Abstract
Purpose :
Retinitis pigmentosa (RP) is a blinding group of diseases caused by mutations in dozens of genes, so a gene agnostic treatment approach is ideal. Neuroinflammation has been implicated in the degeneration of multiple RP models. To support its anabolic drive, activated microglia rely on aerobic glycolysis and pyruvate kinase M2 (PKM2). This study examines the pharmacological activation of PKM2 to metabolically reprogram microglia in the rd10 RP model.
Methods :
To pharmacologically activate PK activity, intraperitoneal (IP) injections of a novel PKM2 activator, MCTI-566, were administered to rd10 mice. Overall PK activity was determined via an enzyme-coupled assay. Immunohistochemistry was used to demonstrate expression of PKM2, as well as to identify the anatomic location of IBA1+ microglia and their colocalization with PKM2. Western blot analysis was utilized to determine the abundance of PKM2. Quantitative Real-Time PCR was used to analyze changes in the expression of inflammatory cytokines. Flow cytometry assessed the effects of MCTI-566 treatment on immune cells.
Results :
Using IP administration of MCTI-566 versus vehicle, we increased overall pyruvate kinase by ~3-fold (p=0.003), while overall protein levels of PKM2 remained comparable between treatment groups. Immunohistochemistry revealed the presence of subretinal microglia and their expression of PKM2 in rd10 mice. The overall retinal myeloid cell population significantly decreased with treatment of MCTI-566 on flow cytometry when selecting for CD11b+ (p=0.0018), CD11b+/Iba1+ (p=0.0027), and CD11b+/CD45+/Ly6C+ (p=0.0018). Furthermore, inflammatory cytokine expression with MCTI-566 treatment was significantly increased for Cxcr2 (p=0.02), Il11 (p=0.03), Il27 (p=0.048), Pf4 (p=0.003), and decreased for Ccl4 (p=0.001), Ccl6 (p=0.009), Ccr4 (p=0.03), Il17a (p=0.01), Il1b (p=0.048), Spp1 (p=0.0001), Tnfsf11 (p=0.02), Mif (p=0.002).
Conclusions :
We previously showed that enhancing retinal PK activity using pharmacologic activation of PKM2 is neuroprotective in a model of acute nutrient deprivation. Our new data suggest that independent of photoreceptors, small molecule PKM2 activators can reprogram microglia and changed the pro-inflammatory environment in the rd10 model of RP. Therefore, the use of small molecule PKM2 activators as a non-gene-specific therapy may prevent the immunologic perturbations in RP.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.