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.

Background: Autophagy is an intracellular degradation process crucial for homeostasis. During autophagy, a double-membrane autophagosome fuses with lysosome through SNARE machinery STX17 to form autolysosome for degradation of damaged organelle. Whereas defective autophagy enhances cholesterol accumulation in the lysosome and impaired autophagic flux that results Niemann-Pick type C1 (NPC1) disease. However, exact interconnection between NPC1 and autophagic flux remain obscure due to the existence of controversial reports.Results: This study aimed at a comparison of the effects of three autophagic inhibitor drugs, including chloroquine, U18666A, and bafilomycin A1, on the intracellular cholesterol transport and autophagy flux. Chloroquine, an autophagic flux inhibitor; U1866A, a NPC1 inhibitor, and bafilomycin A, a lysosomotropic agent are well known to inhibit autophagy by different mechanism. Here we showed that treatment with U1866A and bafilomycin A induces lysosomal cholesterol accumulation that prevented autophagic flux by decreasing autophagosome–lysosome fusion. We also demonstrated that accumulation of cholesterol within the lysosome did not affect lysosomal pH. Although the clearance of accumulated cholesterol by cyclodextrin restored the defective autophagosome–lysosome fusion, the autophagy flux restoration was possible only when lysosomal acidification was not altered. In addition, a failure of STX17 trafficking to autophagosomes plays a key role in prevention of autophagy flux caused by intracellular cholesterol transport inhibitors.Conclusions: Our data provide a new insight that the impaired autophagy flux does not necessarily result in lysosomal cholesterol accumulation even though it prevents autophagosome–lysosome fusion.

Peroxisomes generate reactive oxygen species (ROS) and also play a role in protecting cells from the damaging effects of such radicals. Dysfunctional peroxisomes are recognized by receptors and degraded by a selective type of macroautophagy called pexophagy. Oxidative stress is one of the signals that activates pexophagy through multiple signaling pathways. Conversely, impaired pexophagy results in the accumulation of damaged peroxisomes, which in turn leads to elevated ROS levels and oxidative stress, resulting as cellular dysfunction and the progression of diseases such as neurodegeneration, cancer, and metabolic disorders. This review explores the molecular mechanisms driving pexophagy and its regulation by oxidative stress with a particular focus on ROS. This highlights the role of peroxisomal proteins and ROS-mediated signaling pathways in regulating pexophagy. In addition, emerging evidence suggests that the dysregulation of pexophagy is closely linked to neurological disorders, underscoring its potential as a therapeutic target. Understanding the intricate crosstalk between pexophagy and oxidative stress provides new insights into the maintenance of cellular homeostasis and offers promising directions for addressing neurological disorders that are tightly associated with pexophagy and oxidative stress.

Defects in the PEX5 gene impair the import of peroxisomal matrix proteins, leading to nonfunctional peroxisomes and other associated pathological defects such as Zellweger syndrome. Although PEX5 regulates autophagy process in a stress condition, the mechanisms controlling autophagy by PEX5 under nutrient deprivation are largely unknown. Herein, we show a novel function of PEX5 in the regulation of autophagy via Transcription Factor EB (TFEB). Under serum-starved conditions, when PEX5 is depleted, the mammalian target of rapamycin (mTORC1) inhibitor TSC2 is downregulated, which results in increased phosphorylation of the mTORC1 substrates, including 70S6K, S6K, and 4E-BP-1. mTORC1 activation further suppresses the nuclear localization of TFEB, as indicated by decreased mRNA levels of TFEB, LIPA, and LAMP1. Interestingly, peroxisomal mRNA and protein levels are also reduced by TFEB inactivation, indicating that TFEB might control peroxisome biogenesis at a transcriptional level. Conversely, pharmacological inhibition of mTOR resulting from PEX5 depletion during nutrient starvation activates TFEB by promoting nuclear localization of the protein. In addition, mTORC1 inhibition recovers the damaged-peroxisome biogenesis. These data suggest that PEX5 may be a critical regulator of lysosomal gene expression and autophagy through the mTOR-TFEB-autophagy axis under nutrient deprivation.

Fenofibrate, an activator of peroxisome proliferator-activated receptors (PPARs), has been shown to protect the kidneys and brain cells from oxidative stress; however, its role in preventing hearing loss has not been reported to date, at least to the best of our knowledge. In this study, we demonstrated the protective effects of fenofibrate against gentamicin (GM)-induced ototoxicity. We found that the auditory brainstem response threshold which was increased by GM was significantly reduced by pre-treatment with fenofibrate in rats. In cochlear explants, the disruption of hair cell layers by GM was also markedly attenuated by pre-treatment with fenofibrate. In addition, fenofibrate almost completely abolished GM-induced reactive oxygen species generation, which seemed to be mediated at least in part by the restoration of the expression of PPAR‑α‑dependent antioxidant enzymes, including catalase and superoxide dismutase (SOD)-1. Of note, fenofibrate markedly increased the expression of heme oxygenase-1 (HO-1) which was also induced to a certain degree by GM alone. The induced expression of HO-1 by fenofibrate appeared to be essential for mediating the protective effects of fenofibrate, as the inhibition of HO-1 activity significantly diminished the protective effects of fenofibrate against the GM-mediated death of sensory hair cells in cochlea explant culture, as well as in zebrafish neuromasts. These results suggest that fenofibrate protects sensory hair cells from GM-induced toxicity by upregulating PPAR‑α-dependent antioxidant enzymes, including HO-1. Our results provide insight into the preventive therapy for hearing loss caused by aminoglycoside antibiotics.