Mechanisms of autophagy control through micrornas under cellular stress

Macroautophagy (autophagy) is an evolutionarily conserved stress response mechanism that is necessary for the maintenance of cellular homeostasis. Autophagic activity in cells is regulated by various upstream signaling pathways including mTOR. Stress-mediated inhibition of mTOR complex 1 (mTORC1) results in the nuclear translocation of the TFE/MITF family of transcriptional factors, and triggers an autophagy- and lysosomal-related gene transcription program. In this thesis work, we introduce a specific and rate-limiting role for MITF in autophagy regulation that requires transcriptional activation of MIR211. Under stress conditions including starvation and mTOR inhibition, a MITF-MIR211 axis constitutes a novel feedforward loop that controls autophagic activity in cells. Direct targeting and downregulation of mTORC2 binding partner RICTOR by MIR211 attenuated mTORC1 signal through AKTmediated crosstalk. Under these conditions, the transcription factor MITF translocated from cytosol to the nucleus, and amplified autophagic activity. All together, the outcome of this thesis is the identification of MITF-MIR211 axis as a novel autophagy amplification mechanism required for optimal autophagy activation under cellular stress conditions

MITF-MIR211 axis is a novel autophagy amplifier system during cellular stress

Macroautophagy (autophagy) is an evolutionarily conserved recycling and stress response mechanism. Active at basal levels in eukaryotes, autophagy is upregulated under stress providing cells with building blocks such as amino acids. A lysosome-integrated sensor system composed of RRAG GTPases and MTOR complex 1 (MTORC1) regulates lysosome biogenesis and autophagy in response to amino acid availability. Stress-mediated inhibition of MTORC1 results in the dephosphorylation and nuclear translocation of the TFE/MITF family of transcriptional factors, and triggers an autophagy- and lysosomal-related gene transcription program. The role of family members TFEB and TFE3 have been studied in detail, but the importance of MITF proteins in autophagy regulation is not clear so far. Here we introduce for the first time a specific role for MITF in autophagy control that involves upregulation of MIR211. We show that, under stress conditions including starvation and MTOR inhibition, a MITF-MIR211 axis constitutes a novel feed-forward loop that controls autophagic activity in cells. Direct targeting of the MTORC2 component RICTOR by MIR211 led to the inhibition of the MTORC1 pathway, further stimulating MITF translocation to the nucleus and completing an autophagy amplification loop. In line with a ubiquitous function, MITF and MIR211 were co-expressed in all tested cell lines and human tissues, and the effects on autophagy were observed in a cell-type independent manner. Thus, our study provides direct evidence that MITF has rate-limiting and specific functions in autophagy regulation. Collectively, the MITF-MIR211 axis constitutes a novel and universal autophagy amplification system that sustains autophagic activity under stress conditions.No sponso

Artificial intelligence assisted patient blood and urine droplet pattern analysis for non‑invasive and accurate diagnosis of bladder cancer

Bladder cancer is one of the most common cancer types in the urinary system. Yet, current bladder cancer diagnosis and follow-up techniques are time-consuming, expensive, and invasive. In the clinical practice, the gold standard for diagnosis remains invasive biopsy followed by histopathological analysis. In recent years, costly diagnostic tests involving the use of bladder cancer biomarkers have been developed, however these tests have high false-positive and false-negative rates limiting their reliability. Hence, there is an urgent need for the development of cost-effective, and non-invasive novel diagnosis methods. To address this gap, here we propose a quick, cheap, and reliable diagnostic method. Our approach relies on an artificial intelligence (AI) model to analyze droplet patterns of blood and urine samples obtained from patients and comparing them to cancer-free control subjects.The AI-assisted model in this study uses a deep neural network, a ResNet network, pre-trained on ImageNet datasets. Recognition and classification of complex patterns formed by dried urine or blood droplets under different conditions resulted in cancer diagnosis with a high specificity and sensitivity.Our approach can be systematically applied across droplets, enabling comparisons to reveal shared spatial behaviors and underlying morphological patterns. Our results support the fact that AI-based models have a great potential for non-invasive and accurate diagnosis of malignancies, including bladder cancer

MIR376A is a regulator of starvation-induced autophagy

Background: Autophagy is a vesicular trafficking process responsible for the degradation of long-lived, misfolded or abnormal proteins, as well as damaged or surplus organelles. Abnormalities of the autophagic activity may result in the accumulation of protein aggregates, organelle dysfunction, and autophagy disorders were associated with various diseases. Hence, mechanisms of autophagy regulation are under exploration. Methods: Over-expression of hsa-miR-376a1 (shortly MIR376A) was performed to evaluate its effects on autophagy. Autophagy-related targets of the miRNA were predicted using Microcosm Targets and MIRanda bioinformatics tools and experimentally validated. Endogenous miRNA was blocked using antagomirs and the effects on target expression and autophagy were analyzed. Luciferase tests were performed to confirm that 3’ UTR sequences in target genes were functional. Differential expression of MIR376A and the related MIR376B was compared using TaqMan quantitative PCR. Results: Here, we demonstrated that, a microRNA (miRNA) from the DlkI/Gtl2 gene cluster, MIR376A, played an important role in autophagy regulation. We showed that, amino acid and serum starvation-induced autophagy was blocked by MIR376A overexpression in MCF-7 and Huh-7 cells. MIR376A shared the same seed sequence and had overlapping targets with MIR376B, and similarly blocked the expression of key autophagy proteins ATG4C and BECN1 (Beclin 1). Indeed, 3’ UTR sequences in the mRNA of these autophagy proteins were responsive to MIR376A in luciferase assays. Antagomir tests showed that, endogenous MIR376A was participating to the control of ATG4C and BECN1 transcript and protein levels. Moreover, blockage of endogenous MIR376A accelerated starvation-induced autophagic activity. Interestingly, MIR376A and MIR376B levels were increased with different kinetics in response to starvation stress and tissue-specific level differences were also observed, pointing out to an overlapping but miRNA-specific biological role. Conclusions: Our findings underline the importance of miRNAs encoded by the DlkI/Gtl2 gene cluster in stress-response control mechanisms, and introduce MIR376A as a new regulator of autophagy

Highly luminescent and cytocompatible cationic Ag2S NIR-emitting quantum dots for optical imaging and gene transfection

The development of non-toxic theranostic nanoparticles capable of delivering a therapeutic cargo and providing a means for diagnosis is one of the most challenging tasks in nano-biotechnology. Gene therapy is a very important mode of therapy and polyethyleneimine (PEI) is one of the most successful vehicles for gene transfection, yet poses significant toxicity. Optical imaging utilizing quantum dots is one of the newer but fast growing diagnostic modalities, which requires non-toxic, highly luminescent materials, preferentially active in the near infrared region. Ag2S NIRQDs fit to this profile perfectly. Here, we demonstrate the aqueous synthesis of cationic Ag2S NIRQDs with a mixed coating of 2-mercaptopropionic acid (2MPA) and PEI (branched, 25 kDa), which are highly luminescent in the NIR-I window (lambda(em) = 810-840 nm) as new theranostic nanoparticles. Synergistic stabilization of the QD surface via the simultaneous use of a small molecule and a polymeric material provided the highest quantum yield, 150% (with respect to LDS 798 at pH 7.4), reported in the literature for Ag2S. These cationic particles show a dramatic improvement in cytocompatibility even without PEGylation, a strong optical signal easily detected by confocal laser microscopy and effective conjugation and transfection of the green fluorescence protein plasmid (pGFP) to HeLa and MCF-7 cell lines (40% efficiency). Overall, these Ag2S NIRQDs show great potential as new theranostics

Regulation of autophagy by microRNAs

To cope with stress factors including nutrient deprivation, toxins, abnormal protein accumulation, cells developed mechanisms to repair damage caused by stress or, in cases where the damage is irrepairable, cell death responses. Autophagy and apoptosis are among the most important stress and death response mechanisms. Autophagy is defined by sequestration and transport of cytoplasm pieces and organelles by double- or multi-membrane vesicles and, degradation of the cargo following fusion with lysosomes. Consequently, autophagy plays an important role in elimination of long-lived proteins, damaged organelles, mutant proteins and even intracellular bacteria and viruses, helping to survive stressfull conditions. Paradoxically, depending stress and cell type, autophagy may kill cells by a non-apoptotic programmed cell death mechanism called “autophagic cell death”. Recent studies including our publications suggest that miRNAs add a novel layer of regulation to autophagy pathways. Molecular details of autophagy regulation by miRNAs started to emerge in the last 2 years. In this chapter we will summarize acccumulating data on this novel mechanism of autophagy regulation

Alteration in autophagic-lysosomal potential during ageing and neurological diseases: the microRNA perspective

Macroautophagy (shortly autophagy) is an evolutionary conserved degradation pathway that targets cytoplasmic substrates including long-lived proteins, protein aggregates and damaged organelles, and leads to their degradation in lysosomes. Beyond its role in adaptation to cellular stresses such as nutrient deprivation, hypoxia and toxins; recent studies attributed a central role to autophagy in aging and lifespan determination. Moreover, alterations and abnormalities of autophagy may contribute to a number of important health problems, including cancer, myopathies, metabolic disorders, and the focus of this review, ageing-related neurodegenerative diseases. Some disease-related, mutant and aggregation-prone proteins may be cleared by autophagy, on the other hand, disregulation of the autophagy pathways may also contribute to neurotoxicity observed in degenerative pathologies. microRNAs (miRNAs) are endogenous regulators of gene expression, and their deregulation was reported in several ageing-related conditions. Studies in the last few years introduced miRNAs as novel and potent regulators of autophagy. In this review article, we will summarize the connection between autophagy, ageing and Alzheimer’s, Parkinson’s and Huntington’s diseases, and discuss the role of autophagy-related miRNAs in this context

Regulation of autophagy by miRNAs

To cope with stress factors including nutrient deprivation, toxins, abnormal protein accumulation, cells developed mechanisms to repair damage caused by stress or, in cases where the damage is irrepairable, cell death responses. Autophagy and apoptosis are among the most important stress and death response mechanisms. Autophagy is defined by sequestration and transport of cytoplasm pieces and organelles by double- or multi-membrane vesicles and, degradation of the cargo following fusion with lysosomes. Consequently, autophagy plays an important role in elimination of long-lived proteins, damaged organelles, mutant proteins and even intracellular bacteria and viruses, helping to survive stressfull conditions. Paradoxically, depending stress and cell type, autophagy may kill cells by a non-apoptotic programmed cell death mechanism called “autophagic cell death”. Recent studies including our publications suggest that miRNAs add a novel layer of regulation to autophagy pathways. Molecular details of autophagy regulation by miRNAs started to emerge in the last 2 years. In this chapter we will summarize acccumulating data on this novel mechanism of autophagy regulation

Ag2S-Based NIR-emitting Quantum Dots as New Theranostic Materials

Near Infrared (NIR) emitting semiconductor quantum dots (QDs) have attracted great interest as a new class of fluorescent probes for cellular, molecular and in-vivo imaging applications, due to their stable and size-tunable absorption range, large molar extinction coefficient, long luminescence lifetime and higher penetration depth into the tissues than visible light [1, 2]. Recently, Ag2S quantum dots emerged as promising new particles because of the lower cytotoxicity compared to previously reported NIR QDs such as PbS [3], PbSe [4], CdHgTe [5]. Variety of nanoparticles are being designed and tested for gene transfection. QDs provide both a vector basis and a means for optical detection. Therefore, there is a considerable amount of effort in developing QD based gene vectors. In this study, we report the synthesis of Ag2S based theranostic materials, discuss their physical properties and cytotoxicity and demonstrate their superior transfection efficiency

Ag2S-Based NIR-emitting Quantum Dots as New Theranostic Materials

Near Infrared (NIR) emitting semiconductor quantum dots (QDs) have attracted great interest as a new class of fluorescent probes for cellular, molecular and in-vivo imaging applications, due to their stable and size-tunable absorption range, large molar extinction coefficient, long luminescence lifetime and higher penetration depth into the tissues than visible light [1, 2]. Recently, Ag2S quantum dots emerged as promising new particles because of the lower cytotoxicity compared to previously reported NIR QDs such as PbS [3], PbSe [4], CdHgTe [5]. Variety of nanoparticles are being designed and tested for gene transfection. QDs provide both a vector basis and a means for optical detection. Therefore, there is a considerable amount of effort in developing QD based gene vectors. In this study, we report the synthesis of Ag2S based theranostic materials, discuss their physical properties and cytotoxicity and demonstrate their superior transfection efficiency