Evidence of dysregulation of the CREB/CRE transcriptional pathway in animal models of Huntington's disease (HD) suggests that strategies designed to augment CRE-mediated transcription may be of therapeutic value. Here, we investigated the consequences of CREB activation and repression in chemical and transgenic mouse models of HD. In the 3-nitropropionic acid (3-NP) model, CREB phospho-activation in the striatum was potently repressed within the neurotoxic "core" region prior to cell death. Conversely, marked expression of phospho-CREB, as well the CREB-regulated cytoprotective gene Bcl-2, was detected in the "penumbral" region. To examine potential contributory roles for the CREB/CRE transcriptional pathway in striatal degeneration, we used both CREB loss- (A-CREB) and gain- (VP16-CREB) of-function transgenic mouse strains. 3-NP-induced striatal lesion size and motor dysfunction were significantly increased in A-CREB mice compared to controls. Conversely, striatal damage and motor deficits were diminished in VP16-CREB mice. Furthermore, transgenic A-CREB significantly accelerated motor impairment in the YAC128 mouse model of HD. Together, these results indicate that CREB functionality is lost during the early stages of striatal cell stress and that the repression of CREB-mediated transcription contributes to the pathogenic process.

LK-3, an amphipathic dimeric peptide linked by two disulfide bonds, and related isomeric bundles were synthesized, and their cell penetrating abilities were investigated. The measurements using size exclusion chromatography and dynamic light scattering show that LK-3 and its isomers form cell penetrating oligomers. Calculations, performed for various types of peptide isomers, elucidate a strong correlation between the amphipathic character of dimers and cell penetration ability. The results suggest that the amphipathicities of LK-3 and related bundle dimers are responsible for their oligomerization propensities which in turn determine their cell penetrating abilities. The observations made in this study provide detailed information about the mechanism of cell uptake of LK-3 and suggest a plausible insight of the early stage of nanoparticle formation of the cell penetrating amphipathic peptides.

Human ribosomal protein S3 (hRpS3) is a component of the 40S ribosomal subunit that associated in protein synthesis. hRpS3 has additional ribosomal functions such as DNA repair, transcription, metastasis, and apoptosis via interaction with numerous signaling molecules and has different modifications. Cyclin-dependent kinases (CDKs) are heterodimeric serine/threonine protein kinases that regulate cell cycle progression. Among its members, the Cdk1-cyclin B complex is known to control cell progression in the G2/M phase, while Cdk2-cyclin E/A complexes function in G1/S and S/G2 transition. In our previous study, we observed interaction between hRpS3 and Cdk1. The present study investigated the interaction between hRpS3 and Cdk2. Cdk2 phosphorylated hRps3 at amino acid residues S6 and T221 during the S-phase. Furthermore, hRpS3 knockdown delayed cell cycle progression by modulating the expression of cell cycle-related proteins, including cyclin B1 and cyclin E1. These findings suggest that hRpS3 is involved in Cdk2-mediated cell cycle regulation.

Crosstalk between G-protein signaling and glutamatergic transmission within the brain reward circuits is critical for long-term emotional effects (depression and anxiety), cravings, and negative withdrawal symptoms associated with opioid addiction. A previous study showed that Regulator of G-protein signaling 4 (RGS4) may be implicated in opiate action in the nucleus accumbens (NAc). However, the mechanism of the NAc-specific RGS4 actions that induce the behavioral responses to opiates remains largely unknown. The present study used a short hairpin RNA (shRNA)-mediated knock-down of RGS4 in the NAc of the mouse brain to investigate the relationship between the activation of ionotropic glutamate receptors and RGS4 in the NAc during morphine reward. Additionally, the shRNA-mediated RGS4 knock-down was implemented in NAc/striatal primary-cultured neurons to investigate the role that striatal neurons have in the morphine-induced activation of ionotropic glutamate receptors. The results of this study show that the NAc-specific knockdown of RGS4 significantly increased the behaviors associated with morphine and did so by phosphorylation of the GluR1 (Ser831) and NR2A (Tyr1325) glutamate receptors in the NAc. Furthermore, the knock-down of RGS4 enhanced the phosphorylation of the GluR1 and NR2A glutamate receptors in the primary NAc/striatal neurons during spontaneous morphine withdrawal. These findings show a novel molecular mechanism of RGS4 in glutamatergic transmission that underlies the negative symptoms associated with morphine administration.

Absence seizures are caused by abnormal synchronized oscillations in the thalamocortical (TC) circuit, which result in widespread spike-and-wave discharges (SWDs) on electroencephalography (EEG) as well as impairment of consciousness. Thalamic reticular nucleus (TRN) and TC neurons are known to interact dynamically to generate TC circuitry oscillations during SWDs. Clinical studies have suggested the association of Plcβ1 with early-onset epilepsy, including absence seizures. However, the brain regions and circuit mechanisms related to the generation of absence seizures with Plcβ1 deficiency are unknown. In this study, we found that loss of Plcβ1 in mice caused spontaneous complex-type seizures, including convulsive and absence seizures. Importantly, TRN-specific deletion of Plcβ1 led to the development of only spontaneous SWDs, and no other types of seizures were observed. Ex vivo slice patch recording demonstrated that the number of spikes, an intrinsic TRN neuronal property, was significantly reduced in both tonic and burst firing modes in the absence of Plcβ1 . We conclude that the loss of Plcβ1 in the TRN leads to decreased excitability and impairs normal inhibitory neuronal function, thereby disrupting feedforward inhibition of the TC circuitry, which is sufficient to cause hypersynchrony of the TC system and eventually leads to spontaneous absence seizures. Our study not only provides a novel mechanism for the induction of SWDs in Plcβ1 -deficient patients but also offers guidance for the development of diagnostic and therapeutic tools for absence epilepsy.

Chemokines are small secreted signaling proteins produced by a broad range of cells, including immune cells. Several studies have recently suggested potential roles of chemokines and their receptors in the pathophysiology of autism spectrum disorders (ASDs). SAM3 is a novel brain-specific chemokine-like molecule with an unknown physiological function. We explored the relevance of chemokines in the development of ASD in mice, with a focus on SAM3. We generated Sam3 gene knockout (KO) mice and characterized their behavioral phenotypes, with a focus on those relevant to ASD. Sam3-deficient mice displayed all three core phenotypes of ASD: impaired responses to social novelty, defects in social communication, and increased repetitive behavior. In addition, they showed increased anxiety. Interestingly, gender differences were identified for several behaviors: only male Sam3 KO mice exhibited increased anxiety and increased repetitive behaviors. Sam3 KO mice did not exhibit changes in other behaviors, including locomotor activities, fear learning and memory, and object recognition memory. These findings indicate that a deficiency of SAM3, a novel brain-specific chemokine-like molecule, may lead to the pathogenesis of ASDs and suggest the possibility that SAM3, a soluble factor, could be a novel therapeutic target for ASD treatment.

Cav1.3 has been suggested to mediate hippocampal neurogenesis of adult mice and contribute to hippocampal-dependent learning and memory processes. However, the mechanism of Cav1.3 contribution in these processes is unclear. Here, roles of Cav1.3 of mouse dorsal hippocampus during newborn cell development were examined. We find that knock-out (KO) of Cav1.3 resulted in the reduction of survival of newborn neurons at 28 days old after mitosis. The retroviral eGFP expression showed that both dendritic complexity and the number and length of mossy fiber bouton (MFB) filopodia of newborn neurons at ≥ 14 days old were significantly reduced in KO mice. Both contextual fear conditioning (CFC) and object-location recognition tasks were impaired in recent (1 day) memory test while passive avoidance task was impaired only in remote (≥ 20 days) memory in KO mice. Results using adeno-associated virus (AAV)-mediated Cav1.3 knock-down (KD) or retrovirus-mediated KD in dorsal hippocampal DG area showed that the recent memory of CFC was impaired in both KD mice but the remote memory was impaired only in AAV KD mice, suggesting that Cav1.3 of mature neurons play important roles in both recent and remote CFC memory while Cav1.3 in newborn neurons is selectively involved in the recent CFC memory process. Meanwhile, AAV KD of Cav1.3 in ventral hippocampal area has no effect on the recent CFC memory. In conclusion, the results suggest that Cav1.3 in newborn neurons of dorsal hippocampus is involved in the survival of newborn neurons while mediating developments of dendritic and axonal processes of newborn cells and plays a role in the memory process differentially depending on the stage of maturation and the type of learning task.

Human prostaglandin E2 receptor 4 (EP4) is one of the four subtypes of prostaglandin E2 (PGE2) receptors and belongs to the rhodopsin-type G protein-coupled receptor (GPCR) family. Particularly, EP4 is expressed in various cancer cells and is involved in cancer-cell proliferation by a G protein signaling cascade. To prepare an active form of EP4 for biochemical characterization and pharmaceutical application, this study designed a recombinant protein comprising human EP4 fused to the P9 protein (a major envelope protein of phi6 phage) and overexpressed the P9-EP4 fusion protein in the membrane fraction of E. coli. The solubilized P9-EP4 with sarkosyl (a strong anionic detergent) was purified by affinity chromatography. The purified protein was stabilized with amphiphilic polymers derived from poly-γ-glutamate. The polymer-stabilized P9-EP4 showed specific interaction with the alpha subunits of Gs or Gi proteins, and a high content of α-helical structure by a circular dichroism spectroscopy. Furthermore, the polymer-stabilized P9-EP4 showed strong heat resistance compared with P9-EP4 in detergents. The functional preparation of EP4 and its stabilization with amphiphilic polymers could facilitate both the biochemical characterization and pharmacological applications targeting EP4.

Established fear memory becomes vulnerable to disruption after memory retrieval and extinction; this labile state is critical for inhibiting the return of fear memory. However, the labile state has a very narrow time window after retrieval, and underlying molecular mechanisms are not well known. To that end, we isolated the hippocampus immediately after fear memory retrieval and performed proteomics. We identified Neurobeachin (NBEA), an autism-related regulator of synaptic protein trafficking, to be upregulated after contextual fear memory retrieval. NBEA protein expression was rapid and transient after fear memory retrieval at the synapse. Nbea mRNA was enriched at the synapses, and the rapid induction of NBEA expression was blocked by inhibition of the mammalian target of rapamycin (mTOR)-dependent signaling pathway. Mice with cornu ammonis 1 (CA1)-specific Nbea shRNA knockdown showed normal fear acquisition and contextual fear memory but impaired extinction, suggesting an important role of Nbea in fear memory extinction processes. Consistently, Nbea heterozygotes showed normal fear acquisition and fear memory recall but showed impairment in extinction. Our data suggest that NBEA is necessary either for induction of memory lability or for the physiological process of memory extinction.

Human DNA topoisomerase II-binding protein 1 (hTopBP1) plays an important role in DNA replication and the DNA damage checkpoint pathway. The human mutY homolog (hMYH) is a base excision repair DNA glycosylase that excises adenines or 2-hydroxyadenines that are mispaired with guanine or 7,8-dihydro-8-oxoguanine (8-oxoG). hTopBP1 and hMYH were involved in ATR-mediated Chk1 activation, moreover, both of them were associated with ATR and hRad9 which known as checkpoint-involved proteins. Therefore, we investigated whether hTopBP1 interacted with hMYH, and what the function of their interaction is.

Silk has been a luxurious commodity throughout modern human history and sericulture has played an important role in ancient global trade as well as technological and cultural developments. Archaeological findings suggest that prior to domestication of the mulberry silkworm (Bombyx mori) silks were obtained from a range of silk-producing moth species with regional specificity. However, investigating the origins of sericulture is difficult as classification of silks by species-type has proved technically challenging. We therefore investigated a range of methods for solubilising modern and archaeological silks and developed a mass spectrometry-based proteomics method that was able to successfully differentiate modern Bombyx, Antheraea, and Samia-produced silks down to the species level. We subsequently analysed archaeological silk materials excavated from the ancient city of Palmyra. Solubilisation behaviour and proteomic analysis provided evidence that the Palmyra silks were constructed from wild silk derived from Antheraea mylitta, the Indian Tasar silkworm. We believe this is the first species-level biochemical evidence that supports archaeological theories about the production and trade of Indian wild silks in antiquity.

Active plant immune response involving programmed cell death called the hypersensitive response (HR) is elicited by microbial effectors delivered through the type III secretion system (T3SS). The marine bacterium Hahella chejuensis contains two T3SSs that are similar to those of animal pathogens, but it was able to elicit HR-like cell death in the land plant Nicotiana benthamiana. The cell death was comparable with the transcriptional patterns of H. chejuensis T3SS-1 genes, was mediated by SGT1, a general regulator of plant resistance, and was suppressed by AvrPto1, a type III-secreted effector of a plant pathogen that inhibits HR. Thus, type III-secreted effectors of a marine bacterium are capable of inducing the nonhost HR in a land plant it has never encountered before. This suggests that plants may have evolved to cope with a potential threat posed by alien pathogen effectors. Our work documents an exceptional case of nonhost HR and provides an expanded perspective for studying plant nonhost resistance.

The gamma-amino-butyric acid (GABA) system is a critical mediator of fear extinction process. GABA can induce "phasic" or "tonic" inhibition in neurons through synaptic or extrasynaptic GABAA receptors, respectively. However, role of the thalamic "tonic GABA inhibition" in cognition has not been explored. We addressed this issue in extinction of conditioned fear in mice.

All gammaherpesviruses express homologues of antiapoptotic B-cell lymphoma-2 (BCL-2) to counter the clearance of infected cells by host antiviral defense machineries. To gain insights into the action mechanisms of these viral BCL-2 proteins, we carried out structural and biochemical analyses on the interactions of M11, a viral BCL-2 of murine gamma-herpesvirus 68, with a fragment of proautophagic Beclin1 and BCL-2 homology 3 (BH3) domain-containing peptides derived from an array of proapoptotic BCL-2 family proteins. Mainly through hydrophobic interactions, M11 bound the BH3-like domain of Beclin1 with a dissociation constant of 40 nanomole, a markedly tighter affinity compared to the 1.7 micromolar binding affinity between cellular BCL-2 and Beclin1. Consistently, M11 inhibited autophagy more efficiently than BCL-2 in NIH3T3 cells. M11 also interacted tightly with a BH3 domain peptide of BAK and those of the upstream BH3-only proteins BIM, BID, BMF, PUMA, and Noxa, but weakly with that of BAX. These results collectively suggest that M11 potently inhibits Beclin1 in addition to broadly neutralizing the proapoptotic BCL-2 family in a similar but distinctive way from cellular BCL-2, and that the Beclin1-mediated autophagy may be a main target of the virus.

Status epilepticus (SE) triggers neuronal death, reactive gliosis and remodeling of synaptic circuitry, thus leading to profound pathological alterations in CNS physiology. These processes are, in part, regulated by the rapid upregulation of both cytotoxic and cytoprotective genes. One pathway that may couple SE to transcriptionally dependent alterations in CNS physiology is the CREB (cAMP response element-binding protein)/CRE (cAMP response element) cascade. Here, we utilized the pilocarpine model of SE on a mouse strain transgenic for a CRE-reporter construct (beta-galactosidase) to begin to characterize how seizure activity regulates the activation state of the CREB/CRE pathway in both glia and neurons of the hippocampus. SE triggered a rapid (4-8 h post-SE) but transient increase in CRE-mediated gene expression in the neuronal sublayers. In contrast to neurons, SE induced a lasting increase (up to 20 days) in CRE-mediated transcription in both reactive astrocytes and microglia. CRE-mediated gene expression correlated with expression of the pro-inflammatory enzyme cyclooxygenase-2 (COX-2). To examine the role of CREB in SE-induced COX-2 expression, we generated a transgenic mouse strain that expresses A-CREB, a potent repressor of CREB-dependent transcription. In these animals, the capacity of SE to stimulate COX-2 expression was markedly attenuated, indicating that CREB is a key intermediate in SE-induced COX-2 expression. Collectively these data show that SE triggers two waves of CREB-mediated gene expression, a transient wave in neurons and a long-lasting wave in reactive glial cells, and that CREB couples SE to COX-2 expression.

Adenylate kinase 2 (AK2), which balances adenine nucleotide pool, is a multi-functional protein. Here we show that AK2 negatively regulates tumour cell growth. AK2 forms a complex with dual-specificity phosphatase 26 (DUSP26) phosphatase and stimulates DUSP26 activity independently of its AK activity. AK2/DUSP26 phosphatase protein complex dephosphorylates fas-associated protein with death domain (FADD) and regulates cell growth. AK2 deficiency enhances cell proliferation and induces tumour formation in a xenograft assay. This anti-growth function of AK2 is associated with its DUSP26-stimulating activity. Downregulation of AK2 is frequently found in tumour cells and human cancer tissues showing high levels of phospho-FADD(Ser194). Moreover, reconstitution of AK2 in AK2-deficient tumour cells retards both cell proliferation and tumourigenesis. Consistent with this, AK2(+/-) mouse embryo fibroblasts exhibit enhanced cell proliferation with a significant alteration in phospho-FADD(Ser191). These results suggest that AK2 is an associated activator of DUSP26 and suppresses cell proliferation by FADD dephosphorylation, postulating AK2 as a negative regulator of tumour growth.

The establishment and maintenance of social dominance are critical for social stability and the survival and health of individual animals. Stress lead to depression and a decrease in the social status of depressed persons is a risk factor for suicide. Therefore, we explored the mechanistic and behavioral links among stress, depression, and social dominance and found that mice subjected to chronic restraint stress (CRS), an animal model of stress-induced depression, showed decreased social dominance as measured by a dominance tube test. Importantly, this submissive behavior was occluded by the antidepressant, fluoxetine, a selective serotonin reuptake inhibitor. It is known that social dominance is controlled by synaptic efficacy in the medial prefrontal cortex (mPFC) and that AMPA-type glutamate receptor (AMPA-R) is a key molecule for synaptic efficacy. We found that the phosphorylation on AMPA-R was bidirectionally changed by CRS and fluoxetine in the mPFC of mice with CRS. Moreover, we found a strong correlation between social dominance and AMPA-R phosphorylation that regulates synaptic efficacy by modulating the synaptic targeting of AMPA-R. Our correlational analysis of the behavior and biochemistry of the CRS model suggests that AMPA-R phosphorylation in the mPFC may serve as a biomarker of social dominance related to stress.

Fibrillar aggregates of amyloid-β (Aβ) are the main component of plaques lining the cerebrovasculature in cerebral amyloid angiopathy. As the predominant Aβ isoform in vascular deposits, Aβ40 is a valuable target in cerebral amyloid angiopathy research. However, the slow process of Aβ40 aggregation in vitro is a bottleneck in the search for Aβ-targeting molecules. In this study, we sought a method to accelerate the aggregation of Aβ40 in vitro, to improve experimental screening procedures. We evaluated the aggregating ability of bicine, a biological buffer, using various in vitro methods. Our data suggest that bicine promotes the aggregation of Aβ40 with high speed and reproducibility, yielding a mixture of aggregates with significant β-sheet-rich fibril formation and toxicity.

Persistent poor sleep quality leads to impaired cognitive performance and an inability to perform daily activities. Biomarker-assisted diagnosis is important for the early treatment of poor sleep quality; however, diagnostic biomarkers for poor sleep quality remain unidentified. Circulating microRNAs (miRNAs) have been reported to be linked to the pathogenesis of poor sleep quality, indicating their possible role in sleep problem diagnosis. The present study aimed to identify potential miRNA biomarkers for poor sleep quality.

N-methyl-D-aspartate receptor (NMDAR) modulators have recently received increased attention as potential therapeutics for posttraumatic stress disorder (PTSD). Here, we tested a novel NMDAR-positive modulator, NYX-783, in the following two rodent models of PTSD: an auditory fear-conditioning model and a single-prolonged stress (SPS) model. We examined the ability of NYX-783 to reduce subsequent fear-based behaviors by measuring enhanced fear extinction and reduced spontaneous recovery (spontaneous return of fear) in male mice. NYX-783 administration significantly reduced spontaneous recovery in both PTSD models and enhanced fear extinction in the SPS model. Furthermore, NYX-783 increased the NMDA-induced inward currents of excitatory and inhibitory neurons in the infralimbic medial prefrontal cortex (IL mPFC) and that the GluN2B subunit of NMDARs on pyramidal neurons in the IL mPFC is required for its effect on spontaneous recovery. The downstream expression of brain-derived neurotrophic factor was required for NYX-783 to achieve its behavioral effect. These results elucidate the cellular targets of NYX-783 and the molecular mechanisms underlying the inhibition of spontaneous recovery. These preclinical findings support the hypothesis that NYX-783 may have therapeutic potential for PTSD treatment and may be particularly useful for inhibiting spontaneous recovery.