Mitochondrial translation deficiency impairs NAD+‐mediated lysosomal acidification

Mitochondrial translation dysfunction is associated with neurodegenerative and cardiovascular diseases. Cells eliminate defective mitochondria by the lysosomal machinery via autophagy. The relationship between mitochondrial translation and lysosomal function is unknown. In this study, mitochondrial translation‐deficient hearts from p32‐knockout mice were found to exhibit enlarged lysosomes containing lipofuscin, suggesting impaired lysosome and autolysosome function. These mice also displayed autophagic abnormalities, such as p62 accumulation and LC3 localization around broken mitochondria. The expression of genes encoding for nicotinamide adenine dinucleotide (NAD + ) biosynthetic enzymes—Nmnat3 and Nampt—and NAD + levels were decreased, suggesting that NAD + is essential for maintaining lysosomal acidification. Conversely, nicotinamide mononucleotide (NMN) administration or Nmnat3 overexpression rescued lysosomal acidification. Nmnat3 gene expression is suppressed by HIF1α, a transcription factor that is stabilized by mitochondrial translation dysfunction, suggesting that HIF1α‐Nmnat3‐mediated NAD + production is important for lysosomal function. The glycolytic enzymes GAPDH and PGK1 were found associated with lysosomal vesicles, and NAD + was required for ATP production around lysosomal vesicles. Thus, we conclude that NAD + content affected by mitochondrial dysfunction is essential for lysosomal maintenance. SYNOPSIS Mitochondrial dysfunction has been shown to affect lysosomal functions. This study reveals that defective mitochondrial translation leads to decreased HIF1α‐Nmnat3‐dependent NAD + production, thereby inhibiting localized ATP production at the lysosomal membranes and vATPase function in mouse heart. Cardiomyocyte‐specific knockout (KO) of mitochondrial protein p32 results in increased HIF1α expression in mouse heart. HIF1α suppresses Nicotinamide mononucleotide adenylyltransferase 3 (Nmnat3) transcription, leading to reduced level of NAD + in cardiomyocytes. Glycolytic enzymes GAPDH and PGK1 associate with lysosomes to promote local ATP production. Inhibition of NAD + synthesis in p32 KO cells decreases GAPDH/PGK1‐dependent ATP production around lysosomes and their acidification. NMN treatment or Nmnat3 overexpression rescues lysosomal acidification in p32 KO cells. Graphical Abstract Mitochondrial dysfunction leads to decreased HIF1α‐Nmnat3‐dependent NAD + production, inhibiting localized ATP production at the lysosomal membranes and vATPa