The GTPase Arl8B Plays a Principle Role in the Positioning of Interstitial Axon Branches by Spatially Controlling Autophagosome and Lysosome Location.

Interstitial axon branching is an essential step during the establishment of neuronal connectivity. However, the exact mechanisms on how the number and position of branches are determined are still not fully understood. Here, we investigated the role of Arl8B, an adaptor molecule between lysosomes and kinesins. In chick retinal ganglion cells (RGCs), downregulation of Arl8B reduces axon branch density and shifts their location more proximally, while Arl8B overexpression leads to increased density and more distal positions of branches. These alterations correlate with changes in the location and density of lysosomes and autophagosomes along the axon shaft. Diminishing autophagy directly by knock-down of atg7, a key autophagy gene, reduces branch density, while induction of autophagy by rapamycin increases axon branching, indicating that autophagy plays a prominent role in axon branch formation. In vivo, local inactivation of autophagy in the retina using a mouse conditional knock-out approach disturbs retino-collicular map formation which is dependent on the formation of interstitial axon branches. These data suggest that Arl8B plays a principal role in the positioning of axon branches by spatially controlling autophagy, thus directly controlling formation of neural connectivity in the brain.SIGNIFICANCE STATEMENT The formation of interstitial axonal branches plays a prominent role in numerous places of the developing brain during neural circuit establishment. We show here that the GTPase Arl8B controls density and location of interstitial axon branches, and at the same time controls also density and location of the autophagy machinery. Upregulation or downregulation of autophagy in vitro promotes or inhibits axon branching. Local disruption of autophagy in vivo disturbs retino-collicular mapping. Our data suggest that Arl8B controls axon branching by controlling locally autophagy. This work is one of the first reports showing a role of autophagy during early neural circuit development and suggests that autophagy in general plays a much more prominent role during brain development than previously anticipated.

Glial contribution to cyclodextrin-mediated reversal of cholesterol accumulation in murine NPC1-deficient neurons in vivo.

Niemann-Pick type C disease is a rare and fatal lysosomal storage disorder presenting severe neurovisceral symptoms. Disease-causing mutations in genes encoding either NPC1 or NPC2 protein provoke accumulation of cholesterol and other lipids in specific structures of the endosomal-lysosomal system and degeneration of specific cells, notably neurons in the central nervous system (CNS). 2-hydroxypropyl-beta-cyclodextrin (CD) emerged as potential therapeutic approach based on animal studies and clinical data, but the mechanism of action in neurons has remained unclear. To address this topic in vivo, we took advantage of the retina as highly accessible part of the CNS and intravitreal injections as mode of drug administration. Coupling CD to gold nanoparticles allowed us to trace its intracellular location. We report that CD enters the endosomal-lysosomal system of neurons in vivo and enables the release of lipid-laden lamellar inclusions, which are then removed from the extracellular space by specific types of glial cells. Our data suggest that CD induces a concerted action of neurons and glial cells to restore lipid homeostasis in the central nervous system.

Extracellular phosphorylation drives the formation of neuronal circuitry.

Until recently, the existence of extracellular kinase activity was questioned. Many proteins of the central nervous system are targeted, but it remains unknown whether, or how, extracellular phosphorylation influences brain development. Here we show that the tyrosine kinase vertebrate lonesome kinase (VLK), which is secreted by projecting retinal ganglion cells, phosphorylates the extracellular protein repulsive guidance molecule b (RGMb) in a dorsal-ventral descending gradient. Silencing of VLK or RGMb causes aberrant axonal branching and severe axon misguidance in the chick optic tectum. Mice harboring RGMb with a point mutation in the phosphorylation site also display aberrant axonal pathfinding. Mechanistic analyses show that VLK-mediated RGMb phosphorylation modulates Wnt3a activity by regulating LRP5 protein gradients. Thus, the secretion of VLK by projecting neurons provides crucial signals for the accurate formation of nervous system circuitry. The dramatic effect of VLK on RGMb and Wnt3a signaling implies that extracellular phosphorylation likely has broad and profound effects on brain development, function and disease.

Synthesis of Advanced Mesoporous Materials by Partial Pseudomorphic Transformation.

The structure of porous silica particles can be reorganized without alteration of the particle size and shape by the process of pseudomorphic transformation. Partial pseudomorphic transformation leads to ordered mesoporous silica with bimodal pore size distributions and bottleneck pores. Compared to the classical pathways of mesoporous silica synthesis, pseudomorphic transformation implies less compromise between pore structure and particle shape, while enabling the preparation of complex pore architectures.

Estimating the location and size of retinal injections from orthogonal images of an intact retina.

BACKGROUND: To study the mapping from the retina to the brain, typically a small region of the retina is injected with a dye, which then propagates to the retina's target structures. To determine the location of the injection, usually the retina is dissected out of the eye, flattened and then imaged, causing tears and stretching of the retina. The location of the injection is then estimated from the image of the flattened retina. Here we propose a new method that avoids dissection of the retina. RESULTS: We have developed IntactEye, a software package that uses two orthogonal images of the intact retina to locate focal injections of a dye. The two images are taken while the retina is still inside the eye. This bypasses the dissection step, avoiding unnecessary damage to the retina, and speeds up data acquisition. By using the native spherical coordinates of the eye, we avoid distortions caused by interpreting a curved structure in a flat coordinate system. Our method compares well to the projection method and to the Retistruct package, which both use the flattened retina as a starting point. We have tested the method also on synthetic data, where the injection location is known. Our method has been designed for analysing mouse retinas, where there are no visible landmarks for discerning retinal orientation, but can also be applied to retinas from other species. CONCLUSIONS: IntactEye allows the user to precisely specify the location and size of a retinal injection from two orthogonal images taken of the eye. We are solving the abstract problem of locating a point on a spherical object from two orthogonal images, which might have applications outside the field of neuroscience.

Incorporation of a FRET dye pair into mesoporous materials: a comparison of fluorescence spectra, FRET activity and dye accessibility.

Fluorescein and rhodamine B modified mesoporous silica particles were synthesized by post-grafting and co-condensation approaches. The materials exhibited different pore size distributions, particle shapes and sizes. The materials were characterized by nitrogen sorption, scanning electron microscopy and fluorescence spectroscopy. The Förster resonance energy transfer between the selected dye pair was explored for the different materials by exposure to various concentrations of gaseous ammonia. A logarithmic increase in rhodamine B emission with increasing ammonia concentration was observed for both post-grafted and co-condensed materials. The dye accessibility by ammonia gas in the silica framework of mesoporous materials was evaluated by using a flow cell gas sensor setup built in-house. Response to ammonia gas and recovery with nitrogen gas are explained by comparing the structure properties and dye loading of the materials. The post-grafted dye modified silica showed better performance in terms of reversibility and recovery.

Innovative vision rehabilitation method for hemianopsia: Comparing pre- and post audio-luminous biofeedback training for ocular motility improving visual functions and quality of life.

Background: Homonymous hemianopsia (HH) corresponds to vision loss in one hemi-field secondary to retro-chiasmal injury. Patients with HH experience difficulties in scanning and orientation in their environment. Near vision daily activities such as reading can also be impaired. There is an unmet need for standardized vision rehabilitation protocols for HH. We investigated the effectiveness of biofeedback training (BT), used for vision rehabilitation in patients with central vision loss, in individuals with HH. Methods: In this prospective pilot pre/post study, 12 participants, with HH consecutive to brain injury, performed 5 weekly BT sessions for 20 min each under supervision using the Macular Integrity Assessment microperimeter. BT consisted of relocation of the retinal locus 1-4° toward the blind hemi-field. Outcomes measured post-BT were paracentral retinal sensitivity, visual acuity (near vision), fixation stability, contrast sensitivity, reading speed, and visual functioning questionnaire. Statistical analysis was performed using Bayesian paired t-tests. Results: Paracentral retinal sensitivity significantly increased by 2.7 ± 0.9 dB in the treated eye in 9/11 of the participants. Significant improvements with medium-to-large effect size were observed for fixation stability (8/12 participants), contrast sensitivity (6/12 participants) and near vision visual acuity (10/12 participants). Reading speed increased by 32.5 ± 32.4 words per minute in 10/11 participants. Quality of vision scores improved significantly with large effect size for visual ability, visual information and mobility. Conclusion: BT led to encouraging improvements in visual functions and functional vision in individuals with HH. Further confirmation with larger trials is required.

Genetic dissection of EphA receptor signaling dynamics during retinotopic mapping.

Retinal ganglion cells (RGCs) project axons from their cell bodies in the eye to targets in the superior colliculus of the midbrain. The wiring of these axons to their synaptic targets creates an ordered representation, or "map," of retinal space within the brain. Many lines of experiments have demonstrated that the development of this map requires complementary gradients of EphA receptor tyrosine kinases and their ephrin-A ligands, yet basic features of EphA signaling during mapping remain to be resolved. These include the individual roles played by the multiple EphA receptors that make up the retinal EphA gradient. We have developed a set of ratiometric "relative signaling" (RS) rules that quantitatively predict how the composite low-nasal-to-high-temporal EphA gradient is translated into topographic order among RGCs. A key feature of these rules is that the component receptors of the gradient--in the mouse, EphA4, EphA5, and EphA6--must be functionally equivalent and interchangeable. To test this aspect of the model, we generated compound mutant mice in which the periodicity, slope, and receptor composition of the gradient are systematically altered with respect to the levels of EphA4, EphA5, and a closely related receptor, EphA3, that we ectopically express. Analysis of the retinotopic maps of these new mouse mutants establishes the general utility of the RS rules for predicting retinocollicular topography, and demonstrates that individual EphA gene products are approximately equivalent with respect to axon guidance and target selection.

Multifunctionalized electrospun silk fibers promote axon regeneration in central nervous system.

The repair of central nerves remains a major challenge in regenerative neurobiology. Regenerative guides possessing critical features such as cell adhesion, physical guiding and topical stimulation are needed. To generate such a guide, silk protein materials are prepared using electrospinning. The silk is selected for this study due to its biocompatibility and ability to be electrospun for the formation of aligned biofunctional nanofibers. The addition of Brain Derived Neurotrophic Factor (BDNF), Ciliary Neurotrophic Factor (CNTF) or both to the electrospun fibers enable enhanced function without impact to the structure or the surface morphology. Only a small fraction of the loaded growth factors is released over time allowing the fibers to continue to provide these factors to the cells for extended periods of time. The entrapped factors remain active and available to the cells as rat retinal ganglion cells (RGCs) exhibit longer axonal growth when in contact with the biofunctionalized fibers. Compare to non-functionalized fibers, the growth of neurites increased 2 fold on fibers containing BDNF, 2.5 fold with fibers containing CNTF and by almost 3-fold on fibers containing both factors. The results demonstrate the potential of aligned and functionalized electrospun silk fibers to promote nerve growth in the central nervous system, underlying the great potential of complex biomaterials in neuroregenerative strategies following axotomy and nerve crush traumas.

An Audiovisual 3D-Immersive Stimulation Program in Hemianopia Using a Connected Device.

BACKGROUND Homonymous hemianopia is a loss of conscious vision in one hemifield, strongly affecting everyday life. Audiovisual stimulation programs improve visual perception in the blind hemifield; however, they use large equipment operated in clinical settings. Such treatments require frequent visits at the clinic, hampering the patient's adherence and compliance. In one hemianopia patient, we tested a 4-week dynamic audiovisual rehabilitation program in the stand-alone, remotely controlled, virtual-reality, head-mounted display Oculus Go and measured the effect on visual perception. CASE REPORT A 15-year-old Caucasian male was diagnosed with a right homonymous hemianopia with splitting of central fixation after a traumatic occipital contusion at age 7 months. Visual assessment showed impaired binocular contrast sensitivity and retinal sensitivity. Fixation stability and visual fields were strongly affected. After a 4-week audiovisual rehabilitation program, including 3 hours 20 minutes of stimulation, the contrast sensitivity, fixation stability, and paracentral visual perception were significantly enhanced, improving quality of life. CONCLUSIONS This pioneering work reports the use of virtual-reality in a head-mounted display to provide an audiovisual stimulation protocol for low-vision rehabilitation in a hemianopia patient. Real-time data recording and remote control of the stimulation program demonstrate that such rehabilitation treatment can be performed by the patient at home without interruption of care, decreasing the burden of disease. Beneficial effects on visual function were measured according to clinical guidelines of low-vision assessment. Improvement in visual function and quality of life challenge the prevailing belief that post-acute vision loss is both permanent and unchangeable.

Case Report: Visual Rehabilitation in Hemianopia Patients. Home-Based Visual Rehabilitation in Patients With Hemianopia Consecutive to Brain Tumor Treatment: Feasibility and Potential Effectiveness.

Background/Objectives: Visual field loss is frequent in patients with brain tumors, worsening their daily life and exacerbating the burden of disease, and no supportive care strategies exist. In this case series, we sought to characterize the feasibility and potential effectiveness of a home-based visual rehabilitation program in hemianopia patients using immersive virtual-reality stimulation. Subjects/Methods: Two patients, one with homonymous hemianopia and the other with bitemporal hemianopia, consecutive to pediatric brain tumors, with no prior visual rehabilitation performed 15 min of home-based audiovisual stimulation every 2 days for 6 weeks (case 2) and 7 weeks (case 1) between February and August 2020. Patients used a virtual-reality, stand-alone, and remotely controlled device loaded with a non-commercial audiovisual stimulation program managed in real time from the laboratory. Standard visual outcomes assessed in usual care in visual rehabilitation were measured at the clinic. Following a mixed method approach in this pragmatic study of two cases, we collected quantitative and qualitative data on feasibility and potential effectiveness and compared the results pre- and post-treatment. Results: Implementation and wireless delivery of the audiovisual stimulation, remote data collection, and analysis for cases 1 and 2 who completed 19/20 and 20/20 audiovisual stimulation sessions at home, respectively, altogether indicated feasibility. Contrast sensitivity increased in both eyes for cases 1 and 2. Visual fields, measured by binocular Esterman and monocular Humphrey full-field analyses, improved in case 1. A minor increase was observed in case 2. Cases 1 and 2 enhanced reading speed. Case 2 strongly improved quality of life scores. Conclusion: This is the first report of a home-based virtual-reality visual rehabilitation program for adult patients with hemianopia consecutive to a pediatric brain tumor. We show the feasibility in real-world conditions and potential effectiveness of such technology on visual perception and quality of life.

A relative signalling model for the formation of a topographic neural map.

The highly ordered wiring of retinal ganglion cell (RGC) neurons in the eye to their synaptic targets in the superior colliculus of the midbrain has long served as the dominant experimental system for the analysis of topographic neural maps. Here we describe a quantitative model for the development of one arm of this map--the wiring of the nasal-temporal axis of the retina to the caudal-rostral axis of the superior colliculus. The model is based on RGC-RGC competition that is governed by comparisons of EphA receptor signalling intensity, which are made using ratios of, rather than absolute differences in, EphA signalling between RGCs. Molecular genetic experiments, exploiting a combinatorial series of EphA receptor knock-in and knockout mice, confirm the salient predictions of the model, and show that it both describes and predicts topographic mapping.

New insights on the modeling of the molecular mechanisms underlying neural maps alignment in the midbrain.

We previously identified and modeled a principle of visual map alignment in the midbrain involving the mapping of the retinal projections and concurrent transposition of retinal guidance cues into the superior colliculus providing positional information for the organization of cortical V1 projections onto the retinal map (Savier et al., 2017). This principle relies on mechanisms involving Epha/Efna signaling, correlated neuronal activity and axon competition. Here, using the 3-step map alignment computational model, we predict and validate in vivo the visual mapping defects in a well-characterized mouse model. Our results challenge previous hypotheses and provide an alternative, although complementary, explanation for the phenotype observed. In addition, we propose a new quantification method to assess the degree of alignment and organization between maps, allowing inter-model comparisons. This work generalizes the validity and robustness of the 3-step map alignment algorithm as a predictive tool and confirms the basic mechanisms of visual map organization.

Changes in cholesterol absorption and cholesterol synthesis caused by ezetimibe and/or simvastatin in men.

This study evaluates changes in cholesterol balance in hypercholesterolemic subjects following treatment with an inhibitor of cholesterol absorption or cholesterol synthesis or coadministration of both agents. This was a randomized, double blind, placebo-controlled, four-period crossover study to evaluate the effects of coadministering 10 mg ezetimibe with 20 mg simvastatin (ezetimibe/simvastatin) on cholesterol absorption and synthesis relative to either drug alone or placebo in 41 subjects. Each treatment period lasted 7 weeks. Ezetimibe and ezetimibe/simvastatin decreased fractional cholesterol absorption by 65% and 59%, respectively (P < 0.001 for both relative to placebo). Simvastatin did not significantly affect cholesterol absorption. Ezetimibe and ezetimibe/simvastatin increased fecal sterol excretion (corrected for dietary cholesterol), which also represents net steady state cholesterol synthesis, by 109% and 79%, respectively (P < 0.001). Ezetimibe, simvastatin, and ezetimibe/simvastatin decreased plasma LDL-cholesterol by 20, 38, and 55%, respectively. The coadministered therapy was well tolerated. The decreases in net cholesterol synthesis and increased fecal sterol excretion yielded nearly additive reductions in LDL-cholesterol for the coadministration of ezetimibe and simvastatin.

A molecular mechanism for the topographic alignment of convergent neural maps.

Sensory processing requires proper alignment of neural maps throughout the brain. In the superficial layers of the superior colliculus of the midbrain, converging projections from retinal ganglion cells and neurons in visual cortex must be aligned to form a visuotopic map, but the basic mechanisms mediating this alignment remain elusive. In a new mouse model, ectopic expression of ephrin-A3 (Efna3) in a subset of retinal ganglion cells, quantitatively altering the retinal EFNAs gradient, disrupts cortico-collicular map alignment onto the retino-collicular map, creating a visuotopic mismatch. Genetic inactivation of ectopic EFNA3 restores a wild-type cortico-collicular map. Theoretical analyses using a new mapping algorithm model both map formation and alignment, and recapitulate our experimental observations. The algorithm is based on an initial sensory map, the retino-collicular map, which carries intrinsic topographic information, the retinal EFNAs, to the superior colliculus. These EFNAs subsequently topographically align ingrowing visual cortical axons to the retino-collicular map.

Biologicals dramatic advances in the treatment of psoriasis.

Innovations in biotechnology have made possible the development of several new systemic therapies for psoriasis - the "biologicals", a new group of compounds including monoclonal antibodies, fusion proteins and recombinant proteins. These novel biotechnological advances potentially offer designer drugs, which interfere with specific targets in the pathophysiological network of psoriasis and are thus much safer. The therapeutic strategy of biologicals is based on the knowledge derived from pathogenetic studies, which have focused on targeting disease relevant T-cell- or mediator-systems. Important targets include inactivation of soluble mediators such as tumor-necrosis-factor-alpha, the blockade of receptors for cytokines, adhesion molecules and the interference with T-cell activation by antigen-presenting cells. In addition, recombinant cytokines are able to modulate the immunological balance of this chronic inflammatory skin disease. Currently, up to forty agents are under investigation for the treatment of psoriasis. Four of these agents, alefacept, efalizumab, etanercept and infliximab have already impacted on routine clinical practice. Current developments in the treatment of psoriasis with biologicals are reviewed.

Absorption of riociguat (BAY 63-2521): bioavailability, food effects, and dose proportionality.

Riociguat (BAY 63-2521) is the first member of a novel class of compounds, the soluble guanylate cyclase (sGC) stimulators. Riociguat has a dual mode of action: it sensitizes sGC to endogenous nitric oxide (NO) and stimulates sGC independent of NO availability. To characterize the biopharmaceutical properties of riociguat, including absolute bioavailability, food interactions, and dose proportionality, riociguat (intravenous/oral) was administered to healthy male subjects in 3 open-label, randomized, crossover studies: absolute bioavailability (1 mg; [Formula: see text]), food effect (2.5 mg; [Formula: see text]), and dose proportionality (0.5-2.5 mg; [Formula: see text]). Absolute bioavailability was 94% (95% confidence interval [CI], 83%-107%). Riociguat absorption was delayed by a high-fat breakfast with little effect on the extent of absorption (area under the concentration-time curve [AUC]fed∶AUCfasted, 88% [90% CI, 82%-95%]). Exposure to riociguat was dose proportional over all doses (common slope of AUC, 1.09 [90% CI, 1.04-1.14]; maximum concentration, 0.98 [90% CI, 0.93-1.04]). Intraindividual variability was low; interindividual variability was moderate to high. Riociguat was well tolerated, and adverse events were consistent with the mode of action. In conclusion, riociguat shows complete oral absorption, no clinically relevant food effects, and a dose-proportional increase in systemic exposure (0.5-2.5 mg). These data support the suitability of the individualized dose adjustment scheme employed in the phase 3 clinical studies.

Riociguat (BAY 63-2521) and aspirin: a randomized, pharmacodynamic, and pharmacokinetic interaction study.

In preclinical studies, drugs that increase cyclic guanosine monophosphate levels have been shown to influence platelet function/aggregation; however, the effect of riociguat on human platelets is unclear. Aspirin, a platelet inhibitor, is likely to be given concomitantly in patients receiving riociguat. It is therefore important to establish clinically whether (1) riociguat affects platelet function and (2) aspirin and riociguat interact. This randomized, open-label, crossover study investigated potential pharmacodynamic and pharmacokinetic interactions between these drugs in healthy male volunteers (N = 18). There were 3 treatment regimens: a single morning dose of riociguat 2.5 mg, aspirin 500 mg on 2 consecutive mornings, and both treatments together, with riociguat given on the second morning. Fifteen participants were available for pharmacodynamic/pharmacokinetic analysis. There was no effect of riociguat alone on bleeding time, platelet aggregation, and serum thromboxane B2 levels. The effects of aspirin on these parameters were not influenced by concomitant administration of riociguat. The pharmacokinetic profile of riociguat showed interindividual variability, which was independent of aspirin coadministration. Six of 17 participants available for safety evaluation reported at least 1 treatment-emergent adverse event. All adverse events were of mild severity, apart from 1 report of moderate headache. No serious adverse events occurred. In conclusion, riociguat demonstrated no clinically relevant pharmacodynamic or pharmacokinetic interactions with aspirin at the doses used in this study in healthy men; coadministration of riociguat and aspirin should therefore not require any dose adjustment for either drug.