ANKS3 Co-Localises with ANKS6 in Mouse Renal Cilia and Is Associated with Vasopressin Signaling and Apoptosis In Vivo in Mice

International audienceMutations in Ankyrin repeat and sterile alpha motif domain containing 6 (ANKS6) play a causative role in renal cyst formation in the PKD/Mhm(cy/+) rat model of polycystic kidney disease and in nephronophthisis in humans. A network of protein partners of ANKS6 is emerging and their functional characterization provides important clues to understand the role of ANKS6 in renal biology and in mechanisms involved in the formation of renal cysts. Following experimental confirmation of interaction between ANKS6and ANKS3 using a Yeast two hybrid system, we demonstrated that binding between the two proteins occurs through their sterile alpha motif (SAM) and that the amino acid 823 in rat ANSK6 is key for this interaction. We further showed their interaction by co-immunoprecipitation and showed in vivo in mice that ANKS3 is present in renal cilia. Downregulated expression of Anks3 in vivo in mice by Locked Nucleic Acid (LNA) modified antisense oligonucleotides was associated with increased transcription of vasopressin-induced genes, suggesting changes in renal water permeability, and altered transcription of genes encoding proteins involved in cilium structure, apoptosis and cell proliferation. These data provide experimental evidence of ANKS3-ANKS6 direct interaction through their SAM domain and co-localisation in mouse renal cilia, and shed light on molecular mechanisms indirectly mediated by ANKS6 in the mouse kidney, that may be affected by altered ANKS3-ANKS6 interaction. Our results contribute to improved knowledge of the structure and function of the network of proteins interacting with ANKS6, which may represent therapeutic targets in cystic diseases

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Organ expression, physiological interaction and renal location of ANKS3 and ANKS6 in the mouse.

<p>ANKS3 and ANKS6 are strongly expressed in the mouse kidney.<i>Anks3</i> (<b>A</b>) and <i>Anks6</i> (<b>B</b>) mRNA expression in kidney, liver, pancreas and spleen was evaluated by quantitative RT-PCR. Data are shown as means ± SEM. Quantification of cDNA from 3 mice was normalized to <i>Actb</i> gene expression level. One-way ANOVA was applied to assess statistical significance. P<0.0001 significantly different to renal expression of <i>Anks3</i> and <i>Anks6</i>. (<b>C</b>) Co-localisation of ANKS3 and ANKS6 in the kidney. Representative photomicrographs of 6 kidney sections. ANKS3 staining (revealed by Alexa Fluor 488 in green, left column) co-localises (merge staining in central column) with ANKS6 (right vertical column, revealed by Alexa Fluor 594 in red) in glomeruli (upper panel, scale bar: 90μm) and in tubules (lower panel, scale bar: 300μm. (<b>D</b>) Immunoprecipitation experiments performed with goat anti-ANKS6 antibody, goat anti-ANKS3 antibody or goat IgGs on protein lysates and followed by an immunoblot performed with rabbit anti-ANKS3 antibody (top) and rabbit anti-ANKS6 antibody (bottom). Note that ANKS6 antibody co-immunoprecipitates ANKS3 in kidney protein lysates.</p

<i>Anks3</i> expression knock-down <i>in vivo</i> stimulates expression of genes involved in the vasopressin signaling pathway.

<p>(<b>A</b>-<b>B</b>) Renal mRNA expression of <i>Vit32</i>, <i>Aqp1</i>, <i>Aqp2</i> and <i>Aqp3</i> was evaluated by quantitative RT-PCR in mice four days (n = 3)(<b>A</b>) and twelve days (n = 5)(<b>B</b>) after the final injection of Locked Nucleic Acid modified AntiSense Oligonucleotides (LNA ASO). Quantification of each cDNA was performed in duplicate and normalized to <i>Gusb</i> gene expression level. (<b>C</b>) AQP2 expression in kidney tissues. Representative Western blots performed with protein extracts from 5 kidneys treated with scrambled (SCR) or ANKS3 ASO (top) and quantitative analysis of blots normalized to β-actin expression (bottom) show that AQP2 expression is significantly increased in mice twelve days after the final injection of ANKS3 ASO when compared to mice injected with SCR ASO. (<b>D</b>, <b>E</b>) Immunofluorescence staining of the papilla for AQP2 (<b>D</b>) and AVPR2 (<b>E</b>) show a dramatic increase of the proteins in kidneys of ANKS3 ASO treated mice twelve days after the final injection compared to SCR ASO controls. Scale bar: 300 μm. Data are means ± SEM. Non-parametric Mann-Whitney U test was used to assess differences between ANKS3 ASO and control mice injected with saline (Ctr)- or SCR ASO. *P<0.05; **P<0.01 significantly different to controls.</p

<i>In vivo</i> LNA ASO-induced downregulation of<i>Anks3</i> expression in mouse kidney.

<p>(<b>A</b>) Presence of ANKS3 in kidneys treated with saline (CTR) and scrambled AntiSense Oligonucleotides (SCR ASO) 12 days post injection and with ANKS3 Locked Nucleic Acid modified AntiSense Oligonucleotides (ANKS3 LNA ASOs). Representative photomicrographs of kidney sections from 5 mice four and twelve days after the last injection of LNA ASOs (D4 post inj., D12 post inj.). Kidneys were stained with ANKS3 (revealed by Alexa Fluor 488 in green) and the LNA ASOs were localised with the Alexa Fluor 647 marker in red. (<b>B-D</b>) <i>Anks3</i> renal decreased expression does not affect expression of <i>Anks6</i>. Abundance of <i>Anks3</i> transcripts (<b>B</b>) and protein (<b>C</b>), and <i>Anks6</i> transcripts (<b>D</b>) were evaluated in mouse kidneys four days (D4 post inj., n = 3) and twelve days (D12 post inj., n = 5) after the final injection. cDNA quantification was performed in duplicate and normalized to <i>Actb</i> gene expression level. (<b>C</b>) Western blots (upper panel) and quantitative protein analysis (lower panel) performed with extracts from kidneys of 5 mice treated with saline (Ctr), scramble ASO (SCR ASO) or ANKS3 ASO four and twelve days after the last injection showed that expression of ANKS3 normalized to that of β-actin is significantly decreased in mice treated with ANKS3 ASO. Data are mean ± SEM. Non-parametric Mann-Whitney U test was used to assess differences between ANKS3 ASO and SCR ASO treated mice.**P<0.01; *P<0.05 significantly different to control mice treated with saline (Ctr) or SCR ASO.</p

The mutation (R823W) in PKD/Mhm(<i>cy/cy</i>) rats does not alter ANKS6-ANKS3 renal co-localisation.

<p>Immunohistochemical staining of renal cortex and medulla was performed in 3 week-old wild-type and PKD/Mhm(<i>cy/cy</i>) rats for ANKS3 (upper panel) and ANKS6 (lower panel).</p

Identification of ANKS3, a partner of ANKS6.

<p>(<b>A</b>) Schematic representation of ANKS6 protein and the bait construct (pGBD-B-Anks6-SAM) used to perform the yeast two hybrid screen. (<b>B</b>) Schematic representation of ANKS3 protein for comparison with cDNA sequences of prey 1, 2, 3 and 4, recovered from the yeast two hybrid screen. (<b>C</b>) Interaction of ANKS6 with the 4 preys in interaction assays in yeast. The bait constructions containing either the Ankyrin repeats region of ANKS6 (pGBD-B-Anks6-ANK), or the Serine Rich region (pGBD-B-Anks6-MID), or the SAM domain (pGBD-B-Anks6-SAM) are shown on the left. The ability of these domains to interact with the preys is shown on the right. (<b>D</b>) Effect of the mutation R823W in the SAM domain of ANKS6 on the interaction between the preys and ANKS6. The construction pGBD-ANKS6-SAM and its mutated version (pGBD-ANKS6-SAM-R823W) are shown on the left. The ability of the SAM and SAM-R823W domains of the ANKS6 protein to interact with the preys is shown on the right.</p

Induction of apoptosis and proliferation in <i>in vivo Anks3</i> knock-down mice.

<p>(<b>A</b>) Immunofluorescence TUNEL was used to evaluate the percentage of apoptotic cells in mouse kidneys four days (D4 post inj.) and twelve days (D12 post inj.) after the final injection of scrambled (SCR) or ANKS3 AntiSense Oligonucleotides (ASO). Apoptosis was significantly increased in SCR ASO kidneys and in ANKS3ASO kidneys 12 days after the last injection.(<b>B</b>, <b>C</b>) Abundance of caspase 9 (<b>B</b>) and caspase 3 (<b>C</b>) proteins was evaluated by Western blot twelve days after final Locked Nucleic Acid modified (LNA) ASO injection (n = 5). Protein quantification was tested in duplicate and normalized to the level of ß-actin protein. (<b>D</b>) Percentage of PCNA positive cells in kidneys from mice that received SCR and ANKS3 ASO, 4 and 12 days after the last injection. (<b>E</b>) <i>p53</i> mRNA expression was evaluated by quantitative RT-PCR four (n = 3) and twelve days (n = 5) post treatment in mice injected with LNA ASO, saline (CTR) and SCR ASO. Data are means ± SEM. Quantification of each cDNA was performed in duplicate and normalized to <i>Gusb</i> gene expression. Non-parametric Mann-Whitney U test was used to assess differences between ANKS3 ASO and control mice.*P<0.05 significantly different to control mice.</p

Molecular genetics of the transcription factor GLIS3 identifies its dual function in beta cells and neurons

Calderari S.: Present adress à UMR 1198, BDR I NRA Jouy en JosasThe GLIS family zinc finger 3 isoform (GLIS3) is a risk gene for Type 1 and Type 2 diabetes, glaucoma and Alzheimer's disease endophenotype. We identified GLIS3 binding sites in insulin secreting cells (INS1) (FDR q\textless0.05; enrichment range 1.40-9.11 fold) sharing the motif wrGTTCCCArTAGs, which were enriched in genes involved in neuronal function and autophagy and in risk genes for metabolic and neuro-behavioural diseases. We confirmed experimentally Glis3-mediated regulation of the expression of genes involved in autophagy and neuron function in INS1 and neuronal PC12 cells. Naturally-occurring coding polymorphisms in Glis3 in the Goto-Kakizaki rat model of type 2 diabetes were associated with increased insulin production in vitro and in vivo, suggestive alteration of autophagy in PC12 and INS1 and abnormal neurogenesis in hippocampus neurons. Our results support biological pleiotropy of GLIS3 in pathologies affecting β-cells and neurons and underline the existence of trans‑nosology pathways in diabetes and its co-morbidities