Disruption of the pentose phosphate pathway in Müller cells makes them more susceptible to light stress by interfering with the oxidative stress-induced NRF2 response

Abstract

Purpose : The Pentose Phosphate Pathway (PPP), a metabolic offshoot of the glycolytic pathway, is essential for cell survival by providing protective metabolites and molecules. Transketolase (TKT) is the critical enzyme that controls the extent of PPP “traffic flow”. Here, we explored the role of PPP in maintaining the health of human Müller cells.

Methods : Immunofluorescence staining (IF) established that TKT was predominantly expressed by Müller cells in the human retina. We validated TKT expression further in human primary Müller cells (huPMCs) by Western Blot (WB). We inhibited the expression of TKT in huPMCs by small interfering RNA (siRNA) to disrupt PPP. The knockdown efficiency was verified by WB, IF and a TKT activity assay. We then explored the metabolic changes after PPP disruption using 1,2-C¹³ glucose as a tracer. We stressed the huPMCs, with or without TKT knockdown, by exposing the cells to 32k lux white light for 4 hours with 5 lux dim light as control. We evaluated the cell viability by AlamarBlue assay, cell death by LDH assay and metabolic states by ATP, NADPH assays. Finally, we performed a transcriptomic analysis of huPMCs after treatments, followed by protein level validation by WB. We used bioinformatic analysis to reveal the molecular pathways with prominent changes in knockdown and control groups.

Results : The Müller cell was the primary cell type expressing TKT in the retina. huPMCs also expressed TKT. siRNA treatment reduced both protein expression and enzyme activity of TKT. TKT knockdown inhibited de novo synthesis of pyruvate from glucose, while the proportion of consumed glucose that went into TCA cycle increased. The cell viability of huPMC was significantly reduced after TKT knockdown by light stress with no detectable cell death. Likewise, ATP levels and the NADPH/NADP⁺ ratio dropped after photic stress, exacerbated by TKT knockdown. According to Ingenuity Pathway Analysis, the NRF2 pathway was activated after photic stress, while one of the downstream targets, NAD(P)H Quinone Dehydrogenase 1, was reduced by TKT knockdown.

Conclusions : Müller cells express TKT abundantly. Knockdown of TKT not only disrupted the PPP but also impaired overall glucose utilization by Müller cells. Knockdown of TKT made the cells more vulnerable to light stress, possibly by impairing NRF2 anti-oxidative responses.

This is a 2021 ARVO Annual Meeting abstract.