Sodium phenylbutyrate reduces repetitive self-grooming behavior and rescues social and cognitive deficits in mouse models of autism.

Autism spectrum disorder (ASD) is a neurodevelopment disorder characterized by deficits in social interaction and restrictive, repetitive, and stereotypical patterns of behavior. However, there is no pharmacological drug that is currently used to target these core ASD symptoms. Sodium phenylbutyrate (NaPB) is a well-known long-term treatment of urea cycle disorders in children. In this study, we assessed the therapeutic effects of NaPB, which is a chemical chaperone as well as histone deacetylase inhibitor on a BTBR T + Itpr3tf/J (BTBR) mice model of ASD. We found that acute and chronic treatment of NaPB remarkably improved, not only core ASD symptoms, including repetitive behaviors and sociability deficit, but also cognitive impairment in the BTBR mice. NaPB substantially induced histone acetylation in the brain of the BTBR mice. Intriguingly, the therapeutic effects of NaPB on autistic-like behaviors, such as repetitive behaviors, impaired sociability, and cognitive deficit also showed in the valproic acid (VPA)-induced mouse model of autism. In addition, pentylenetetrazole (PTZ)-induced seizure was significantly attenuated by NaPB treatment in C57BL/6J and BTBR mice. These findings suggest that NaPB may provide a novel therapeutic approach for the treatment of patients with ASD.

Novel Amide Derivatives as Potent Tyrosinase Inhibitors; In-vitro, In-vivo Antimelanogenic Activity and Computational Studies.

BACKGROUND: Tyrosinase is involved in the melanin biosynthesis and the abnormal accumulation of melanin pigments leading to hyperpigmentation disorders. Controlling the melanogenesis could be an important strategy for treating abnormal pigmentation. METHODS: In the present study, a series of amide derivatives (3a-e and 5a-e) were synthesized aiming to inhibit tyrosinase activity and melanin production. All derivatives were screened for tyrosinase inhibition in a cell-free system. The possible interactions of amide derivatives with tyrosinase enzyme and effect of these interactions on tyrosinase structure were checked by molecular docking in silico and by Circular Dichroism (CD) studies, respectively. The most potent amide derivative (5c) based on cell-free experiments, was further tested for cellular ROS inhibition and for tyrosinase activity using mouse skin melanoma (B16F10) cells. RESULTS: The tyrosinase inhibitory concentration (IC50) for tested compounds was observed between the range of 68 to 0.0029 µg/ml with a lowest IC50 value of compound 5c which outperforms the reference arbutin and kojic acid. The cellular tyrosinase activity and melanin quantification assay demonstrate that 15µg/ml of 5c attenuates 36% tyrosinase, 24% melanin content of B16F10 cells without significant cell toxicity. Moreover, the zebrafish in vivo assay reveals that 5c effectively reduces melanogenesis without perceptible toxicity. Furthermore, the molecular docking demonstrates that compound 5c interacts with copper ions and multiple amino acids in the active site of tyrosinase with best glide score (-5.387 kcal/mol), essential for mushroom tyrosinase inhibition and the ability to diminish the melanin synthesis in-vitro and in-vivo. CONCLUSION: Thus, we propose compound 5c as a potential candidate to control tyrosinase rooted hyperpigmentation in the future.

Effects of histone acetyltransferase inhibitors on L-DOPA-induced dyskinesia in a murine model of Parkinson's disease.

Histone acetylation is a key regulatory factor for gene expression in cells. Modulation of histone acetylation by targeting of histone acetyltransferases (HATs) effectively alters many gene expression profiles and synaptic plasticity in the brain. However, the role of HATs on L-DOPA-induced dyskinesia of Parkinson's disease (PD) has not been reported. Our aim was to determine whether HAT inhibitors such as anacardic acid, garcinol, and curcumin from natural plants reduce severity of L-DOPA-induced dyskinesia using a unilaterally 6-hydroxydopamine (6-OHDA)-lesioned PD mouse model. Anacardic acid 2 mg/kg, garcinol 5 mg/kg, or curcumin 100 mg/kg co-treatment with L-DOPA significantly reduced the axial, limb, and orofacial (ALO) score indicating less dyskinesia with administration of HAT inhibitors in 6-OHDA-lesioned mice. Additionally, L-DOPA's efficacy was not altered by the compounds in the early stage of treatment. The expression levels of c-Fos, Fra-2, and Arc were effectively decreased by administration of HAT inhibitors in the ipsilateral striatum. Our findings indicate that HAT inhibitor co-treatment with L-DOPA may have therapeutic potential for management of L-DOPA-induced dyskinesia in patients with PD.

Mind Bomb-Binding Partner RanBP9 Plays a Contributory Role in Retinal Development.

Ran-binding protein family member, RanBP9 has been reported in various basic cellular mechanisms and neuropathological conditions including schizophrenia. Previous studies have reported that RanBP9 is highly expressed in the mammalian brain and retina; however, the role of RanBP9 in retinal development is largely unknown. Here, we present the novel and regulatory roles of RanBP9 in retinal development of a vertebrate animal model, zebrafish. Zebrafish embryos exhibited abundant expression of ranbp9 in developing brain tissues as well as in the developing retina. Yeast two-hybrid screening demonstrated the interaction of RanBP9 with Mind bomb, a component of Notch signaling involved in both neurogenesis and neural disease autism. The interaction is further substantiated by co-localization studies in cultured cells. Knockdown of ranbp9 resulted in retinal dysplasia with defective proliferation of retinal cells, downregulation of neuronal differentiation marker huC, elevation of neural proliferation marker her4, and alteration of cell cycle marker p57kip2. Expression of the Müller glial cell marker glutamine synthase was also affected in knockdown morphants. Our results suggest that Mind bomb-binding partner RanBP9 plays a role during retinal cell development of zebrafish embryogenesis.

Expression patterns of prune2 is regulated by Notch and retinoic acid signaling pathways in the zebrafish embryogenesis.

PRUNE2 has been identified as a susceptible gene for Alzheimer's disease and a marker for leiomyosarcomas. Isoforms of Prune2 regulate neuronal cell differentiation and synaptogenesis. Although expression pattern of Prune2 has been reported in the murine brain, its expression patterns and regulation along vertebrate embryogenesis remain to be further investigated. We thus defined the expression profiles and transcriptional regulation of prune2 in zebrafish embryos. prune2 exhibits maternal expression, but is increased in later embryonic stages, and expressed in the telencephalon, epiphysis cluster, nucleus of the tract of the post optic commissure, spinal cord, cerebellum, tegmentum, anterior lateral line ganglion, posterior lateral line ganglion and rhombomeres 2 through 5. Two color WISH with a post-mitotic neuron specific marker, huC defined that prune2 is expressed in the post mitotic neurons. The level of prune2 transcripts is upregulated in Notch signaling homozygous mutant, mib1-/-(mibta52b), indicating that Notch signaling regulates transcription of prune2. Interestingly, in silico analysis of prune2 promoter found retinoic acid (RA) response elements (AGGTCAcaTGACCA) located at -3 to -16 relative to the first exon. It turned out that RA signaling altered the expression pattern of prune2 in the hindbrain. We further propose that Prune2 might be a putative regulator for CNS development in zebrafish embryogenesis.

Gadd45ß ameliorates L-DOPA-induced dyskinesia in a Parkinson's disease mouse model.

The dopamine precursor 3,4-dihydroxyphenyl-l-alanine (L-DOPA) is currently the most efficacious pharmacotherapy for Parkinson's disease (PD). However, long-term L-DOPA treatment leads to the development of abnormal involuntary movements (AIMs) in patients and animal models of PD. Recently, involvement of growth arrest and DNA damage-inducible 45ß (Gadd45ß) was reported in neurological and neurobehavioral dysfunctions. However, little is known about the role of Gadd45ß in the dopaminergic nigrostriatal pathway or L-DOPA-induced dyskinesia (LID). To address this issue, we prepared an animal model of PD using unilateral 6-hydroxydopamine (6-OHDA) lesions in the substantia nigra of Gadd45ß(+/+) and Gadd45ß(-/-) mice. Dyskinetic symptoms were triggered by repetitive administration of L-DOPA in these 6-OHDA-lesioned mice. Whereas dopamine denervation in the dorsal striatum decreased Gadd45ß mRNA, chronic L-DOPA treatment significantly increased Gadd45ß mRNA expression in the 6-OHDA-lesioned striatum of wild-type mice. Using unilaterally 6-OHDA-lesioned Gadd45ß(+/+) and Gadd45ß(-/-) mice, we found that mice lacking Gadd45ß exhibited long-lasting increases in AIMs following repeated administration of L-DOPA. By contrast, adeno-associated virus-mediated expression of Gadd45ß in the striatum reduced AIMs in Gadd45ß knockout mice. The deficiency of Gadd45ß in LID increased expression of ΔFosB and c-Fos in the lesioned striatum 90 min after the last administration of L-DOPA following 11days of daily L-DOPA treatments. These data suggest that the increased expression of Gadd45ß induced by repeated administration of L-DOPA may be beneficial in patients with PD.

Physcion-8-O-beta-D-glucopyranoside enhances the commitment of mouse mesenchymal progenitors into osteoblasts and their differentiation: Possible involvement of signaling pathways to activate BMP gene expression.

Here, we show the involvement of signaling pathways to induce the gene expression of bone morphogenetic protein (BMP) in the osteogenic activity of physcion-8-O-beta-D-glucopyranoside (physcion-Glu); it stimulated osteoblast differentiation in mouse osteoblast MC3T3-E1 subclone 4 cells and induced BMP-2 gene expression and activation of Akt and ERK/MAP kinases. Physcion-Glu-induced BMP-2 expression and mineralization were attenuated by LY294002, an inhibitor of PI3K that lies upstream of Akt and MAP kinases, suggesting that physcion-Glu induces osteoblast differentiation via PI3K-Akt/MAP kinase signaling pathways, which play important roles in inducing BMP-2 gene expression. Physcion-Glu also enhanced BMP-2-induced commitment of mouse bi-potential mesenchymal precursor C2C12 cells into osteoblasts while inducing the transcription of several osteogenic BMP isoforms, such as BMP-2, -4, -7, and -9. Osteogenic synergy between BMP-2 and physcion-Glu was supported by the fact that noggin inhibited BMP-2 and physcion-Glu-induced alkaline phosphatase expression and activity. Considering that physcion-Glu induced Runx2 activity and the nuclear translocation of p-Smad, physcion-Glu could act by enhancing the BMP signaling pathway that induces Smad activation and translocation to activate Runx2. In conclusion, physcion-Glu could enhance the commitment of mesenchymal progenitors into osteoblasts and their differentiation by activating signaling pathways to induce BMP gene expression.

Anti-resorptive saurolactam exhibits in vitro anti-inflammatory activity via ERK-NF-kappaB signaling pathway.

Natural products and their derivatives have historically been an invaluable a source of therapeutic agents. In this report, we demonstrated the anti-inflammatory activity of saurolactam, a compound isolated from the aerial portions of the Chinese lizard, Saururus chinensis. In RAW264.7 macrophage cells, saurolactam significantly inhibited the lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase and cyclooxygenase-2 and, consequently, inhibited the release of NO and prostaglandin E2. Moreover, real-time PCR and multiplex cytokine assays showed that saurolactam (10 microM) significantly inhibited the LPS-induced mRNA and protein expression levels of pro-inflammatory genes, including interleukin (IL)-1beta, IL-6, and tumor necrosis factor-alpha. Finally, western blot analysis showed that saurolactam dose-dependently inhibited LPS-induced extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinase activation and nuclear factor (NF)-kappaB translocation into the nucleus. The inhibitory activity of saurolactam on the activation of NF-kappaB was confirmed by a NF-kappaB luciferase reporter gene assay. In conclusion, we propose that the in vitro anti-inflammatory activity of saurolactam is produced by blocking ERK/MAP kinase and NF-kappaB activation.

Activation of the thyroid-stimulating hormone beta-subunit gene by LIM homeodomain transcription factor Lhx2.

Although there is evidence that the LIM homeodomain transcription factor, Lhx2, can stimulate transcription of the glycoprotein hormone alpha-subunit gene, the role of Lhx2 in regulating TSH beta-subunit has not been established. In the present studies, the ability of Lhx2 to regulate transcription of the TSH beta-subunit gene was examined. In the thyrotrope-derived TalphaT1 cell line, Lhx2 expression was found to be induced by treatment with either TRH or cAMP, consistent with the possibility that Lhx2 may play a role in mediating the ability of this signaling pathway to stimulate TSH gene expression. Transient, forced overexpression of Lhx2 stimulated activity of a TSH beta-subunit reporter gene. Deletion studies provided evidence that the -177 to -79 region of the TSH beta-subunit promoter was necessary for stimulation of reporter gene activity by Lhx2. A gel mobility shift assay provided the evidence that Lhx2 can bind to this region of DNA. DNase I footprinting studies demonstrated that two distinct regions of the TSHbeta promoter, -118 to -108 and -86 to -68, are protected by Lhx2 from nuclease digestion. These regions contain repeats of the sequence, 5'-(G/T)CAAT(T/A)-3'. Mutation of this sequence, especially in the -86 to -68 region, substantially decreased Lhx2 responsiveness of the TSH beta-subunit reporter gene. In addition, a DNA fragment containing the -177 to -79 region of the TSHbeta promoter was found to confer Lhx2 responsiveness to a minimal promoter. These results provide multiple lines of evidence consistent with a role for Lhx2 in modulating expression of the TSH beta-subunit gene.

Snx5, as a Mind bomb-binding protein, is expressed in hematopoietic and endothelial precursor cells in zebrafish.

Notch signaling has an evolutionarily conserved function for cell fate determination and stem cell maintenance. Previously, we identified a novel component of the Notch signaling pathway in zebrafish, mind bomb, which encodes an E3 ubiquitin ligase essential for Notch signal activation. Further studies showed that Mind bomb(-/-) mouse embryos exhibited pan-Notch phenotypes in various tissues, suggesting that Mind bomb function is conserved in mammals. Therefore we sought to understand the various molecular partners of Mind bomb using yeast two-hybrid screening. In this search we identified Sorting nexin 5 (Snx5) as a novel interacting partner of Mind bomb. Furthermore we demonstrated that Snx5 colocalizes with Mind bomb in early endosomal compartments, suggesting that Snx5 is important for Mind bomb trafficking. In addition, we identified zebrafish orthologue of Snx5 and showed that snx5 is predominantly expressed in hematopoietic and endothelial precursor cells in zebrafish. We also found defects in hematopoiesis and blood vessel development in snx5 morpholino-injected embryos. Taken together, we show that Snx5, a novel interacting partner of Mind bomb, may have an essential role for cell fate determination in early development.

Isolation and expression of Napor/CUG-BP2 in embryo development.

The human neuroblastoma apoptosis-related RNA-binding protein NAPOR is an ELAV-like RNA-binding protein with three characteristic RNA recognition motifs (RRMs). We report here the cloning and characterization of a zebrafish Napor that has a high sequence homology to human NAPOR protein. Whole-mount in situ hybridization analysis revealed that zebrafish napor is dynamically expressed in early development. In addition to its maternal expression, napor transcripts were detected in adaxial mesoderm cells and lateral neural plate cells at early somite stages. By 10-somite stage, napor expression was restricted to the central nervous system, having a specific expression domain of rhombomere 5 in the hindbrain. In 24 hpf embryo, napor was expressed in subsets of differentiating neural cells in the forebrain and hindbrain as well as somitic muscle cells. The number of napor-expressing neural cells was greatly increased in the mind bomb mutant that has neurogenic phenotype resulting from deficits in the Notch signaling pathway. Furthermore, overexpression of napor by RNA microinjection resulted in severe defects in nervous system and gastrulation, suggesting the need for tight control of napor gene regulation during embryo development.

Gonadotropin-releasing hormone-induced stimulation of the rat secretogranin II promoter involves activation of CREB.

To investigate the events involved in regulation of the secretogranin II (SgII) gene, luciferase reporter constructs were transfected into gonadotrope-derived, alphaT3-1 cells. DNA between -91 and -60 relative to the transcription start site was found to be required for GnRH induced SgII reporter gene activation. This region contains a consensus cAMP response element (CRE) and disruption of this CRE reduced GnRH responsiveness of the SgII promoter. CREB was shown to bind to the SgII CRE and transfection studies with a dominant-negative CREB mutant provided evidence that CREB is required for GnRH responsiveness of the SgII promoter. An expression vector for an inhibitor of the cAMP-dependent protein kinase was found to reduce the ability of cAMP or GnRH to activate the SgII-luciferase reporter gene. These studies offer evidence that GnRH-induced activation of the SgII promoter in the alphaT3-1 cell line requires cAMP-dependent protein kinase activity and a functional CRE within the 5'-flanking region of the gene.

Specification of an anterior neuroectoderm patterning by Frizzled8a-mediated Wnt8b signalling during late gastrulation in zebrafish.

Wnts have been shown to provide a posteriorizing signal that has to be repressed in the anterior neuroectoderm for normal anteroposterior (AP) patterning. We have previously identified a zebrafish frizzled8a (fz8a) gene expressed in the presumptive anterior neuroectoderm as well as prechordal plate at the late gastrula stage. We have investigated the role of Fz8a-mediated Wnt8b signalling in anterior brain patterning in zebrafish. We show that in zebrafish embryos: (1) Wnt signalling has at least two different stage-specific posteriorizing activities in the anterior neuroectoderm, one before mid-gastrulation and the other at late gastrulation; (2) Fz8a plays an important role in mediating anterior brain patterning; (3) Wnt8b and Fz8a can functionally interact to transmit posteriorizing signals that determine the fate of the posterior diencephalon and midbrain in late gastrula embryos; and (4) Wnt8b can suppress fz8a expression in the anterior neuroectoderm and potentially affect the level and/or range of Wnt signalling. In conclusion, we suggest that a gradient of Fz8a-mediated Wnt8b signalling may play crucial role in patterning the posterior diencephalon and midbrain regions in the late gastrula.