Topoisomerase IIA in adult NSCs regulates SVZ neurogenesis by transcriptional activation of Usp37.

Topoisomerase IIA (TOP2a) has traditionally been known as an important nuclear enzyme that resolves entanglements and relieves torsional stress of DNA double strands. However, its function in genomic transcriptional regulation remains largely unknown, especially during adult neurogenesis. Here, we show that TOP2a is preferentially expressed in neurogenic niches in the brain of adult mice, such as the subventricular zone (SVZ). Conditional knockout of Top2a in adult neural stem cells (NSCs) of the SVZ significantly inhibits their self-renewal and proliferation, and ultimately reduces neurogenesis. To gain insight into the molecular mechanisms by which TOP2a regulates adult NSCs, we perform RNA-sequencing (RNA-Seq) plus chromatin immunoprecipitation sequencing (ChIP-Seq) and identify ubiquitin-specific protease 37 (Usp37) as a direct TOP2a target gene. Importantly, overexpression of Usp37 is sufficient to rescue the impaired self-renewal ability of adult NSCs caused by Top2a knockdown. Taken together, this proof-of-principle study illustrates a TOP2a/Usp37-mediated novel molecular mechanism in adult neurogenesis, which will significantly expand our understanding of the function of topoisomerase in the adult brain.

Rnf220/Zc4h2-mediated monoubiquitylation of Phox2 is required for noradrenergic neuron development.

Noradrenaline belongs to the monoamine system and is involved in cognition and emotional behaviors. Phox2a and Phox2b play essential but non-redundant roles during development of the locus coeruleus (LC), the main noradrenergic (NA) neuron center in the mammalian brain. The ubiquitin E3 ligase Rnf220 and its cofactor Zc4h2 participate in ventral neural tube patterning by modulating Shh/Gli signaling, and ZC4H2 mutation is associated with intellectual disability, although the mechanisms for this remain poorly understood. Here, we report that Zc4h2 and Rnf220 are required for the development of central NA neurons in the mouse brain. Both Zc4h2 and Rnf220 are expressed in developing LC-NA neurons. Although properly initiated at E10.5, the expression of genes associated with LC-NA neurons is not maintained at the later embryonic stages in mice with a deficiency of either Rnf220 or Zc4h2 In addition, we show that the Rnf220/Zc4h2 complex monoubiquitylates Phox2a/Phox2b, a process required for the full transcriptional activity of Phox2a/Phox2b. Our work reveals a role for Rnf220/Zc4h2 in regulating LC-NA neuron development, and this finding may be helpful for understanding the pathogenesis of ZC4H2 mutation-associated intellectual disability.

Satb2 Regulates EphA7 to Control Soma Spacing and Self-Avoidance of Cortical Pyramidal Neurons.

Soma spacing and dendritic arborization during brain development are key events for the establishment of proper neural circuitry and function. Transcription factor Satb2 is a molecular node in regulating the development of the cerebral cortex, as shown by the facts that Satb2 is required for the regionalization of retrosplenial cortex, the determination of callosal neuron fate, and the regulation of soma spacing and dendritic self-avoidance of cortical pyramidal neurons. In this study, we explored downstream effectors that mediate the Satb2-implicated soma spacing and dendritic self-avoidance. First, RNA-seq analysis of the cortex revealed differentially expressed genes between control and Satb2 CKO mice. Among them, EphA7 transcription was dramatically increased in layers II/III of Satb2 CKO cortex. Overexpression of EphA7 in the late-born cortical neurons of wild-type mice via in utero electroporation resulted in soma clumping and impaired self-avoidance of affected pyramidal neurons, which resembles the phenotypes caused by knockdown of Satb2 expression. Importantly, the phenotypes by Satb2 knockdown was rescued by reducing EphA7 expression in the cortex. Finally, ChIP and luciferase reporter assays indicated a direct suppression of EphA7 expression by Satb2. These findings provide new insights into the complexity of transcriptional regulation of the morphogenesis of cerebral cortex.

Effects of maternal urban particulate matter SRM 1648a exposure on birth outcomes and offspring growth in mice.

The association between exposure to particulate matter (PM) during pregnancy and abnormal birth outcomes is still inconclusive. This study aims to provide more evidence for this public health concern by investigating birth outcomes and the growth of offspring in mice exposed to PM during pregnancy. C57BL/6 J pregnant mice were exposed to PM via nasal drip at three doses or solvent control. The dam weight gain was recorded during pregnancy. The number of pups, pup weight, and placental weight were recorded at embryonic day 18.5 (E18.5) necropsy. For mice that gave birth naturally, we calculated the gestation length and measured the body weight of offspring once a week from the 1st to the 6th week after birth. The results showed that there were no significant differences in maternal body weight gain, conception rate, pregnancy duration, and litter size among different groups. There were no significant differences in fetal weight, placental weight, and fetal/placental weight ratio at E18.5. Weight gain in offspring was reduced after birth. The average body weight of offspring in the high-dose group was significantly lower than that in the control group at weeks 5 in female pups. There were no significant differences in the body weight of male offspring among groups from 1st to the 6th. Together, our study indicated that maternal exposure to PM did not significantly impact birth outcomes of C57BL/6 J mice but affected growth trajectories in offspring after birth in a dose- and fetal sex-dependent manner.

RBP-J deficiency promoted the proliferation and differentiation of CD133-positive cells in both in vitro and in vivo studies.

Although Notch signalling pathway could control the proliferation and differentiation of neural stem cells (NSCs), it is largely unknown about the effect of Notch signalling pathway on the neurogenesis of CD133-positive cells. By using the primary cultured ependymal cells and the transgenic mouse, we found that CD133 immunoreactivity was exclusively localized in the ependymal layer of ventricles; moreover, most CD133-positive cells were co-labelled with Nestin. In addition, recombination signal binding protein J (RBP-J), a key nuclear effector of Notch signalling pathway, was highly active in CD133-positive cells. CD133-positive cells can differentiate into the immature and mature neurons; in particular, the number of CD133-positive cells differentiating into the immature and mature neurons was significantly increased following the deficiency or interference of RBP-J in vivo or in vitro. By using real-time qPCR and Western blot, we found that RBP-J and Hes1 were downregulated, whereas Notch1 was upregulated in the expression levels of mRNAs and proteins following the deficiency or interference of RBP-J. These results demonstrated RBP-J deficiency promoted the proliferation and differentiation of CD133-positive cells. Therefore, we speculated that RBP-J could maintain CD133-positive cells in the characteristics of NSCs possibly by regulating Notch1/RBP-J/Hes1 pathway. It will provide a novel molecular insight into the function of RBP-J as well as facilitate a future investigation of CD133-positive cells with respect to their potential application in neurodegenerative disorder.

ZFP804A mutant mice display sex-dependent schizophrenia-like behaviors.

Genome-wide association studies uncovered the association of ZNF804A (Zinc-finger protein 804A) with schizophrenia (SZ). In vitro data have indicated that ZNF804A might exert its biological roles by regulating spine and neurite morphogenesis. However, no in vivo data are available for the role of ZNF804A in psychiatric disorders in general, SZ in particular. We generated ZFP804A mutant mice, and they showed deficits in contextual fear and spatial memory. We also observed the sensorimotor gating impairment, as revealed by the prepulse inhibition test, but only in female ZFP804A mutant mice from the age of 6 months. Notably, the PPI difference between the female mutant and control mice was no longer existed with the administration of Clozapine or after the ovariectomy. Hippocampal long-term potentiation was normal in both genders of the mutant mice. Long-term depression was absent in male mutants, but facilitated in the female mutants. Protein levels of hippocampal serotonin-6 receptor and GABAB1 receptor were increased, while those of cortical dopamine 2 receptor were decreased in the female mutants with no obvious changes in the male mutants. Moreover, the spine density was reduced in the cerebral cortex and hippocampus of the mutant mice. Knockdown of ZFP804A impaired the neurite morphogenesis of cortical and hippocampal neurons, while its overexpression enhanced neurite morphogenesis only in the cortical neurons in vitro. Our data collectively support the idea that ZFP804A/ZNF804A plays important roles in the cognitive functions and sensorimotor gating, and its dysfunction may contribute to SZ, particularly in the female patients.

Myosin X Interaction with KIF13B, a Crucial Pathway for Netrin-1-Induced Axonal Development.

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.

The rostromedial tegmental nucleus is essential for non-rapid eye movement sleep.

The rostromedial tegmental nucleus (RMTg), also called the GABAergic tail of the ventral tegmental area, projects to the midbrain dopaminergic system, dorsal raphe nucleus, locus coeruleus, and other regions. Whether the RMTg is involved in sleep-wake regulation is unknown. In the present study, pharmacogenetic activation of rat RMTg neurons promoted non-rapid eye movement (NREM) sleep with increased slow-wave activity (SWA). Conversely, rats after neurotoxic lesions of 8 or 16 days showed decreased NREM sleep with reduced SWA at lights on. The reduced SWA persisted at least 25 days after lesions. Similarly, pharmacological and pharmacogenetic inactivation of rat RMTg neurons decreased NREM sleep. Electrophysiological experiments combined with optogenetics showed a direct inhibitory connection between the terminals of RMTg neurons and midbrain dopaminergic neurons. The bidirectional effects of the RMTg on the sleep-wake cycle were mimicked by the modulation of ventral tegmental area (VTA)/substantia nigra compacta (SNc) dopaminergic neuronal activity using a pharmacogenetic approach. Furthermore, during the 2-hour recovery period following 6-hour sleep deprivation, the amount of NREM sleep in both the lesion and control rats was significantly increased compared with baseline levels; however, only the control rats showed a significant increase in SWA compared with baseline levels. Collectively, our findings reveal an essential role of the RMTg in the promotion of NREM sleep and homeostatic regulation.

Central 5-hydroxytryptamine (5-HT) mediates colonic motility by hypothalamus oxytocin-colonic oxytocin receptor pathway.

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.

The Small GTPase Rac1 Contributes to Extinction of Aversive Memories of Drug Withdrawal by Facilitating GABAA Receptor Endocytosis in the vmPFC.

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.

Brain-derived Neurotrophic Factor Promotes the Migration of Olfactory Ensheathing Cells Through TRPC Channels.

Olfactory ensheathing cells (OECs) are a unique type of glial cells with axonal growth-promoting properties in the olfactory system. Organized migration of OECs is essential for neural regeneration and olfactory development. However, the molecular mechanism of OEC migration remains unclear. In the present study, we examined the effects of brain-derived neurotrophic factor (BDNF) on OEC migration. Initially, the "scratch" migration assay, the inverted coverslip and Boyden chamber migration assays showed that BDNF could promote the migration of primary cultured OECs. Furthermore, BDNF gradient attracted the migration of OECs in single-cell migration assays. Mechanistically, TrkB receptor expressed in OECs mediated BDNF-induced OEC migration, and BDNF triggered calcium signals in OECs. Finally, transient receptor potential cation channels (TRPCs) highly expressed in OECs were responsible for BDNF-induced calcium signals, and required for BDNF-induced OEC migration. Taken together, these results demonstrate that BDNF promotes the migration of cultured OECs and an unexpected finding is that TRPCs are required for BDNF-induced OEC migration. GLIA 2016;64:2154-2165.

Both Myosin-10 isoforms are required for radial neuronal migration in the developing cerebral cortex.

During embryonic development of the mammalian cerebral cortex, postmitotic cortical neurons migrate radially from the ventricular zone to the cortical plate. Proper migration involves the correct orientation of migrating neurons and the transition from a multipolar to a mature bipolar morphology. Herein, we report that the 2 isoforms of Myosin-10 (Myo10) play distinct roles in the regulation of radial migration in the mouse cortex. We show that the full-length Myo10 (fMyo10) isoform is located in deeper layers of the cortex and is involved in establishing proper migration orientation. We also demonstrate that fMyo10-dependent orientation of radial migration is mediated at least in part by the netrin-1 receptor deleted in colorectal cancer. Moreover, we show that the headless Myo10 (hMyo10) isoform is required for the transition from multipolar to bipolar morphologies in the intermediate zone. Our study reveals divergent functions for the 2 Myo10 isoforms in controlling both the direction of migration and neuronal morphogenesis during radial cortical neuronal migration.

Notch-Rbpj signaling is required for the development of noradrenergic neurons in the mouse locus coeruleus.

The locus coeruleus (LC) is the main source of noradrenaline in the brain and is implicated in a broad spectrum of physiological and behavioral processes. However, genetic pathways controlling the development of noradrenergic neurons in the mammalian brain are largely unknown. We report here that Rbpj, a key nuclear effector in the Notch signaling pathway, plays an essential role in LC neuron development in the mouse. Conditional inactivation of Rbpj in the dorsal rhombomere (r) 1, where LC neurons are born, resulted in a dramatic increase in the number of Phox2a- and Phox2b-expressing early-differentiating LC neurons, and dopamine-ß-hydroxylase- and tyrosine-hydroxylase-expressing late-differentiating LC neurons. In contrast, other neuronal populations derived from the dorsal r1 were either reduced or unchanged. In addition, a drastic upregulation of Ascl1, an essential factor for noradrenergic neurogenesis, was observed in dorsal r1 of conditional knockout mice. Through genomic sequence analysis and EMSA and ChIP assays, a conserved Rbpj-binding motif was identified within the Ascl1 promoter. A luciferase reporter assay revealed that Rbpj per se could induce Ascl1 transactivation but this effect was counteracted by its downstream-targeted gene Hes1. Moreover, our in vitro gene transfection and in ovo electroporation assays showed that Rbpj upregulated Ascl1 expression when Hes1 expression was knocked down, although it also exerted a repressive effect on Ascl1 expression in the presence of Hes1. Thus, our results provide the first evidence that Rbpj functions as a key modulator of LC neuron development by regulating Ascl1 expression directly, and indirectly through its target gene Hes1.

Myosin X regulates netrin receptors and functions in axonal path-finding.

Netrins regulate axon path-finding during development, but the underlying mechanisms are not well understood. Here, we provide evidence for the involvement of the unconventional myosin X (Myo X) in netrin-1 function. We find that Myo X interacts with the netrin receptor deleted in colorectal cancer (DCC) and neogenin, a DCC-related protein. Expression of Myo X redistributes DCC to the cell periphery or to the tips of neurites, whereas its silencing prevents DCC distribution in neurites. Moreover, expression of DCC, but not neogenin, stimulates Myo X-mediated formation and elongation of filopodia, suggesting that Myo X function may be differentially regulated by DCC and neogenin. The involvement of Myo X in netrin-1 function was further supported by the effects of inhibiting Myo X function in neurons. Cortical explants derived from mouse embryos expressing a motor-less Myo X exhibit reduced neurite outgrowth in response to netrin-1 and chick commissural neurons expressing the motor-less Myo X, or in which Myo X is silenced using microRNA (miRNA), show impaired axon projection in vivo. Taken together, these results identify a novel role for Myo X in regulating netrin-1 function.

Neonatal intrahippocampal injection of lipopolysaccharide induces deficits in social behavior and prepulse inhibition and microglial activation in rats: Implication for a new schizophrenia animal model.

Several lines of evidence have suggested that the dysregulation of immune system is involved in the pathogenesis of schizophrenia. Microglia are the resident macrophage of the brain and the major player in innate immunity in the brain. We hypothesized that microglia activation may be closely associated with the neuropathology of schizophrenia. Neonatal intrahippocampal injection of lipopolysaccharide (LPS), an activator of microglia, was performed in rats at postnatal day 7 (PD7), and they were separately treated with saline or minocycline for consecutive 3days. Behavioral changes (locomotor activity, social interaction and prepulse inhibition) were examined in adulthood, and the number of microglia was assessed using immunohistochemistry at PD9, PD21 and PD67. The adult rats in LPS-injected group showed obvious behavioral alterations (deficits in social behavior and prepulse inhibition) and a persistently dramatic increase of number of activated microglial cells in the hippocampus, cerebral cortex and thalamus compared to those in saline-injected group. Interestingly, pretreatment with minocycline could significantly rescue the behavioral deficits and prevent microglia activation. Our results suggest that neonatal intrahippocampal LPS injection may serve as a potential schizophrenia animal model, and inhibition of microglia activation may be a potential treatment strategy for schizophrenia.

RNF220-mediated K63-linked polyubiquitination stabilizes Olig proteins during oligodendroglial development and myelination.

Maldevelopment of oligodendroglia underlies neural developmental disorders such as leukodystrophy. Precise regulation of the activity of specific transcription factors (TFs) by various posttranslational modifications (PTMs) is required to ensure proper oligodendroglial development and myelination. However, the role of ubiquitination of these TFs during oligodendroglial development is yet unexplored. Here, we find that RNF220, a known leukodystrophy-related E3 ubiquitin ligase, is required for oligodendroglial development. RNF220 depletion in oligodendrocyte lineage cells impedes oligodendrocyte progenitor cell proliferation, differentiation, and (re)myelination, which consequently leads to learning and memory defects. Mechanistically, RNF220 targets Olig1/2 for K63-linked polyubiquitination and stabilization during oligodendroglial development. Furthermore, in a knock-in mouse model of leukodystrophy-related RNF220R365Q mutation, the ubiquitination and stabilization of Olig proteins are deregulated in oligodendroglial cells. This results in pathomimetic oligodendroglial developmental defects, impaired myelination, and abnormal behaviors. Together, our evidence provides an alternative insight into PTMs of oligodendroglial TFs and how this essential process may be implicated in the etiology of leukodystrophy.

Ventricular Netrin-1 deficiency leads to defective pyramidal decussation and mirror movement in mice.

The corticospinal tract (CST) is the principal neural pathway responsible for conducting voluntary movement in the vertebrate nervous system. Netrin-1 is a well-known guidance molecule for midline crossing of commissural axons during embryonic development. Families with inherited Netrin-1 mutations display congenital mirror movements (CMM), which are associated with malformations of pyramidal decussation in most cases. Here, we investigated the role of Netrin-1 in CST formation by generating conditional knockout (CKO) mice using a Gfap-driven Cre line. A large proportion of CST axons spread laterally in the ventral medulla oblongata, failed to decussate and descended in the ipsilateral spinal white matter of Ntn1Gfap CKO mice. Netrin-1 mRNA was expressed in the ventral ventricular zone (VZ) and midline, while Netrin-1 protein was transported by radial glial cells to the ventral medulla, through which CST axons pass. The level of transported Netrin-1 protein was significantly reduced in Ntn1Gfap CKO mice. In addition, Ntn1Gfap CKO mice displayed increased symmetric movements. Our findings indicate that VZ-derived Netrin-1 deletion leads to an abnormal trajectory of the CST in the spinal cord due to the failure of CST midline crossing and provides novel evidence supporting the idea that the Netrin-1 signalling pathway is involved in the pathogenesis of CMM.

Satb2 is required for dendritic arborization and soma spacing in mouse cerebral cortex.

Self-avoidance is a mechanism by which dendrites from the same neuron repel one another in order to establish uniform coverage of the dendritic field. The importance of self-avoidance for the development of complex arborization patterns has been highlighted by studies of Drosophila sensory and mouse retinal neurons. However, it is unclear whether branch patterning in the mammalian central nervous system is also governed by this strategy. We reduced Satb2 expression in a population of layer II/III pyramidal neurons in vivo by RNA interference and found that the somas of Satb2-deficient neurons clumped together, and their dendrites failed to expand laterally but instead formed fascicles. Furthermore, experiments showed that reducing Satb2 caused the adhesion of not only neighboring Satb2-deficient neurons but also neighboring wild-type neurons. Our results indicate a cell autonomous and non-cell autonomous role for Satb2 in regulating the adhesive and/or repulsive properties of cerebral pyramidal neurons.

Zbtb20 is essential for the specification of CA1 field identity in the developing hippocampus.

The development of hippocampal circuitry depends on the proper assembly of correctly specified and fully differentiated hippocampal neurons. Little is known about factors that control the hippocampal specification. Here, we show that zinc finger protein Zbtb20 is essential for the specification of hippocampal CA1 field identity. We found that Zbtb20 expression was initially activated in the hippocampal anlage at the onset of corticogenesis, and persisted in immature hippocampal neurons. Targeted deletion of Zbtb20 in mice did not compromise the progenitor proliferation in the hippocampal and adjacent transitional ventricular zone, but led to the transformation of the hippocampal CA1 field into a transitional neocortex-like structure, as evidenced by cytoarchitectural, neuronal migration, and gene expression phenotypes. Correspondingly, the subiculum was ectopically located adjacent to the CA3 in mutant. Although the field identities of the mutant CA3 and dentate gyrus (DG) were largely maintained, their projections were severely impaired. The hippocampus of Zbtb20 null mice was reduced in size, and exhibited increased apoptotic cell death during postnatal development. Our data establish an essential role of Zbtb20 in the specification of CA1 field identity by repressing adjacent transitional neocortex-specific fate determination.