Autophagy and apoptosis proteins in ex-vivo dissected TM tissuein severe glaucoma
while autophagy predominant over apoptosis in HTM cells after severe oxidative stress
Table 1 gives the demographic and clinical characteristics of patients included in the study. Of 1813 proteins identified, we found a total of 797 unique elements in POAG and PACG compared to controls with a total of 12 identifiable and quantifiable proteins in POAG and 7 in PACG proteins were found to be differentially expressed compared to controls, Figure 1. We also found myocilin and AQP1 apart from other proteins which were specific markers for TM tissue, Table 2.Categorising proteins according to functional relevance,we could see overall distribution of the 797 proteins in diverse cellular functional categories with most proteins in POAG and PACG were involved in response to stimulus and defence response, cell death and cell movement organisation. Most of them were extracellular or membrane proteins (as opposed to membrane proteins in controls) involved in protein binding and catalytic activity or structural molecule activity, Fig. S1.
Both POAG and PACG TM tissues had several proteins which were significantly different from that of controls of which most were either ECM related or cell death proteins, Fig S1 and S2. Both apoptosis and autophagy proteins were overexpressed in severe POAG >PACG samples compared to controls. Notably, apoptotic protein, caspase14, was under-expressed in severe POAG and PACG specimens while autophagy related LAMC1 protein was overexpressed in severe POAG >severe PACG, Fig S1. We also found endoplasmic reticulum stress markers namely, translation initiation factor2α (eIF2) and ER chaperone GRP78 (glucose-regulated protein) to be overexpressed in POAG and PACG compared to controls, Table 2.
Stress induction on in-vitro HTM cell lines with reduced cell survival was seen to start at 400-1000uM concentration at 48-72 hours and this response correlated with maximum cell death at 72 hours, Fig 2.Flow cytometry results also showed parallel changes with maximum apoptotic cells at 48 hours with some necrotic cells also seen at 24-48 hours.
AnalyzingHTM cell gene expression at 48 hrs (similar to moderate glaucoma) and 72 hours (maximum cell death similar to severe glaucoma stage) of oxidative stress, autophagy genes were parallelly up-regulated at 48 hours of exposure>72 hours which was in contrast to apoptosis related genes showing down-regulation at 72 hours, Fig.3. Apoptosis genes were up-regulated at 48 hours of H2O2 exposure while several genes like BCL, FASL and CASP7 were down-regulated after 72 hours of exposure simulating changes in HTM tissue specimens in severe glaucoma.Necrosis genes were also seen to maximally up regulated at 48 hours compared to 72 hours where most genes were down regulated or minimally functional, Fig 3.
Switch to Autophagy in peripheral blood of severe glaucoma and epigenetic control of cell death
Flow cytometryon PBMC showed significantly higher (>2 times higher) pro-apoptotic and apoptotic PBMC cells in glaucoma cases and POAG > PACG eyes, Fig S3.Interestingly, autophagy genes showed an abrupt up-regulation of genes like AKT1 and ATG16seen in severe glaucoma as opposed to down-regulation in moderate POAG and PACG, Fig S4.In contrast, the PBMC cells in severe glaucoma showed apoptotic genes to be down regulated in severe glaucoma in both POAG and PACG, Fig S4 (E-G) with necrosis genes showing minimal changes between glaucoma stages, Figure S4 (H, I).
These findings suggests that in PBMC, autophagy may be the predominant mechanisms of cell death in severe glaucoma wherein autophagy related process may be the driving force chosen for cell death over apoptosis. This mirrored proteome results in TM dissected tissues and HTM experiments where autophagy related proteins were over-expressed in severe glaucoma. To evaluate if epigenetic regulatory mechanisms may explain this switch in preference of cell death process with different glaucoma stages, we evaluated the histone modifications in PBMC and also on in-vitro cell cultures after stress.
Concurring with several histone proteins over-expressed in TM tissues (Supplemental data), immunoblotting showed increased expression of H3K14ac in severe POAG and PACG, Fig S4 (J,K) while other methylation changes were seen over-expressed only in severe POAG and minimal in other stages of POAG or PACG. Similar results were seen in HTM cell cultures with H3K14ac seen at 72 hours of stress exposure akin to severe glaucoma in PBMC, Fig 3 (D,E).