Differential brain expression pattern of Sez6 alternative splicing isoform with deleted transmembrane domain.

Seizure-related gene 6 (Sez6) is a transmembrane protein specifically localized on neuronal dendrites and responsible for dendritic branching and synapse formation. Alternative splicing produces three isoforms of Sez6 mRNAs: the dominant isoform encodes a transmembrane-type protein, whereas the two recessive isoforms encode transmembrane and secretory proteins. In the present study, to clarify the differential functions of these isoforms, the expression patterns resulting from Sez6 splicing isoforms were investigated in the mouse brain as well as in cultured neurons. The whole brains were sliced into coronal sections of 1-mm thickness, and brain areas were punched out from these coronal sections. The mRNA levels of each Sez6 isoform in the prefrontal cortex, cingulate cortex, striatum, hippocampus, and amygdala, where Sez6 expression has been reported previously, were analyzed using a qPCR technique, and primary neurons cultured under different treatment conditions were assessed in terms of increased Sez6 gene expression. Our results show that the splicing patterns of Sez6 were modulated in a brain area-specific manner. In particular, the striatum showed a characteristic splicing pattern of recessive isoforms. Moreover, neuronal activation by convulsant drug stimulation increased recessive isoforms like the dominant isoform in cultured cortical neurons at 5 or 10 days in vitro. In conclusion, alternative splicing of Sez6, as well as of other proteins expressed specifically in the brain, results in brain area-specific expression patterns. Furthermore, the alternative splicing of Sez6 may be modulated by drugs that elevate Sez6 gene expression.

Susceptibility of common family Anatidae bird species to clade 2.3.4.4e H5N6 high pathogenicity avian influenza virus: an experimental infection study.

BACKGROUND: There were large outbreaks of high pathogenicity avian influenza (HPAI) caused by clade 2.3.4.4e H5N6 viruses in the winter of 2016-2017 in Japan, which caused large numbers of deaths among several endangered bird species including cranes, raptors, and birds in Family Anatidae. In this study, susceptibility of common Anatidae to a clade 2.3.4.4e H5N6 HPAI virus was assessed to evaluate their potential to be a source of infection for other birds. Eurasian wigeons (Mareca penelope), mallards (Anas platyrhynchos), and Northern pintails (Anas acuta) were intranasally inoculated with 106, 104, or 102 50% egg infectious dose (EID50) of clade 2.3.4.4e A/teal/Tottori/1/2016 (H5N6). RESULTS: All birds survived for 10 days without showing any clinical signs of infection. Most ducks inoculated with ≥ 104 EID50 of virus seroconverted within 10 days post-inoculation (dpi). Virus was mainly shed via the oral route for a maximum of 10 days, followed by cloacal route in late phase of infection. Virus remained in the pancreas of some ducks at 10 dpi. Viremia was observed in some ducks euthanized at 3 dpi, and ≤ 106.3 EID50 of virus was recovered from systemic tissues and swab samples including eyeballs and conjunctival swabs. CONCLUSIONS: These results indicate that the subject duck species have a potential to be a source of infection of clade 2.3.4.4e HPAI virus to the environment and other birds sharing their habitats. Captive ducks should be reared under isolated or separated circumstances during the HPAI epidemic season to prevent infection and further viral dissemination.

Contribution of mutation I142M in fusion protein and Q44R in matrix protein of Newcastle disease virus to virulence in ducks.

Although verogenic Newcastle disease viruses (NDVs) generally cause subclinical infection in waterfowls such as ducks, NDVs with high virulence in waterfowl have been sporadically reported. We previously reported that the NDV d5a20b strain, which is obtained by serial passaging of the velogenic 9a5b strain in domestic ducks, showed increased virulence in ducks (Hidaka et al., 2021). The d5a20b strain had 11 amino acid substitutions in its P/V, M, F, HN, and L proteins as compared to 9a5b. In the present study, we generated a series of recombinant (r) NDVs with these amino acid substitutions to identify the molecular basis of virulence of NDV in ducks, and evaluated their influences on virulence and in vitro viral properties. Each of the single amino acid substitutions in either the F protein I142M or the M protein Q44R contributed to the enhancement of intracerebral and intranasal pathogenicity in domestic ducks. The cell-cell fusion activity of the virus with F I142M was five times higher than that of the parental r9a5b. The virus with M Q44R rapidly replicated in duck embryo fibroblasts. Additionally, the rM+F+HN strain, which has the same amino acid sequences as d5a20b in M, F, and HN proteins, showed the highest level of virulence and replication efficiency among the generated recombinant viruses, nearly comparable to rd5a20b. These results suggest that multiple factors are involved in the high growth ability of NDV in duck cells, leading to increased virulence in vivo.

Separation from a bonded partner alters neural response to inflammatory pain in monogamous rodents.

Pain experience is known to be modified by social factors, but the brain mechanisms remain unspecified. We recently established an animal model of social stress-induced hyperalgesia (SSIH) using a socially monogamous rodent, the prairie vole, in which males separated from their female partners (loss males) became anxious and displayed exacerbated inflammatory pain behaviors compared to males with partners (paired males). In the present study, to explore the neural pathways involved in SSIH, a difference in neuronal activation in pain-related brain regions, or "pain matrix", during inflammatory pain between paired and loss males was detected using Fos immunoreactivity (Fos-ir). Males were paired with a female and pair bonding was confirmed in all subjects using a partner preference test. During formalin-induced inflammatory pain, both paired and loss males showed a significant induction of Fos-ir throughout the analyzed pain matrix components compared to basal condition (without injection), and no group differences in immunoreactivity were found among the injected males in many brain regions. However, the loss males had significantly lower Fos-ir following inflammatory pain in the medial prefrontal cortex and nucleus accumbens shell than the paired males, even though base Fos-ir levels were comparable between groups. Notably, both regions with different Fos-ir are major components of the dopamine and oxytocin systems, which play critical roles in both pair bonding and pain regulation. The present results suggest the possibility that pain exacerbation by social stress emerges through alteration of signaling in social brain circuitry.

Prior infection with antigenically heterologous low pathogenic avian influenza viruses interferes with the lethality of the H5 highly pathogenic strain in domestic ducks.

Low and highly pathogenic avian influenza viruses (LPAIVs and HPAIVs, respectively) have been co-circulating in poultry populations in Asian, Middle Eastern, and African countries. In our avian-flu surveillance in Vietnamese domestic ducks, viral genes of LPAIV and HPAIV have been frequently detected in the same individual. To assess the influence of LPAIV on the pathogenicity of H5 HPAIV in domestic ducks, an experimental co-infection study was performed. One-week-old domestic ducks were inoculated intranasally and orally with phosphate-buffered saline (PBS) (control) or 106 EID50 of LPAIVs (A/duck/Vietnam/LBM678/2014 (H6N6) or A/Muscovy duck/Vietnam/LBM694/2014 (H9N2)). Seven days later, these ducks were inoculated with HPAIV (A/Muscovy duck/Vietnam/LBM808/2015 (H5N6)) in the same manner. The respective survival rates were 100% and 50% in ducks pre-infected with LBM694 or LBM678 strains and both higher than the survival of the control group (25%). The virus titers in oral/cloacal swabs of each LPAIV pre-inoculation group were significantly lower at 3-5 days post-HPAIV inoculation. Notably, almost no virus was detected in swabs from surviving individuals of the LBM678 pre-inoculation group. Antigenic cross-reactivity among the viruses was not observed in the neutralization test. These results suggest that pre-infection with LPAIV attenuates the pathogenicity of HPAIV in domestic ducks, which might be explained by innate and/or cell-mediated immunity induced by the initial infection with LPAIV.

The chicken-derived velogenic Newcastle disease virus can acquire high pathogenicity in domestic ducks via serial passaging.

Velogenic Newcastle disease virus (NDV) strains, which show high mortality in chickens, generally do not cause severe disease in waterfowl such as ducks. To elucidate the difference in the pathogenic mechanisms of NDV between chickens and ducks, a chicken-derived velogenic strain (9a5b) was passaged in domestic ducks five times in their air sacs, followed by 20 times in their brains. Eventually, 9a5b acquired higher intracerebral and intranasal pathogenicity in ducks. The intracerebral pathogenicity index (ICPI) value increased from 1.10 to 1.88. All one-week-old ducks intranasally inoculated with the passaged virus (d5a20b) died by 5 days post-inoculation, whereas 70% of the ducks inoculated with parental 9a5b survived for 8 days. The d5a20b strain replicated in broader systemic tissues in ducks compared with the 9a5b strain. The velogenic profile of 9a5b in chickens was maintained after passaging in ducks. The d5a20b suppressed IFN-ß gene expression in duck embryo fibroblasts and replicated more rapidly than 9a5b. A total of 11 amino acid substitutions were found in the P, V, M, F, HN, and L proteins of d5a20b. These results suggest that chicken-derived velogenic NDVs have the potential to become virulent in both chickens and ducks during circulation in domesticated waterfowl populations. RESEARCH HIGHLIGHTSChicken-derived NDV acquired high pathogenicity in ducks with serial passaging.The passaged NDV showed intracerebral and intranasal pathogenicity in ducks.The passaged NDV efficiently replicated in systemic tissues in ducks.Of 11 amino acid substitutions some or all are likely involved in pathogenicity.

Effects of post-weaning social isolation on social behaviors and oxytocinergic activity in male and female rats.

AIMS: Post-weaning social deprivation is known to induce behavioral and neuronal alterations associated with anxiety and stress responses in adulthood. However, the effects of social deprivation on the development of sociability are poorly understood. We examined the effects of social deprivation on subsequent social behaviors and oxytocinergic activity using socially-isolated (approximately two months post-weaning) male and female rats. MAIN METHODS: The behaviors were analyzed using a social preference test and a social approach test. Immunohistochemical investigations were conducted in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) to examine the effects of social isolation on oxytocinergic activity in these regions. Oxytocinergic activity was measured by quantifying the number of oxytocin neurons expressing Fos following exposure to a novel conspecific. In all of the experiments of this study, ovariectomized females were used for social stimuli. KEY FINDINGS: The behavioral results show that isolation-reared females, but not males, displayed impaired social preference and decreased social approach towards ovariectomized females, compared with the pair-reared group, suggesting low priority of processing social versus non-social stimuli and low motivation for contact with a stranger, respectively. The immunohistochemical results show that social isolation decreased both the number and the ratio of Fos-positive cells in oxytocin neurons in the PVN in females, but not in males, following exposure to ovariectomized females. In the SON, the Fos-positive ratio was decreased in isolation-reared females, but not in males, compared with the pair-reared group. SIGNIFICANCE: Post-weaning social isolation changed social behaviors and oxytocinergic activity in female rats, suggesting that in female rats post-weaning social experiences contribute to the development of sociability. These findings could impact the treatment of social dysfunction in humans.

Vulnerability or resilience of motopsin knockout mice to maternal separation stress depending on adulthood behaviors.

BACKGROUND: Both environmental and genetic conditions contribute to the robust development of neuronal circuits and adulthood behaviors. Loss of motopsin gene function causes severe intellectual disability in humans and enhanced social behavior in mice. Furthermore, childhood maltreatment is a risk factor for some psychiatric disorders, and children with disabilities have a higher risk of abuse than healthy children. MATERIALS AND METHODS: In this study, we investigated the effects of maternal separation (MS) on adulthood behaviors of motopsin knockout (KO) and wild-type (WT) mice. RESULTS: The MS paradigm decreased the duration that WT mice stayed in the center area of an open field, but not for motopsin KO mice; however, it decreased the novel object recognition index in both genotypes. In the marble burying test, motopsin KO mice buried fewer marbles than WT mice, regardless of the rearing conditions. The MS paradigm slightly increased and reduced open arm entry in the elevated plus maze by WT and motopsin KO mice, respectively. In the three-chamber test, the rate of sniffing the animal cage was increased by the MS paradigm only for motopsin KO mice. After the three-chamber test, motopsin KO mice had fewer cFos-positive cells in the prelimbic cortex, which is involved in emotional response, than WT mice. In the infralimbic cortex, the MS paradigm decreased the number of cFos-positive cells in motopsin KO mice. CONCLUSION: Our results suggest that motopsin deficiency and childhood adversity independently affect some behaviors, but they may interfere with each other for other behaviors. Defective neuronal circuits in the prefrontal cortex may add to this complexity.

Spatio-temporal regulations and functions of neuronal alternative RNA splicing in developing and adult brains.

Alternative pre-mRNA splicing is a fundamental mechanism that generates molecular diversity from a single gene. In the central nervous system (CNS), key neural developmental steps are thought to be controlled by alternative splicing decisions, including the molecular diversity underlying synaptic wiring, plasticity, and remodeling. Significant progress has been made in understanding the molecular mechanisms and functions of alternative pre-mRNA splicing in neurons through studies in invertebrate systems; however, recent studies have begun to uncover the potential role of neuronal alternative splicing in the mammalian CNS. This article provides an overview of recent findings regarding the regulation and function of neuronal alternative splicing. In particular, we focus on the spatio-temporal regulation of neurexin, a synaptic adhesion molecule, by neuronal cell type-specific factors and neuronal activity, which are thought to be especially important for characterizing neural development and function within the mammalian CNS. Notably, there is increasing evidence that implicates the dysregulation of neuronal splicing events in several neurological disorders. Therefore, understanding the detailed mechanisms of neuronal alternative splicing in the mammalian CNS may provide plausible treatment strategies for these diseases.

N-Glycosylation modulates filopodia-like protrusions induced by sez-6 through regulating the distribution of this protein on the cell surface.

Seizure-related gene 6 (sez-6) is a trans-membrane protein expressed by neuronal cells that modulates dendritic branching. It has three clusters of eleven possible N-glycosylation sites in the extracellular domain region: sugar chain (SC)1-3, SC4-7, and SC8-11. Recent reports suggest that N-glycosylation modulates the membrane trafficking and function of trans-membrane proteins. Here, we studied the role of N-glycosylation in sez-6 function. We transfected mutants lacking one, two, or all N-glycosylation clusters into neuro2a cells. A mutant lacking all N-glycosylation was transported to the cell membrane. Mutants lacking one cluster (sez-6 ΔSC1-3, ΔSC4-7, ΔSC8-11) were evenly distributed on the cell membrane and secreted into the conditioned medium, as in wild-type sez-6; in contrast, the unglycosylated mutant, sez-6 ΔSC1-11, and mutants having only one cluster (sez-6 SC1-3, SC8-11) were localized in some portions on the cell membrane. Despite sez-6 SC4-7 having only one cluster, it was transported like the wild type. Among mutants behaving like the wild type, sez-6 ΔSC1-3 and ΔSC4-7 reduced neurite formation. Interestingly, mutants lacking SC4-7 (sez-6 ΔSC4-7) did not affect the formation of filopodia-like protrusions. In contrast, other mutants as well as the wild type induced it, suggesting that SC4-7 is crucial for filopodia-like protrusions. Our results indicate that N-glycosylation regulates cell morphology through modulating the cell surface distribution of sez-6 protein.

A mental retardation gene, motopsin/prss12, modulates cell morphology by interaction with seizure-related gene 6.

A serine protease, motopsin (prss12), plays a significant role in cognitive function and the development of the brain, since the loss of motopsin function causes severe mental retardation in humans and enhances social behavior in mice. Motopsin is activity-dependently secreted from neuronal cells, is captured around the synaptic cleft, and cleaves a proteoglycan, agrin. The multi-domain structure of motopsin, consisting of a signal peptide, a proline-rich domain, a kringle domain, three scavenger receptor cysteine-rich domains, and a protease domain at the C-terminal, suggests the interaction with other molecules through these domains. To identify a protein interacting with motopsin, we performed yeast two-hybrid screening and found that seizure-related gene 6 (sez-6), a transmembrane protein on the plasma membrane of neuronal cells, bound to the proline-rich/kringle domain of motopsin. Pull-down and immunoprecipitation analyses indicated the interaction between these proteins. Immunocytochemical and immunohistochemical analyses suggested the co-localization of motopsin and sez-6 at neuronal cells in the developmental mouse brain and at motor neurons in the anterior horn of human spinal cords. Transient expression of motopsin in neuro2a cells increased the number and length of neurites as well as the level of neurite branching. Interestingly, co-expression of sez-6 with motopsin restored the effect of motopsin at the basal level, while sez-6 expression alone showed no effects on cell morphology. Our results suggest that the interaction of motopsin and sez-6 modulates the neuronal cell morphology.

A novel monoclinic phase of impurity-doped CaGa(2)S(4) as a phosphor with high emission intensity.

In the solid-state synthesis of impurity-doped CaGa(2)S(4), calcium tetra-thio-digallate(III), a novel phosphor material (denominated as the X-phase), with monoclinic symmetry in the space group P2(1)/a, has been discovered. Its emission intensity is higher than that of the known ortho-rhom-bic polymorph of CaGa(2)S(4) crystallizing in the space group Fddd. The asymmetric unit of the monoclinic phase consists of two Ca, four Ga and eight S sites. Each of the Ca and Ga atoms is surrounded by seven and four sulfide ions, respectively, thereby sharing each of the sulfur sites with the nearest neighbours. In contrast, the corresponding sites in the ortho-rhom-bic phase are surrounded by eight and four S atoms, respectively. The photoluminescence peaks from Mn(2+) and Ce(3+) in the doped X-phase, both of which are supposed to replace Ca(2+) ions, have been observed to shift towards the high energy side in comparison with those in the ortho-rhom-bic phase. This suggests that the crystal field around the Mn(2+) and Ce(3+) ions in the X-phase is weaker than that in the ortho-rhom-bic phase.