Seeding Activity of Skin Misfolded Tau as a Biomarker for Tauopathies.

Background: Tauopathies are a group of age-related neurodegenerative diseases characterized by the accumulation of pathologically phosphorylated tau protein in the brain, leading to prion-like propagation and aggregation. They include Alzheimer's disease (AD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and Pick's disease (PiD). Currently, reliable diagnostic biomarkers that directly reflect the capability of propagation and spreading of misfolded tau aggregates in peripheral tissues and body fluids are lacking. Methods: We utilized the seed-amplification assay (SAA) employing ultrasensitive real-time quaking-induced conversion (RT-QuIC) to assess the prion-like seeding activity of pathological tau in the skin of cadavers with neuropathologically confirmed tauopathies, including AD, PSP, CBD, and PiD, compared to normal controls. Results: We found that the skin prion-SAA demonstrated a significantly higher sensitivity (75-80%) and specificity (95-100%) for detecting tauopathy, depending on the tau substrates used. Moreover, increased tau-seeding activity was also observed in biopsy skin samples from living AD and PSP patients examined. Analysis of the end products of skin-tau SAA confirmed that the increased seeding activity was accompanied by the formation of tau aggregates with different physicochemical properties related to two different tau substrates used. Conclusions: Overall, our study provides proof-of-concept that the skin tau-SAA can differentiate tauopathies from normal controls, suggesting that the seeding activity of misfolded tau in the skin could serve as a diagnostic biomarker for tauopathies.

Large-scale validation of skin prion seeding activity as a biomarker for diagnosis of prion diseases.

Definitive diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD) relies on the examination of brain tissues for the pathological prion protein (PrPSc). Our previous study revealed that PrPSc-seeding activity (PrPSc-SA) is detectable in skin of sCJD patients by an ultrasensitive PrPSc seed amplification assay (PrPSc-SAA) known as real-time quaking-induced conversion (RT-QuIC). A total of 875 skin samples were collected from 2 cohorts (1 and 2) at autopsy from 2-3 body areas of 339 cases with neuropathologically confirmed prion diseases and non-sCJD controls. The skin samples were analyzed for PrPSc-SA by RT-QuIC assay. The results were compared with demographic information, clinical manifestations, cerebrospinal fluid (CSF) PrPSc-SA, other laboratory tests, subtypes of prion diseases defined by the methionine (M) or valine (V) polymorphism at residue 129 of PrP, PrPSc types (#1 or #2), and gene mutations in deceased patients. RT-QuIC assays of the cohort #1 by two independent laboratories gave 87.3% or 91.3% sensitivity and 94.7% or 100% specificity, respectively. The cohort #2 showed sensitivity of 89.4% and specificity of 95.5%. RT-QuIC of CSF available from 212 cases gave 89.7% sensitivity and 94.1% specificity. The sensitivity of skin RT-QuIC was subtype dependent, being highest in sCJDVV1-2 subtype, followed by VV2, MV1-2, MV1, MV2, MM1, MM1-2, MM2, and VV1. The skin area next to the ear gave highest sensitivity, followed by lower back and apex of the head. Although no difference in brain PrPSc-SA was detected between the cases with false negative and true positive skin RT-QuIC results, the disease duration was significantly longer with the false negatives [12.0 ± 13.3 (months, SD) vs. 6.5 ± 6.4, p < 0.001]. Our study validates skin PrPSc-SA as a biomarker for the detection of prion diseases, which is influenced by the PrPSc types, PRNP 129 polymorphisms, dermatome sampled, and disease duration.

Remission of social behavior impairment by oral administration of a precursor of NAD in CD157, but not in CD38, knockout mice.

Nicotinamide adenine dinucleotide (NAD) is a substrate of adenosine diphosphate (ADP)-ribosyl cyclase and is catalyzed to cyclic ADP-ribose (cADPR) by CD38 and/or CD157. cADPR, a Ca2+ mobilizing second messenger, is critical in releasing oxytocin from the hypothalamus into the brain. Although NAD precursors effectively play a role in neurodegenerative disorders, muscular dystrophy, and senescence, the beneficial effects of elevating NAD by NAD precursor supplementation on brain function, especially social interaction, and whether CD38 is required in this response, has not been intensely studied. Here, we report that oral gavage administration of nicotinamide riboside, a perspective NAD precursor with high bioavailability, for 12 days did not show any suppressive or increasing effects on sociability (mouse's interest in social targets compared to non-social targets) in both CD157KO and CD38KO male mice models in a three-chamber test. CD157KO and CD38KO mice displayed no social preference (that is, more interest towards a novel mouse than a familiar one) behavior. This defect was rescued after oral gavage administration of nicotinamide riboside for 12 days in CD157KO mice, but not in CD38KO mice. Social memory was not observed in CD157KO and CD38KO mice; subsequently, nicotinamide riboside administration had no effect on social memory. Together with the results that nicotinamide riboside had essentially no or little effect on body weight during treatment in CD157KO mice, nicotinamide riboside is less harmful and has beneficial effect on defects in recovery from social behavioral, for which CD38 is required in mice.

Receptor for advanced glycation end-products and child neglect in mice: A possible link to postpartum depression.

The receptor for advanced glycation end-products (RAGE), a pattern recognition molecule, has a role in the remodeling of vascular endothelial cells mainly in lungs, kidney and brain under pathological conditions. We recently discovered that RAGE binds oxytocin (OT) and transports it to the brain from circulation on neurovascular endothelial cells. We produced knockout mice of the mouse homologue of the human RAGE gene, Ager, designated RAGE KO mice. In RAGE KO mice, while hyperactivity has been reported in male mice, maternal behavior was impaired in female mice. After an additional stress, deficits in pup care were observed in RAGE KO mother mice. This resulted in pup death within 1-2 days, suggesting that RAGE plays a critical role during the postpartum period. Thus, RAGE seems to be important in the manifestation of normal maternal behavior in dams. In this review, we summarize the significance of brain OT transport by RAGE and propose that RAGE-dependent OT can dampen stress signals during pregnancy, delivery and early postpartum periods. To the best of our knowledge, there have been no previous articles on these RAGE-dependent results. Based on these results in mice, we discuss a potential critical role of RAGE in emotion swings at the puerperium (peripartum) and postpartum periods in women.

Oxytocin Dynamics in the Body and Brain Regulated by the Receptor for Advanced Glycation End-Products, CD38, CD157, and Nicotinamide Riboside.

Investigating the neurocircuit and synaptic sites of action of oxytocin (OT) in the brain is critical to the role of OT in social memory and behavior. To the same degree, it is important to understand how OT is transported to the brain from the peripheral circulation. To date, of these, many studies provide evidence that CD38, CD157, and receptor for advanced glycation end-products (RAGE) act as regulators of OT concentrations in the brain and blood. It has been shown that RAGE facilitates the uptake of OT in mother's milk from the digestive tract to the cell surface of intestinal epithelial cells to the body fluid and subsequently into circulation in male mice. RAGE has been shown to recruit circulatory OT into the brain from blood at the endothelial cell surface of neurovascular units. Therefore, it can be said that extracellular OT concentrations in the brain (hypothalamus) could be determined by the transport of OT by RAGE from the circulation and release of OT from oxytocinergic neurons by CD38 and CD157 in mice. In addition, it has recently been found that gavage application of a precursor of nicotinamide adenine dinucleotide, nicotinamide riboside, for 12 days can increase brain OT in mice. Here, we review the evaluation of the new concept that RAGE is involved in the regulation of OT dynamics at the interface between the brain, blood, and intestine in the living body, mainly by summarizing our recent results due to the limited number of publications on related topics. And we also review other possible routes of OT recruitment to the brain.

Oral Supplementation with L-Carnosine Attenuates Social Recognition Deficits in CD157KO Mice via Oxytocin Release.

The outcomes of supplementation with L-carnosine have been investigated in clinical trials in children with autism spectrum disorder (ASD). However, reports on the effects of L-carnosine in humans have been inconsistent, and the efficacy of L-carnosine supplementation for improving ASD symptoms has yet to be investigated in animal studies. Here, we examined the effects of oral supplementation with L-carnosine on social deficits in CD157KO mice, a murine model of ASD. Social deficits in CD157KO mice were assessed using a three-chamber social approach test. Oral supplementation with L-carnosine attenuated social behavioral deficits. The number of c-Fos-positive oxytocin neurons in the supraoptic nucleus and paraventricular nucleus was increased with L-carnosine supplementation in CD157KO mice after the three-chamber social approach test. We observed an increase in the number of c-Fos-positive neurons in the basolateral amygdala, a brain region involved in social behavior. Although the expression of oxytocin and oxytocin receptors in the hypothalamus was not altered by L-carnosine supplementation, the concentration of oxytocin in cerebrospinal fluid was increased in CD157KO mice by L-carnosine supplementation. These results suggest that L-carnosine supplementation restores social recognition impairments by augmenting the level of released oxytocin. Thus, we could imply the possibility of a safe nutritional intervention for at least some types of ASD in the human population.

An improved sample extraction method reveals that plasma receptor for advanced glycation end-products (RAGE) modulates circulating free oxytocin in mice.

The receptor for advanced glycation end-products (RAGE) binds oxytocin (OT) and transports it from the blood to the brain. As RAGE's OT-binding capacity was lost in RAGE knockout (KO) mice, we predicted that circulating concentrations of unbound (free) OT should be elevated compared to wild-type (WT) mice. However, this hypothesis has not yet been investigated. Unfortunately, the evaluation of the dynamics of circulating free and bound plasma OT is unclear in immunoassays, in part because of interference from plasma proteins. A radioimmunoassay (RIA) is considered the gold standard method for overcoming this issue, but is more challenging to implement; thus, commercially available enzyme-linked immunosorbent assays (ELISAs) are more commonly used. Here, we developed a pre-treatment method to remove the interference-causing components from plasma before performing ELISA. The acetonitrile protein precipitation (PPT) approach was reliable, with fewer steps needed to measure free OT concentrations than by solid-phase extraction of plasma samples. PPT-extracted plasma samples yielded higher concentrations of OT in RAGE KO mice than in WT mice using ELISA. After peripheral OT injection, free OT plasma levels spiked immediately then rapidly declined in WT mice, but remained high in KO mice. These results suggest that plasma samples with PPT pre-treatment appear to be superior and that circulating soluble RAGE can most likely serve as a buffer for plasma OT, which indicates a novel physiological function of RAGE.

Oxytocin ameliorates impaired social behavior in a Chd8 haploinsufficiency mouse model of autism.

BACKGROUND: Autism spectrum disorder (ASD) is characterized by the core symptoms of impaired social interactions. Increasing evidence suggests that ASD has a strong genetic link with mutations in chromodomain helicase DNA binding protein 8 (CHD8), a gene encoding a chromatin remodeler. It has previously been shown that Chd8 haplodeficient male mice manifest ASD-like behavioral characteristics such as anxiety and altered social behavior. Along with that, oxytocin (OT) is one of the main neuropeptides involved in social behavior. Administration of OT has shown improvement of social behavior in genetic animal models of ASD. The present study was undertaken to further explore behavioral abnormalities of Chd8 haplodeficient mice of both sexes, their link with OT, and possible effects of OT administration. First, we performed a battery of behavioral tests on wild-type and Chd8+/∆SL female and male mice. Next, we measured plasma OT levels and finally studied the effects of intraperitoneal OT injection on observed behavioral deficits. RESULTS: We showed general anxiety phenotype in Chd8+/∆SL mice regardless of sex, the depressive phenotype in Chd8+/∆SL female mice only and bidirectional social deficit in female and male mice. We observed decreased level of OT in Chd+/∆SL mice, possibly driven by males. Mice injected by OT demonstrated recovery of social behavior, while reduced anxiety was observed only in male mice. CONCLUSIONS: Here, we demonstrated that abnormal social behaviors were observed in both male and female Chd8+/∆SL mice. The ability of peripheral OT administration to affect such behaviors along with altered plasma OT levels indicated a possible link between Chd8 + /∆SL and OT in the pathogenesis of ASD as well as the possible usefulness of OT as a therapeutic tool for ASD patients with CHD8 mutations.

Transport of oxytocin to the brain after peripheral administration by membrane-bound or soluble forms of receptors for advanced glycation end-products.

Oxytocin (OT) is a neuropeptide hormone. Single and repetitive administration of OT increases social interaction and maternal behaviour in humans and mammals. Recently, it was found that the receptor for advanced glycation end-products (RAGE) is an OT-binding protein and plays a critical role in the uptake of OT to the brain after peripheral OT administration. Here, we address some unanswered questions on RAGE-dependent OT transport. First, we found that, after intranasal OT administration, the OT concentration increased in the extracellular space of the medial prefrontal cortex (mPFC) of wild-type male mice, as measured by push-pull microperfusion. No increase of OT in the mPFC was observed in RAGE knockout male mice. Second, in a reconstituted in vitro blood-brain barrier system, inclusion of the soluble form of RAGE (endogenous secretory RAGE [esRAGE]), an alternative splicing variant, in the luminal (blood) side had no effect on the transport of OT to the abluminal (brain) chamber. Third, OT concentrations in the cerebrospinal fluid after i.p. OT injection were slightly higher in male mice overexpressing esRAGE (esRAGE transgenic) compared to those in wild-type male mice, although this did not reach statistical significance. Although more extensive confirmation is necessary because of the small number of experiments in the present study, the reported data support the hypothesis that RAGE may be involved in the transport of OT to the mPFC from the circulation. These results suggest that the soluble form of RAGE in the plasma does not function as a decoy in vitro.

Receptor for advanced glycation end-products (RAGE) plays a critical role in retrieval behavior of mother mice at early postpartum.

Receptor for advanced glycation end-products (RAGE) is a pattern recognition molecule belonging to the immunoglobulin superfamily, and it plays a role in the remodeling of endothelial cells under pathological conditions. Recently, it was shown that RAGE is a binding protein for oxytocin (OT) and a transporter of OT to the brain on neurovascular endothelial cells via blood circulation. Deletion of the mouse RAGE gene, Ager (RAGE KO), induces hyperactivity in male mice. Impairment of pup care by mother RAGE KO mice after stress exposure results in the death of neonates 1-2 days after pup birth. Therefore, to understand the role of RAGE during the postpartum period, this study aims to examine parental behavior in female RAGE KO mice and ultrasonic vocalizations in pups. RAGE KO mothers without stress before delivery raised their pups and displayed hyperactivity at postpartum day (PPD) 3. KO dams showed impaired retrieval or interaction behavior after additional stress, such as body restraint stress or exposure to a novel environment, but such impaired behavior disappeared at PPD 7. Postnatal day 3 pups emitted ultrasonic vocalizations at >60 kHz as a part of the mother-pup relationship, but the number and category of calls by RAGE KO pups were significantly lower than wild-type pups. The results indicate that RAGE is important in the manifestation of normal parental behavior in dams and for receiving maternal care by mouse pups; moreover, brain OT recruited by RAGE plays a role in damping of signals of additional external stress and endogenous stress during the early postpartum period. Thus, RAGE-dependent OT may be critical for initiating and maintaining the normal mother-child relationship.

Distinct physical condition and social behavior phenotypes of CD157 and CD38 knockout mice during aging.

The ability of CD38 and CD157 to utilize nicotinamide adenine dinucleotide (NAD) has received much attention because the aging-induced elevation of CD38 expression plays a role in the senescence-related decline in NAD levels. Therefore, it is of interest to examine and compare the effects of age-associated changes on the general health and brain function impairment of Cd157 and Cd38 knockout (CD157 KO and CD38 KO) mice. The body weight and behaviors were measured in 8-week-old (young adult) or 12-month-old (middle-aged) male mice of both KO strains. The locomotor activity, anxiety-like behavior, and social behavior of the mice were measured in the open field and three-chamber tests. The middle-aged CD157 KO male mice gained more body weight than young adult KO mice, while little or no body weight gain was observed in the middle-aged CD38 KO mice. Middle-aged CD157 KO mice displayed increased anxiety-like behavior and decreased sociability and interaction compared with young adult KO mice. Middle-aged CD38 KO mice showed less anxiety and hyperactivity than CD157 KO mice, similar to young adult CD38 KO mice. The results reveal marked age-dependent changes in male CD157 KO mice but not in male CD38 KO mice. We discuss the distinct differences in aging effects from the perspective of inhibition of NAD metabolism in CD157 and CD38 KO mice, which may contribute to differential behavioral changes during aging.

Nicotinamide riboside supplementation corrects deficits in oxytocin, sociability and anxiety of CD157 mutants in a mouse model of autism spectrum disorder.

Oxytocin (OT) is a critical molecule for social recognition and memory that mediates social and emotional behaviours. In addition, OT acts as an anxiolytic factor and is released during stress. Based on the activity of CD38 as an enzyme that produces the calcium-mobilizing second messenger cyclic ADP-ribose (cADPR), CD157, a sister protein of CD38, has been considered a candidate mediator for the production and release of OT and its social engagement and anti-anxiety functions. However, the limited expression of CD157 in the adult mouse brain undermined confidence that CD157 is an authentic and/or actionable molecular participant in OT-dependent social behaviour. Here, we show that CD157 knockout mice have low levels of circulating OT in cerebrospinal fluid, which can be corrected by the oral administration of nicotinamide riboside, a recently discovered vitamin precursor of nicotinamide adenine dinucleotide (NAD). NAD is the substrate for the CD157- and CD38-dependent production of cADPR. Nicotinamide riboside corrects social deficits and fearful and anxiety-like behaviours in CD157 knockout males. These results suggest that elevating NAD levels with nicotinamide riboside may allow animals with cADPR- and OT-forming deficits to overcome these deficits and function more normally.

CD38, CD157, and RAGE as Molecular Determinants for Social Behavior.

Recent studies provide evidence to support that cluster of differentiation 38 (CD38) and CD157 meaningfully act in the brain as neuroregulators. They primarily affect social behaviors. Social behaviors are impaired in Cd38 and Cd157 knockout mice. Single-nucleotide polymorphisms of the CD38 and CD157/BST1 genes are associated with multiple neurological and psychiatric conditions, including autism spectrum disorder, Parkinson's disease, and schizophrenia. In addition, both antigens are related to infectious and immunoregulational processes. The most important clues to demonstrate how these molecules play a role in the brain are oxytocin (OT) and the OT system. OT is axo-dendritically secreted into the brain from OT-containing neurons and causes activation of OT receptors mainly on hypothalamic neurons. Here, we overview the CD38/CD157-dependent OT release mechanism as the initiation step for social behavior. The receptor for advanced glycation end-products (RAGE) is a newly identified molecule as an OT binding protein and serves as a transporter of OT to the brain, crossing over the blood-brain barrier, resulting in the regulation of brain OT levels. We point out new roles of CD38 and CD157 during neuronal development and aging in relation to nicotinamide adenine dinucleotide+ levels in embryonic and adult nervous systems. Finally, we discuss how CD38, CD157, and RAGE are crucial for social recognition and behavior in daily life.

Vascular RAGE transports oxytocin into the brain to elicit its maternal bonding behaviour in mice.

Oxytocin sets the stage for childbirth by initiating uterine contractions, lactation and maternal bonding behaviours. Mice lacking secreted oxcytocin (Oxt -/-, Cd38 -/-) or its receptor (Oxtr -/-) fail to nurture. Normal maternal behaviour is restored by peripheral oxcytocin replacement in Oxt -/- and Cd38 -/-, but not Oxtr -/- mice, implying that circulating oxcytocin crosses the blood-brain barrier. Exogenous oxcytocin also has behavioural effects in humans. However, circulating polypeptides are typically excluded from the brain. We show that oxcytocin is transported into the brain by receptor for advanced glycation end-products (RAGE) on brain capillary endothelial cells. The increases in oxcytocin in the brain which follow exogenous administration are lost in Ager -/- male mice lacking RAGE, and behaviours characteristic to abnormalities in oxcytocin signalling are recapitulated in Ager -/- mice, including deficits in maternal bonding and hyperactivity. Our findings show that RAGE-mediated transport is critical to the behavioural actions of oxcytocin associated with parenting and social bonding.

Sex Differences in Salivary Oxytocin and Cortisol Concentration Changes during Cooking in a Small Group.

Background: Oxytocin (OT), a neuropeptide, has positive effects on social and emotional processes during group activities. Because cooking is an integrated process in the cognitive, physical, and socio-emotional areas, cooking in a group is reported to improve emotion and cognition. However, evidence for efficacy in group cooking has not been well established at the biological level. Methods: To address this shortcoming, we first measured salivary levels of OT and cortisol (CORT), a biomarker of psychological stress, before and after group cooking for approximately 1 h by people who know each other in healthy married or unmarried men and women. We then compared the initial OT and CORT concentrations with those during individual non-cooking activities in isolation. Results: Baseline OT concentrations before group and non-group sessions did not significantly differ and OT levels increased after both types of activity in men and women. In men, however, the percentage changes of OT levels in the first over the second saliva samples were significantly small during cooking compared with those in individual activities. In women, however, such a difference was not observed. In contrast, the mean salivary CORT concentrations after group cooking were significantly decreased from the baseline level in both sexes, though such decreases were not significant after individual activity sessions. The sex-specific differences were marital-status independent. Conclusion: These results indicate that OT and CORT concentrations after two activity sessions by a familiar group changed in opposite directions in a sex-specific manner. This suggests that, because cooking is experience-based, we need to consider the sex-specific features of group cooking if we apply it for intervention.

Intestinal transepithelial permeability of oxytocin into the blood is dependent on the receptor for advanced glycation end products in mice.

Plasma oxytocin (OT) originates from secretion from the pituitary gland into the circulation and from absorption of OT in mother's milk into the blood via intestinal permeability. However, the molecular mechanism underlying the absorption of OT remains unclear. Here, we report that plasma OT concentrations increased within 10 min after oral delivery in postnatal day 1-7 mice. However, in Receptors for Advanced Glycation End Products (RAGE) knockout mice after postnatal day 3, an identical OT increase was not observed. In adult mice, plasma OT was also increased in a RAGE-dependent manner after oral delivery or direct administration into the intestinal tract. Mass spectrometry evaluated that OT was absorbed intact. RAGE was abundant in the intestinal epithelial cells in both suckling pups and adults. These data highlight that OT is transmitted via a receptor-mediated process with RAGE and suggest that oral OT supplementation may be advantageous in OT drug development.

An immunohistochemical, enzymatic, and behavioral study of CD157/BST-1 as a neuroregulator.

BACKGROUND: Recent rodent and human studies provide evidence in support of the fact that CD157, well known as bone marrow stromal cell antigen-1 (BST-1) and a risk factor in Parkinson's disease, also meaningfully acts in the brain as a neuroregulator and affects social behaviors. It has been shown that social behaviors are impaired in CD157 knockout mice without severe motor dysfunction and that CD157/BST1 gene single nucleotide polymorphisms are associated with autism spectrum disorder in humans. However, it is still necessary to determine how this molecule contributes to the brain's physiological and pathophysiological functions. METHODS: To gain fresh insights about the relationship between the presence of CD157 in the brain and its enzymatic activity, and aberrant social behavior, CD157 knockout mice of various ages were tested. RESULTS: CD157 immunoreactivity colocalized with nestin-positive cells and elements in the ventricular zones in E17 embryos. Brain CD157 mRNA levels were high in neonates but low in adults. Weak but distinct immunoreactivity was detected in several areas in the adult brain, including the amygdala. CD157 has little or no base exchange activity, but some ADP-ribosyl cyclase activity, indicating that CD157 formed cyclic ADP-ribose but much less nicotinic acid adenine dinucleotide phosphate, with both mobilizing Ca2+ from intracellular Ca2+ pools. Social avoidance in CD157 knockout mice was rescued by a single intraperitoneal injection of oxytocin. CONCLUSIONS: CD157 may play a role in the embryonic and adult nervous systems. The functional features of CD157 can be explained in part through the production of cyclic ADP-ribose rather than nicotinic acid adenine dinucleotide phosphate. Further experiments are required to elucidate how the embryonic expression of CD157 in neural stem cells contributes to behaviors in adults or to psychiatric symptoms.