Localization Of Transcription Factor EB Research Articles

Pathogenic variants in TMEM184B cause a neurodevelopmental syndrome via alteration of metabolic signaling.

Transmembrane protein 184B (TMEM184B) is an endosomal 7-pass transmembrane protein with evolutionarily conserved roles in synaptic structure and axon degeneration. We report six pediatric patients who have de novo heterozygous variants in TMEM184B. All individuals harbor rare missense or mRNA splicing changes and have neurodevelopmental deficits including intellectual disability, corpus callosum hypoplasia, seizures, and/or microcephaly. TMEM184B is predicted to contain a pore domain, wherein many human disease-associated variants cluster. Structural modeling suggests that all missense variants alter TMEM184B protein stability. To understand the contribution of TMEM184B to neural development in vivo, we suppressed the TMEM184B ortholog in zebrafish and observed microcephaly and reduced anterior commissural neurons, aligning with patient symptoms. Ectopic TMEM184B expression resulted in dominant effects for K184E and G162R. However, in vivo complementation studies demonstrate that all other variants tested result in diminished protein function and indicate a haploinsufficiency basis for disease. Expression of K184E and other variants increased apoptosis in cell lines and altered nuclear localization of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, suggesting disrupted nutrient signaling pathways. Together, our data indicate that TMEM184B variants cause cellular metabolic disruption likely through divergent molecular effects that all result in abnormal neural development.

Lysosome dynamics during human endometrial stromal cells decidualization: effect of para-nonylphenol.

During the process of decidualization, the stromal cells of the endometrium change dynamically to create a favorable environment for embryo implantation. Lysosome activity has often been associated with physiological changes in the endometrium during the pre-implantation period and early pregnancy. In this study, the effect of para-nonylphenol (p-NP), an endocrine disruptor, on human immortalized endometrial stromal cells (tHESCs) was investigated. After exposure to p-NP (1 nM and 1 pM), the cells were examined for the decidualization markers Connexin-43, insulin like growth factor binding protein 1 (IGFBP1) and Prolactin. In addition, the effect of p-NP on lysosome biogenesis and exocytosis was investigated by examining the expression and localization of the transcription factor EB (TFEB) and that of the lysosomal-associated membrane protein 1 (LAMP-1). Finally, we evaluated the effect of p-NP on ECM remodeling using a fibronectin assay. Our results showed that p-NP reduced the expression of Prolactin protein, increased the nuclear localization of TFEB, and induced the increase and translocation of the lysosomal protein LAMP-1 to the membrane of tHESCs. The data indicate an impairment of decidualization and suggest an increase in lysosomal biogenesis and exocytosis, which is supported by the higher release of active cathepsin D by tHESCs. Given the importance of cathepsins in the processing and degradation of the ECM during trophoblast invasiveness and migration into the decidua, our results appear to be clear evidence of the negative effects of p-NP on endometrial processes that are fundamental to reproductive success and the establishment of pregnancy.

Transcriptional dysregulation of autophagy in the muscle of a mouse model of Duchenne muscular dystrophy

It has been reported that autophagic activity is disturbed in the skeletal muscles of dystrophin-deficient mdx mice and patients with Duchenne muscular dystrophy (DMD). Transcriptional regulations of autophagy by FoxO transcription factors (FoxOs) and transcription factor EB (TFEB) play critical roles in adaptation to cellular stress conditions. Here, we investigated whether autophagic activity is dysregulated at the transcription level in dystrophin-deficient muscles. Expression levels of autophagy-related genes were globally decreased in tibialis anterior and soleus muscles of mdx mice compared with those of wild-type mice. DNA microarray data from the NCBI database also showed that genes related to autophagy were globally downregulated in muscles from patients with DMD. These downregulated genes are known as targets of FoxOs and TFEB. Immunostaining showed that nuclear localization of FoxO1 and FoxO3a was decreased in mdx mice. Western blot analyses demonstrated increases in phosphorylation levels of FoxO1 and FoxO3a in mdx mice. Nuclear localization of TFEB was also reduced in mdx mice, which was associated with elevated phosphorylation levels of TFEB. Collectively, the results suggest that autophagy is disturbed in dystrophin-deficient muscles via transcriptional downregulation due to phosphorylation-mediated suppression of FoxOs and TFEB.

Secreted frizzled-related protein 2 ameliorates diabetic cardiomyopathy by activating mitophagy

ObjectivesSecreted frizzled-related protein 2 (SFRP2), a novel adipokine that used to be considered an inhibitor of the canonical Wnt pathway, may play a protective role in metabolic disorders. However, its effect on diabetic cardiomyopathy was still unclear. Accumulating evidence indicates that mitophagy can protect cardiac function in the diabetic heart. The present study aimed to explore the roles of SFRP2 on diabetic cardiomyopathy, focusing on the effects and mechanisms for regulating mitophagy. MethodsWild-type H9c2 cells, Sfrp2 overexpression and knockdown H9c2 cells were exposed to a glucolipotoxic milieu. Reactive oxygen species (ROS) production, cell viability, apoptosis, mitophagy and lysosomal activity were detected. The interaction of SFRP2 with frizzled 5 (FZD5), and its effect on expression and intracellular localization of transcription factor EB (TFEB) and β-catenin were also explored. Diabetic rats and Sfrp2 overexpression diabetic rats were constructed to further document the findings from the in vitro study. ResultsThe expression of SFRP2 was low and mitophagy was inhibited in H9c2 cells in a glucolipotoxic milieu. Sfrp2 overexpression activated mitophagy and reduced H9c2 cells injury, whereas Sfrp2 deficiency inhibited mitophagy and worsened this injury. Consistent with the in vitro findings, Sfrp2 overexpression ameliorated the impairment in cardiac function of diabetic rats by activating mitophagy. Sfrp2 overexpression upregulated the expression of calcineurin and TFEB, but did not affect β-catenin in vitro and in vivo. The calcineurin inhibitor tacrolimus can inhibit mitophagy and worsen cell injury in Sfrp2 overexpression H9c2 cells. Furthermore, we found that FZD5 is required for the SFRP2-induced activation of the calcineurin/TFEB pathway and interacts with SFRP2 in H9c2 cells. Transfection with small interfering RNA targeting FZD5 opposed the effects of Sfrp2 overexpression on mitophagy and cell survival in a glucolipotoxic environment. ConclusionsSFRP2 can protect the diabetic heart by interacting with FZD5 and activating the calcineurin/TFEB pathway to upregulate mitophagy in H9c2 cells.

Intermittent hypoxia therapy ameliorates beta-amyloid pathology via TFEB-mediated autophagy in murine Alzheimer's disease

BackgroundAlzheimer's disease (AD) is the most prevalent neurodegenerative disorder. Impaired autophagy in plaque-associated microglia (PAM) has been reported to accelerate amyloid plaque deposition and cognitive impairment in AD pathogenesis. Recent evidence suggests that the transcription factor EB (TFEB)-mediated activation of the autophagy–lysosomal pathway is a promising treatment approach for AD. Moreover, the complementary therapy of intermittent hypoxia therapy (IHT) has been shown to upregulate autophagy and impart beneficial effects in patients with AD. However, the effect of IHT on PAM remains unknown.Methods8-Month-old APP/PS1 mice were treated with IHT for 28 days. Spatial learning memory capacity and anxiety in mice were investigated. AD pathology was determined by the quantity of nerve fibers and synapses density, numbers of microglia and neurons, Aβ plaque deposition, pro-inflammatory factors, and the content of Aβ in the brain. TFEB-mediated autophagy was determined by western blot and qRT-PCR. Primary microglia were treated with oligomeric Aβ 1–42 (oAβ) combined with IHT for mechanism exploration. Differential genes were screened by RNA-seq. Autophagic degradation process of intracellular oAβ was traced by immunofluorescence.ResultsIn this study, we found that IHT ameliorated cognitive function by attenuating neuronal loss and axonal injury in an AD animal model (APP/PS1 mice) with beta-amyloid (Aβ) pathology. In addition, IHT-mediated neuronal protection was associated with reduced Aβ accumulation and plaque formation. Using an in vitro PAM model, we further confirmed that IHT upregulated autophagy-related proteins, thereby promoting the Aβ autophagic degradation by PAM. Mechanistically, IHT facilitated the nuclear localization of TFEB in PAM, with TFEB activity showing a positive correlation with Aβ degradation by PAM in vivo and in vitro. In addition, IHT-induced TFEB activation was associated with the inhibition of the AKT–MAPK–mTOR pathway.ConclusionsThese results suggest that IHT alleviates neuronal damage and neuroinflammation via the upregulation of TFEB-dependent Aβ clearance by PAM, leading to improved learning and memory in AD mice. Therefore, IHT may be a promising non-pharmacologic therapy in complementary medicine against AD.Graphical

Promoting TFEB nuclear localization with curcumin analog C1 attenuates sensory hair cell injury and delays age-related hearing loss in C57BL/6 mice

Sensory hair cell (HC) injuries, especially outer hair cell (OHC) loss, are well-documented to be the primary pathology of age-related hearing loss (AHL). Recent studies have demonstrated that autophagy plays an important role in HC injury in the inner ear. In our previous works, a decline in autophagy levels and HC loss were found to occur simultaneously in the inner ears of aged C57BL/6 mice, and the administration of rapamycin promoted autophagy levels, which reduced OHC loss and delayed AHL, but the underlying mechanism of autophagy in AHL has not been well elucidated. Transcription factor EB (TFEB), an autophagy regulator and the downstream target of mammalian target of rapamycin (mTOR), is involved in the pathological development of neurodegenerative disease. This study would address the link between autophagy and TFEB in aged C57BL/6 mouse cochleae and clarify the effect of the TFEB activator curcumin analog C1 (C1) in aged cochleae. Decreased TFEB nuclear localization (p = 0.0371) and autophagy dysfunction (p = 0.0273) were observed in the cochleae of aged C57BL/6 mice that exhibited AHL, HCs loss and HCs senescence. Treatment with C1 promoted TFEB nuclear localization and restored autophagy, subsequently alleviating HC injury and delaying AHL. The protective effect of C1 on HEI-OC1 cells against autophagy disorder and aging induced by D-galactose was abolished by chloroquine, which is one of the commonly used autophagy inhibitors. Overall, our results demonstrated that the capacity to perform autophagy is mediated by the nuclear localization of TFEB in aged C57BL/6 mouse cochleae. C1 promotes the nuclear localization of TFEB, subsequently alleviating HC injury and delaying AHL by restoring the impaired autophagy function. TFEB may serve as a new therapeutic target for AHL treatment.

Melatonin inhibits atherosclerosis progression via galectin-3 downregulation to enhance autophagy and inhibit inflammation.

Autophagy deficiency in macrophages exacerbates inflammation in atherosclerosis (AS),and recently, galectin-3 (Gal-3) has been implicated as a critical promoter of inflammation in AS. Further, melatonin (Mel) exerts an autophagy-promoting effect in many chronic inflammatory diseases. In this study, we aimed to investigate whether Mel inhibits AS progression by downregulating Gal-3 to enhance autophagy and inhibit inflammation. Thus, we performed in vivo and in vitro experiments using high-fat diet (HFD)-fed ApoE-/- mice and THP-1 macrophages, respectively. Smart-seq of AS plaque macrophages revealed that the differentially expressed genes (DEGs) downregulated by Mel were enriched in immune-related processes, and changes in inflammation status were confirmed based on lower levels of proinflammatory factors in Mel-treated HFD-fed ApoE-/- mice and THP-1 macrophages. Further, via transcriptome-based multiscale network pharmacology platform (TMNP), the upstream target genes of the smart-seq DEGs were identified, and Gal-3 showed a high score. Gal-3 was downregulated both in vivo and in vitro by Mel treatment. Besides, the enrichment of the target genes predicted via the TMNP method indicated that autophagy considerably affected the DEGs. Mel treatment as well as Gal-3 knockdown downregulated most inflammatory response-related proteins could attribute to enhancing autophagy. Mechanistically, Mel treatment inhibited Gal-3 leading to lowering the activity of the nuclear transcription factor-kappa B (NF-κB) pathway, and promoting the nuclear localization of transcription factor EB (TFEB). However, increased secretion of Gal-3 activated the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway and impaired autophagy via binding to CD98.Thus, Mel promoted autophagy and restrained inflammation by downregulating Gal-3, implying that it holds promise as a treatment for AS.

High Cholesterol-Induced Bone Loss Is Attenuated by Arctiin via an Action in Osteoclasts.

High cholesterol-induced bone loss is highly associated with oxidative stress, which leads to the generation of oxysterols, such as 7-ketocholesterol (7-KC). Here, we conducted in vivo and in vitro experiments to determine whether arctiin prevents high cholesterol diet-induced bone loss by decreasing oxidative stress. First, arctiin was orally administered to atherogenic diet (AD)-fed C57BL/6J male mice at a dose of 10 mg/kg for 6 weeks. Micro-computerized tomography (μCT) analysis showed that arctiin attenuated AD-induced boss loss. For our in vitro experiments, the anti-oxidant effects of arctiin were evaluated in 7-KC-stimulated osteoclasts (OCs). Arctiin decreased the number and activity of OCs and inhibited autophagy by disrupting the nuclear localization of transcription factor EB (TFEB) and downregulating the oxidized TFEB signaling pathway in OCs upon 7-KC stimulation. Furthermore, arctiin decreased the levels of reactive oxygen species (ROS) by enhancing the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), catalase, and heme oxygenase 1 (HO-1), all of which affected OC differentiation. Conversely, silencing of Nrf2 or HO-1/catalase attenuated the effects of arctiin on OCs. Collectively, our findings suggested that arctiin attenuates 7-KC-induced osteoclastogenesis by increasing the expression of ROS scavenging genes in the Nrf2/HO-1/catalase signaling pathway, thereby decreasing OC autophagy. Moreover, arctiin inhibits the oxidation and nuclear localization of TFEB, thus protecting mice from AD-induced bone loss. Our findings thus demonstrate the therapeutic potential of arctiin for the prevention of cholesterol-induced bone loss.

Doxorubicin Induces Bone Loss by Increasing Autophagy through a Mitochondrial ROS/TRPML1/TFEB Axis in Osteoclasts.

Doxorubicin (DOX), a widely used chemotherapeutic agent, has been linked to an increased risk of bone damage in human patients and induces bone loss in mice. DOX induces autophagy, which contributes to bone homeostasis and excess autophagy in osteoclasts (OCs), resulting in bone loss. We hypothesized that DOX-induced bone loss is caused by the induction of autophagy in OCs. In vitro, DOX significantly increased the area of OCs and bone resorption activity, whereas it decreased OC number through apoptosis. DOX enhanced the level of LC3II and acidic vesicular organelles-containing cells in OCs, whereas an autophagy inhibitor, 3-methyladenine (3-MA), reversed these, indicating that enhanced autophagy was responsible for the effects of DOX. Increased mitochondrial reactive oxygen species (mROS) by DOX oxidized transient receptor potential mucolipin 1 (TRPML1) on the lysosomal membrane, which led to nuclear localization of transcription factor EB (TFEB), an autophagy-inducing transcription factor. In vivo, micro-computerized tomography analysis revealed that the injection of 3-MA reversed DOX-induced bone loss, and tartrate-resistant acid phosphatase staining showed that 3-MA reduced the area of OCs on the bone surface, which was enhanced upon DOX administration. Collectively, DOX-induced bone loss is at least partly attributable to autophagy upregulation in OCs via an mROS/TRPML1/TFEB axis.

Feeding activates FGF15‐SHP‐TFEB‐mediated lipophagy in the gut

Lysosome‐mediated macroautophagy, including lipophagy, is activated under nutrient deprivation but is repressed after feeding. We show that, unexpectedly, feeding activates intestinal autophagy/lipophagy in a manner dependent on both the orphan nuclear receptor, small heterodimer partner (SHP/NR0B2), and the gut hormone, fibroblast growth factor‐15/19 (FGF15/19). Furthermore, postprandial intestinal triglycerides (TGs) and apolipoprotein‐B48 (ApoB48), the TG‐rich chylomicron marker, were elevated in SHP‐knockout and FGF15‐knockout mice. Genomic analyses of the mouse intestine indicated that SHP partners with the key lysosomal activator, transcription factor‐EB (TFEB) to upregulate the transcription of autophagy/lipolysis network genes after feeding. FGF19 treatment activated lipophagy, reducing TG and ApoB48 levels in HT29 intestinal cells, which was dependent on TFEB. Mechanistically, feeding‐induced FGF15/19 signaling increased the nuclear localization of TFEB and SHP via PKC beta/zeta‐mediated phosphorylation, leading to increased transcription of the TFEB/SHP target lipophagy genes, Ulk1 and Atgl. Collectively, these results demonstrate that paradoxically after feeding, FGF15/19‐activated SHP and TFEB activate gut lipophagy, limiting postprandial TGs. As excess postprandial lipids cause dyslipidemia and obesity, the FGF15/19‐SHP‐TFEB axis that reduces intestinal TGs via lipophagic activation provides promising therapeutic targets for obesity‐associated metabolic disease.

Elucidation of TFEB nuclear translocation dynamics in human cells by means of Quantitative Modelling and Microfluidics

The Transcription Factor EB (TFEB) plays a pivotal role in the transcriptional regulation of lysosomal biogenesis and autophagy in response to starvation. TFEB activity is regulated by the kinase mTOR. In nutrient-rich conditions, mTOR phosphorylates TFEB by sequestering it in the cytoplasm. During starvation, mTOR is inhibited and unphosphorylated TFEB translocates into the nucleus, where it regulates the expression of its target genes. We performed an experiment monitoring in real-time the nuclear localization of TFEB in individual cells growing in a microfluidics device following alternating pulses of starvation and refeeding. The results revealed the presence of an “overshoot” dynamics with TFEB translocating to the nucleus upon starvation but then partially retranslocating to the cytoplasm. To elucidate the mechanism behind its nuclear shuttling dynamics, we first developed a two-compartment dynamical model (nucleus and cytoplasm) with two different species (dephosphorylated and phosphorylated TFEB) for each of the two compartments. The transport and de/phosphorylation kinetics are described with first order kinetics whose parameters depend on the nutrient status, while the total TFEB is kept constant. This model was not capable to recapitulate the observed overshoot dynamics. Consequently, we investigated two alternative models, hypothesizing that upon starvation, a nuclear export channel is activated either by TFEB itself (negative feedback loop - NFL) or as a direct consequence of mTOR inhibition (incoherent feedforward loop -IFFL). The new models were both able to qualitatively recapitulate the observed dynamics. Finally, we propose an experiment able to distinguish between the two alternative models.

Increase in the sensitivity to PLX4720 through inhibition of transcription factor EB-dependent autophagy in BRAF inhibitor-resistant cells.

Long-term treatment with oncogenic BRAF inhibitors confers resistance to BRAF inhibitor monotherapy. In this study, a combination treatment strategy with autophagy inhibitors was proposed to increase the sensitivity of BRAF mutant containing A375P melanoma cells that have developed resistance to BRAF inhibitors. We found that the A375P/Multi-drug resistance (A375P/Mdr) cells, which are resistant to both BRAF inhibitors and MEK inhibitors, exhibited a higher basal autophagic flux compared to their parental A375P cells, as determined by tandem mRFP-GFP-tagged LC3 imaging assay and LC3 conversion. In addition, transcription factor EB (TFEB), which acts as a transcription factor regulating the transcription of autophagy-related genes, was much more localized in the nucleus in A375P/Mdr cells than in A375P cells, indicating that the increase in basal autophagic flux was TFEB-dependent. In particular, the overexpression of an activated form of TFEB (TFEBAA) caused a modest increase in PLX4720 resistance in A375P/Mdr cells. Interestingly, treatment with early stage autophagy inhibitors reversed BRAF inhibitor-induced resistance, whereas late autophagy inhibition did not. In contrast, inhibition of ER stress by 4-phenylbutyric acid suppressed basal autophagic flux. Moreover, ER stress inhibition significantly remarkably inhibited the nuclear localization of TFEB, resulting in an increase in the sensitivity of A375P/Mdr cells to PLX4720. Taken together, these results suggest that autophagy may be an important mechanism of acquired resistance to BRAF inhibitors. Thus, targeting autophagy may be suitable for the treatment of tumors resistant to BRAF inhibitor.

The human vault RNA enhances tumorigenesis and chemoresistance through the lysosome in hepatocellular carcinoma

ABSTRACT The small non-coding VTRNA1-1 (vault RNA 1–1) is known to confer resistance to apoptosis in several malignant cell lines and to also modulate the macroautophagic/autophagic flux in hepatocytes, thus highlighting its pro-survival role. Here we describe a new function of VTRNA1-1 in regulating in vitro and in vivo tumor cell proliferation, tumorigenesis and chemoresistance. Knockout (KO) of VTRNA1-1 in human hepatocellular carcinoma cells reduced nuclear localization of TFEB (transcription factor EB), leading to a downregulation of the coordinated lysosomal expression and regulation (CLEAR) network genes and lysosomal compartment dysfunction. We demonstrate further that impaired lysosome function due to loss of VTRNA1-1 potentiates the anticancer effect of conventional chemotherapeutic drugs. Finally, loss of VTRNA1-1 reduced drug lysosomotropism allowing higher intracellular compound availability and thereby significantly reducing tumor cell proliferation in vitro and in vivo. These findings reveal a so far unknown role of VTRNA1-1 in the intracellular catabolic compartment and describe its contribution to lysosome-mediated chemotherapy resistance.

PARsylated transcription factor EB (TFEB) regulates the expression of a subset of Wnt target genes by forming a complex with \u03b2-catenin-TCF/LEF1

Wnt signaling is mainly transduced by β-catenin via regulation of the β-catenin destruction complex containing Axin, APC, and GSK3β. Transcription factor EB (TFEB) is a well-known master regulator of autophagy and lysosomal biogenesis processes. TFEB’s nuclear localization and transcriptional activity are also regulated by various upstream signals. In this study, we found that Wnt signaling induces the nuclear localization of TFEB and the expression of Wnt target genes is regulated by TFEB-β-catenin-TCF/LEF1 as well as β-catenin-TCF/LEF1 complexes. Our biochemical data revealed that TFEB is a part of the β-catenin destruction complex, and destabilization of the destruction complex by knockdown of either Axin or APC causes nuclear localization of TFEB. Interestingly, RNA-sequencing analysis revealed that about 27% of Wnt3a-induced genes were TFEB dependent. However, these “TFEB mediated Wnt target genes” were different from TFEB target genes involved in autophagy and lysosomal biogenesis processes. Mechanistically, we found that Tankyrase (TNKS) PARsylates TFEB with Wnt ON signaling, and the nuclear localized PARsylated TFEB forms a complex with β-catenin-TCF/LEF1 to induce the “TFEB mediated Wnt target genes”. Finally, we found that in various types of cancer, the levels of TFEB mediated Wnt target genes exhibit strong correlations with the level of Axin2, which represents the activity of Wnt signaling. Overall, our data suggest that Wnt signaling induces the expression of a subset of genes that are distinct from previously known genes regulated by the β-catenin-TCF/LEF1 complex or TFEB, by forming a transcription factor complex consisting of PARsylated TFEB and β-catenin-TCF/LEF1.

20-Deoxyingenol alleviates osteoarthritis by activating TFEB in chondrocytes

Osteoarthritis (OA) is an age-related degenerative disease and currently cannot be cured. Transcription factor EB (TFEB) is one of the major transcriptional factors that regulates autophagy and lysosomal biogenesis. TFEB has been shown to be an effective therapeutic target for many diseases including OA. The current study explores the therapeutic effects of 20-Deoxyingenol (20-DOI) on OA as well as its working mechanism on TFEB regulation. The in vitro study showed that 20-DOI may suppress apoptosis and senescence induced by oxidative stress in chondrocytes; it may also promote the nuclear localization of TFEB in chondrocytes. Knock-down of TFEB compromised the effects of 20-DOI on apoptosis and senescence. The in vivo study demonstrated that 20-DOI may postpone the progression of OA in mouse destabilization of the medial meniscus (DMM) model; it may also suppress apoptosis and senescence and promote the nuclear localization of TFEB in chondrocytes in vivo. This work suggests that 20-Deoxyingenol may alleviate osteoarthritis by activating TFEB in chondrocytes, while 20-DOI may become a potential drug for OA therapy.

Pseudoginsenoside F11 ameliorates the dysfunction of the autophagy-lysosomal pathway by activating calcineurin-mediated TFEB nuclear translocation in neuron during permanent cerebral ischemia

We have previously found that transcription factor EB (TFEB), as a master regulator of autophagy and lysosome biogenesis, provides neuroprotective effects on cerebral ischemia-induced neuronal damage by activation of autophagy-lysosomal pathway (ALP). We have also reported that Pseudoginsenoside F11 (PF11), an ocotillol-type saponin isolated from Panax quinquefolium L., significantly attenuates the ischemic injury of rats subjected to permanent middle cerebral artery occlusion (pMCAO), possibly by alleviating the autophagic/lysosomal defects. The present study aims to investigate whether the beneficial effect of PF11 on ALP dysfunction induced by permanent ischemic stroke is based on its regulation of TFEB nuclear translocation in pMCAO rats and the oxygen-glucose-deprived (OGD) primary neurons. Meanwhile, the role of calcineurin, a serine/threonine protein phosphatase, during this process in which PF11 regulated TFEB transcriptional activity was also explored. The data showed that PF11 exerted significant protective effects on pMCAO-induced injury and decreased OGD-induced neuronal death. The nuclear localization of TFEB was decreased at 24 h after pMCAO. Notably, PF11 (6, 12 mg/kg, i.v.) significantly increased TFEB nuclear expression and Tfeb mRNA level at 24 h following pMCAO. OGD treatment promoted TFEB aggregation and nuclear translocation until 6 h, and the nuclear localization of TFEB was decreased at 12 h. Similarly, PF11 (30, 100 μM) could also promote the translocation of TFEB into nuclear in primary neurons at 12 h after OGD treatment. Moreover, PF11 attenuated OGD-induced lysosomal dysfunction and abnormal accumulation of autophagosomes and substrates. These in vitro effects could be abolished by neuronal-specific knocking down of TFEB via transfecting primary neurons with lentivirus encoding shTfeb. Further studies indicated that cyclosporine (10 μM), an inhibitor of calcineurin, could significantly diminish the effects of PF11 on TFEB nuclear translocation and ALP dysfunction in OGD-treated neurons. In summary, these results demonstrate that PF11 attenuates the dysfunction of ALP in permanent cerebral ischemia by promoting the calcineurin-mediated nuclear translocation of TFEB and further identifies an autophagic mechanism of PF11 against cerebral ischemia.

Trehalose alleviates apoptosis by protecting the autophagy-lysosomal system in alveolar macrophages during human silicosis

BackgroundAlveolar macrophages (AMs) are the primary targets of silicosis. Blockade of autophagy may aggravate the apoptosis of AMs. Trehalose (Tre), a transcription factor EB (TFEB) activator, may impact the autophagy-lysosomal system in AMs during silicosis. However, the mechanism by which Tre acts upon AMs in silicosis is unknown. MethodsWe collected AMs from twenty male workers exposed to silica and divided them into observer and silicosis patient groups. AMs from the two groups were then exposed to Tre. Western blot was used to measure the expression of autophagy-associated proteins. Lysosomal-associated membrane protein 1 (LAMP1) expression was observed using immunofluorescence and western blot. Apoptosis of the AMs was detected by TUNEL assay and western blot. ResultsTre induced localization of TFEB to the nucleus in the AMs of both groups. After Tre exposure, LAMP1 levels increased and LC3 levels decreased in the AMs of both groups, suggesting that Tre may increase the function of the autophagy-lysosomal system. The LC3-II/I ratio in the Tre-exposed AMs was lower than in the AMs not exposed to Tre. The LC3-II/I ratio in AMs subjected to Tre plus Bafilomycin (Baf) was higher than the ratio in cells exposed to Tre or Baf individually. Additionally, p62 levels decreased after Tre stimulation in the AMs of both groups. This indicates that Tre may accelerate the process of autophagic degradation. We also found decreased levels of cleaved caspase-3 after Tre treatment in the AMs of both groups. However, p-mTOR (Ser2448) and p-mTOR (Ser2481) levels did not change significantly after Tre treatment, suggesting that the mTOR signaling pathway was not affected by Tre treatment. ConclusionOur findings suggest that the restoration of autophagy-lysosomal function by Tre may be a potential protective strategy against silicosis.

PGC-1α overexpression increases transcription factor EB nuclear localization and lysosome abundance in dystrophin-deficient skeletal muscle.

Duchenne muscular dystrophy (DMD) is caused by the absence of functional dystrophin protein and results in progressive muscle wasting. Dystrophin deficiency leads to a host of dysfunctional cellular processes including impaired autophagy. Autophagic dysfunction appears to be due, at least in part, to decreased lysosomal abundance mediated by decreased nuclear localization of transcription factor EB (TFEB), a transcription factor responsible for lysosomal biogenesis. PGC‐1α overexpression decreased disease severity in dystrophin‐deficient skeletal muscle and increased PGC‐1α has been linked to TFEB activation in healthy muscle. The purpose of this study was to determine the extent to which PGC‐1α overexpression increased nuclear TFEB localization, increased lysosome abundance, and increased autophagosome degradation. We hypothesized that overexpression of PGC‐1α would drive TFEB nuclear translocation, increase lysosome biogenesis, and improve autophagosome degradation. To address this hypothesis, we delivered PGC‐1α via adeno‐associated virus (AAV) vector injected into the right limb of 3‐week‐old mdx mice and the contralateral limbs received a sham injection. At 6 weeks of age, this approach increased PGC‐1α transcript by 60‐fold and increased TFEB nuclear localization in gastrocnemii from PGC‐1α treated limbs by twofold compared to contralateral controls. Furthermore, lamp2, a marker of lysosome abundance, was significantly elevated in muscles from limbs overexpressing PGC‐1α. Lastly, increased LC3II and similar p62 in PGC‐1α overexpressing‐limbs compared to contralateral limbs are supportive of increased degradation of autophagosomes. These data provide mechanistic insight into PGC‐1α‐mediated benefits to dystrophin‐deficient muscle, such that increased TFEB nuclear localization in dystrophin‐deficient muscle leads to increased lysosome biogenesis and autophagy.

Dysregulation of TFEB contributes to manganese-induced autophagic failure and mitochondrial dysfunction in astrocytes

ABSTRACT Epidemiological and clinical studies have long shown that exposure to high levels of heavy metals are associated with increased risks of neurodegenerative diseases. It is widely accepted that autophagic dysfunction is involved in pathogenesis of various neurodegenerative disorders; however, the role of heavy metals in regulation of macroautophagy/autophagy is unclear. Here, we show that manganese (Mn) induces a decline in nuclear localization of TFEB (transcription factor EB), a master regulator of the autophagy-lysosome pathway, leading to autophagic dysfunction in astrocytes of mouse striatum. We further show that Mn exposure suppresses autophagic-lysosomal degradation of mitochondria and induces accumulation of unhealthy mitochondria. Activation of autophagy by rapamycin or TFEB overexpression ameliorates Mn-induced mitochondrial respiratory dysfunction and reactive oxygen species (ROS) generation in astrocytes, suggesting a causal relation between autophagic failure and mitochondrial dysfunction in Mn toxicity. Taken together, our data demonstrate that Mn inhibits TFEB activity, leading to impaired autophagy that is causally related to mitochondrial dysfunction in astrocytes. These findings reveal a previously unappreciated role for Mn in dysregulation of autophagy and identify TFEB as a potential therapeutic target to mitigate Mn toxicity. Abbreviations BECN1: beclin 1; CTSD: cathepsin D; DMEM: Dulbecco’s Modified Eagle Medium; GFAP: glial fibrillary acid protein; GFP: green fluorescent protein; HBSS: hanks balanced salt solution; LAMP: lysosomal-associated membrane protein; LDH: lactate dehydrogenase; Lys Inh: lysosomal inhibitors; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAPK: mitogen-activated protein kinase; Mn: manganese; MTOR: mechanistic target of rapamycin kinase; OCR: oxygen consumption rate; PBS: phosphate-buffered saline; PFA: paraformaldehyde; PI: propidium iodide; ROS: reactive oxygen species; s.c.: subcutaneous; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TFEB: transcription factor EB

Autophagy and Inflammasome Activation in Dilated Cardiomyopathy.

Background: The clinical outcome of patients affected by dilated cardiomyopathy (DCM) is heterogeneous, since its pathophysiology is only partially understood. Interleukin 1β levels could predict the mortality and necessity of cardiac transplantation of DCM patients. Objective: To investigate mechanisms triggering sterile inflammation in dilated cardiomyopathy (DCM). Methods: Hearts explanted from 62 DCM patients were compared with 30 controls, employing immunohistochemistry, cellular and molecular biology, as well as metabolomics studies. Results: Although misfolded protein accumulation and aggresome formation characterize DCM hearts, aggresomes failed to trigger the autophagy lysosomal pathway (ALP), with consequent accumulation of both p62SQSTM1 and dysfunctional mitochondria. In line, DCM hearts are characterized by accumulation of lipoperoxidation products and activation of both redox responsive pathways and inflammasome. Consistently with the fact that mTOR signaling may impair ALP, we observed, an increase in DCM activation, together with a reduction in the nuclear localization of Transcription Factor EB -TFEB- (a master regulator of lysosomal biogenesis). These alterations were coupled with metabolomic alterations, including accumulation of branched chain amino acids (BCAAs), known mTOR activators. Consistently, reduced levels of PP2Cm, a phosphatase that regulates the key catabolic step of BCAAs, coupled with increased levels of miR-22, a regulator of PP2Cm levels that triggers senescence, characterize DCM hearts. The same molecular defects were present in clinically relevant cells isolated from DCM hearts, but they could be reverted by downregulating miR-22. Conclusion: We identified, in human DCM, a complex series of events whose key players are miR-22, PP2Cm, BCAA, mTOR, and ALP, linking loss of proteostasis with inflammasome activation. These potential therapeutic targets deserve to be further investigated.