Depletion of Ric-8B leads to reduced mTORC2 activity

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Research Article

Maíra H. Nagai, 

Victor P. S. Xavier, 

Luciana M. Gutiyama, 

Cleiton F. Machado, 

Alice H. Reis, 

Elisa R. Donnard, 

Pedro A. F. Galante, 

Jose G. Abreu, 

William T. Festuccia, 

Bettina Malnic


Published: May 11, 2020


?This is an uncorrected proof.

AbstractmTOR, a serine/threonine protein kinase that is involved in a series of critical cellular processes, can be found in two functionally distinct complexes, mTORC1 and mTORC2. In contrast to mTORC1, little is known about the mechanisms that regulate mTORC2. Here we show that mTORC2 activity is reduced in mice with a hypomorphic mutation of the Ric-8B gene. Ric-8B is a highly conserved protein that acts as a non-canonical guanine nucleotide exchange factor (GEF) for heterotrimeric Gαs/olf type subunits. We found that Ric-8B hypomorph embryos are smaller than their wild type littermates, fail to close the neural tube in the cephalic region and die during mid-embryogenesis. Comparative transcriptome analysis revealed that signaling pathways involving GPCRs and G proteins are dysregulated in the Ric-8B mutant embryos. Interestingly, this analysis also revealed an unexpected impairment of the mTOR signaling pathway. Phosphorylation of Akt at Ser473 is downregulated in the Ric-8B mutant embryos, indicating a decreased activity of mTORC2. Knockdown of the endogenous Ric-8B gene in cultured cell lines leads to reduced phosphorylation levels of Akt (Ser473), further supporting the involvement of Ric-8B in mTORC2 activity. Our results reveal a crucial role for Ric-8B in development and provide novel insights into the signals that regulate mTORC2.
Author summary
Gene inactivation in mice can be used to identify genes that are involved in important biological processes and that may contribute to disease. We used this approach to study the Ric-8B gene, which is highly conserved in mammals, including humans. We found that Ric-8B is essential for embryogenesis and for the proper development of the nervous system. Ric-8B mutant mouse embryos are smaller than their wild type littermates and show neural tube defects at the cranial region. This approach also allowed us to identify the biological pathways that potentially contribute to the observed phenotypes, and uncover a novel role for Ric-8B in the mTORC2 signaling pathway. mTORC2 plays particular important roles in the adult brain, and has been implicated in neurological disorders. Our mutant mice provide a model to study the complex molecular and cellular processes underlying the interplay between Ric-8B and mTORC2 in neuronal function.

Citation: Nagai MH, Xavier VPS, Gutiyama LM, Machado CF, Reis AH, Donnard ER, et al. (2020) Depletion of Ric-8B leads to reduced mTORC2 activity. PLoS Genet 16(5):

https://doi.org/10.1371/journal.pgen.1008255Editor: J. Silvio Gutkind, University of California San Diego, UNITED STATESReceived: June 13, 2019; Accepted: February 24, 2020; Published: May 11, 2020Copyright: © 2020 Nagai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Data Availability: All relevant data are within the manuscript and its Supporting Information files.Funding: MHN received support from Fundação de Amparo à Pesquisa do Estado de São Paulo (#14/15495-8, MN). BM and VPSX receive support from Fundação de Amparo à Pesquisa do Estado de São Paulo (#16/24471-0 (BM) and #2019/05166-0 (VPSX)). BM and LMG received support from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Competing interests: The authors have declared that no competing interests exist.

IntroductionRic-8B (resistant to inhibitors of cholinesterase 8B) is a highly conserved protein which interacts with Gαs class subunits from heterotrimeric G proteins [1,2]. In vitro, Ric-8B can work as a guanine nucleotide exchange factor (GEF) for both Gαs and Gαolf [1,3]. While Gαs is ubiquitously expressed, Gαolf is restrictedly expressed in the olfactory neurons and in a few regions of the brain, such as the striatum [4–6]. Ric-8B expression in adult mice is highly predominant in the same tissues where Gαolf is expressed indicating that these two proteins are functional partners in vivo [2]. Consistent with the role of a GEF, Ric-8B is able to amplify odorant receptor signaling or dopamine receptor signaling through Gαolf in cultured cells [2,7–9]. Also, a series of studies indicate that Ric-8B regulates Gα protein abundance in the cells, and suggest that Ric-8B may serve as a chaperone that promotes Gα protein stability and the formation of functional G protein complexes [7,8,10–13].
In addition to the full-length Ric-8B, an alternatively spliced version of Ric-8B lacking exon 9, denominated Ric-8BΔ9, is also highly expressed in the olfactory epithelium. Differently from full-length Ric-8B, Ric-8BΔ9 does not bind to Gαs and does not show GEF activity, or does it very inefficiently [2,3]. Studies have shown that both Ric-8B and Ric-8BΔ9 are able to interact with the different Gγ subunit types, Gγ13, Gγ7 and Gγ8 [10]. Chan and colleagues showed that Ric-8BΔ9, but not full-length Ric-8B, can bind Gβ1γ2 [3]. These results suggest that besides acting on the Gαs subunits, the Ric-8B proteins may also play a role in Gβγ signaling.
Despite the restricted pattern of expression in adult mice, previous studies have shown that complete knockout of the Ric-8B gene results in mice that are not viable and that die early during embryogenesis (between E4 and E8.5) [7]. Here we investigated the physiological roles of Ric-8B during development using a gene trapped allele of Ric-8B that shows reduced levels of Ric-8B expression. We found that the Ric-8B mutant embryos are small, fail to close the neural tube at the cephalic region and die around E10.5. In the embryo, Ric-8B gene expression is predominant in the nervous system, more specifically in the neural folds in the cephalic region and in the ventral region of the neural tube. Increased apoptosis is observed in the region of the neural tube defects in the Ric-8B mutant embryos. Comparative transcriptome analysis unexpectedly revealed that mTOR signaling is impaired in the Ric-8B mutant embryos. mTOR is a serine/threonine protein kinase that acts as the catalytic core of two distinct complexes: mTORC1 and mTORC2. mTORC1 mainly controls cell growth and metabolism, promotes protein synthesis and is the best characterized complex to date [14,15]. mTORC2, on the other hand, has been implicated in the regulation of cytoskeletal organization, cell survival and cell migration [14–19]. Both complexes, mTORC1 and mTORC2, have been linked to the control of protein synthesis, although the role of mTORC2 is not as clearly defined as that of mTORC1 [20,21]. We found that mTORC2 activity, but not mTORC1 activity, is downregulated in the mutant embryo. Similar effects were observed in HEK293T and HepG2 cells which were knocked down for Ric-8B. Altogether these results show that Ric-8B is essential for embryogenesis. They also show that depletion of Ric-8B reduces mTORC2 activity.

Generation of Ric-8B gene trap mice
In order to generate mice that are deficient for Ric-8B we obtained two Baygenomics ES cell lines [22], which contain a gene trap vector in the Ric-8B gene. In the RRH188 cell line, the vector is inserted in the intron between exons 3 and 4, and in the RRA103 cell line the vector is inserted in the intron between exons 7 and 8 (Fig 1A). We used these two ES cell lines to produce chimeric mice, but we could only obtain chimeras from the RRH188 cell line. The insertion of the gene trap vector leads to the expression of a chimeric mRNA containing exons 1, 2 and 3 of the Ric-8B gene in frame with the β-geo sequence [23]. The resulting Ric-8B fusion protein is likely to be nonfunctional, because it only contains amino acids 1–246 from Ric-8B. Chimeric males were crossed with C57BL/6 females and the agouti-colored offspring were analyzed for transmission of the gene trap vector. As expected, approximately 50% of these mice were heterozygous for the gene trap insertion. These mice develop normally with no signs of deficits, when compared to their wild type siblings.
Fig 1. Ric-8B gene trap mice.(A) The genomic structure of the Ric-8B gene is shown, with its ten exons (1–10) and nine introns (between the exons) and the insertion sites of the gene trap vector in the ES cell lines RRH188 and RRA103. The insertion of the gene trap vector in intron 3 in the RRH188 cell line leads to the expression of chimeric mRNA containing exons 1, 2 and 3 in frame with the β-geo sequence. The locations of the primers used for PCR-based genotyping are indicated. SA: splice acceptor site. The Ric-8BΔ9 isoform lacks exon 9 (indicated in grey), is indicated. B) Multiplex PCR-based genotyping of the embryos obtained from intercrossing of heterozygous mice using one forward primer (188intronF2) and two reverse primers (VectorR2 and 188intronR2). A 312 bp PCR product is expected for the mutant allele, and a 582 bp PCR product is expected for the wild type allele. Representative analyzed embryos showed the following genotypes: Ric-8Bwt/wt (wild type, lanes 1, 3 and 5), Ric-8Bwt/bgeo (heterozygote, lanes 2 and 4) and Ric-8Bbgeo/bgeo (homozygous, lanes 6 and 7). (C) Ric-8B gene expression in the mouse embryo. RT-PCR was conducted to amplify Ric-8B and Ric-8BΔ9 transcripts from RNA prepared from wild type mouse embryos at different developmental stages. The PCR product sizes expected using the pair of primers that flank the ninth exon (A) are 462 bp (Ric-8B) and 342 bp (Ric-8BΔ9). (D) RT-PCR was conducted to amplify the regions between exon 3 and exon 5 (1) and exon 7 and exon 10 (2) of Ric-8B and actin (3) from embryos with different genotypes as indicated. (E) Real-time PCR was conducted to compare the expression levels of the Ric-8B gene in wild type (Ric-8Bwt/wt), heterozygotes (Ric-8Bwt/bgeo) and homozygous (Ric-8Bbgeo/bgeo) mutant embryos. Transcript levels were normalized to β-actin levels and are shown relative to the expression levels in wild type embryos. (F) Western blot analysis of protein extracts from embryos with the different genotypes using anti-Ric-8B antibodies. α-tubulin was used as a loading control.


Ric-8B mutant mice are embryonic lethal
Heterozygous mice were intercrossed to generate homozygous mutant mice. Genotyping of the offspring revealed the absence of mice homozygous for the Ric-8B gene mutation (Table 1). In order to determine the time of embryonic death, embryos from heterozygous intercrosses were genotyped at different developmental stages (Fig 1B, Table 1). We found that homozygous embryos die around embryonic day 10.5.
As mentioned above, two major transcript forms of Ric-8B are expressed in the olfactory epithelium, a full-length Ric-8B isoform and Ric-8BΔ9, an isoform which lacks exon 9 (Fig 1A) [2]. RT-PCR experiments showed that the Ric-8B gene is expressed in the mouse embryo at early stages of development, and that Ric-8BΔ9 is the predominant isoform present in the embryo, while both isoforms are equally abundant in the olfactory epithelium of adult mice (Fig 1C, [2]).
RT-PCR analysis demonstrated that even though detectable, the levels of Ric-8B mRNA are much lower in the homozygotes (Ric-8Bbgeo/bgeo) than in the wild type (Ric-8Bwt/wt) or heterozygous (Ric-8Bwt/bgeo) embryos (Fig 1D and 1E). Accordingly, Western blot analysis of extracts prepared from whole-embryos demonstrated that the levels of Ric-8B protein are lower in the homozygous embryos than in wild type or heterozygous embryos (Fig 1F). The residual levels present in the homozygous embryos is likely to result from the gene knockout technology used, that is, a small fraction of the mRNA can be produced by splicing out the intron containing the gene trap vector leading to the production of wild type Ric-8B mRNAs [23].

Ric-8B gene expression in the adult mouse
We used the β-geo gene reporter, which is expressed under the control of the Ric-8B promoter, to monitor the Ric-8B gene expression in the heterozygous (Ric-8Bwt/bgeo) mice. Strong blue staining is detected in the olfactory epithelium, but not in the olfactory bulb, vomeronasal organ or brain (Fig 2A). Staining can also be detected in the septal organ of Masera, an isolated small patch of sensory epithelium located at the ventral base of the nasal septum [24], which is known to contain olfactory sensory neurons that express Gαolf as well as other canonical olfactory sensory neuron genes [25]. Although no staining was observed in the medial view of the brain (Fig 2A), when the brain is sectioned in a parasagittal plane, blue staining of the striatum is revealed (Fig 2B). X-gal staining of sections cut through the nasal cavity shows that β-galactosidase activity is present throughout the olfactory epithelium and in the region where the neurons of the septal organ are located (Fig 2C, 2D and 2F). These results are in agreement with analysis of Ric-8B gene expression by in situ hybridization [2], and indicate that the expression of the β-geo reporter is indistinguishable from that of the endogenous Ric-8B gene.
Fig 2. Ric-8B expression in the adult Ric-8Bwt/bgeo mouse as revealed by X-gal staining.(A) Sagittal whole-mount view of the nasal cavity and brain stained with X-gal. Blue staining can be detected in the olfactory epithelium (OE) and septal organ (SO), but not in the olfactory bulb (OB) nor in the vomeronasal organ (VNO). (B) The brain region is cut in a parasagittal plane, revealing blue staining of the striatum. (C-G) X-gal staining of sections cut through the nasal cavity of Ric-8B wt/bgeo mice (C, D and F) or Ric-8B wt/wt mice (E and G). X-gal staining is present throughout the olfactory epithelium (C, D and F), and in the septal organ region (arrow in D). In the same experimental conditions, no staining is observed in the wild type tissues. Sections in A-G were taken from the regions indicated in the schematic representation of the mouse brain.

https://doi.org/10.1371/journal.pgen.1008255.g002We also examined the Ric-8B gene expression in different tissues of the heterozygous mice. No staining was observed in any of the several analyzed tissues (see S1 Fig). Altogether, these results confirm the previous findings that the expression of the Ric-8B gene is predominantly expressed in a few tissues in the adult mouse. Lower levels of Ric-8B gene expression are however also detectable in several other tissues, as shown in publicly available gene expression data, such as the EMBL-EBI expression Atlas (https://www.ebi.ac.uk/gxa/home) and Mouse ENCODE transcriptome data (https://www.ncbi.nlm.nih.gov/gene/237422/).

Ric-8B is required for embryonic growth and development of the cranial neural tube
Analysis of the heterozygous embryos showed that expression from the Ric-8B gene promoter is restricted to the cephalic neural folds and neural tube regions (Fig 3B, 3E and 3E’). Notably, the levels of β-galactosidase expression are significantly higher in the Ric-8Bbgeo/bgeo embryos when compared to heterozygote embryos (Fig 3C, 3F and 3F’). These results are expected, since the Ric-8Bbgeo/bgeo embryos carry two copies of the β-geo reporter gene while the Ric-8Bwt/bgeo embryos have only one copy.
Fig 3. Ric-8B mutant embryos are smaller and show defective neural tube closure in the cephalic region.(A-F´) Wild type (Ric-8Bwt/wt), heterozygous (Ric-8Bwt/bgeo) or homozygous (Ric-8Bbgeo/bgeo) embryos at E8.5 or E9.5 stained with X-gal are shown (not shown in scale). Ventral (D-F) and dorsal (D’-F’) views of the embryos are shown. In the Ric-8Bbgeo/bgeo embryos, the cephalic neural folds are not fused (white arrows) but the other regions of the neural tube are normally closed (F, F’). (G-I) Wild type (Ric-8Bwt/wt), heterozygous (Ric-8Bwt/bgeo) or homozygous (Ric-8Bbgeo/bgeo) embryos were examined by scanning electron microscopy at E9.5. G’, H’ and I’ are magnified regions from G, H and I, respectively. P (prosencephalon), BA (first branchial arch). Scale bars in G- I represent 100 μm, in G’-I’, 50 μm. (J) Representative images of a wild type and a homozygous mutant embryo (shown in scale).

https://doi.org/10.1371/journal.pgen.1008255.g003At E8.5–9.5, heterozygous embryos (Ric-8Bwt/bgeo, Fig 3B, 3E and 3E’) are morphologically similar to that of the wild type embryos (Ric-8Bwt/wt, Fig 3A, 3D and 3D’). However, from E8.5, great part of homozygous mutant embryos (Ric-8Bbgeo/bgeo; Fig 3B and 3C) are slightly smaller and show phenotypic abnormalities in the prosencephalon. The reduced size of homozygote embryos is emphasized in later stages (Fig 3J), and the failure in the closure of the cephalic neural tube becomes evident at E9.5 (Fig 3F and 3F’). In order to better visualize the fusion of the cephalic neural folds we performed scanning electron microscopy. Wild type as well as heterozygous E9.5 embryos show normally closed neural tube (Fig 3G and 3G’, 3H and 3H’). The Ric-8Bbgeo/bgeo embryos, however, do not display fused midline hemispheres (Fig 3I and 3I’). These embryos are not able to close the neural tube and usually display open rhombencephalic, mesencephalic and prosencephalic vesicles (Fig 3I and 3I’). It is important to note that this ‘open brain’ phenotype is highly penetrant, shown by ~86% of the analyzed embryos.
Expression of the Ric-8B gene along the embryonic anterior-posterior axis was analyzed in transverse sections of X-gal stained embryos. Staining in Ric-8Bwt/bgeo embryos is highly restricted to the notochord, dorsal neural tube in the region of the presumptive brain, and to the ventral region of the neural tube, including the floor plate of the spinal cord (Fig 4A, 4B and 4E–4H). The same regions are strongly stained in Ric-8Bbgeo/bgeo embryos, however, less intense staining is also detected all over the neural tube and regions of the adjacent mesoderm (Fig 4C, 4D and 4I–4L). As mentioned above, while the neural tube is normally closed in the Ric-8Bwt/bgeo embryos, it fails to close in the brain region in Ric-8Bbgeo/bgeo embryos (arrows in Fig 4G and 4K).
Fig 4. Ric-8B expression in the embryo is predominant in the nervous system.Upper panels: sagittal views of a Ric-8Bwt/bgeo embryo (A) or a Ric-8B bgeo/bgeo embryo (C) at E8.5. The lines indicate the levels at which the sections were cut (shown in B and D). In the Ric-8Bwt/bgeo embryo (A and B), blue staining is detected in the neural folds in the brain region (asterisk) and in the notochord (nc). In the Ric-8Bbgeo/bgeo embryo (C and D) blue staining is detected in the same regions as in the Ric-8Bwt/bgeo embryo, however the staining is stronger and expanded. Lower panels: sagittal and ventral views of a Ric-8Bwt/bgeo embryo (E and F) or a Ric-8Bbgeo/bgeo embryo (I and J) at E9.5. In the Ric-8Bwt/bgeo embryo (E-H), blue staining is detected in the neural fold fusion in the brain region (arrows) and in the floor plate region (fp). Note that the neural tube is closed (arrow in G). (H) Higher magnification of the neural tube in the Ric-8Bwt/bgeo embryo showing strong blue staining in the floor plate region and weaker staining in the notochord. In the Ric-8Bbgeo/bgeo embryo (I- L) blue staining is detected in the same regions as in the Ric-8Bwt/bgeo embryo; however, the staining is stronger and expanded. Note that the neural tube is not closed in the anterior region of the head (arrows in K) but are normally closed at the more caudal regions. (L) Higher magnification of the neural tube in the Ric-8Bbgeo/bgeo embryo showing strong blue staining in both the floor plate (fp) and notochord (nc) regions. Me (mesenchyme).

https://doi.org/10.1371/journal.pgen.1008255.g004Ric-8B expression pattern in the notochord (Fig 4B) and the floor plate (Fig 4H, S4A Fig) is highly reminiscent of the sonic hedgehog (Shh) expression at the same embryonic stages [26]. However, we found that Shh signaling is not grossly altered in the homozygous Ric-8B mutant embryos (S4B and S4C Fig). In situ hybridization on sections of an E10.5 wild type embryo shows that Gαolf is not co-expressed with Ric-8B in the floor plate, while Gαs is highly expressed all over the neural tube (S5A Fig). These results suggest that Gαs may be the target for Ric-8B in the mouse embryo, instead of Gαolf.
The expression of the β-geo reporter gene in the neural folds and roof plate at E8.5-E9.5 (Fig 4) strongly suggest that the deficiency of Ric-8B gene expression is leading to the failure of neural tube closure. Previous studies have shown that this phenotype can result from a variety of embryonic disturbances [27,28], such as abnormalities in the contraction of apical actin microfilaments within neuroepithelial cells [27], or reduced/ increased apoptosis of neuroepithelial cells [27]. We analyzed the distribution of polymerized actin in the neural tubes of wild type and mutant E9.5 embryos, however, no significant differences between wild type and mutant embryos were observed (Fig 5A). Disturbances in apoptosis were analyzed by immunostaining for activated caspase-3 in E9.5 embryo sections. We found an increased number of apoptotic cells in the neural tube of Ric-8Bbgeo/bgeo embryos, as well as in the cranial mesenchyme (Fig 5B).
Fig 5. Increased apoptosis in Ric-8Bbgeo/bgeo embryos and MEFs.(A) Transverse sections of E9.5 embryos stained with rhodamine-conjugated phalloidin (red) show that actin filament (F-actin) is highly concentrated in the apical region of the neural tube in wild type, heterozygous and mutant embryos. (B) Transverse sections of E9.5 embryos stained for active caspase 3 (green). Ric-8Bbgeo/bgeo embryos show an increased number of apoptotic cells in the mesenchyme and neuroepithelium when compared to wild type or heterozygous embryos. DAPI was used to stain the nuclei. Forebrain (fb); mesenchyme (me); midbrain (mb); hindbrain (hb); neuroepithelium (ne). The approximate localizations of the sections are indicated to the right. (C) MEFs were generated from Ric-8Bwt/wt (n=2) or Ric-8Bbgeo/bgeo (n=1) embryos were double stained for BrdU (red) and activated caspase-3 (green). The percentages of cells labeled in each case are indicated in the graph.

https://doi.org/10.1371/journal.pgen.1008255.g005We also tested the impact of Ric-8B gene depletion on apoptosis in vitro, by using mouse embryonic fibroblasts (MEFs) generated from the Ric-8B mutant embryos. Even though Ric-8B is predominantly expressed in the nervous system (as shown in Figs 3 and 4), MEFs prepared from wild type embryos also express Ric-8B. Noticeably, MEF preparations from Ric-8Bbgeo/bgeo embryos died within few days in culture. We found that, while the number of dividing cells seems to be unaltered, as revealed by bromodeoxyuridine (BrdU) staining, the number of cells immuno stained for activated caspase-3 is increased in MEFs generated from Ric-8Bbgeo/bgeo embryos, when compared to MEFs generated from Ric-8Bwt/wt embryos (Fig 5C). These results indicate that depletion of Ric-8B leads to increased apoptosis.

Cell signaling pathways altered in the Ric-8B mutant embryos
To gain insight into the molecular mechanisms impacted by the mutation in the Ric-8B gene, we sequenced and compared the transcriptomes of mutant and wild type E10.5 embryos. A total of 947 coding genes were found to be differentially expressed (FDR Read More

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