48,031 research outputs found

    Connexins : substrates and regulators of autophagy

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    Connexins mediate intercellular communication by assembling into hexameric channel complexes that act as hemichannels and gap junction channels. Most connexins are characterized by a very rapid turn-over in a variety of cell systems. The regulation of connexin turn-over by phosphorylation and ubiquitination events has been well documented. Moreover, different pathways have been implicated in connexin degradation, including proteasomal and lysosomal-based pathways. Only recently, autophagy emerged as an important connexin-degradation pathway for different connexin isoforms. As such, conditions well known to induce autophagy have an immediate impact on the connexin-expression levels. This is not only limited to experimental conditions but also several pathophysiological conditions associated with autophagy (dys) function affect connexin levels and their presence at the cell surface as gap junctions. Finally, connexins are not only substrates of autophagy but also emerge as regulators of the autophagy process. In particular, several connexin isoforms appear to recruit pre-autophagosomal autophagy-related proteins, including Atg16 and PI3K-complex components, to the plasma membrane, thereby limiting their availability and capacity for regulating autophagy

    Tracking autophagy during proliferation and differentiation of trypanosoma brucei

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    Autophagy is a lysosome-dependent degradation mechanism that sequesters target cargo into autophagosomal vesicles. The Trypanosoma brucei genome contains apparent orthologues of several autophagy-related proteins including an ATG8 family. These ubiquitin-like proteins are required for autophagosome membrane formation, but our studies show that ATG8.3 is atypical. To investigate the function of other ATG proteins, RNAi compatible T. brucei were modified to function as autophagy reporter lines by expressing only either YFP-ATG8.1 or YFP-ATG8.2. In the insect procyclic lifecycle stage, independent RNAi down-regulation of ATG3 or ATG7 generated autophagy-defective mutants and confirmed a pro-survival role for autophagy in the procyclic form nutrient starvation response. Similarly, RNAi depletion of ATG5 or ATG7 in the bloodstream form disrupted autophagy, but did not impede proliferation. Further characterisation showed bloodstream form autophagy mutants retain the capacity to undergo the complex cellular remodelling that occurs during differentiation to the procyclic form and are equally susceptible to dihydroxyacetone-induced cell death as wild type parasites, not supporting a role for autophagy in this cell death mechanism. The RNAi reporter system developed, which also identified TOR1 as a negative regulator controlling YFP-ATG8.2 but not YFP-ATG8.1 autophagosome formation, will enable further targeted analysis of the mechanisms and function of autophagy in the medically relevant bloodstream form of T. brucei

    Exploiting macrophage autophagy-lysosomal biogenesis as a therapy for atherosclerosis

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    Macrophages specialize in removing lipids and debris present in the atherosclerotic plaque. However, plaque progression renders macrophages unable to degrade exogenous atherogenic material and endogenous cargo including dysfunctional proteins and organelles. Here we show that a decline in the autophagy-lysosome system contributes to this as evidenced by a derangement in key autophagy markers in both mouse and human atherosclerotic plaques. By augmenting macrophage TFEB, the master transcriptional regulator of autophagy-lysosomal biogenesis, we can reverse the autophagy dysfunction of plaques, enhance aggrephagy of p62-enriched protein aggregates and blunt macrophage apoptosis and pro-inflammatory IL-1ÎČ levels, leading to reduced atherosclerosis. In order to harness this degradative response therapeutically, we also describe a natural sugar called trehalose as an inducer of macrophage autophagy-lysosomal biogenesis and show trehalose's ability to recapitulate the atheroprotective properties of macrophage TFEB overexpression. Our data support this practical method of enhancing the degradative capacity of macrophages as a therapy for atherosclerotic vascular disease

    Autophagy in Microglia and Alzheimer's disease

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    Alzheimer’s disease (AD) is the most common neurodegenerative disease, characterized by amyloid-beta plaques, neurofibrillary tangles and neuroinflammation. Autophagy has been associated with several neurodegenerative diseases. Recently, autophagy has been linked to the regulation of the inflammatory response in macrophages. My thesis investigates how an impairment of autophagy influences the inflammatory response of microglia. We used Beclin1 heterozygous (Becn1+/-) mice as a model of impaired autophagy. Beclin1 plays a role in the initiation of autophagy and was shown to be decreased in microglia isolated from AD patients compared to healthy controls. In vitro, acutely stimulated microglia from neonatal Becn1+/- mice exhibited increased expression of the proinflammatory cytokines IL-1beta and IL-18 compared to wild type microglia. Both IL-1beta and IL-18 are processed by the NLRP3 inflammasome pathway. The investigation of this pathway showed an elevated number of cells with inflammasomes and increased levels of the inflammasome components NLRP3 and cleaved Caspase1 in Becn1+/- microglia. Super resolution microscopy revealed a very close association of NLRP3 aggregates and LC3-positive autophagosomes. Interestingly, despite suggestions that the murine CALCOCO2 does not function as an autophagic adaptor, we discovered CALCOCO2 colocalised with NLRP3 and that its downregulation by siRNA knockdown increased IL-1beta release. These data support the notion that selective autophagy can impact microglia activation by modulating IL-1beta and IL-18 production via NLRP3 degradation. These in vitro data present a mechanism how impaired autophagy could contribute to neuroinflammation in AD. In vivo analysis of Becn1+/-.APPPS1 mice also demonstrated enhanced IL-1beta levels, but no differences in amyloid beta pathology, nor phagocytic capacity. The constitutive heterozygosity of Beclin1 might be responsible for the milder effects in vivo. Therefore, we performed studies utilizing more sophisticated models targeting immune cells specifically. The first model, Aldh1l1-iCre.Becn1-flox, targets Becn1 deletion specifically in astrocytes in the central nervous system after injection with the drug tamoxifen. Peripherally, Aldh1l1 is also expressed by hepatocytes. The Aldh1l1-iCre.Becn1-flox mice suffered from peripheral damage in the liver 10 days after tamoxifen injection, and can therefore not be used in further studies. The second model, Cx3Cr1-iCre.Becn1-flox, targets Becn1 deletion specifically in microglia in the central nervous system, and will be crossed to the APPPS1 mice to create a tool to study the role of Beclin1 in microglia in neuroinflammation and neurodegeneration. This new tool and the data generated in this work will support a new direction of research, to unravel the therapeutic potential of autophagy-dependent inflammation in neurodegenerative diseases.Die Alzheimer-Krankheit (AD) ist die hĂ€ufigste neurodegenerative Erkrankung, die durch Amyloid-Beta-Plaques, neurofibrillĂ€re Verwicklungen und Neuroinflammation gekennzeichnet ist. Autophagie wurde mit mehreren neurodegenerativen Erkrankungen in Verbindung gebracht. Vor Kurzem wurde Autophagie mit der Regulierung der EntzĂŒndungsreaktion in Makrophagen in Verbindung gebracht. Meine Dissertation untersucht, wie eine BeeintrĂ€chtigung der Autophagie die EntzĂŒndungsreaktion von Mikroglia beeinflusst. Wir haben Beclin1-heterozygote (Becn1+/-) MĂ€use als Modell fĂŒr eingeschrĂ€nkte Autophagie verwendet. Beclin1 spielt eine Rolle bei der Initiierung der Autophagie und es wurde gezeigt, dass es bei aus AD-Patienten isolierten Mikrogliazellen im Vergleich zu gesunden Kontrollen abnimmt. Akut stimulierte Mikroglia aus neonatalen Becn1+/– MĂ€usen zeigten in vitro eine erhöhte Expression der proinflammatorischen Zytokine IL-1beta und IL-18 im Vergleich zu Wildtyp-Mikroglia. Sowohl IL-1beta als auch IL-18 werden vom NLRP3-Inflammasom-Weg verarbeitet. Die Untersuchung dieses Weges zeigte eine erhöhte Anzahl von Zellen mit Inflammasomen und erhöhte Spiegel der Inflammasomenkomponenten NLRP3 und gespaltenen Caspase1 in Becn1+/– Mikroglia. Super-Resolution-Mikroskopie zeigte eine sehr enge Lokalisation von NLRP3-Aggregaten und LC3-positiven Autophagosomen. Interessanterweise haben wir trotz der Kritik, dass das murine CALCOCO2 nicht als autophagischer Adapter fungiert, entdeckt, dass CALCOCO2 mit NLRP3 kolokalisiert und dass die Herunterregulierung durch siRNA die IL-1beta-Freisetzung erhöhte. Diese Daten stĂŒtzen die Ansicht, dass selektive Autophagie die Mikroglia-Aktivierung beeinflussen kann, indem die IL-1beta- und IL-18-Produktion durch NLRP3-Abbau moduliert wird. Diese in vitro Daten stellen einen Mechanismus dar, wie eine gestörte Autophagie zur Neuroinflammation bei AD beitragen kann. In vivo Analysen von Becn1+/–.APPPS1 MĂ€usen zeigten ebenfalls erhöhte IL-1beta-Spiegel, jedoch keine Unterschiede in der Amyloid-Beta-Pathologie und auch keine in Bezug auf die PhagozytosekapazitĂ€t. Die konstitutive Heterozygotie von Beclin1 könnte fĂŒr die geringen Auswirkungen in vivo verantwortlich sein. Daher etablierten zwei neue Modelle, die speziell auf Immunzellen abzielten. Das erste Modell, Aldh1l1-iCre.Becn1-Flox, zielt auf die Becn1-Deletion spezifisch in Astrozyten im zentralen Nervensystem nach Injektion des Arzneimittels Tamoxifen ab. In der Peripherie wird Aldh1l1 auch von Hepatozyten exprimiert. Die Aldh1l1-iCre.Becn1-Flox MĂ€use erlitten 10 Tage nach Tamoxifen-Injektion eine periphere SchĂ€digung der Leber und können daher nicht in weiteren Studien verwendet werden. Das zweite Modell, Cx3Cr1-iCre.Becn1-flox, zielt auf die Becn1-Deletion speziell in Mikroglia im Zentralnervensystem ab und wird mit den APPPS1-MĂ€usen gekreuzt, um ein Modell fĂŒr die Untersuchung der Rolle von Beclin1 in Mikroglia bei Neuroinflammation und Neurodegeneration darzustellen. Dieses neue Mausmodell und die in dieser Arbeit generierten Daten werden eine neue Richtung der Forschung unterstĂŒtzen, um das therapeutische Potenzial autophagieabhĂ€ngiger EntzĂŒndungen bei neurodegenerativen Erkrankungen zu ermitteln

    Age-related deficits in skeletal muscle recovery following disuse are associated with neuromuscular junction instability and ER stress, not impaired protein synthesis.

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    Age-related loss of muscle mass and strength can be accelerated by impaired recovery of muscle mass following a transient atrophic stimulus. The aim of this study was to identify the mechanisms underlying the attenuated recovery of muscle mass and strength in old rats following disuse-induced atrophy. Adult (9 month) and old (29 month) male F344BN rats underwent hindlimb unloading (HU) followed by reloading. HU induced significant atrophy of the hindlimb muscles in both adult (17-38%) and old (8-29%) rats, but only the adult rats exhibited full recovery of muscle mass and strength upon reloading. Upon reloading, total RNA and protein synthesis increased to a similar extent in adult and old muscles. At baseline and upon reloading, however, proteasome-mediated degradation was suppressed leading to an accumulation of ubiquitin-tagged proteins and p62. Further, ER stress, as measured by CHOP expression, was elevated at baseline and upon reloading in old rats. Analysis of mRNA expression revealed increases in HDAC4, Runx1, myogenin, Gadd45a, and the AChRs in old rats, suggesting neuromuscular junction instability/denervation. Collectively, our data suggests that with aging, impaired neuromuscular transmission and deficits in the proteostasis network contribute to defects in muscle fiber remodeling and functional recovery of muscle mass and strength

    Targeting quiescent leukemic stem cells using second generation autophagy inhibitors

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    In chronic myeloid leukemia (CML), tyrosine kinase inhibitor (TKI) treatment induces autophagy that promotes survival and TKI-resistance in leukemic stem cells (LSCs). In clinical studies hydroxychloroquine (HCQ), the only clinically approved autophagy inhibitor, does not consistently inhibit autophagy in cancer patients, so more potent autophagy inhibitors are needed. We generated a murine model of CML in which autophagic flux can be measured in bone marrow-located LSCs. In parallel, we use cell division tracing, phenotyping of primary CML cells, and a robust xenotransplantation model of human CML, to investigate the effect of Lys05, a highly potent lysosomotropic agent, and PIK-III, a selective inhibitor of VPS34, on the survival and function of LSCs. We demonstrate that long-term haematopoietic stem cells (LT-HSCs: Lin−Sca-1+c-kit+CD48−CD150+) isolated from leukemic mice have higher basal autophagy levels compared with non-leukemic LT-HSCs and more mature leukemic cells. Additionally, we present that while HCQ is ineffective, Lys05-mediated autophagy inhibition reduces LSCs quiescence and drives myeloid cell expansion. Furthermore, Lys05 and PIK-III reduced the number of primary CML LSCs and target xenografted LSCs when used in combination with TKI treatment, providing a strong rationale for clinical use of second generation autophagy inhibitors as a novel treatment for CML patients with LSC persistence

    Parkinsonian toxin-induced oxidative stress inhibits basal autophagy in astrocytes via NQO2/quinone oxidoreductase 2: Implications for neuroprotection

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    open12openJanda, Elzbieta; Lascala, Antonella; Carresi, Cristina; Parafati, Maddalena; Aprigliano, Serafina; Russo, Vanessa; Savoia, Claudia; Ziviani, Elena; Musolino, VINCENZO MARIA; Morani, Federica; Isidoro, Ciro; Mollace, VincenzoJanda, Elzbieta; Lascala, Antonella; Carresi, Cristina; Parafati, Maddalena; Aprigliano, Serafina; Russo, Vanessa; Savoia, Claudia; Ziviani, Elena; Musolino, VINCENZO MARIA; Morani, Federica; Isidoro, Ciro; Mollace, Vincenz

    Interaction of mycobacterium tuberculosis with the host cells: a focus in the molecular mechanism involved in trafficking and autophagy

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    Tuberculosis (TB) is an ancient disease remaining a serious health threat worldwide. It is caused by Mycobacterium tuberculosis (Mtb), an acid-fast bacilli, non-sporulated, slow-growing, immobile and aerobic. The pathogenesis of the disease is based on its ability to multiply and survive within phagocytic cells of the host, particularly macrophages and monocytes. The majority (90 %) of infected humans have a ?latent infection?, meaning they efficiently contain but do not spread the bacteria; they are infected but asymptomatic and not contagious. However the remaining 10 % have a lifetime risk of reactivating the infection and developing active tuberculosis (Sakamoto, 2012). The great destructive impact on public health, the co-infection with the human immunodeficiency virus (HIV) and the appearance of drug resistant strains of Mtb are demanding the development of new tools for prevention and treatment.During the last decade a greater understanding on the human immune response to Mtb infection as well as the contribution of factors linked to the pathogenesis of the disease has been achieved. Although the knowledge about the human immune response against Mtb as well as the contribution of factors linked to the pathogenesis of the disease have markedly increased in the last year, a deeper understanding of its immunopathogenesis will lead to the identification of new drugs and the development of effective vaccines.Fil: Zarelli, Valeria Eugenia Paola. Consejo Nacional de Investigaciones CientĂ­ficas y TĂ©cnicas. Centro CientĂ­fico TecnolĂłgico Conicet - Mendoza. Instituto de HistologĂ­a y EmbriologĂ­a de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias MĂ©dicas. Instituto de HistologĂ­a y EmbriologĂ­a de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Giai, Constanza. Consejo Nacional de Investigaciones CientĂ­ficas y TĂ©cnicas. Centro CientĂ­fico TecnolĂłgico Conicet - Mendoza. Instituto de HistologĂ­a y EmbriologĂ­a de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias MĂ©dicas. Instituto de HistologĂ­a y EmbriologĂ­a de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Colombo, Maria Isabel. Consejo Nacional de Investigaciones CientĂ­ficas y TĂ©cnicas. Centro CientĂ­fico TecnolĂłgico Conicet - Mendoza. Instituto de HistologĂ­a y EmbriologĂ­a de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias MĂ©dicas. Instituto de HistologĂ­a y EmbriologĂ­a de Mendoza Dr. Mario H. Burgos; Argentin
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