Autophagy in Microglia and Alzheimer's disease

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 häufigste neurodegenerative Erkrankung, die durch Amyloid-Beta-Plaques, neurofibrilläre Verwicklungen und Neuroinflammation gekennzeichnet ist. Autophagie wurde mit mehreren neurodegenerativen Erkrankungen in Verbindung gebracht. Vor Kurzem wurde Autophagie mit der Regulierung der Entzündungsreaktion in Makrophagen in Verbindung gebracht. Meine Dissertation untersucht, wie eine Beeinträchtigung der Autophagie die Entzündungsreaktion von Mikroglia beeinflusst. Wir haben Beclin1-heterozygote (Becn1+/-) Mäuse als Modell für eingeschränkte 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+/– Mäusen 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 stützen 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 Mäusen zeigten ebenfalls erhöhte IL-1beta-Spiegel, jedoch keine Unterschiede in der Amyloid-Beta-Pathologie und auch keine in Bezug auf die Phagozytosekapazität. Die konstitutive Heterozygotie von Beclin1 könnte für 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 Mäuse erlitten 10 Tage nach Tamoxifen-Injektion eine periphere Schädigung 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-Mäusen gekreuzt, um ein Modell für 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 unterstützen, um das therapeutische Potenzial autophagieabhängiger Entzündungen bei neurodegenerativen Erkrankungen zu ermitteln

A 3-D in vitro co-culture model of mammary gland involution.

Involution is a process whereby the mammary gland undergoes extensive tissue remodelling involving exquisitely coordinated cell death, extracellular matrix degradation and adipose tissue regeneration following the weaning of offspring. These processes are mediated in part through Jak/Stat signalling pathways, which can be deregulated in breast cancer. Synthetic in vitro analogues of the breast could become important tools for studying tumorigenic processes, or as personalized drug discovery platforms and predictors of therapeutic response. Ideally, such models should support 3D neo-tissue formation, so as to recapitulate physiological organ function, and be compatible with high-throughput screening methodologies. We have combined cell lines of epithelial, stromal and immunological origin within engineered porous collagen/hyaluronic acid matrices, demonstrating 3D-specific molecular signatures. Furthermore seeded cells form mammary-like branched tissues, with lobuloalveolar structures that undergo inducible involution phenotypes reminiscent of the native gland under hormonal/cytokine regulation. We confirm that autophagy is mediated within differentiated mammary epithelial cells in a Stat-dependent manner at early time points following the removal of a prolactin stimulus (H/WD). In addition, epithelial cells express markers of an M2 macrophage lineage under H/WD, a process that is attenuated with the introduction of the monocyte/macrophage cell line RAW 264.7. Thus, such 3D models are suitable platforms for studying cell-cell interactions and cell death mechanisms in relation to cancer

Fucosylation of LAMP-1 and LAMP-2 by FUT1 correlates with lysosomal positioning and autophagic flux of breast cancer cells.

Alpha1,2-fucosyltransferases, FUT1 and FUT2, which transfer fucoses onto the terminal galactose of N-acetyl-lactosamine via α1,2-linkage have been shown to be highly expressed in various types of cancers. A few studies have shown the involvement of FUT1 substrates in tumor cell proliferation and migration. Lysosome-associated membrane protein 1, LAMP-1, has been reported to carry alpha1,2-fucosylated Lewis Y (LeY) antigens in breast cancer cells, however, the biological functions of LeY on LAMP-1 remain largely unknown. Whether or not its family member, LAMP-2, displays similar modifications and functions as LAMP-1 has not yet been addressed. In this study, we have presented evidence supporting that both LAMP-1 and 2 are substrates for FUT1, but not FUT2. We have also demonstrated the presence of H2 and LeY antigens on LAMP-1 by a targeted nanoLC-MS(3) and the decreased levels of fucosylation on LAMP-2 by MALDI-TOF analysis upon FUT1 knockdown. In addition, we found that the expression of LeY was substantial in less invasive ER+/PR+/HER- breast cancer cells (MCF-7 and T47D) but negligible in highly invasive triple-negative MDA-MB-231 cells, of which LeY levels were correlated with the levels of LeY carried by LAMP-1 and 2. Intriguingly, we also observed a striking change in the subcellular localization of lysosomes upon FUT1 knockdown from peripheral distribution of LAMP-1 and 2 to a preferential perinuclear accumulation. Besides that, knockdown of FUT1 led to an increased rate of autophagic flux along with diminished activity of mammalian target of rapamycin complex 1 (mTORC1) and enhanced autophagosome-lysosome fusion. This may be associated with the predominantly perinuclear distribution of lysosomes mediated by FUT1 knockdown as lysosomal positioning has been reported to regulate mTOR activity and autophagy. Taken together, our results suggest that downregulation of FUT1, which leads to the perinuclear localization of LAMP-1 and 2, is correlated with increased rate of autophagic flux by decreasing mTOR signaling and increasing autolysosome formation

An analog of glibenclamide selectively enhances autophagic degradation of misfolded α1-antitrypsin Z

The classical form of α1-antitrypsin deficiency (ATD) is characterized by intracellular accumulation of the misfolded variant α1-antitrypsin Z (ATZ) and severe liver disease in some of the affected individuals. In this study, we investigated the possibility of discovering novel therapeutic agents that would reduce ATZ accumulation by interrogating a C. elegans model of ATD with high-content genome-wide RNAi screening and computational systems pharmacology strategies. The RNAi screening was utilized to identify genes that modify the intracellular accumulation of ATZ and a novel computational pipeline was developed to make high confidence predictions on repurposable drugs. This approach identified glibenclamide (GLB), a sulfonylurea drug that has been used broadly in clinical medicine as an oral hypoglycemic agent. Here we show that GLB promotes autophagic degradation of misfolded ATZ in mammalian cell line models of ATD. Furthermore, an analog of GLB reduces hepatic ATZ accumulation and hepatic fibrosis in a mouse model in vivo without affecting blood glucose or insulin levels. These results provide support for a drug discovery strategy using simple organisms as human disease models combined with genetic and computational screening methods. They also show that GLB and/or at least one of its analogs can be immediately tested to arrest the progression of human ATD liver disease.</div

CD5L promotes M2 macrophage polarization through autophagy-mediated upregulation of ID3

CD5L (CD5 molecule-like) is a secreted glycoprotein that controls key mechanisms in inflammatory responses, with involvement in processes such as infection, atherosclerosis, and cancer. In macrophages, CD5L promotes an anti-inflammatory cytokine profile in response to TLR activation. In the present study, we questioned whether CD5L is able to influence human macrophage plasticity, and drive its polarization toward any specific phenotype. We compared CD5L-induced phenotypic and functional changes to those caused by IFN/LPS, IL4, and IL10 in human monocytes. Phenotypic markers were quantified by RT-qPCR and flow cytometry, and a mathematical algorithm was built for their analysis. Moreover, we compared ROS production, phagocytic capacity, and inflammatory responses to LPS. CD5L drove cells toward a polarization similar to that induced by IL10. Furthermore, IL10- and CD5L-treated macrophages showed increased LC3-II content and colocalization with acidic compartments, thereby pointing to the enhancement of autophagy-dependent processes. Accordingly, siRNA targeting ATG7 in THP1 cells blocked CD5L-induced CD163 and Mer tyrosine kinase mRNA and efferocytosis. In these cells, gene expression profiling and validation indicated the upregulation of the transcription factor ID3 by CD5L through ATG7. In agreement, ID3 silencing reversed polarization by CD5L. Our data point to a significant contribution of CD5L-mediated autophagy to the induction of ID3 and provide the first evidence that CD5L drives macrophage polarization.Peer ReviewedPostprint (published version

Loss of Wdfy3 in mice alters cerebral cortical neurogenesis reflecting aspects of the autism pathology.

Autism spectrum disorders (ASDs) are complex and heterogeneous developmental disabilities affecting an ever-increasing number of children worldwide. The diverse manifestations and complex, largely genetic aetiology of ASDs pose a major challenge to the identification of unifying neuropathological features. Here we describe the neurodevelopmental defects in mice that carry deleterious alleles of the Wdfy3 gene, recently recognized as causative in ASDs. Loss of Wdfy3 leads to a regionally enlarged cerebral cortex resembling early brain overgrowth described in many children on the autism spectrum. In addition, affected mouse mutants display migration defects of cortical projection neurons, a recognized cause of epilepsy, which is significantly comorbid with autism. Our analysis of affected mouse mutants defines an important role for Wdfy3 in regulating neural progenitor divisions and neural migration in the developing brain. Furthermore, Wdfy3 is essential for cerebral expansion and functional organization while its loss-of-function results in pathological changes characteristic of ASDs

Human cachexia induces changes in mitochondria, autophagy and apoptosis in the skeletal muscle

Cachexia is a wasting syndrome characterized by the continuous loss of skeletal muscle mass due to imbalance between protein synthesis and degradation, which is related with poor prognosis and compromised quality of life. Dysfunctional mitochondria are associated with lower muscle strength and muscle atrophy in cancer patients, yet poorly described in human cachexia. We herein investigated mitochondrial morphology, autophagy and apoptosis in the skeletal muscle of patients with gastrointestinal cancer-associated cachexia (CC), as compared with a weight-stable cancer group (WSC). CC showed prominent weight loss and increased circulating levels of serum C-reactive protein, lower body mass index and decreased circulating hemoglobin, when compared to WSC. Electron microscopy analysis revealed an increase in intermyofibrillar mitochondrial area in CC, as compared to WSC. Relative gene expression of Fission 1, a protein related to mitochondrial fission, was increased in CC, as compared to WSC. LC3 II, autophagy-related (ATG) 5 and 7 essential proteins for autophagosome formation, presented higher content in the cachectic group. Protein levels of phosphorylated p53 (Ser46), activated caspase 8 (Asp384) and 9 (Asp315) were also increased in the skeletal muscle of CC. Overall, our results demonstrate that human cancer-associated cachexia leads to exacerbated muscle-stress response that may culminate in muscle loss, which is in part due to disruption of mitochondrial morphology, dysfunctional autophagy and increased apoptosis. To the best of our knowledge, this is the first report showing quantitative morphological alterations in skeletal muscle mitochondria in cachectic patients

Modulation of Mitochondrial Function and Autophagy Mediates Carnosine Neuroprotection Against Ischemic Brain Damage

Background and Purpose—Despite the rapidly increasing global burden of ischemic stroke, no therapeutic options for neuroprotection against stroke currently exist. Recent studies have shown that autophagy plays a key role in ischemic neuronal death, and treatments that target autophagy may represent a novel strategy in neuroprotection. We investigated whether autophagy is regulated by carnosine, an endogenous pleiotropic dipeptide that has robust neuroprotective activity against ischemic brain damage. Methods—We examined the effect of carnosine on mitochondrial dysfunction and autophagic processes in rat focal ischemia and in neuronal cultures. Results—Autophagic pathways such as reduction of phosphorylated mammalian target of rapamycin (mTOR)/p70S6K and the conversion of microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II were enhanced in the ischemic brain. However, treatment with carnosine significantly attenuated autophagic signaling in the ischemic brain, with improvement of brain mitochondrial function and mitophagy signaling. The protective effect of carnosine against autophagy was also confirmed in primary cortical neurons. Conclusions—Taken together, our data suggest that the neuroprotective effect of carnosine is at least partially mediated by mitochondrial protection and attenuation of deleterious autophagic processes. Our findings shed new light on the mechanistic pathways that this exciting neuroprotective agent influences

Tracking autophagy during proliferation and differentiation of trypanosoma brucei

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