Current Molecular Medicine - Volume 13, Issue 6, 2013

Neuroplasticity depends on the precise timing of gene expression, which requires accurate control of mRNA stability and rapid elimination of abnormal mRNA. Nonsense-mediated mRNA decay (NMD) is an RNA surveillance mechanism that ensures the speedy degradation of mRNAs carrying premature termination codons (PTCs). This mechanism relies on several key Exon Junction Complex (EJC) factors to distinguish PTCs from normal stop codons. NMD degrades not only aberrant transcripts carrying PTCs, but also normal transcripts harboring a normal stop codon [1]. Intriguingly, mutations in an NMD factor, Upf3b, have been found in patients with autism [2, 3]. A binding partner of Upf3b, RBM8a, is located in the 1q21.1 copy-number variation (CNV) associated with mental retardation, autism [4], schizophrenia [5], and microcephaly [6]. However, the functions of EJC factors and their roles in behavioral regulation are still elusive. RBM8a protein is a core component of the EJC that plays an important role in NMD. Recent genetic study indicated that RBM8a gain-of-function significantly associated with intellectual disability [7]. In this study we investigated the effect of RBM8a overexpression on affective behaviors in mice. Lentivirus expressing RBM8a was infused into the hippocampus of adult mice to conduct behavioral studies including social interaction, open field, elevated plus maze, and forced swimming tests. Our results showed that overexpression of RBM8a in the mouse dentate gyrus (DG) leads to increased anxiety-like behavior, abnormal social interaction and decreased immobile time in forced swimming test (FST). To examine the underlying mechanism, we found that overexpressing RBM8a in cultured primary neurons lead to significant higher frequency of miniature excitatory postsynaptic currents (mEPSCs). To explore the underlying mechanism of RBM8a mediated behavioral changes, RNAimmunoprecipitation (RNA-IP) detected that RBM8a binds to CaMK2, GluR1 and Egr1 mRNA, suggesting that RBM8a may target neuronal genes to regulate behaviors. This is the first study that demonstrates the key role of RBM8a on the emotional behaviors in mice. These results reveal new neural mechanisms by which NMD modulates behaviors and potentially provide a better understanding of pathophysiology underlying psychiatric disorders.

Antimicrobial peptides (AMPs) are significant components of the innate immune system and play indispensable roles in the resistance to bacterial infection. Here, we investigated the antimicrobial activity of lycosin-I, a 24-residue cationic anticancer peptide derived from Lycosa singorensis with high structural similarity to several cationic and amphiphilic antimicrobial peptides. The antimicrobial activity of lycosin-I against 27 strains of microbes including bacteria and fungi was examined and compared with that of the Xenopus-derived AMP magainin 2 using a microdilution assay. Lycosin-I inhibited the growth of most microorganisms at low micromolar concentrations, and was a more potent inhibitor than magainin 2. Lycosin-I showed rapid, selective and broad-spectrum bactericidal activity and a synergistic effect with traditional antibiotics. In vivo, it showed potent bactericidal activity in a mouse thigh infection model. High Mg2+ concentrations reduced the antibacterial effect of lycosin-I, implying that the peptide might directly interact with the bacterial cell membrane. Uptake of the fluorogenic dye SYTOX and changes in the surface of lycosin-Itreated bacterial cells observed by scanning electron microscopy confirmed that lycosin-I permeabilized the cell membrane, resulting in the rapid bactericidal effect. Taken together, our findings indicate that lycosin-I is a promising peptide with the potential for the development of novel antibacterial agents.

Failure after glaucoma filtration surgery is attributed to fibrosis at the operated site. To understand the wound healing process after glaucoma filtration surgery, we have developed a mouse model for glaucoma filtration surgery which closely mimics the clinical response. In this study, we describe a systematic analysis of the wound healing response in vivo. Our data revealed that the post-surgical tissue response was separable into two distinguishable phases. The early “acute inflammatory” phase was characterized by significantly increased transcript expression of Vegfa, Cxcl1, Cxcl5, Ccl2, Ccl3, Ccl4, Gmcsf and specific Mmps as well as greater infiltration of monocytes/macrophages and T cells. The late “fibrotic” phase was characterized by an increased expression of Tgfb2 and extracellular matrix genes as well as a notable reduction of infiltrating inflammatory cells. Significantly, more mitotic cells were observed at both time points post-surgery. Subconjunctival fibroblasts may be involved in both phases since they have the capacity to reiterate the in vivo gene expression profiles upon either pro-inflammatory or pro-fibrotic cytokine stimulation. Given that the cellular and molecular targets that govern the early and late phases of wound healing are distinct and time sensitive, a multi-targeted therapeutic approach to sequentially inhibit inflammation and fibrogenesis at the critical time point may lead to improved surgical outcomes in glaucoma filtration surgery.

Age-related macular degeneration (AMD) is a leading cause of visual impairment in aging populations in industrialized countries. Here we investigated whether the genotype of vascular endothelial growth factor A (VEGFA) gene is associated with response to anti-VEGF therapy. 223 eyes with neovascular AMD were treated with intravitreal anti-VEGF therapy. Responders were defined as patients who had an improvement in best corrected visual acuity (BCVA) of at least 5 letters or one line on the EDTRS visual acuity chart along with resolution of intraretinal or subretinal fluid over 12 months. Patients who did not meet the definition of responders were classified as poor-responders. The vision of responders (n = 148) improved while the vision of poor-responders (n = 75) worsened (P <0.001). Responders on average had a decrease in central foveal thickness (CFT), while poor-responders had an increase in CFT (P <0.001). Compared with the responder group, the poor-responder group had a higher frequency of the risk (T) allele (Allelic P = 0.019) and TT genotype (P = 0.002 under a recessive model) for the VEGFA-rs943080 polymorphism. VEGFA expression was 1.8-fold higher in cells with the VEGFA rs943080 TT genotype than in cells with the VEGFA rs943080 CC genotype (P = 0.012). Age, gender, smoking, diabetes mellitus, and hypertension did not play a significant role in treatment response, but BMI was found to be significantly different between responders and poorresponders (P = 0.033). In conclusion, we demonstrated a potential pharmacogenetic relationship between the VEGFA gene and treatment response to anti-VEGF therapy. The studies are registered at ClinicalTrials.gov under the identifiers NCT00474695 (http://clinicaltrials. gov/ct2/show/NCT00474695) and NCT01464723 (http://clinicaltrials.gov/ct2/show/NCT01464723).

Oxidative stress is one of the main contributors in the pathogenesis of diabetic retinopathy. The aim of this study is to investigate the effects of SS31 which is a mitochondria-targeted antioxidant peptide on the retinas of streptozotocin (STZ)-induced diabetic rats. Two weeks after induction of diabetes, SS31 (3 mg/kg) or the same volume of normal saline (N.S) was injected subcutaneously into the back of diabetic rats every day. Four months later, the integrity of inner blood retinal barrier (iBRB) was measured by Evans blue perfusion. The expression and distribution of claudin-5, occludin, acrolein, 8-OHdG and nitrotyrosine in the rat retinas were detected by immunofluorescent staining. Retinal ultrastructures were observed by transmission electron microscopy. The protein level of VEGFR2, Trx-2, Bcl-2, Bax, caspase-3, p53, and NF-κB in the rat retinas were assayed by western blot. Four months after subcutaneous injection, the diabetic rats treated with SS31 had better structures of retinal ganglion cells, thinner capillary basement membrane, less iBRB leakage, more uniform staining of claudin-5 and occludin in the retinal vessels, lower levels of acrolein, 8-OHdG, nitrotyrosine, Bax, caspase-3, p53, and NF-κB, and higher levels of Trx-2 and Bcl-2 in the retinas than those treated with N.S. In conclusion, SS31 could protect the retinal structures and inhibit the breakdown of iBRB by reducing oxidative damage, increasing Trx-2 and Bcl-2 expression, and decreasing p53, NF-κB, Bax, caspase-3, and VEGFR2 expression in the retinas of diabetic rats. SS31 could be a potential new treatment for diabetic retinopathy and other oxidative stress-related diseases.

Objective: Intravitreal glucocorticoids and anti-vascular endothelial growth factor (VEGF) therapies are novel strategies for the treatment of advanced diabetic retinopathy, a condition with inflammatory and neuropathic elements. In contrast with anti-VEGF therapy, glucocorticoids may also exert neuroprotective effects. How glucocorticoids protect retinal neurons is unknown. The aims of the study are to investigate the anti-apoptotic actions of glucocorticoids on diabetic retinal neurons, and characterize the signalling pathways involved. Research Design and Methods: The regulation of gene expression of the four p38 mitogen-activated protein kinase (MAPK) isoforms (α, β, δ and γ) and the glucocorticoid receptor (GR) in the retinas was evaluated using quantitative RT-PCR, Western blot and immunohistochemistry. Phosphorylation of all isoforms p38MAPK (Thr180/Tyr182) and GR (S-211) was further evaluated. Apoptosis was confirmed by immunolocalization of active CASPASE-3 and the subsequent cleavage of poly (ADP-ribose) polymerase (PARP) following intravitreal injection of triamcinolone acetonide (IVTA), in an early diabetic rat model (26 days after induction of diabetes). Results: IVTA significantly down-regulated mRNA expression of Caspase 3. Activation of CASPASE-3, the subsequent cleavage of PARP-1 and phosphorylation of p38MAPK induced by diabetes were attenuated by IVTA treatment, concomitantly with activation by phosphorylation of the glucocorticoid receptor (GR S-211). Conclusions: IVTA activates the GR and exerts neural protective effects on retinal neurons. Inhibition of the p38MAPK pathway and activation of GR play a critical anti-apoptotic role in retinal neurons of diabetes following IVTA treatment. Both the anti-inflammatory and anti-apoptotic effects of glucocorticoids may be mediated through inhibition of the p38MAPK pathway in diabetic retinopathy.

The extracellular signal-regulated kinase (ERK) is one of the three major types of mitogen-activated protein kinases. Previous studies showed that ERKs mediate various signaling pathways for cell proliferation, differentiation, survival and transformation in mammals. In the present study, we use goldfish as a model system and demonstrate that ERK kinases play important roles in promoting embryonic survival and regulate development of eye and trunk in vertebrates. ERKs are highly expressed in multiple tissues including lens epithelial cells, lens fiber cells, retina, brain, muscle and heart of adult goldfish. Injection of the dominant negative ERK mutant (DNM-ERK) into the fertilized eggs of goldfish significantly inhibited ERK activity at blastula stage, and completely blocked ERK activity at gastrula and later stages. As a result, the blastula cells were induced into apoptosis, and majority of the injected embryos were lethal at embryonic stages. At the molecular level, inhibition of ERK activity by DNM-ERKs suppressed phosphorylation of Bad at Ser-112 to promote apoptosis. Similar results were observed when MEK activity was inhibited by U0126 treatment. The survived embryos display significant abnormality in the phenotypes of both eye and trunk. Associated with the abnormality in the eye development, phosphorylation in Pax-6 and expression of HSF4 were significantly decreased and expression of the β-crystallin gene was also downregulated. These results provide novel information regarding the roles of ERKs in regulating vertebrate development.

It is well established that the tumor suppressor p53 plays major roles in regulating apoptosis and cell cycle progression. In addition, recent studies have demonstrated that p53 is actively involved in regulating cell differentiation in muscle, the circulatory system and various carcinoma tissues. We have recently shown that p53 also controls lens differentiation. Regarding the mechanism, we reveal that p53 directly regulates c-Maf and Prox1, two important transcription factors to control cell differentiation in the ocular lens. In the present study, we present further evidence to show that p53 can regulate lens differentiation by controlling expression of the differentiation genes coding for the lens crystallins. First, the αA and βA3/A1 gene promoters or introns all contain putative p53 binding sites. Second, gel mobility shifting assays revealed that the p53 protein in nuclear extracts from lens epithelial cells directly binds to the p53 binding sites found in these crystallin gene promoters or introns. Third, exogenous wild type p53 induces dose-dependent expression of the luciferase reporter gene driven by different crystallin gene promoters and the exogenous dominant negative mutant p53 causes dose-dependent inhibition of the same crystallin genes. Fourth, ChIP assays revealed that p53 binds to crystallin gene promoters in vivo. Finally, in the p53 knockout mouse lenses, expression levels of various crystallins were found down-regulated in comparison with those from the wild type mouse lenses. Together, our results reveal that p53 directly regulates expression of different sets of genes to control lens differentiation.

Four-and-a-half LIM proteins FHL1-3 play important roles in cardiovascular pathophysiology. However, their roles in heart development remain unclear. Here, we report that fhlA, the zebrafish homolog of human FHL1, was found to be expressed around the 22-somite stage. After 24 hpf, expression was restricted to the heart. fhlA knockdown caused an enlarged cardiac chamber phenotype with up-regulated expression of the cardiac markers, but fhlA overexpression reduced the sizes of the cardiac chambers and down-regulated expression of the markers. The morphology associated with the cmlc2, amhc, and vmhc expression patterns at the 22 somite and 24 hpf stages included a broadened domain in embryos lacking fhlA and a smaller domain in embryos overexpressing fhlA. The changes in the sizes of the chambers were attributed to the changes in the number of ventricular and atrial cells. Loss of fhlA caused a longer heart period and pause between heartbeats in M-modes than in controls, but fhlA overexpression caused shorter systolic and diastolic intervals. Abnormal cardiac chambers and physiological function were found to be largely rescued. We also showed the expression of fhlA in the heart to be increased by retinoic acid (RA) and decreased by the RA synthase inhibitor DEAB. Both fhlA and RA signaling caused a phenotype characterized by the morphological alterations in the chamber sizes, suggesting that the role of fhlA in heart development is probably regulated by RA signaling. Taken together, these results showed that fhlA regulates the size of the heart chamber by reducing the number of cardiac cells.

Genetic mutations in GATA4, a transcriptional factor, have been found to cause congenital heart diseases. The underlying mechanism, however, remains largely unknown. We previously reported 7 heterozygous variants in patients with ventricular septal defects (VSD). Here we functionally characterized a de novo mutation p.S335X and demonstrated that this mutation led to the pre-termination of its translation, producing a truncated GATA4 lacking a conservative region at C-terminus. Truncated GATA4 did not disturb its subcellular localization; however, it delayed the cardiomyocyte differentiation in P19cl6 model and prohibited Bcl2 expression that led to apoptosis proved by fragmented genomic DNA and positive TUNEL staining in H9C2 cells. By ChIP assay, we showed that GATA4 without C-terminus reduced its DNA binding affinity and suppressed the expressions of its target genes. These findings suggest that C-terminus of GATA4 is critical to maintain DNA binding, and genetic mutations in this region may affect genes important for myocyte apoptosis and differentiation associated with congenital heart defects.

N-acetyl-L-cysteine (NAC) improves antioxidant potentials of RBCs to provide protection against oxidative stress induced hemolysis. The antioxidant mechanism of NAC to reduce oxidative stress in RBC, studied through inactivation of pro-oxidant MetHb. NAC causes irreversible inactivation of the MetHb in an H2O2 dependent manner, and the inactivation follows the pseudo- first- order kinetics. The kinetic constants are ki = 8.5μM, kinact = 0.706 min-1 and t1/2 = 0.9 min. Spectroscopic studies indicate that MetHb accepts NAC as a substrate and oxidizes through a single electron transfer mechanism to the NACox. The single e- oxidation product of NAC has been identified as the 5, 5’- dimethyl-1- pyrroline N- oxide (DMPO) adduct of the sulfur centered radical (aN = 15.2 G and aH=16. 78 G). Binding studies indicate that NACox interacts at the heme moiety and NAC oxidation through MetHb is essential for NAC binding. Heme-NAC adduct dissociated from MetHb and identified (m/z 1011.19) as 2:1 ratio of NAC/heme in the adduct. TEMPO and PBN treatment reduces NAC binding to MetHb and protects against inactivation confirms the role of thiyl radical in the inactivation process. Furthermore, scavenging thiyl radicals by TEMPO abolish the protective effect of NAC in hemolysis. Current work highlights antioxidant mechanism of NAC through NAC thiyl radical generation, and MetHb inactivation to exhibit protection in RBC against oxidative stress induced hemolysis.

Human Bone Marrow Mesenchymal Stem cells (hMSC) are a promising candidate for cytotherapy. However, the therapeutic potential is limited since the therapy requires ex-vivo cell culturing in which deterioration in cellular viability and aging is observed with time. Telomerase ribonucleoprotein complex re-elongates telomeres and therefore promotes genomic integrity, proliferation and lifespan. Recently we showed that increasing telomerase reverse transcriptase (TERT) expression by novel compound confers resistance from apoptosis induced by oxidative stress. Here we investigated the possibility that a controlled induction of human TERT (hTERT) levels by chemical compounds (AGS-499 and AGS-500) might improve the functionality of hMSC derived from healthy and neurodegenerative diseased individuals. We demonstrate that AGS treatments of hMSC increased telomerase activity and hTERT levels in a time and dose dependent manner. Prolonged treatments with the compounds increased the average telomeres length, without altering population doublings (PD) or inducing chromosomal aberrations. AGS treatments of hMSC protected the cells from apoptosis and DNA damages induced by H2O2, and from the toxicity induced by long term exposure to DMSO. These AGS effects were shown to be mediated by telomerase since they were not observed when TERT was depleted from hMSC or in mouse embryonic stem cells derived from TERT knockout mice. Furthermore, AGS compounds did not alter the functionality of hMSC as examined by their ability to differentiate into various lineages in the presence of the compounds. These results suggest that pharmaceutical increase of telomerase may confer a beneficial therapeutic advantage in regenerative medicine when hMSC therapy is applied.

The purpose of this study was to test the hypothesis that chronic stress in a negative social and psychological state plays a critical role in pancreatic cancer development and progression. In this study, we created a new stress model system to determine the effects of chronic stress on pancreatic cancer progression. Here, we show that chronic stress not only causes depression in mice, most likely attributed to an elevated level of epinephrine, but also induces pancreatic cancer progression. We provide evidence that the pancreatic cancer progression induced by chronic stress could be blocked to a significant degree by β2-AR inhibitor ICI118 551 or HIF-1α inhibitor 2-methoxyestradiol. Moreover, establishment of pancreatic cancer in mice exposed to chronic stress was accompanied by up-regulation of the expression of MMP-2, MMP-9, and VEGF, mediated by a HIF- 1α-dependent β-AR signaling pathway. Our data suggest that the β2-AR-HIF-1α axis regulates stress-induced pancreatic tumor growth and angiogenesis. This study may have a therapeutic or preventive potential for the patients with pancreatic cancer who are especially prone to psychosocial stress challenges.

Hypertension is traditionally considered a disease in which elevated blood pressure contributes to inflammation and activation of the immune system, leading to cardiovascular injury and end-organ damage. Here, we discuss the effects of aldosterone on the immune system and aldosterone’s contribution to vascular pathogenesis. Studies in human have suggested a broader role for aldosterone, beyond elevating blood pressure. Recent clinical data support the notion that aldosterone can directly alter the function of the immune system and cause vascular-damaging inflammation. Clinical observations have been reproduced in experimental models of hypertension, further supporting the idea that an aberrant immune response contributes to the onset of hypertension. Such studies have shown that myeloid cells are required to induce the disease and IL-17-producing CD4+ T cells may contribute to maintaining aldosterone-mediated hypertension. In addition, regulatory T cells diminish the inflammatory damage caused by aldosterone during hypertension. This is a very active area of research that could lead to new therapeutic targets for treating hypertension.