RNA-binding protein Hermes/RBPMS inversely affects synapse density and axon arbor formation in retinal ganglion cells in vivo.
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ABSTRACT:
SUBMITTER: Hornberg H
PROVIDER: S-EPMC4603358 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Primary open-angle glaucoma (POAG) is one of the three principal subtypes of glaucoma and among the leading cause of blindness worldwide. POAG is defined by cell death of the retinal ganglion cells (RGCs) and surrounding neuronal cells at higher or normal intraocular pressure (IOP). Coded by one of the three genes responsible for POAG, WD repeat-containing protein 36 (WDR36) has two domains with a similar folding. To address whether WDR36 is functionally important in the retina, we developed four transgenic mice strains overexpressing a wild-type (Wt) and three mutant variants of D606G, deletion of amino acids at positions 605-607 (Del605-607) and at 601-640 (Del601-640) equivalent to the location of the D658G mutation observed in POAG patients. A triple amino acid deletion of mouse Wdr36 at positions 605-607 corresponding to the deletion at positions 657-659 in humans developed progressive retinal degeneration at the peripheral retina with normal IOP. RGCs and connecting amacrine cell synapses were affected at the peripheral retina. Axon outgrowth rate of cultured RGC directly isolated from transgenic animal was significantly reduced by the Wdr36 mutation compared with Wt. Molecular modeling of wild and mutant mouse Wdr36 revealed that deletion at positions 605-607 removed three residues and a hydrogen bond, required to stabilize anti-parallel ?-sheet of the 6th ?-propeller in the second domain. We concluded that WDR36 plays an important functional role in the retina homeostasis and mutation to this gene can cause devastating retinal damage. These data will improve understanding of the functional property of WDR36 in the retina and provide a new animal model for glaucoma therapeutics.
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Project description:PurposeThe ability to accurately quantify immunohistochemically labeled retinal ganglion cells (RGCs) on wholemounts is an important histopathological determinant in experimental retinal research. Traditionally, this has been performed by manual or semi-automated counting of RGCs. Here, we describe an automated software that accurately and efficiently counts immunolabeled RGCs with the ability to batch process images and perform whole-retinal analysis to permit isodensity map generation.MethodsRetinal wholemounts from control rat eyes, and eyes subjected to either chronic ocular hypertension or N-methyl-D-aspartate (NMDA)-induced excitotoxicity, were labeled by immunohistochemistry for two different RGC-specific markers, Brn3a and RNA-binding protein with multiple splicing (RBPMS). For feasibility of manual counting, images were sampled from predefined retinal sectors, totaling 160 images for Brn3a and 144 images for RBPMS. The automated program was initially calibrated for each antibody prior to batch analysis to ensure adequate cell capture. Blinded manual RGC counts were performed by three independent observers.ResultsThe automated counts of RGCs labeled for Brn3a and RBPMS closely matched manual counts. The automated script accurately quantified both physiological and damaged retinas. Efficiency in counting labeled RGC wholemount images is accelerated 40-fold with the automated software. Whole-retinal analysis was demonstrated with integrated retinal isodensity map generation.ConclusionsThis automated cell counting software dramatically accelerates data acquisition while maintaining accurate RGC counts across different immunolabels, methods of injury, and spatial heterogeneity of RGC loss. This software likely has potential for wider application.Translational relevanceThis study provides a valuable tool for preclinical RGC neuroprotection studies that facilitates the translation of neuroprotection to the clinic.
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