Itaconate uptake via SLC13A3 improves hepatic antibacterial innate immunity.

Itaconate is an immunoregulatory metabolite produced by the mitochondrial enzyme immune-responsive gene 1 (IRG1) in inflammatory macrophages. We recently identified an important mechanism by which itaconate is released from inflammatory macrophages. However, it remains unknown whether extracellular itaconate is taken up by non-myeloid cells to exert immunoregulatory functions. Here, we used a custom-designed CRISPR screen to identify the dicarboxylate transporter solute carrier family 13 member 3 (SLC13A3) as an itaconate importer and to characterize the role of SLC13A3 in itaconate-improved hepatic antibacterial innate immunity. Functionally, liver-specific deletion of Slc13a3 impairs hepatic antibacterial innate immunity in vivo and in vitro. Mechanistically, itaconate uptake via SLC13A3 induces transcription factor EB (TFEB)-dependent lysosomal biogenesis and subsequently improves antibacterial innate immunity in mouse hepatocytes. These findings identify SLC13A3 as a key itaconate importer in mouse hepatocytes and will aid in the development of potent itaconate-based antibacterial therapeutics.

Dimorphic effect of TFE3 in determining mitochondrial and lysosomal content in muscle following denervation.

BACKGROUND: Muscle atrophy is a common consequence of the loss of innervation and is accompanied by mitochondrial dysfunction. Mitophagy is the adaptive process through which damaged mitochondria are removed via the lysosomes, which are regulated in part by the transcription factor TFE3. The role of lysosomes and TFE3 are poorly understood in muscle atrophy, and the effect of biological sex is widely underreported. METHODS: Wild-type (WT) mice, along with mice lacking TFE3 (KO), a transcriptional regulator of lysosomal and autophagy-related genes, were subjected to unilateral sciatic nerve denervation for up to 7 days, while the contralateral limb was sham-operated and served as an internal control. A subset of animals was treated with colchicine to capture mitophagy flux. RESULTS: WT females exhibited elevated oxygen consumption rates during active respiratory states compared to males, however this was blunted in the absence of TFE3. Females exhibited higher mitophagy flux rates and greater lysosomal content basally compared to males that was independent of TFE3 expression. Following denervation, female mice exhibited less muscle atrophy compared to male counterparts. Intriguingly, this sex-dependent muscle sparing was lost in the absence of TFE3. Denervation resulted in 45% and 27% losses of mitochondrial content in WT and KO males respectively, however females were completely protected against this decline. Decreases in mitochondrial function were more severe in WT females compared to males following denervation, as ROS emission was 2.4-fold higher. In response to denervation, LC3-II mitophagy flux was reduced by 44% in females, likely contributing to the maintenance of mitochondrial content and elevated ROS emission, however this response was dysregulated in the absence of TFE3. While both males and females exhibited increased lysosomal content following denervation, this response was augmented in females in a TFE3-dependent manner. CONCLUSIONS: Females have higher lysosomal content and mitophagy flux basally compared to males, likely contributing to the improved mitochondrial phenotype. Denervation-induced mitochondrial adaptations were sexually dimorphic, as females preferentially preserve content at the expense of function, while males display a tendency to maintain mitochondrial function. Our data illustrate that TFE3 is vital for the sex-dependent differences in mitochondrial function, and in determining the denervation-induced atrophy phenotype.

Daphmacrimines A-K, Daphniphyllum alkaloids from Daphniphyllum macropodum Miq.

Daphmacrimines A-K (1-11) were isolated from the leaves and stems of Daphniphyllum macropodum Miq. Their structures and stereochemistries were determined by extensive techniques, including HRESIMS, NMR, ECD, IR, and single-crystal X-ray crystallography. Daphmacrimines A-D (1-4) are unprecedented Daphniphyllum alkaloids with a 2-oxazolidinone ring. Daphmacrimine I (9) contains a nitrile group, which is relatively rare in naturally occurring alkaloids. The abilities of daphmacrimines A-D and daphmacrimines G-K to enhance lysosomal biogenesis were evaluated through LysoTracker Red staining. Daphmacrimine K (11) can induce lysosomal biogenesis and promote autophagic flux.

ABCG2 is an itaconate exporter that limits antibacterial innate immunity by alleviating TFEB-dependent lysosomal biogenesis.

Itaconate is a metabolite that synthesized from cis-aconitate in mitochondria and transported into the cytosol to exert multiple regulatory effects in macrophages. However, the mechanism by which itaconate exits from macrophages remains unknown. Using a genetic screen, we reveal that itaconate is exported from cytosol to extracellular space by ATP-binding cassette transporter G2 (ABCG2) in an ATPase-dependent manner in human and mouse macrophages. Elevation of transcription factor TFEB-dependent lysosomal biogenesis and antibacterial innate immunity are observed in inflammatory macrophages with deficiency of ABCG2-mediated itaconate export. Furthermore, deficiency of ABCG2-mediated itaconate export in macrophages promotes antibacterial innate immune defense in a mouse model of S. typhimurium infection. Thus, our findings identify ABCG2-mediated itaconate export as a key regulatory mechanism that limits TFEB-dependent lysosomal biogenesis and antibacterial innate immunity in inflammatory macrophages, implying the potential therapeutic utility of blocking itaconate export in treating human bacterial infections.

SMURF1 controls the PPP3/calcineurin complex and TFEB at a regulatory node for lysosomal biogenesis.

Macroautophagy/autophagy is a homeostatic process in response to multiple signaling, such as the lysosome-dependent recycling process of cellular components. Starvation-induced MTOR inactivation and PPP3/calcineurin activation were shown to promote the nuclear translocation of TFEB. However, the mechanisms via which signals from endomembrane damage are transmitted to activate PPP3/calcineurin and orchestrate autophagic responses remain unknown. This study aimed to show that autophagy regulator SMURF1 controlled TFEB nuclear import for transcriptional activation of the lysosomal biogenesis. We showed that blocking SMURF1 affected lysosomal biogenesis in response to lysosomal damage by preventing TFEB nuclear translocation. It revealed galectins recognized endolysosomal damage, and led to recruitment of SMURF1 and the PPP3/calcineurin apparatus on lysosomes. SMURF1 interacts with both LGALS3 and PPP3CB to form the LGALS3-SMURF1-PPP3/calcineurin complex. Importantly, this complex further stabilizes TFEB, thereby activating TFEB for lysosomal biogenesis. We determined that LLOMe-mediated TFEB nuclear import is dependent on SMURF1 under the condition of MTORC1 inhibition. In addition, SMURF1 is required for PPP3/calcineurin activity as a positive regulator of TFEB. SMURF1 controlled the phosphatase activity of the PPP3CB by promoting the dissociation of its autoinhibitory domain (AID) from its catalytic domain (CD). Overexpression of SMURF1 showed similar effects as the constitutive activation of PPP3CB. Thus, SMURF1, which bridges environmental stress with the core autophagosomal and autolysosomal machinery, interacted with endomembrane sensor LGALS3 and phosphatase PPP3CB to control TFEB activation.Abbreviations: ATG: autophagy-related; LLOMe: L-Leucyl-L-Leucine methyl ester; ML-SA1: mucolipin synthetic agonist 1; MTOR: mechanistic target of rapamycin kinase; PPP3CB: protein phosphatase 3 catalytic subunit beta; RPS6KB1/p70S6K: ribosomal protein S6 kinase B1; SMURF1: SMAD specific E3 ubiquitin protein ligase 1; TFEB: transcription factor EB.

Iridoid glycosides from Morinda officinalis induce lysosomal biogenesis and promote autophagic flux to attenuate oxidative stress.

Morinda officinalis is a traditional Chinese tonic herb, and have been used in the treatment of multiple diseases. Here, three iridoid glycosides isolated from M. officinalis were evaluated for their roles in the autophagy-lysosomal pathway. All three iridoid glycosides could induce TFEB/TFE3-mediated lysosomal biogenesis and trigger autophagy. Interestingly, they promoted the nuclear import of TFEB/TFE3 without affecting their nuclear export, suggesting their role in the maintenance of lysosomal homeostasis. The results from this study shed light on the identification of autophagy activators from M. officinalis and provide a basis for developing them in the treatment of oxidative stress-involved diseases.

Structural and Comparative Analyses of Insects Suggest the Presence of an Ultra-Conserved Regulatory Element of the Genes Encoding Vacuolar-Type ATPase Subunits and Assembly Factors.

Gene and genome comparison represent an invaluable tool to identify evolutionarily conserved sequences with possible functional significance. In this work, we have analyzed orthologous genes encoding subunits and assembly factors of the V-ATPase complex, an important enzymatic complex of the vacuolar and lysosomal compartments of the eukaryotic cell with storage and recycling functions, respectively, as well as the main pump in the plasma membrane that energizes the epithelial transport in insects. This study involves 70 insect species belonging to eight insect orders. We highlighted the conservation of a short sequence in the genes encoding subunits of the V-ATPase complex and their assembly factors analyzed with respect to their exon-intron organization of those genes. This study offers the possibility to study ultra-conserved regulatory elements under an evolutionary perspective, with the aim of expanding our knowledge on the regulation of complex gene networks at the basis of organellar biogenesis and cellular organization.

Altered endosomal-lysosomal biogenesis in melanoma.

Cutaneous melanoma is the deadliest form of skin neoplasm and its high mortality rates could be averted by early accurate detection. While the detection of melanoma is currently reliant upon melanin visualisation, research into melanosome biogenesis, as a key driver of pathogenesis, has not yielded technology that can reliably distinguish between atypical benign, amelanotic and melanotic lesions. The endosomal-lysosomal system has important regulatory roles in cancer cell biology, including a specific functional role in melanosome biogenesis. Herein, the involvement of the endosomal-lysosomal system in melanoma was examined by pooled secondary analysis of existing gene expression datasets. A set of differentially expressed endosomal-lysosomal genes was identified in melanoma, which were interconnected by biological function. To illustrate the protein expression of the dysregulated genes, immunohistochemistry was performed on samples from patients with cutaneous melanoma to reveal candidate markers. This study demonstrated the dysregulation of Syntenin-1, Sortilin and Rab25 may provide a differentiating feature between cutaneous melanoma and squamous cell carcinoma, while IGF2R may indicate malignant propensity in these skin cancers.

Activating transcription factor 4 regulates mitochondrial content, morphology, and function in differentiating skeletal muscle myotubes.

Mitochondrial function is widely recognized as a major determinant of health, emphasizing the importance of understanding the mechanisms promoting mitochondrial quality in various tissues. Recently, the mitochondrial unfolded protein response (UPRmt) has come into focus as a modulator of mitochondrial homeostasis, particularly in stress conditions. In muscle, the necessity for activating transcription factor 4 (ATF4) and its role in regulating mitochondrial quality control (MQC) have yet to be determined. We overexpressed (OE) and knocked down ATF4 in C2C12 myoblasts, differentiated them to myotubes for 5 days, and subjected them to acute (ACA) or chronic (CCA) contractile activity. ATF4 mediated myotube formation through the regulated expression of myogenic factors, mainly Myc and myoblast determination protein 1 (MyoD), and suppressed mitochondrial biogenesis basally through peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1α). However, our data also show that ATF4 expression levels are directly related to mitochondrial fusion and dynamics, UPRmt activation, as well as lysosomal biogenesis and autophagy. Thus, ATF4 promoted enhanced mitochondrial networking, protein handling, and the capacity for clearance of dysfunctional organelles under stress conditions, despite lower levels of mitophagy flux with OE. Indeed, we found that ATF4 promoted the formation of a smaller pool of high-functioning mitochondria that are more responsive to contractile activity and have higher oxygen consumption rates and lower reactive oxygen species levels. These data provide evidence that ATF4 is both necessary and sufficient for mitochondrial quality control and adaptation during both differentiation and contractile activity, thus advancing the current understanding of ATF4 beyond its canonical functions to include the regulation of mitochondrial morphology, lysosomal biogenesis, and mitophagy in muscle cells.

Effect of Lacking ZKSCAN3 on Autophagy, Lysosomal Biogenesis and Senescence.

Transcription factors can affect autophagy activity by promoting or inhibiting the expression of autophagic and lysosomal genes. As a member of the zinc finger family DNA-binding proteins, ZKSCAN3 has been reported to function as a transcriptional repressor of autophagy, silencing of which can induce autophagy and promote lysosomal biogenesis in cancer cells. However, studies in Zkscan3 knockout mice showed that the deficiency of ZKSCAN3 did not induce autophagy or increase lysosomal biogenesis. In order to further explore the role of ZKSCAN3 in the transcriptional regulation of autophagic genes in human cancer and non-cancer cells, we generated ZKSCAN3 knockout HK-2 (non-cancer) and Hela (cancer) cells via the CRISPR/Cas9 system and analyzed the differences in gene expression between ZKSCAN3 deleted cells and non-deleted cells through fluorescence quantitative PCR, western blot and transcriptome sequencing, with special attention to the differences in expression of autophagic and lysosomal genes. We found that ZKSCAN3 may be a cancer-related gene involved in cancer progression, but not an essential transcriptional repressor of autophagic or lysosomal genes, as the lacking of ZKSCAN3 cannot significantly promote the expression of autophagic and lysosomal genes.

Deceiving the big eaters: Salmonella Typhimurium SopB subverts host cell xenophagy in macrophages via dual mechanisms.

Salmonella, a stealthy facultative intracellular pathogen, utilises an array of host immune evasion strategies. This facilitates successful survival via replicative niche establishment in otherwise hostile environments such as macrophages. Salmonella survives in and utilises macrophages for effective dissemination, ultimately leading to systemic infection. Bacterial xenophagy or macro-autophagy is an important host defense mechanism in macrophages. Here, we report for the first time that the Salmonella pathogenicity island-1 (SPI-1) effector SopB is involved in subverting host autophagy via dual mechanisms. SopB is a phosphoinositide phosphatase capable of altering the phosphoinositide dynamics of the host cell. Here, we demonstrate that SopB mediates escape from autophagy by inhibiting the terminal fusion of Salmonella-containing vacuoles (SCVs) with lysosomes and/or autophagosomes. We also report that SopB downregulates overall lysosomal biogenesis by modulating the Akt-transcription factor EB (TFEB) axis via restricting the latter's nuclear localisation. TFEB is a master regulator of lysosomal biogenesis and autophagy. This reduces the overall lysosome content inside host macrophages, further facilitating the survival of Salmonella in macrophages and systemic dissemination of Salmonella.

Trigonochinene E promotes lysosomal biogenesis and enhances autophagy via TFEB/TFE3 in human degenerative NP cells against oxidative stress.

BACKGROUND: Macroautophagy (henceforth autophagy) is the major form of autophagy, which delivers intracellular cargo to lysosomes for degradation. Considerable research has revealed that the impairment of lysosomal biogenesis and autophagic flux exacerbates the development of autophagy-related diseases. Therefore, reparative medicines restoring lysosomal biogenesis and autophagic flux in cells may have therapeutic potential against the increasing prevalence of these diseases. PURPOSE: The aim of the present study was thus to explore the effect of trigonochinene E (TE), an aromatic tetranorditerpene isolated from Trigonostemon flavidus, on lysosomal biogenesis and autophagy and to elucidate the potential underlying mechanism. METHODS: Four human cell lines, HepG2, nucleus pulposus (NP), HeLa and HEK293 cells were applied in this study. The cytotoxicity of TE was evaluated by MTT assay. Lysosomal biogenesis and autophagic flux induced by 40 µM TE were analyzed using gene transfer techniques, western blotting, real-time PCR and confocal microscopy. Immunofluorescence, immunoblotting and pharmacological inhibitors/activators were applied to determine the changes in the protein expression levels in mTOR, PKC, PERK, and IRE1α signaling pathways. RESULTS: Our results showed that TE promotes lysosomal biogenesis and autophagic flux by activating the transcription factors of lysosomes, transcription factor EB (TFEB) and transcription factor E3 (TFE3). Mechanistically, TE induces TFEB and TFE3 nuclear translocation through an mTOR/PKC/ROS-independent and endoplasmic reticulum (ER) stress-mediated pathway. The PERK and IRE1α branches of ER stress are crucial for TE-induced autophagy and lysosomal biogenesis. Whereas TE activated PERK, which mediated calcineurin dephosphorylation of TFEB/TFE3, IRE1α was activated and led to inactivation of STAT3, which further enhanced autophagy and lysosomal biogenesis. Functionally, knockdown of TFEB or TFE3 impairs TE-induced lysosomal biogenesis and autophagic flux. Furthermore, TE-induced autophagy protects NP cells from oxidative stress to ameliorate intervertebral disc degeneration (IVDD). CONCLUSIONS: Here, our study showed that TE can induce TFEB/TFE3-dependent lysosomal biogenesis and autophagy via the PERK-calcineurin axis and IRE1α-STAT3 axis. Unlike other agents regulating lysosomal biogenesis and autophagy, TE showed limited cytotoxicity, thereby providing a new direction for therapeutic opportunities to use TE to treat diseases with impaired autophagy-lysosomal pathways, including IVDD.

ZLN005 improves the survival of polymicrobial sepsis by increasing the bacterial killing via inducing lysosomal acidification and biogenesis in phagocytes.

Polymicrobial sepsis still has a high mortality rate despite the development of antimicrobial agents, elaborate strategies to protect major organs, and the investment of numerous medical resources. Mitochondrial dysfunction, which acts as the center of energy metabolism, is clearly the basis of pathogenesis. Drugs that act on PGC1α, the master regulator of mitochondrial biosynthesis, have shown useful effects in the treatment of sepsis; therefore, we investigated the efficacy of ZLN005, a PGC1α agonist, and found significant improvement in overall survival in an animal model. The mode of action of this effect was examined, and it was shown that the respiratory capacity of mitochondria was enhanced immediately after administration and that the function of TFEB, a transcriptional regulator that promotes lysosome biosynthesis and mutually enhances PGC1α, was enhanced, as was the physical contact between mitochondria and lysosomes. ZLN005 strongly supported immune defense in early sepsis by increasing lysosome volume and acidity and enhancing cargo degradation, resulting in a significant reduction in bacterial load. ZLN005 rapidly acted on two organelles, mitochondria and lysosomes, against sepsis and interactively linked the two to improve the pathogenesis. This is the first demonstration that acidification of lysosomes by a small molecule is a mechanism of action in the therapeutic strategy for sepsis, which will have a significant impact on future drug discovery.

TFE3-mediated impairment of lysosomal biogenesis and defective autophagy contribute to fluoride-induced hepatotoxicity.

Excessive fluoride exposure can cause liver injury, but the specific mechanisms need further investigation. We aimed to explore the role of impaired lysosomal biogenesis and defective autophagy in fluoride-induced hepatotoxicity and its potential mechanisms, focusing on the role of transcription factor E3 (TFE3) in regulating hepatocyte lysosomal biogenesis. To this end, we established a Sprague-Dawley (SD) rat model exposed to sodium fluoride (NaF) and a rat liver cell line (BRL3A) model exposed to NaF. The results showed that NaF exposure diminished liver function and led to apoptosis as well as autophagosome accumulation and impaired autophagic degradation. In addition, NaF exposure caused compromised lysosome biogenesis and decreased lysosomal degradation, and inhibited TFE3 nuclear translocation. Notably, the mTOR inhibitors rapamycin (RAPA) and Ad-TFE3 promoted lysosomal biogenesis and enhanced lysosomal degradation function. Furthermore, RAPA and Ad-TFE3 reduced NaF-induced apoptosis by alleviating impaired autophagic degradation. In conclusion, NaF impairs lysosomal biogenesis by inhibiting TFE3 nuclear translocation, decreasing lysosomal degradation function, resulting in impaired autophagic degradation, and ultimately inducing apoptosis. Therefore, TFE3 may be a promising therapeutic target for fluoride-induced hepatotoxicity.

Effect of the Ketone Body, D-ß-Hydroxybutyrate, on Sirtuin2-Mediated Regulation of Mitochondrial Quality Control and the Autophagy-Lysosomal Pathway.

Mitochondrial activity and quality control are essential for neuronal homeostasis as neurons rely on glucose oxidative metabolism. The ketone body, D-ß-hydroxybutyrate (D-BHB), is metabolized to acetyl-CoA in brain mitochondria and used as an energy fuel alternative to glucose. We have previously reported that D-BHB sustains ATP production and stimulates the autophagic flux under glucose deprivation in neurons; however, the effects of D-BHB on mitochondrial turnover under physiological conditions are still unknown. Sirtuins (SIRTs) are NAD+-activated protein deacetylases involved in the regulation of mitochondrial biogenesis and mitophagy through the activation of transcription factors FOXO1, FOXO3a, TFEB and PGC1α coactivator. Here, we aimed to investigate the effect of D-BHB on mitochondrial turnover in cultured neurons and the mechanisms involved. Results show that D-BHB increased mitochondrial membrane potential and regulated the NAD+/NADH ratio. D-BHB enhanced FOXO1, FOXO3a and PGC1α nuclear levels in an SIRT2-dependent manner and stimulated autophagy, mitophagy and mitochondrial biogenesis. These effects increased neuronal resistance to energy stress. D-BHB also stimulated the autophagic-lysosomal pathway through AMPK activation and TFEB-mediated lysosomal biogenesis. Upregulation of SIRT2, FOXOs, PGC1α and TFEB was confirmed in the brain of ketogenic diet (KD)-treated mice. Altogether, the results identify SIRT2, for the first time, as a target of D-BHB in neurons, which is involved in the regulation of autophagy/mitophagy and mitochondrial quality control.

Lysosomal quality control: molecular mechanisms and therapeutic implications.

Lysosomes are essential catabolic organelles with an acidic lumen and dozens of hydrolytic enzymes. The detrimental consequences of lysosomal leakage have been well known since lysosomes were discovered during the 1950s. However, detailed knowledge of lysosomal quality control mechanisms has only emerged relatively recently. It is now clear that lysosomal leakage triggers multiple lysosomal quality control pathways that replace, remove, or directly repair damaged lysosomes. Here, we review how lysosomal damage is sensed and resolved in mammalian cells, with a focus on the molecular mechanisms underlying different lysosomal quality control pathways. We also discuss the clinical implications and therapeutic potential of these pathways.

The inhibition of TRPML1/TFEB leads to lysosomal biogenesis disorder, contributes to developmental fluoride neurotoxicity.

Fluoride is capable of inducing developmental neurotoxicity; regrettably, the mechanism is obscure. We aimed to probe the role of lysosomal biogenesis disorder in developmental fluoride neurotoxicity-specifically, the regulating effect of the transient receptor potential mucolipin 1 (TRPML1)/transcription factor EB (TFEB) signaling pathway on lysosomal biogenesis. Sprague-Dawley rats were given fluoridated water freely, during pregnancy to the parental rats to 2 months after delivery to the offspring. In addition, neuroblastoma SH-SY5Y cells were treated with sodium fluoride (NaF), with or without mucolipin synthetic agonist 1 (ML-SA1) or adenovirus TFEB (Ad-TFEB) intervention. Our findings revealed that NaF impaired learning and memory as well as memory retention capacities in rat offspring, induced lysosomal biogenesis disorder, and decreased lysosomal degradation capacity, autophagosome accumulation, autophagic flux blockade, apoptosis, and pyroptosis. These changes were evidenced by the decreased expression of TRPML1, nuclear TFEB, LAMP2, CTSB, and CTSD, as well as increased expression of LC3-II, p62, cleaved PARP, NLRP3, Caspase1, and IL-1ß. Furthermore, TRPML1 activation and TFEB overexpression both restored TFEB nuclear protein expression and promoted lysosomal biogenesis while enhancing lysosomal degradation capacity, recovering autophagic flux, and attenuating NaF-induced apoptosis and pyroptosis. Taken together, these results show that NaF promotes the progression of developmental fluoride neurotoxicity by inhibiting TRPML1/TFEB expression and impeding lysosomal biogenesis. Notably, the activation of TRPML1/TFEB alleviated NaF-induced developmental neurotoxicity. Therefore, TRPML1/TFEB may be promising markers of developmental fluoride neurotoxicity.

Lysosome signaling in cell survival and programmed cell death for cellular homeostasis.

Recent developments in lysosome biology have transformed our view of lysosomes from static garbage disposals that can also act as suicide bags to decidedly dynamic multirole adaptive operators of cellular homeostasis. Lysosome-governed signaling pathways, proteins, and transcription factors equilibrate the rate of catabolism and anabolism (autophagy to lysosomal biogenesis and metabolite pool maintenance) by sensing cellular metabolic status. Lysosomes also interact with other organelles by establishing contact sites through which they exchange cellular contents. Lysosomal function is critically assessed by lysosomal positioning and motility for cellular adaptation. In this setting, mechanistic target of rapamycin kinase (MTOR) is the chief architect of lysosomal signaling to control cellular homeostasis. Notably, lysosomes can orchestrate explicit cell death mechanisms, such as autophagic cell death and lysosomal membrane permeabilization-associated regulated necrotic cell death, to maintain cellular homeostasis. These lines of evidence emphasize that the lysosomes serve as a central signaling hub for cellular homeostasis.

Identification of active natural products that induce lysosomal biogenesis by lysosome-based screening and biological evaluation.

Lysosomal biogenesis is an essential adaptive process by which lysosomes exert their function in maintaining cellular homeostasis. Defects in lysosomal enzymes and functions lead to lysosome-related diseases, including lysosomal storage diseases and neurodegenerative disorders. Thus, activation of the autophagy-lysosomal pathway, especially induction of lysosomal biogenesis, might be an effective strategy for the treatment of lysosome-related diseases. In this study, we established a lysosome-based screening system to identify active compounds from natural products that could promote lysosomal biogenesis. The subcellular localizations of master transcriptional regulators of lysosomal genes, TFEB, TFE3 and ZKSCAN3 were examined to reveal the potential mechanisms. More than 200 compounds were screened, and we found that Hdj-23, a triterpene isolated from Walsura cochinchinensis, induced lysosomal biogenesis via activation of TFEB/TFE3. In summary, this study introduced a lysosome-based live cell screening strategy to identify bioactive compounds that promote lysosomal biogenesis, which would provide potential candidate enhancers of lysosomal biogenesis and novel insight for treating lysosome-related diseases.

Molecular mechanism of acetylsalicylic acid in improving learning and memory impairment in APP/PS1 transgenic mice by inhibiting the abnormal cell cycle re-entry of neurons.

Alzheimer's disease (AD) is a neurodegenerative disorder accompanied by the loss and apoptosis of neurons. Neurons abnormally enter the cell cycle, which results in neuronal apoptosis during the course of AD development and progression. However, the mechanisms underlying cell cycle re-entry have been poorly studied. Using neuroblastoma (N) 2a SW and APP/PS1 transgenic (Tg) mice as in vitro and in vivo AD models, we found that the expression of cyclin-dependent kinase (CDK)1/2/4 and cyclin A2/B1/D3/E1 was increased while the protein expression of p18 and p21 was decreased, which led to enhanced cell cycle re-entry in a ß-amyloid protein (Aß)-dependent mechanism. By preparing and treating with the temperature-sensitive chitosan-encapsulated drug delivery system (CS), the abnormal expression of CDK1/2/4, cyclin A2/B1/D3/E1 and p18/21 was partially restored by acetylsalicylic acid (ASA), which decreased the apoptosis of neurons in APP/PS1 Tg mice. Moreover, CDK4 and p21 mediated the effects of ASA on activating transcription factor (TF) EB via peroxisome proliferator-activated receptor (PPAR) α, thus leading to the uptake of Aß by astrocytes in a low-density lipoprotein receptor (Ldlr)-dependent mechanism. Moreover, the mechanisms of Aß-degrading mechanisms are activated, including the production of microtubule-associated protein light chain (LC) 3II and Lamp2 protein by ASA in a PPARα-activated TFEB-dependent manner. All these actions contribute to decreasing the production and deposition of Aß, thus leading to improved cognitive decline in APP/PS1 Tg mice.