Abstract
Purpose :
Rescuing mitochondrial quality control (MQC) has emerged as a promising strategy for retinal degenerative conditions, where Müller glia may play a fundamental role by supporting the removal of dysfunctional mitochondria through mitophagy. Here we report a novel Mito-QC Müller cell line (MQ-MG2) for the rapid and efficient screening of mitophagy inducers.
Methods :
MQ-MG2 were cloned from spontaneously immortalised Primary Muller cells (PMCs) derived from mitophagy reporter (Mito-QC) mice. The phenotypic characteristics of Müller glia were evaluated by immunocytochemistry, Western blotting, metabolic flux assay (Seahorse), and their neural stem-cell potential in response to fibroblast growth factor-2 (40 ng/mL), retinoic acid (500nM) or commercial differentiation media. The versatility of MQ-MG2 as a drug screening platform was tested using mitophagy inductors of putative molecular pathways (PINK1-dependent and PINK1-independent) and validated in vivo using Mito-QC mice.
Results :
MQ-MG2 were cultured over 60 passages while retaining expression of Mito-QC reporter and key phenotypic markers of Müller glia (glutamine synthase, GFAP, and IL-33). At the bioenergetic level, MQ-MG2 displayed lower basal- and ATP-linked respiration than PMCs but higher reserve capacity. Glycolytic profiles remained comparable to PMCs, as indicated by (baseline and stressed) extracellular acidification rates. MQ-MG2 showed neural stem-cell properties, as demonstrated by the expression of characteristic markers (Pax6, Nestin, Notch1, Sox2, βIII-Tubulin) and their ability to adopt neuronal morphology and form neuro-spheres. MQ-MG2 exhibited excellent sensitivity for the screening of mitophagy inducers, including known PINK1-kinase activators (Niclosamide, N6-Furfuryl-Adenosine) and newly identified small molecules. The versatility of MQ-MG2 as a drug discovery platform was further confirmed in vivo, where oral administration of PIA-01 (PINK1-independent activator) amplified mitophagy at the outer retina dose-dependently.
Conclusions :
MQ-MG2 enables rapid and reliable detection of mitophagy-activating drugs for ocular applications including stem-cell biology. This cell line overcomes primary culture limitations, including variability due to cellular senescence and heterogeneity, while reducing the need for animals in ocular drug discovery.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.