MicroRNA Signature of Epithelial-Mesenchymal Transition in Group B Streptococcal Infection of the Placental Chorioamniotic Membranes.

BACKGROUND: Infection-induced preterm birth is a major cause of neonatal mortality and morbidity and leads to preterm premature rupture of placental chorioamniotic membranes. The loss of amniotic epithelial cells and tensile strength preceding membrane rupture is poorly understood. We hypothesized that intrauterine bacterial infection induces changes in microRNA (miRNA) expression, leading to amniotic epithelial cell loss and membrane weakening. METHODS: Ten pregnant pigtail macaques received choriodecidual inoculation of either group B Streptococcus (GBS) or saline (n = 5/group). Placental chorioamniotic membranes were studied using RNA microarray and immunohistochemistry. Chorioamniotic membranes from women with preterm premature rupture of membranes (pPROM) and normal term pregnancies were studied using transmission electron microscopy. RESULTS: In our model, an experimental GBS infection was associated with changes in the miRNA profile in the chorioamniotic membranes consistent with epithelial to mesenchymal transition (EMT) with loss of epithelial (E-cadherin) and gain of mesenchymal (vimentin) markers. Similarly, loss of desmosomes (intercellular junctions) was seen in placental tissues from women with pPROM. CONCLUSIONS: We describe EMT as a novel mechanism for infection-associated chorioamniotic membrane weakening, which may be a common pathway for many etiologies of pPROM. Therapy based on anti-miRNA targeting of EMT may prevent pPROM due to perinatal infection.

Flow of energy in the outer retina in darkness and in light.

Structural features of neurons create challenges for effective production and distribution of essential metabolic energy. We investigated how metabolic energy is distributed between cellular compartments in photoreceptors. In avascular retinas, aerobic production of energy occurs only in mitochondria that are located centrally within the photoreceptor. Our findings indicate that metabolic energy flows from these central mitochondria as phosphocreatine toward the photoreceptor's synaptic terminal in darkness. In light, it flows in the opposite direction as ATP toward the outer segment. Consistent with this model, inhibition of creatine kinase in avascular retinas blocks synaptic transmission without influencing outer segment activity. Our findings also reveal how vascularization of neuronal tissue can influence the strategies neurons use for energy management. In vascularized retinas, mitochondria in the synaptic terminals of photoreceptors make neurotransmission less dependent on creatine kinase. Thus, vasculature of the tissue and the intracellular distribution of mitochondria can play key roles in setting the strategy for energy distribution in neurons.

Roles of glucose in photoreceptor survival.

Vertebrate photoreceptor neurons have a high demand for metabolic energy, and their viability is very sensitive to genetic and environmental perturbations. We investigated the relationship between energy metabolism and cell death by evaluating the metabolic effects of glucose deprivation on mouse photoreceptors. Oxygen consumption, lactate production, ATP, NADH/NAD(+), TCA cycle intermediates, morphological changes, autophagy, and viability were evaluated. We compared retinas incubated with glucose to retinas deprived of glucose or retinas treated with a mixture of mitochondrion-specific fuels. Rapid and slow phases of cell death were identified. The rapid phase is linked to reduced mitochondrial activity, and the slower phase reflects a need for substrates for cell maintenance and repair.

Disruption of kinesin II function using a dominant negative-acting transgene in Xenopus laevis rods results in photoreceptor degeneration.

PURPOSE: Kinesin II is a motor protein that moves on microtubules and whose importance in ciliary and flagellar transport has been well documented. In the current study, the role of kinesin II in rod photoreceptors was examined by expressing a dominant negative-acting transgene that disrupts kinesin II function in Xenopus laevis rods of transgenic tadpoles. METHODS: A previously characterized dominant negative-acting kinesin II transgene tagged with enhanced green fluorescent protein (EGFP) driven by the Xenopus rod opsin promoter was used to make Xenopus transgenic tadpoles to disrupt kinesin II function specifically in rod photoreceptors. Transgenic tadpole retinas were examined to ascertain transgene expression pattern and morphologic phenotype. Rod-to-cone ratios were determined in experimental and control retinas. RESULTS: Visualized by its EGFP tag, the kinesin II transgene was expressed in rods in a mosaic pattern in the retina. Subcellular localization of transgenic kinesin II was similar to that of endogenous kinesin II subunit photoreceptor expression-that is, it was localized to the connecting cilium, inner segment, and synapse. However, in kinesin II transgene-expressing animals, fluorescence was transient. Ocular fluorescence was lost 6 days after its first detection. The disappearance of fluorescence was due to degeneration of rods expressing the transgene. Retinas of 7- to 9-day old kinesin II transgenic tadpoles had significantly fewer rods than did control retinas. CONCLUSIONS: The observation that rod degeneration is produced by expression of a dominant negative-acting kinesin II transgene in Xenopus rods is consistent with previous studies in mice, suggesting that kinesin II function is required for photoreceptor survival.

Myosin 3A transgene expression produces abnormal actin filament bundles in transgenic Xenopus laevis rod photoreceptors.

Myo3A, a class III myosin, localizes to the distal (plus) ends of inner segment actin filament bundles that form the core of microvillus-like calycal processes encircling the base of the photoreceptor outer segment. To investigate Myo3A localization and function, we expressed green fluorescent protein-tagged bass Myo3A and related constructs in transgenic Xenopus rods using a modified opsin promoter. Tagged intact Myo3A localized to rod calycal processes, as previously reported for native bass Myo3A. Transgenic rods developed abnormally large calycal processes and subsequently degenerated. Modified Myo3A expression constructs demonstrated that calycal process localization required an active motor domain and the tail domain. Expressed tail domain alone localized to actin bundles along the entire inner segment length, rather than to the distal end. This tail domain localization required the conserved C-terminal domain (3THDII) previously shown to possess an actin-binding motif. Our findings suggest that Myo3A plays a role in the morphogenesis and maintenance of calycal processes of vertebrate photoreceptors.