Tree shrews (Tupaia belangeri) exhibit novelty preference in the novel location memory task with 24-h retention periods

Novelty preference is pervasive in mammalian species, and describes an inherent tendency to preferentially explore novelty. The novel location memory task studied here assesses the ability of animals to form accurate memories of a spatial configuration, consisting of several identical objects placed within an arena. Tree shrews were first familiarized with a particular object configuration during several sessions, and then an object was displaced during a test session. Tree shrews exhibited enhanced exploration when confronted with this novel configuration. The most reliable indicator associated with novelty preference was an enhancement in directed exploration towards the novel object, although we also observed a non-specific overall increase in exploration in one experiment. During the test session, we also observed an exploration of the location, which had previously been occupied by the displaced object, an effect termed empty quadrant. Our behavioral findings suggest multiple stages of spatial memory formation in tree shrews that are associated with various forms of behavioral responses to novelty. Reduced novelty preference has been linked to major depressive disorder in human patients. Given the established social conflict depression model in tree shrews, we anticipate that the study of the neural circuits of novelty preference and their malfunction during depression may have implications for understanding or treating depression in humans

Role of Bicaudal C1 in renal gluconeogenesis and its novel interaction with the CTLH complex

<div><p>Altered glucose and lipid metabolism fuel cystic growth in polycystic kidneys, but the cause of these perturbations is unclear. Renal cysts also associate with mutations in Bicaudal C1 (Bicc1) or in its self-polymerizing sterile alpha motif (SAM). Here, we found that Bicc1 maintains normoglycemia and the expression of the gluconeogenic enzymes FBP1 and PEPCK in kidneys. A proteomic screen revealed that Bicc1 interacts with the C-Terminal to Lis-Homology domain (CTLH) complex. Since the orthologous Gid complex in <i>S</i>. <i>cerevisae</i> targets FBP1 and PEPCK for degradation, we mapped the topology among CTLH subunits and found that SAM-mediated binding controls Bicc1 protein levels, whereas Bicc1 inhibited the accumulation of several CTLH subunits. Under the conditions analyzed, Bicc1 increased FBP1 protein levels independently of the CTLH complex. Besides linking Bicc1 to cell metabolism, our findings reveal new layers of complexity in the regulation of renal gluconeogenesis compared to lower eukaryotes.</p></div

Protein levels of CTLH subunits in Bicc1^-/- kidneys.

<p>(A-D) Western blot analysis of (A) RanBP9, (B) Twa1, (C) WDR26 and (D) GID4 in newborn WT and Bicc1^-/- kidney extracts. Bars represent mean ± SEM. **p <0.01. (E) RT-qPCR analysis of WDR26 and GID4 mRNAs in WT and Bicc1^-/- kidneys (n = 3 per genotype). GAPDH mRNA was used for normalization.</p

Mass spectrometric identification of CTLH complex subunits co-purified with TAP-tagged Bicc1-SH.

<p>Mass spectrometric identification of CTLH complex subunits co-purified with TAP-tagged Bicc1-SH.</p

Bicc1 is a target of the CTLH complex.

<p>(A) Western blot and RT-qPCR analysis of endogenous Bicc1 in WT mIMCD3 cells treated for 48 hrs with scrambled or WDR26 and Twa1 siRNAs. Quantifications of three experiments show the average fold change in Bicc1 protein (bottom left) and mRNA levels (bottom right) relative to scramble-treated cells in three independent experiments. TATA-box binding protein (TBP) mRNA was used for normalization. (B) Bicc1 protein levels in mIMCD3 cells after treatment with Bafilomycin A1 (BafA1, 100 nM), MG132 [10 μM] or both for 4 hrs. Bars represent mean ± SEM fold changes in Bicc1 protein levels relative to untreated cells in two experiments. *p<0.05. (C) Coimmunostaining of HA-Bicc1 and endogenous RanBP9 in HEK293T imaged at high laser intensities. Bars 20 μm.</p

Mass spectrometric identification of CTLH complex subunits co-purified with TAP-tagged Bicc1 MutD.

<p>Mass spectrometric identification of CTLH complex subunits co-purified with TAP-tagged Bicc1 MutD.</p

Endogenous Bicc1 in mIMCD3 cells increases FBP1 protein levels independently of its interaction with the CTLH complex.

<p>(A) Western blot analysis of FBP1 in HEK293T cells transfected with the indicated dose of HA-Bicc1. Bars represent mean ± SEMs. *p <0.05. (B) Western blot of FBP1 in mIMCD3 48hrs after transfection of scrambled or Bicc1 siRNA. Below: Bars represent mean ± SEMs. ***p <0.001. (C) Western blot and RT-qPCR analysis (right) of FBP1 in mIMCD3 cells (WT) and a representative CRISPR-edited clone (sgBicc1) (n = 3 per genotype). For RT-qPCR GAPDH mRNA was used for normalization. Bars represent mean ± SEMs. **p <0.01 (D) Western blot of FBP1, WDR26 and Twa1 in mIMCD3 transfected with siScr or siWDR26 or siTWA1 siRNAs for 48 hrs. Error bars represent mean ± SEM of two experiments. *p <0.05. (E) Western blots of FBP1, Bicc1 and WDR26 in LLC-PK1 proximal tubule cells transfected with siScr or two different siWDR26 siRNAs (siA, siB) for 48 hrs. Data are representative of two experiments. (F) Western blots of HA-Bicc1 and FBP1 in HEK293T cells transfected with increasing doses of HA-Bicc1 MutD for 24 hrs. Top: Alanine substitutions in Bicc1 MutD (D913;K915;E916/AAA). Charged residues (red and blue) in end helix (EH) and mid loop (ML) surfaces of the dimerization interface in the SAM domain and their conservation determined by ClustalW (grey shading), as well as the positions of 5 α helices are indicated.</p

Activity of tagged Bicc1 proteins and coimmunoprecipitation of mammalian CTLH complex subunits.

<p>(A) Bicc1-SH tagged with 4 internal and 2 C-terminal StrepII and 1 HA epitopes. Positions of 3 KH (K-Homology domain), 2 KH-like domains (KHL) and one sterile alpha motif (SAM) are indicated. (B) Top panel: Western blot of Bicc1-SH (arrowhead) in Flp-In T-rex cell extracts treated with or without doxycycline for 24 hrs. Traces of Bicc1-SH leaked into the last lane. Densitometric quantification of expression levels relative to endogenous Bicc1 (arrow) in mIMCD3 cells (100%) after normalization to γ-tubulin is shown below. Bottom panel: Western blot of Bicc1-SH induced by the indicated concentrations of doxycycline in comparison to transiently transfected HA-Bicc1. (C) Expression of AC6 and PKIα 3'UTR luciferase reporters in Flp-In T-rex HEK293T cells transfected with HA-Bicc1 or empty vector (mock) or induced with doxycycline to express Bicc1-SH. Luciferase values are relative to co-transfected β-galactosidase. Bars represent mean ± SEM. *p <0.05; **p <0.01. (D) Anti-Flag (DDK) coimmunoprecipitation of HA-Bicc1 and endogenous RanBP9, Twa1 and MKLN1 in HEK293T extracts. Mock transfected cells served as negative control. (E) Anti-MKLN1 coimmunoprecipitation of HA-Bicc1 and endogenous RanBP9 in HEK293T cells. Pre-immune IgG: Negative control. Input and IP samples were on the same gel but shown at different exposure times. (F) Coimmunoprecipitation of RanBP9, WDR26, and Twa1 with endogenous Bicc1 in mIMCD3 cells extract using anti-Bicc1 or pre-immune IgG (negative control).</p

Bicc1 binds the CTLH complex via ARMC8 and dependent on SAM polymer interfaces in competition with ANKS3.

<p>(A) Density fractionation of CTLH complex in extracts of empty vector- (upper graph) or HA-Bicc1-transfected HEK293T cells (below). Graphs show percentages of the total of each subunit per fraction. Grey areas denote LMW fractions 1 to 9. Results represent mean ± SEM from 2 experiments. (B) Anti-HA co-immunoprecipitation of CTLH subunits with HA-Bicc1 or its truncated derivatives Bicc1ΔKH and Bicc1ΔSAM in HEK293T cells. Inputs correspond to 2% of the total extract. Mock-transfected cells were used as negative control. (C) Pull down of endogenous Twa1 by a GST fusion protein of Bicc1 SAM domain or GST alone (control) in HEK293T cells transfected with or without HA-Bicc1. Inputs and IPs on the same gel were cropped because of different exposure times. (D) Anti-Flag co-immunoprecipitation assays in HEK293T cell extracts reveal no CTLH complex binding to Flag-tagged ANKS3 above background levels seen with preimmune IgG. (E) Co-immunoprecipitation of CTLH subunits by HA-Bicc1 alone or together with cotransfected Flag-ANKS3 or C-terminally truncated Flag-ANKS3ΔC, or by Bicc1 MutD. Inputs correspond to 2% of the total extract. Inputs and IPs on the same gel were cropped because of different exposure times. (F) Summary of human CTLH complex subunit interactions and binding to Bicc1 MutD mapped by yeast two-hybrid assays. Arrows depict the relative strength of binding in two independent experiments. (G) Recruitment of CTLH complexes via both ARMC8 and polymerization-competent Bicc1 SAM domains accelerates the clearance of LMW Bicc1 oligomers (left), whereas SAM domain polymerization stabilizes Bicc1 in large scaffolds to mediate silencing of specific target mRNAs and to stimulate FBP1 accumulation (right).</p