Differential functional consequences of GRIN2A mutations associated with schizophrenia and neurodevelopmental disorders.

Human genetic studies have revealed rare missense and protein-truncating variants in GRIN2A, encoding for the GluN2A subunit of the NMDA receptors, that confer significant risk for schizophrenia (SCZ). Mutations in GRIN2A are also associated with epilepsy and developmental delay/intellectual disability (DD/ID). However, it remains enigmatic how alterations to the same protein can result in diverse clinical phenotypes. Here, we performed functional characterization of human GluN1/GluN2A heteromeric NMDA receptors that contain SCZ-linked GluN2A variants, and compared them to NMDA receptors with GluN2A variants associated with epilepsy or DD/ID. Our findings demonstrate that SCZ-associated GRIN2A variants were predominantly loss-of-function (LoF), whereas epilepsy and DD/ID-associated variants resulted in both gain- and loss-of-function phenotypes. We additionally show that M653I and S809R, LoF GRIN2A variants associated with DD/ID, exert a dominant-negative effect when co-expressed with a wild-type GluN2A, whereas E58Ter and Y698C, SCZ-linked LoF variants, and A727T, an epilepsy-linked LoF variant, do not. These data offer a potential mechanism by which SCZ/epilepsy and DD/ID-linked variants can cause different effects on receptor function and therefore result in divergent pathological outcomes.

Effect of Electric Field on α-Synuclein Fibrils: Revealed by Molecular Dynamics Simulations.

The self-association of amylogenic proteins to the fibril form is considered a pivotal factor in the pathogenesis of neurodegenerative diseases, including Parkinson's disease (PD). PD causes unintended or uncontrollable movements in its common symptoms. α-synuclein is the major cause of PD development and thus has been the main target of numerous studies to suppress and sequester its expression or effectively degrade it. Nonetheless, to date, there are no efficient and proven ways to prevent pathological protein aggregation. Recent investigations proposed applying an external electric field to interrupt the fibrils. This method is a non-invasive approach that has a certain benefit over others. We performed molecular dynamics (MD) simulations by applying an electric field on highly toxic fibrils of α-synuclein to gain a molecular-level insight into fibril disruption mechanisms. The results revealed that the applied external electric field induces substantial changes in the conformation of the α-synuclein fibrils. Furthermore, we show the threshold value for electric field strength required to completely disrupt the α-synuclein fibrils by opening the hydrophobic core of the fibril. Thus, our findings might serve as a valuable foundation to better understand molecular-level mechanisms of the α-synuclein fibrils disaggregation process under an applied external electric field.

Distribution of TRPM8-expressing trigeminal nerve fibers in the pons and medulla oblongata of the mouse brain.

Transient receptor potential melastatin 8 (TRPM8), a cold-mediated ion channel, is well known to be expressed in primary sensory neurons; however, limited information is currently available on the distribution of TRPM8-expressing trigeminal nerve fibers in the brainstem. The present study showed the distribution of TRPM8-expressing fibers in the pons and medulla oblongata of the TRPM8 KO mice engineered by knocking in EGFP at the frame of the start codon of TRPM8. In addition, TRPM8-expressing fibers were also observed in the brachium pontis, middle cerebellar peduncle, the sensory root of the trigeminal nerve, and spinal trigeminal tract (sp5). Furthermore, TRPM8-expressing nerve fibers surrounded the somata of HuC/D-positive neurons in the sp5. Moreover, the distribution of TRPM8-expressing fibers from rostral to caudal was visualized in sagittal sections of the mouse brain. The present results also revealed that a high number of TRPM8-expressing fibers colocalized with CTB-labeled fibers in the sp5 following an injection of CTB into the whisker compared to mice's eye and ear. These results show the distribution pathway of TRPM8-expressing fibers in the pons and medulla oblongata and possible involvement in peripheral signaling from the trigeminal nerve.

Role of TRPM8 in cold avoidance behaviors and brain activation during innocuous and nocuous cold stimuli.

Transient receptor potential melastatin 8 (TRPM8) is a cold-sensing cation channel; however, its role in the transferal of information on peripheral cold sensation to the brain remains unclear. Therefore, we herein investigated cold avoidance behaviors and the neuronal activation of the hypothalamus and cerebral cortex in TRPM8 knockout (KO) mice to innocuous and nocuous cold stimuli. An innocuous cold stimulation at 15 °C decreased the duration of sleeping and increased that of rearing, climbing, and eating in WT mice, but it did not alter the duration of these behaviors in TRPM8 KO animals. The innocuous cold stimulation also increased the frequency of rearing, climbing, walking, and eating in WT mice, but it did not change that of these behaviors in TRPM8 KO animals. In contrast, a nocuous cold stimulation at 9 °C decreased the duration of sleeping and increased that of rearing and climbing in both WT and TRPM8 KO mice. The nocuous cold stimulation increased the frequency of rearing, climbing, and walking in WT and TRPM8 KO mice. Quantitative Fos immunohistochemistry showed that both innocuous and nocuous cold stimulations increased the number of Fos-positive neurons in temperature- and metabolism-associated hypothalamic regions in WT mice, but not in TRPM8 KO animals. The number of Fos-positive neurons was markedly increased in the primary motor and somatosensory cortices in WT and TRPM8 KO mice following the nocuous cold stimulation, but only increased in WT mice after the innocuous cold stimulation. Collectively, the present results indicate that TRPM8 plays a crucial role in activating autonomic hypothalamic neuronal circuits under innocuous and nocuous cold stimuli.

Role of TRPM8 in switching between fever and hypothermia in adult mice during endotoxin-induced inflammation.

Transient receptor potential melastatin 8 (TRPM8) functions in the sensing of noxious and innocuous colds; however, its significance in pathogen-induced thermoregulation remains unclear. In the present study, we investigated the role of TRPM8 in the regulation of endotoxin-induced body temperature control. The peripheral administration of low-dose lipopolysaccharide (LPS) at 50 â€‹µg/kg generated fever in wild-type (WT) mice, whereas it caused hypothermia in TRPM8 knockout (KO) animals. LPS-induced sickness responses such as decrease in body weight, and food and water intake were not different between WT and TRPM8 KO mice. TRPM8 KO mice exhibited more severe hypothermia and lower locomotor activity following the peripheral administration of high-dose LPS at 5 â€‹mg/kg compared with WT ones. An intracerebroventricular (i.c.v.) injection of either LPS at 3.6 â€‹µg/kg or interleukin-1ß at 400 â€‹ng/kg elicited hypothermia in TRPM8 KO mice, in contrast to fever in WT animals. The peripheral administration of zymosan at 3 â€‹mg/kg also induced hypothermia in contrast to fever in WT mice. An i.c.v. injection of prostaglandin E2 at 16 or 160 â€‹nmol/kg induced normal fever in both WT and TRPM8 KO mice. Infrared thermography showed significant decline of the interscapular skin temperature that estimates temperature of the brown adipose tissue, regardless of no alteration of its temperature in WT animals. Fos immunohistochemistry showed stronger Fos activation of hypothalamic thermoregulation-associated nuclei in TRPM8 KO mice compared with WT animals following the peripheral administration of low-dose LPS. Therefore, the present study indicates that TRPM8 is necessary for switching between fever and hypothermia during endotoxin-induced inflammation.

Effects of leptin on proliferation of astrocyte- and tanycyte-like neural stem cells in the adult mouse medulla oblongata.

Astrocyte- and tanycyte-like neural stem cells (NSCs) were recently detected in the area postrema (AP) and central canal (CC) of the adult medulla oblongata, respectively. The present study aimed to examine dynamical behaviors of the astrocyte- and tanycyte-like NSCs of the mouse medulla oblongata to leptin. The neurosphere assay identified astrocytes in the AP and tanycytes in the CC as NSCs based on their self-renewing neurospherogenic potential. Both NSCs in neurosphere cultures were multipotent cells that generate astrocytes, oligodendrocytes, and neurons. Astrocyte-like NSCs actively proliferated and tanycyte-like NSCs were quiescent under physiologically-relevant in vivo conditions. Chronic leptin treatment promoted proliferation of astrocyte-like NSCs in the AP both in vitro and in vivo. Leptin receptors were expressed in astrocyte-like, but not tanycyte-like NSCs. Food deprivation significantly diminished proliferation of astrocyte-like NSCs. Therefore, the present study indicates that proliferation of astrocyte-like, but not tanycyte-like NSCs is regulated by nutritional conditions.

Chronic running and a corticosterone treatment attenuate astrocyte-like neural stem cell proliferation in the area postrema of the adult mouse brain.

The discovery of neural stem cells (NSCs) in the adult mammalian brain has provided insights into an extra level of brain plasticity. The proliferation and differentiation of NSCs is modulated by various physiological, pathological, and pharmacological stimuli. NSCs were recently detected in the medulla oblongata of adult rodents and humans; however, their functional significance currently remains unknown. In the present study, we examined the effects of chronic wheel-running and a corticosterone (CORT) treatment on the proliferation of astrocyte-like NSCs in the area postrema (AP) and dentate gyrus (DG). Chronic running significantly decreased the number of bromodeoxyuridine (BrdU)-labeled astrocyte-like NSCs in the AP of adult mice, but markedly increased that of BrdU+ NSCs/neural progenitor cells in the DG. The chronic CORT treatment markedly reduced the number of BrdU+ astrocyte-like NSCs in the AP, but not in the DG. These results demonstrate that the proliferation of astrocyte-like NSCs in the medulla oblongata is decreased by chronic running and a CORT treatment.

TRPV1 is crucial for thermal homeostasis in the mouse by heat loss behaviors under warm ambient temperature.

Thermal homeostasis in mammalians is a self-regulating process by which biological systems maintain an internal thermal stability, even under different temperature conditions; however, the molecular mechanisms involved under warm ambient temperature remain unclear. Here, we aimed to clarify functional significance of transient receptor potential vanilloid receptor 1 (TRPV1) under warm ambient temperature. TRPV1 KO mice exhibited transient hyperthermia when exposed to 30.0 and 32.5 °C, whereas wild-type (WT) mice did not. TRPV1 KO mice exhibited prolonged and prominent hyperthermia upon exposure to 35.0 °C, whereas WT mice showed transient hyperthermia. Hyperthermia also occurs in WT mice that received intracerebroventricular injection of TRPV1 antagonist AMG9810 upon exposure to 35.0 °C. Heat loss behaviors, sleeping and body licking, were deficient in TRPV1 KO mice exposed to warm temperatures. Therefore, the present results indicate that central TRPV1 is crucial for maintaining a constant body temperature via the initiation of heat loss behaviors under warm ambient temperature.

Proliferation of endothelial cells in the choroid plexus of normal and hydrocephalic mice.

The choroid plexus (CP), located at the walls of the brain ventricles, produces and secretes cerebrospinal fluid (CSF). Hydrocephalus is a neurological disorder in which the CP abnormally secretes excess amounts of CSF into the ventricles. There is currently no information on the vascular dynamics of the CP in adult brains under normal and hydrocephalic conditions. In the present study, we reported the continuous proliferation of endothelial cells in the CP of normal mice, which depended on vascular endothelial cell growth factor (VEGF). The proliferation of endothelial cells increased in mice with intraventricular hemorrhage, which was attenuated by a pretreatment with the toll-like receptor 4 (TLR4) inhibitor VIPER. Moreover, the intracerebroventricular infusion of the TLR4 agonist, lipopolysaccharide, increased endothelial cell proliferation in the CP and induced ventriculomegaly. The present results provide insights into the importance of the TLR4-initiated and VEGF-dependent proliferation of endothelial cells in the pathogenesis of hydrocephalus.

Depletion of microglia and macrophages with clodronate liposomes attenuates zymosan-induced Fos expression and hypothermia in the adult mouse.

Toll-like receptor 2 (TLR2) recognizes a wide range of microbial molecules and plays critical roles in the initiation of innate immune responses. In the present study, we aimed to investigate whether the depletion of microglia and macrophages with clodronate liposomes (Clod-Lips) attenuates the activation of mouse brain circuits for TLR2-mediated inflammation and hypothermia. The peripheral administration of the TLR2 agonist zymosan induced nuclear factor-κB activation in microglia and macrophages and Fos expression in astrocytes/tanycytes and neurons in the circumventricular organs (CVOs). The depletion of microglia and macrophages with Clod-Lips markedly decreased zymosan-induced Fos expression in astrocytes/tanycytes and neurons in the CVOs. The treatment with Clod-Lips significantly attenuated zymosan-induced hypothermia. These results indicate that microglia and macrophages in the CVOs participate in the initiation and transmission of inflammatory responses after the peripheral administration of zymosan.

Effect of fluoxetine on proliferation and/or survival of microglia and oligodendrocyte progenitor cells in the fornix and corpus callosum of the mouse brain.

BACKGROUND: Fluoxetine is one of the most widely prescribed antidepressants and a selective inhibitor of presynaptic 5-HT transporters. The fornix is the commissural and projection fiber that transmits signals from the hippocampus to other parts of the brain and opposite site of hippocampus. The corpus callosum (CC) is the largest of the commissural fibers that link the cerebral cortex of the left and right cerebral hemispheres. These brain regions play pivotal roles in cognitive functions, and functional abnormalities in these regions have been implicated in the development of various brain diseases. The purpose of the present study was to investigate the effects of fluoxetine on the proliferation and/or survival of microglia and oligodendrocyte progenitor cells (OPCs) in the fornix and CC, the white matter connecting cortical-limbic system, of the adult mouse brain. METHODS: The effects of fluoxetine on the proliferation and/or survival of microglia and OPCs were examined in lipopolysaccharide (LPS)-treated and normal mice. Proliferating cells were detected in mice that drank water containing the thymidine analog, bromodeoxyuridine (BrdU), using immunohistochemistry. RESULT: Fluoxetine significantly attenuated LPS-induced increases in the number of BrdU-labeled microglia and morphological activation from the ramified to ameboid shape, and decreased the number of BrdU-labeled OPCs under basal conditions. CONCLUSIONS: The present results indicate that fluoxetine exerts inhibitory effects on LPS-induced increases in the proliferation and/or survival and morphological activation of microglia and basal proliferation and/or survival of OPCs in the fornix and CC of adult mice.

Neural stem cell phenotype of tanycyte-like ependymal cells in the circumventricular organs and central canal of adult mouse brain.

Tanycyte is a subtype of ependymal cells which extend long radial processes to brain parenchyma. The present study showed that tanycyte-like ependymal cells in the organum vasculosum of the lamina terminalis, subfornical organ and central canal (CC) expressed neural stem cell (NSC) marker nestin, glial fibrillar acidic protein and sex determining region Y. Proliferation of these tanycyte-like ependymal cells was promoted by continuous intracerebroventricular infusion of fibroblast growth factor-2 and epidermal growth factor. Tanycytes-like ependymal cells in the CC are able to form self-renewing neurospheres and give rise mostly to new astrocytes and oligodendrocytes. Collagenase-induced small medullary hemorrhage increased proliferation of tanycyte-like ependymal cells in the CC. These results demonstrate that these tanycyte-like ependymal cells of the adult mouse brain are NSCs and suggest that they serve as a source for providing new neuronal lineage cells upon brain damage in the medulla oblongata.

Transient increase of microglial C1q expression in the circumventricular organs of adult mouse during LPS-induced inflammation.

The circumventricular organs (CVOs) are the brain regions that lack the blood-brain barrier and allow free entry of blood-derived molecules, offering specialized niche to initiate rapid and early neuroinflammatory responses in the brain. Complement component 1q (C1q) is shown to be the first recognition component of the complement pathway and has a crucial function in the brain under pathological conditions. In the present study, we found that C1q expression in CX3CR1-positive microglia was increased in the CVOs and their neighbouring brain regions of adult mice at 1 day after a single administration of 1 mg/kg lipopolysaccharide (LPS), whereas it returned to control levels at 3 days after LPS stimulation. C1q expression was also seen to localize at synapsin-positive presynaptic axonal terminals in various brain regions. Thus, the present study demonstrates a transient upregulation of microglial C1q expression in the CVOs and their adjacent brain regions, indicating that a transient upregulation of C1q is possibly concerned with physiological responses at early phase of brain inflammation. SIGNIFICANCE OF THE STUDY: The circumventricular organs (CVOs) are specialized brain regions that lack the blood-brain barrier (BBB) and initiate neuroinflammatory responses in the brains. The present study showed that the expression of complement protein C1q was highly increased in microglia of the CVOs and their adjacent brain regions. Moreover, C1q expression was observed to localize specifically at presynaptic axonal terminals in the CVOs and their neighbouring brain regions. Thus, the present study indicates that C1q is possibly correlated with physiological responses at early phase of brain inflammation.

Activation of microglia and macrophages in the circumventricular organs of the mouse brain during TLR2-induced fever and sickness responses.

Toll-like receptor 2 (TLR2) recognizes cell wall components from Gram-positive bacteria. Until now, however, little has been known about the significance of brain TLR2 in controlling inflammation and thermoregulatory responses during systemic Gram-positive bacterial infection. In the present study, the TLR2 immunoreactivity was seen to be prominent in the microglia/macrophages of the circumventricular organs (CVOs) of the mouse brain. The intraperitoneal injection of Pam3CSK4, a TLR2 agonist, induced nuclear factor-κ B activation in the microglia/macrophages of the CVOs. The injection of Pam3CSK4 also produced the expression of Fos at astrocytes and neurons in the CVOs and the regions neighboring the CVOs. The Pam3CSK4 injection induced fever and sickness responses. Pretreatment with lipopolysaccharide, a TLR4 agonist, augmented the Pam3CSK4-induced fever together with the increased TLR2 immunoreactivity. These results indicate that the TLR2 in microglia/macrophages of the CVOs are possibly associated with initiating and transmitting inflammatory responses in the brain.

VEGF- and PDGF-dependent proliferation of oligodendrocyte progenitor cells in the medulla oblongata after LPC-induced focal demyelination.

The myelin sheath is critical in maintaining normal functions of the adult central nervous system (CNS) and the loss of the myelin sheath results in various neurological diseases. Although remyelination is the intrinsic repair system against demyelination that new myelin sheath is formed around axons in the adult CNS, little has been reported on remyelination system in the medulla oblongata. In the present study, we showed that the proliferation of oligodendrocyte progenitor cells (OPCs) was increased in the medulla oblongata by lysophosphatidylcholine (LPC)-induced focal demyelination, but that of NSCs was not changed. The inhibition of vascular endothelial growth factor (VEGF)- and platelet-derived growth factor (PDGF)-signaling suppressed the proliferation of OPCs by LPC-induced demyelination. Thus, the present study indicates that resident OPCs contribute to focal remyelination and VEGF and PDGF signaling is required for the proliferation of OPCs in the medulla oblongata of the adult mouse.

Requirement of extracellular Ca2+ binding to specific amino acids for heat-evoked activation of TRPA1.

KEY POINTS: We found that extracellular Ca2+ , but not other divalent cations (Mg2+ and Ba2+ ) or intracellular Ca2+ , is involved in heat-evoked activation of green anole (ga) TRPA1. Heat-evoked activation of chicken (ch) and rat snake (rs) TRPA1 does not depend solely on extracellular Ca2+ . Neutralization of acidic amino acids on the outer surface of TRPA1 by extracellular Ca2+ is important for heat-evoked large activation of gaTRPA1, chTRPA1 and rsTRPA1. ABSTRACT: Transient receptor potential ankyrin 1 (TRPA1) is a homotetrameric non-selective cation-permeable channel that has six transmembrane domains and cytoplasmic N- and C-termini. The N-terminus is characterized by an unusually large number of ankyrin repeats. Although the 3-dimensional structure of human TRPA1 has been determined, and TRPA1 channels from insects to birds are known to be activated by heat stimulus, the mechanism for temperature-dependent TRPA1 activation is unclear. We previously reported that extracellular Ca2+ , but not intracellular Ca2+ , plays an important role in heat-evoked TRPA1 activation in green anole lizards (gaTRPA1). Here we focus on extracellular Ca2+ -dependent heat sensitivity of gaTRPA1 by comparing gaTRPA1 with heat-activated TRPA1 channels from rat snake (rsTRPA1) and chicken (chTRPA1). In the absence of extracellular Ca2+ , rsTRPA1 and chTRPA1 are activated by heat and generate small inward currents. A comparison of extracellular amino acids in TRPA1 identified three negatively charged amino acid residues (glutamate and aspartate) near the outer pore vestibule that are involved in heat-evoked TRPA1 activation in the presence of extracellular Ca2+ . These results suggest that neutralization of acidic amino acids by extracellular Ca2+ is important for heat-evoked activation of gaTRPA1, chTRPA1, and rsTRPA1, which could clarify mechanisms of heat-evoked channel activation.

Heat and AITC activate green anole TRPA1 in a membrane-delimited manner.

Transient receptor potential ankyrin 1 (TRPA1) is a member of the large TRP super family of ion channels and functions as a Ca(2+)-permeable nonselective cation channel that is activated by various noxious stimuli. TRPA1 was initially identified as a potential mediator of noxious cold stimuli in mammalian nociceptive sensory neurons, while TRPA1s from nonmammalian vertebrates (snakes, green anole lizards, and frogs) were recently reported to be activated by heat, but not cold stimulus. In this study, we examined detailed properties of the green anole TRPA1 channel (gaTRPA1) related to thermal and chemical stimulation in whole-cell and single-channel recordings. Heat activates gaTRPA1 with a temperature threshold for activation of 35.8 °C, while heat together with allyl isothiocyanate (AITC), a chemical agonist, had synergistic effects on gaTRPA1 channel activation in that either the temperature threshold or activating AITC concentration was reduced in the presence of the other stimulus. Significant heat-evoked gaTRPA1 activation was observed in the presence but not absence of extracellular Ca(2+). gaTRPA1 channels were also activated by heat and AITC in excised membrane patches with an inside-out configuration. By comparing the kinetics of heat- and AITC-evoked single-channel currents, we defined similarities and differences of gaTRPA1 channel responses to heat and AITC. We observed similar current-voltage relationship and unitary amplitudes for heat- and AITC-evoked currents and found that heat-activated currents showed shorter durations of both open and closed times. Our results suggest that the gaTRPA1 channel is directly activated by heat and chemical stimuli.