The Role of Chaperone-Mediated Autophagy in Tissue Homeostasis and Disease Pathogenesis.

Chaperone-mediated autophagy (CMA) is a selective proteolytic pathway in the lysosomes. Proteins are recognized one by one through the detection of a KFERQ motif or, at least, a KFERQ-like motif, by a heat shock cognate protein 70 (Hsc70), a molecular chaperone. CMA substrates are recognized and delivered to a lysosomal CMA receptor, lysosome-associated membrane protein 2A (LAMP-2A), the only limiting component of this pathway, and transported to the lysosomal lumen with the help of another resident chaperone HSp90. Since approximately 75% of proteins are reported to have canonical, phosphorylation-generated, or acetylation-generated KFERQ motifs, CMA maintains intracellular protein homeostasis and regulates specific functions in the cells in different tissues. CMA also regulates physiologic functions in different organs, and is then implicated in disease pathogenesis related to aging, cancer, and the central nervous and immune systems. In this minireview, we have summarized the most important findings on the role of CMA in tissue homeostasis and disease pathogenesis, updating the recent advances for this Special Issue.

Chaperone-Mediated Autophagy in Pericytes: A Key Target for the Development of New Treatments against Glioblastoma Progression.

Glioblastoma (GB) cells physically interact with peritumoral pericytes (PCs) present in the brain microvasculature. These interactions facilitate tumor cells to aberrantly increase and benefit from chaperone-mediated autophagy (CMA) in the PC. GB-induced CMA leads to major changes in PC immunomodulatory phenotypes, which, in turn, support cancer progression. In this review, we focus on the consequences of the GB-induced up-regulation of CMA activity in PCs and evaluate how manipulation of this process could offer new strategies to fight glioblastoma, increasing the availability of treatments for this cancer that escapes conventional therapies. We finally discuss the use of modified PCs unable to increase CMA in response to GB as a cell therapy alternative to minimize undesired off-target effects associated with a generalized CMA inhibition.

Chaperone-Mediated Autophagy Ablation in Pericytes Reveals New Glioblastoma Prognostic Markers and Efficient Treatment Against Tumor Progression.

Background: The lack of knowledge of the progression mechanisms of glioblastoma (GB), the most aggressive brain tumor, contributes to the absence of successful therapeutic strategies. Our team has recently demonstrated a crucial new role for chaperone-mediated autophagy (CMA) in pericytes (PC)-acquired immunosuppressive function, which prevents anti-tumor immune responses and facilitates GB progression. The possible impact that GB-induced CMA in PC has on other functions that might be useful for future GB prognosis/treatment, has not been explored yet. Thus, we proposed to analyze the contribution of CMA to other GB-induced changes in PC biology and determine if CMA ablation in PC is a key target mechanism for GB treatment. Methods: Studies of RNA-seq and secretome analysis were done in GB-conditioned PC with and without CMA (from knockout mice for LAMP-2A) and compared to control PC. Different therapeutic strategies in a GB mouse model were compared. Results: We found several gene expression pathways enriched in LAMP2A-KO PC and affected by GB-induced CMA in PC that correlate with our previous findings. Phagosome formation, cellular senescence, focal adhesion and the effector function to promote anti-tumor immune responses were the most affected pathways, revealing a transcriptomic profiling of specific target functions useful for future therapies. In addition, several molecules associated with tumor mechanisms and related to tumor immune responses such as gelsolin, periostin, osteopontin, lumican and vitamin D, were identified in the PC secretome dependent on GB-induced CMA. The CMA ablation in PC with GB cells showed an expected immunogenic phenotype able to phagocyte GB cells and a key strategy to develop future therapeutic strategies against GB tumor progression. A novel intravenous therapy using exofucosylated CMA-deficient PC was efficient to make PC reach the tumor niche and facilitate tumor elimination. Conclusion: Our results corroborate previous findings on the impaired immunogenic function of PC with GB-induced CMA, driving to other altered PC functions and the identifications of new target markers related to the tumor immune responses and useful for GB prognosis/therapy. Our work demonstrates CMA ablation in PC as a key target mechanism to develop a successful therapy against GB progression.

Glioblastoma ablates pericytes antitumor immune function through aberrant up-regulation of chaperone-mediated autophagy.

The contractile perivascular cells, pericytes (PC), are hijacked by glioblastoma (GB) to facilitate tumor progression. PC's protumorigenic function requires direct interaction with tumor cells and contributes to the establishment of immunotolerance to tumor growth. Cancer cells up-regulate their own chaperone-mediated autophagy (CMA), a process that delivers selective cytosolic proteins to lysosomes for degradation, with pro-oncogenic effects. However, the possible impact that cancer cells may have on CMA of surrounding host cells has not been explored. We analyzed the contribution of CMA to the GB-induced changes in PC biology. We have found that CMA is markedly up-regulated in PC in response to the oxidative burst that follows PC-GB cell interaction. Genetic manipulations to block the GB-induced up-regulation of CMA in PC allows them to maintain their proinflammatory function and to support the induction of effective antitumor T cell responses required for GB clearance. GB-induced up-regulation of CMA activity in PC is essential for their effective interaction with GB cells that help tumor growth. We show that CMA inhibition in PC promotes GB cell death and the release of high immunogenic levels of granulocyte-macrophage colony stimulating factor (GM-CSF), through deregulation of the expression of cell-to-cell interaction proteins and protein secretion. A GB mouse model grafted in vivo with CMA-defective PC shows reduced GB proliferation and effective immune response compared to mice grafted with control PC. Our findings identify abnormal up-regulation of CMA as a mechanism by which GB cells elicit the immunosuppressive function of PC and stabilize GB-PC interactions necessary for tumor cell survival.

Autophagy in the Immunosuppressive Perivascular Microenvironment of Glioblastoma.

Glioblastoma (GB) has been shown to up-regulate autophagy with anti- or pro-oncogenic effects. Recently, our group has shown how GB cells aberrantly up-regulate chaperone-mediated autophagy (CMA) in pericytes of peritumoral areas to modulate their immune function through cell-cell interaction and in the tumor's own benefit. Thus, to understand GB progression, the effect that GB cells could have on autophagy of immune cells that surround the tumor needs to be deeply explored. In this review, we summarize all the latest evidence of several molecular and cellular immunosuppressive mechanisms in the perivascular tumor microenvironment. This immunosuppression has been reported to facilitate GB progression and may be differently modulated by several types of autophagy as a critical point to be considered for therapeutic interventions.

Glioblastoma progression is assisted by induction of immunosuppressive function of pericytes through interaction with tumor cells.

The establishment of immune tolerance during Glioblastoma Multiforme (GBM) progression, is characterized by high levels expression of anti-inflammatory cytokines, which suppress the function of tumor assocciated myeloid cells, and the activation and expansion of tumor antigen specific T cells. However, the mechanisms underlying the failed anti-tumor immune response around the blood vessels during GBM, are poorly understood. The consequences of possible interactions between cancer cells and the perivascular compartment might affect the tumor growth. In this work we show for the first time that GBM cells induce immunomodulatory changes in pericytes in a cell interaction-dependent manner, acquiring an immunosuppresive function that possibly assists the evasion of the anti-tumor immune response and consequently participates in tumor growth promotion. Expression of high levels of anti-inflammatory cytokines was detected in vitro and in vivo in brain pericytes that interacted with GBM cells (GBC-PC). Furthermore, reduction of surface expression of co-stimulatory molecules and major histocompatibility complex molecules in GBC-PC correlated with a failure of antigen presentation to T cells and the acquisition of the ability to supress T cell responses. In vivo, orthotopic xenotransplant of human glioblastoma in an immunocompetent mouse model showed significant GBM cell proliferation and tumor growth after the establishment of interspecific immunotolerance that followed GMB interaction with pericytes.

Chaperone-mediated autophagy regulates T cell responses through targeted degradation of negative regulators of T cell activation.

Chaperone-mediated autophagy (CMA) targets soluble proteins for lysosomal degradation. Here we found that CMA was activated in T cells in response to engagement of the T cell antigen receptor (TCR), which induced expression of the CMA-related lysosomal receptor LAMP-2A. In activated T cells, CMA targeted the ubiquitin ligase Itch and the calcineurin inhibitor RCAN1 for degradation to maintain activation-induced responses. Consequently, deletion of the gene encoding LAMP-2A in T cells caused deficient in vivo responses to immunization or infection with Listeria monocytogenes. Impaired CMA activity also occurred in T cells with age, which negatively affected their function. Restoration of LAMP-2A in T cells from old mice resulted in enhancement of activation-induced responses. Our findings define a role for CMA in regulating T cell activation through the targeted degradation of negative regulators of T cell activation.

Tle4 regulates epigenetic silencing of gamma interferon expression during effector T helper cell tolerance.

In response to suboptimal activation, T cells become hyporesponsive, with a severely reduced capacity to proliferate and produce cytokines upon reencounter with antigen. Chromatin analysis of T cells made tolerant by use of different in vitro and in vivo approaches reveals that the expression of gamma interferon (IFN-γ) is epigenetically silenced in anergic effector TH1 cells. In those T cells, calcium signaling triggers the expression of Tle4, a member of the Groucho family of corepressors, which is then recruited to a distal regulatory element in the Ifng locus and causes the establishment of repressive epigenetic marks at the Ifng gene regulatory elements. Consequently, impaired Tle4 activity results in a markedly reduced capacity to inhibit IFN-γ production in tolerized T cells. We propose that Blimp1-dependent recruitment of Tle4 to the Ifng locus causes epigenetic silencing of the expression of the Ifng gene in anergic TH1 cells. These results define a novel function of Groucho family corepressors in peripheral T cells and demonstrate that specific mechanisms are activated in tolerant T helper cells to directly repress expression of effector cytokines, supporting the hypothesis that stable epigenetic imprinting contributes to the maintenance of the tolerance-associated hyporesponsive phenotype in T cells.

Induction and stability of the anergic phenotype in T cells.

One of the mechanisms that are in place to control the activation of mature T cells that bear self-reactive antigen receptors is anergy, a long-term state of hyporesponsiveness that is established in T cells in response to suboptimal stimulation. T cells receive signals that result not only from antigen recognition and costimulation but also from other sources, including cytokine receptors, inhibitory receptors or metabolic sensors. Integration of those signals will determine T cell fate. Under conditions that induce anergy, T cells activate a program of gene expression that leads to the production of proteins that block T cell receptor signaling and inhibit cytokine gene expression. In this review we will examine those signals that determine functional outcome following antigen encounter, review current knowledge of the factors that ensure signaling inhibition and epigenetic gene silencing in anergic cells and explore the mechanisms that lead to the reversal of anergy and the reacquisition of effector functions.

Autophagy and the regulation of the immune response.

Autophagy is a highly conserved mechanism of lysosomal-mediated protein degradation that plays a crucial role in maintaining cellular homeostasis by recycling amino acids, reducing the amount of damaged proteins and regulating protein levels in response to extracellular signals. In the last few years specific functions for different forms of autophagy have been identified in many tissues and organs. In the Immune System, autophagy functions range from the elimination infectious agents and the modulation of the inflammatory response, to the selection of antigens for presentation and the regulation of T cell homeostasis and activation. Here, we review the recent advances that have allowed us to better understand why autophagy is a crucial process in the regulation of the innate and adaptive immune responses.

Age-related oxidative stress compromises endosomal proteostasis.

A hallmark of aging is an imbalance between production and clearance of reactive oxygen species and increased levels of oxidatively damaged biomolecules. Herein, we demonstrate that splenic and nodal antigen-presenting cells purified from aging mice accumulate oxidatively modified proteins with side-chain carbonylation, advanced glycation end products, and lipid peroxidation. Furthermore, we show that the endosomal accumulation of oxidatively modified proteins interferes with the efficient processing of exogenous antigens and degradation of macroautophagy-delivered proteins. In support of a causative role for oxidized products in the inefficient immune response, a decrease in oxidative stress improved the adaptive immune response to immunizing antigens. These findings underscore a previously unrecognized negative effect of age-dependent changes in cellular proteostasis on the immune response.

Selective autophagy in the maintenance of cellular homeostasis in aging organisms.

Altered cellular homeostasis, accumulation of damaged non-functional organelles and presence of protein inclusions are characteristics shared by almost all types of differentiated cells in aged organisms. Cells rely on quality control mechanisms to prevent the occurrence of these events and the subsequent cellular compromise associated with them. What goes wrong in aging cells? Growing evidence supports gradual malfunctioning with age of the cellular quality control systems. In this review, we focus on autophagy, a catabolic process that contributes to the maintenance of cellular homeostasis through the degradation of unwanted and damaged components in lysosomes. We describe recent advances on the molecular characterization of this process, its different variants and the multiplicity of functions attributed to them. Autophagic dysfunction has been identified in severe human disorders, many of which worsen with age. We comment on the contribution of an adequate autophagic function to longevity, and the negative impact on health-span of the age-dependent decline in autophagic function.

Serine residues in the LAT adaptor are essential for TCR-dependent signal transduction.

The adaptor protein LAT has a prominent role in the transduction of intracellular signals elicited by the TCR/CD3 complex. Upon TCR engagement, LAT becomes tyrosine-phosphorylated and thereby, recruits to the membrane several proteins implicated in the activation of downstream signaling pathways. However, little is known about the role of other conserved motifs present in the LAT sequence. Here, we report that the adaptor LAT contains several conserved serine-based motifs, which are essential for proper signal transduction through the TCR. Mutation of these serine motifs in the human T cell line Jurkat prevents proper calcium influx, MAPK activation, and IL-2 production in response to TCR/CD3 stimulation. Moreover, this mutant form of LAT has a reduced ability to bind to PLC-γ1 and SLP-76, although phosphorylation of tyrosine residues 132, 171, and 191 is not decreased, raising a possible role for the serine-based motifs of LAT for the binding of important partners. The functional role of LAT serine-based motifs in signal transduction could be mediated by an effect on tyrosine phosphorylation, as their mutation significantly diminishes the phosphorylation of tyrosine residue 226. In addition, these serine motifs seem to have a regulatory role, given that upon their mutation, ZAP-70 shows enhanced phosphorylation. Therefore, the LAT serine-based motifs likely regulate signaling pathways that are essential for T cell physiology.

Macroautophagy regulates energy metabolism during effector T cell activation.

Macroautophagy is a highly conserved mechanism of lysosomal-mediated protein degradation that plays a key role in maintaining cellular homeostasis by recycling amino acids, reducing the amount of damaged proteins, and regulating protein levels in response to extracellular signals. We have found that macroautophagy is induced after effector T cell activation. Engagement of the TCR and CD28 results in enhanced microtubule-associated protein 1 light chain 3 (LC3) processing, increased numbers of LC3-containing vesicles, and increased LC3 flux, indicating active autophagosome formation and clearance. The autophagosomes formed in stimulated T cells actively fuse with lysosomes to degrade their cargo. Using a conditional KO mouse model where Atg7, a critical gene for macroautophagy, is specifically deleted in T cells, we have found that macroautophagy-deficient effector Th cells have defective IL-2 and IFN-γ production and reduced proliferation after stimulation, with no significant increase in apoptosis. We have found that ATP generation is decreased when autophagy is blocked, and defects in activation-induced cytokine production are restored when an exogenous energy source is added to macroautophagy-deficient T cells. Furthermore, we present evidence showing that the nature of the cargo inside autophagic vesicles found in resting T cells differs from the cargo of autophagosomes in activated T cells, where mitochondria and other organelles are selectively excluded. These results suggest that macroautophagy is an actively regulated process in T cells that can be induced in response to TCR engagement to accommodate the bioenergetic requirements of activated T cells.

Mechanisms of self-inactivation in anergic T cells

Las células T autorreactivas que escapan al proceso de selección negativa en el timo han de ser inactivadas o eliminadas en la periferia. En respuesta a una estimulación parcial o subóptima, las células T se vuelvenanérgicas e incapaces de proliferar y producir citocinas en respuesta aencuentros posteriores con el antígeno. Las señales mediadas por calcio tienen un papel importante en la inducción de anergia, por medio de la activación de un programa de auto-inactivación intrínseco a la célula dependiente de calcio/calcineurina/NFAT. Esta revisión se centra en la descripción de nuestros conocimientos actuales acerca de los mecanismos reguladores de la expresión de un programa de expresión génica específico dela anergia de las células T, y cómo las proteínas codificadas por esos genesimponen un estado funcional falta de respuesta a nuevos estimulos. Estose lleva a cabo mediante la localización y la modulación de la actividadde sucesos cruciales para la activación de las células T, incluyendo fenó-menos como la atenuación de las señales del receptor de linfocitos T (TCR)y la inhibición de la transcripción de citocinas (AU)

Self-reactive T cells that escape negative selection in the thymus mustbe inactivated or eliminated in the periphery. In response to a partial orsuboptimal stimulation, T cells become anergic and unable to proliferate and express cytokines in response subsequent re-encounters with antigen. Calcium signaling plays a central role in the induction of anergy, causing the activation of a calcium/calcineurin/NFAT-dependent cell-intrinsic program of self-inactivation. This review will focus on our currentknowledge on the mechanisms that regulate the expression of an anergyspecific program of gene expression in T cells, and how the proteins encoded by those genes impose a state of functional unresponsiveness by targeting and modulating the activity of crucial events required for the activation of T cells, which include downregulation of TCR signaling andinhibition of cytokine transcription (AU)

Transcriptional regulation by poly(ADP-ribose) polymerase-1 during T cell activation.

BACKGROUND: Accumulating evidence suggests an important role for the enzyme poly(ADP-ribose) polymerase-1 (PARP-1) as an integral part of the gene expression regulatory machinery during development and in response to specific cellular signals. PARP-1 might modulate gene expression through its catalytic activity leading to poly(ADP-ribosyl)ation of nuclear proteins or by its physical association with relevant proteins. Recently, we have shown that PARP-1 is activated during T cell activation. However, the proposed role of PARP-1 in reprogramming T cell gene expression upon activation remains largely unexplored. RESULTS: In the present study we use oligonucleotide microarray analysis to gain more insight into the role played by PARP-1 during the gene expression reprogramming that takes place in T cells upon activation with anti-CD3 stimulation alone, or in combination with anti-CD28 co-stimulation. We have identified several groups of genes with expression modulated by PARP-1. The expression of 129 early-response genes to anti-CD3 seems to be regulated by PARP-1 either in a positive (45 genes) or in a negative manner (84 genes). Likewise, in the presence of co-stimulation (anti-CD3 + anti-CD28 stimulation), the expression of 203 genes is also regulated by PARP-1 either up (173 genes) or down (30 genes). Interestingly, PARP-1 deficiency significantly alters expression of genes associated with the immune response such as chemokines and genes involved in the Th1/Th2 balance. CONCLUSION: This study provides new insights into changes in gene expression mediated by PARP-1 upon T cell activation. Pathway analysis of PARP-1 as a nuclear signalling molecule in T cells would be of relevance for the future development of new therapeutic approaches targeting PARP-1 in the acquired immune response.

Regulation of NFAT by poly(ADP-ribose) polymerase activity in T cells.

The nuclear factor of activated T cells (NFAT) family of transcription factors is pivotal for T lymphocyte functionality. All relevant NFAT activation events upon T cells stimulation such as nuclear translocation, DNA binding, and transcriptional activity have been shown to be dictated by its phosphorylation state. Here, we provide evidence for a novel post-translational modification that regulates NFAT. Indeed, NFATc1 and NFATc2 are poly(ADP-ribosyl)ated by poly-ADP-ribose polymerase-1 (PARP-1). Moreover, we have also found a physical interaction between PARP-1 and both NFATc1 and NFATc2. Interestingly, PARP is activated during T cell stimulation in the absence of DNA damage, leading to ADP-ribose polymers formation and transfer to nuclear acceptor proteins. Our data suggest that poly(ADP-ribosyl)ation modulates the activation of NFAT in T cells, as PARP inhibition causes an increase in NFAT-dependent transactivation and a delay in NFAT nuclear export. Poly(ADP-ribosyl)ation will expedited NFAT export from the nucleus directly or by priming/facilitating NFAT phosphorylation. Altogether, these data point to PARP-1 and poly(ADP-ribosyl)ation as a novel regulatory mechanism of NFAT at nuclear level, suggesting a potential use of PARP as a new therapeutic target in the modulation of NFAT.

Effects of living cyanobacteria, cyanobacterial extracts and pure microcystins on growth and ultrastructure of microalgae and bacteria.

In this study, we demonstrate the inhibitory effect of both cyanobacterial extracts and pure microcystins on the growth of microalgae and bacteria. This inhibitory effect was more persistent in pure microcystins than in the extracts, which lost their properties eight days after exposure. In addition, the effects on bacteria were longerlasting than those on microalgae. The microalgae exposed to both extracts and cultures of microcystin producing species showed morphological and ultrastructural alterations, even in cases where there was no clear effect on growth. The implications for colonisation and benthic communities structure and development are discussed in the context of biomonitoring.