Biochemistry/Molecular Biology - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyReserve University, Cleveland, OH; 2 Ophthalmology and VisualSciences, Case Western Reserve University, Cleveland, OH.Purpose: It is impossible to overstate the ubiquity of miRNAmediatedgene regulation in the biology of plants and animals.miRNAs have been implicated in virtually every physiologicalprocess and disease pathology in which they have been studied.Despite this widespread functional relevance, surprisingly little isknown about the physiological impact of miRNAs in the retina.Inactivation of the miRNA processing enzyme, Dicer, in thedeveloping retina leads to defects in cell fate determination andsurvival of progenitors, as well as progressive retinal degeneration.However, it’s unclear which retinal cell types require miRNA generegulation for survival and function or whether miRNAs are requiredfor survival or function of the mature retina. The aim of this study isto address the impact of loss of miRNA gene regulation in mature rodphotoreceptors.Methods: To address these questions we have developed a rodspecificconditional Dicer knockout mouse model. We have analyzedthe morphology of mutant mice using light microscopy,immunohistochemistry, and electron microscopy. We are alsoanalyzing the function of the visual system using electroretinogram,single cell electrophysiological approaches, and retinoid analysis.Results: These animals develop progressive retinal degeneration,beginning with disorganization of rod outer segments around andculminating with degeneration of nearly all photoreceptors. ERGanalysis shows progressive loss of rod function, followed by loss ofcone-driven visual responses. Electrophysiological and highresolution imaging studies are underway to gain insight into themechanism of photoreceptor death in these mice.Conclusions: The observed retinal degeneration demonstrates thatmiRNAs are essential for the survival and function of mature rodphotoreceptors. In addition, these results suggest that miRNAs otherthan the photoreceptor specific miR-183/96/182 cluster are essentialfor rod survival and vision.Commercial Relationships: Thomas Sundermeier, None; NingZhang, None; Debarshi Mustafi, None; Hideo Kohno, None;Krzysztof Palczewski, QLT Inc (F), Polgenix Inc (E), Visum Inc(P), Amegen Inc (F)Support: NIH Fellowship 1F32EY022830Program Number: 6114 Poster Board Number: B0185Presentation Time: 10:30 AM - 12:15 PMTransient Upregulation and then Downregulation of BACE 1 andPS-1 in ER Stress Induced Apoptosis of Retinal Ganglion Cells invitroBingqian Liu, Jian Ge. State Key Laboratory of Ophthalmology,Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou,China.Purpose: To investigate the level of beta-secretase 1 (BACE-1),presenilin-1 (PS-1) and full length amyloid precursor protein (APP)in Endoplasmic Reticulum (ER) stress induced retinal ganglion cellapoptosis in vitro.Methods: Tunicamycin (Tm, 2μg/ml) were added for 48 hours toinduce ER stress in retinal ganglion cells (RGC-5 cell line) culturedin Dulbecco’s modified Eagle’s medium supplemented with 10%fetal bovine serum. Protein levels of 78 kDa glucose-regulatedprotein (GRP-78), full length APP, BACE1, PS-1, and cleavedcaspase 3 were examined by western blot analysis.Results: ER stress marker GRP-78 was increased in RGC-5 cellstreated with Tm in a time-dependent manner. Protein level of fulllength APP was decreased steadily after Tm treatment. BACE-1, andPS-1 were both increased significantly at 2 hour time point in Tmgroup compared to vehicle control. Then, BACE-1 decreased rapidly,while PS-1 decreased slowly, to an undetectable level. Cleavedcaspase-3 was increased significantly under ER stress.Conclusions: BACE-1 and PS-1 were initially increased and thendecreased in ER stress induced apoptosis of retinal ganglion cells invitro. Full length APP might be cleaved by the transient upregulationof BACE-1 and PS-1. Further study is needed to determine whetherthere is a negative feedback between the production of amyloid betaand the BACE-1 and PS-1 activities in this process.Commercial Relationships: Bingqian Liu, None; Jian Ge, NoneSupport: National Natural Science Foundation of China 81000376.Program Number: 6115 Poster Board Number: B0186Presentation Time: 10:30 AM - 12:15 PMProgranulin Mutant Mice Exhibit Accumulation ofIntraneuronal Lipofuscin Aggregates in the RetinaBrian P. Hafler 1 , Zoe A. Klein 2 , Stephen Strittmatter 2 .1 Ophthalmology and Visual Science, Yale School of Medicine, NewHaven, CT; 2 Program in Cellular Neuroscience, Neurodegeneration,and Repair, Yale School of Medicine, New Haven, CT.Purpose: Patients lacking progranulin develop neuronal ceroidlipofuscinosis, a neurodegenerative lysosomal disorder characterizedby abnormal lipofuscin accumulation. Previous investigations haveshown that mice lacking progranulin, a secreted glycoproteinencoded by the granulin (GRN) gene, develop abnormalintraneuronal lipofuscin accumulation in the brain. To determine ifprogranulin mutant mice have a similar phenotype in the retina asobserved in humans with mutations in the progranulin gene, GRN-/-retinas were examined for lipofuscin accumulation.Methods: In order to address whether the age-accelerated lipofuscinaccumulation in the brains of GRN /- mice was present in the retina,we examined the retinas of progranulin deficient mice. Thegeneration of GRN-/- mice has been described previously (Kayasugaet al., 1997). GRN -/- genotype was detected using PCR with primersdirected against GRN and western blot for progranulin protein. GRN-/- mice were examined for lipofuscin using confocal microscopyautofluorescence.Results: As expected, GRN-/- mice lacked progranulin compared tocontrols using western blot. Given than humans deficient forprogranulin develop retinal dystrophy, GRN-/- mice were examinedfor lipofuscin in 12 month old mice. Retinas from GRN-/- mice wereexamined using autofluorescence, and interestingly, there wasintracellular accumulation of highly autofluorescent lipofuscinmaterial in neurons as compared to GRN+/+ mice.Conclusions: These data indicate that GRN-/- mice exhibit anaccumulation of intraneuronal autofluorescent lipofuscin aggregates.As humans with the neurodegenerative disease neuronal ceroidlipofuscinosis linked to progranulin gene mutations on chromosome17 have excessive accumulation of lipofuscin in the retina, this studyis, to our knowledge, is the first example demonstrating a similarretinal phenotype of GRN-/- mice providing a new model toinvestigate the retinal pathology found in this disorder.Commercial Relationships: Brian P. Hafler, None; Zoe A. Klein,None; Stephen Strittmatter, NoneSupport: Department of Ophthalmology and Visual Science, YaleSchool of Medicine, and Kavli Institute for Neuroscience at YaleUniversityProgram Number: 6116 Poster Board Number: B0187Presentation Time: 10:30 AM - 12:15 PMC1qa deficiency exacerbates RGC loss in a mouse glaucomamodel©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.