Glaucoma is a chronic optic neuropathy characterized by degeneration of the Retinal Ganglion Cells (RGCs) and the optic nerve. As of 2024, glaucoma is the second cause of irreversible blindness on a global scale. The pathogenesis of the disease is largely uncharacterized yet, but, several recent studies have pointed out phenotypical and molecular alterations of circulating immune cells, in particular T-lymphocytes, in preclinical models and in human subjects.

Therefore, we have here undertaken a characterization of the proteome of peripheral blood mononuclear cells (PBMC) of subjects diagnosed with primary open angle glaucoma (POAG) and cataract, enrolled as healthy controls.

The study was authorized by the local ethic committee and subjects asked to signa written informed consent. PBMCs (n=12 POAG, n=12 cataract; age: 71.8±7.5y POAG, 70.1±12.7 cataract; comparable gender ratio) were isolated from peripheral blood by Ficoll-Histopaque sedimentation and lysed in urea buffer. 200 µg of proteins were reduced, alkylated and digested with lysil-c endopeptidase (1:2000) and trypsin (1:50 ratio). Peptides (750 ng) were injected in a Orbitrap Exploris 240 online with a nanoUHPLC. The analysis was run by label free quantification using three different acquisition modalities: DDA, DIA and DIA-GPF. Proteins were searched by Proteome Discoverer (DDA) and DIA-NN (for DIA library free searches). In the case of GPF-refined library generation, staggered GPF (n=6) files were submitted to DIA-NN. Output data were analysed by R.

The following discussion refers to DIA data under library free searches. Proteins were filtered according to: q-value (Lib.Q.Value in the output matrix) ≤0.01; identification with at least 1 proteotypic peptide; identification of the protein in ≥ 8 out of 12 subjects of the same experimental group. Quality controls (e.g., Coefficient of Variation, distribution of data, blood proteins contamination, PCA plot etc.) were checked as optimal.

Even applying strict criteria, the approach identified 4001 proteins common to POAG and cataract groups. Additional 465 proteins were identified as POAG-specific and 118 as cataract-specific. Volcano plot (0.5<log2Fold Change<0.5, p<0.05) indicated that 353 proteins were found as upregulated in POAG PBMCs, while 112 proteins were identified as downregulated in the same experimental group. GeneOntology-enrichment and KEGG pathway analysis showed that POAG PBMCs suffered from possible alterations referable to mechanisms of immune system polarization, transcription and translation processes, extracellular proteolysis, autophagy, as well as mitochondrial and redox metabolism. Main proteins were studied by Western blotting to confirm proteomics data.

Further studies are needed to figure out the distribution of PBMC subsets along with their polarization and metabolic trajectories to address the role of these factors in mediating the neuroinflammation state that accompanies glaucoma progression.