Specific Silencing of the REST Target Genes in Insulin-Secreting Cells Uncovers Their Participation in Beta Cell Survival

The absence of the transcriptional repressor RE-1 Silencing Transcription Factor (REST) in insulin-secreting beta cells is a major cue for the specific expression of a large number of genes. These REST target genes were largely ascribed to a function of neurotransmission in a neuronal context, whereas their role in pancreatic beta cells has been poorly explored. To identify their functional significance, we have generated transgenic mice expressing REST in beta cells (RIP-REST mice), and previously discovered that REST target genes are essential to insulin exocytosis. Herein we characterized a novel line of RIP-REST mice featuring diabetes. In diabetic RIP-REST mice, high levels of REST were associated with postnatal beta cell apoptosis, which resulted in gradual beta cell loss and sustained hyperglycemia in adults. Moreover, adenoviral REST transduction in INS-1E cells led to increased cell death under control conditions, and sensitized cells to death induced by cytokines. Screening for REST target genes identified several anti-apoptotic genes bearing the binding motif RE-1 that were downregulated upon REST expression in INS-1E cells, including Gjd2, Mapk8ip1, Irs2, Ptprn, and Cdk5r2. Decreased levels of Cdk5r2 in beta cells of RIP-REST mice further confirmed that it is controlled by REST, in vivo. Using siRNA-mediated knockdown in INS-1E cells, we showed that Cdk5r2 protects beta cells against cytokines and palmitate-induced apoptosis. Together, these data document that a set of REST target genes, including Cdk5r2, is important for beta cell survival

Comparative transcriptome profiling of amyloid precursor protein family members in the adult cortex

<p>Abstract</p> <p>Background</p> <p>The β-amyloid precursor protein (APP) and the related β-amyloid precursor-like proteins (APLPs) undergo complex proteolytic processing giving rise to several fragments. Whereas it is well established that Aβ accumulation is a central trigger for Alzheimer's disease, the physiological role of APP family members and their diverse proteolytic products is still largely unknown. The secreted APPsα ectodomain has been shown to be involved in neuroprotection and synaptic plasticity. The γ-secretase-generated APP intracellular domain (AICD) functions as a transcriptional regulator in heterologous reporter assays although its role for endogenous gene regulation has remained controversial.</p> <p>Results</p> <p>To gain further insight into the molecular changes associated with knockout phenotypes and to elucidate the physiological functions of APP family members including their proposed role as transcriptional regulators, we performed DNA microarray transcriptome profiling of prefrontal cortex of adult wild-type (WT), APP knockout (APP^-/-), APLP2 knockout (APLP2^-/-) and APPsα knockin mice (APP^α/α) expressing solely the secreted APPsα ectodomain. Biological pathways affected by the lack of APP family members included neurogenesis, transcription, and kinase activity. Comparative analysis of transcriptome changes between mutant and wild-type mice, followed by qPCR validation, identified co-regulated gene sets. Interestingly, these included heat shock proteins and plasticity-related genes that were both down-regulated in knockout cortices. In contrast, we failed to detect significant differences in expression of previously proposed AICD target genes including <it>Bace1</it>, <it>Kai1</it>, <it>Gsk3b</it>, <it>p53</it>, <it>Tip60</it>, and <it>Vglut2</it>. Only <it>Egfr </it>was slightly up-regulated in APLP2^-/-mice. Comparison of APP^-/-and APP^α/αwith wild-type mice revealed a high proportion of co-regulated genes indicating an important role of the C-terminus for cellular signaling. Finally, comparison of APLP2^-/-on different genetic backgrounds revealed that background-related transcriptome changes may dominate over changes due to the knockout of a single gene.</p> <p>Conclusion</p> <p>Shared transcriptome profiles corroborated closely related physiological functions of APP family members in the adult central nervous system. As expression of proposed AICD target genes was not altered in adult cortex, this may indicate that these genes are not affected by lack of APP under resting conditions or only in a small subset of cells.</p

Quality control of fibrinogen secretion in the molecular pathogenesis of congenital afibrinogenemia

Congenital afibrinogenemia is a rare bleeding disorder characterized by the absence in circulation of fibrinogen, a hexamer composed of two sets of three polypeptides (Aalpha, Bbeta and gamma). Each polypeptide is encoded by a distinct gene, FGA, FGB and FGG, all three clustered in a region of 50 kb on 4q31. A subset of afibrinogenemia mutations has been shown to specifically impair fibrinogen secretion, but the underlying molecular mechanisms remained to be elucidated. Here, we show that truncation of the seven most C-terminal residues (R455-Q461) of the Bbeta chain specifically inhibits fibrinogen secretion. Expression of additional mutants and structural modelling suggests that neither the last six residues nor R455 is crucial per se for secretion, but prevent protein misfolding by protecting hydrophobic residues in the betaC core. Immunofluorescence and immuno-electron microscopy studies indicate that secretion-impaired mutants are retained in a pre-Golgi compartment. In addition, expression of Bbeta, gamma and angiopoietin-2 chimeric molecules demonstrated that the betaC domain prevents the secretion of single chains and complexes, whereas the gammaC domain allows their secretion. Our data provide new insight into the mechanisms accounting for the quality control of fibrinogen secretion and confirm that mutant fibrinogen retention is one of the pathological mechanisms responsible for congenital afibrinogenemia

Targeting pannexin1 improves seizure outcome.

Imbalance of the excitatory neurotransmitter glutamate and of the inhibitory neurotransmitter GABA is one of several causes of seizures. ATP has also been implicated in epilepsy. However, little is known about the mechanisms involved in the release of ATP from cells and the consequences of the altered ATP signaling during seizures. Pannexin1 (Panx1) is found in astrocytes and in neurons at high levels in the embryonic and young postnatal brain, declining in adulthood. Panx1 forms large-conductance voltage sensitive plasma membrane channels permeable to ATP that are also activated by elevated extracellular K(+) and following P2 receptor stimulation. Based on these properties, we hypothesized that Panx1 channels may contribute to seizures by increasing the levels of extracellular ATP. Using pharmacological tools and two transgenic mice deficient for Panx1 we show here that interference with Panx1 ameliorates the outcome and shortens the duration of kainic acid-induced status epilepticus. These data thus indicate that the activation of Panx1 in juvenile mouse hippocampi contributes to neuronal hyperactivity in seizures

Different mechanisms preclude mutant CLDN14 proteins from forming tight junctions in vitro

Mutations in claudin 14 (CLDN14) cause nonsyndromic DFNB29 deafness in humans. The analysis of a murine model indicated that this phenotype is associated with degeneration of hair cells, possibly due to cation overload. However, the mechanism linking these alterations to CLDN14 mutations is unknown. To investigate this mechanism, we compared the ability of wild-type and missense mutant CLDN14 to form tight junctions. Ectopic expression in L mouse fibroblasts (LM cells) of wild-type CLDN14 protein induced the formation of tight junctions, while both the c.254T>A (p.V85D) mutant, previously identified in a Pakistani family, and the c.301 G>A (p.G101R) mutant, identified in this study through the screen of 183 Spanish and Greek patients affected with sporadic nonsyndromic deafness, failed to form such junctions. However, the two mutant proteins differed in their ability to localize at the plasma membrane. We further identified hitherto undescribed exons of CLDN14 that are utilized in alternative spliced transcripts. We demonstrated that different mutations of CLDN14 impaired by different mechanisms the ability of the protein to form tight junctions. Our results indicate that the ability of CLDN14 to be recruited to these junctions is crucial for the hearing process

Diabetic RIP-REST transgenic mice are hyperglycemic and show poor insulin secretion.

<p><i>A</i>. Blood glucose levels were assessed at different ages in male and female diabetic RIP-REST transgenic (dark diamonds; n = 7) and wild type mice (open diamonds; n = 5). Diabetic RIP-REST mice feature hyperglycemia from weaning onward. Results are mean ± SD. ***<i>P</i><0.001 versus values of wild type mice. <i>B</i>. Diabetic RIP-REST mice (black circles, n = 3) and wild type littermates (open squares, n = 4) were subjected to <i>in situ</i> pancreatic perfusion at 1.5 ml/min rate. After a 30-min equilibration period at basal 1.4 mmol/l glucose, the pancreas was perfused sequentially at different glucose concentrations, first at 1.4 mmol/l for 20 min, next at 8.0 mmol/l for 20 min, then at 16.0 mmol/l for 20 min, followed by a 30-min stimulation at 8.0 mmol/l plus 1 nmol/l GLP-1, and finally at 1.4 mmol/l for 15 min. Results are mean ± SD.</p

Identification of RE-1-containing genes potentially involved in beta cell survival.

<p><i>A</i>. Alignment of identified RE-1 sequences in human (hum) or murine (mus) genes with the consensus RE-1. Left alignment identifies members of the insulin-like growth factor transduction pathway: <i>Igfbp7</i>: insulin-like growth factor binding protein 7; <i>IRS</i>: insulin receptor substrate; <i>Akt3</i>: PKB gamma; <i>CTNND2</i>: delta2 catenin; <i>Ctnnb1</i>: beta catenin; <i>MAPK10</i>: JNK3; <i>MAP4K3</i>: MEK kinase kinase 3. Upper right alignment identifies members of the cyclin-related family of mitogenic factors: <i>Cdk5r2</i>: cyclin-dependent kinase 5 regulatory subunit 2; <i>CKS2</i>: CDC28 protein kinase regulatory subunit 2; <i>CNNG1</i>: cyclin G1; <i>Cnnm2</i>: cyclin M2. Lower right alignment identifies <i>Api5</i>: apoptosis inhibitor 5 and <i>Nrg1</i>: neuregulin1. <i>B</i>. Quantitative RT-PCR analysis of mRNA levels from control INS-1E cells (white bars) and INS-1E cells infected with GFP-expressing adenovirus (gray bars) or <i>REST</i>-expressing adenovirus (black bars). <i>Ptprn</i>: protein tyrosine phosphatase, receptor type, N; <i>Irs2</i>: insulin receptor substrate2; <i>Cdk5r2</i>: cyclin-dependent kinase 5 regulatory subunit 2; <i>Nrg1</i>: neuregulin1; <i>Ctnnd2</i>: delta2 catenin; <i>Api5</i>: apoptosis inhibitor 5. Results are mean ± SD of six independent experiments. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 versus INS-1E cells transduced with GFP. <i>C</i>. Quantitative RT-PCR analysis of mRNA levels from islets of WT (white bars) (n = 5) and RIP-REST (black bars) (n = 5) mice. <i>Gjd2</i>: Connexin36; <i>Mapk8ip1</i>: Ib1; <i>Cdk5r2</i>: cyclin-dependent kinase 5 regulatory subunit 2; <i>Ptprn</i>: protein tyrosine phosphatase, receptor type, N; <i>Irs2</i>: insulin receptor substrate2; <i>Nrg1</i>: neuregulin1; <i>Ctnnd2</i>: delta2 catenin. Results are mean ± s.e.m. **<i>P</i><0.01 versus values of wild type animals. D. Left panel, immunoblotting of total proteins from non infected (NI) and infected (Ad-GFP, Ad-REST) INS-1E cells showing decreased protein levels of Cdk5r2 upon REST expression. The right panel shows the corresponding quantifications of REST protein levels in INS-1E cells after infection. Data are mean ± SEM of 4 independent experiments. **<i>P</i><0.01, ***<i>P</i><0.001 versus the respective Ad-GFP-infected and NI conditions.</p

<i>REST</i> expression is associated with a major loss of beta cell mass.

<p><i>A</i>. Immunostaining for insulin (Ins, red) and glucagon (Glu, green) reveals, when compared to wild type mice (WT, left panel), a major loss of insulin-positive cells, within a low number of disorganized islets in pancreas of adult diabetic RIP-REST mice (middle panel). REST nuclear staining (green, right panel) indicates that only a few surviving beta cells still express REST. Blue staining is the DAPI labeling of nuclei. Scale bar, 25 μm. <i>B</i>. Immunostaining for insulin (Ins, red) and glucagon (Glu, green) shows that, even if disorganized when compared to islets from wild type animals (WT, left panel), islets from diabetic RIP-REST mice at postnatal day 7 (P7) show a significant number of insulin-positive cells (middle panel). REST nuclear staining (green, right panel) indicates that a majority of beta cells express REST. Blue staining is the DAPI labeling of nuclei. Scale bar, 50 μm. <i>C</i>. Quantification of beta-(Ins+ cells) and alpha- (Gluc+ cells) cell mass in P2, P7 and adult diabetic RIP-REST (black bars, n = 3 each) and wild type mice (white bars, n = 3 each). A 30, 45 and 90% reduction in beta cell mass is observed in P2, P7, and adult diabetic RIP-REST mice, respectively, when compared with corresponding mass of controls. Results are mean ± SD. *<i>P</i><0.05, ***<i>P</i><0.001 versus values of wild type mice. <i>D</i>. Confocal analysis of insulin (Ins, red) and glucagon (Glu, green) immunofluorescence shows scarce double insulin-and glucagon-expressing cells (arrows) in pancreatic sections of adult diabetic RIP-REST mice. Blue staining is the DAPI labeling of nuclei. Scale bar, 25 μm.</p

Beta cell loss in diabetic RIP-REST mice occurs through apoptosis.

<p><i>A</i>. Nuclear PCNA (green) and insulin (red) staining show same level of proliferating beta cells (arrows) in both wild type (WT, left panel) and diabetic RIP-REST mice (right panel) at P2. Blue staining is the DAPI labeling of nuclei. Scale bar, 50 μm. <i>B</i>. Representative images of nuclear TUNEL (green) and insulin (red) staining showing apoptotic nuclei of beta cells in sections of P2 diabetic RIP-REST mice. No apoptotic beta cells were detected in wild type animals (not shown). Blue staining is the DAPI labeling of nuclei. Scale bar, 25 μm. C. Quantification of apoptotic nuclei in non-infected INS-1E cells (NI) and INS-1E cells transfected with a control (Ad-GFP) or REST-expressing adenovirus (Ad-REST) at different multiplicity of infection (MOI) as indicated, and treated 24 h with a mix of cytokines (black bars) or not (white bars). Results are mean ± SD of six independent experiments. *<i>P</i><0.05 versus respective controls in treated or untreated conditions. # <i>P</i><0.05 versus MOI 2 of Ad-REST infection.</p

CDK5R2 protects beta cells against cytokines and palmitate.

<p><i>A</i>. Left panel, immunoblotting of total proteins from INS-1E cells, 72 h after transfection with a negative siRNA (siNeg) or siRNAs against <i>Cdk5r2</i> (siCdk5r2#1 and siCdk5r2#2). Right panel, quantification of the corresponding Cdk5r2 protein levels. Results are mean ± SEM of three independent experiments. **<i>P</i><0.01 versus values of siNeg-transduced INS-1E cells. <i>B</i>. Quantification of apoptotic nuclei in non-transfected INS-1E cells (NT) and INS-1E cells transfected with a negative siRNA (siNeg) or with siRNAs against <i>Cdk5r2</i> (siCdk5r2#1 and siCdk5r2#2), treated 24 h with a mix of cytokines (black bars) or not (white bars). Results are mean ± SEM of six independent experiments. **<i>P</i><0.01 versus controls. <i>C</i>. Immunoblotting of total proteins from INS-1E cells transfected with a negative siRNA (siNeg) or siRNAs against <i>Cdk5r2</i> (siCdk5r2#1 and siCdk5r2#2), treated 24 h with a mix of cytokines (Cyt) or not (Ctrl). The cleavage of caspase-9 and −3, induced by cytokines, is higher in siCdk5r2- than in siNeg-treated cells. <i>D</i>. Quantification of apoptotic nuclei in non-transfected INS-1E cells (NT) and INS-1E cells transfected with a negative siRNA (siNeg) or siRNAs against <i>Cdk5r2</i> (siCdk5r2#1 and siCdk5r2#2), exposed 15 h to control medium (white bars) or palmitate (black bars). Results are mean ± SEM of six independent experiments. **<i>P</i><0.01 versus controls.</p