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Aims/Purpose: The controlled activation of mitophagy has emerged as an exciting therapeutic angle in the treatment of ocular conditions, including diabetic retinopathy and age‐related macular degeneration. Here, we report a novel Muller cell line (MQ‐MG2) for the rapid screening of mitophagy‐activating drugs. Methods: MQ‐MG2 were cloned from spontaneously immortalized primary Muller cells (PMCs) derived from mitophagy reporter (Mito‐QC) mice. Muller glia characteristics were assessed by immunocytochemistry, western blotting, and metabolic flux (Seahorse). The versatility of MQ‐MG2 to detect mitophagy‐activating drugs was tested via pharmacological activation (PINK1‐dependent and PINK1‐independent) and further validated in vivo using Mito‐QC mice. Results: MQ‐MG2 were expanded over 50 passages while retaining expression of Mito‐QC reporter. MQ‐MG2 showed key phenotypic markers of Muller glia, including glutamine synthetase, glial fibrillary acidic protein, Aquaporin 4, and Interleukin‐33. At the bioenergetic level, MQ‐MG2 showed lower basal‐ and ATP‐linked respiration than PMCs, while displaying superior reserve capacity (p < 0.001). Indicative of equivalent glycolytic profiles, extracellular acidification rates (baseline and stressed) remained comparable to PMCs. Importantly, MQ‐MG2 showed excellent sensitivity to screen for mitophagy‐activating drugs, including novel small‐molecules acting through (putative) PINK1‐independent pathways. The versatility of MQ‐MG2 as a drug discovery platform was validated in vivo, where oral administration of PINK1‐independent activator (PIA‐01) amplified mitophagy at the outer retina dose‐dependently. Conclusions: MQ‐MG2 enables rapid and reliable detection of mitophagy‐activating drugs for ocular application. This platform overcomes the limitations of primary cultures, including variability due to cellular senescence and heterogeneity, while minimizing the need for animals in drug discovery. [ABSTRACT FROM AUTHOR]

Aims/Purpose: We hypothesize that exacerbated mitochondrial fusion abrogates mitophagy in diabetic retinopathy [DR]. Our aim was to rescue this process pharmacologically to prevent vision loss in a mouse model of type‐1 diabetes. Methods: We generated a drug screening platform mimicking mitochondrial hyperfusion in DR. To this end, retinal Müller cells (mouse primary and MIO‐M1 cell line) were antagonized for mitochondrial fission using a Drp1 inhibitor peptide under elevated glucose (30.5 mM) conditions. Pharmacological 'hits' rescuing mitophagy in conditions of diabetes‐induced hyperfusion were assessed for their capability to improve mitochondrial health (membrane potential [ΔΨm]) and bioenergetics (Seahorse). The bio‐activity of lead compounds were corroborated in vivo using diabetic mitophagy reporter mice (Mito‐QC Ins2Akita), while effectiveness to alleviate retinal degeneration was evaluated via electroretinography (ERG), retinal thickness (SD‐OCT) and morphometric analysis of retinal neurons. Results: Among all drugs screened, we identified PA‐01 (a PINK1‐mitophagy activator) as capable of safely rescuing mitophagy under conditions of diabetes‐induced mitochondrial hyperfusion. Accordingly, PA‐01 rescued mitochondrial health (ΔΨm) and bioenergetics in Müller glia cultures, by optimizing metabolic flux (basal‐ and ATP‐linked respiration). The bio‐activity of PA‐01 was demonstrated in vivo, where long‐term oral administration rescued mitochondrial hyperfusion and mitophagy in the diabetic retina, while further preventing neurodegeneration. This was reflected in i) improved scotopic ERG responses (increased a‐wave and b‐wave amplitudes), ii) improved SD‐OCT retinal thickness and iii) neuroprotection to photoreceptors, synaptic terminals and amacrine cells. Conclusions: Exacerbated mitochondrial fusion contributes to DR pathology by inhibiting mitophagy in Müller glia. Rescuing this process pharmacologically holds promising therapeutic potential to alleviate vision loss in DR. [ABSTRACT FROM AUTHOR]

Aims/Purpose: The accumulation of dysfunctional mitochondria has been implicated in the pathogenesis of diabetic eye disease through the impairment of mitochondrial quality control (MQC). This study aimed to investigate whether mitophagy‐activating drugs can alleviate pathology in the anterior and posterior ocular tissues of type‐1 diabetic mice. Methods: Mitophagy was evaluated in corneal and retinal tissues from diabetic mitophagy reporter mice (mitoQC‐Ins2Akita). The therapeutic potential of targeting mitophagy was evaluated by treating diabetic Ins2Akita mice with PIA‐01 (a PINK1‐independent mitophagy activator). Treatment was delivered orally between 4 and 8 months of diabetes. Ocular degenerative changes were evaluated by scotopic electroretinography (ERG), along with immunohistochemical analysis of retinal neurons and corneal cytoarchitecture. Results: Indicative of deteriorated MQC, mitophagy and mitochondrial transcription factor A (TFAM) positive mitochondrial nucleoids (which contain mtDNA) significantly decreased in the cornea and retina by 8 months of diabetes. Oral delivery of PIA‐01 restored TFAM+ mitochondrial nucleoids, leading to improved visual outcomes in Ins2Akita mice. In the retina, this was reflected by improved ERG responses (e.g. increased scotopic a‐wave and b‐wave amplitudes), while also protecting retinal neurons, including cone‐photoreceptors, synaptic structures, amacrine cells and retinal ganglion cells. This protection extended to the anterior segment, where PIA‐01 rescued corneal cytoarchitecture (basal epithelium and stroma) in diabetic mice. Conclusions: Diabetes progresses with deterioration of MQC in anterior and posterior ocular tissues. Rescuing MQC via mitophagy‐activating drugs may allow for multisystem therapies to control diabetic eye disease. [ABSTRACT FROM AUTHOR]