REST Target Genes and Beta Cell Survival Figure 5. Identification of RE-1-containing genes potentially involved in beta cell survival. A. 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: Igfbp7: insulin-like growth factor binding protein 7; IRS: insulin receptor substrate; Akt3: PKB gamma; CTNND2: delta2 catenin; Ctnnb1: beta catenin; MAPK10: JNK3; MAP4K3: MEK kinase kinase 3. Upper right alignment identifies members of the cyclin-related family of mitogenic factors: Cdk5r2: cyclin-dependent kinase 5 regulatory subunit 2; CKS2: CDC28 protein kinase regulatory subunit 2; CNNG1: cyclin G1; Cnnm2: cyclin M2. Lower right alignment identifies Api5: apoptosis inhibitor 5 and Nrg1: neuregulin1. B. 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 REST-expressing adenovirus (black bars). Ptprn: protein tyrosine phosphatase, receptor type, N; Irs2: insulin receptor substrate2; Cdk5r2: cyclin-dependent kinase 5 regulatory subunit 2; Nrg1: neuregulin1; Ctnnd2: delta2 catenin; Api5: apoptosis inhibitor 5. Results are mean 6 SD of six independent experiments. *P,0.05, **P,0.01, ***P,0.001 versus INS-1E cells transduced with GFP. C. Quantitative RT-PCR analysis of mRNA levels from islets of WT (white bars) (n = 5) and RIP-REST (black bars) (n = 5) mice. Gjd2: Connexin36; Mapk8ip1: Ib1; Cdk5r2: cyclin-dependent kinase 5 regulatory subunit 2; Ptprn: protein tyrosine phosphatase, receptor type, N; Irs2: insulin receptor substrate2; Nrg1: neuregulin1; Ctnnd2: delta2 catenin. Results are mean 6 s.e.m. **P,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 6 SEM of 4 independent experiments. **P,0.01, ***P,0.001 versus the respective Ad-GFP-infected and NI conditions. doi:10.1371/journal.pone.0045844.g005 CDK5 and its activators on insulin secretion. Whereas inhibition of CDK5R1 activity was reported to promote insulin secretion [32], the association of CDK5 to CDK5R2, but not CDK5R1, has also been shown to induce insulin release [33]. The role of CDK5 in neuronal survival is also a matter of debate. Deregulation of CDK5 activity by p25 has been involved in numerous neurodegenerative diseases [34,35], while Cdk5-loss-of- function studies have revealed its pro-survival function in neurons under normal [36–39] or stress conditions [36,37,40,41]. Similar- ly, it is not clear whether CDK5 plays a prosurvival activity or not in pancreatic beta cells. While its inhibition promoted PDX-1 nuclear localization and increased beta cell survival in response to glucotoxicity [42], a recent publication showed that decreased levels of CDK5 triggered apoptosis via loss of activation of focal adhesion kinase and decreased PI3K/AKT signaling [43]. So far, however, the role of CDK5R2 in beta cell survival has not been explored. In the latter study, the authors proposed that CDK5R1 may be the activator form of CDK5 that is mediating the prosurvival activity, neglecting the role of CDK5R2. Here, we show that Cdk5r2 belongs to the cluster of RE-1-containing genes, and therefore that it is crucial and specific to neurons and pancreatic beta cells. Moreover, even though the silencing of Cdk5r2 is not inducing beta cell death under normal conditions, we provide evidence that its knock-down by two distinct siRNAs leads to enhanced sensitivity of INS-1E cells exposed to pro-inflamma- tory cytokines or palmitate, two mediators of beta cell apoptosis in T1D and T2D, respectively [44]. In summary, the present findings extend our previous observa- tions that ectopic expression of REST in beta cells allows for novel insights into the specific traits that make a beta cell what it is [12]. Our work further demonstrates the need for healthy beta cells to express the whole set of RE-1-containing genes. Specifically, we show here that high chronic levels of REST in beta cells leads to diabetes, due to prolonged repression of multiple REST-target genes known to contribute to beta cell survival. We further identify a hitherto neglected anti-apoptotic player, CDK5R2, which we show to be essential for beta cell survival under conditions that trigger apoptosis. Given the large number of estimated REST targets, it is anticipated that several other genes, which remain to be identified and validated, may play an important role in beta cell life and function under the native conditions of lack of REST expression. Materials and Methods Transgenic mice Transgenic mice specifically expressing REST in beta cells were obtained by pronuclear injection of C57Bl6/J zygotes as previously described [13]. Our institutional review committee for animal experiments approved all the procedures for mice care, surgery and euthanasia. Pancreas perfusion and insulin secretion Mice were anesthetized i.p with 100 mg/kg b:w sodium pentothal, and prepared for pancreas perfusion as previously described [45]. The pancreas was perfused at 1.5 ml/min at 37uC with modified Krebs-Ringer HEPES buffer supplemented with the indicated concentrations of glucose. The pancreatic effluent of the first 30 min of perfusion with basal glucose (1.4 mmol/l) was not collected. After this equilibration period, the effluent was collected in 1-min fractions from a catheter placed in the portal vein. The insulin content of each fraction was determined by radioimmu- noassay. Immunohistochemistry and confocal microscopy Pancreas were fixed in 4% (w/v) paraformaldehyde, equilibrat- ed overnight in 15% (w/v) sucrose, embedded in 15% sucrose- 15% (w/v) gelatin and quickly frozen in methylbutane/liquid nitrogen. Cryosections were permeabilized for 20 min in 0.1% Triton X-100, then blocked in 1.5% BSA in PBS for 30 min, and incubated overnight at 4uC with either polyclonal rabbit antibodies against human REST [46], glucagon (Dako, Baar, Switzerland, 1/500), proliferating cell nuclear antigen (PCNA, Dako, 1/200) or polyclonal guinea-pig antibodies against insulin (Zymed Lab. Inc., San Francisco, USA, 1/500). Primary antibodies were detected using appropriate fluorescein or rhoda- mine-conjugated antibodies. A supplementary antigen-retrieval treatment was required for anti-PCNA, which consisted of boiling the slides in citrate buffer (10 mM Na-citrate, pH 6) for 10 min before incubation with the blocking solution. Semi-quantitative immunodetection of REST was performed using secondary antibodies coupled to horseradish peroxidase from the rabbit Vectastain ABC kit, using 3-amino-9-ethylcarbazole (AEC) as substrate, according to the manufacturer’s instructions (Vector Lab. Inc., Burlingame, CA, USA). Sections were viewed on either a Leica DM5500 fluorescence microscope or a Leica SP2 upright confocal microscope (Leica, Nidau, Switzerland). TUNEL labeling was performed using the in situ cell death detection kit, according to the manufacturer’s instructions (Roche Diagnostics, Rotkreuz, Switzerland). Image processing and cell mass quantification For semi-quantitative assessment of REST abundance in beta cells of transgenic mice, images have been processed as follow using the ImageJ software: images were first converted to a 32 bit format and subjected to background substraction. A binary image was then created from a duplicated cropped region of interest (i.e PLOS ONE | www.plosone.org 8 September 2012 | Volume 7 | Issue 9 | e45844