The cerebellum is responsible for coordinating motor functions and has a unique laminated architecture. Purkinje cells are inhibitory neurons and represent the only output from the cerebellar cortex. Tyrosine hydroxylase (TH) is the key enzyme for the synthesis of catecholamines, including dopamine and noradrenaline, and it is normally not expressed in cerebellar neurons.

Paeoniflorin, the major component of Paeonia lactiflora pall, has previously been reported to prevent thrombosis. Plasminogen activator urokinase (uPA) is a serine protease that markedly facilitates normal thrombosis resolution. Paeoniflorin and uPA have been linked to the mitogen‑activated protein kinase (MAPK) signaling pathway. In the current study, the influence of paeoniflorin on the expression of uPA was investigated and the underlying regulatory mechanism was preliminarily determined. The prothrombotic state of the model animals treated with paeoniflorin were assessed by enzyme‑linked immunosorbent assay (ELISA). Additionally, the cytotoxicity of paeoniflorin on human umbilical vein endothelial cell (HUVEC) cultures was estimated using a methyl thiazolyl tetrazolium assay and the possible pathways involved in the interaction between paeoniflorin and uPA were evaluated using western blot analysis. The ELISA results demonstrated that the levels of 6‑keto prostaglandin F1a, fibronectin and uPA were significantly upregulated by treatment with paeoniflorin compared with control (P<0.05). By contrast, the expression of fibrinogen, D‑dimer and thromboxane B2 were inhibited. With an increase in the concentration of paeoniflorin the cell viability of HUVECs decreased gradually. The results of western blot analysis demonstrated that paeoniflorin increased the phosphorylation of MAPK 14 (p38) and MAPK 8 (JNK). The present study demonstrated that paeoniflorin has the potential to improve the prethrombotic state and recanalize thrombosis by increasing the expression of uPA, which may be mediated via regulation of the p38 and JNK MAPK signaling pathways. However, this treatment effect was dependent on the concentration of paeoniflorin used, an unsuitable concentration of the agent would result in a negative effect on the anti‑thrombosis pathways.

The Hedgehog (Hh) signaling pathway plays important roles in the tumorigenesis of multiple cancers and is a key target for drug discovery. In a screen of natural products extracted from Chinese herbs, we identified eight ent-Kaurane diterpenoids and two triterpene dilactones as novel Hh pathway antagonists. Epistatic analyses suggest that these compounds likely act at the level or downstream of Smoothened (Smo) and upstream of Suppressor of Fused (Sufu). The ent-Kauranoid-treated cells showed elongated cilia, suppressed Smo trafficking to cilia, and mitotic defects, while the triterpene dilactones had no effect on the cilia and ciliary Smo. These ent-Kaurane diterpenoids provide new prototypes of Hh inhibitors, and are valuable probes for deciphering the mechanisms of Smo ciliary transport and ciliogenesis.

Gut-derived 5-hydroxytryptamine (5-HT) is well known for its role in mediating colonic motility function. However, it is not very clear whether brain-derived 5-HT is involved in the regulation of colonic motility. In this study, we used central 5-HT knockout (KO) mice to investigate whether brain-derived 5-HT mediates colonic motility, and if so, whether it involves oxytocin (OT) production in the hypothalamus and OT receptor in the colon. Colon transit time was prolonged in KO mice. The OT levels in the hypothalamus and serum were decreased significantly in the KO mice compared to wild-type (WT) controls. OT increased colonic smooth muscle contraction in both KO and WT mice, and the effects were blocked by OT receptor antagonist and tetrodotoxin but not by hexamethonium or atropine. Importantly, the OT-induced colonic smooth muscle contraction was decreased significantly in the KO mice relative to WT. The OT receptor expression of colon was detected in colonic myenteric plexus of mice. Central 5-HT is involved in the modulation of colonic motility which may modulate through its regulation of OT synthesis in the hypothalamus. Our results reveal a central 5-HT - hypothalamus OT - colonic OT receptor axis, providing a new target for the treatment of brain-gut dysfunction.

Lung cancer is the most frequent cancer type and the leading cause of tumor-associated deaths worldwide. TP53 is an important tumor suppressor gene and is frequently inactivated in lung cancer. E3 ligases targeting p53, such as MDM2, are involved in the development of lung cancer. The E3 ligase HUWE1, which targets many tumor-associated proteins including p53, has been reported to be highly expressed in lung cancer; however, its role in lung tumorigenesis is unclear. Methods: The expression of HUWE1 and p53 in lung cancer cells was modulated and the phenotypes were assessed by performing soft agar colony forming assays, cell cycle analysis, BrdU incorporation assays, and xenograft tumor growth assays. The effect on tumorigenesis in genetically-engineered mice was also analyzed. The mechanism through which HUWE1 sustained lung cancer cell malignancy was confirmed by western blotting. HUWE1 expression in clinical lung cancer was identified by immunohistochemistry and validated by analyzing lung adenocarcinoma and lung squamous carcinoma samples from the Cancer Genome Atlas (TCGA) database. Finally, we assessed the association between HUWE1 expression and patient outcome using online survival analysis software including survival information from the caBIG, GEO, and TCGA database. Results: Inactivation of HUWE1 in a human lung cancer cell line inhibited proliferation, colony-forming capacity, and tumorigenicity. Mechanistically, this phenotype was driven by increased p53, which was due to attenuated proteasomal degradation by HUWE1. Up-regulation of p53 inhibited cancer cell malignancy, mainly through the induction of p21 expression and the down-regulation of HIF1α. Huwe1 deletion completely abolished the development of EGFRVIII-induced lung cancer in Huwe1 conditional knockout mice. Furthermore, survival analysis of lung cancer patients showed that increased HUWE1 expression is significantly associated with worse prognosis. Conclusion: Our data suggest that HUWE1 plays a critical role in lung cancer and that the HUWE1-p53 axis might be a potential target for lung cancer therapy.

Itch, chronic itch in particular, can have a significant negative impact on an individual's quality of life. However, the molecular mechanisms underlying itch processing in the central nervous system remain largely unknown.

Early-life sensory input plays a crucial role in brain development. Although deprivation of orofacial sensory input at perinatal stages disrupts the establishment of the barrel cortex and relevant callosal connections, its long-term effect on adult behavior remains elusive. In this study, we investigated the behavioral phenotypes in adult mice with unilateral transection of the infraorbital nerve (ION) at postnatal day 3 (P3). Although ION-transected mice had normal locomotor activity, motor coordination, olfaction, anxiety-like behaviors, novel object memory, preference for social novelty and sociability, they presented deficits in social memory and spatial memory compared with control mice. In addition, the social memory deficit was associated with reduced oxytocin (OXT) levels in the hypothalamus and could be partially restored by intranasal administration of OXT. Thus, early sensory deprivation does result in behavioral alterations in mice, some of which may be associated with the disruption of oxytocin signaling.

Extinction of aversive memories has been a major concern in neuropsychiatric disorders, such as anxiety disorders and drug addiction. However, the mechanisms underlying extinction of aversive memories are not fully understood. Here, we report that extinction of conditioned place aversion (CPA) to naloxone-precipitated opiate withdrawal in male rats activates Rho GTPase Rac1 in the ventromedial prefrontal cortex (vmPFC) in a BDNF-dependent manner, which determines GABAA receptor (GABAAR) endocytosis via triggering synaptic translocation of activity-regulated cytoskeleton-associated protein (Arc) through facilitating actin polymerization. Active Rac1 is essential and sufficient for GABAAR endocytosis and CPA extinction. Knockdown of Rac1 expression within the vmPFC of rats using Rac1-shRNA suppressed GABAAR endocytosis and CPA extinction, whereas expression of a constitutively active form of Rac1 accelerated GABAAR endocytosis and CPA extinction. The crucial role of GABAAR endocytosis in the LTP induction and CPA extinction is evinced by the findings that blockade of GABAAR endocytosis by a dynamin function-blocking peptide (Myr-P4) abolishes LTP induction and CPA extinction. Thus, the present study provides first evidence that Rac1-dependent GABAAR endocytosis plays a crucial role in extinction of aversive memories and reveals the sequence of molecular events that contribute to learning experience modulation of synaptic GABAAR endocytosis.SIGNIFICANCE STATEMENT This study reveals that Rac1-dependent GABAAR endocytosis plays a crucial role in extinction of aversive memories associated with drug withdrawal and identifies Arc as a downstream effector of Rac1 regulations of synaptic plasticity as well as learning and memory, thereby suggesting therapeutic targets to promote extinction of the unwanted memories.

The evolutionarily conserved Dbx homeodomain-containing proteins play important roles in the development of vertebrate central nervous system. In mouse, Dbx and Nkx6 have been suggested to be cross-repressive partners involved in the patterning of ventral neural tube. Here, we have isolated Xenopus Dbx2 and studied its developmental expression and function during neural development. Like XDbx1, from mid-neurula stage on, XDbx2 is expressed in stripes between the primary motoneurons and interneurons. At the tailbud stages, it is detected in the middle region of the neural tube. XDbx2 acts as a transcriptional repressor in vitro and over-expression of XDbx2 inhibits primary neurogenesis in Xenopus embryos. Over-expression of XDbx genes represses the expression of XNkx6.2 and vise versa. Knockdown of either XDbx1, XDbx2 or both by specific morpholinos induces lateral expansion of XNkx6.2 expression domains. These data reveal conserved roles for Dbx in primary neurogenesis and dorsoventral neural patterning in Xenopus.

The highly ordered vertebrate retina is composed of seven cell types derived from a common pool of retinal progenitor cells (RPCs), and is a good model for the studies of cell differentiation and interaction during neural development. Notch signaling plays a pivotal role in retinogenesis in mammals, but the full scope of the functions of Notch pathway, and the underlying molecular mechanisms, remain unclear.

Myosin X (Myo X) transports cargos to the tips of filopodia for cell adhesion, migration, and neuronal axon guidance. Deleted in Colorectal Cancer (DCC) is one of the Myo X cargos that is essential for Netrin-1-regulated axon pathfinding. The function of Myo X in axon development in vivo and the underlying mechanisms remain elusive. Here, we provide evidence for the role of Myo X in Netrin-1-DCC-regulated axon development in developing mouse neocortex. The knockout (KO) or knockdown (KD) of Myo X in cortical neurons of embryonic mouse brain impairs axon initiation and contralateral branching/targeting. Similar axon deficits are detected in Netrin-1-KO or DCC-KD cortical neurons. Further proteomic analysis of Myo X binding proteins identifies KIF13B (a kinesin family motor protein). The Myo X interaction with KIF13B is induced by Netrin-1. Netrin-1 promotes anterograde transportation of Myo X into axons in a KIF13B-dependent manner. KIF13B-KD cortical neurons exhibit similar axon deficits. Together, these results reveal Myo X-KIF13B as a critical pathway for Netrin-1-promoted axon initiation and branching/targeting.SIGNIFICANCE STATEMENT Netrin-1 increases Myosin X (Myo X) interaction with KIF13B, and thus promotes axonal delivery of Myo X and axon initiation and contralateral branching in developing cerebral neurons, revealing unrecognized functions and mechanisms underlying Netrin-1 regulation of axon development.

It has been reported that allopregnanolone (APα) promotes the neurogenesis of the neural progenitor cells (NPCs) in the subventricular zone (SVZ) and prevents the decrease of dopaminergic neurons in 6-hydroxydopamine (6-OHDA)-treated mice by binding to γ-aminobutyric acid A receptor (GABAAR) and then opening voltage-gated L-type Ca2+ channel, but the underlying mechanisms remain elusive. The aim of this study was to explore the possible involvement of GABAAR and calcium/calmodulin-dependent protein kinase II delta 3 (CaMKIIδ3) in this process.

The ubiquitin E3 ligase RNF220 and its co-factor ZC4H2 are required for multiple neural developmental processes through different targets, including spinal cord patterning and the development of the cerebellum and the locus coeruleus. Here, we explored the effects of loss of ZC4H2 and RNF220 on the proliferation and differentiation of neural stem cells (NSCs) derived from mouse embryonic cortex. We showed that loss of either ZC4H2 or RNF220 inhibits the proliferation and promotes the differentiation abilities of NSCs in vitro. RNA-Seq profiling revealed 132 and 433 differentially expressed genes in the ZCH2-/- and RNF220-/- NSCs, compared to wild type (WT) NSCs, respectively. Specifically, Cend1, a key regulator of cell cycle exit and differentiation of neuronal precursors, was found to be upregulated in both ZCH2-/- and RNF220-/- NSCs at the mRNA and protein levels. The targets of Cend1, such as CyclinD1, Notch1 and Hes1, were downregulated both in ZCH2-/- and RNF220-/- NSCs, whereas p53 and p21 were elevated. ZCH2-/- and RNF220-/- NSCs showed G0/G1 phase arrest compared to WT NSCs in cell cycle analysis. These results suggested that ZC4H2 and RNF220 are likely involved in the regulation of neural stem cell proliferation and differentiation through Cend1.

ZC4H2 encodes a C4H2 type zinc-finger nuclear factor, the mutation of which has been associated with disorders with various clinical phenotypes in human, including developmental delay, intellectual disability and dystonia. ZC4H2 has been suggested to regulate spinal cord patterning in zebrafish as a co-factor for RNF220, an ubiquitin E3 ligase involved in Gli signaling. Here we showed that ZC4H2 and RNF220 knockout animals phenocopy each other in spinal patterning in both mouse and zebrafish, with mispatterned progenitor and neuronal domains in the ventral spinal cord. We showed evidence that ZC4H2 is required for the stability of RNF220 and also proper Gli ubiquitination and signaling in vivo. Our data provides new insights into the possible etiology of the neurodevelopmental impairments observed in ZC4H2-associated syndromes.

Sonic Hedgehog (Shh) signaling plays crucial roles in patterning the ventral neural tube, which is transformed into opposing gradients of repressor and activator forms of Glis. Here, we show that the fine-tuning of the shape of the Gli gradients through non-proteolytic ubiquitination-mediated nuclear exportation plays an important role in the control of local neural cell fate. Loss of RNF220, a ventral neural-specific ubiquitin E3 ligase, leads to ventral expansion of the intermediate V0 and dorsal expansion of the ventral V3 neurons, while reducing the V1, V2, and motor neurons between them. We show that RNF220 interacts with all Glis, either in their activator or repressor forms; induces their K63-linked ubiquitination; and promotes their nuclear export, likely by unmasking a nuclear export signal in the zinc finger domain. We propose that RNF220 works to refine the Gli gradients during neural patterning by limiting the effective Gli levels in the nucleus.

Development of thoracolumbar vertebra (TLV) and rib primordium (RP) is a common evolutionary feature across vertebrates, although whole-organism analysis of the expression dynamics of TLV- and RP-related genes has been lacking. Here, we investigated the single-cell transcriptome landscape of thoracic vertebra (TV), lumbar vertebra (LV), and RP cells from a pig embryo at 27 days post-fertilization (dpf) and identified six cell types with distinct gene expression signatures. In-depth dissection of the gene expression dynamics and RNA velocity revealed a coupled process of osteogenesis and angiogenesis during TLV and RP development. Further analysis of cell type-specific and strand-specific expression uncovered the extremely high level of HOXA10 3'-UTR sequence specific to osteoblasts of LV cells, which may function as anti-HOXA10-antisense by counteracting the HOXA10-antisense effect to determine TLV transition. Thus, this work provides a valuable resource for understanding embryonic osteogenesis and angiogenesis underlying vertebrate TLV and RP development at the cell type-specific resolution, which serves as a comprehensive view on the transcriptional profile of animal embryo development.

PTEN is known as a tumor suppressor and plays essential roles in brain development. Here, we report that PTEN in primary sensory neurons is involved in processing itch and thermal information in adult mice. Deletion of PTEN in the dorsal root ganglia (DRG) is achieved in adult Drg11-CreER: PTENflox/flox (PTEN CKO) mice with oral administration of tamoxifen, and CKO mice develop pathological itch and elevated itch responses on exposure to various pruritogens. PTEN deletion leads to ectopic expression of TRPV1 and MrgprA3 in IB4+ non-peptidergic DRG neurons, and the TRPV1 is responsive to capsaicin. Importantly, the elevated itch responses are no longer present in Drg11-CreER: PTENflox/flox: TRPV1flox/flox (PTEN: TRPV1 dCKO) mice. In addition, thermal stimulation is enhanced in PTEN CKO mice but blunted in dCKO mice. PTEN-involved regulation of itch-related gene expression in DRG neurons provides insights for understanding molecular mechanism of itch and thermal sensation at the spinal level.

Allopregnanolone (APα), as a functional neurosteroid, exhibits the neuroprotective effect on neurodegenerative diseases such as Parkinson's disease (PD) through γ-aminobutyric acid A receptor (GABAAR), but it has not been completely understood about its molecular mechanisms. In order to investigate the neuroprotective effect of APα, as well as to clarify its possible molecular mechanisms, SH-SY5Y neuronal cell lines were incubated with 6-hydroxydopamine (6-OHDA), which has been widely used as an in vitro model for PD, along with APα alone or in combination with GABAAR antagonist (bicuculline, Bic), intracellular Ca2+ chelator (EGTA) and voltage-gated L-type Ca2+ channel blocker (Nifedipine). The viability, proliferation, and differentiation of SH-SY5Y cells, the expression levels of calmodulin (CaM), Ca2+/calmodulin-dependent protein kinase II δ3 (CaMKIIδ3), cyclin-dependent kinase-1 (CDK1) and brain-derived neurotrophic factor (BDNF), as well as the interaction between CaMKIIδ3 and CDK1 or BDNF, were detected by morphological and molecular biological methodology. Our results found that the cell viability and the number of tyrosine hydroxylase (TH), bromodeoxyuridine (BrdU) and TH/BrdU-positive cells in 6-OHDA-treated SH-SY5Y cells were significantly decreased with the concomitant reduction in the expression levels of aforementioned proteins, which were ameliorated following APα administration. In addition, Bic could further increase the number of TH or BrdU-positive cells as well as the expression levels of aforementioned proteins except for TH/BrdU-double positive cells, while EGTA and Nifedipine could attenuate the expression levels of CaM, CaMKIIδ3 and BDNF. Moreover, there existed a direct interaction between CaMKIIδ3 and CDK1 or BDNF. As a result, APα-induced an increase in the number of TH-positive SH-SY5Y cells might be mediated through GABAAR via Ca2+/CaM/CaMKIIδ3/BDNF (CDK1) signaling pathway, which would ultimately facilitate to elucidate PD pathogenesis and hold a promise as an alternative therapeutic target for PD.

We present the complete mitochondrial genome of Toxotes chatareus yielded by the next-generation sequencing data in this study. The complete mitochondrial genome of T. chatareus has 16,543 bp and contained 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and a single control region (D-loop). The overall base composition was A 28.75%, C 29.80%, G 15.77%, T 25.68% and its gene arrangement was similar with other Carangaria mitochondrial genomes. Additionally, the phylogenetic relationships of 13 Carangaria species based on the complete mitochondrial genome was analyzed using the neighbor-joining method. The result showed T. chatareus was clustered with L. lactarius suggesting the close phylogenetic affinity they owned. Together, the complete mitochondrial genome of T. chatareus would be beneficial for the study of phylogenetic relationship, taxonomic classification and phylogeography of the Carangaria.

Dopaminergic (DA) neurons in the arcuate nucleus (ARC) of the hypothalamus play essential roles in the secretion of prolactin and the regulation of energy homeostasis. However, the gene regulatory network responsible for the development of the DA neurons remains poorly understood. Here we report that the transcription factor special AT-rich binding protein 2 (Satb2) is required for the development of ARC DA neurons. Satb2 is expressed in a large proportion of DA neurons without colocalization with proopiomelanocortin (POMC), orexigenic agouti-related peptide (AgRP), neuropeptide-Y (NPY), somatostatin (Sst), growth hormone-releasing hormone (GHRH), or galanin in the ARC. Nestin-Cre;Satb2flox/flox (Satb2 CKO) mice show a reduced number of ARC DA neurons with unchanged numbers of the other types of ARC neurons, and exhibit an increase of serum prolactin level and an elevated metabolic rate. The reduction of ARC DA neurons in the CKO mice is observed at an embryonic stage and Dlx1 is identified as a potential downstream gene of Satb2 in regulating the development of ARC DA neurons. Together, our study demonstrates that Satb2 plays a critical role in the gene regulatory network directing the development of DA neurons in ARC.