Insulin Receptor Associates with Promoters Genome-wide and Regulates Gene Expression.

Insulin receptor (IR) signaling is central to normal metabolic control and dysregulated in prevalent chronic diseases. IR binds insulin at the cell surface and transduces rapid signaling via cytoplasmic kinases. However, mechanisms mediating long-term effects of insulin remain unclear. Here, we show that IR associates with RNA polymerase II in the nucleus, with striking enrichment at promoters genome-wide. The target genes were highly enriched for insulin-related functions including lipid metabolism and protein synthesis and diseases including diabetes, neurodegeneration, and cancer. IR chromatin binding was increased by insulin and impaired in an insulin-resistant disease model. Promoter binding by IR was mediated by coregulator host cell factor-1 (HCF-1) and transcription factors, revealing an HCF-1-dependent pathway for gene regulation by insulin. These results show that IR interacts with transcriptional machinery at promoters and identify a pathway regulating genes linked to insulin's effects in physiology and disease.

APC is an RNA-binding protein, and its interactome provides a link to neural development and microtubule assembly.

Adenomatous polyposis coli (APC) is a microtubule plus-end scaffolding protein important in biology and disease. APC is implicated in RNA localization, although the mechanisms and functional significance remain unclear. We show APC is an RNA-binding protein and identify an RNA interactome by HITS-CLIP. Targets were highly enriched for APC-related functions, including microtubule organization, cell motility, cancer, and neurologic disease. Among the targets is ß2B-tubulin, known to be required in human neuron and axon migration. We show ß2B-tubulin is synthesized in axons and localizes preferentially to dynamic microtubules in the growth cone periphery. APC binds the ß2B-tubulin 3' UTR; experiments interfering with this interaction reduced ß2B-tubulin mRNA axonal localization and expression, depleted dynamic microtubules and the growth cone periphery, and impaired neuron migration. These results identify APC as a platform binding functionally related protein and RNA networks, and suggest a self-organizing model for the microtubule to localize synthesis of its own subunits.

This message will self-destruct: NMD regulates axon guidance.

The navigation of axons to their final destination can involve a sequence of steps that require different sets of guidance receptors. In this issue, Colak et al. show that regulated intra-axonal protein synthesis coupled to nonsense-mediated mRNA decay (NMD) controls a switch in Robo3.2 expression that is critical for navigation.

Transmembrane receptor DCC associates with protein synthesis machinery and regulates translation.

Extracellular signals regulate protein translation in many cell functions. A key advantage of control at the translational level is the opportunity to regulate protein synthesis within specific cellular subregions. However, little is known about mechanisms that may link extracellular cues to translation with spatial precision. Here, we show that a transmembrane receptor, DCC, forms a binding complex containing multiple translation components, including eukaryotic initiation factors, ribosomal large and small subunits, and monosomes. In neuronal axons and dendrites DCC colocalizes in particles with translation machinery, and newly synthesized protein. The extracellular ligand netrin promoted DCC-mediated translation and disassociation of translation components. The functional and physical association of a cell surface receptor with the translation machinery leads to a generalizable model for localization and extracellular regulation of protein synthesis, based on a transmembrane translation regulation complex.

Control of Selective mRNA Translation in Neuronal Subcellular Compartments in Health and Disease.

In multiple cell types, mRNAs are transported to subcellular compartments, where local translation enables rapid, spatially localized, and specific responses to external stimuli. Mounting evidence has uncovered important roles played by local translation in vivo in axon survival, axon regeneration, and neural wiring, as well as strong links between dysregulation of local translation and neurologic disorders. Omic studies have revealed that >1000 mRNAs are present and can be selectively locally translated in the presynaptic and postsynaptic compartments from development to adulthood in vivo A large proportion of the locally translated mRNAs is specifically upregulated or downregulated in response to distinct extracellular signals. Given that the local translatome is large, selectively translated, and cue-specifically remodeled, a fundamental question concerns how selective translation is achieved locally. Here, we review the emerging regulatory mechanisms of local selective translation in neuronal subcellular compartments, their mRNA targets, and their orchestration. We discuss mechanisms of local selective translation that remain unexplored. Finally, we describe clinical implications and potential therapeutic strategies in light of the latest advances in gene therapy.

Evidence of an Annexin A4 mediated plasma membrane repair response to biomechanical strain associated with glaucoma pathogenesis.

Glaucoma is a common neurodegenerative blinding disease that is closely associated with chronic biomechanical strain at the optic nerve head (ONH). Yet, the cellular injury and mechanosensing mechanisms underlying the resulting damage have remained critically unclear. We previously identified Annexin A4 (ANXA4) from a proteomic analyses of human ONH astrocytes undergoing pathological biomechanical strain that mimics glaucomatous conditions. Annexins are a family of calcium-dependent phospholipid binding proteins with key functions in plasma membrane repair (PMR); an active mechanism to limit and mend cellular injury that involves membrane and cytoskeletal reorganizations. However, a role for direct membrane damage and PMR has not been well studied in the context of biomechanical strain, such as that associated with glaucoma. Here we report that this moderate strain surprisingly damages cell membranes to increase permeability in a calcium-dependent manner, and induces rapid aggregation of ANXA4 at injury sites. ANXA4 loss-of-function increases permeability, while exogenous ANXA4 reduces it. Furthermore, ANXA4 aggregation is associated with F-actin dynamics in vitro, and remarkably this interaction and aggregation signature is also observed in the glaucomatous ONH in patient samples. Together these studies link moderate biomechanical strain with direct membrane damage and actin dynamics, and identify an active PMR role for ANXA4 in new model of cell injury associated with glaucoma pathogenesis.

2017 National Optometry Workforce Survey.

SIGNIFICANCE: Planning for the effective delivery of eye care, on all levels, depends on an accurate and detailed knowledge of the optometric workforce and an understanding of demographic/behavioral trends to meet future needs of the public. PURPOSE: The purposes of this study were to assess the current and future supply of doctors of optometry and to examine in-depth trends related to (1) demographic shifts, (2) sex-based differences, (3) differences in practice behaviors in between self-employed and employed optometrists, and (4) the concept of additional capacity within the profession. METHODS: The 2017 National Optometry Workforce Survey (31 items) was distributed to 4050 optometrists, randomly sampled from a population of 45,033 currently licensed and practicing optometrists listed in the American Optometric Association's Optometry Master Data File. A stratified sampling method was applied to the population of optometrists using primary license state, age, and sex as variables to ensure a representative sample. RESULTS: With a response rate of 29% (1158 responses), the sample ensured a 95% confidence interval with a margin of error of <5%. Key results include finding no significant differences between men and women for hours worked (38.9 vs. 37.5), productivity (patient visits per hour, 2.0 vs. 1.9), or career options/professional growth satisfaction with 65% for both. The data indicate a likely range of additional patient capacity of 2.29 to 2.57 patients per week (5.05 to 5.65 million annually profession-wide). CONCLUSIONS: The optometric workforce for the next decade is projected to grow 0.6 to 0.7% more annually than the U.S. population. The study found additional capacity for the profession more limited than previously suggested. Findings also illustrate an evolving/equitable workforce based on sex, in terms of both productivity and satisfaction. The trend toward employed versus self-employed was marked with 44% reporting they are employed, up from 29% in 2012.

IMP2 axonal localization, RNA interactome, and function in the development of axon trajectories.

RNA-based regulatory mechanisms play important roles in the development and plasticity of neural circuits and neurological disease. Developing axons provide a model well suited to the study of RNA-based regulation, and contain specific subsets of mRNAs that are locally translated and have roles in axon pathfinding. However, the RNA-binding proteins involved in axon pathfinding, and their corresponding mRNA targets, are still largely unknown. Here we find that the RNA-binding protein IMP2 (Igf2bp2) is strikingly enriched in developing axon tracts, including in spinal commissural axons. We used the HITS-CLIP approach to perform a genome-wide identification of RNAs that interact directly with IMP2 in the native context of developing mouse brain. This IMP2 interactome was highly enriched for mRNA targets related to axon guidance. Accordingly, IMP2 knockdown in the developing spinal cord led to strong defects in commissural axon trajectories at the midline intermediate target. These results reveal a highly distinctive axonal enrichment of IMP2, show that it interacts with a network of axon guidance-related mRNAs, and reveal that it is required for normal axon pathfinding during vertebrate development.

All roads lead to glaucoma: Induced retinal injury cascades contribute to a common neurodegenerative outcome.

Glaucoma describes a distinct optic neuropathy with complex etiology and a variety of associated risk factors, but with similar pathological endpoints. Risk factors such as age, increased intraocular pressure (IOP), low mean arterial pressure, and autoimmune disease, can all be associated with death of retinal ganglion cells (RGCs) and optic nerve head remodeling. Today, IOP management remains the standard of care, even though IOP elevation is not pathognomonic of glaucoma, and patients can continue to lose vision despite effective IOP control. A contemporary view of glaucoma as a complex, neurodegenerative disease has developed, along with the recognition of a need for new disease modifying retinal treatment strategies and improved outcomes. However, the distinction between risk factors triggering the disease process and retinal injury responses is not always clear. In this review, we attempt to distinguish between the various triggers, and their association with subsequent key RGC injury mechanisms. We propose that distinct glaucomatous risk factors result in similar retinal and optic nerve injury cascades, including oxidative and metabolic stress, glial reactivity, and altered inflammatory responses, which induce common molecular signals to induce RGC apoptosis. This organization forms a coherent disease framework and presents conserved targets for therapeutic intervention that are not limited to specific risk factors.

Structural Characterization of a Heparan Sulfate Pentamer Interacting with LAR-Ig1-2.

Leukocyte common antigen-related (LAR) protein is one of the type IIa receptor protein tyrosine phosphatases (RPTPs) that are important for signal transduction in biological processes, including axon growth and regeneration. Glycosaminoglycan chains, including heparan sulfate (HS) and chondroitin sulfate (CS), act as ligands that regulate LAR signaling. Here, we report the structural characterization of the first two immunoglobulin domains (Ig1-2) of LAR interacting with an HS pentasaccharide (GlcNS6S-GlcA-GlcNS3,6S-IdoA2S-GlcNS6S-OME, fondaparinux) using multiple solution-based NMR methods. In the course of the study, we extended an assignment strategy useful for sparsely labeled proteins expressed in mammalian cell culture supplemented with a single type of isotopically enriched amino acid ([15N]-Lys in this case) by including paramagnetic perturbations to NMR resonances. The folded two-domain structure for LAR-Ig1-2 seen in previous crystal structures has been validated in solution using residual dipolar coupling data, and a combination of chemical shift perturbation on titration of LAR-Ig1-2 with fondaparinux, saturation transfer difference (STD) spectra, and transferred nuclear Overhauser effects (trNOEs) have been employed in the docking program HADDOCK to generate models for the LAR-fondaparinux complex. These models are further analyzed by postprocessing energetic analysis to identify key binding interactions. In addition to providing insight into the ligand interaction mechanisms of type IIa RPTPs and the origin of opposing effects of CS and HS ligands, these results may assist in future design of therapeutic compounds for nervous system repair.

Biomechanical insult switches PEA-15 activity to uncouple its anti-apoptotic function and promote erk mediated tissue remodeling.

Biomechanical insult contributes to many chronic pathological processes, yet the resulting influences on signal transduction mechanisms are poorly understood. The retina presents an excellent mechanotransduction model, as mechanical strain on sensitive astrocytes of the optic nerve head (ONH) is intimately linked to chronic tissue remodeling and excavation by matrix metalloproteinases (MMPs), and apoptotic cell death. However, the mechanism by which these effects are induced by biomechanical strain is unclear. We previously identified the small adapter protein, PEA-15 (phosphoprotein enriched in astrocytes), through proteomic analyses of human ONH astrocytes subjected to pathologically relevant biomechanical insult. Under resting conditions PEA-15 is regulated through phosphorylation of two key serine residues to inhibit extrinsic apoptosis and ERK1/2 signaling. However, we surprisingly observed that biomechanical insult dramatically switches PEA-15 phosphorylation and function to uncouple its anti-apoptotic activity, and promote ERK1/2-dependent MMP-2 and MMP-9 secretion. These results reveal a novel cell autonomous mechanism by which biomechanical strain rapidly modifies this signaling pathway to generate altered tissue injury responses.

Relationship between retinal blood flow and arterial oxygen.

KEY POINTS: Vascular reactivity, the response of the vessels to a vasoactive stimulus such as hypoxia and hyperoxia, can be used to assess the vascular range of adjustment in which the vessels are able to compensate for changes in PO2. Previous studies in the retina have not accurately quantified retinal vascular responses and precisely targeted multiple PaO2 stimuli at the same time as controlling the level of carbon dioxide, thus precluding them from modelling the relationship between retinal blood flow and oxygen. The present study modelled the relationship between retinal blood flow and PaO2, showing them to be a combined linear and hyperbolic function. This model demonstrates that the resting tonus of the vessels is at the mid-point and that they have great vascular range of adjustment, compensating for decreases in oxygen above a PETCO2 of 32-37 mmHg but being limited below this threshold. Retinal blood flow (RBF) increases in response to a reduction in oxygen (hypoxia) but decreases in response to increased oxygen (hyperoxia). However, the relationship between blood flow and the arterial partial pressure of oxygen has not been quantified and modelled in the retina, particularly in the vascular reserve and resting tonus of the vessels. The present study aimed to determine the limitations of the retinal vasculature by modelling the relationship between RBF and oxygen. Retinal vascular responses were measured in 13 subjects for eight different blood gas conditions, with the end-tidal partial pressure of oxygen (PETCO2) ranging from 40-500 mmHg. Retinal vascular response measurements were repeated twice; using the Canon laser blood flowmeter (Canon Inc., Tokyo, Japan) during the first visit and using Doppler spectral domain optical coherence tomography during the second visit. We determined that the relationship between RBF and PaO2 can be modelled as a combination of hyperbolic and linear functions. We concluded that RBF compensated for decreases in arterial oxygen content for all stages of hypoxia used in the present study but can no longer compensate below a PETCO2 of 32-37 mmHg. These vessels have a great vascular range of adjustment, increasing diameter (8.5% arteriolar and 21% total venous area) with hypoxia (40 mmHg P ETC O2; P < 0.001) and decreasing diameter (6.9% arteriolar and 23% total venous area) with hyperoxia (500 mmHg PETCO2; P < 0.001) to the same extent. This indicates that the resting tonus is near the mid-point of the adjustment ranges at resting PaO2 where sensitivity is maximum.

MicroRNA-132 is enriched in developing axons, locally regulates Rasa1 mRNA, and promotes axon extension.

Developing axons can locally synthesize proteins, with roles in axon growth, guidance, and regeneration, but the mechanisms that regulate axonal mRNA translation are not well understood. MicroRNAs (miRNAs) are important regulators of translation but have still been little characterized in developing axons. Here we study mouse dorsal root ganglion (DRG) axons and show that their extension is impaired by conditional deficiency of the miRNA-processing enzyme Dicer in vitro and in vivo. A screen for axonal localization identifies a specific set of miRNAs preferentially enriched within the developing axon. High axonal expression and preferential localization were observed for miR-132, a miRNA previously known for roles in dendrites and dysregulation in major neurologic diseases. miR-132 knockdown reduced extension of cultured DRG axons, whereas overexpression increased extension. Mechanistically, miR-132 regulated the mRNA for the Ras GTPase activator Rasa1, a novel target in neuronal function. Moreover, miR-132 regulation of Rasa1 translation was seen in severed axons, demonstrating miRNA function locally within the axon. miR-132 expression in DRGs peaked in the period of maximum axon growth in vivo, consistent with its effect on axon growth, and suggesting a role as a developmental timer. Together, these findings identify miR-132 as a positive regulator of developing axon extension, acting through repression of Rasa1 mRNA, in a mechanism that operates locally within the axon.

PGC-1α signaling coordinates susceptibility to metabolic and oxidative injury in the inner retina.

Retinal ganglion cells (RGCs), used as a common model of central nervous system injury, are particularly vulnerable to metabolic and oxidative damage. However, molecular mechanisms underlying this sensitivity have not been determined in vivo. PGC-1α (encoded by PPARGC1A) regulates adaptive metabolism and oxidative stress responses in a tissue- and cell-specific manner. Aberrant PGC-1α signaling is implicated in neurodegeneration, but the mechanism underlying its role in central nervous system injury remains unclear. We provide evidence from a mouse model that PGC-1α expression and activity are induced in adult retina in response to metabolic and oxidative challenge. Deletion of Ppargc1a dramatically increased RGC loss, in association with dysregulated expression of PGC-1α target metabolic and oxidative stress response genes, including Hmox1 (encoding HO-1), Tfam, and Vegfa. Vehicle-treated and naive Ppargc1a(-/-) mice also showed mild RGC loss, and surprisingly prominent and consistent retinal astrocyte reactivity. These cells critically regulate metabolic homeostasis in the inner retina. We show that PGC-1α signaling (not previously studied in glia) regulates detoxifying astrocyte responses to hypoxic and oxidative stresses. Finally, PGC-1α expression was modulated in the inner retina with age and in a model of chronic optic neuropathy. These data implicate PGC-1α signaling as an important regulator of astrocyte reactivity and RGC homeostasis to coordinate pathogenic susceptibility to metabolic and oxidative injury in the inner retina.

Type III neuregulin 1 regulates pathfinding of sensory axons in the developing spinal cord and periphery.

Sensory axons must develop appropriate connections with both central and peripheral targets. Whereas the peripheral cues have provided a classic model for neuron survival and guidance, less is known about the central cues or the coordination of central and peripheral connectivity. Here we find that type III Nrg1, in addition to its known effect on neuron survival, regulates axon pathfinding. In type III Nrg1(-/-) mice, death of TrkA(+) nociceptive/thermoreceptive neurons was increased, and could be rescued by Bax elimination. In the Bax and type III Nrg1 double mutants, axon pathfinding abnormalities were seen for TrkA(+) neurons both in cutaneous peripheral targets and in spinal cord central targets. Axon guidance phenotypes in the spinal cord included penetration of axons into ventral regions from which they would normally be repelled by Sema3A. Accordingly, sensory neurons from type III Nrg1(-/-) mice were unresponsive to the repellent effects of Sema3A in vitro, which might account, at least in part, for the central projection phenotype, and demonstrates an effect of type III Nrg1 on guidance cue responsiveness in neurons. Moreover, stimulation of type III Nrg1 back-signaling in cultured sensory neurons was found to regulate axonal levels of the Sema3A receptor neuropilin 1. These results reveal a molecular mechanism whereby type III Nrg1 signaling can regulate the responsiveness of neurons to a guidance cue, and show that type III Nrg1 is required for normal sensory neuron survival and axon pathfinding in both central and peripheral targets.

Optical origins of opposing facial expression actions.

Darwin theorized that emotional expressions originated as opposing functional adaptations for the expresser, not as distinct categories of social signals. Given that two thirds of the eye's refractive power comes from the cornea, we examined whether opposing expressive behaviors that widen the eyes (e.g., fear) or narrow the eyes (e.g., disgust) may have served as an optical trade-off, enhancing either sensitivity or acuity, thereby promoting stimulus localization ("where") or stimulus discrimination ("what"), respectively. An optical model based on eye apertures of posed fear and disgust expressions supported this functional trade-off. We then tested the model using standardized optometric measures of sensitivity and acuity. We demonstrated that eye widening enhanced stimulus detection, whereas eye narrowing enhanced discrimination, each at the expense of the other. Opposing expressive actions around the eye may thus reflect origins in an optical principle, shaping visual encoding at its earliest stage-how light is cast onto the retina.

Proteomics analyses of human optic nerve head astrocytes following biomechanical strain.

We investigate the role of glial cell activation in the human optic nerve caused by raised intraocular pressure, and their potential role in the development of glaucomatous optic neuropathy. To do this we present a proteomics study of the response of cultured, optic nerve head astrocytes to biomechanical strain, the magnitude and mode of strain based on previously published quantitative models. In this case, astrocytes were subjected to 3 and 12% stretches for either 2 h or 24 h. Proteomic methods included nano-liquid chromatography, tandem mass spectrometry, and iTRAQ labeling. Using controls for both stretch and time, a six-plex iTRAQ liquid chromatography- tandem MS (LC/MS/MS) experiment yielded 573 proteins discovered at a 95% confidence limit. The pathways included transforming growth factor ß1, tumor necrosis factor, caspase 3, and tumor protein p53, which have all been implicated in the activation of astrocytes and are believed to play a role in the development of glaucomatous optic neuropathy. Confirmation of the iTRAQ analysis was performed by Western blotting of various proteins of interest including ANXA 4, GOLGA2, and αB-Crystallin.