Cellular stresses trigger autophagy to remove damaged macromolecules and organelles. Lysosomes ‘host' multiple stress-sensing mechanisms that trigger the coordinated biogenesis of autophagosomes and lysosomes. For example, transcription factor (TF)EB, which regulates autophagy and lysosome biogenesis, is activated following the inhibition of mTOR, a lysosome-localized nutrient sensor. Here we show that reactive oxygen species (ROS) activate TFEB via a lysosomal Ca2+-dependent mechanism independent of mTOR. Exogenous oxidants or increasing mitochondrial ROS levels directly and specifically activate lysosomal TRPML1 channels, inducing lysosomal Ca2+ release. This activation triggers calcineurin-dependent TFEB-nuclear translocation, autophagy induction and lysosome biogenesis. When TRPML1 is genetically inactivated or pharmacologically inhibited, clearance of damaged mitochondria and removal of excess ROS are blocked. Furthermore, TRPML1's ROS sensitivity is specifically required for lysosome adaptation to mitochondrial damage. Hence, TRPML1 is a ROS sensor localized on the lysosomal membrane that orchestrates an autophagy-dependent negative-feedback programme to mitigate oxidative stress in the cell.

Mutations in the SCN1A gene have been identified in epilepsy patients with widely variable phenotypes and modes of inheritance and in asymptomatic carriers. This raises challenges in evaluating the pathogenicity of SCN1A mutations. We systematically reviewed all SCN1A mutations and established a database containing information on functional alterations. In total, 1,257 mutations have been identified, of which 81.8% were not recurrent. There was a negative correlation between phenotype severity and missense mutation frequency. Further analyses suggested close relationships among genotype, functional alteration, and phenotype. Missense mutations located in different sodium channel regions were associated with distinct functional changes. Missense mutations in the pore region were characterized by the complete loss of function, similar to haploinsufficiency. Mutations with severe phenotypes were more frequently located in the pore region, suggesting that functional alterations are critical in evaluating pathogenicity and can be applied to patient management. A negative correlation was found between phenotype severity and familial incidence, and incomplete penetrance was associated with missense and splice site mutations, but not truncations or genomic rearrangements, suggesting clinical genetic counseling applications. Mosaic mutations with a load of 12.5-25.0% were potentially pathogenic with low penetrance, suggesting the need for future studies on less pathogenic genomic variations.

Upon nutrient starvation, autophagy digests unwanted cellular components to generate catabolites that are required for housekeeping biosynthesis processes. A complete execution of autophagy demands an enhancement in lysosome function and biogenesis to match the increase in autophagosome formation. Here, we report that mucolipin-1 (also known as TRPML1 or ML1), a Ca 2+ channel in the lysosome that regulates many aspects of lysosomal trafficking, plays a central role in this quality-control process. By using Ca 2+ imaging and whole-lysosome patch clamping, lysosomal Ca 2+ release and ML1 currents were detected within hours of nutrient starvation and were potently up-regulated. In contrast, lysosomal Na + -selective currents were not upregulated. Inhibition of mammalian target of rapamycin (mTOR) or activation of transcription factor EB (TFEB) mimicked a starvation effect in fed cells. The starvation effect also included an increase in lysosomal proteostasis and enhanced clearance of lysosomal storage, including cholesterol accumulation in Niemann-Pick disease type C (NPC) cells. However, this effect was not observed when ML1 was pharmacologically inhibited or genetically deleted. Furthermore, overexpression of ML1 mimicked the starvation effect. Hence, lysosomal adaptation to environmental cues such as nutrient levels requires mTOR/TFEB-dependent, lysosome-to-nucleus regulation of lysosomal ML1 channels and Ca 2+ signaling.

Diabetes mellitus (DM) affects bone metabolism and leads to osteoporosis; however, its pathogenetic mechanisms remain unknown. We found that high glucose (HG) conditions induced the production of reactive oxygen species (ROS) and the expression of proteins related to MAPKs [phosphorylated (p)‐ERK, p‐JNK, and p‐p38], NF‐κB(NF‐κB, p‐IαB, and IKK), and NACHT‐LRR‐PYD domains‐containing protein 3 (NALP3) (NLRP3) [apoptosis‐associated speck‐like protein containing a caspase activation and recruitment domain (ASC), caspase‐1, IL‐18, IL‐1β, and NLRP3] in osteoclasts (OCs) in vitro. Further analysis showed that in HG‐induced OCs, ROS is an upstream signal for MAPKs, NF‐κB, and the NLRP3 inflammasome. Moreover, MAPKs mediated the activation of NF‐κB and NLRP3, whereas NF‐κB up‐regulated the NLRP3 inflammasome response. Interestingly, HG inducement enhanced the bone resorption of OCs but inhibited their efferocytosis, whereas insulin and lipoxin A4 (4) treatment reversed this phenomenon. In streptozotocin‐induced diabetic rats in vivo, the numbers and the bone‐resorption capacity of OCs as well as the serum levels of TRACP‐5b were significantly increased, and the expression of MAPK‐, NF‐κB‐, and NLRP3 inflammasome‐related proteins in the proximal tibia were also significantly elevated; however, treatment with insulin and LXA4 reversed this elevation. Together, these results demonstrated that the activation of ROS/MAPKs/NF‐κB/NLRP3 and the inhibition of efferocytosis in OCs are the main causes of osteoporosis in DM.—An, Y., Zhang, H., Wang, C., Jiao, F., Xu, H., Wang, X., Luan, W., Ma, F., Ni, L., Tang, X., Liu, M., Guo, W., Yu, L. Activation of ROS/MAPKs/NF‐κB/NLRP3 and inhibition of efferocytosis in osteoclast‐mediated diabetic osteoporosis. FASEB J. 33, 12515–12527 (2019). http://www.fasebj.org