Gene deletion-induced autophagy deficiency leads to neural tube defects (NTDs), similar to those in diabetic pregnancy. Here we report the key autophagy regulators modulated by diabetes in the murine developing neuroepithelium. Diabetes predominantly leads to exencephaly, induces neuroepithelial cell apoptosis and suppresses autophagy in the forebrain and midbrain of NTD embryos. Deleting the Prkca gene, which encodes PKCα, reverses diabetes-induced autophagy impairment, cellular organelle stress and apoptosis, leading to an NTD reduction. PKCα increases the expression of miR-129-2, which is a negative regulator of autophagy. miR-129-2 represses autophagy by directly targeting PGC-1α, a positive regulator for mitochondrial function, which is disturbed by maternal diabetes. PGC-1α supports neurulation by stimulating autophagy in neuroepithelial cells. These findings identify two negative autophagy regulators, PKCα and miR-129-2, which mediate the teratogenicity of hyperglycaemia leading to NTDs. We also reveal a function for PGC-1α in embryonic development through promoting autophagy and ameliorating hyperglycaemia-induced NTDs.

BACKGROUND Maternal type 1 and 2 diabetes mellitus are strongly associated with high rates of severe structural birth defects, including congenital heart defects. Studies in type 1 diabetic embryopathy animal models have demonstrated that cellular stress-induced apoptosis mediates the teratogenicity of maternal diabetes leading to congenital heart defect formation. However, the mechanisms underlying maternal type 2 diabetes mellitus–induced congenital heart defects remain largely unknown. OBJECTIVE We aim to determine whether oxidative stress, endoplasmic reticulum stress, and excessive apoptosis are the intracellular molecular mechanisms underlying maternal type 2 diabetes mellitus–induced congenital heart defects. STUDY DESIGN A mouse model of maternal type 2 diabetes mellitus was established by feeding female mice a high-fat diet (60% fat). After 15 weeks on the high-fat diet, the mice showed characteristics of maternal type 2 diabetes mellitus. Control dams were either fed a normal diet (10% fat) or the high-fat diet during pregnancy only. Female mice from the high-fat diet group and the 2 control groups were mated with male mice that were fed a normal diet. At E12.5, embryonic hearts were harvested to determine the levels of lipid peroxides and superoxide, endoplasmic reticulum stress markers, cleaved caspase 3 and 8, and apoptosis. E17.5 embryonic hearts were harvested for the detection of congenital heart defect formation using India ink vessel patterning and histological examination. RESULTS Maternal type 2 diabetes mellitus significantly induced ventricular septal defects and persistent truncus arteriosus in the developing heart, along with increasing oxidative stress markers, including superoxide and lipid peroxidation; endoplasmic reticulum stress markers, including protein levels of phosphorylated-protein kinase RNA-like endoplasmic reticulum kinase, phosphorylated-IRE1α, phosphorylated-eIF2α, C/EBP homologous protein, and binding immunoglobulin protein; endoplasmic reticulum chaperone gene expression; and XBP1 messenger RNA splicing, as well as increased cleaved caspase 3 and 8 in embryonic hearts. Furthermore, maternal type 2 diabetes mellitus triggered excessive apoptosis in ventricular myocardium, endocardial cushion, and outflow tract of the embryonic heart. CONCLUSION Similar to those observations in type 1 diabetic embryopathy, maternal type 2 diabetes mellitus causes heart defects in the developing embryo manifested with oxidative stress, endoplasmic reticulum stress, and excessive apoptosis in heart cells.

Autism results in significant morbidity and mortality in children. The functional and molecular changes in the autistic brains are unclear. The present study utilized autistic brain tissues from the National Institute of Child Health and Human Development's Brain Tissue Bank for the analysis of cellular and molecular changes in autistic brains. Three key brain regions, the hippocampus, the cerebellum, and the frontal cortex, in six cases of autistic brains and six cases of non-autistic brains from 6 to 16 years old deceased children, were analyzed. The current study investigated the possible roles of endoplasmic reticulum (ER) stress, oxidative stress, and apoptosis as molecular mechanisms underlying autism. The activation of three signals of ER stress (protein kinase R-like endoplasmic reticulum kinase, activating transcription factor 6, inositol-requiring enzyme 1 alpha) varies in different regions. The occurrence of ER stress leads to apoptosis in autistic brains. ER stress may result from oxidative stress because of elevated levels of the oxidative stress markers: 4-Hydroxynonenal and nitrotyrosine-modified proteins in autistic brains. These findings suggest that cellular stress and apoptosis may contribute to the autistic phenotype. Pharmaceuticals and/or dietary supplements, which can alleviate ER stress, oxidative stress and apoptosis, may be effective in ameliorating adverse phenotypes associated with autism.

SUMMARY Vascular endothelium plays a crucial role in vascular homeostasis and tissue fluid balance. To target endothelium for robust genome editing, we developed poly(ethylene glycol) methyl ether- block -poly(lactide-co-glycolide) (PEG- b -PLGA) copolymer-based nanoparticle formulated with polyethyleneimine. A single i.v. administration of mixture of nanoparticles and plasmid DNA expressing Cas9 controlled by CDH5 promoter and guide RNA ( U6 promoter) induced highly efficient genome editing in endothelial cells (ECs) of the vasculatures, including lung, heart, aorta, and peripheral vessels in adult mice. Western blotting and immunofluorescent staining demonstrated an ~80% decrease of protein expression selectively in ECs, resulting in a phenotype similar to that of genetic knockout mice. Nanoparticle delivery of plasmid DNA could induce genome editing of two genes or genome editing and transgene expression in ECs simultaneously. Thus, nanoparticle delivery of plasmid DNA is a powerful tool to rapidly and efficiently alter expression of gene(s) in ECs for cardiovascular research and potential gene therapy.