Complement Expression in the Retina is not Influenced by Short-term Pressure Elevation

Purpose: To determine whether short-term pressure elevation affects complement gene expression in the retina in vitro and in vivo. Methods: Muller cell (TR-MUL5) cultures and organotypic retinal cultures from adult mice and monkeys were sub- jected to either 24-h or 72-h of pressure at 0, 15, 30, and 45 mmHg above ambient. C57BL/6 mice were subjected to microbead-induced intraocular pressure (IOP) elevation for 7 days. RNA and protein were extracted and used for analysis of expression levels of complement component genes and complement component 1, q subcomponent (C1q) and comple- ment factor H (CFH) immunoblotting. Results: mRNA levels of complement genes and C1q protein levels in Muller cell cultures remained the same for all pressure levels after exposure for either 24 or 72 h. In primate and murine organotypic cultures, pressure elevation did not produce changes in complement gene expression or C1q and CFH protein levels at either the 24-h or 72-h time points. Pressure-related glial fibrillary acidic protein (GFAP) mRNA expression changes were detected in primate retinal organotypic cultures (analysis of variance [ANOVA]; p0.05 for both) with contralateral control and naïve control eyes. Conclusions: Short-term elevation of pressure in vitro as well as short-term (1 week) IOP elevation in vivo does not seem to dramatically alter complement system gene expression in the retina. Prolonged expression to elevated pressure may be necessary to affect the complement system expression

Single-cell profiling reveals an endothelium-mediated immunomodulatory pathway in the eye choroid

The activity and survival of retinal photoreceptors depend on support functions performed by the retinal pigment epithelium (RPE) and on oxygen and nutrients delivered by blood vessels in the underlying choroid. By combining single-cell and bulk RNA sequencing, we categorized mouse RPE/choroid cell types and characterized the tissue-specific transcriptomic features of choroidal endothelial cells. We found that choroidal endothelium adjacent to the RPE expresses high levels of Indian Hedgehog and identified its downstream target as stromal GLI1+ mesenchymal stem cell-like cells. In vivo genetic impairment of Hedgehog signaling induced significant loss of choroidal mast cells, as well as an altered inflammatory response and exacerbated visual function defects after retinal damage. Our studies reveal the cellular and molecular landscape of adult RPE/choroid and uncover a Hedgehog-regulated choroidal immunomodulatory signaling circuit. These results open new avenues for the study and treatment of retinal vascular diseases and choroid-related inflammatory blinding disorders.Funding for this study was provided by National Institutes of Health grants EY08538 and GM34107 (E. Rodriguez-Boulan); EY027038 (R.F. Mullins); 1R21CA224391-01A1 (J.H. Zippin); and 1R01CA194547, 1U24CA210989, and P50CA211024 (O. Elemento); National Cancer Institute grant R01CA192176 and cancer center support grant P30 CA008748-48 (A.L. Joyner); Comunidad Autónoma de Madrid grant 2017-T1/BMD-5247 (I. Benedicto); Agencia Nacional Argentina de Promoción Cient´ıfica y Tecnológica grant PICT 2014-3687 and Fundación Sales (G.A. Rabinovich); a Pew Latin American Fellowship (G.L. Lehmann); Calder Research Scholar Award Vitiligo/Pigment Cell Disorders (J.H. Zippin); Starr Foundation Tri-Institutional Stem Cell Initiative award 2013-028; NYSTEM contract C32596GG; and Research to Prevent Blindness and Dyson Foundation departmental grants. The CNIC is supported by the Instituto de Salud Carlos III, the Ministerio de Ciencia e Innovación, and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S

Κυττοαρχιτεκτονική οργάνωση της οπτικής καλύπτρας πτηνού βάση της έκφρασης της συνθάσης του μονοξειδίου του αζώτου κατά την ανάπτυξη και μετά τη διατομή του οπτικού νεύρου

This study aimed to define the plasticity induced changes to the cytoarchitectonic organisation of the primary visual areas following monocular deprivation, to developing birds. Specifically, the result of early axotomy of the optic nerve on the expression of Nitric Oxide Synthase (NOS) by the cells of the avian optic tectum was studied. This agent (NO) has been the object of intensive research in the plasticity field since its expression has been shown to be subject to modulation as a result of trauma to the nervous system. The localisation of NOS was performed using the NADPH-diaphorase histochemical process. The evaluation of the results was performed by stereological analysis of the sectioned tissue, using the optical disector method. Following the axotomy of the optic nerve, an increase of the number of NOS expressing neurons was detected at the visually deprived tectum. The increase appears first at the dorso caudal part of the tectum and proceeds towards the rostro ventral. An increase in the number of positive cells, having the opposite direction, appears also to the side ipsilateral to the axotomy. The morphological analysis of the NOS positive cells further supports the above stated. These results suggest a significant role for NO in the shaping of the normal cytoarchitecture of the avian optic tectum. Even more significant is its role to the reorganisation, of the mono- and polysynaptic paths, that takes place during placticity procedures. These procedures aim in the replacement of the visual abilities of the organism as well as the utilisation of the sensory deprived tectum.Η εργασία αυτή έγινε με σκοπό την μελέτη των πλαστικών αλλαγών στην κυττοαρχιτεκτονική οργάνωση, των πρωτογενών σταθμών οπτικής πληροφορίας, μετά από μονόφθαλμη στέρηση, σε νεαρά ζώα. Ειδικότερα, μελετήθηκαν οι επιδράσεις που έχει η διατομή του οπτικού νεύρου, στην έκφραση της συνθάσης της ελεύθερης ρίζας του μονοξειδίου του αζώτου (ΝΟ) στα κύτταρα της οπτικής καλύπτρας πτηνού. Το μόριο αυτό έχει γίνει αντικείμενο εντατικής έρευνας στον τομέα της πλαστικότητας, αφού έχει δειχθεί πως η έκφρασή του υπόκειται σε σημαντικές αλλαγές σαν αποτέλεσμα τραυματισμών στο νευρικό σύστημα. Ο εντοπισμός του ενζύμου της συνθάσης του ΝΟ (NOS) στα κύτταρα έγινε με την χρήση ιστοχημείας NADPH-διαφοράσης. Για την εκτίμηση των αποτελεσμάτων επιλέχθηκε η στερεολογική ανάλυση του διατμημένου ιστού με την μέθοδο του disector. Μετά από την διατομή του οπτικού νεύρου παρατηρήθηκε αύξηση των κυττάρων που εκφράζουν NOS στην οπτικά στερημένη πλευρά της καλύπτρας. Η αύξηση αυτή εμφανίζεται από το οπίσθιο ραχιαίο τμήμα της καλύπτρας και εξαπλώνεται προς το εμπρόσθιο κοιλιακό. Στην ομόπλευρη προς την διατομή πλευρά εμφανίζεται αύξηση του αριθμού των θετικών κυττάρων με την αντίθετη όμως κατεύθυνση. Η ανάλυση της μορφολογίας των NOS θετικών κυττάρων συνηγορεί στα παραπάνω. Τα αποτελέσματα συνηγορούν στον σημαντικό ρόλο του NO στην διαμόρφωση της κανονικής κυττοαρχιτεκτονικής της οπτικής καλύπτρας των πτηνών. Ακόμη πιο σημαντικός φαίνεται να είναι ο ρόλος της στην αναδιοργάνωση των μονοσυναπτικών και πολυσυναπτικών μονοπατιών που λαμβάνει χώρα κατά τις διαδικασίες πλαστικότητας. Η διαδικασία αυτή σκοπό έχει την όσο το δυνατό, αναπλήρωση των οπτικών δυνατοτήτων του οργανισμού καθώς και την αξιοποίηση του αισθητικά στερημένου τμήματος της καλύπτρα

Κυττοαρχιτεκτονική οργάνωση της οπτικής καλύπτρας πτηνού βάση της έκφρασης της συνθάσης του μονοξειδίου του αζώτου κατά την ανάπτυξη και μετά τη διατομή του οπτικού νεύρου

This study aimed to define the plasticity induced changes to the cytoarchitectonic organisation of the primary visual areas following monocular deprivation, to developing birds. Specifically, the result of early axotomy of the optic nerve on the expression of Nitric Oxide Synthase (NOS) by the cells of the avian optic tectum was studied. This agent (NO) has been the object of intensive research in the plasticity field since its expression has been shown to be subject to modulation as a result of trauma to the nervous system. The localisation of NOS was performed using the NADPH-diaphorase histochemical process. The evaluation of the results was performed by stereological analysis of the sectioned tissue, using the optical disector method. Following the axotomy of the optic nerve, an increase of the number of NOS expressing neurons was detected at the visually deprived tectum. The increase appears first at the dorso caudal part of the tectum and proceeds towards the rostro ventral. An increase in the number of positive cells, having the opposite direction, appears also to the side ipsilateral to the axotomy. The morphological analysis of the NOS positive cells further supports the above stated. These results suggest a significant role for NO in the shaping of the normal cytoarchitecture of the avian optic tectum. Even more significant is its role to the reorganisation, of the mono- and polysynaptic paths, that takes place during placticity procedures. These procedures aim in the replacement of the visual abilities of the organism as well as the utilisation of the sensory deprived tectum.Η εργασία αυτή έγινε με σκοπό την μελέτη των πλαστικών αλλαγών στην κυττοαρχιτεκτονική οργάνωση, των πρωτογενών σταθμών οπτικής πληροφορίας, μετά από μονόφθαλμη στέρηση, σε νεαρά ζώα. Ειδικότερα, μελετήθηκαν οι επιδράσεις που έχει η διατομή του οπτικού νεύρου, στην έκφραση της συνθάσης της ελεύθερης ρίζας του μονοξειδίου του αζώτου (ΝΟ) στα κύτταρα της οπτικής καλύπτρας πτηνού. Το μόριο αυτό έχει γίνει αντικείμενο εντατικής έρευνας στον τομέα της πλαστικότητας, αφού έχει δειχθεί πως η έκφρασή του υπόκειται σε σημαντικές αλλαγές σαν αποτέλεσμα τραυματισμών στο νευρικό σύστημα. Ο εντοπισμός του ενζύμου της συνθάσης του ΝΟ (NOS) στα κύτταρα έγινε με την χρήση ιστοχημείας NADPH-διαφοράσης. Για την εκτίμηση των αποτελεσμάτων επιλέχθηκε η στερεολογική ανάλυση του διατμημένου ιστού με την μέθοδο του disector. Μετά από την διατομή του οπτικού νεύρου παρατηρήθηκε αύξηση των κυττάρων που εκφράζουν NOS στην οπτικά στερημένη πλευρά της καλύπτρας. Η αύξηση αυτή εμφανίζεται από το οπίσθιο ραχιαίο τμήμα της καλύπτρας και εξαπλώνεται προς το εμπρόσθιο κοιλιακό. Στην ομόπλευρη προς την διατομή πλευρά εμφανίζεται αύξηση του αριθμού των θετικών κυττάρων με την αντίθετη όμως κατεύθυνση. Η ανάλυση της μορφολογίας των NOS θετικών κυττάρων συνηγορεί στα παραπάνω. Τα αποτελέσματα συνηγορούν στον σημαντικό ρόλο του NO στην διαμόρφωση της κανονικής κυττοαρχιτεκτονικής της οπτικής καλύπτρας των πτηνών. Ακόμη πιο σημαντικός φαίνεται να είναι ο ρόλος της στην αναδιοργάνωση των μονοσυναπτικών και πολυσυναπτικών μονοπατιών που λαμβάνει χώρα κατά τις διαδικασίες πλαστικότητας. Η διαδικασία αυτή σκοπό έχει την όσο το δυνατό, αναπλήρωση των οπτικών δυνατοτήτων του οργανισμού καθώς και την αξιοποίηση του αισθητικά στερημένου τμήματος της καλύπτρα

Marfan syndrome caused by a novel FBN1

Mutations in fibrillin-1 (FBN1) cause a wide spectrum of disorders, including Marfan syndrome, which have in common defects in fibrillin-1 microfibrils. Ectopia lentis and myopia are frequently observed ocular manifestations of Marfan syndrome. Glaucoma is also associated with Marfan syndrome, though the form of glaucoma has not been well-characterized. In this report, ocular examination of a patient diagnosed with Marfan syndrome based on family history and aortic dilatation was performed, including measurement of facility of aqueous humor outflow by tonography. The patient did not have ectopia lentis at the age of 42 years. Based on optic nerve appearance, reduced outflow facility, elevated IOP with open angles and clear signs of pigment dispersion, the patient was diagnosed with pigmentary glaucoma. The patient was heterozygous for a novel truncating mutation in FBN1, p.Leu72Ter. Histology of normal human eyes revealed abundant expression of elastic fibers and fibrillin-1 in aqueous humor outflow structures. This is the first report of a patient with Marfan syndrome that is caused by a confirmed FBN1 mutation with associated pigmentary glaucoma. In addition to identifying a novel mutation of FBN1 and broadening the spectrum of associated ocular phenotypes in Marfan syndrome, our findings suggest that pigmentary glaucoma may involve defects in fibrillin-1 microfibrils. © 2013 Wiley Periodicals, Inc

Gene Expression Changes in Areas of Focal Loss of Retinal Ganglion Cells in the Retina of DBA/2J Mice

The present paper reports findings from microarray experiments on gene expression changes in areas of focal loss of retinal ganglion cells in the DBA/2 mouse model of glaucoma. The results were further confirmed by RT-PCR and immunoblot analysis and highlight functional gene networks that are involved in the spontaneous development of glaucoma in the DBA/2 mouse

A cellular and molecular spatial atlas of dystrophic muscle.

Asynchronous skeletal muscle degeneration/regeneration is a hallmark feature of Duchenne muscular dystrophy (DMD); however, traditional -omics technologies that lack spatial context make it difficult to study the biological mechanisms of how asynchronous regeneration contributes to disease progression. Here, using the severely dystrophic D2-mdx mouse model, we generated a high-resolution cellular and molecular spatial atlas of dystrophic muscle by integrating spatial transcriptomics and single-cell RNAseq datasets. Unbiased clustering revealed nonuniform distribution of unique cell populations throughout D2-mdx muscle that were associated with multiple regenerative timepoints, demonstrating that this model faithfully recapitulates the asynchronous regeneration observed in human DMD muscle. By probing spatiotemporal gene expression signatures, we found that propagation of inflammatory and fibrotic signals from locally damaged areas contributes to widespread pathology and that querying expression signatures within discrete microenvironments can identify targetable pathways for DMD therapy. Overall, this spatial atlas of dystrophic muscle provides a valuable resource for studying DMD disease biology and therapeutic target discovery

Germline Mutations in CIDEB and Protection against Liver Disease

BACKGROUND Exome sequencing in hundreds of thousands of persons may enable the identification of rare protein-coding genetic variants associated with protection from human diseases like liver cirrhosis, providing a strategy for the discovery of new therapeutic targets. METHODS We performed a multistage exome sequencing and genetic association analysis to identify genes in which rare protein-coding variants were associated with liver phenotypes. We conducted in vitro experiments to further characterize associations. RESULTS The multistage analysis involved 542,904 persons with available data on liver aminotransferase levels, 24,944 patients with various types of liver disease, and 490,636 controls without liver disease. We found that rare coding variants in APOB, ABCB4, SLC30A10, and TM6SF2 were associated with increased aminotransferase levels and an increased risk of liver disease. We also found that variants in CIDEB, which encodes a structural protein found in hepatic lipid droplets, had a protective effect. The burden of rare predicted loss-of-function variants plus missense variants in CIDEB (combined carrier frequency, 0.7%) was associated with decreased alanine aminotransferase levels (beta per allele, -1.24 U per liter; 95% confidence interval [CI], -1.66 to -0.83; P=4.8×10-9) and with 33% lower odds of liver disease of any cause (odds ratio per allele, 0.67; 95% CI, 0.57 to 0.79; P=9.9×10-7). Rare coding variants in CIDEB were associated with a decreased risk of liver disease across different underlying causes and different degrees of severity, including cirrhosis of any cause (odds ratio per allele, 0.50; 95% CI, 0.36 to 0.70). Among 3599 patients who had undergone bariatric surgery, rare coding variants in CIDEB were associated with a decreased nonalcoholic fatty liver disease activity score (beta per allele in score units, -0.98; 95% CI, -1.54 to -0.41 [scores range from 0 to 8, with higher scores indicating more severe disease]). In human hepatoma cell lines challenged with oleate, CIDEB small interfering RNA knockdown prevented the buildup of large lipid droplets. CONCLUSIONS Rare germline mutations in CIDEB conferred substantial protection from liver disease