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Transcript of 29082012
Omega 3 FA with 22 carbon chains
Most abundant PUFA in photoreceptors
Maintain fluidity and optimal environment The Effects of Docosahexaenoic Acid (DHA) Supplementation on Protein Expression in a Mouse Model of Stargardt-like Macular Dystrophy (STGD3) Mandy Hong
Center for Neuroscience
August 29, 2012 What problem are we addressing? Introduction How can we address it? Questions? Hypothesis Function Anatomy Protein Expression Lipid Profile Results Conclusions What problem are we addressing? Age-related Macular Degeneration Progressive, degenerative disease affecting central vision
Age of onset around 60 years
Over 2 million Canadians affected How can we address this problem? AMD is multifactorial
Genetics Stargardt-like Macular Dystrophy (STGD3) Follows a similar clinical profile as dry AMD
Decrease in visual acuity
Central retina atrophy
early onset of disease
ELOVL4 gene Impaired elongation of very long chain fatty acids Loss of endoplasmic retention signal Disrupt photoreceptor function Aggregate formation of mislocalized ELOVL4 protein
Saturation of proteasomes and iniation of UPR
Disruption of cellular function and apoptosis Transgenic mice expressing the human ELOVL4 gene demonstrate similar disease progression as in human STGD3 pathology and dry AMD
Lipofuscin accumulation in RPE
Death of rods
Death of cones How can we address this problem? Dietary Intervention Docosahexaenoic Acid Literary Support How does DHA exert its therapeutic effects in the context of retinal degeneration? Function Protein Expression Function Positive correlation between DHA intake and retinal function Protein Expression Down-regulation in proteins involved in cell-mediated response, inflammation and neurotropic factors Experimental Design Experimental Design 6 experimental groups Genotype: WT and TG
Diet: chow, DHA-, DHA+
Groups: WTchow, TGchow,WT-, TG-, WT+, and TG+ Timeline E0 P15 P21 P30 P60 P90 Dietary Intervention ERG ERG PR counts Protein expression FA profile Onset of rod loss Wean Eyes open Function - Electroretinography (ERG) Animals are dark adapted for 1 h and anaesthetized using ketamine: xylazine mixture. reference ground recording Various intensities of light is presented to the animal using the colour dome to elicit electrical activity from retinal nerve cells.
ERG waveforms are acquired for both scotopic and photopic conditions. Anatomy - Photoreceptor Counts 2 cross sections per animal are imaged from the center, middle, and periphery of the retina.
Counts are made using ImageJ analysis and averages are made to produce a single number. Enucleated eyes are immersed in 4% PFA after lens are removed.
Remaining eyecup is subjected to sucrose gradient and embedded in resin, flash frozen and stored at -80 oC.
The tissue is cut at 20um and mounted on slides with DAPI. Protein Expression - Western blotting Elucidate specific targets and pathways through which DHA supplementation affects retinal degeneration Many proteins found in drusens are associated with the complement system
of these proteins, several are modified by adducts produced by oxidative damage Hollyfield et al (2010) Antibody availability and viability?? FA profile - Gas Chromatography Lipids are extracted from retinas and phospholipids separated on Analtech silica-gel G thin-layer chromatography plates.
Fatty acid methyl esters are separated by automated gas-liquid chromatography.
These analytical conditions separate all saturated, mono-, di- and polyunsaturated fatty acids from C12 to C36 in chain length. Function Anatomy Function - Scotopic Intensity Response Function - Photopic Intensity Response Function - Flicker Response 1 month 3 month Anatomy - Photoreceptor Counts Center Protein Expression and Fatty Acid Profile Western-blotting have not been performed
currently looking for proteins of interest Lipid extraction from retinas have been performed
awaiting data from Winnipeg to be analyzed PIR SIR Flicker PR counts Scotopic Intensity Response Reduction in wave amplitudes for TG animals across all diets
effect conserved from 1 month
earliest detection Distinction between genotypes more defined in a-wave amplitude compared to b-wave.
photoreceptor injury more detrimental than bipolar cells
reduction in a-wave may contribute to smaller b-waves DHA+ groups produced the largest amplitudes within their own genotypes
improvements in membrane fluidity and signal transduction No main effects observed for implicit time
effect of genotype and diet occurs at a larger scale than at the biochemical level Photopic Intensity Response No distinctions or trends that define genotype or diet
changes in rods occur before cones
confirmation of earlier findings Flicker Response 1 m 3 m Changes occur between age groups, but not between genotype or diet
reduced efficiency in visual cycle recovery
DHA not involved in this specific process Photoreceptor Counts Significantly reduced numbers in TG animals on DHA+ diet
DHA may have a negative effect on degenerating retinas HOW?? Carboxyethylpyrrole Summary of effects of DHA scotopic wave amplitudes
no effect on flicker fusion
cell numbers photoreceptor function
??? Implications Results What is CEP? implicit time A protein adduct that is generated from oxidation of DHA
Associated with oxidative stress in AMD patients Increased DHA and oxidative products, CEP, contribute to retinal degeneration... But how does it improve function as well?? Fat-1 mice is a transgenic model that converts omega 6 into 3 FA DHA levels
large wave amplitudes
CEP and GFAP Increased wave amplitudes may be due to altered FA content in photoreceptors and contribute to increased resistance. V=IR Summary of effects of DHA scotopic wave amplitudes
no effect on flicker fusion
cell numbers photoreceptor integrity
??? Implications Results resistance oxidative stress DHA supplementation? DHA intake should be monitored. Imbalance of omega-6/ omega-3 in Western diets. Reduce omega-6 intake
and maintain healthy intake of DHA