A novel AAV9-dual microRNA-vector targeting GRIK2 in the hippocampus as a treatment for mesial temporal lobe epilepsy.

Mesial temporal lobe epilepsy (mTLE) is the most prevalent type of epilepsy in adults. First and subsequent generations of anti-epileptic therapy regimens fail to decrease seizures in a large number of patients suffering from mTLE, leaving surgical ablation of part of the hippocampus as the only therapeutic option to potentially reach seizure freedom. GluK2 has recently been identified as a promising target for the treatment of mTLE using gene therapy. Here, we engineered an adeno-associated virus serotype 9 vector expressing a cluster of two synthetic microRNAs (miRNAs), expressed from the human synapsin promoter, that target GRIK2 mRNA. Intra-hippocampal delivery of this vector in a mouse model of mTLE significantly reduced GRIK2 expression and daily seizure frequency. This treatment also improved the animals' health, reduced their anxiety, and restored working memory. Focal administration of the vector to the hippocampus of cynomolgus monkeys in GLP toxicology studies led to the selective transduction of hippocampal neurons with little exposure elsewhere in the brain and no transduction outside the central nervous system. Expression of miRNAs in hippocampal neurons resulted in substantially decreased GRIK2 mRNA expression. These data suggest that the intra-hippocampal delivery of a GMP-grade AAV9 encoding a synthetic miRNAs targeting GRIK2 is a promising treatment strategy for mTLE.

Nuclear-localized, iron-bound superoxide dismutase-2 antagonizes epithelial lineage programs to promote stemness of breast cancer cells via a histone demethylase activity.

The dichotomous behavior of superoxide dismutase-2 (SOD2) in cancer biology has long been acknowledged and more recently linked to different posttranslational forms of the enzyme. However, a distinctive activity underlying its tumor-promoting function is yet to be described. Here, we report that acetylation, one of such posttranslational modifications (PTMs), increases SOD2 affinity for iron, effectively changing the biochemical function of this enzyme from that of an antioxidant to a demethylase. Acetylated, iron-bound SOD2 localizes to the nucleus, promoting stem cell gene expression via removal of suppressive epigenetic marks such as H3K9me3 and H3K927me3. Particularly, H3K9me3 was specifically removed from regulatory regions upstream of Nanog and Oct-4, two pluripotency factors involved in cancer stem cell reprogramming. Phenotypically, cells expressing nucleus-targeted SOD2 (NLS-SOD2) have increased clonogenicity and metastatic potential. FeSOD2 operating as H3 demethylase requires H2O2 as substrate, which unlike cofactors of canonical demethylases (i.e., oxygen and 2-oxoglutarate), is more abundant in tumor cells than in normal tissue. Therefore, our results indicate that FeSOD2 is a demethylase with unique activities and functions in the promotion of cancer evolution toward metastatic phenotypes.

The APC/CFZY-1/Cdc20 Complex Coordinates With OMA-1 to Regulate the Oocyte-to-Embryo Transition in Caenorhabditis elegans.

During oocyte maturation and the oocyte-to-embryo transition, key developmental regulators such as RNA-binding proteins coordinate translation of particular messenger RNA (mRNAs) and related developmental processes by binding to their cognate maternal mRNAs. In the nematode Caenorhabditis elegans, these processes are regulated by a set of CCCH zinc finger proteins. Oocyte maturation defective-1 (OMA-1) and OMA-2 are two functionally redundant CCCH zinc finger proteins that turnover rapidly during the first embryonic cell division. These turnovers are required for proper transition from oogenesis to embryogenesis. A gain-of-function mutant of OMA-1, oma-1(zu405), stabilizes and delays degradation of OMA-1, resulting in delayed turnover and mis-segregation of other cell fate determinants, which eventually causes embryonic lethality. We performed a large-scale forward genetic screen to identify suppressors of the oma-1(zu405) mutant. We show here that multiple alleles affecting functions of various anaphase promoting complex/cyclosome (APC/C) subunits, including MAT-1, MAT-2, MAT-3, EMB-30, and FZY-1, suppress the gain-of-function mutant of OMA-1. Transcriptome analysis suggested that overall transcription in early embryos occurred after introducing mutations in APC/C genes into the oma-1(zu405) mutant. Mutations in APC/C genes prevent OMA-1 enrichment in P granules and correct delayed degradation of downstream cell fate determinants including pharynx and intestine in excess-1 (PIE-1), posterior segregation-1 (POS-1), muscle excess-3 (MEX-3), and maternal effect germ-cell defective-1 (MEG-1). We demonstrated that only the activator FZY-1, but not FZR-1, is incorporated in the APC/C complex to regulate the oocyte-to-embryo transition. Our findings suggested a genetic relationship linking the APC/C complex and OMA-1, and support a model in which the APC/C complex promotes P granule accumulation and modifies RNA binding of OMA-1 to regulate the oocyte-to-embryo transition process.

Mitochondrial Superoxide Dismutase: What the Established, the Intriguing, and the Novel Reveal About a Key Cellular Redox Switch.

Significance: Reactive oxygen species (ROS) are now widely recognized as central mediators of cell signaling. Mitochondria are major sources of ROS. Recent Advances: It is now clear that mitochondrial ROS are essential to activate responses to cellular microenvironmental stressors. Mediators of these responses reside in large part in the cytosol. Critical Issues: The primary form of ROS produced by mitochondria is the superoxide radical anion. As a charged radical anion, superoxide is restricted in its capacity to diffuse and convey redox messages outside of mitochondria. In addition, superoxide is a reductant and not particularly efficient at oxidizing targets. Because there are many opportunities for superoxide to be neutralized in mitochondria, it is not completely clear how redox cues generated in mitochondria are converted into diffusible signals that produce transient oxidative modifications in the cytosol or nucleus. Future Directions: To efficiently intervene at the level of cellular redox signaling, it seems that understanding how the generation of superoxide radicals in mitochondria is coupled with the propagation of redox messages is essential. We propose that mitochondrial superoxide dismutase (SOD2) is a major system converting diffusion-restricted superoxide radicals derived from the electron transport chain into highly diffusible hydrogen peroxide (H2O2). This enables the coupling of metabolic changes resulting in increased superoxide to the production of H2O2, a diffusible secondary messenger. As such, to determine whether there are other systems coupling metabolic changes to redox messaging in mitochondria as well as how these systems are regulated is essential.

SOD2 acetylation on lysine 68 promotes stem cell reprogramming in breast cancer.

Mitochondrial superoxide dismutase (SOD2) suppresses tumor initiation but promotes invasion and dissemination of tumor cells at later stages of the disease. The mechanism of this functional switch remains poorly defined. Our results indicate that as SOD2 expression increases acetylation of lysine 68 ensues. Acetylated SOD2 promotes hypoxic signaling via increased mitochondrial reactive oxygen species (mtROS). mtROS, in turn, stabilize hypoxia-induced factor 2α (HIF2α), a transcription factor upstream of "stemness" genes such as Oct4, Sox2, and Nanog. In this sense, our findings indicate that SOD2K68Ac and mtROS are linked to stemness reprogramming in breast cancer cells via HIF2α signaling. Based on these findings we propose that, as tumors evolve, the accumulation of SOD2K68Ac turns on a mitochondrial pathway to stemness that depends on HIF2α and may be relevant for the progression of breast cancer toward poor outcomes.

Manganese superoxide dismutase and glutathione peroxidase-1 contribute to the rise and fall of mitochondrial reactive oxygen species which drive oncogenesis.

Reactive oxygen species (ROS) largely originating in the mitochondria play essential roles in the metabolic and (epi)genetic reprogramming of cancer cell evolution towards more aggressive phenotypes. Recent studies have indicated that the activity of superoxide dismutase (SOD2) may promote tumor progression by serving as a source of hydrogen peroxide (H2O2). H2O2 is a form of ROS that is particularly active as a redox agent affecting cell signaling due to its ability to freely diffuse out of the mitochondria and alter redox active amino acid residues on regulatory proteins. Therefore, there is likely a dichotomy whereas SOD2 can be considered a protective antioxidant, as well as a pro-oxidant during cancer progression, with these effects depending on the accumulation and detoxification of H2O2. Glutathione peroxidase-1 GPX1, is a selenium-dependent scavenger of H2O2 which partitions between the mitochondria and the cytosol. Epidemiologic studies indicated that allelic variations in the SOD2 and GPX1 genes alter the distribution and relative concentrations of SOD2 and GPX1 in mitochondria, thereby affecting the dynamic between the production and elimination of H2O2. Experimental and epidemiological evidence supporting a conflicting role of SOD2 in tumor biology, and epidemiological evidence that SOD2 and GPX1 can interact to affect cancer risk and progression indicated that it is the net accumulation of mitochondrial H2O2 (mtH2O2) resulting from of the balance between the activities SOD2 and anti-oxidants such as GPX1 that determines whether SOD2 prevents or promotes oncogenesis. In this review, research supporting the idea that GPX1 is a gatekeeper restraining the oncogenic power of mitochondrial ROS generated by SOD2 is presented. This article is part of a Special Issue entitled Mitochondria in Cancer, edited by Giuseppe Gasparre, Rodrigue Rossignol and Pierre Sonveaux.

SOD2 and the Mitochondrial UPR: Partners Regulating Cellular Phenotypic Transitions.

ATP and reactive oxygen species (ROS) are signaling molecules that control cellular function and phenotype. Mitochondria produce both ATP and ROS. Since the electrons needed to generate either ATP or ROS originate from NADH/FADH2, the mechanism through which electrons flow towards oxygen determines yields and whether ATP or ROS prevails. Alterations in the electron flow impact cells dramatically, such as by supporting specialization (which requires high ATP) or imposing dedifferentiation. High ROS, facilitated by enzymes such as superoxide dismutase 2 (SOD2) that enhance mitochondrial hydrogen peroxide (mtH2O2), are normally linked to dedifferentiation of somatic cells. Here we propose that combined high mtH2O2 and mitochondrial unfolded protein response (UPR(mt)) activation are essential for somatic dedifferentiation programs and the acquisition of stem-like properties in reparative processes and disease.

Linking the circadian rhythm gene Arntl2 to interleukin 21 expression in type 1 diabetes.

The circadian rhythm-related aryl hydrocarbon receptor nuclear translocator-like 2 (Arntl2) gene has been identified as a candidate gene for the murine type 1 diabetes locus Idd6.3. Previous studies suggested a role in expansion of CD4(+)CD25(-) T cells, and this then creates an imbalance in the ratio between T-effector and CD4(+)CD25(+) T-regulator cells. Our transcriptome analyses identify the interleukin 21 (IL21) gene (Il21) as a direct target of ARNTL2. ARNTL2 binds in an allele-specific manner to the RNA polymerase binding site of the Il21 promoter and inhibits its expression in NOD.C3H congenic mice carrying C3H alleles at Idd6.3. IL21 is known to promote T-cell expansion, and in agreement with these findings, mice with C3H alleles at Idd6.3 produce lower numbers of CD4(+)IL21(+) and CD4(+) and CD8(+) T cells compared with mice with NOD alleles at Idd6.3. Our results describe a novel and rather unexpected role for Arntl2 in the immune system that lies outside of its predicted function in circadian rhythm regulation.

Downregulation of the circadian rhythm related gene Arntl2 suppresses diabetes protection in Idd6 NOD.C3H congenic mice.

1. Our previous studies of the murine genetic locus Idd6 revealed the aryl hydrocarbon receptor nuclear translocator-like protein 2 (Arntl2) as a candidate gene for type 1 diabetes; and in Idd6 NOD.C3H congenic mice, Arntl2 upregulation is linked to decreased diabetes development. 2. In the present study, shRNA plasmids capable of suppressing Arntl2 expression were developed and given to diabetes resistant NOD.C3H congenic mice by hydrodynamic tail vein injection. The effects of Arntl2 suppression on diabetes incidence and immune cell numbers were investigated. 3. Diabetes incidence was increased by Arntl2 mRNA interference in the congenic strain and this was associated with an increase in CD4(+) T cells and a decrease in regulatory T cells in the peripheral immune system. 4. These results provide additional support for the protective role of the Arntl2 gene located in locus Idd6 in diabetes progression in NOD.C3H congenic mice.

Inhibition of type 1 diabetes by upregulation of the circadian rhythm-related aryl hydrocarbon receptor nuclear translocator-like 2.

The genetic locus Idd6 is involved in type 1 diabetes development in the non-obese diabetic (NOD) mouse through its effect on the immune system and in particular, on T cell activities. Analysis of congenic strains for Idd6 has established the Aryl hydrocarbon receptor nuclear translocator-like 2 (Arntl2) as a likely candidate gene. In this study we investigate the role of Arntl2 in the autoimmune disease and T cell activation. An Arntl2 expressing plasmid was transfected into CD4(+) T cells by nucleofection. Expression levels of cytokines and CD4(+) T cell activation markers, cell death, apoptosis, and cell proliferation rates were characterized in ex vivo experiments whilst in vivo the transfected cells were transferred into NOD.SCID mice to monitor diabetes development. The results demonstrate that Arntl2 overexpression leads to inhibition of CD4(+) T cell proliferation and decreases in their diabetogenic activity without influence on the expression levels of cytokines, CD4(+) T cell activation markers, cell death, and apoptosis. Our findings suggest that Arntl2 at the Idd6 locus may act via the inhibition of CD4(+) T cell proliferation and the reduction in the diabetogenic activity of CD4(+) T cells to protect against autoimmune type 1 diabetes in the NOD mice.

Insulin expression in livers of diabetic mice mediated by hydrodynamics-based administration.

AIM: Transfer and expression of insulin gene in vivo are an alternative strategy to improve glycemic control in type 1 diabetes. Hydrodynamics-based procedure has been proved to be very efficient to transfer naked DNA to mouse livers. The basal hepatic insulin production mediated by this rapid tail vein injection was studied to determine its effect on the resumption of glycemic control in type 1 diabetic mice. METHODS: Engineered insulin cDNA was inserted into plasmid vectors under a CMV promoter, and transferred into STZ induced diabetic mice by hydrodynamic procedure. Glucose levels, body weight of treated mice, insulin levels, immunohistology of the liver, and quantity of insulin mRNA in the liver were assayed to identify the improvement of hyperglycemic complication after plasmid administration. Sleeping Beauty, a transposon system, was also used to prolong the insulin expression in the liver. RESULTS: After plasmid administration, Plasma insulin was significantly increased in the diabetic mice and the livers were insulin-positive by immunostaining. At the same time the hyperglycemic complication was improved. The blood glucose levels of mice were reduced to normal. Glucose tolerance of the treated diabetic mice was improved. Body weight loss was also ameliorated. The rapid tail vein injection did not cause any fatal result. CONCLUSION: Our results suggested that insulin gene could be efficiently transferred into the livers of diabetic mice via rapid tail vein injection and it resulted in high level of insulin expression. The basal hepatic insulin production mediated by hydrodynamics-based administration improved the glycemic control in type 1 diabetes dramatically and ameliorated diabetic syndromes. Hydrodynamics-based administration offers a simple and efficient way in the study of gene therapy for type 1 diabetes.

Conditions affecting hydrodynamics-based gene delivery into mouse liver in vivo.

1. It has been demonstrated that the hydrodynamics-based procedure has high efficiency to deliver foreign genes into the liver. The widespread use of this procedure in gene function studies and as a treatment option for liver and other organ diseases puts considerable importance on the investigation of various conditions that affect hydrodynamics-based gene delivery into mouse liver in vivo. 2. Various conditions, including the volume, speed and solution of the injection and the state, gender and strain of the animal were manipulated to evaluate their effect on the expression levels in mice of human factor IX (hFIX) 8 h after tail vein injection of the plasmid pCMV-hFIX. 3. It was found that an injection volume of 2-2.5 mL and an injection speed of 5-7 s were very effective in delivering DNA into the mouse liver. Using Ringer's solution as an injection fluid increased the efficiency of hFIX expression. 4. Anaesthetized mice expressed higher hFIX than conscious mice. Males expressed higher hFIX than females. The ICR mouse strain demonstrated higher expression of the foreign gene than did the C57 strain. 5. The effects of these specific factors on hFIX expression may be caused by variations in hydrostatic pressure, the degree of liver damage and liver size. 6. It can be concluded that there are optimal conditions for hFIX expression in the liver. This information may be helpful for the application of hydrodynamics-based procedures in the investigation of gene expression and gene therapy.

Construction of a recombinant vector based on AAV carrying human endothelial nitric-oxide synthase gene.

AIM: To construct an AAV based vector carrying human endothelial nitric-oxide synthase (eNOS) cDNA and study its expression in vitro for future gene therapy. METHODS: eNOS cDNA was inserted into the EcoR I site of pSNAV-1 containing the cytomegalovirus (CMV) promoter and inverted terminal repeat sequences of adeno-associated virus. The constructed vector was transfected into BHK and C2C12 cells. eNOS cDNA and mRNA were detected by polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR), respectively. RESULTS: By restriction enzyme digestion analysis, it was proved that eNOS cDNA was inserted into pSNAV-1 in a proper direction. PCR detection demonstrated that pSNAV-eNOS was transferred into both BHK and C2C12 cells. RT-PCR analysis showed that these pSNAV-eNOS transfected cells could express eNOS mRNA. CONCLUSION: pSNAV-eNOS was successfully constructed with the ability to express human eNOS mRNA in cultured mammalian cells.