Добірка наукової літератури з теми "Transcription factor EB (TFEB)"

The kinase PINK1 and ubiquitin ligase Parkin can regulate the selective elimination of damaged mitochondria through autophagy (mitophagy). Because of the demand on lysosomal function by mitophagy, we investigated a role for the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, in this process. We show that during mitophagy TFEB translocates to the nucleus and displays transcriptional activity in a PINK1- and Parkin-dependent manner. MITF and TFE3, homologues of TFEB belonging to the same microphthalmia/transcription factor E (MiT/TFE) family, are similarly regulated during mitophagy. Unlike TFEB translocation after starvation-induced mammalian target of rapamycin complex 1 inhibition, Parkin-mediated TFEB relocalization required Atg9A and Atg5 activity. However, constitutively active Rag guanosine triphosphatases prevented TFEB translocation during mitophagy, suggesting cross talk between these two MiT/TFE activation pathways. Analysis of clustered regularly interspaced short palindromic repeats–generated TFEB/MITF/TFE3/TFEC single, double, and triple knockout cell lines revealed that these proteins partly facilitate Parkin-mediated mitochondrial clearance. These results illuminate a pathway leading to MiT/TFE transcription factor activation, distinct from starvation-induced autophagy, which occurs during mitophagy.

In response to the increased energy demands of contractions, skeletal muscle adapts remarkably well through acutely regulating metabolic pathways to maintain energy balance and in the longer term by regulating metabolic reprogramming, such as remodeling and expanding the mitochondrial network. This long-term adaptive response involves modulation of gene expression at least partly through the regulation of specific transcription factors and transcriptional coactivators. The AMPK-peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) pathway has long been known to orchestrate contraction-mediated adaptive responses, although AMPK- and PGC1α-independent pathways have also been proposed. Transcription factor EB (TFEB) and TFE3, known as important regulators of lysosomal biogenesis and autophagic processes, have emerged as new metabolic coordinators. The activity of TFEB/TFE3 is regulated through posttranslational modifications (i.e., phosphorylation) and spatial organization. Under nutrient and energy stress, TFEB and TFE3 are dephosphorylated and translocate to the nucleus, where they activate transcription of their target genes. It has recently been reported that exercise promotes nuclear translocation and activation of TFEB/TFE3 in mouse skeletal muscle through the Ca2+-stimulated protein phosphatase calcineurin. Skeletal muscle-specific ablation of TFEB exhibits impaired glucose homeostasis and mitochondrial biogenesis with reduced metabolic flexibility during exercise, and global TFE3 depletion results in diminished endurance and abolished exercise-induced metabolic benefits. Transcriptomic analysis of the muscle-specific TFEB-null mice has demonstrated that TFEB regulates the expression of genes involved in glucose metabolism and mitochondrial homeostasis. This review aims to summarize and discuss emerging roles for TFEB/TFE3 in metabolic and adaptive responses to exercise and contractile activity in skeletal muscle.

The autophagy-lysosome pathway is a major protein degradation pathway stimulated by multiple cellular stresses, including nutrient or growth factor deprivation, hypoxia, misfolded proteins, damaged organelles, and intracellular pathogens. Recent studies have revealed that transcription factor EB (TFEB) and transcription factor E3 (TFE3) play a pivotal role in the biogenesis and functions of autophagosome and lysosome. Here we report that three translation inhibitors (cycloheximide, lactimidomycin, and rocaglamide A) can facilitate the nuclear translocation of TFEB/TFE3 via dephosphorylation and 14-3-3 dissociation. In addition, the inhibitor-mediated TFEB/TFE3 nuclear translocation significantly increases the transcriptional expression of their downstream genes involved in the biogenesis and function of autophagosome and lysosome. Furthermore, we demonstrated that translation inhibition increased autophagosome biogenesis but impaired the degradative autolysosome formation because of lysosomal dysfunction. These results highlight the previously unrecognized function of the translation inhibitors as activators of TFEB/TFE3, suggesting a novel biological role of translation inhibition in autophagy regulation.

The transcription factor EB (TFEB) promotes protein degradation by the autophagy and lysosomal pathway (ALP) and overexpression of TFEB was suggested for the treatment of ALP-related diseases that often affect the heart. However, TFEB-mediated ALP induction may perturb cardiac stress response. We used adeno-associated viral vectors type 9 (AAV9) to overexpress TFEB (AAV9-Tfeb) or Luciferase-control (AAV9-Luc) in cardiomyocytes of 12-week-old male mice. Mice were subjected to transverse aortic constriction (TAC, 27G; AAV9-Luc: n = 9; AAV9-Tfeb: n = 14) or sham (AAV9-Luc: n = 9; AAV9-Tfeb: n = 9) surgery for 28 days. Heart morphology, echocardiography, gene expression, and protein levels were monitored. AAV9-Tfeb had no effect on cardiac structure and function in sham animals. TAC resulted in compensated left ventricular hypertrophy in AAV9-Luc mice. AAV9-Tfeb TAC mice showed a reduced LV ejection fraction and increased left ventricular diameters. Morphological, histological, and real-time PCR analyses showed increased heart weights, exaggerated fibrosis, and higher expression of stress markers and remodeling genes in AAV9-Tfeb TAC compared to AAV9-Luc TAC. RNA-sequencing, real-time PCR and Western Blot revealed a stronger ALP activation in the hearts of AAV9-Tfeb TAC mice. Cardiomyocyte-specific TFEB-overexpression promoted ALP gene expression during TAC, which was associated with heart failure. Treatment of ALP-related diseases by overexpression of TFEB warrants careful consideration.

Background/Aims: This study determined the role and mechanism of action of transcription factor EB (TFEB) in H2O2-induced neuronal apoptosis. Methods: SH-SY5Y cells were treated with Akt inhibitor/activator and different concentrations of H2O2. Cell apoptosis was detected by flow cytometric analysis. Akt and TFEB phosphorylation and PARP cleavage were determined by Western blotting. HEK293T cells were transfected with different truncated TFEB mutants and HA-Akt-WT; SH-SY5Y cells were transfected with Flag-vector, Flag-TFEB, Flag-TFEB-S467A or Flag-TFEB-S467D; and TFEB interaction with Akt was determined by co-immunoprecipitation and GST pull-down assays. Results: A low concentration of H2O2 induces TFEB phosphorylation at Ser467 and nuclear translocation, facilitating neuronal survival, whereas a high concentration of H2O2 promotes SH-SY5Y cell apoptosis via suppressing TFEB Ser467 phosphorylation and nuclear translocation. The TFEB-S467D mutant is more easily translocated into the nucleus than the non-phosphorylated TFEB-S467A mutant. Further, Akt physically binds to TFEB via its C-terminal tail interaction with the HLH domain of TFEB and phosphorylates TFEB at Ser467. Mutation of TFEB-Ser467 can prevent the phosphorylation of TFEB by Akt, preventing inhibition of oxidative stress-induced apoptosis. Conclusions: Oxidative stress induces neuronal apoptosis through suppressing TFEB phosphorylation at Ser467 by Akt, providing a novel therapeutic strategy for neurodegenerative diseases.

To determine the roles of transcription factor EB (TFEB) in colorectal cancer (CRC), we collected samples of tumor tissues and normal tissues from 40 patients with CRC. The expression of TFEB in these samples was analyzed by using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Furthermore, we explored the expression of TFEB mRNA in CCD-18Co normal cells and HT-29, HCT-8, C2BBe1 cancer cells. HT-29, HCT-8, and C2BBe1 cancer cells were transfected with a TFEB-specific small interference RNA (siRNA) and scrambled siRNA, then the TFEB expression was confirmed by Western blot. The migration and invasion abilities of cells transfected with TFEB-siRNA were examined by transwell method and wound-healing assay. The subsequent effect of TFEB silencing on the tumor growth was also detected in mice xenograft model in vivo. Our study found that TFEB expression was significantly increased ( P < 0.05) in colorectal tumor tissues compared with normal tissues. Consistent with TFEB expression in tissues, compared with the normal CCD-18Co cells, TFEB mRNA expression was also significantly augmented in CRC cells. TFEB protein expression was markedly reduced in HT-29, HCT-8, and C2BBe1 cells after TFEB-siRNA transfection. In addition, inhibition of TFEB expression resulted in decrease of cells migration and invasion abilities. In vivo study, compared with the negative control group, the tumor weight, and volume were also reduced after inhibiting the TFEB expression. Our research suggested that TFEB expression is related to the occurrence and development of colorectal adenocarcinoma. The migration and invasion abilities of cancer cells, the weight and volume of tumor were all decreased when inhibiting TFEB expression. Thus, TFEB serves as an important factor in the development of CRC by modulating cancer cell migration and invasion, showing the potential therapeutic target of CRC in clinical.

Niemann–Pick type C disease (NPCD) is a lysosomal storage disease (LSD) characterized by abnormal cholesterol accumulation in lysosomes, impaired autophagy flux, and lysosomal dysfunction. The activation of transcription factor EB (TFEB), a master lysosomal function regulator, reduces the accumulation of lysosomal substrates in LSDs where the degradative capacity of the cells is compromised. Genistein can pass the blood–brain barrier and activate TFEB. Hence, we investigated the effect of TFEB activation by genistein toward correcting the NPC phenotype. We show that genistein promotes TFEB translocation to the nucleus in HeLa TFEB-GFP, Huh7, and SHSY-5Y cells treated with U18666A and NPC1 patient fibroblasts. Genistein treatment improved lysosomal protein expression and autophagic flux, decreasing p62 levels and increasing those of the LC3-II in NPC1 patient fibroblasts. Genistein induced an increase in β-hexosaminidase activity in the culture media of NPC1 patient fibroblasts, suggesting an increase in lysosomal exocytosis, which correlated with a decrease in cholesterol accumulation after filipin staining, including cells treated with U18666A and NPC1 patient fibroblasts. These results support that genistein-mediated TFEB activation corrects pathological phenotypes in NPC models and substantiates the need for further studies on this isoflavonoid as a potential therapeutic agent to treat NPCD and other LSDs with neurological compromise.

The oncogenic Transcription Factor EB (TFEB), a member of MITF-TFE family, is known to be the most important regulator of the transcription of genes responsible for the control of lysosomal biogenesis and functions, autophagy, and vesicles flux. TFEB activation occurs in response to stress factors such as nutrient and growth factor deficiency, hypoxia, lysosomal stress, and mitochondrial damage. To reach the final functional status, TFEB is regulated in multimodal ways, including transcriptional rate, post-transcriptional regulation, and post-translational modifications. Post-transcriptional regulation is in part mediated by miRNAs. miRNAs have been linked to many cellular processes involved both in physiology and pathology, such as cell migration, proliferation, differentiation, and apoptosis. miRNAs also play a significant role in autophagy, which exerts a crucial role in cell behaviour during stress or survival responses. In particular, several miRNAs directly recognise TFEB transcript or indirectly regulate its function by targeting accessory molecules or enzymes involved in its post-translational modifications. Moreover, the transcriptional programs triggered by TFEB may be influenced by the miRNA-mediated regulation of TFEB targets. Finally, recent important studies indicate that the transcription of many miRNAs is regulated by TFEB itself. In this review, we describe the interplay between miRNAs with TFEB and focus on how these types of crosstalk affect TFEB activation and cellular functions.

Cisplatin, a widely used anticancer agent, can cause nephrotoxicity, including both acute kidney injury (AKI) and chronic kidney diseases, by accumulating in renal tubular epithelial cells (TECs). Mitochondrial pathology plays an important role in the pathogenesis of AKI. Based on the regulatory role of transcription factor EB (TFEB) in mitochondria, we investigated whether TFEB is involved in cisplatin-induced TEC damage. The results show that the expression of TFEB decreased in a concentration-dependent manner in both mouse kidney tissue and HK-2 cells when treated with cisplatin. A knockdown of TFEB aggravated cisplatin-induced renal TEC injury, which was partially reversed by TFEB overexpression in HK-2 cells. It was further observed that the TFEB knockdown also exacerbated cisplatin-induced mitochondrial damage in vitro, and included the depolarization of membrane potential, mitochondrial fragmentation and swelling, and the production of reactive oxygen species. In contrast, TFEB overexpression alleviated cisplatin-induced mitochondrial damage in TECs. These findings suggest that decreased TFEB expression may be a key mechanism of mitochondrial dysfunction in cisplatin-induced AKI, and that upregulation of TFEB has the potential to act as a therapeutic target to alleviate mitochondrial dysfunction and cisplatin-induced TEC injury. This study is important for developing therapeutic strategies to manipulate mitochondria through TFEB to delay AKI progression.

Abstract Pancreatic ductal adenocarcinoma (PDAC), which comprises 85% of all the pancreatic cancers, is one of the most aggressive and deadly tumor that exists today with less than 8% of survival 5 years after diagnosis. It is predicted to become the second cause of cancer death by 2030. A typical characteristic of this tumor is the presence of prominent stroma component mainly produced by Pancreatic Stellate Cells (PSCs) in response to soluble factors released by cancer cells. PSCs activation and differentiation into CAF (Cancer-associated fibroblasts) is a reversible process, which impacts on both tumor progression and drug resistance. Transcription factor EB (TFEB) is mostly known as a master regulator of autophagy and lysosomal biogenesis. A recent study demonstrated that TFEB inhibits epithelial-to-mesenchymal transition (EMT) and myofibroblast differentiation in epicardial cells by upregulating TGIF1, a TGFβ pathway repressor. Based on this observation, we investigated if TFEB might have an inhibitory role in PSCs activation, taking into consideration the crucial role of TGFβ signaling in this process. The TFEB expression, measured by immunostaining, was significantly downregulated in CAFs in human PDAC samples compared to PSCs in healthy pancreas. In PSCs, co-cultured with PDAC cell lines, CAF markers were upregulated while TFEB expression was downregulated. The overexpression of TFEB in PSCs suppressed CAF markers upregulation induced by co-culture with PDAC cells at both mRNA and protein levels. The bulk RNAseq analysis showed the differential expression of genes required for extracellular matrix production, fibrotic response and inflammation in PSCs co-cultured with PDAC cell lines versus PSCs alone, with and without TFEB overexpression. These in vitro data suggest a role for TFEB in PSCs activation and stroma formation which will be further investigated in PDAC mouse models. Citation Format: Alejandra Diaz Alcalde, Edoardo Vallariello, Elena Astanina, Emanuele Middonti, Federico Bussolino. Transcription factor EB modulates fibrotic response in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2355.

Skeletal muscle is the most abundant tissue in the whole organism representing more than 40% of the total body mass. This organ is responsible for the 30% of metabolic rate in basal condition, suggesting its great relevance not only for locomotor activity, but also for the control of whole body metabolism. Indeed skeletal muscle is a highly dynamic tissue that modulates its metabolism and mass as a consequence of different physiopathological conditions. One stimulus that triggers major adaptations is exercise, which is also well known to activate autophagy (Grumati, Coletto, Schiavinato, et al., 2011). Physical exercise elicits several beneficial effects acting on mitochondrial content/function, fatty acid oxidation and glucose uptake; however it is considered a disruptive trigger for myofiber homeostasis that needs to be counterbalanced through the activation of transcriptionally regulated pathways ready to contrast mechanical and metabolic stresses produced during contraction. The role of FoxOs transcription factors and TFEB in regulating protein breakdown and autophagy is known (Milan et al., 2015; Settembre et al., 2011). However the role of TFEB in skeletal muscle and its possible effects in controlling exercise-dependent adaptations in this tissue were not proved. TFEB has been proposed as the key factor that coordinates autophagy to lysosomal biogenesis in cell culture, with different evidences showing the regulation of its activity. In particular it is known that an mTORC1 phosphorylation is able to prevent TFEB function by retaining it in the cytoplasm. However, there were no evidences concerning the possible phosphatases involved in TFEB activation. Using a cellular high content screening able to monitor TFEB nuclear translocation during starvation, we identified PPP3CB, the catalytic subunit of calcineurin, as one of the highest hit for TFEB nuclear relocalization. We demonstrated that calcineurin activity is necessary and sufficient to push TFEB in the nucleus, where it can complete its function. Nevertheless, calcineurin is known to be active in skeletal muscle during contraction as a consequence of calcium oscillations. For this reason we wondered whether calcineurin activity could affect TFEB translocation also in adult skeletal muscle during exercise. Using muscles transfected with a TFEB-GFP reporter, we demonstrated that calcineurin activity is necessary and sufficient to promote TFEB nuclear translocation even in adult skeletal muscle during coxntraction. However, the physiological meaning of this nuclear translocation in skeletal muscle remained to be addressed. To answer this question we used gain and loss of function approaches, by mean of viral infection of TFEB overesxpressing vectors, muscle specific TFEB knockout animals and tamoxifen inducible muscle specific TFEB transgenic animals. From microarray analysis of muscles overexpressing and lacking TFEB, we realized that the major pathways affected by genetic manipulation are related to mitochondrial biogenesis and function, lipid utilization and glucose homeostasis. Thus we started to dissect the function of TFEB in skeletal muscle proving that its activation is required for mitochondrial biogenesis that is indeed increased in transgenic muscle. We also found an augmented mitochondrial number and size in transgenic muscle, with only a small percentage of dysfunctional mitochondria in KO animals. These changes were paralleled by a TFEB signature in gene expression of genes involved in mitochondrial biogenesis and functionality. Moreover, these morphometric and gene expression evidences correlate with increased mitochondrial respiration and higher activity of respiratory chain complexes. For this reason transgenic muscles produce more ATP than normal mice, while KO muscles have a lower ATP synthesis because mitochondria present a leak in mitochondrial membrane that dissipate membrane potential. Nevertheless, in order to understand if TFEB is able to promote this mitochondrial program independently from PGC1α, we checked the expression of NRF1/2, TFAM and other genes involved in mitochondrial biogenesis in a model of PGC1α ablation during TFEB overexpression. These data, and complexes activity measurements, demonstrate that TFEB is able per se to activate the transcriptional program directly binding to NRF1 and NRF2 genes promoters without the need of the transcriptional co-activator. At this point, we challenged mice with exercise finding that transgenic mice are more resistant to exhaustive contraction than control; conversely muscle specific TFEB-KO animals display pronounced exercise intolerance due to their lack in ATP production. In order to better explain this latter finding, thanks to metabolic measurements we realized that KO muscles rely more on glucose oxidation both in basal condition and during the first phases of exercise thus explaining the observed exercise intolerance triggered by glycogen storage depletion. Furthermore lactate quantification in serum before and after exercise suggests that KO animal depend more on anaerobic glycolysis with respect to control and transgenic counterpart. To deeply investigate the role of glucose oxidation that seems the cause of exercise intolerance, we monitored glycogen levels in muscle of KO animals in resting condition, revealing a reduction of glycogen storage. For this reason after the early stages of exercise TFEB-KO animals need to rapidly shift their metabolism to fatty acid oxidation that however cannot support energy demand because of the presence of dysfunctional mitochondria. Altogether these findings indicate that TFEB is impinging more on metabolism rather than autophagy, that indeed is not affected by TFEB genetic modulation; more in detail TFEB seems to significantly modulate muscular glucose homeostasis that is altered in KO animals. Reduced glucose uptake and glycogen synthesis during EU clamps explains why glycogen storages are depleted in KO animals, while the transgenic counterpart present more glycogen accumulation. This phenotypic effect is paralleled by a change in glucose related genes expression, with higher levels of glucose transporters and glycogen synthesis regulator in transgenic muscles, even in the absence of PGC1α. Nevertheless TFEB overexpression is also able to drive factors such as nNOS and AMPK activity, thus modulating not only the expression but also the signalling pathways related to glucose homeostasis. In conclusion all these findings strongly support a new vision of TFEB as master regulator of metabolic flexibility during physical exercise in a PGC1α-independent fashion.
Il muscolo scheletrico è il tessuto più abbondante dell’organismo e rappresenta più del 40% della massa corporea. Questo organo è responsabile del 30% della spesa energetica a riposo, suggerendo la sua importanza non solo a livello di locomozione ma anche nel controllo del metabolismo a livello sistemico. Infatti il muscolo scheletrico è un tessuto estremamente dinamico, capace di modulare il suo metabolismo in seguito a stimoli di diversa natura. Uno stimolo che attiva maggiori adattamenti metabolici è l’esercizio, che è noto attivare anche l’autofagia. L’esercizio fisico stimola molti effetti benefici sul contenuto e funzionalità mitocondriale, ossidazione degli acidi grassi e assorbimento del glucosio; tuttavia, è considerato uno stimolo che danneggia la normale omeostasi delle fibre muscolari per cui necessita di essere controbilanciato dall’attivazione di meccanismi trascrizionalmente controllati che contrastano gli stress meccanici e metabolici prodotti durante la contrazione. Il ruolo dei fattori di trascrizione FoxO e TFEB nel regolare la degradazione proteica e l’autofagia è largamente conosciuto. Tuttavia, il ruolo di TFEB nel muscolo scheletrico e i suoi possibili effetti nel regolare gli adattamenti derivanti dall’esercizio in questo tessuto non sono ancora chiari. TFEB è stato proposto come fattore chiave nel coordinare autofagia e biogenesi lisosomiale in cellule in coltura, con diverse evidenze che dimostrano la regolazione della sua attività. In particolare è noto come la fosforilazione operata da mTORC1 sia in grado di prevenire l’attivazione di TFEB sequestrandolo nel citoplasma. Tuttavia, non esistono dati riguardanti le possibili fosfatasi coinvolte nell’attivazione di TFEB. Mediante l’utilizzo di uno High Content Screening in grado di monitorare la traslocazione di TFEB nel nucleo durante la starvation, abbiamo identificato il gene PPP3CB, codificante la subunità catalitica della calcineurina, come uno dei migliori geni coinvolti nella rilocalizzazione di TFEB. Abbiamo dimostrato che l’attività della calcineurina è necessaria e sufficiente per spingere TFEB nel nucleo, dove può espletare la sua funzione. Tuttavia, la calcineurina è noto essere attiva nel muscolo scheletrico durante la contrazione come conseguenza dei transienti di calcio. Per questo motivo ci siamo chiesti se l’attività della calcineurina possa influenzare la traslocazione di TFEB nel nucleo anche nel muscolo scheletrico durante l’esercizio fisico. Utilizzando un reporter TFEB-GFP abbiamo dimostrato che l’attività della calcineurina è necessaria e sufficiente a promuovere la traslocazione nucleare di TFEB anche nel muscolo scheletrico durante la contrazione. Tuttavia il significato fisiologico di questo avvenimento rimane da essere spiegato. Per rispondere a questa domanda abbiamo usato degli approcci di gain e loss of function utilizzando infezioni virali con vettori per l’overespressione di TFEB, una linea di topi con delezione muscolo specifica di TFEB e un’altra linea in cui l’overespressione di TFEB può essere attivata in muscolo grazie al tamoxifen. Da uno studio di espressione genica in muscoli overesprimenti TFEB e TFEB deficienti, abbiamo trovato che le vie di segnale principalmente coinvolte dalle manipolazioni genetiche erano quelle correlate alla biogenesi mitocondriale, utilizzo dei lipidi e omeostasi del glucosio. Abbiamo perciò cominciato a dissezionare il ruolo di TFEB nel muscolo scheletrico provando che la sua attivazione è richiesta per la biogenesi mitocondriale, che è per l'appunto aumentata nei muscoli transgenici. Infatti, in questi abbiamo trovato un aumento nel numero e nella dimensione dei mitocondri, mentre abbiamo riportato solo una piccola percentuale di mitocondri disfunzionali nei muscoli knockout. Questi cambiamenti sono accompagnati da un’attivazione dei geni TFEB-dipendenti responsabili per la biogenesi e funzionalità mitocondriale. Inoltre, questi cambiamenti morfometrici e di espressione genica correlano con un aumento nella respirazione mitocondriale e nell’attività dei complessi della catena respiratoria. Per questo motivo i muscoli transgenici producono più ATP dei wildtype, mentre i muscoli KO presentano una ridotta sintesi di ATP a causa di una disfunzionalità della membrana mitocondriale che dissipa il gradiente protonico. Tuttavia, per capire se questi cambiamenti dipendono direttamente da TFEB indipendentemente da PGC1α, abbiamo monitorato l’espressione di NRF1/2, TFAM e altri geni coinvolti nella biogenesi mitocondriale in un modello in cui PGC1α è deleto e TFEB overespresso. Questi dati di espressione uniti alle misure delle attività dei complessi dimostrano che TFEB è in grado di indurre autonomamente la biogenesi mitocondriale legandosi direttamente ai promotori dei geni NRF1 e NRF2. A questo punto abbiamo sottoposto a esercizio i topi riscontrando che gli animali transgenici resistono maggiormente all’attività fisica; al contrario i topi KO presentano una marcata intolleranza all’esercizio a causa della scarsa produzione di ATP. Per spiegare meglio questo fenomeno, grazie a misurazioni di parametri metabolici abbiamo riscontrato che i topi KO fanno affidamento maggiormente nell’ossidazione del glucosio sia a riposo che durante le fasi iniziali dell’esercizio fisico, spiegando l’intolleranza con la fine delle riserve di glicogeno. Inoltre, le quantificazioni del lattato nel siero prima e dopo l’esercizio suggeriscono che i muscoli KO dipendono maggiormente dalla glicolisi anaerobia a differenza delle controparti wildtype e transgenica. A questo punto, per investigare più in dettaglio il ruolo dell’ossidazione del glucosio che sembra essere alla base dell’intolleranza all’esercizio, abbiamo misurato i livelli di glucosio intramuscolare negli animali KO, notando che a riposo questi presentano una riduzione considerevole delle riserve. Per questo motivo gli animali KO, dopo i primi momenti di esercizio, sono costretti a cambiare il loro metabolismo verso una maggiore ossidazione degli acidi grassi che comunque non riesce a supportare la domanda energetica a causa dei mitocondri disfunzionali. Tutte queste evidenze indicano che TFEB controlla più il metabolismo rispetto all’autofagia la quale non è influenzata dalla modulazione genetica di TFEB; più in dettaglio TFEB sembra controllare direttamente il metabolismo del glucosio che è alterato negli animali TFEB-deficienti. Un ridotto assorbimento del glucosio e una ridotta sintesi del glicogeno durante gli EU-clamps spiegano perché le riserve di glicogeno sono ridotte negli animali KO mentre la controparte transgenica ne accumula in più. Questi effetti fenotipici sono accompagnati da un cambiamento nell’espressione di geni connessi all’omeostasi del glucosio, con maggiore presenza di trascritti per i trasportatori di glucosio and regolatori della sintesi del glicogeno nei muscoli transgenici, anche in assenza di PGC1α. Inoltre, l’overespressione di TFEB è in grado di modulare anche l’attività di nNOS e AMPK, influenzando l’omeostasi del glucosio non solo dal punto di visto trascrizionale, ma impattando anche sulle vie di segnale ad esso correlate. In conclusione tutte queste scoperte sostengono fortemente una nuova visione di TFEB come un fattore chiave nella regolazione della flessibilità metabolica durante l’esercizio fisico in modo indipendente da PGC1α.

Plusieurs études ont suggéré une implication des glycogène synthase kinases 3 (GSK3) dans la carcinogenèse, notamment du pancréas. Des études ont rapporté des résultats contradictoires quant à l’impact des GSK3 sur la survie cellulaire. Au niveau du pancréas, il a été observé que l’inhibition des GSK3 inhibe la croissance entre autres via la régulation de la voie JNK ou NFkB. Les inhibiteurs des GSK3 sont présentement à l’étude comme traitement de différentes pathologies, notamment pour le cancer pancréatique. Une meilleure compréhension des voies de signalisation régulées par les GSK3 sera donc nécessaire. Nous avons entrepris ces travaux afin de mieux comprendre les mécanismes impliqués dans la régulation de la survie des cellules pancréatiques tumorales par les GSK3. Nous avons démontré que l’inhibition des GSK3 induit l’apoptose et l’autophagie dans les cellules pancréatiques tumorales humaines. L’inhibition des GSK3 stimule l’autophagie autant dans les cellules pancréatiques tumorales que non tumorales, alors que l’apoptose est induite spécifiquement dans les cellules tumorales. Contrairement à l’apoptose, l’autophagie est induite indépendamment de la voie JNK-cJUN suite à l’inhibition des GSK3. Nos résultats démontrent que l’inhibition des GSK3 mène à l’inactivation de la voie mTORC1 qui pourrait contribuer à l’induction de l’autophagie. D’autre part, nos travaux ont démontré pour la première fois que les GSK3 régulent le facteur de transcription EB (TFEB) dans les cellules pancréatiques tumorales. En effet, l’inhibition des GSK3 entraîne la déphosphorylation de TFEB, notamment sur la Ser211, la dissociation des 14-3- 3 et sa translocation nucléaire. Nos résultats suggèrent que la régulation de TFEB par les GSK3 impliquerait des Ser/Thr phosphatases et pourrait être indépendante de l’activité mTORC1. L’inhibition de l’autophagie ou la déplétion de l’expression de TFEB sensibilise les cellules pancréatiques tumorales à l’apoptose induite suite à l’inhibition des GSK3 suggérant un rôle pro-survie de l’autophagie et de TFEB dans ces cellules. Enfin, l’inhibition des GSK3 semble mener à l’inhibition de la glycolyse qui contribuerait à l’induction de l’apoptose. En résumé, nos résultats démontrent que l’inhibition des GSK3 induit à la fois des signaux pro-apoptotiques et pro-survie suggérant que l’équilibre entre ces signaux dicterait l’impact des GSK3 sur la survie des cellules pancréatiques tumorales humaines.

Les lysosomes jouent un rôle central dans la régulation des processus anaboliques et cataboliques, la signalisation cellulaire ainsi que dans la mise en œuvre des programmes transcriptionnels au sein des cellules. Ils favorisent l’adaptation des cellules cancéreuses lors des variations du microenvironnement en leur fournissant les métabolites essentiels et l’énergie nécessaire à leur survie et à leur prolifération. Un acteur majeur dans la réponse adaptative des lysosomes est le facteur de transcription EB (TFEB). TFEB coordonne l’expression de gènes associés à la fonction et à la biogenèse des lysosomes, y compris ceux impliqués dans l’autophagie, un processus catabolique majeur des cellules qui dépend des lysosomes. TFEB et l’autophagie fonctionnent comme des mécanismes adaptatifs visant à rétablir l’homéostasie cellulaire en réponse à un stress. Cependant, la biogenèse des lysosomes et l'augmentation de leur taille induite par TFEB peuvent rendre les cellules cancéreuses plus vulnérables aux composés ciblant les lysosomes. Cette vulnérabilité ouvre la porte au développement de nouvelles stratégies pour lutter contre le cancer en ciblant simultanément les lysosomes et en activant TFEB. L’objectif initial de cette étude a été de découvrir de nouveaux agents pharmacologiques agonistes de TFEB, manifestant une cytotoxicité significative contre les cellules cancéreuses. Par un criblage de la bibliothèque Prestwick comprenant 1200 composés approuvés par la « Food and Drug Administration » (FDA), nous avons identifié deux antidépresseurs, la sertraline et l’indatraline, qui agissent en tant que puissants activateurs de la translocation de TFEB vers le noyau. Les deux composés induisent une accumulation de cholestérol au sein des lysosomes, entraînant la perméabilisation de leurs membranes et une perturbation du flux autophagique. L’analyse de modélisation moléculaire a révélé que les deux composés pourraient inhiber le trafic du cholestérol en se liant au site de fixation du cholestérol des transporteurs, Niemann-Pick type C1 (NPC1) et NPC2. Dans les cellules cancéreuses, la sertraline et l’indatraline provoquent une mort cellulaire immunogénique, en transformant les cellules mourantes en vaccins prophylactiques capables de protéger contre la croissance tumorale chez la souris. Dans un contexte thérapeutique, une dose unique de ces composés était suffisante pour ralentir de façon significative la croissance tumorale de manière dépendante des lymphocytes T. Ces résultats caractérisent la sertraline et l’indatraline comme des agents immunostimulants qui agissent à travers un mécanisme novateur connectant l’accumulation du cholestérol lysosomal aux dommages lysosomaux, entraînant ainsi la mort immunogénique des cellules cancéreuses. Ces résultats soutiennent le repositionnement de ces deux molécules en tant qu’agents immunostimulants pour le traitement du cancer et encouragent l’extension de cette étude à d’autres inhibiteurs du transport lysosomal du cholestérol
Lysosomes serve as an intracellular platform that coordinates anabolic and catabolic processes, cell signaling, and transcriptional programs. These organelles allow the adaptation of cancer cells to a changing microenvironment by supplying them with essential metabolites and energy for their survival and proliferation. A major player in the lysosomal adaptive response is the transcription factor EB (TFEB), which is part of the microphthalmia/transcription factor E (MIT/TFE) family of transcription factors. TFEB plays a pivotal role in driving the expression of several genes associated with lysosome function and biogenesis, including those participating in autophagy. The latter is a critical lysosomal catabolic process in the cell. While TFEB and autophagy function as adaptive mechanisms to reestablish cellular homeostasis in response to stressors, TFEB-induced lysosomal biogenesis and enlargement can render cancer cells more vulnerable to compounds targeting lysosomes. This vulnerability opens the door for developing new strategies to combat cancers by simultaneously targeting the lysosome and activating TFEB. This study initially aimed to uncover novel pharmacological agents that function as agonists of TFEB and exhibit substantial cytotoxicity against cancer cells. By conducting cell-based drug screening of the Prestwick library, consisting of 1200 Food and Drug Administration (FDA)-approved compounds, we identified two antidepressants, sertraline and indatraline, as potent inducers of TFEB nuclear translocation. Both compounds promoted cholesterol accumulation within lysosomes, resulting in lysosomal membrane permeabilization, disruption of autophagy, and cell death. Molecular docking analysis unveiled that indatraline and sertraline may inhibit cholesterol traffic by binding to the same cavity where cholesterol typically binds to the lysosomal cholesterol transporters, Niemann-Pick type C1 (NPC1) and NPC2. In cancer cells, sertraline and indatraline elicited immunogenic cell death, converting dying cells into prophylactic vaccines that were able to protect against tumor growth in mice. In a therapeutic setting, a single dose of each compound was sufficient to significantly reduce the outgrowth of established tumors in a T cell-dependent manner. These results identify sertraline and indatraline as immunostimulatory agents that operate through a novel mechanism that connects lysosomal cholesterol accumulation to lysosomal membrane permeabilization, ultimately leading to immunogenic cell death. These results support the repositioning of sertraline and indatraline as immunostimulatory agents for cancer treatment and encourage the broadening of this study to other lysosomal cholesterol transport inhibitors

Die Komposition der Skelettmuskulatur resultiert aus der fein abgestimmten Balance von Proteinauf- und Abbaumechanismen. Die Skelettmuskelatrophie kann in verschiedenen Situationen entstehen bzw. von diversen Krankheiten ausgelöst werden (Altern, Hunger, Krebs, Nervenschädigung, Kachexie) und ist meist die Folge von gesteigertem Proteinabbau, der die Proteinsynthese überwiegt. Der Muskelabbau ist physiologisch teilweise sinnvoll und dient der Notversorgung von lebenswichtigen Organen mit Lipiden, Aminosäuren und Glukose. Insgesamt ist eine funktionsfähige Muskulatur sehr wichtig, sowohl für Gesunde als auch Erkrankte, da bei Muskelatrophie auslösenden Erkrankungen das Gesamtüberleben wesentlich verringert ist und die Lebensqualität der Patienten enorm reduziert ist. Der Abbau von strukturellen Muskelproteinen wurde hauptsächlich dem Ubiquitin-Proteasom System zugeschrieben, dessen Regulation und von seinen einzelnen Enzymen muss genauestens verstanden sein, um in der Zukunft zielgerichtete Therapien entwickeln zu können. Eines der zentralen Enzyme in der Skelett- und Herzmuskelatrophie ist die E3 Ubiquitin Ligase MuRF1. In nahezu allen Modellen für Muskelatrophie wurde eine starke Zunahme der Expression von MuRF1 beschrieben. Betrachtet man die sehr zentrale Rolle von MuRF1 im UPS, dort vermittelt MuRF1 den Abbau von strukturellen Proteinen des Sarkomers, und der beobachteten starken Regulation bei diversen Atrophie-Modellen, wird klar, wie wichtig das Verständnis der transkriptionellen Regulation von MuRF1 selbst ist. In den letzten Jahren wurden bereits einige Transkriptionsfaktoren identifiziert, die an der Regulation von MuRF1 bei verschiedenen Atrophie-Modellen beteiligt sind, die Studien zeigten aber auch, dass noch nicht alle Modelle erklärt werden konnten. Um die verbleibenden Wissenslücken zu füllen, wurde in dieser Studie nach neuen transkriptionellen Regulatoren von MuRF1 gesucht und deren Beteiligung an bereits bekannten Signalwegen analysiert.
Skeletal muscle mass is permanently balanced as a result of fine tuned protein synthesis and degradation mechanisms. Skeletal muscle atrophy occurs when protein degradation exceeds protein synthesis, which happens in a variety of conditions, such as aging, starvation, cancer, cachexia or denervation. Degradation of muscle mass can sometimes be useful, e.g. as source for lipids, amino acids and glucose in case of critical malnutrition as well as several other physiological conditions. But a solid composition and thereby functional maintenance of muscles is necessary for healthy individuals as well as individuals suffering from atrophy releasing diseases as to retain their mobility and to preserve full heart functions. Since degradation of structural proteins in muscle tissue has been addressed mainly to the ubiquitin-proteasome-system, the regulation of the participating components needs to be understood in detail to develop constructive treatments and therapies for atrophy prevention. One of the key enzymes in skeletal and heart muscle atrophy is the E3 ubiquitin ligase MuRF1. Its expression levels and protein content was found to be elevated in almost every know atrophy model. MuRF1 is very critical for the muscles composition and thus their functional integrity, as it marks and initiates degradation of structural and contractile proteins via the UPS. Since MuRF1 plays a prominent role in muscle atrophy, its transcriptional regulation needs to be well understood to develop effective therapies for all the different atrophy models MuRF1 has been linked to. Several transcription factors have been identified to regulate MuRF1 at different ratios and in diverse atrophy models. Importantly, they do not explain all MuRF1 inducing events observed. To fill some of the remaining knowledge gaps, the studies aims were to find new transcriptional regulators for MuRF1 and to analyze potential involvements of the obtained candidates in pathways affecting skeletal muscle atrophy.

La malaltia de Parkinson (MP) és un trastorn neurodegeneratiu crònic que es caracteritza per una pèrdua progressiva de les neurones dopaminèrgiques de la substància nigra pars compacta (SNpc). Tot i que s’han dut a terme diverses estratègies per intentar aturar la progressió de la MP, cap d’elles ha demostrat ser de suficient eficàcia. El possible paper del factor de transcripció EB (TFEB) com a diana terapèutica en la MP va guanyar importància quan es va descobrir que el TFEB controla la biogènesi lisosomal i l’autofàgia, i que la seva activació podria contrarestar el defecte lisosomal i l’agregació de proteïnes, dos fenòmens descrits des de fa temps que estan implicats en les malalties neurodegeneratives, incloent la MP. Malgrat això, la majoria de les presumptes dianes del TFEB que s’han descrit fins a dia d’avui estan implicades en diversos processos biològics que no estan relacionats amb el sistema autofàgic-lisosomal. Per tant, aprofundir el nostre coneixement sobre la funció del TFEB en neurones podria ser crucial per desenvolupar una bona estratègia terapèutica per la MP i altres trastorns neurològics. En aquesta tesi, s’ha determinat l’efecte de sobreexpressar el TFEB per mitjà d’un vector viral adeno-associat en les neurones dopaminèrgiques de la substància nigra de ratolí i s’han estudiat els processos moleculars activats en condicions de sobreexpressió de TFEB que podrien oferir beneficis importants en el context de la MP. En aquest sentit, s’ha demostrat que la sobreexpressió de TFEB induïa un efecte neurotròfic, el qual no estava descrit, i que alhora anava acompanyat d’una millora en la funcionalitat dopaminèrgica, de l’activació de vies de senyalització implicades en supervivència i de canvis mitochondrials que, tots plegats, podrien contribuir en fer més resistents a les neurones front la mort cel·lular. Per explorar en més profunditat aquest tema, es va estudiar el potencial terapèutic del TFEB en un context parkinsonià, l’induït per la neurotoxina MPTP. D’aquesta manera, es va demostrar que la sobreexpressió de TFEB era capaç d’aturar la neurodegeneració induïda pel MPTP tant a nivell de cos neuronal com de projecció dopaminèrgica a nivell de l’estriat i que, al mateix temps, era capaç de restaurar l’activitat/funció i el fenotip neuronal en el model MPTP de la MP. A més, la sobreexpressió de TFEB va ser capaç de contrarestar els processos deleteris com la depleció lisosomal i la mort cel·lular mitjançada pel mitocondri, els quals estan relacionats tant amb la neurotoxicitat induïda pel MPTP com amb la MP. Per altra banda, també es va descobrir que activar la via autofàgica-lisosomal a través d’un knockdown del principal repressor de l’autofàgia, ZKSCAN3, no va ser suficient per evitar la neurodegeneració o l’atròfia induïda pel MPTP. En conclusió, els resultats d’aquesta tesi revelen nous mecanismes de rellevant importància per l’efecte neuroprotector induït pel TFEB, i remarquen que incrementar l’activitat del TFEB podria ser una bona estratègia per lluitar contra la mort neuronal i restaurar la funcionalitat neuronal en la MP i altres malalties neurodegeneratives.
Parkinson’s disease (PD) is a chronic neurodegenerative disorder mainly characterized by progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Albeit diverse efforts have been carried out to halt the progression of PD, none of them have been fully satisfactory. The possible implication of transcription factor EB (TFEB) as a therapeutic target in PD has gained momentum since it was discovered that TFEB controls lysosomal biogenesis and autophagy and that its activation might counteract lysosomal impairment and protein aggregation, which have long been described in neurodegenerative diseases, including PD. However, the majority of putative direct targets of TFEB described to date is linked to a range of biological processes that are not related to the autophagy-lysosomal system. Therefore, to deepen our knowledge on TFEB function in neurons may be crucial to develop a potential therapeutic strategy for PD and other related neurological disorders. In this thesis, we assessed the effect of overexpressing TFEB by means of an adeno-associated viral vector in mouse substantia nigra dopaminergic neurons and studied several molecular processes activated upon TFEB overexpression that may offer potential benefits in the context of PD. In this line, we demonstrated that TFEB overexpression drove a previously unknown bona fide neurotrophic effect accompanied by an enhanced dopaminergic function, activation of pro-survival signaling pathways and mitochondrial changes that altogether may contribute to render neurons less prone to cell death. To delve further into this concept, we studied the therapeutic potential of TFEB in a parkinsonian context, that induced by the neurotoxin MPTP. In this regard, we showed that TFEB overexpression was indeed able to block MPTP-induced neurodegeneration both at the cell body level as well as striatal dopaminergic terminals and restored neuronal activity/function and phenotype in the MPTP mouse model of PD. Moreover, TFEB overexpression also counteracted the deleterious events like lysosomal depletion and mitochondria-mediated cell death that are linked to MPTP neurotoxicity and PD. Besides, we unraveled that activating the autophagy-lysosomal pathway by knocking down the master repressor of autophagy ZKSCAN3 did not prevent MPTP-induced neurodegeneration or atrophy. Altogether, our results uncover new mechanisms decisive for the neuroprotective effect elicited by TFEB and highlight increasing TFEB activity as a therapeutic approach to fight neuronal death and restore neuronal function in PD and other neurodegenerative diseases.

La maladie d’Alzheimer (MA) se caractérise par l’accumulation dans le cerveau d’agrégats extracellulaires et intraneuronaux (Aβ et Tau). Dans la cellule, la principale voie de dégradation des protéines agrégées est la voie lysosomale-autophagique, qui est altérée de façon précoce chez les patients Alzheimer. Des études récentes de mon laboratoire ont montré que ce dysfonctionnement serait à la fois la cause et la conséquence de l’accumulation du précurseur direct de l’Aβ, appelé C99 ou APP-CTFβ. De par sa toxicité, le C99 semble donc jouer un rôle crucial dans l’étiologie de la maladie. Son accumulation se produit majoritairement dans les compartiments endolysosomaux mais de façon intéressante, un marquage extracellulaire associé au C99 a également été observé à des stades plus avancés de la maladie ou en présence d’un inhibiteur de la γ-sécrétase (enzyme clivant le C99 en Aβ). Le premier axe de mon travail de thèse a donc consisté à étudier l’efficacité d’une restauration du système lysosomal-autophagique sur l’accumulation du C99. Dans ce but, nous avons utilisé une stratégie virale visant à exprimer le facteur de transcription EB (TFEB) dans un modèle murin de la MA (3xTg-AD). Ce facteur est le principal régulateur de la biogenèse lysosomale et de l’autophagie. Deux approches ont été testées cherchant à exprimer le TFEB avant ou après le début de l’accumulation du C99, grâce à une injection de virus exprimant le TFEB, soit en intracérébroventriculaire dès la naissance, soit par stéréotaxie à l’âge de 4 mois. Ces études ont montré une réduction importante de l’accumulation intraneuronale du C99 chez les souris 3xTg-AD, que ce soit via l’approche "préventive" ou "curative". Le deuxième axe de mon travail de thèse a cherché à comprendre l’origine du marquage extracellulaire observé dans le cerveau et associé au C99. Nous avons émis l’hypothèse que ce marquage correspondrait à des exosomes enrichis en C99. Les exosomes sont des vésicules extracellulaires, d’origine endosomale et sécrétées par les cellules, ayant déjà été décrites comme transportant des protéines neurotoxiques. Grâce à des approches pharmacologiques, immunocytochimiques et génétiques, nous avons confirmé cette hypothèse et mis en évidence la présence de C99 et de son dérivé le C83 (APP-CTFα) dans les exosomes purifiés à partir de modèles cellulaires ou murins de la MA, sous forme monomérique et oligomérique. Nos travaux montrent également que la charge des exosomes en oligomères est fortement amplifiée en présence d’une inhibition de la γ-sécrétase, expliquant ainsi le marquage extracellulaire. En conclusion, mes travaux de thèse (1) proposent une potentielle stratégie thérapeutique, basée sur l’activation du TFEB et visant à empêcher l’accumulation du C99, (2) montrent la présence de monomères et d’oligomères de C99 dans les exosomes ainsi qu’un lien entre la γ-sécrétase et l’oligomérisation. Les futures études devront déterminer le rôle exact de ces exosomes enrichis en C99
Alzheimer’s disease (AD) is characterized by the pathological accumulation of extracellular and intracellular aggregates (Aβ and Tau) in the brain. AD is also associated with an early alteration of the major degradation pathway of aggregated proteins, the autophagic-lysosomal pathway. Recent works have suggested that this defectcouldbothbeacauseandaconsequenceofearlyintraneuronalaccumulation of C99 (also named as APP-CTFβ), the direct precursor of Aβ. Due to its toxicity, C99 could be a possible key player of AD etiology. The accumulation of this product occurs mainly within organelles of the endolysosomal network, but our recent observations also indicate an extracellular accumulation of C99 in later stages of the disease, or in conditions where the Aβ-generating enzyme, γ-secretase, is blocked. The first aim of my PhD project was to investigate the possible beneficial effect of restoringlysosomal-autophagicfunctiononC99accumulation. Tothisend, weused a viral strategy to overexpress TFEB, a master regulator of both lysosome biogenesis and autophagy, in a mouse model of AD (3xTg-AD mouse). Two approaches were tested aiming to express TFEB either before or after the beginning of C99 accumulation, by injecting AAV-TFEBs into the ventricles of newborn mice or by stereotaxic injection into 3 month-old mice, respectively. These studies have shown a strong TFEB-mediated reduction of C99 accumulation when using both the preventive and curative approach. The aim of the second part of my PhD work was to understand the reasons of the extracellular accumulation of C99. We postulated that this C99-associated immunostaining could correspond to exosomal-associated C99. Exosomes are nanosizedvesiclesofendocyticoriginthatarereleasedfromcellsandknowntotransport neurotoxic proteins. In our study based on pharmacological, immunocytochemical and genetic approaches, we have confirmed this hypothesis and have shown the presence of C99, and of its direct derived-fragment C83 (APP-CTFα), existing as both monomers and oligomers, in exosomes purified from AD cell and mouse models. Moreover, our data have shown that the levels of these APP-CTFs are strongly increased by γ-secretase inhibition, thus explaining the higher levels of extracellular staining in γ-secretase treated animals. In conclusion, my PhD work shows 1) a new potential therapeutic strategy based on TFEB activation aiming to reduce early C99 accumulation and 2) the presence of monomeric and oligomeric C99 in exosomes in AD models and a link between γ-secretase inhibition and oligomerisation. Future studies are needed to elucidate the exact role of these C99-enriched exosomes in AD

This thesis reports the research I conducted on aspects of the pharmacology and biological activities of two natural stilbenes, Resveratrol (Rv) and Pterostilbene (Pt), major polyphenolic components of grapevines and blueberries respectively. Over the years, these two molecules have drawn attention from the scientific community thanks to their beneficial bioactivities, relevant for many areas of health care. Numerous papers describe their protective roles against the metabolic syndrome, cancer development and neurodegeneration. These striking effects nowadays are not exclusively attributed to their redox properties but also to their capacity to modulate, directly or indirectly, the activities of key proteins of interconnected cellular signalling networks. Importantly, no noxious side effects have been reported. However, in spite of their positive features, Rv and Pt are not free of shortcomings. They are subjected to an intensive phase II metabolism, largely limiting their bioavailability. Moreover, while much research has been carried out to elucidate the molecular mechanisms of action of Rv, those of Pt still largely remain to be established. During my PhD program, I addressed both these themes. I describe below: 1) the attempts to increase the bioavailability of Rv and Pt 2) my investigations on intracellular processes affected by Pt as well as the demonstration of its therapeutic potential in two different in vivo models 1) A variety of approaches has been developed to overcome the poor bioavailability of many drugs. My research group turned to the development of prodrugs for oral administration. In the case of polyphenols, a useful prodrug implies the use of protecting groups to mask the characteristic hydroxyl groups and to avoid metabolic transformations by phase II conjugative enzymes. These substituents, technically defined as pro-moieties, are attached to the backbone of the molecule through a chemical bond which can be broken in the relevant physiological environment. The choice of the pro-moiety is important for the process of absorption from the intestinal tract. The type of linkage is crucial to achieve “cargo” regeneration with appropriate kinetics. We created prodrugs for oral administration. Thus, they ought to exhibit relative stability in the gastro-intestinal tract and lability in other biological compartments (body fluids and organs). When I joined the group of Dr. Mario Zoratti, N,N-disubstituted carbamoyl derivatives of Rv bearing a PEG 350 or a sugar pro-moiety had been created. Although they increased the poor water solubility of the natural compound, these prodrugs were too stable under physiological conditions. Thus, we moved to the N-monosubstituted carbamoyl bond, expected to be less stable, for new prodrugs. Importantly, this type of chemical bond is essentially stable in acid, while it can be more easily lysed at neutral or basic pH values. A first set of compounds incorporated amino acids as pro-moieties. We hypothesized that in addition to increasing water solubility, amino acids might be recognized and transported by specific carrier systems, expressed in the enterocytes, and thus favor the intestinal absorption of the molecules. In another set of Nmonosubstituted carbamoyl derivatives polyhydroxylated groups (dihydroxypropyl or 6-deoxygalactose) were attached to all three, two or one phenolic oxygen(s) of Rv. In this case, the hope was that sugar transporters might intervene to facilitate absorption through the intestinal epithelium. All synthetized compounds displayed hydrolysis kinetics suitable for their use as prodrugs. However, absorption after oral administration to rats turned out to be rather unsatisfactory, with no evidence of an involvement of membrane carriers. In no case did galactosylated Rv derivatives reach the bloodstream of rats. The best results were obtained by administering monosubstituted aminoacid derivatives. We speculate that the extended, three-pronged structure of the fully protecting prodrugs might interfere with recognition and transport. All synthesis procedures and the characterization and pharmacokinetic evaluation of these families of compounds have been already published. The papers are included in this thesis as chapters 1 and 2. Once completed the assessment of Rv prodrugs, I started a new research project involving Pterostilbene, a more compact and possibly more effective compound than Resveratrol. This represented the main theme of my PhD program investigations. As Pt was still poorly defined from a pharmacological point of view, we first evaluated its distribution in blood and organs after oral administration (see chapter 3). Thus, we developed an appropriate method for the quantification of the molecule and its metabolites in organs after administration to rats. The resulting protocol preserves stability and allows the quantitative extraction and clarification from biological matrices of Pt itself and its metabolites, and their quantitative analysis by HPLC/UV. We used this method to determine the levels of these compounds in the blood and several other organs as a function of time after administration as a single intra-gastric dose of 88µmoles/kg of body weight. Pterostilbene levels in several organs turned out to be much higher than those measured in blood, reaching nmoles/gr (μM)-range concentrations. Moreover, Pt-4’-Sulfate was identified as the major metabolic species. Its levels were higher than those of Pt in all tissues examined except the brain. In light of this, we next aimed to prevent metabolic modifications by phase II conjugative enzymes. I exploited the experience derived from the work with the prodrugs of Rv and in collaboration with the research group of Prof. Paradisi of the Department of Chemical Sciences of the University of Padua, we developed and characterized a collection of Pt prodrugs bearing amino acids as pro-moieties, linked to the Pt 4’-OH via an Nmonosubstituted carbamate ester group. The rates of hydrolysis of all compounds fell in a range suitable for their use. Derivatives with hydrophobic aminoacid side chains were notable for the high levels of Pterostilbene they regenerated in blood after intragastric administration. We selected the most promising one and we measured its levels in rat organs, following the same methodology developed for Pterostilbene. This time, the pro-drug itself was the major specie measured in all organs considered (except the brain) but, most importantly, the levels of (regenerated) Pterostilbene were drastically increased and those of its sulfate decreased in comparison with the administration of the polyphenol as such (see chapter 4). 2) As mentioned above, the intracellular processes underlying Pt benefits are still largely undefined. Several papers suggest that the induction of autophagy may contribute importantly. I decided to investigate this aspect. Autophagy is a catabolic pathway whose dysregulation is associated with various pathological conditions. As discovered by Prof. Ballabio and co-workers it is mainly controlled by transcription factor EB (TFEB), normally inhibited by mTORC1. Thus, I evaluated the effect of Pt on the autophagy master regulator. We demonstrated that this polyphenol stimulates TFEB activity by promoting its translocation to the nucleus as well as its expression. Accordingly, Pt induced an increase of LC3B protein, an autophagy marker, and an up-regulation of TFEB lysosomal target genes. These investigations have been extended to two Pt major metabolites, Pt-4’-Sulfate and DiHydroPt, as they are the main species formed upon ingestion of the natural compound. Interestingly, while the former was ineffective, the reduced form showed an activity similar to that of the parent compound but required higher concentrations. Further studies have been carried out to clarify the upstream signalling cascade. FRET-sensor based measurements have shown that Pt is able to modestly increase the concentration of cAMP, followed by the activation of CREB. This cyclic nucleotide has been previously reported to indirectly activate AMPK, a well-recognized mTORC1 antagonist. Thus, the enhancement of this cellular axis may be responsible for the activation of TFEB. Accordingly, we observed a reduction of the activity of the mammalian target of the rapamycin. However, pharmacological interventions expected to drastically increase cAMP and AMPK activity were less effective than Pt, suggesting that this phenolic compound promotes TFEB nuclear migration by modulating more than one signalling pathway. In addition to mTORC1 inhibition, the activation of TFEB is mediated by Calcineurin. Recently, it has been demonstrated that both endogenous and exogenous ROS may promote the activity of this phosphatase. We observed in in vitro studies with cultured cells that indeed Pt increased the production of these species by mitochondria and that this phenomenon is related to TFEB migration. In light of this, it is possible to speculate that this may represent an alternative way through which this phenolic compound perturbs TFEB intracellular localization (see chapter 5). The establishment of the capability of Pt to induce autophagy in cultured cells led me to test Pt as a potential therapeutic treatment for Collagen VI (ColVI) muscular dystrophies. These disorders are mainly characterized by the presence of dysfunctional mitochondria. The concomitant deficiency of the autophagic process results in the exacerbation of the pathological conditions since the apoptotic death of myofibers occurs. As known from the literature, the use of an antisense morpholino is nowadays the best strategy to obtain zebrafish animal models with a strong phenotype of ColVI-related muscle dystrophy. Therefore, we injected into fertilized eggs a designed oligonucleotide, specifically directed against the ColVI exon 9 splicing region. This results in a frame-shift deletion of the N-terminal region of the ColVIa1 triple helical domain and therefore in a strong impairment of the organization of the muscular fibers. The data I have obtained using this model indicate that Pt treatment induces a recovery of muscular structure by more than 30%, as well as a clear recovery of motor activity.This part of the project has been carried out in collaboration with Prof. Paolo Bernardi and Dr. Marco Schiavone of the University of Padua. Although these results do not provide evidence that the amelioration of the dystrophic phenotype is due to the induction of autophagy, it is credible to suppose that the same cascade of events enhanced in vitro by Pt may be relevant also in vivo. Supporting this hypothesis, Pt provoked a significant increase of a mCherry protein, whose expression is under control of cAMP responsive elements (CRE) in a zebrafish transgenic reporter (this model has been generated by Dr. Patrizia Porazzi and Prof. Natascia Tiso of the University of Padua). Further investigations need to be performed (see chapter 6). Finally, during my graduate training I have also participated in another project still ongoing in my research laboratory. The pharmacokinetic study we performed in rats showed that Pt was particularly abundant in the brain. This observation is consistent with several papers showing a role of this phenolic compound in ameliorating performance of old rodents in behavioral tests. However, also in this context, the mechanisms accounting for these effects have been poorly characterized. My in vitro data, confirmed later in zebrafish, suggest that Pt may activate CREB following an increase of cAMP. This transcription factor has been demonstrated to play a crucial role in memory consolidation as it promotes neurogenesis in the dentate gyrus, a subarea of the hippocampus, in adult individuals. Memory, among other functions, undergoes deterioration in old age. In light of this, we set up an experimental work (see chapter 7), in collaboration with Prof. Nicoletta Berardi and the research group of Dr. Alessandro Sale, aimed to evaluate the molecular changes in the dentate gyrus and the hippocampus related to the recovery of cognitive impairments in old rats by Pt, if any. The results confirmed that after chronic administration of Pt aged animal presented a remarkable improvement in memory-related behavioral tasks. Moreover, although statistics need to be improved by adding more animals, Western blot and gene expression analyses suggest that the treatment up-regulates the activity of transcription factor CREB. An increase of mitochondrial mass has been also measured. These effects were more evident in the dentate gyrus if compared to the remaining hippocampus and strongly support the occurrence of neuronal remodeling processes. However, no differences in PSD95 levels have been observed. Further markers of synaptic plasticity will need to be taken into account in future investigations. Concluding, the studies I carried out have outlined one of the mechanisms accounting for the striking properties of the Pt, characterized its organ pharmacokinetics in the rat and allowed to boost its bioavailability. Thus, they may be helpful for the development of Pt-based therapeutic/preventive treatments.
Questa tesi riguarda la ricerca che ho condotto su aspetti della farmacologia e delle attività biologiche di due stilbeni, il Resveratrolo (Rv) e lo Pterostilbene (Pt), componenti polifenoliche principali rispettivamente della vite e dei mirtilli. Negli ultimi anni queste due molecole hanno attirato l’attenzione della comunità scientifica grazie alle loro attività biologiche di rilievo per molte settori della medicina. Numerosi studi descrivono le loro azioni protettive nei confronti di sindromi metaboliche, contro il cancro e la neurodegenerazione. Al giorno d’oggi, questi effetti non vengono attribuiti tanto alle loro proprietà antiossidanti quanto alla loro capacità di modulare, direttamente o indirettamente, l’attività di proteine-chiave in vie di segnalazione intracellulare tra loro interconnesse. Un punto importante è che non sono stati riportati effetti collaterali negativi del loro utilizzo. Nonostante questi aspetti positivi, Rv e Pt presentano dei punti deboli. Entrambi sono soggetti ad un intenso metabolismo di fase II che ne limita significativamente la biodisponibilità. Inoltre, mentre molto lavoro è stato condotto con il fine di elucidare i meccanismi di azione del Rv, quelli dello Pt rimangono ancora in gran parte da chiarire. Durante il mio corso di dottorato, mi sono occupata di entrambe queste problematiche. Descrivo di seguito: 1) I tentativi effettuati per aumentare la biodisponibilità del Rv e dello Pt 2) Gli studi dei processi intracellulari innescati dallo Pt e la valutazione del suo potenziale terapeutico in due modelli in vivo 1) Per far fronte al problema della scarsa biodisponibilità sono stati adottati diversi metodi. Il mio gruppo di ricerca è ricorso alla produzione di pro-farmaci. Nel caso dei polifenoli, un buon pro-farmaco implica l’utilizzo di gruppi protettori per mascherare i gruppi ossidrilici tipici di queste molecole e per evitare le modifiche metaboliche da parte degli enzimi coniugativi di fase II. Tali sostituenti, chiamati “pro-moieties”, sono legati chimicamente alla struttura stilbenica in modo reversibile nelle condizioni fisiologiche d’impiego. Essi assumono particolare importanza per l’assorbimento dei composti, mentre la tipologia del legame è cruciale affinché le molecole attive vengano rigenerate con cinetiche adeguate. Noi abbiamo voluto creare dei pro-farmaci da somministrare oralmente. Pertanto essi dovevano essere relativamente stabili nel tratto gastro-intestinale e più labili negli altri compartimenti biologici (fluidi corporei e organi). Quando ho iniziato la mia esperienza nel gruppo di ricerca del Dott. Mario Zoratti, erano stati sintetizzati dei derivati del Rv come esteri carbammici N,N-bisostituiti recanti come sostituenti (pro-moieties) PEG 350 o gruppi glicosidici. Questi profarmaci aumentavano la scarsa solubilità in acqua del composto fenolico, ma si sono rivelati troppo stabili in ambiente fisiologico per essere utilizzati come pro-drugs. Abbiamo quindi deciso di creare dei derivati carbamoilici N-monosostituiti che si prevedeva sarebbero stati meno stabili. Un aspetto importante è che questa struttura chimica è essenzialmente stabile in acido, mentre può essere idrolizzata a pH vicino alla neutralità o basico. In un primo set di composti si sono utilizzati come promoieties degli aminoacidi. Oltre ad aumentare la solubilità in acqua, ci si aspettava che gli aminoacidi potessero essere riconosciuti da specifici sistemi di trasporto espressi a livello degli enterociti favorendo l’assorbimento intestinale della molecola. In un altro gruppo di derivati carbamoilici N-monosostituiti, tutti e tre, due o uno solo degli ossigeni fenolici del Resveratrolo sono stati decorati con gruppi poliossidrilati (diidrossipropil o 6-deossigalattosil). In questo caso si sperava che i trasportatori di zuccheri potessero intervenire per facilitarne l’assorbimento attraverso l’epitelio intestinale. Come ci si attendeva, tutti i composti sintetizzati venivano idrolizzati con cinetiche compatibili con la loro utilizzazione come pro-drugs. L’assorbimento dopo somministrazione orale invece è risultato essere insoddisfacente, senza alcuna evidenza di un coinvolgimento di traslocatori di membrana. Il Rv galattosilato non ha raggiunto la circolazione sistemica in nessun caso. I risultati migliori sono stati ottenuti con i derivati amminoacidici mono-sostituiti. Ipotizziamo quindi che la struttura estesa e tripartita dei prodrugs con protezione completa interferisca con il riconoscimento ed il trasporto da parte dei sistemi di membrana. Tutte le procedure sintetiche, la caratterizzazione e la valutazione farmacocinetica di queste famiglie di composti sono già state pubblicate. Gli articoli relativi sono inclusi in questa tesi come capitoli 1 e 2. Dopo aver completato il lavoro coi pro-farmaci del Rv, ho iniziato un nuovo progetto riguardante lo Pt. Questo ha rappresentato il principale tema delle ricerche del mio programma di PhD. Poiché lo Pt era stato poco studiato da un punto di vista farmacologico, ne abbiamo dapprima valutato la distribuzione nel sangue e negli organi (capitolo 3). Abbiamo quindi messo a punto un metodo per la quantificazione di questa molecola e suoi metaboliti negli organi di ratto. Il protocollo che abbiamo sviluppato ha permesso di preservare la stabilità del composto e di ottenere un’estrazione quantitativa dello Pt e dei suoi metaboliti dalle matrici biologiche. Le analisi HPLC/UV hanno rivelato che, in seguito ad una somministrazione singola di una dose pari a 88µmoli/Kg di peso corporeo, i livelli di Pt in diversi organi erano maggiori rispetto a quelli riscontrati nel sangue, raggiungendo valori nell’ambito di varie nmoli/gr (µM). Inoltre, lo Pt-4’-solfato è stato identificato come la principale specie metabolica. I suoi livelli erano più alti rispetto a quelli dello Pt in tutti gli organi presi in considerazione tranne che nel cervello. Alla luce di questi risultati, abbiamo lavorato ulteriormente per cercare di limitare le modificazioni metaboliche da parte degli enzimi di fase II. Ho quindi sfruttato l’esperienza acquisita dal lavoro con i pro-farmaci del Rv e in collaborazione con il gruppo di ricerca della Prof. Paradisi del Dipartimento di Scienze Chimiche dell’Università di Padova, abbiamo sintetizzato e caratterizzato una serie di derivati dello Pt recanti amminoacidi come pro-moieties, collegate all’ossidrile in posizione 4’ tramite un legame carbammico mono-sostituito. Le cinetiche di idrolisi di questi composti sono risultate adatte per il loro utilizzo. I derivati con catene laterali idrofobiche si sono distinti per gli elevati livelli di Pterostilbene rigenerato nel sangue dopo somministrazione intra-gastrica ai ratti. Abbiamo pertanto selezionato il più promettente tra questi composti e ne abbiamo controllato la distribuzione negli organi, seguendo lo stesso protocollo utilizzato per lo Pt. In questo caso, il pro-farmaco per se è stato identificato come la specie predominante in tutti gli organi eccetto che nel cervello, ma soprattutto, i livelli di Pt rilasciati sono risultati notevolmente maggiori, e quelli di solfato minori, se paragonati a quelli ottenuti somministrando la molecola originaria (capitolo 4). 2) Come accennato in precedenza, i meccanismi intracellulari alla base degli effetti benefici dello Pt non sono stati ancora completamente delucidati. Alcune ricerche suggeriscono che le sue proprietà potrebbero essere attribuite all’induzione dell’autofagia. Ho quindi deciso di indagare questo aspetto (capitolo 5). L’autofagia è una via di degradazione la cui errata regolazione è associata a varie condizioni patologiche. Il Prof. Ballabio ed i suoi collaboratori hanno dimostrato che questo processo è regolato principalmente dal Fattore di Trascrizione EB (TFEB), inibito da mTORC1. Ho quindi voluto verificare gli effetti dello Pt su questo fattore di trascrizione. Ho dimostrato che questo polifenolo è in grado di stimolare l’attività di TFEB inducendo la sua migrazione nel nucleo e promuovendo la sua espressione. In accordo con quanto osservato, lo Pt ha provocato un aumento della lipidazione della proteina LC3 e dei livelli di espressione di alcuni geni lisosomiali target di TFEB. È stata valutata anche l’efficacia dei due metaboliti principali dello Pt, Pt-4’-solfato e DiidroPt, poiché essi rappresentano le specie principali ritrovate in circolo dopo somministrazione dello Pt. Mentre il primo composto è risultato essere inerte, il secondo si è mostrato attivo come lo Pt, ma a concentrazioni più alte. Ulteriori studi sono stati effettuati per esplorare le vie di segnalazione a monte di questi fenomeni. Misurazioni basate sull’utilizzo di sonde FRET hanno messo in evidenza che lo Pt incrementa la concentrazione di cAMP e attiva CREB. Come riportato in precedenti lavori, questo nucleotide ciclico può indurre indirettamente l’attivazione dell’AMPK, noto antagonista di mTORC1. La stimolazione di questo asse di segnalazione cellulare potrebbe quindi essere alla base dell’attivazione del TFEB. In accordo con questa ipotesi, abbiamo osservato una riduzione dell’attività chinasica di mTORC1. Trattamenti farmacologici volti ad aumentare le concentrazioni di cAMP o attivare l’AMPK si sono tuttavia rivelati meno efficaci dello Pt indicando che questo polifenolo induce la migrazione di TFEB al nucleo modulando diverse vie di segnalazione cellulare. La traslocazione nucleare di TFEB, oltre ad essere sotto il controllo di mTORC1, può anche essere regolata dalla Calcineurina. Di recente, è stato dimostrato che questa fosfatasi può essere attivata da ROS esogeni ed endogeni. Nel contesto di queste ricerche, ho evidenziato che lo Pt aumenta la produzione mitocondriale di queste specie e che questo fenomeno è correlato alla migrazione del TFEB. Alla luce di ciò, è possibile ipotizzare che questa rappresenti una via alternativa attraverso la quale lo Pterostilbene influenza la localizzazione subcellulare del fattore di trascrizione (capitolo 5). Lo stabilirsi dell’azione pro-autofagica dello Pt in cellule in coltura mi ha spinto a saggiare il suo potenziale terapeutico per il trattamento delle distrofie muscolari da carenza di Collagene VI. Queste malattie sono caratterizzate principalmente dall’accumulo di mitocondri non funzionali. Concomitanti difetti del processo autofagico aggravano ulteriormente le condizioni patologiche e conducono alla morte di tipo apoptotico delle miofibrille. Come indicato dalla letteratura scientifica, l’utilizzo di un morfolino antisenso è attualmente la strategia migliore per ottenere un fenotipo marcato di distrofie muscolari da carenza di Collagene VI in zebrafish. Abbiamo quindi iniettato specifici oligonucleotidi antisenso diretti contro l’esone 9 dell’mRNA codificante per il Collagene VI nelle uova fecondate di tali pesci. In questo modo abbiamo indotto una delezione “in frame” della regione N-terminale del dominio a tripla elica della molecola di Collagene VI e una profonda alterazione della struttura delle fibre muscolari. I dati che ho ottenuto indicano che il trattamento con lo Pt induce un recupero pari a più del 30% nella struttura delle fibre muscolari ed un notevole aumento dell’attività motoria. Questa parte del progetto è stata svolta in collaborazione con il Prof. Bernardi e il Dott. Marco Schiavone del Dipartimento di Scienze Biomediche dell’Università di Padova. Nonostante i nostri risultati non dimostrino direttamente che il miglioramento delle condizioni patologiche sia dovuto all’induzione dell’autofagia, è plausibile che la stessa serie di eventi che si verificano in vitro possano aver luogo anche in vivo. A supporto di questa ipotesi, abbiamo verificato che lo Pt è in grado di aumentare i livelli di una proteina mCherry la cui espressione è posta sotto il controllo di elementi influenzati dal cAMP (CRE) in una linea transgenica reporter di zebrafish (questo modello è stato generato dalla Dott.ssa Patrizia Porazzi e dalla Prof.ssa Natascia Tiso del Dipartimento di Biologia dell’Università di Padova). Tali fenomeni necessitano di essere ulteriormente approfonditi (capitolo 6). Infine, durante il mio percorso di dottorato, ho preso parte ad un altro progetto ancora in corso nel mio laboratorio di ricerca. Le analisi farmacocinetiche condotte in ratti hanno riportato che lo Pt è particolarmente abbondante nel cervello. Questa evidenza è in linea con diversi studi che dimostrano la capacità del polifenolo di migliorare le prestazioni di animali anziani in test comportamentali. I meccanismi molecolari alla base di questi effetti sono stati poco caratterizzati anche in questo contesto. Il lavoro che ho condotto in vitro, e confermato in zebrafish, dimostra che lo Pt è in grado di indurre un aumento di cAMP attivando CREB. È stato dimostrato che questo fattore di trascrizione svolge un ruolo cruciale per il consolidamento della memoria perché in grado di promuovere la neurogenesi in soggetti adulti a livello del giro dentato, una parte dell’ippocampo. La memoria, insieme ad altre funzioni cognitive, peggiora notevolmente con l’invecchiamento. Alla luce di ciò, in collaborazione con la Prof.ssa Nicoletta Berardi ed il gruppo di ricerca del Dott. Alessandro Sale (Istituto di Neuroscienze - Pisa), abbiamo messo a punto un lavoro sperimentale volto a valutare cambiamenti molecolari nel giro dentato e nell’ippocampo in seguito ad un eventuale miglioramento cognitivo in ratti anziani da parte dello Pt (capitolo 7). I risultati che abbiamo ottenuto hanno confermato che, dopo somministrazione cronica di questo composto, gli animali dimostrano un miglioramento della memoria. Inoltre, nonostante la statistica necessiti di essere ampliata aggiungendo più individui, sia analisi Western blot che RT-qPCR suggeriscono che tale trattamento ha indotto una up-regolazione di CREB. Allo stesso tempo, è stato misurato un aumento della massa mitocondriale. Questi effetti sono più evidenti a livello del giro dentato piuttosto che nella parte restante dell’ippocampo e suggeriscono fortemente il verificarsi di processi di rimodellamento neuronale. Nonostante ciò, non sono state riportate differenze nei livelli di PSD95. Per le prossime analisi saranno presi in considerazione marker diversi di plasticità sinaptica. In conclusione, le ricerche che ho condotto hanno permesso di delineare uno dei meccanismi responsabili delle proprietà dello Pt e di incrementare la sua biodisponibilità. Pertanto, essi potrebbero esse utili per lo sviluppo di futuri trattamenti terapeutici o preventivi.

博士
國立清華大學
化學系
104
Except of the simplified view of lysosomes as the final compartments of degradation process, lysosomes are increasingly regarded as upstream organelles in the control of cell functions. Therefore, lysosome homeostasis should be tightly regulated to match the catabolic needs as well as to maintain lysosomal pathways. Here we use light-induced mitophagy substrates to disturb lysosome homeostatsis and reveal how lysosome biogenesis responses quantitatively to different levels of degradation stress. We observed that TFEB-mediated lysosomal genes activation is upregulated in dose-dependent manner upon variants mitophagy degradation stresses. This stress-response coordination is quantitatively modulated by mTOR inactivation. We further show that mitophagy-dependent mTOR inactivation is mediated by spatially recruiting mTOR activity regulators, DEPDC5 and FLCN, on autolysosome, a hybrid organelle of autophagosome and lysosome. Also, blocking autophagosome-lysosome fusion, by knocking down STX17, makes TFEB activation response decoupling from stress. These results suggest lysosome and autolysosome are functionally different in respect of mTOR activity and TFEB activation. Through fusing with autophagosomes, lysosomes sense degradation stress and decode into fine-tuning TFEB activation in the maintenance of self-regulated homeostasis.

Tuberous Sclerosis Complex (TSC) is a rare, autosomal dominant genetic disease that results from the loss-of-function mutations of either the TSC1 or TSC2 genes. It is a multisystemic disorder with manifestations in several organs including the lungs, kidneys, brain, skin, and heart. Loss of either TSC1 or TSC2 causes hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1) pathway resulting in cell proliferation and the continuous activation of multiple anabolic pathways that lead to tumor growth. Transcription factor EB (TFEB) is one of the many downstream targets of the mTORC1 pathway and is a master transcriptional regulator of lysosomal biogenesis. In the classical paradigm of TFEB-mTORC1 interaction, TFEB is negatively regulated by mTORC1. Interestingly, TFEB is upregulated and fully functional in Tsc1- and Tsc2- deficient, mTORC1 hyperactivated cells suggesting an alternate regulation involved in the context of TSC. The objective of this research was to investigate how TFEB upregulation and resulting lysosomal dysregulation in Tsc1- and Tsc2-deficient cells drives the pathogenesis of Tuberous Sclerosis Complex. By western blot analysis, elevated levels of TFEB were detected in Tsc1- and Tsc2-deficient cells compared to Tsc1- and Tsc2- expressing cells and multiple lysosomal proteins also showed increased expression in these cell lines. In Tsc1- and Tsc2-expressing MEFs, TFEB was phosphorylated and retained in the cytoplasm. Immunofluorescence imaging of TFEB in Tsc1- and Tsc2-deficient cells demonstrated nuclear localization. This was in contrast to previous studies, in which TFEB was retained in the cytoplasm upon mTORC1 activation. Immunohistochemistry staining of TFEB and the lysosomal protein, NPC1 (NPC Intracellular Cholesterol Transporter 1), in human TSC-associated renal cell carcinoma (RCC) also showed increased staining of TFEB and NPC1 in RCC compared to normal kidney tissue. This histological finding was confirmed in two mouse models of renal TSC, TSC2 +/- AJ mice and KSP-CreERT2 Tsc2fl/fl mice, which revealed strong expression of NPC1 in cyst-lining cells, specifically in the apical region, suggesting increased lysosome number and activity as a contributing factor to cystogenesis. These results suggest a novel mechanism in the development of Tuberous Sclerosis Complex through the dysregulation of lysosomal activity. Further investigation into the mechanism of nuclear localization of TFEB and its upregulation in Tsc1- and Tsc2- deficient cells may provide insight into the pathogenesis of the disease and could indicate a new therapeutic target for treating TSC and its associated disorders.
2022-06-07T00:00:00Z