Optic Nerve Lipidomics Reveal Impaired Glucosylsphingosine Lipids Pathway in Glaucoma

Abstract

Purpose To determine major differences in lipid profile between human control and glaucomatous optic nerve. To assess major enzymes in lipid pathway if aberration is revealed for a lipid class by profiling. Methods Optic nerve (ON) samples were obtained from human cadaveric donors [control ( n = 11) and primary open-angle glaucoma (POAG; n = 12)]; the lipids were extracted using Bligh and Dyer methods. Control and glaucoma donors were all Caucasians age 72.3 ± 5.9 and 70.3 ± 10.5 (inclusive of both sexes), respectively. Lipids were extracted after weighing the tissue; the protein amounts in the corresponding aqueous phase of organic solvent extraction were recorded. High-resolution mass spectrometry was performed using a Q-exactive mass spectrometer coupled with an EASY-nLC 1000 liquid chromatograph instrument. Bioinformatics and statistical analysis were performed using LipidSearch v.4.1 and MetaboAnalyst 4.0/STATA 14.2. Protein amounts were determined using Bradford's method. Western blot, ELISA, and immunohistochemistry utilized established protocols and were performed for protein quantification and localization, respectively. Additional donor tissues were utilized for Western blot, ELISA, and immunohistochemistry. Results Principal component analysis (PCA) placed control and glaucomatous ONs in two distinct groups based on analysis of lipid profiles. Total lipid, total phospholipids, total ceramide, and total sphingolipids were similar (without significant difference) between control and glaucoma. However, we found a significant increase in glucosylsphingosine in glaucoma compared to control samples. We found similar levels of glucocerebrosidase (GBA), ceramide glucosyltransferase (UGCG), decreased nonlysosomal glucocerebrosidase (GBA2), and increased lysosomal and nonlysosomal acylsphingosine amidohydrolase (ASAH1 and ASAH2) levels in glaucomatous ON compared to control. Conclusions We found significant differences in glucosylsphingosine lipids, consistent with decreased GBA and GBA2 and increased ASAH1 and ASAH2 immunoreactivity in glaucoma, suggesting the potential impairment of sphingolipid enzymatic pathways in lysosomal and nonlysosomal cellular compartments.

Ocular phenotypes in a mouse model of impaired glucocerebrosidase activity

“…Specifically, glaucoma has been reported for PD and mucopolysaccharidoses 15 , 41 , 42 . Further, there are reports of elevated GluSph levels in ocular tissue of glaucoma patients, suggesting an association of GBA pathway dysfunction and ocular disease even in patients without GBA1 mutations 44 . However, it is unknown whether an impairment of GCase activity would be sufficient to cause ocular pathology in any patient population.…”

Alteration in Lysophospholipids and Converting Enzymes in Glaucomatous Optic Nerves

“…43 To further study the role of lipids in CNS diseases, we have performed high-resolution mass spectrometry on human ON extracted lipids. 13 We analyzed glucosylsphingosine lipids in the ON in our previous studies, and due to lack of any comprehensive studies on ON LPLs, we chose to focus here on LPLs for their importance in neuronal signaling, function, and pathophysiology. 37,38,42 Analysis of mass spectrometry lipidomic data revealed a decrease in levels of LPA, LPC, and LPE in the human glaucomatous ON compared with control (Fig.…”

“…Patients were considered viable glaucoma donors if the patients had at least two static perimetry recordings (within 1-2 years) and showed distinct vision loss over time, as described previously. 13 Many donor eyes also had measurements of NFL thickness, and other clinical parameters that aid in diagnosis of glaucoma. Acceptable control eyes had no medical history of optic neuropathy.…”

“…Previously we have profiled lipids of the ON and, due to lipids' tremendous diversity, have focused only on glucosylsphingosine lipids and impairment of sphingolipid enzymatic pathways. 13 We aim here to investigate lysophospholipids (LPLs) present in the ON.…”

Elusive Roles of the Different Ceramidases in Human Health, Pathophysiology, and Tissue Regeneration

“…Treatment with ACDase inhibitors decreases the accumulation of α-synuclein in cases of GBA1 mutation [ 81 ]. Similar lipid patterns are observed in the optic nerves of glaucoma patients, where Asah1 and Asah2 genes are overexpressed, but non-lysosomal GCase-GBA2 is inhibited, resulting in a lower total lipid content and significantly higher concentrations of Glc-Sph [ 83 ].…”