Abstract
Purpose :
Retinal Müller glial cells (MGCs) are known to secrete factors protecting retinal neurons from metabolic and neurodegenerative diseases like diabetes. Previous studies from our lab have shown that the molecular chaperone αA-crystallin and particularly, its phosphomimetic form (T148D) is a potent neuroprotector for retinal neurons. We also showed that this molecular chaperone is highly expressed by MGCs under stress but its link to retinal neuron survival remained unclear. Thus, the present study was carried out to understand the specific role of αA-crystallin in retinal MGCs.
Methods :
Primary MGCs obtained from Ko-aA-crystallin mice were transfected with plasmids encoding either wild-type (WT), phosphomimetic (T148D), or non-phosphorylatable mutants (T148A) of αA-crystallin. The cells were then either exposed to serum starvation or high glucose with TNF-a (100ng/ml). The conditioned media obtained from the primary MGCs were first analyzed to determine their specific content, and then supplemented to either differentiated retinoblastoma cells (R28 and SH-Y5Y) or primary ganglion cells to test their impact on cell viability. aA-crystallin recombinant proteins were produced and used to supplement culture media of primary ganglion cells and assess its neuroprotective potential through dose-response and survival assays.
Results :
Our results demonstrate for the first time that primary MGCs secrete aA-crystallin. It also demonstrates that media from MGCs overexpressing WT (45%) or T148D (38%), but not T148A aA-crystallin (21%) significantly diminish metabolic-stress induced cell death in differentiated retinal cell lines as well as in primary ganglion cells as compared to empty vector (EV) control. Mechanistically, we showed that this protective effect was associated with a reduced translocation of the pro-apoptotic effector Bax into the mitochondria by WT (31%) and T148D (79%) aA-crystallin as compared to EV. Further, we showed that exogenously added WT and T148D recombinant aA-crystallin, but not T148A greatly promoted ganglion cell survival exposed to metabolic stress.
Conclusions :
The data strongly suggest that primary MGCs not only express but also secrete aA-crystallin in response to stress, and that the secreted αA-crystallin plays an important neuroprotective paracrine role in the retina under metabolic stress.
This is a 2020 ARVO Annual Meeting abstract.