Age-Related Macular Degeneration
Rosalie Crouch, Ph.D., Medical University of South Carolina, Department of Ophthalmology
Anne Hanneken, M.D., The Scripps Research Institute, LaJolla
This project seeks new insights into retinoid biology directly in human tissue specimens. Understanding the molecular basis of age-related macular degeneration (AMD) will provide a basis for development of new treatments and approaches to management of the disease. To date, the application of imaging mass spectrometry (IMS) to retinoid metabolites and the significant advances made in histology-directed imaging and high spatial resolution imaging have had a major impact on the understanding of AMD [1-6]. IMS revealed a lack of A2E in the macula of the human retina, including retinas with dry and wet AMD. Sample preparation advancements made it possible to obtain high quality images of retinal cross-sections, and high spatial resolution imaging distinguished among the adjacent cell layers, including the retinal pigment epithelium, which is one cell thick (Figure 1) . Continued identification of cell layer specific metabolites and lipids will enable molecular signatures of the pathology to be determined, shedding new light on the etiology of wet and dry AMD.
Figure 1. High spatial resolution image of a retinal cross-section showing distinct cell layers. J Am Soc Mass Spectrom 25 (8):1394-1403.
Optic Nerve Regeneration After Crush Remodels the Injury Site: Molecular Insights From Imaging Mass Spectrometry
Joseph Caprioli, M.D., University of California, Los Angeles
Mammalian central nervous system axons fail to regenerate after injury. Contributing factors include limited intrinsic growth capacity and an inhibitory glial environment. Inflammation-induced optic nerve regeneration (IIR) is thought to boost retinal ganglion cell (RGC) intrinsic growth capacity through progrowth gene expression, but effects on the inhibitory glial environment of the optic nerve are unexplored. To investigate progrowth molecular changes associated with reactive gliosis during IIR, we developed an imaging mass spectrometry (IMS)-based approach that identifies discriminant molecular signals in and around optic nerve crush (ONC) sites. ONC was performed in rats, and IIR was established by intravitreal injection of a yeast cell wall preparation. Optic nerves were collected at various postcrush intervals, and longitudinal sections were analyzed with matrix-assisted laser desorption/ionization (MALDI) IMS and data mining. Immunohistochemistry and confocal microscopy were used to compare IIR increased the area of the crush site that was occupied by a dense cellular infiltrate and mass spectral features consistent with lysosome-specific lipids. IIR also increased immunohistochemical labeling for microglia and macrophages. IIR enhanced clearance of lipid sulfatide myelin-associated inhibitors of axon growth and accumulation of simple GM3 gangliosides in a spatial distribution consistent with degradation of plasma membrane from degenerated axons. IIR promotes a robust phagocytic response that improves clearance of myelin and axon debris. This growth-permissive molecular remodeling of the crush injury site extends our current understanding of IIR to include mechanisms extrinsic to the RGC.
Abstract: Stark et al., Optic Nerve Regeneration After Crush Remodels the Injury Site: Molecular Insights From Imaging Mass Spectrometry, Invest Ophthalmol Vis Sci. 2018. (7)
Bis(monoacylglycero)phosphate lipids in the retinal pigment epithelium implicate lysosomal/endosomal dysfunction in a model of Stargardt disease and human retinas
Rosalie Crouch, Ph.D., Medical University of South Carolina, and Yiannis Koutalos Ph.D., Medical University of South Carolina
Stargardt disease is a juvenile onset retinal degeneration, associated with elevated levels of lipofuscin and its bis-retinoid components, such as N-retinylidene-N-retinylethanolamine (A2E). However, the pathogenesis of Stargardt is still poorly understood and targeted treatments are not available. Utilizing high spatial and high mass resolution matrix assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS), we determined alterations of lipid profiles specifically localized to the retinal pigment epithelium (RPE) in Abca4 −/− Stargardt model mice compared to their relevant background strain. Extensive analysis by LC-MS/MS in both positive and negative ion mode was required to accurately confirm the identity of one highly expressed lipid class, bis(monoacylgylercoro)phosphate (BMP) lipids, and to distinguish them from isobaric species. The same BMP lipids were also detected in the RPE of healthy human retina. BMP lipids have been previously associated with the endosomal/lysosomal storage diseases Niemann-Pick and neuronal ceroid lipofuscinosis and have been reported to regulate cholesterol levels in endosomes. These results suggest that perturbations in lipid metabolism associated with late endosomal/lysosomal dysfunction may play a role in the pathogenesis of Stargardt disease and is evidenced in human retinas.
Abstract: Anderson et al., Bis(monoacylglycero)phosphate lipids in the retinal pigment epithelium implicate lysosomal/endosomal dysfunction in a model of Stargardt disease and human retinas. Sci Rep. 2017 (8)
1. Ablonczy Z, Gutierrez DB, Grey AC, Schey KL, Crouch RK (2012) Molecule-specific imaging and quantitation of A2E in the RPE. Advances in experimental medicine and biology. 723:75-81.PMCID: 3817715. Return to text.
2. Ablonczy Z, Higbee D, Anderson DM, Dahrouj M, Grey AC, Gutierrez D, Koutalos Y, Schey KL, Hanneken A, Crouch RK (2013) Lack of correlation between the spatial distribution of A2E and lipofuscin fluorescence in the human retinal pigment epithelium. Invest Ophthalmol Vis Sci 54 (8):5535-5542. PMCID: 3747789. Return to text.
3. Ablonczy Z, Higbee D, Grey AC, Koutalos Y, Schey KL, Crouch RK (2013) Similar molecules spatially correlate with lipofuscin and N-retinylidene-N-retinylethanolamine in the mouse but not in the human retinal pigment epithelium. Arch Biochem biophys 539 (2):196-202. PMCID: 3818512. Return to text.
4. Ablonczy Z, Smith N, Anderson DM, Grey AC, Spraggins J, Koutalos Y, Schey KL, Crouch RK (2014) The utilization of fluorescence to identify the components of lipofuscin by imaging mass spectrometry. Proteomics 14 (7-8):936-944. PMCID: 4017854. Return to text.
5. Anderson DM, Ablonczy Z, Koutalos Y, Spraggins J, Crouch RK, Caprioli RM, Schey KL (2014) High resolution MALDI imaging mass spectrometry of retinal tissue lipids. J Am Soc Mass Spectrom 25 (8):1394-1403. PMCID: 4180438. Return to text.
6. Grey AC, Crouch RK, Koutalos Y, Schey KL, Ablonczy Z (2011) Spatial localization of A2E in the retinal pigment epithelium. Invest Ophthalmol Vis Sci 52 (7):3926-3933. PMCID: 21357388. Return to text.
7. Stark et al., Optic Nerve Regeneration After Crush Remodels the Injury Site: Molecular Insights From Imaging Mass Spectrometry, Invest Ophthalmol Vis Sci. 2018. Return to text.
8. Anderson et al., Bis(monoacylglycero)phosphate lipids in the retinal pigment epithelium implicate lysosomal/endosomal dysfunction in a model of Stargardt disease and human retinas. Sci Rep. 2017. Return to text.