Author Correction: Endothelial activation of caspase-9 promotes neurovascular injury in retinal vein occlusion.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Endothelial activation of caspase-9 promotes neurovascular injury in retinal vein occlusion.

Central nervous system ischemic injury features neuronal dysfunction, inflammation and breakdown of vascular integrity. Here we show that activation of endothelial caspase-9 after hypoxia-ischemia is a critical event in subsequent dysfunction of the blood-retina barrier, using a panel of interrelated ophthalmic in vivo imaging measures in a mouse model of retinal vein occlusion (RVO). Rapid nonapoptotic activation of caspase-9 and its downstream effector caspase-7 in endothelial cells promotes capillary ischemia and retinal neurodegeneration. Topical eye-drop delivery of a highly selective caspase-9 inhibitor provides morphological and functional retinal protection. Inducible endothelial-specific caspase-9 deletion phenocopies this protection, with attenuated retinal edema, reduced inflammation and preserved neuroretinal morphology and function following RVO. These results reveal a non-apoptotic function of endothelial caspase-9 which regulates blood-retina barrier integrity and neuronal survival, and identify caspase-9 as a therapeutic target in neurovascular disease.

Accumulation of Neurofascin at Nodes of Ranvier Is Regulated by a Paranodal Switch.

The paranodal junctions flank mature nodes of Ranvier and provide a barrier between ion channels at the nodes and juxtaparanodes. These junctions also promote node assembly and maintenance by mechanisms that are poorly understood. Here, we examine their role in the accumulation of NF186, a key adhesion molecule of PNS and CNS nodes. We previously showed that NF186 is initially targeted/accumulates via its ectodomain to forming PNS (hemi)nodes by diffusion trapping, whereas it is later targeted to mature nodes by a transport-dependent mechanism mediated by its cytoplasmic segment. To address the role of the paranodes in this switch, we compared accumulation of NF186 ectodomain and cytoplasmic domain constructs in WT versus paranode defective (i.e., Caspr-null) mice. Both pathways are affected in the paranodal mutants. In the PNS of Caspr-null mice, diffusion trapping mediated by the NF186 ectodomain aberrantly persists into adulthood, whereas the cytoplasmic domain/transport-dependent targeting is impaired. In contrast, accumulation of NF186 at CNS nodes does not undergo a switch; it is predominantly targeted to both forming and mature CNS nodes via its cytoplasmic domain and requires intact paranodes. Fluorescence recovery after photobleaching analysis indicates that the paranodes provide a membrane diffusion barrier that normally precludes diffusion of NF186 to nodes. Linkage of paranodal proteins to the underlying cytoskeleton likely contributes to this diffusion barrier based on 4.1B and ßII spectrin expression in Caspr-null mice. Together, these results implicate the paranodes as membrane diffusion barriers that regulate targeting to nodes and highlight differences in the assembly of PNS and CNS nodes.SIGNIFICANCE STATEMENT Nodes of Ranvier are essential for effective saltatory conduction along myelinated axons. A major question is how the various axonal proteins that comprise the multimeric nodal complex accumulate at this site. Here we examine how targeting of NF186, a key nodal adhesion molecule, is regulated by the flanking paranodal junctions. We show that the transition from diffusion-trapping to transport-dependent accumulation of NF186 requires the paranodal junctions. We also demonstrate that these junctions are a barrier to diffusion of axonal proteins into the node and highlight differences in PNS and CNS node assembly. These results provide new insights into the mechanism of node assembly and the pathophysiology of neurologic disorders in which impaired paranodal function contributes to clinical disability.

Localized Myosin II Activity Regulates Assembly and Plasticity of the Axon Initial Segment.

The axon initial segment (AIS) is the site of action potential generation and a locus of activity-dependent homeostatic plasticity. A multimeric complex of sodium channels, linked via a cytoskeletal scaffold of ankyrin G and beta IV spectrin to submembranous actin rings, mediates these functions. The mechanisms that specify the AIS complex to the proximal axon and underlie its plasticity remain poorly understood. Here we show phosphorylated myosin light chain (pMLC), an activator of contractile myosin II, is highly enriched in the assembling and mature AIS, where it associates with actin rings. MLC phosphorylation and myosin II contractile activity are required for AIS assembly, and they regulate the distribution of AIS components along the axon. pMLC is rapidly lost during depolarization, destabilizing actin and thereby providing a mechanism for activity-dependent structural plasticity of the AIS. Together, these results identify pMLC/myosin II activity as a common link between AIS assembly and plasticity.

Control of embryonic stem cell identity by BRD4-dependent transcriptional elongation of super-enhancer-associated pluripotency genes.

Transcription factors and chromatin-remodeling complexes are key determinants of embryonic stem cell (ESC) identity. Here, we demonstrate that BRD4, a member of the bromodomain and extraterminal domain (BET) family of epigenetic readers, regulates the self-renewal ability and pluripotency of ESCs. BRD4 inhibition resulted in induction of epithelial-to-mesenchymal transition (EMT) markers and commitment to the neuroectodermal lineage while reducing the ESC multidifferentiation capacity in teratoma assays. BRD4 maintains transcription of core stem cell genes such as OCT4 and PRDM14 by occupying their super-enhancers (SEs), large clusters of regulatory elements, and recruiting to them Mediator and CDK9, the catalytic subunit of the positive transcription elongation factor b (P-TEFb), to allow Pol-II-dependent productive elongation. Our study describes a mechanism of regulation of ESC identity that could be applied to improve the efficiency of ESC differentiation.

Pediatric educational needs assessment for urban and rural emergency medical technicians.

OBJECTIVE: The objective of the study was to identify past experiences, present needs, barriers, and desired methods of training for urban and rural emergency medical technicians. METHODS: This 62-question pilot-tested written survey was administered at the 2008 Oregon EMS and 2009 EMS for Children conferences. Respondents were compared with registration lists and the state emergency medical services (EMS) database to assess for nonresponder bias. Agencies more than 10 miles from a population of 40,000 were defined as rural. RESULTS: Two hundred nineteen (70%) of 313 EMS personnel returned the surveys. Respondents were 3% first responders, 27% emergency medical technician basics, 20% intermediates, and 47% paramedics. Sixty-eight percent were rural, and 32% were urban. Sixty-eight percent reported fewer than 10% pediatric transports. Overall, respondents rated their comfort caring for pediatric patients as 3.1 on a 5-point Likert scale (95% confidence interval, 3.1-3.2). Seventy-two percent reported a mean rating of less than "comfortable" (4 on the scale) across 17 topics in pediatric care, which did not differ by certification level. Seven percent reported no pediatric training in the last 2 years, and 76% desired more. The "quality of available trainings" was ranked as the most important barrier to training; 26% of rural versus 7% of urban EMS personnel ranked distance as the most significant barrier (P < 0.01). Fifty-one percent identified highly realistic simulations as the method that helped them learn best. In the past 2 years, 19% had trained on a highly realistic pediatric simulator. One to 3 hours was the preferred duration for trainings. CONCLUSIONS: Except for distance as a barrier, there were no significant differences between urban and rural responses. Both urban and rural providers desire resources, in particular, highly realistic simulation, to address the infrequency of pediatric transports and limited training.