Transcriptomic networks of gba3 governing specification of the dopaminergic neurons in zebrafish embryos.

BACKGROUND: Molecular networks associated with dopaminergic (DA) neurogenesis remain undefined within mammalian models. To address this issue, the transient zebrafish model lmx1al: EGFP was generated, which expresses GFP in the DA precursor cells as well as in the DA neurons of the ventral diencephalon (VD). We found that the novel pseudogene gba3 has not been well studied in zebrafish neurogenesis. OBJECTIVE: Crucial networks associated with gba3 transcripts were investigated because the biological functions of these networks have not been reported in DA neurogenesis in zebrafish. METHODS: RNA isolation and sequencing were employed with GFP-expressing cells from 20-, 22-, and 24 h post-fertilization (hpf), while subsequent transcriptomic analysis generated differentially expressed genes with DA neurogenesis (DEG-DA) list. Hierarchical cluster analysis provided absolute guidance for the collection of gba3, an essential transcript that is strictly spatiotemporally expressed during DA neurogenesis, which was proven with whole-mount in situ hybridization (WISH) and knockdown and rescue of the gba3 transcripts in zebrafish embryos. RESULTS: The gba3 transcripts were restricted to the midbrain at 24 hpf and the midbrain and pectoral fins at 30 hpf in zebrafish embryos. Functional studies with knockdown of gba3 found a diminished state in the midbrain and midbrain-hindbrain boundary (MHB) and an underdeveloped condition in the anteroposterior and dorsolateral axis relative to the wild type (WT) at 24 hpf. However, it was recovered after forced expression of gba3 transcripts at 24 hpf. Molecular markers for the DA precursors and mature neurons lmx1al, nurr1, th, and pitx3 were analyzed in the gba3 MOs. The levels of transcripts lmx1al, nurr1, and th were significantly reduced in the midbrain ventral diencephalon (VD) and hindbrain of gba3 morphants compared to the WT at 24 hpf, while expression patterns of pitx3 transcripts showed no differences in the identical regions between gba3 MOs and the controls. CONCLUSIONS: We discuss transcriptional networks in which transcripts of gba3 plausibly govern the specification of dopaminergic neurogenesis in zebrafish embryos.

Kapd Is Essential for Specification of the Dopaminergic Neurogenesis in Zebrafish Embryos.

To define novel networks of Parkinson's disease (PD) pathogenesis, the substantia nigra pars compacta of A53T mice, where a death-promoting protein, FAS-associated factor 1 was ectopically expressed for 2 weeks in the 2-, 4-, 6-, and 8-month-old mice, and was subjected to transcriptomic analysis. Compendia of expression profiles and a hierarchical clustering heat map of differentially expressed genes associated with PD were bioinformatically generated. Transcripts level of a particular gene was fluctuated by 20, 60, and 0.75 fold in the 4-, 6-, and 8-month-old mice compared to the 2 months old. Because the gene contained Kelch domain, it was named as Kapd (Kelch-containing protein associated with PD). Biological functions of Kapd were systematically investigated in the zebrafish embryos. First, transcripts of a zebrafish homologue of Kapd, kapd were found in the floor plate of the neural tube at 10 h post fertilization (hpf), and restricted to the tegmentum, hypothalamus, and cerebellum at 24 hpf. Second, knockdown of kapd caused developmental defects of DA progenitors in the midbrain neural keel and midbrain? hindbrain boundary at 10 hpf. Third, overexpression of kapd increased transcripts level of the dopaminergic immature neuron marker, shha in the prethalamus at 16.5 hpf. Finally, developmental consequences of kapd knockdown reduced transcripts level of the markers for the immature and mature DA neurons, nkx2.2, olig2, otx2b, and th in the ventral diencephalon of the midbrain at 18 hpf. It is thus most probable that Kapd play a key role in the specification of the DA neuronal precursors in zebrafish embryos.

Trim46 contributes to the midbrain development via Sonic Hedgehog signaling pathway in zebrafish embryos.

TRIM46 is a RING finger E3 ligase which belongs to TRIM (tripartite motif-containing) protein family. TRIM46 is required for neuronal polarity and axon specification by driving the formation of parallel microtubule arrays, whereas its embryological functions remain to be determined yet. Expression patterns and biological functions of trim46a, a zebrafish homologue of TRIM46, were studied in zebrafish embryo. First, maternal transcripts of trim46a were present at 1 cell stage whereas zygotic messages were abundant in the eyes, MHB (Midbrain-Hindbrain Boundary) and hindbrain at 24 hpf (hours post fertilization). Second, transcriptional regulatory region of trim46a contains cis-acting elements binding a transcriptional factor Foxa2. Transcription of foxa2 is positively regulated by Sonic Hedgehog (SHH), and treatment of cyclopamine, an SHH inhibitor, represses transcription of foxa2 in 4 hpf through 24 hpf embryos. Third, the transcriptional repression of foxa2 inhibited transcription of trim46a to cause developmental defects in the midbrain and MHB. Finally, spatiotemporal expression patterns of a midbrain marker otx2b in the developmental defects confirmed inhibition of SHH by cyclopamine caused underdevelopment of the midbrain and MHB at 24 hpf. We propose a signaling network where trim46a contributes to development of the midbrain and MHB via Foxa2, a downstream element of SHH signaling in zebrafish embryogenesis.

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.

Metformin regulates astrocyte reactivity in Parkinson's disease and normal aging.

Parkinson's disease (PD) is a neurodegenerative disease characterized by the progressive loss of dopaminergic neurons in the substantia nigra, leading to motor symptoms. Despite the remarkable improvements in the management of PD in recent decades, many patients remain significantly disabled. Metformin is a primary medication for the management of type 2 diabetes. We previously showed that co-treatment with metformin and 3,4-dihydroxyphenyl-l-alanine (l-DOPA) prevented the development of l-DOPA-induced dyskinesia in a 6-hydroxydopamine (6-OHDA)-lesioned animal model of PD. However, effects of metformin on PD- and aging-induced genes in reactive astrocytes remain unknown. In this study, we assessed the effect of metformin on motor function, neuroprotection, and reactive astrocytes in the 6-OHDA-induced PD animal model. In addition, the effects of metformin on the genes expressed by specific types of astrocytes were analyzed in PD model and aged mice. Here, we showed that metformin treatment effectively improves the motor symptoms in the 6-OHDA-induced PD mouse model, whereas metformin had no effect on tyrosine hydroxylase-positive neurons. The activation of AMPK and BDNF signaling pathways was induced by metformin treatment on the 6-OHDA-lesioned side of the striatum. Metformin treatment caused astrocytes to alter reactive genes in a PD animal model. Moreover, aging-induced genes in reactive astrocytes were effectively regulated or suppressed by metformin treatment. Taken together, these results suggest that metformin should be evaluated for the treatment of Parkinson's disease and related neurologic disorders characterized by astrocyte activation.

Trim45 is essential to the development of the diencephalon and eye in zebrafish embryos.

Trim45 is one of the RING (really interesting new gene) finger containing E3 ligase, which belongs to TRIM (Tripartite motif) protein family. Its molecular biological functions have been well characterized but not in light of developmental aspects. Here, we are reporting its expression patterns and developmental functions in zebrafish embryos. First, maternal transcripts of trim45 were found at one cell stage while its zygotic messages appeared at 30% epiboly. trim45 transcripts were restricted to the optical tectum, hypothalamus, hindbrain, and pharyngeal endoderm at 24 hpf (hour post-fertilization), and further to the retinal ganglion cell layer and cranial ganglion at 36 hpf. Second, ectopic expression of trim45 by injecting its mRNAs into embryos at one cell stage caused significant expansion of the diencephalon and eye fields at 24 hpf. In contrast, knock-down of trim45 with anti-sense trim45 morpholinos reduced the size of the two tissues at 24 hpf. Finally, the spatial distribution of the transcripts from olig2 and rx1/rx3, markers for the midbrain and eye respectively, were significantly decreased in the thalamus and eye fields respectively at 24 hpf. Based upon these observations, we proposed possible roles of Trim45 in the development of the diencephalon and eye in zebrafish embryos.

march5 Governs the Convergence and Extension Movement for Organization of the Telencephalon and Diencephalon in Zebrafish Embryos.

MARCH5 is a RING finger E3 ligase involved in mitochondrial integrity, cellular protein homeostasis, and the regulation of mitochondrial fusion and fission. To determine the function of MARCH5 during development, we assessed transcript expression in zebrafish embryos. We found that march5 transcripts were of maternal origin and evenly distributed at the 1-cell stage, except for the mid-blastula transition, with expression predominantly in the developing central nervous system at later stages of embryogenesis. Overexpression of march5 impaired convergent extension movement during gastrulation, resulting in reduced patterning along the dorsoventral axis and alterations in the ventral cell types. Overexpression and knockdown of march5 disrupted the organization of the developing telencephalon and diencephalon. Lastly, we found that the transcription of march5 was tightly regulated by the transcriptional regulators CHOP, C/EBPα, Staf, Znf143a, and Znf76. These results demonstrate the essential role of March5 in the development of zebrafish embryos.

Znf76 is associated with development of the eyes, midbrain, MHB, and hindbrain in zebrafish embryos.

ZNF76 is a transcriptional repressor that targets the TATA-binding protein (TBP) and plays an essential role during brain development; however, its function during embryogenesis remains unclear. Here, we report the expression pattern and potential functions of znf76 in zebrafish embryos. Maternal transcripts of znf76 were detected at low levels in embryos at the 1-cell stage, with zygotic transcripts appearing at the sphere stage. At the bud stage, the distribution of znf76 transcripts was polarized to the anterior and posterior regions of the embryos, and znf76 transcripts were further restricted to the trigeminal placode and proctodeum posterior gut of the embryos at 18 h postfertilization (hpf). znf76 transcripts were localized to the midbrain-hindbrain boundary (MHB), hindbrain, and developing eyes at 24 hpf. Ectopic expression of znf76 with 5'-capped znf76 mRNA microinjected into embryos at the 1-cell stage caused phenotypic defects in the eyes, MHB, hindbrain, and spinal cord. Overexpression of znf76 resulted in a drastic reduction of pax2a, fgf8a, and rx1 transcripts in the optic stalk, MHB, and eyes, respectively. Taken together, these data indicate that Znf76 governs developmental processes in the MHB, hindbrain, and eyes in zebrafish embryos. We also discuss the Fgf8 signaling networks associated with the Znf76 function.

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.

Peli1b governs the brain patterning via ERK signaling pathways in zebrafish embryos.

Pellino proteins are associated with immune and stress responses through their effects on NF-κB signaling and B-cell development, and through their role as a scaffold in TLR/IL-1R signaling pathways. However, their function during embryonic development is unclear. Here, we report the developmental expression patterns and functions of peli1b, which encodes a zebrafish ortholog of human Pellino1. Maternal peli1b transcripts were present in zebrafish embryos at the 1-cell stage and zygotic transcripts appeared in the shield area at 6 hours post fertilization (hpf), particularly in the neural plate of the dorsal region. peli1b transcripts were concentrated in the somites, lens, myogenic cells, lateral plate mesoderm, and presomitic mesoderm at 12 hpf, but expression shifted to the telencephalon, diencephalon, hindbrain, and rhombomeres (r1-7) at 24 hpf. Distribution of peli1b transcripts was further restricted to the telencephalon, diencephalon, hindbrain, eyes, and pectoral fins at 48 hpf. Knock-down of peli1b with a peli1b antisense morpholino resulted in significant developmental defects and a reduction in size of the telencephalon, diencephalon, rhombomeres (r1-7), and spinal cord at 24 hpf. When peli1b-knock-down embryos were analyzed for zic3, a marker associated with the central nervous system, we found lower levels of zic3 transcripts in the shield area at 6 hpf and in the posterior diencephalon, dorsal neural plate, midbrain, and hindbrain at 14 hpf. Finally, the ERK3/4 inhibitor SB203580 also induced a significant reduction in the level of zic3 transcripts in the neural plate at 6 hpf and in the posterior diencephalon, dorsal neural plate, midbrain, hindbrain, segmental plate, dorsal spinal cord, and dorsal posterior neural plate at 14 hpf. It is thus likely that the association between Peli1b and brain development in zebrafish embryos occurs via ERK3/4 pathways.

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.

Rnf152 Is Essential for NeuroD Expression and Delta-Notch Signaling in the Zebrafish Embryos.

We report the biological functions of a zebrafish homologue of RING-finger protein 152 (rnf152) during embryogenesis. rnf152 was initially identified as a brain-enriched E3 ligase involved in early embryogenesis of zebrafish. Expression of rnf152 was ubiquitous in the brain at 24 hpf but restricted to the eyes, midbrain-hindbrain boundary (MHB), and rhombomeres at 48 hpf. Knockdown of rnf152 in zebrafish embryos caused defects in the eyes, MHB, and rhombomeres (r1-7) at 24 hpf. These defects in rnf152-deficient embryos were analyzed by whole-mount in situ hybridization (WISH) using neuroD, deltaD, notch1a, and notch3 probes. NeuroD expression was abolished in the marginal zone, outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL) of the eyes at 27 hpf. Furthermore, deltaD and notch1a expression was remarkably reduced in the ONL, INL, subpallium, tectum, cerebellum, and rhombomeres (r1-7) at 24 hpf, whereas notch3 expression was reduced in the tectum, cerebellum, and rhombomeres at 24 hpf. Finally, we confirmed that expression of Notch target genes, her4 and ascl1a, also decreased significantly in these areas at 24 hpf. Thus, we propose that Rnf152 is essential for development of the eyes, midbrain and hindbrain, and that Delta-Notch signaling is involved.

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.

Sinup is essential for the integrity of centrosomes and mitotic spindles in zebrafish embryos.

Fish lineage-specific gene, sinup [Siaz-interacting nuclear protein], modulates neural plate formation in embryogenesis and shares homology with human TPX2 protein, a member of the vertebrate mitogen-activating protein family. In spite of the presence of the TPX2 domain in Sinup, its cellular function has been unknown. As an initial approach to this question, we expressed Sinup by injecting sinup-EGFP mRNAs into zebrafish embryos at the one- to two-cell stage. First of all, Sinup-EGFP was associated with centrosomes and mitotic spindles. In particular, Sinup was localized to the spindle poles and midbody microtubules during the period between anaphase and cytokinesis. Second, various deleted mutants of Sinup-EGFP failed to be associated with the centrosomes and mitotic spindles. Third, a Sinup mutant, where the 144th Serine residue was converted to alanine, not only disturbed the mitotic spindle organization, such as multipolar spindles, fragmented spindle poles, and flattened spindles, but also arrested the cell cycle at metaphase and cell movement. Finally, Sinup is phosphorylated by Aurora A and the 144th Serine mutant of Sinup is partially phosphorylated by Aurora A kinase. We thus propose that Sinup is an essential element for the integrity of centrosomes and mitotic spindle fibers as well as for the normal process of cell cycle and cellular movement in vertebrate embryos.

Spatiotemporal expression pattern of the zebrafish aquaporin 8 family during early developmental stages.

Aquaporin 8 (Aqp8) is a transmembrane protein that is selectively permeated by water and some small solutes, and physiologically contributes to acid-base equilibrium in the gastrointestinal tract. Here, we described the characterization and spatiotemporal expression pattern of zebrafish aqp8 (zaqp8) gene family, including zaqp8a.1, zaqp8a.2, and zaqp8b, during the early developmental stages. The expression of zaqp8a.1 started first in the lateral plate mesoderm at the 12-somite stage (ss) and then expanded sequentially to the dorsal aorta, intersegmental blood vessels and then to the dorsal longitudinal anastomotic vessel at 24 h post fertilization (hpf). At 28 hpf, expression of zaqp8a.1 was also detected in the embryonic heart tube. Four days post fertilization (dpf), strong zaqp8a.1 expression was detected in the gastrointestinal tract and liver. By 72 hpf, the expression of zaqp8a.2 was first detected in the primitive gut region but not detected in the liver. The expression of zaqp8b was first detected in the intermediate mesoderm at 10 ss. From 24 hpf to 6 dpf, the proximal convoluted segment of the embryonic kidney was marked by zaqp8b expression Overall, these differential expression patterns of aqp8a.1, aqp8a.2, and aqp8b suggest that they possibly play distinct roles throughout the embryonic development by controlling or maintaining organ-specific cellular water homeostasis. Our study provides new evidence that organogenesis requires differential roles of Aqp8 proteins in zebrafish.

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.

STEP signaling pathway mediates psychomotor stimulation and morphine withdrawal symptoms, but not for reward, analgesia and tolerance.

Striatal-enriched protein tyrosine phosphatase (STEP) is abundantly expressed in the striatum, which strongly expresses dopamine and opioid receptors and mediates the effects of many drugs of abuse. However, little is known about the role of STEP in opioid receptor function. In the present study, we generated STEP-targeted mice carrying a nonsense mutation (C230X) in the kinase interaction domain of STEP by screening the N-ethyl-N-nitrosourea (ENU)-driven mutant mouse genomic DNA library and subsequent in vitro fertilization. It was confirmed that the C230X nonsense mutation completely abolished functional STEP protein expression in the brain. STEP(C230X-/-) mice showed attenuated acute morphine-induced psychomotor activity and withdrawal symptoms, whereas morphine-induced analgesia, tolerance and reward behaviors were unaffected. STEP(C230X-/-) mice displayed reduced hyperlocomotion in response to intrastriatal injection of the µ-opioid receptor agonist DAMGO, but the behavioral responses to δ- and κ-opioid receptor agonists remained intact. These results suggest that STEP has a key role in the regulation of psychomotor action and physical dependency to morphine. These data suggest that STEP inhibition may be a critical target for the treatment of withdrawal symptoms associated with morphine.

Proper Activity of Histone H3 Lysine 4 (H3K4) Methyltransferase Is Required for Morphogenesis during Zebrafish Cardiogenesis.

While increasing evidence indicates the important function of histone methylation during development, how this process influences cardiac development in vertebrates has not been explored. Here, we elucidate the functions of two histone H3 lysine 4 (H3K4) methylation enzymes, SMYD3 and SETD7, during zebrafish heart morphogenesis using gene expression profiling by whole mount in situ hybridization and antisense morpholino oligonucleotide (MO)-based gene knockdown. We find both smyd3 and setd7 are highly expressed within developing zebrafish heart and knock-down of these genes led to severe defects in cardiac morphogenesis without altering the expressions pattern of heart markers, including cmlc2, vmhc, and amhc. Furthermore, double knock-down by coinjection of smyd3 and setd7 MOs caused the synergistic defects in heart development. As similar to knock-down effect, overexpression of these genes also caused the heart morphogenesis defect in zebrafish. These results indicate that histone modifying enzymes, SMYD3 and SETD7, appear to function synergistically during heart development and their proper functioning is essential for normal heart morphogenesis during development.

Siah ubiquitin ligases modulate nodal signaling during zebrafish embryonic development.

Siah2 is a zebrafish homologue of mammalian Siah family. Siah acts as an E3 ubiquitin ligase that binds proteins destined for degradation. Extensive homology between siah and Drosophila Siah homologue (sina) suggests their important physiological roles during embryonic development. However, detailed functional studies of Siah in vertebrate development have not been carried out. Here we report that Siah2 specifically augments nodal related gene expression in marginal blastomeres at late blastula through early gastrula stages of zebrafish embryos. Siah2 dependent Nodal signaling augmentation is confirmed by cell-based reporter gene assays using 293T cells and 3TPluciferase reporter plasmid. We also established a molecular hierarchy of Siah as a upstream regulator of FoxH1/Fast1 transcriptional factor in Nodal signaling. Elevated expression of nodal related genes by overexpression of Siah2 was enough to override the inhibitory effects of atv and lft2 on the Nodal signaling. In particular, E3 ubiquitin ligase activity of Siah2 is critical to limit the duration and/or magnitude of Nodal signaling. Additionally, since the embryos injected with Siah morpholinos mimicked the atv overexpression phenotype at least in part, our data support a model in which Siah is involved in mesendoderm patterning via modulating Nodal signaling.