Phase separation of C19orf12 regulates BNIP3 protein quality control and maintains neuronal mitophagy

Mutations in C19orf12, an orphan gene with elusive function, cause mitochondrial membrane protein-associated neurodegeneration (MPAN). Despite the intriguing mitochondrial deficits, the mechanisms underlying the loss of function of C19orf12 in MPAN pathogenesis remain unclear. In this study, we aim to explore the functional impacts of C19orf12 mutations on mitophagy in MPAN models in vitro and in vivo. Our findings suggest that C19orf12 regulates the turnover of mitophagy receptor BNIP3 proteins through the lysosomal degradation pathway. Disruption of this process leads to the accumulation of oxidized BNIP3 proteins on mitochondria that are ineffective in initiating mitophagy. Mechanistically, C19orf12 participates in protein condensate formation by liquid-liquid phase separation to facilitate BNIP3 protein turnover on the mitochondrial membrane. Along with mitophagy deficits, a rodent MPAN model exhibits motor deficits and core pathological features of MPAN, including iron accumulation, axonal spheroids, and neuroinflammation. This study underscores the pivotal role of C19orf12 in regulating the quality control of BNIP3 protein to control mitophagy, highlighting the significance of impaired mitophagy in the pathogenesis of MPAN.Abbreviations: ATP: adenosine triphosphate; BafA: bafilomycin A1; BNIP3: BCL2 interacting protein 3; BTZ: bortezomib; C19orf12: chromosome 19 open reading frame 12; CFP: cyan fluorescent protein; CHX: cycloheximide; DNP: dinitrophenyl; FCCP: carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; FRAP: fluorescence recovery after photobleaching; GFP: green fluorescent protein; H&E stain: haematoxylin and eosin stain; LCD: low complexity domain; LC/MS: liquid chromatography-mass spectrometry; LLPS: liquid-liquid phase seperation; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; mito-SRAI: mitochondrial signal-retaining autophagy indicator; MG132: cbz-leu-leu-leucinal; MMP: mitochondrial membrane potential; MPAN: mitochondrial membrane protein-associated neurodegeneration; NBIA: neurodegeneration with brain iron accumulation;PLA: proximity ligation assay; RFP: red fluorescent protein; ROS: reactive oxygen species; STX17: syntaxin 17; TFAM: transcription factor A, mitochondrial; TOLLES: TOLerance of lysosomal EnvironmentS; YPet: YFP for energy transfer