Abnormal social behaviors and altered gene expression rates in a mouse model for Potocki-Lupski syndrome

The Potocki-Lupski syndrome (PTLS) is associated with a microduplication of 17p11.2. Clinical features include multiple congenital and neurobehavioral abnormalities and autistic features. We have generated a PTLS mouse model, Dp(11)17/+, that recapitulates some of the physical and neurobehavioral phenotypes present in patients. Here, we investigated the social behavior and gene expression pattern of this mouse model in a pure C57BL/6-Tyrc-Brd genetic background. Dp(11)17/+ male mice displayed normal home-cage behavior but increased anxiety and increased dominant behavior in specific tests. A subtle impairment in the preference for a social target versus an inanimate target and abnormal preference for social novelty (the preference to explore an unfamiliar mouse versus a familiar one) was also observed. Our results indicate that these animals could provide a valuable model to identify the specific gene(s) that confer abnormal social behaviors and that map within this delimited genomic deletion interval. In a first attempt to identify candidate genes and for elucidating the mechanisms of regulation of these important phenotypes, we directly assessed the relative transcription of genes within and around this genomic interval. In this mouse model, we found that candidates genes include not only most of the duplicated genes, but also normal-copy genes that flank the engineered interval; both categories of genes showed altered expression levels in the hippocampus of Dp(11)17/+ mic

Podocyte-Specific Overexpression of Wild Type or Mutant Trpc6 in Mice Is Sufficient to Cause Glomerular Disease

Mutations in the TRPC6 calcium channel (Transient receptor potential channel 6) gene have been associated with familiar forms of Focal and Segmental Glomerulosclerosis (FSGS) affecting children and adults. In addition, acquired glomerular diseases are associated with increased expression levels of TRPC6. However, the exact role of TRPC6 in the pathogenesis of FSGS remains to be elucidated. In this work we describe the generation and phenotypic characterization of three different transgenic mouse lines with podocyte-specific overexpression of the wild type or any of two mutant forms of Trpc6 (P111Q and E896K) previously related to FSGS. Consistent with the human phenotype a non-nephrotic range of albuminuria was detectable in almost all transgenic lines. The histological analysis demonstrated that the transgenic mice developed a kidney disease similar to human FSGS. Differences of 2–3 folds in the presence of glomerular lesions were found between the non transgenic and transgenic mice expressing Trpc6 in its wild type or mutant forms specifically in podocytes. Electron microscopy of glomerulus from transgenic mice showed extensive podocyte foot process effacement. We conclude that overexpression of Trpc6 (wild type or mutated) in podocytes is sufficient to cause a kidney disease consistent with FSGS. Our results contribute to reinforce the central role of podocytes in the etiology of FSGS. These mice constitute an important new model in which to study future therapies and outcomes of this complex disease

Tubular overexpression of gremlin induces renal damage susceptibility in mice.

A growing number of patients are recognized worldwide to have chronic kidney disease. Glomerular and interstitial fibrosis are hallmarks of renal progression. However, fibrosis of the kidney remains an unresolved challenge, and its molecular mechanisms are still not fully understood. Gremlin is an embryogenic gene that has been shown to play a key role in nephrogenesis, and its expression is generally low in the normal adult kidney. However, gremlin expression is elevated in many human renal diseases, including diabetic nephropathy, pauci-immune glomerulonephritis and chronic allograft nephropathy. Several studies have proposed that gremlin may be involved in renal damage by acting as a downstream mediator of TGF-β. To examine the in vivo role of gremlin in kidney pathophysiology, we generated seven viable transgenic mouse lines expressing human gremlin (GREM1) specifically in renal proximal tubular epithelial cells under the control of an androgen-regulated promoter. These lines demonstrated 1.2- to 200-fold increased GREM1 expression. GREM1 transgenic mice presented a normal phenotype and were without proteinuria and renal function involvement. In response to the acute renal damage cause by folic acid nephrotoxicity, tubule-specific GREM1 transgenic mice developed increased proteinuria after 7 and 14 days compared with wild-type treated mice. At 14 days tubular lesions, such as dilatation, epithelium flattening and hyaline casts, with interstitial cell infiltration and mild fibrosis were significantly more prominent in transgenic mice than wild-type mice. Tubular GREM1 overexpression was correlated with the renal upregulation of profibrotic factors, such as TGF-β and αSMA, and with increased numbers of monocytes/macrophages and lymphocytes compared to wild-type mice. Taken together, our results suggest that GREM1-overexpressing mice have an increased susceptibility to renal damage, supporting the involvement of gremlin in renal damage progression. This transgenic mouse model could be used as a new tool for enhancing the knowledge of renal disease progression

Generation and validation of transgenic mice with specific tubular GREM1 overexpression.

<p>(a) Illustration of the pKAP GREM1-c-myc-IRES-eGFP plasmid. Restriction sites used for transgene isolation are indicated with EV (<i>Eco</i>RV) and A (<i>Ase</i>I). (b) <b>eGFP and c-myc detection in renal tubular epithelial cells of transgenic mice.</b> Immunofluorescence against eGFP and immunohistochemistry for c-myc (peroxidase immunostaining) to detect these proteins in the kidney tissue of transgenic males from line A and WT mice (400x). (c) Kidneys were dissected from WT and transgenic male mice of lines A, B, C, D and E, and isolated proteins were subjected to western blotting using an antibody against c-myc (1∶1000); anti β-actin (1∶2500) was used as a loading control. GREM1 expression was determined by densitometric analysis of the c-myc/β-actin ratio and normalized to transgenic line C expression.</p

Correlation of TGF-β and GREM1 expression in transgenic line A homozygous mice.

<p>TGF-β gene expression was measured in FA-injected GREM1 transgenic mice. We observed a strongly positive correlation between TGF-β and GREM1 expression (p≤0.029, R  =  0.67; 4-9 mice per group).</p

Effect of FA administration on GREM1 expression in transgenic line A homozygous mice.

<p>Gremlin expression in transgenic mice was examined 7 and 14 days after treatment with FA. The GREM1 relative expression in homozygous mice of transgenic line A was increased at 7 days after injection with FA and remained increased at 14 days. Data are shown as the mean ± SEM of 4-9 mice per group * p≤0.0049 TG-FA vs TG-Veh, used as control because no WT littermates of the transgenic homozygotes mice were available.</p

FA injection induces interstitial cell infiltration in transgenic line A homozygous mice.

<p>The inflammatory cell infiltration was characterized by immunohistochemistry with anti-F4/80 (monocytes/macrophages) and anti-CD3 (T cells) antibodies. (A and B). Representative immunostaining of one mouse from each group (x400 magnification). (C) Quantification of positive IHC signals were quantified for (a) F4/80, (b) CD3 and (c) PCNA using KS300 image analyzer software. All parameters were significantly increased in transgenic mice. Data are shown as the mean ± SEM of 14–18 mice per group * p < 0.05; ** p < 0.01 vs WT-FA.</p

Molecular characterization of transgenic mice.

<p>Qualitative and quantitative data from the transgenic lines are shown. Copy number and GREM1 expression levels were determined using real-time PCR. The eGFP signal was qualitatively analyzed. c-myc levels were quantified by densitometric analysis in each line and normalized to line C. Ectopic expression is indicated as the number of the eight analyzed extrarenal organs that demonstrated increased for GREM1 expression compared with wild-type expression. M, male; F, female; n.d., no data. *** p < 0.001.</p

In vitro validation of the GREM1 plasmid.

<p>(a) EBNA293 cells were transfected with pCDNA3-GREM1-c-myc-IRES-eGFP, as described in the methods. Immunofluorescence shows that eGFP (green) and c-myc (red) are expressed in the same transfected EBNA293 cell. Nuclei were stained with DAPI (blue) (1000x). (b) In HK-2 cells transfected with pCDNA3-GREM1-c-myc-IRES-eGFP, GREM-1 expression was evaluated by immunocytochemistry using an antibody against GREM-1, followed by a secondary TRICT antibody (red staining). The figure shows eGFP and GREM-1 expressed in the same cell (800x). (c) Confocal immunofluorescence of HK-2 transfected cells, showing the loss of E-cadherin and induction of vimentin in eGFP-positive GREM-1-expressing cells (1600X). E-cadherin and vimentin immunostaining was detected with secondary anti-FITC antibodies (green).</p