Targeted deletion of RIC8A in mouse neural precursor cells interferes with the development of the brain,eyes, and muscles

Autosomal recessive disorders such as Fukuyama congenital muscular dystrophy, Walker–Warburg syndrome, and the muscle–eye–brain disease are characterized by defects in the development of patient's brain, eyes, and skeletal muscles. These syndromes are accompanied by brain malformations like type II lissencephaly in the cerebral cortex with characteristic overmigrations of neurons through the breaches of the pial basement membrane. The signaling pathways activated by laminin receptors, dystroglycan and integrins, control the integrity of the basement membrane, and their malfunctioning may underlie the pathologies found in the rise of defects reminiscent of these syndromes. Similar defects in corticogenesis and neuromuscular disorders were found in mice when RIC8A was specifically removed from neural precursor cells. RIC8A regulates a subset of G‐protein α subunits and in several model organisms, it has been reported to participate in the control of cell division, signaling, and migration. Here, we studied the role of RIC8A in the development of the brain, muscles, and eyes of the neural precursor‐specific conditional Ric8a knockout mice. The absence of RIC8A severely affected the attachment and positioning of radial glial processes, Cajal‐Retzius’ cells, and the arachnoid trabeculae, and these mice displayed additional defects in the lens, skeletal muscles, and heart development. All the discovered defects might be linked to aberrancies in cell adhesion and migration, suggesting that RIC8A has a crucial role in the regulation of cell–extracellular matrix interactions and that its removal leads to the phenotype characteristic to type II lissencephaly‐associated diseases. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 374–390, 2018     

Ablation of RIC8A Function in Mouse Neurons Leads to a Severe Neuromuscular Phenotype and Postnatal Death

Resistance to inhibitors of cholinesterase 8 (RIC8) is a guanine nucleotide exchange factor required for the intracellular regulation of G protein signalling. RIC8 activates different Gα subunits via non-canonical pathway, thereby amplifying and prolonging the G protein mediated signal. In order to circumvent the embryonic lethality associated with the absence of RIC8A and to study its role in the nervous system, we constructed Ric8a conditional knockout mice using Cre/loxP technology. Introduction of a synapsin I promoter driven Cre transgenic mouse strain (SynCre) into the floxed Ric8a (Ric8a ^F/F) background ablated RIC8A function in most differentiated neuron populations. Mutant SynCre ^+/- Ric8 ^lacZ/F mice were born at expected Mendelian ratio, but they died in early postnatal age (P4-P6). The mutants exhibited major developmental defects, like growth retardation and muscular weakness, impaired coordination and balance, muscular spasms and abnormal heart beat. Histological analysis revealed that the deficiency of RIC8A in neurons caused skeletal muscle atrophy and heart muscle hypoplasia, in addition, the sinoatrial node was misplaced and its size reduced. However, we did not observe gross morphological changes in brains of SynCre ^+/- Ric8a ^lacZ/F mutants. Our results demonstrate that in mice the activity of RIC8A in neurons is essential for survival and its deficiency causes a severe neuromuscular phenotype.     

Expression Pattern and Localization Dynamics of Guanine Nucleotide Exchange Factor RIC8 during Mouse Oogenesis

Targeting of G proteins to the cell cortex and their activation is one of the triggers of both asymmetric and symmetric cell division. Resistance to inhibitors of cholinesterase 8 (RIC8), a guanine nucleotide exchange factor, activates a certain subgroup of G protein α-subunits in a receptor independent manner. RIC8 controls the asymmetric cell division in Caenorhabditis elegans and Drosophila melanogaster, and symmetric cell division in cultured mammalian cells, where it regulates the mitotic spindle orientation. Although intensely studied in mitosis, the function of RIC8 in mammalian meiosis has remained unknown. Here we demonstrate that the expression and subcellular localization of RIC8 changes profoundly during mouse oogenesis. Immunofluorescence studies revealed that RIC8 expression is dependent on oocyte growth and cell cycle phase. During oocyte growth, RIC8 is abundantly present in cytoplasm of oocytes at primordial, primary and secondary preantral follicle stages. Later, upon oocyte maturation RIC8 also populates the germinal vesicle, its localization becomes cell cycle dependent, and it associates with chromatin and the meiotic spindle. After fertilization, RIC8 protein converges to the pronuclei and is also detectable at high levels in the nucleolus precursor bodies of both maternal and paternal pronucleus. During first cleavage of zygote RIC8 localizes in the mitotic spindle and cell cortex of forming blastomeres. In addition, we demonstrate that RIC8 co-localizes with its interaction partners Gα_i1/2:GDP and LGN in meiotic/mitotic spindle, cell cortex and polar bodies of maturing oocytes and zygotes. Downregulation of Ric8 by siRNA leads to interferred translocation of Gα_i1/2 to cortical region of maturing oocytes and reduction of its levels. RIC8 is also expressed at high level in female reproductive organs e.g. oviduct. Therefore we suggest a regulatory function for RIC8 in mammalian gametogenesis and fertility.     

NCAM180 regulates Ric8A membrane localization and potentiates β-adrenergic response

Cooperation between receptors allows integrated intracellular signaling leading to appropriate physiological responses. The Neural Cell Adhesion Molecule (NCAM) has three main isoforms of 120, 140 and 180 kDa, with adhesive and signaling properties, but their respective functions remains to be fully identified. Here we show that the human NCAM180 intracellular domain is a novel interactor of the human guanosine exchange factor (GEF) Ric8A using the yeast two hybrid system and immunoprecipitation. Furthermore, NCAM, Ric8A and G(αs) form a tripartite complex. Colocalization experiments by confocal microscopy revealed that human NCAM180 specifically induces the recruitment of Ric8A to the membrane. In addition, using an in vitro recombinant system, and in vivo by comparing NCAM knock-out mouse brain to NCAM heterozygous and wild type brains, we show that NCAM expression dose dependently regulates Ric8A redistribution in detergent resistent membrane microdomains (DRM). Previous studies have demonstrated essential roles for Ric8 in G(α) protein activity at G protein coupled receptors (GPCR), during neurotransmitter release and for asymmetric cell division. We observed that inhibition of Ric8A by siRNA or its overexpression, decreases or increases respectively, cAMP production following β-adrenergic receptor stimulation. Furthermore, in human HEK293T recombinant cells, NCAM180 potentiates the G(αs) coupled β-adrenergic receptor response, in a Ric8A dependent manner, whereas NCAM120 or NCAM140 do not. Finally, in mouse hippocampal neurons expressing endogenously NCAM, NCAM is required for the agonist isoproterenol to induce cAMP production, and this requirement depends on Ric8A. These data illustrate a functional crosstalk between a GPCR and an IgCAM in the nervous system.     

Biophysical studies support a predicted superhelical structure with armadillo repeats for Ric‐8

Ric‐8 is a highly conserved cytosolic protein (MW 63 KDa) initially identified in C. elegans as an essential factor in neurotransmitter release and asymmetric cell division. Two different isoforms have been described in mammals, Ric‐8A and Ric‐8B; each possess guanine nucleotide exchange activity (GEF) on heterotrimeric G‐proteins, but with different Gα subunits specificities. To gain insight on the mechanisms involved in Ric‐8 cellular functions it is essential to obtain some information about its structure. Therefore, the aim of this work was to create a structural model for Ric‐8. In this case, it was not possible to construct a model based on comparison with a template structure because Ric‐8 does not present sequence similarity with any other protein. Consequently, different bioinformatics approaches that include protein folding and structure prediction were used. The Ric‐8 structural model is composed of 10 armadillo folding motifs, organized in a right‐twisted α‐alpha super helix. In order to validate the structural model, a His‐tag fusion construct of Ric‐8 was expressed in E. coli, purified by affinity and anion exchange chromatography and subjected to circular dichroism analysis (CD) and thermostability studies. Ric‐8 is approximately 80% alpha helix, with a Tm of 43.1°C, consistent with an armadillo‐type structure such as α‐importin, a protein composed of 10 armadillo repeats. The proposed structural model for Ric‐8 is intriguing because armadillo proteins are known to interact with multiple partners and participate in diverse cellular functions. These results open the possibility of finding new protein partners for Ric‐8 with new cellular functions.     

A Ric8/Synembryn Homolog Promotes Gpa1 and Gpa2 Activation To Respectively Regulate Cyclic AMP and Pheromone Signaling in Cryptococcus neoformans

The G protein α subunits Gpa1, Gpa2, and Gpa3 mediate signal transduction and are important in the growth and virulence of Cryptococcus neoformans. To understand how Gpa1 functions without a conventional Gβ subunit, we characterized a resistance to inhibitors of cholinesterase 8 (Ric8) homolog from C. neoformans, which shares amino acid sequence homology with other Ric8 proteins that exhibit guanine nucleotide exchange factor (GEF) activity toward Gα. We found that the ric8 mutant was reduced in capsule size and melanin formation, which could be suppressed by cyclic AMP (cAMP) supplementation or by introducing the activated GPA1^Q284L allele. Consistent with the fact that Ric8 participates in cAMP signaling to regulate virulence, the ric8 mutant was attenuated in virulence toward mice. Interestingly, disruption of RIC8 also resulted in opposing effects on pheromone signaling, as the ric8 mutant showed reduced mating but an enhanced ability to induce the pheromone response in the mating partner. To identify Ric8 functional mechanisms, we examined the interactions between Ric8 and the three Gα proteins. Ric8 interacted with Gpa1 and Gpa2, but not Gpa3. The presence of Gpa1^Q284L negatively affected its interaction with Ric8, whereas the activated Gpa2^Q203L allele abolished the interaction. Collectively, these findings suggest that Ric8 functions as a GEF to facilitate the activation of Gpa1-cAMP signaling and to promote Gpa2, affecting mating efficiency. Our study highlights the distinct and conserved characteristics associated with G protein signaling and contributes to our overall understanding of how G protein α subunits function with or without a canonical Gβ partner in C. neoformans.     

RIC, a calmodulin-binding Ras-like GTPase.

Neuronal activity dramatically increases the concentration of cytosolic Ca2+, which then serves as a second messenger to direct diverse cellular responses. Calmodulin is a primary mediator of Ca2+ signals in the nervous system. In a screen for calmodulin-binding proteins, we identified RIC, a protein related to the Ras subfamily of small GTPases. In addition to the ability to bind calmodulin, a number of unique features distinguished RIC from other Ras-like GTPases, including the absence of a signal for prenylation and a distinct effector (G2) domain. Furthermore, we describe two human proteins, RIN and RIT, which were 71% and 66% identical to RIC respectively, shared related G2 domains with RIC, and lacked prenylation signals, suggesting that the RIC family is conserved from flies to humans. While Ric and RIT were widely expressed, expression of RIN was confined to the neuron system.     

Xenopus laevis RIC‐3 enhances the functional expression of the C. elegans homomeric nicotinic receptor,ACR‐16, in Xenopus oocytes

RIC‐3 enhances the functional expression of certain nicotinic acetylcholine receptors (nAChRs) in vertebrates and invertebrates and increases the availability of functional receptors in cultured cells and Xenopus laevis oocytes. Maximal activity of RIC‐3 may be cell‐type dependent, so neither mammalian nor invertebrate proteins is optimal in amphibian oocytes. We cloned the X. laevis ric‐3 cDNA and tested the frog protein in oocyte expression studies. X. laevis RIC‐3 shares 52% amino acid identity with human RIC‐3 and only 17% with that of Caenorhabditis elegans. We used the C. elegans nicotinic receptor, ACR‐16, to compare the ability of RIC‐3 from three species to enhance receptor expression. In the absence of RIC‐3, the proportion of oocytes expressing detectable nAChRs was greatly reduced. Varying the ratio of acr‐16 to X. laevis ric‐3 cRNAs injected into oocytes had little impact on the total cell current. When X. laevis, human or C. elegans ric‐3 cRNAs were co‐injected with acr‐16 cRNA (1 : 1 ratio), 100 μM acetylcholine induced larger currents in oocytes expressing X. laevis RIC‐3 compared with its orthologues. This provides further evidence for a species‐specific component of RIC‐3 activity, and suggests that X. laevis RIC‐3 is useful for enhancing the expression of invertebrate nAChRs in X. laevis oocytes.     

Leishmania mitochondrial tRNA importers

The RNA Import Complex (RIC) is a multi-subunit protein complex from the mitochondria of the kinetoplastid protozoon Leishmania tropica that induces transport of tRNA across natural and artificial membranes. Leishmania, Trypanosoma and related genera of the order Kinetoplastidae are early diverging, atypical eukaryotes with unique RNA metabolic pathways, including the import of nucleus-encoded tRNAs into the mitochondrion to complement the deletion of all organelle-encoded tRNA genes. Biochemical and genetic studies of RIC are contributing to greater understanding of the mechanism of import. Additionally, RIC was shown to act as an efficient delivery vehicle for tRNA and other small RNAs into mitochondria within intact mammalian cells, indicating its applicability to the management of diseases caused by mitochondrial mutations.     

Loss of CABLES1, a Cyclin-dependent Kinase-interacting Protein that Inhibits Cell Cycle Progression,Results in Germline Expansion at the Expense of Oocyte Quality in Adult Female Mice

Recent studies have shown that cell cycle inhibitors encoded by the Ink4a gene locus constrain the self-renewing activity of adult stem cells of the hematopoietic and nervous systems. Here we report that knockout (KO) of the Cables1 [cyclin-dependent kinase (CDK)-5 and ABL enzyme substrate 1] cell cycle-regulatory gene in mice has minimal to no effect on hematopoietic stem cell (HSC) dynamics. However, female Cables1-null mice exhibit a significant expansion of germ cell (oocyte) numbers throughout adulthood. This is accompanied by a dramatic elevation in the number of atretic immature oocytes within the ovaries and an increase in the incidence of degenerating oocytes retrieved following superovulation of CABLES1-deficient females. These outcomes are not observed in mice lacking p16^INK4a alone or both p16^INK4a and p19^ARF. These data support recent reports that adult female mice can generate new oocytes and follicles but the enhancement of postnatal oogenesis by Cables1 KO appears offset by a reduction in oocyte quality, as reflected by increased elimination of these additional germ cells via apoptosis. This work also reveals cell lineage specificity with respect to the role that specific CDK-interacting proteins play in restraining the activity of adult germline versus somatic stem cells.     

EineAnabaena-artige Cyanophycee mit auffallendem Zellteilungs- und Fadenzerfallsrhythmus

This paper deals with a filamentousCyanophycea from aSphagnum bog whose position in the current system is dubious. A characteristic feature, among others, is a certain rhythm of cell division and bipartition of the filaments. As a consequence, only 8-, 16- and 32-celled filaments are present in the population. An occasional disturbance of the rhythm is transitory. In the longitudinal direction the filaments exhibit mirror symmetry in regard to their middle.

     

Ric1p and Rgp1p form a complex that catalyses nucleotide exchange on Ypt6p

Cells lacking the GTPase Ypt6p have defects in intracellular traffic and are temperature sensitive. Their growth is severely impaired by additional mutation of IMH1, which encodes a non-essential Golgi-associated coiled-coil protein. A screen for mutants that, like ypt6, specifically impair the growth of imh1 cells led to the identification of RIC1. Ric1p forms a tight complex with a previously uncharacterized protein, Rgp1p. The Ric1p-Rgp1p complex binds Ypt6p in a nucleotide-dependent manner, and purified Ric1p-Rgp1 stimulates guanine nucleotide exchange on Ypt6p in vitro. Deletion of RIC1 or RGP1, like that of YPT6, blocks the recycling of the exocytic SNARE Snc1p from early endosomes to the Golgi and causes temperature-sensitive growth, but this defect can be relieved by overexpression of YPT6. Ric1p largely colocalizes with the late Golgi marker Sec7p. Ypt6p shows a similar distribution, but this is altered when RIC1 or RGP1 is mutated. We infer that the Ric1p-Rgp1p complex serves to activate Ypt6p on Golgi membranes by nucleotide exchange, and that this is required for efficient fusion of endosome-derived vesicles with the Golgi.     

Neurogenin2‐d4Venus and Gadd45g‐d4Venus transgenic mice: Visualizing mitotic and migratory behaviors of cells committed to the neuronal lineage in the developing mammalian brain

To achieve highly sensitive and comprehensive assessment of the morphology and dynamics of cells committed to the neuronal lineage in mammalian brain primordia, we generated two transgenic mouse lines expressing a destabilized (d4) Venus controlled by regulatory elements of the Neurogenin2 (Neurog2) or Gadd45g gene. In mid‐embryonic neocortical walls, expression of Neurog2‐d4Venus mostly overlapped with that of Neurog2 protein, with a slightly (1 h) delayed onset. Although Neurog2‐d4Venus and Gadd45g‐d4Venus mice exhibited very similar labeling patterns in the ventricular zone (VZ), in Gadd45g‐d4Venus mice cells could be visualized in more basal areas containing fully differentiated neurons, where Neurog2‐d4Venus fluorescence was absent. Time‐lapse monitoring revealed that most d4Venus⁺ cells in the VZ had processes extending to the apical surface; many of these cells eventually retracted their apical process and migrated basally to the subventricular zone, where neurons, as well as the intermediate neurogenic progenitors that undergo terminal neuron‐producing division, could be live‐monitored by d4Venus fluorescence. Some d4Venus⁺ VZ cells instead underwent nuclear migration to the apical surface, where they divided to generate two d4Venus⁺ daughter cells, suggesting that the symmetric terminal division that gives rise to neuron pairs at the apical surface can be reliably live‐monitored. Similar lineage‐committed cells were observed in other developing neural regions including retina, spinal cord, and cerebellum, as well as in regions of the peripheral nervous system such as dorsal root ganglia. These mouse lines will be useful for elucidating the cellular and molecular mechanisms underlying development of the mammalian nervous system.     

Musashi1‐CreERT2: A new cre line for conditional mutagenesis in neural stem cells

The RNA‐binding protein Musashi1 (Msi1) is one of two mammalian homologues of DrosophilaMusashi, which is required for the asymmetric cell division of sensory organ precursor cells. In the mouse central nervous system (CNS), Msi1 is preferentially expressed in mitotically active progenitor cells in the ventricular zone (VZ) of the neural tube during embryonic development and in the subventricular zone (SVZ) of the postnatal brain. Previous studies showed that cells in the SVZ can contribute to long‐term neurogenesis in the olfactory bulb (OB), but it remains unclear whether Msi1‐expressing cells have self‐renewing potential and can contribute to neurogenesis in the adult. Here, we describe the generation of Msi1‐CreER^T2 knock‐in mice and show by cell lineage tracing that Msi1‐CreER^T2‐expressing cells mark neural stem cells (NSCs) in both the embryonic and adult brain. Msi1‐CreER^T2 mice thus represent a new tool in our arsenal for genetically manipulating NSCs, which will be essential for understanding the molecular mechanisms underlying neural development. genesis, 51:128–134, 2013. © 2012 Wiley Periodicals, Inc.     

Epithelial microRNA‐9a regulates dendrite growth through Fmi‐Gq signaling in Drosophila sensory neurons

microRNA‐9 (miR‐9) is highly expressed in the nervous system across species and plays essential roles in neurogenesis and axon growth; however, little is known about the mechanisms that link miR‐9 with dendrite growth. Using an in vivo model of Drosophila class I dendrite arborization (da) neurons, we show that miR‐9a, a Drosophila homolog of mammalian miR‐9, downregulates the cadherin protein Flamingo (Fmi) thereby attenuating dendrite development in a non‐cell autonomous manner. In miR‐9a knockout mutants, the dendrite length of a sensory neuron ddaE was significantly increased. Intriguingly, miR‐9a is specifically expressed in epithelial cells but not in neurons, thus the expression of epithelial but not neuronal Fmi is greatly elevated in miR‐9a mutants. In contrast, overexpression of Fmi in the neuron resulted in a reduction in dendrite growth, suggesting that neuronal Fmi plays a suppressive role in dendrite growth, and that increased epithelial Fmi might promote dendrite growth by competitively binding to neuronal Fmi. Fmi has been proposed as a G protein‐coupled receptor (GPCR), we find that neuronal G protein Gαq (Gq), but not Go, may function downstream of Fmi to negatively regulate dendrite growth. Taken together, our results reveal a novel function of miR‐9a in dendrite morphogenesis. Moreover, we suggest that Gq might mediate the intercellular signal of Fmi in neurons to suppress dendrite growth. Our findings provide novel insights into the complex regulatory mechanisms of microRNAs in dendrite development, and further reveal the interplay between the different components of Fmi, functioning in cadherin adhesion and GPCR signalling. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 225–237, 2016     

Inactivation of Pde8b enhances memory,motor performance,and protects against age‐induced motor coordination decay

Phosphodiesterases (PDEs) are critical regulatory enzymes in cyclic nucleotide signaling. PDEs have diverse expression patterns within the central nervous system (CNS), show differing affinities for cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), and regulate a vast array of behaviors. Here, we investigated the expression profile of the PDE8 gene family members Pde8a and Pde8b in the mouse brain. We find that Pde8a expression is largely absent in the CNS; by contrast, Pde8b is expressed in select regions of the hippocampus, ventral striatum, and cerebellum. Behavioral analysis of mice with Pde8b gene inactivation (PDE8B KO) demonstrate an enhancement in contextual fear, spatial memory, performance in an appetitive instrumental conditioning task, motor‐coordination, and have an attenuation of age‐induced motor coordination decline. In addition to improvements observed in select behaviors, we find basal anxiety levels to be increased in PDE8B KO mice. These findings indicate that selective antagonism of PDE8B may be an attractive target for enhancement of cognitive and motor functions; however, possible alterations in affective state will need to be weighed against potential therapeutic value .