1. Molecular organization of phosphatidylcholine (PC) molecular species containing very long chain polyunsaturated fatty acids (VLC-PUFAs) in the human retina.
By: Jarrod C. Harman, Bokkyoo Jun, William C. Gordon, Nicolas G. Bazan
Presented by: Jarrod C. Harman

2. Preview MALDI AMD VLC-PUFA PCs
Matrix Assisted Laser Desorption Ionization
Age Related Macular Degeneration
Phosphatidylcholine species containing Very Long Chain- Polyunsaturated Fatty Acid

3. Ion mobility plot produced using DriftScope™ image
MALDI
ToF & IMS
Time of Flight: measures time of travel to determine mass.
Ion mobility scanning: often used with ToF for separation of molecular compounds
Ion mobility plot produced using DriftScope™ image

4. What is AMD? Facts about AMD Wet AMD Dry AMD
Leading Cause of vision loss in people over age 60
Disease causes progressive blurred area in center of vision
AMD affects the macula: allows for fine details of vision
Characterized by growth of abnormal blood vessels
Growth from the choroid commonly referred to as neovascularization
Bleeding from abnormal blood vessels causes scarring
Characterized by the presence of yellow deposits in the macula called drusen
Drusen deposits can grow in size and number with age
Advanced stages exhibit a thinning or loss of the light sensitive cells in the macula

5. METHODS
Eyes were flattened and a horizontal strip of retina and underlying tissue obtained which contained the optic nerve, the macula, and peripheral retina. 
Twenty µm-thick vertical cryo-sections, showing all layers of the retina, RPE, choriocapillaris, and sclera, were attached to glass coverslips
2, 5-dihydroxybenzoic acid matrix was applied by sublimation for positive ion analysis
Waters Synapt G2-S was used to perform MALDI imaging for 30µm spatial resolution.
Ion mobility separation was used in conjunction with ToF to determine lipid/PC molecular species.

6. Phosphatidylcholine
Class of phospholipids that are largely incorporated into biological membranes
Structure is composed of choline head-group attached to a glycerophosphoric spine with 2 fatty acid tails.
VLC-PUFAs currently hypothesized to occupy Sn1 position with DHA at Sn2 position.
Changes in PC species are observed in a wide array of neurological conditions
Sn 1  
Sn 2

7. VLC-PUFA PC Species & AMD
Normal 84 yo CF
merge
Green: m/z 856, PC(18:0/22:6)Photoreceptor layer
m/z: 864.7
Periphery
m/z: 963.7
Macula
Merge
Results
Identified marker for photoreceptor layer (PC 18:0/22:6)
Using MALDI, demonstrated co-localization of VLC-PUFA PC(34:5/22:6) with the photoreceptor marker
AMD macula show decreases in both peripheral PC 32:0, and macular PC 34:1
m/z 856, PC(18:0/22:6)
m/z 1070, PC(34:5/22:6)
PC(34:5/22:6) co-localizes with photoreceptors

8. Results NORMAL AMD Green 856 Red 1070 Blue 994
Normal Retina
Red 1048 m/z
Green 856 m/z
Blue 994 m/z
Normal Retina
AMD Retina
AMD Retina
Note loss of m/z: 1048,  PC(56:11-H+)
A decline in abundance of PC(34:5/22:6) and PC (34:6/22:6) was observed in the central retina of advanced AMD.
Significant losses of the photoreceptor layers were observed in AMD retinas.
Depicted above is a spectrum illustrating the loss of VLC-PUFA PC species in the central AMD retina.
There was an overall reduction in VLC-PUFA-containing PCs in the AMD retina.

9. Conclusion
Here we illustrate molecular organization of various VLC-PUFA-containing PC molecular species within the AMD and normal human retinas.
Using MALDI, we have demonstrated co-localization of VLC-PC (34:5/22:6) within the photoreceptor layers. VLC-PC species are lost in AMD retinas.
This suggests a cause/effect relationship between AMD and the loss of these molecular species.
PC (18:0/22:6) has been localized to photo-receptors, and, therefore, can be used as a specific marker.
When comparing PC (34:5/22:6) and PC (34:6/22:6), we found a decline in abundance in the central retina in advanced AMD.
There is an overall reduction in VLC-PUFA-containing PCs in the AMD retina.

10. Acknowledgments Sources Special Thanks Dr. Nicolas Bazan
Bazan NG, Molina MF, Gordon WC. Docosahexaenoic acid signalolipidomics in nutrition: significance in aging, neuroinflammation, macular degeneration, Alzheimer's, and other neurodegenerative diseases.  Annu Rev Nutr Aug 21;31:
Bazan NG. Homeostatic regulation of photoreceptor cell integrity: significance of the potent mediator neuro-protectin D1 biosynthesized from docosahexaenoic acid: the Proctor Lecture. Invest Ophthalmol Vis Sci Nov;48(11):
Rice DS, Calandria JM, Gordon WC, Jun B, Zhou Y, Gelfman CM, Li S, Jin M, Knott EJ, Chang B, Abuin A, Issa T, Potter D, Platt KA, Bazan NG. Adiponectin receptor 1 conserves docosahexaenoic acid and promotes photoreceptor cell survival. Nat Commun Mar 4;6:6228.
Zemski Berry KA, Gordon WC, Murphy RC, Bazan NG. Spatial organization of lipids in the human retina and optic nerve by MALDI imaging mass spectrometry. J Lipid Res Mar;55(3):
Special Thanks
Dr. Nicolas Bazan
Dr. Bokkyoo Jun
Dr. Bill Gordon
Dr. Filipe Muhale
Neuroscience Center of Excellence
Waters,Inc
NIH
LSUHSC
Supported by grants NEI and NIGMS GM (NGB) and the EENT Foundation