Activation Of Transcription Factor EB Research Articles

Rottlerin Enhances the Autophagic Degradation of Phosphorylated Tau in Neuronal Cells.

Intra-neuronal accumulation of hyper-phosphorylated tau as neurofibrillary tangles (NFT) is a hallmark of Alzheimer's disease (AD). To prevent the aggregation of phosphorylated tau in neurons, decreasing the phosphorylated tau protein levels is important. Here, we examined the biological effects of rottlerin, a phytochemical compound extracted from the Kamala tree, Mallotus philippinensis, on phosphorylated tau levels. Notably, rottlerin decreased the levels of intracellular phosphorylated and total tau. A marked increase in the LC3-II, a hallmark of autophagy, was observed in these cells, indicating that rottlerin strongly induced autophagy. Interestingly, rottlerin induced the phosphorylation of Raptor at S792 through the activation of adenosine-monophosphate activated-protein kinase (AMPK), which likely inhibits the mammalian target of rapamycin complex 1 (mTORC1), thus resulting in the activation of transcription factor EB (TFEB), a master regulator of autophagy. In addition, nuclear factor erythroid 2-related factor 2 (Nrf2) activity increased in the presence of rottlerin. The decrease of phosphorylated tau levels in the presence of rottlerin was ameliorated by the knockdown of TFEB andpartially attenuated by the knockout of the Nrf2 gene. Taken together, rottlerin likely enhances the degradation of phosphorylated tau through autophagy activated by TFEB and Nrf2. Thus, our results suggest that a natural compound rottlerin could be used as a preventive and therapeutic drug for AD.

Puerarin enhances TFEB-mediated autophagy and attenuates ROS-induced pyroptosis after ischemic injury of random-pattern skin flaps

Background and aimNecrosis of random-pattern flaps restricts their application in clinical practice. Puerarin has come into focus due to its promising therapeutic effects in ischemic diseases. Here, we employed Puerarin and investigated its role and potential mechanisms in flap survival. Experimental procedureThe effect of Puerarin on the viability of human umbilical vein endothelial cells (HUVECs) was assessed by CCK-8, EdU staining, migration, and scratch assays. Survival area measurement and laser Doppler blood flow (LDBF) were utilized to assess the viability of ischemic injury flaps. Levels of molecules related to oxidative stress, pyroptosis, autophagy, transcription factor EB (TFEB), and the AMPK-TRPML1-Calcineurin signaling pathway were detected using western blotting, immunofluorescence, dihydroethidium (DHE) staining, RT-qPCR and Elisa. Key resultsThe findings demonstrated that Puerarin enhanced the survivability of ischemic flaps. Autophagy, oxidative stress, and pyroptosis were implicated in the ability of Puerarin in improving flap survival. Increased autophagic flux and augmented tolerance to oxidative stress contribute to Puerarin's suppression of pyroptosis. Additionally, Puerarin modulated the activity of TFEB through the AMPK-TRPML1-Calcineurin signaling pathway, thereby enhancing autophagic flux. Conclusions and implicationsPuerarin promoted flap survival from ischemic injury through upregulation of TFEB-mediated autophagy and inhibition of oxidative stress. Our findings offered valuable support for the clinical application of Puerarin in the treatment of ischemic diseases, including random-pattern flaps.

TXNIP deficiency attenuates renal fibrosis by modulating mTORC1/TFEB-mediated autophagy in diabetic kidney disease

Thioredoxin-interacting protein (TXNIP) is an important regulatory protein for thioredoxin (TRX) that elicits the generation of reactive oxygen species (ROS) by inhibiting the redox function of TRX. Abundant evidence suggests that TXNIP is involved in the fibrotic process of diabetic kidney disease (DKD). However, the potential mechanism of TXNIP in DKD is not yet well understood. In this study, we found that TXNIP knockout suppressed renal fibrosis and activation of mammalian target of rapamycin complex 1 (mTORC1) and restored transcription factor EB (TFEB) and autophagy activation in diabetic kidneys. Simultaneously, TXNIP interference inhibited epithelial-to-mesenchymal transformation (EMT), collagen I and fibronectin expression, and mTORC1 activation, increased TFEB nuclear translocation, and promoted autophagy restoration in HK-2 cells exposed to high glucose (HG). Rapamycin, an inhibitor of mTORC1, increased TFEB nuclear translocation and autophagy in HK-2 cells under HG conditions. Moreover, the TFEB activators, curcumin analog C1 and trehalose, effectively restored HG-induced autophagy, and abrogated HG-induced EMT and collagen I and fibronectin expression in HK-2 cells. Taken together, these findings suggest that TXNIP deficiency ameliorates renal fibrosis by regulating mTORC1/TFEB-mediated autophagy in diabetic kidney diseases.

Altered TFEB subcellular localization in nigral neurons of subjects with incidental, sporadic and GBA-related Lewy body diseases

Transcription factor EB (TFEB) is a master regulator of genes involved in the maintenance of autophagic and lysosomal homeostasis, processes which have been implicated in the pathogenesis of GBA-related and sporadic Parkinson’s disease (PD), and dementia with Lewy bodies (DLB). TFEB activation results in its translocation from the cytosol to the nucleus. Here, we investigated TFEB subcellular localization and its relation to intracellular alpha-synuclein (aSyn) accumulation in post-mortem human brain of individuals with either incidental Lewy body disease (iLBD), GBA-related PD/DLB (GBA-PD/DLB) or sporadic PD/DLB (sPD/DLB), compared to control subjects. We analyzed nigral dopaminergic neurons using high-resolution confocal and stimulated emission depletion (STED) microscopy and semi-quantitatively scored the TFEB subcellular localization patterns. We observed reduced nuclear TFEB immunoreactivity in PD/DLB patients compared to controls, both in sporadic and GBA-related cases, as well as in iLBD cases. Nuclear depletion of TFEB was more pronounced in neurons with Ser129-phosphorylated (pSer129) aSyn accumulation in all groups. Importantly, we observed previously-unidentified TFEB-immunopositive perinuclear clusters in human dopaminergic neurons, which localized at the Golgi apparatus. These TFEB clusters were more frequently observed and more severe in iLBD, sPD/DLB and GBA-PD/DLB compared to controls, particularly in pSer129 aSyn-positive neurons, but also in neurons lacking detectable aSyn accumulation. In aSyn-negative cells, cytoplasmic TFEB clusters were more frequently observed in GBA-PD/DLB and iLBD patients, and correlated with reduced GBA enzymatic activity as well as increased Braak LB stage. Altered TFEB distribution was accompanied by a reduction in overall mRNA expression levels of selected TFEB-regulated genes, indicating a possible early dysfunction of lysosomal regulation. Overall, we observed cytoplasmic TFEB retention and accumulation at the Golgi in cells without apparent pSer129 aSyn accumulation in iLBD and PD/DLB patients. This suggests potential TFEB impairment at the early stages of cellular disease and underscores TFEB as a promising therapeutic target for synucleinopathies.

Role of TFEB in Huntington's Disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by an expansion of the CAG trinucleotide repeat in exon 1 of the huntingtin (HTT) gene. This expansion leads to a polyglutamine (polyQ) tract at the N-terminal end of HTT, which reduces the solubility of the protein and promotes its accumulation. Inefficient clearance of mutant HTT (mHTT) by the proteasome or autophagy-lysosomal system leads to accumulation of oligomers and toxic protein aggregates in neurons, resulting in impaired proteolytic systems, transcriptional dysregulation, impaired axonal transport, mitochondrial dysfunction and cellular energy imbalance. Growing evidence suggests that the accumulation of mHTT aggregates and autophagic and/or lysosomal dysfunction are the major pathogenic mechanisms underlying HD. In this context, enhancing autophagy may be an effective therapeutic strategy to remove protein aggregates and improve cell function. Transcription factor EB (TFEB), a master transcriptional regulator of autophagy, controls the expression of genes critical for autophagosome formation, lysosomal biogenesis, lysosomal function and autophagic flux. Consequently, the induction of TFEB activity to promote intracellular clearance may be a therapeutic strategy for HD. However, while some studies have shown that overexpression of TFEB facilitates the clearance of mHTT aggregates and ameliorates the disease phenotype, others indicate such overexpression may lead to mHTT co-aggregation and worsen disease progression. Further studies are necessary to confirm whether TFEB modulation could be an effective therapeutic strategy against mHTT-mediated toxicity in different disease models.

Autophagic-lysosomal damage induced by swainsonine is protected by trehalose through activation of TFEB-regulated pathway in renal tubular epithelial cells

Swainsonine (SW) is the main toxic component of locoweed. Previous studies have shown that kidney damage is an early pathologic change in locoweed poisoning in animals. Trehalose induces autophagy and alleviates lysosomal damage, while its protective effect and mechanism against the toxic injury induced by SW is not clear. Based on the published literature, we hypothesize that transcription factor EB(TFEB) -regulated is targeted by SW and activating TFEB by trehalose would reverse the toxic effects. In this study, we investigate the mechanism of protective effects of trehalose using renal tubular epithelial cells. The results showed that SW induced an increase in the expression level of microtubule-associated protein light chain 3-II and p62 proteins and a decrease in the expression level of ATPase H+ transporting V1 Subunit A, Cathepsin B, Cathepsin D, lysosome-associated membrane protein 2 and TFEB proteins in renal tubular epithelial cells in a time and dose-dependent manner suggesting TFEB-regulated lysosomal pathway is adversely affected by SW. Conversely, treatment with trehalose, a known activator of TFEB promote TFEB nuclear translocation suggesting that TFEB plays an important role in protection against SW toxicity. We demonstrated in lysosome staining that SW reduced the number of lysosomes and increased the luminal pH, while trehalose could counteract these SW-induced effects. In summary, our results demonstrated for the first time that trehalose could alleviate the autophagy degradation disorder and lysosomal damage induced by SW. Our results provide an interesting method for reversion of SW-induced toxicity in farm animals and furthermore, activation of TFEB by trehalose suggesting novel mechanism of treating lysosomal storage diseases.

Age-related driving mechanisms of retinal diseases and neuroprotection by transcription factor EB-targeted therapy

Abstract Retinal aging has been recognized as a significant risk factor for various retinal disorders, including diabetic retinopathy, age-related macular degeneration, and glaucoma, following a growing understanding of the molecular underpinnings of their development. This comprehensive review explores the mechanisms of retinal aging and investigates potential neuroprotective approaches, focusing on the activation of transcription factor EB. Recent meta-analyses have demonstrated promising outcomes of transcription factor EB-targeted strategies, such as exercise, calorie restriction, rapamycin, and metformin, in patients and animal models of these common retinal diseases. The review critically assesses the role of transcription factor EB in retinal biology during aging, its neuroprotective effects, and its therapeutic potential for retinal disorders. The impact of transcription factor EB on retinal aging is cell-specific, influencing metabolic reprogramming and energy homeostasis in retinal neurons through the regulation of mitochondrial quality control and nutrient-sensing pathways. In vascular endothelial cells, transcription factor EB controls important processes, including endothelial cell proliferation, endothelial tube formation, and nitric oxide levels, thereby influencing the inner blood-retinal barrier, angiogenesis, and retinal microvasculature. Additionally, transcription factor EB affects vascular smooth muscle cells, inhibiting vascular calcification and atherogenesis. In retinal pigment epithelial cells, transcription factor EB modulates functions such as autophagy, lysosomal dynamics, and clearance of the aging pigment lipofuscin, thereby promoting photoreceptor survival and regulating vascular endothelial growth factor A expression involved in neovascularization. These cell-specific functions of transcription factor EB significantly impact retinal aging mechanisms encompassing proteostasis, neuronal synapse plasticity, energy metabolism, microvasculature, and inflammation, ultimately offering protection against retinal aging and diseases. The review emphasizes transcription factor EB as a potential therapeutic target for retinal diseases. Therefore, it is imperative to obtain well-controlled direct experimental evidence to confirm the efficacy of transcription factor EB modulation in retinal diseases while minimizing its risk of adverse effects.

Low-dose cadmium telluride quantum dots trigger M1 polarization in macrophages through mTOR-mediated transcription factor EB activation

The increasing application of quantum dots (QDs) increases interactions with organisms. The inflammatory imbalance is a significant manifestation of immunotoxicity. Macrophages maintain inflammatory homeostasis. Using macrophages differentiated by phorbol 12-myristate 13-acetate-induced THP-1 cells as models, the study found that low-dose (5 μM) cadmium telluride QDs (CdTe-QDs) hindered monocyte-macrophage differentiation. CD11b is a surface marker of macrophage, and the addition of CdTe-QDs during induction resulted in a decrease in CD11b expression. Moreover, exposure of differentiated THP-1 macrophage (dTHP-1) to 5 μM CdTe-QDs led to the initiation of M1 polarization. This was indicated by the increased surface marker CD86 expression, along with elevated level of NF-κB and IL-1β proteins. The potential mechanisms are being explored. The transcription factor EB (TFEB) plays a significant role in immune regulation and serves as a crucial regulator of the autophagic lysosomal pathway. After exposed to CdTe-QDs, TFEB activation-mediated autophagy and M1 polarization were observed to occur simultaneously in dTHP-1. The mTOR signaling pathway contributed to TFEB activation induced by CdTe-QDs. However, mTOR-independent activation of TFEB failed to promote M1 polarization. These results suggest that mTOR-TFEB is an advantageous target to enhance the biocompatibility of CdTe-QDs.

TRIM27 elicits protective immunity against tuberculosis by activating TFEB-mediated autophagy flux

ABSTRACT Infectious diseases, such as Mycobacterium tuberculosis (Mtb)-caused tuberculosis (TB), remain a global threat exacerbated by increasing drug resistance. Host-directed therapy (HDT) is a promising strategy for infection treatment through targeting host immunity. However, the limited understanding of the function and regulatory mechanism of host factors involved in immune defense against infections has impeded HDT development. Here, we identify the ubiquitin ligase (E3) TRIM27 (tripartite motif-containing 27) as a host protective factor against Mtb by enhancing host macroautophagy/autophagy flux in an E3 ligase activity-independent manner. Mechanistically, upon Mtb infection, nuclear-localized TRIM27 increases and functions as a transcription activator of TFEB (transcription factor EB). Specifically, TRIM27 binds to the TFEB promoter and the TFEB transcription factor CREB1 (cAMP responsive element binding protein 1), thus enhancing CREB1-TFEB promoter binding affinity and promoting CREB1 transcription activity toward TFEB, eventually inducing autophagy-related gene expression as well as autophagy flux activation to clear the pathogen. Furthermore, TFEB activator 1 can rescue TRIM27 deficiency-caused decreased autophagy-related gene transcription and attenuated autophagy flux, and accordingly suppressed the intracellular survival of Mtb in cell and mouse models. Taken together, our data reveal that TRIM27 is a host defense factor against Mtb, and the TRIM27-CREB1-TFEB axis is a potential HDT-based TB target that can enhance host autophagy flux. Abbreviations: ATG5: autophagy related 5; BMDMs: bone marrow-derived macrophages; CFU: colony-forming unit; ChIP-seq: chromatin immunoprecipitation followed by sequencing; CREB1: cAMP responsive element binding protein 1; CTSB: cathepsin B; E3: ubiquitin ligase; EMSA: electrophoretic mobility shift assay; HC: healthy control; HDT: host-directed therapy; LAMP: lysosomal associated membrane protein; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCOLN1: mucolipin TPR cation channel 1; Mtb: Mycobacterium tuberculosis; NLS: nuclear localization signal; PBMCs: peripheral blood mononuclear cells; PRKA/PKA: protein kinase cAMP-activated; qRT-PCR: quantitative real-time PCR; RFP: RET finger protein; TB: tuberculosis; TBK1: TANK binding kinase 1; TFEB: transcription factor EB; TRIM: tripartite motif; TSS: transcription start site; ULK1: unc-51 like autophagy activating kinase 1.

Promotion of transcription factor EB-dependent autophagic process by curcumin alleviates arsenic-caused lung oxidative stress and inflammation in mice

Arsenic is a human carcinogen widely distributed in the environment, and arsenic exposure from drinking water has received widespread attention as a global public health problem. Curcumin is a natural bioactive substance with high efficiency and low toxicity extracted from turmeric, which has a variety of biological properties such as anti-oxidation, anti-inflammation, anti-cancer, and immuno-modulatory activities. Curcumin is widely used in daily life as a food additive and dietary supplement. However, its protective effects in lung injuries by chronic arsenic exposure orally remain unexplored. In this study, curcumin treatment not only significantly accelerated arsenic elimination and improved lung tissue morphology, but also decreased arsenic-generated ROS by activating Nrf2 and its down-stream antioxidants. Further, curcumin alleviated inflammatory changes in mice exposed to arsenic for 6 and 12 weeks, as manifested by lung MPO levels, total protein and cellular levels in bronchoalveolar lavage fluid (BALF), serum IL-4 levels, and MAPK/NF-κB expression in lung tissue. Notably, our study also confirmed that curcumin could promote the expression and nuclear translocation of the transcription factor EB (TFEB), as well as activate TFEB-regulated autophagy in lung tissue of arsenic-treated mice, accompanied by inhibition of the AKT-mTOR signaling pathway. Overall, our study here suggests that natural bioactive compound curcumin could alleviate arsenic-induced pulmonary oxidative stress and inflammation in vivo, which is closely related to enhanced TFEB activity and induction of the autophagic process.

Cathelicidin LL-37 promotes wound healing in diabetic mice by regulating TFEB-dependent autophagy

Diabetic patients often experience impaired wound healing. Human cathelicidin LL-37 possesses various biological functions, such as anti-microbial, anti-inflammatory, and pro-wound healing activities. Autophagy has important effects on skin wound healing. However, little is known about whether LL-37 accelerates diabetic wound healing by regulating autophagy. In the study, we aimed to investigate the role of autophagy in LL-37-induced wound healing and uncover the underlying mechanisms involved. A full-thickness wound closure model was established in diabetic mice to evaluate the effects of LL-37 and an autophagy inhibitor (3-MA) on wound healing. The roles of LL-37 and 3-MA in regulating keratinocyte migration were assessed using transwell migration and wound healing assays. The activation of transcription factor EB (TFEB) was measured using western blotting and immunofluorescence (IF) assays of its nuclear translocation. The results showed that LL-37 treatment improved wound healing in diabetic mice, whereas these effects were reversed by 3-MA. In vitro, 3-MA decreased the effects of LL-37 on promoting HaCat keratinocyte migration in the presence of high glucose (HG). Mechanistically, LL-37 promoted TFEB activation and resulted in subsequent activation of autophagy, as evidenced by increased nuclear translocation of TFEB and increased expression of ATG5, ATG7, and beclin 1 (BECN1), whereas these changes were blocked by TFEB knockdown. As expected, TFEB knockdown damaged the effects of LL-37 on promoting keratinocyte migration. Collectively, these results suggest that LL-37 accelerates wound healing in diabetic mice by activating TFEB-dependent autophagy, providing new insights into the mechanism by which LL-37 promotes diabetic wound healing.

TFEB activation triggers pexophagy for functional adaptation during oxidative stress under calcium deficient-conditions

BackgroundCalcium is a ubiquitous intracellular messenger that regulates the expression of various genes involved in cell proliferation, differentiation, and motility. The involvement of calcium in diverse metabolic pathways has been suggested. However, the effect of calcium in peroxisomes, which are involved in fatty acid oxidation and scavenges the result reactive oxygen species (ROS), remains elusive. In addition, impaired peroxisomal ROS inhibit the mammalian target of rapamycin complex 1 (mTORC1) and promote autophagy. Under stress, autophagy serves as a protective mechanism to avoid cell death. In response to oxidative stress, lysosomal calcium mediates transcription factor EB (TFEB) activation. However, the impact of calcium on peroxisome function and the mechanisms governing cellular homeostasis to prevent diseases caused by calcium deficiency are currently unknown.MethodsTo investigate the significance of calcium in peroxisomes and their roles in preserving cellular homeostasis, we established an in-vitro scenario of calcium depletion.ResultsThis study demonstrated that calcium deficiency reduces catalase activity, resulting in increased ROS accumulation in peroxisomes. This, in turn, inhibits mTORC1 and induces pexophagy through TFEB activation. However, treatment with the antioxidant N-acetyl-l-cysteine (NAC) and the autophagy inhibitor chloroquine impeded the nuclear translocation of TFEB and attenuated peroxisome degradation.ConclusionsCollectively, our study revealed that ROS-mediated TFEB activation triggers pexophagy during calcium deficiency, primarily because of attenuated catalase activity. We posit that calcium plays a significant role in the proper functioning of peroxisomes, critical for fatty-acid oxidation and ROS scavenging in maintaining cellular homeostasis. These findings have important implications for signaling mechanisms in various pathologies, including Zellweger’s syndrome and ageing.

Trehalose promotes atherosclerosis regression in female mice.

Atherosclerosis is a chronic inflammatory disease caused by the deposition of lipids within the artery wall. During atherogenesis, efficient autophagy is needed to facilitate efferocytosis and cholesterol efflux, limit inflammation and lipid droplet buildup, and eliminate defective mitochondria and protein aggregates. Central to the regulation of autophagy is the transcription factor EB (TFEB), which coordinates the expression of lysosomal biogenesis and autophagy genes. In recent years, trehalose has been shown to promote TFEB activation and protect against atherogenesis. Here, we sought to investigate the role of autophagy activation during atherosclerosis regression. Atherosclerosis was established in C57BL/6N mice by injecting AAV-PCSK9 and 16 weeks of Western diet feeding, followed by switching to a chow diet to induce atherosclerosis regression. During the regression period, mice were either injected with trehalose concomitant with trehalose supplementation in their drinking water or injected with saline for 6 weeks. Female mice receiving trehalose had reduced atherosclerosis burden, as evidenced by reduced plaque lipid content, macrophage numbers and IL-1β content in parallel with increased plaque collagen deposition, which was not observed in their male counterparts. In addition, trehalose-treated female mice had lower levels of circulating leukocytes, including inflammatory monocytes and CD4+ T cells. Lastly, we found that autophagy flux in male mice was basally higher than in female mice during atherosclerosis progression. Our data demonstrate a sex-specific effect of trehalose in atherosclerosis regression, whereby trehalose reduced lipid content, inflammation, and increased collagen content in female mice but not in male mice. Furthermore, we discovered inherent differences in the autophagy flux capacities between the sexes: female mice exhibited lower plaque autophagy than males, which rendered the female mice more responsive to atherosclerosis regression. Our work highlights the importance of understanding sex differences in atherosclerosis to personalize the development of future therapies to treat cardiovascular diseases.

Effects of α7 nicotinic acetylcholine receptor agonist against α-synuclein-induced neurotoxicity

The α7 neuronal nicotinic acetylcholine receptor (α7 nAChR) is a potential target for the development of Parkinson’s disease (PD) therapeutics. α-Synuclein (α-Syn), a principal component of Lewy bodies (cytoplasmic inclusions), is a major contributor to PD pathophysiology. Previous studies have demonstrated that activating α7 nAChR protects against nigrostriatal dopamine degeneration in acute and chronic PD animal models induced by 6-hydroxydopamine and rotenone, respectively. In the present study, we investigated the effects of PNU282987, a selective α7 nAChR agonist, against α-Syn-induced neurotoxicity in α-SynWT-, α-SynA30P-, and α-SynE46K-N2a cells. PNU282987 exhibited substantial neuroprotection against both wild-type and mutant-type α-Syn-induced toxicity. Furthermore, PNU282987 promoted transcription factor EB activity and reduced intracellular α-Syn protein levels through autophagy induction. These results highlight the therapeutic potential of α7 nAChR activation in diseases characterized by α-Syn aggregation, such as PD.

Tangshen formula protects against podocyte apoptosis via enhancing the TFEB-mediated autophagy-lysosome pathway in diabetic nephropathy

Ethnopharmacological relevanceDiabetic nephropathy (DN) is the leading cause of end-stage kidney disease and currently there are no specific and effective drugs for its treatment. Podocyte injury is a detrimental feature and the major cause of albuminuria in DN. We previously reported Tangshen Formula (TSF), a Chinese herbal medicine, has shown therapeutic effects on DN. However, the underlying mechanisms remain obscure. Aim of the studyThis study aimed to explore the protective effect of TSF on podocyte apoptosis in DN and elucidate the potential mechanism. Materials and methodsThe effects of TSF were assessed in a murine model using male KKAy diabetic mice, as well as in advanced glycation end products-stimulated primary mice podocytes. Transcription factor EB (TFEB) knockdown primary podocytes were employed for mechanistic studies. In vivo and in vitro studies were performed and results assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. ResultsTSF treatment alleviated podocyte apoptosis and structural impairment, decreased albuminuria, and mitigated renal dysfunction in KKAy mice. Notably, TSF extracted twice showed a more significant reduction in proteinuria than TSF extracted three times. Accumulation of autophagic biomarkers p62 and LC3, and aberrant autophagic flux in podocytes of DN mice were significantly altered by TSF therapy. Consistent with the in vivo results, TSF prevented the apoptosis of primary podocytes exposed to AGEs and activated autophagy. However, the anti-apoptosis capacity of TSF was countered by the autophagy–lysosome inhibitor chloroquine. We found that TSF increased the nuclear translocation of TFEB in diabetic podocytes, and thus upregulated transcription of its several autophagic target genes. Pharmacological activation of TFEB by TSF accelerated the conversion of autophagosome to autolysosome and lysosomal biogenesis, further augmented autophagic flux. Conversely, TFEB knockdown negated the favorable effects of TSF on autophagy in AGEs-stimulated primary podocytes. ConclusionsThese findings indicate TSF appears to attenuate podocyte apoptosis and promote autophagy in DN via the TFEB-mediated autophagy-lysosome system. Thus, TSF may be a therapeutic candidate for DN.

Tamoxifen Activates Transcription Factor EB and Triggers Protective Autophagy in Breast Cancer Cells by Inducing Lysosomal Calcium Release: A Gateway to the Onset of Endocrine Resistance.

Among the several mechanisms accounting for endocrine resistance in breast cancer, autophagy has emerged as an important player. Previous reports have evidenced that tamoxifen (Tam) induces autophagy and activates transcription factor EB (TFEB), which regulates the expression of genes controlling autophagy and lysosomal biogenesis. However, the mechanisms by which this occurs have not been elucidated as yet. This investigation aims at dissecting how TFEB is activated and contributes to Tam resistance in luminal A breast cancer cells. TFEB was overexpressed and prominently nuclear in Tam-resistant MCF7 cells (MCF7-TamR) compared with their parental counterpart, and this was not dependent on alterations of its nucleo-cytoplasmic shuttling. Tam promoted the release of lysosomal Ca2+ through the major transient receptor potential cation channel mucolipin subfamily member 1 (TRPML1) and two-pore channels (TPCs), which caused the nuclear translocation and activation of TFEB. Consistently, inhibiting lysosomal calcium release restored the susceptibility of MCF7-TamR cells to Tam. Our findings demonstrate that Tam drives the nuclear relocation and transcriptional activation of TFEB by triggering the release of Ca2+ from the acidic compartment, and they suggest that lysosomal Ca2+ channels may represent new druggable targets to counteract the onset of autophagy-mediated endocrine resistance in luminal A breast cancer cells.

A snake cathelicidin enhances transcription factor EB-mediated autophagy and alleviates ROS-induced pyroptosis after ischaemia-reperfusion injury of island skin flaps.

Ischaemia-reperfusion (I/R) injury is a major contributor to skin flap necrosis, which presents a challenge in achieving satisfactory therapeutic outcomes. Previous studies showed that cathelicidin-BF (BF-30) protects tissues from I/R injury. In this investigation, BF-30 was synthesized and its role and mechanism in promoting survival of I/R-injured skin flaps explored. Survival rate analysis and laser Doppler blood flow analysis were used to evaluate I/R-injured flap viability. Western blotting, immunofluorescence, TdT-mediated dUTP nick end labelling (TUNEL) and dihydroethidium were utilized to examine the levels of apoptosis, pyroptosis, oxidative stress, transcription factor EB (TFEB)-mediated autophagy and molecules related to the adenosine 5'-monophosphate-activated protein kinase (AMPK)-transient receptor potential mucolipin 1 (TRPML1)-calcineurin signalling pathway. The outcomes revealed that BF-30 enhanced I/R-injured island skin flap viability. Autophagy, oxidative stress, pyroptosis and apoptosis were related to the BF-30 capability to enhance I/R-injured flap survival. Improved autophagy flux and tolerance to oxidative stress promoted the inhibition of apoptosis and pyroptosis in vascular endothelial cells. Activation of TFEB increased autophagy and inhibited endothelial cell oxidative stress in I/R-injured flaps. A reduction in TFEB level led to a loss of the protective effect of BF-30, by reducing autophagy flux and increasing the accumulation of reactive oxygen species (ROS) in endothelial cells. Additionally, BF-30 modulated TFEB activity via the AMPK-TRPML1-calcineurin signalling pathway. BF-30 promotes I/R-injured skin flap survival by TFEB-mediated up-regulation of autophagy and inhibition of oxidative stress, which may have possible clinical applications.

In vitro assessment of dietary bioactives for TFEB activation as a possible target to support cognitive and emotional wellbeing

Aging and chronic stress are associated with altered brain plasticity and an increased risk of developing cognitive and emotional health problems. A key cellular mechanism involved is autophagy, which is known to be important for memory encoding and information processing, and is also thought to be important for psychiatric and emotional health. Activation of the autophagic system can be established by the transcription factor EB (TFEB), which regulates both autophagosome formation and lysosomal biogenesis and function. As well-known activators of autophagy like caloric restriction, fasting, or regular exercise require intervention programs that are hard to adhere to, the search for nutritional solutions with similar effects, is increasing. We therefore evaluated pro-autophagic dietary bioactives for effects specifically through TFEB activation and assessed whether combining bioactives can lead to synergistic or additive effects. We also discuss current evidence supporting the use of these natural dietary compounds to promote cognitive and emotional wellbeing. MethodsPrimary cortical astrocytes were treated with 13 different ingredients and analyzed for their effect on TFEB nuclear translocation by high content imaging. Effective ingredients were then evaluated at different concentrations for their effect on TFEB downstream signaling by analyzing mRNA levels of Beclin1, LC3, Lamp-1 and Cathepsin D. Furthermore, the synergistic effects of sub-optimal concentrations of different ingredient combinations were evaluated on the same TFEB downstream signaling markers. Potential signs of toxicity of all ingredients and their combinations were evaluated by analysis of ATP production and nuclear counts. ResultsDHA, EPA, 7,8-DHF, ellagic acid, curcumin and spermidine increased TFEB nuclear localization. DHA and spermidine exhibited the strongest effects when measuring mRNA levels of TFEB downstream signaling markers. The most significant synergies were observed when combining spermidine with ellagic acid or when combining DHA with curcumin. None of the ingredients alone or in combination presented any sign of toxicity at the concentrations tested. Discussion/ConclusionThe results of our study show for the first time a synergistic effect of spermidine and ellagic acid and of DHA and curcumin on TFEB signaling and thus its ability to act on the autophagic system in brain cells. These combinations may hold potential as dietary promoters of cognitive and emotional wellbeing.

Abstract 12784: Ischemic Cardiac Autophagy/Mitophagy is Regulated by TRPML1 and Mitochondrial Reactive Oxygen Species

Introduction: In myocardial infarction (MI), activated autophagy/mitophagy helps degrade cytoplasmic macromolecules/proteins/ organelles (especially damaged mitochondria), recycle breakdown products, and prevent oxidative damage and cell death. It had reported that reactive oxygen species (ROS) activates the transcription factor EB (TFEB) via a lysosomal transient receptor potential mucolipin 1 (TRPML1) Ca 2+ efflux. TFEB is thought to be the master regulator of lysosome biogenesis. Hypothesis: We investigated whether lysosomal TRPML1 was activated by ROS and contributed to ischemic cardiac autophagy/mitophagy. Methods: MI was induced in wild type C57BL/6J mice by ligated left anterior descending coronary artery. Mice were studied at three weeks after MI surgery. Results: The lysosomal-restricted TRPML1 Ca 2+ release channel was significantly upregulated by 84.8% in cardiac border zone (BZ) and 55.4% in a remote zone after MI. As expected, dephosphorylated TFEB (i.e., active TFEB), mitophagy marker Parkin, autophagy marker LC3II, and lysosome marker LAMP1 increased substantially after MI. Autophagy degradation was upregulated as measured by SQSTM1/p62. TRPML1 protein expression was reduced in a dose-dependent manner by TRPML1 shRNA AAV9 (AAV9 was injected 1 week after MI for two weeks). The protein levels of LC3II and LAMP1 significantly decreased in MI BZ after AAV9 transfection. Mitochondrial ROS scavenger (mitoTEMPO, 1 mg/kg/day, i.p.) treatment for two weeks (starting at 1 week after MI) significantly inhibited TRPML1, LAMP1, LC3II and Parkin expression in the MI BZ. Conversely, when TRPML1 was overexpressed in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), the protein levels of LC3II/Parkin were increased. Speaking to human relevance, TRPML1 was significantly increased in human patients with ischemic cardiomyopathy. Conclusion: Autophagy and mitophagy after MI are regulated by TRPML1 and mitochondrial ROS.

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