catalyzing efficacy and domain 3 to binding affinity. Domain 2 is involved in binding affinity, in combination with domain 3, in addition to its own contribution to catalyzing efficacy. This assignment of functions to individual domains is similar to that observed in other PDI domains, which is consistent with the high sequence homology between ERp and PDI domains.