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Peroxisomes are dynamic organelles that participate in a diverse array of cellular processes, including β‐oxidation, which produces a considerable amount of reactive oxygen species (ROS). Although we showed that catalase depletion induces ROS‐mediated pexophagy in cells, the effect of catalase deficiency during conditions that favor ROS generation remains elusive in mice. In this study, we reported that prolonged fasting in catalase‐knockout (KO) mice drastically increased ROS production, which induced liver‐specific pexophagy, an autophagic degradation of peroxisomes. In addition, increased ROS generation induced the production of pro‐inflammatory cytokines in the liver tissues of catalase‐KO mice. Furthermore, there was a significant increase in the levels of aspartate transaminase and alanine transaminase as well as apparent cell death in the liver of catalase‐KO mice during prolonged fasting. However, an intra‐peritoneal injection of the antioxidant N‐acetyl‐l‐cysteine (NAC) and autophagy inhibitor chloroquine inhibited the inflammatory response, liver damage, and pexophagy in the liver of catalase‐KO mice during prolonged fasting. Consistently, genetic ablation of autophagy, Atg5 led to suppression of pexophagy during catalase inhibition by 3‐aminotriazole (3AT). Moreover, treatment with chloroquine also ameliorated the inflammatory response and cell death in embryonic fibroblast cells from catalase‐KO mice. Taken together, our data suggest that ROS‐mediated liver‐specific pexophagy observed during prolonged fasting in catalase‐KO mice may be responsible for the process associated with hepatic cell death.

Recent evidence has linked the lysosomal cholesterol accumulation in Niemann-Pick type C1 with anomalies associated with primary ciliogenesis. Here, we report that perturbed intracellular cholesterol distribution imposed by lysosomal cholesterol accumulation during TMEM135 depletion is closely associated with impaired ciliogenesis. TMEM135 depletion does not affect the formation of the basal body and the ciliary transition zone. TMEM135 depletion severely blunts Rab8 trafficking to the centrioles without affecting the centriolar localization of Rab11 and Rabin8, the upstream regulators of Rab8 activation. Although TMEM135 depletion prevents enhanced IFT20 localization at the centrioles, ciliary vesicle formation is not affected. Furthermore, enhanced IFT20 localization at the centrioles is dependent on Rab8 activation. Supplementation of cholesterol in complex with cyclodextrin rescues Rab8 trafficking to the centrioles and Rab8 activation, thereby recovering primary ciliogenesis in TMEM135-depleted cells. Taken together, our data suggest that TMEM135 depletion prevents ciliary vesicle elongation, a characteristic of impaired Rab8 function. Our study thus reveals a previously uncharacterized effect of erroneous intracellular cholesterol distribution on impairing Rab8 function and primary ciliogenesis.

The primary cilia are evolutionarily conserved microtubule-based cellular organelles that perceive metabolic status and thus link the sensory system to cellular signaling pathways. Therefore, ciliogenesis is thought to be tightly linked to autophagy, which is also regulated by nutrient-sensing transcription factors, such as PPARA (peroxisome proliferator activated receptor alpha) and NR1H4/FXR (nuclear receptor subfamily 1, group H, member 4). However, the relationship between these factors and ciliogenesis has not been clearly demonstrated. Here, we present direct evidence for the involvement of macroautophagic/autophagic regulators in controlling ciliogenesis. We showed that activation of PPARA facilitated ciliogenesis independently of cellular nutritional states. Importantly, PPARA-induced ciliogenesis was mediated by controlling autophagy, since either pharmacological or genetic inactivation of autophagy significantly repressed ciliogenesis. Moreover, we showed that pharmacological activator of autophagy, rapamycin, recovered repressed ciliogenesis in ppara cells. Conversely, activation of NR1H4 repressed cilia formation, while knockdown of NR1H4 enhanced ciliogenesis by inducing autophagy. The reciprocal activities of PPARA and NR1H4 in regulating ciliogenesis were highlighted in a condition where de-repressed ciliogenesis by NR1H4 knockdown was further enhanced by PPARA activation. The in vivo roles of PPARA and NR1H4 in regulating ciliogenesis were examined in greater detail in ppara mice. In response to starvation, ciliogenesis was facilitated in wild-type mice via enhanced autophagy in kidney, while ppara mice displayed impaired autophagy and kidney damage resembling ciliopathy. Furthermore, an NR1H4 agonist exacerbated kidney damage associated with starvation in ppara mice. These findings indicate a previously unknown role for PPARA and NR1H4 in regulating the autophagy-ciliogenesis axis in vivo.

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