Autophagie ist ein intrazellulärer Prozess, bei dem fehlgefaltete Proteine oder beschädigte Zellorganellen abgebaut und ihre Bestandteile recycelt werden. Dieser Prozess findet in allen Zellen des Körpers im Sinne einer ständigen Zellreparatur statt und ist in bestimmten Situationen stark erhöht. Die Autophagie ist bei vielen Krankheiten gestört, darunter Krebs, Herz-Kreislauf-, neurodegenerative und Stoffwechselerkrankungen.
Im Else Kröner Graduiertenkolleg „Autophagie - Recycling, Reparatur, Abwehr (AURA)“ werden Medizinstudierende im Rahmen eines strukturierten Qualifizierungsprogramms an das Thema Autophagie herangeführt und bearbeiten Promotionsprojekte, die sich mit den Mechanismen der Autophagie und ihrer Regulation sowie den Folgen einer gestörten Autophagie beschäftigen. Ziel des Else Kröner Promotionskollegs AURA ist es, Studierende der Medizin für die Forschung zu begeistern, wissenschaftliche Kompetenzen zu vermitteln und das Interesse an einer akademischen Karriere als Clinician Scientist zu wecken sowie die klinisch orientierte Autophagieforschung zu stärken.
Das Programm ist auf zwei Jahre angelegt. Die Aufnahme in das Promotionskolleg AURA setzt eine Unterbrechung des Studiums für zwei Semester voraus, in denen die Doktorandinnen und Doktoranden durch Stipendien unterstützt werden, um sich ganz der Arbeit an ihrem Promotionsprojekt widmen zu können. Daran schließt sich eine studienbegleitende Förderung in geringerem Umfang an, in der das Promotionsvorhaben abgeschlossen werden soll.
Alle Betreuer/innen, die ein thematisch passendes Promotionsprojekt vorstellen möchten, sind ebenfalls herzlich eingeladen. Hierfür bitten wir Sie bitte um die Einreichung eines Projektvorschlages (Template Projektvorschlag, per E-Mail an: ) und die Vorbereitung eines Posters zur Vorstellung im Rahmen des Kick-off Meetings.
Sprecher
Sprecher
Stellvertretende Sprecherin
Ausschreibung
Im Rahmen des von der Else Kröner-Fresenius-Stiftung geförderten Promotionskollegs „Autophagie -Recycling, Reparatur, Abwehr (AURA)“ werden hochmotivierte und engagierte Studierende der Medizin gefördert, die an einem Promotionsvorhaben zum Thema Autophagie und deren Rolle bei verschiedenen Erkrankungen interessiert sind. Die Förderung erstreckt sich über einen Zeitraum von zwei Jahren. Voraussetzung ist eine Unterbrechung des Studiums für zwei Semester. In dieser Zeit erhalten die Promovierenden ein Stipendium in Höhe von 1000 €. Im zweiten Jahr werden die Promovierenden mit einem Stipendium in Höhe von 200 € unterstützt, um die Promotion studienbegleitend abschließen zu können. Während der Förderung wird ein begleitendes Qualifizierungsprogramm angeboten, in dem neben der fachlichen Weiterbildung auch Methodenkompetenz und wichtige Schlüsselqualifikationen vermittelt werden. Details zu Forschungsinhalten, möglichen Projekten und Betreuern finden Sie hier.
Interessierte Studierende werden gebeten, sich mit den Projektverantwortlichen in Verbindung zu setzen. Die Bewerbung erfolgt über einen Projektantrag, der in einem kompetitiven Verfahren von einer Gutachterkommission bewertet wird. Dem Antrag ist ein vom Studiendekanat befürworteter Antrag auf Beurlaubung beizufügen. Während der Beurlaubung können keine Leistungsnachweise erbracht werden.
Bewerbungsunterlagen:
- Projektantrag (Vorgaben der DFG für Anträge auf Sachbeihilfe, einzeilig, Arial 11 pt, maximal 7 Seiten, siehe Template)
- Motivationsschreiben
- Lebenslauf
- Zeugnisse
- Unterstützungsschreiben der Betreuerin/des Betreuers
- Bestätigung der Annahme als Doktorand/Doktorandin bzw. Kopie des Antrages zur Annahme als Doktorand/Doktorandin
- Beantragung der Freistellung vom Studium für zwei Semester (die Beurlaubung ist grundsätzlich vor Beginn des betreffenden Semesters zu beantragen).
Termine:
Bewerbungsschluss 15.7.2024
Bewerbersymposium zwischen 25.8. und 30.8.2024
Beginn der Förderung 1.10.2024
Projektvorschläge
Interaction of autophagy and JAK/STAT3 signaling in osteoarthritis-affected cells
Background and previous work:
Osteoarthritis (OA) is a common degenerative joint disease characterized by articular cartilage degeneration, bone sclerosis and synovial inflammation. OA research is focused on identifying OA-affected metabolic pathways as new targets for OA treatment. Autophagy is an important intracellular homeostatic mechanism for the removal of dysfunctional cellular organelles and substances. Dysfunction of autophagic processes accelerates the progression of osteoarthritis. The interaction between autophagy and JAK/STAT3 signaling has been described in many oncological studies, but little is known about this interaction in human OA. Recent studies showed that inhibition of JAK2 signaling modulated several autophagy markers in an animal model of OA and cell line experiments (Zhang 2023, Zhang 2016). JAK/STAT3 signaling is involved in several pathological processes and plays an important role in inflammatory diseases. Increased activation of STAT3 has been described in OA tissues and contributes to degenerative cartilage processes. Therefore, the JAK/STAT3 signaling pathway is a promising target for OA treatment.
Interleukin-6 (IL-6) is an important activator of the JAK/STAT3 signaling pathway. Publications from the experimental trauma surgery working group (UKJ) have demonstrated the importance of IL-6 for OA pain and OA-related metabolic processes (Eitner 2017, Eitner 2024). In addition, the results showed increased STAT3 activation in human OA chondrocytes. Ongoing experiments are investigating the effect of autophagy activation on IL-6 production in synoviocytes and whether synovial inflammation affects the expression of autophagic markers in synovial tissue. Thus, the analysis of OA metabolism in cultured human synoviocytes and chondrocytes from OA patients, the analysis of the JAK/STAT3 signaling pathway and expression of autophagy markers are well established in this group.
Specific aims:
The aim of the project is to analyze the effect of activation and inactivation of JAK/STAT3 signaling on autophagy in human OA chondrocytes and synoviocytes. In addition, the effect of autophagy activation and inhibition on the production of cartilage-matrix substances and pro-inflammatory mediators will be investigated.
We hypothesize that activation of the JAK/STAT3 signaling pathway inhibits autophagy in human OA chondrocytes, thereby promoting inflammatory and chondrodegenerative processes.
Working programme:
Human OA chondrocytes and synoviocytes will be obtained from patients with end-stage knee OA who undergoing joint arthroplasty. Isolated cells will be cultured and stimulated with modulators of the JAK/STAT3 signaling pathway such as the JAK/STAT3 inhibitors Baricitinib, AG490, Ruxolitinib and Stattic, and the Stat3 activator Interleukin-6. Expression levels of autophagic markers such a Beclin-1, autophagy protein 5 (ATG5), and microtubule-associated protein light chain 3 (LC-3) and JAK/STAT3 signaling molecules will be analyzed by PCR. Phosphorylation of JAK and STAT3 will be analyzed by Western blot experiments. Several potent activators (Rapamycin, SRT 1720) and inhibitors (3-Methyladenine, MRT 67307) of autophagy will be used to analyze the effect of autophagy on the metabolism of chondrocytes and synoviocytes. Cell viability will be determined. The synthesis of e.g. IL-6, matrix-metalloproteinase-1 (MMP1), glycosaminoglycan, and Pro-collagen Type II of stimulated cells will be analyzed by ELISA and PCR.
References:
Eitner 2024: DOI: 10.1016/j.joca.2024.02.006
Eitner 2017: DOI: 10.1097/j.pain.0000000000000972
Zhang 2023: DOI: 10.1007/s10753-023-01840-3
Zhang 2016: DOI: 10.3892/mmr.2016.4970
Principal Investigator:
Dr. Annett Eitner (Department of Trauma, Hand and Reconstructive Surgery, Experimental Trauma Surgery)
Is autophagy in tendons triggered by mechanical stress?
Background and previous work:
Tendinopathy, which is usually caused by overuse, is closely linked to processes of degeneration and inflammation (Millar et. al. 2021 10.1038/s41572-020-00234-1). Both processes show essential influences on biomechanical properties and decrease stability and elasticity of tendons (Galloway et. al. 2013, 10.2106/JBJS.L.01004). Especially the structural changes seem to be mediated by autophagy (Li et. al. 2018, 10.1016/j.lfs.2018.07.049). Autophagy, a physiological process, that enables the cell to degrade intracellularly damaged or non-utilised proteins (Mizushima et. al. 2011, 10.1016/j.cell.2011.10.026), which is particularly important for reorganisation during tissue remodelling. Likewise, the cell fate of stem/progenitor cells, the remodelling and cellular plasticity of tissue are controlled by autophagy (Perrotta et. al. 2020, 10.3389/fcell.2020.602901).
In a recent study we have found that overloading in bioartificial tendons (BATs), a 3D in vitro system, resulted in significantly decreased expression of the tenocyte-specific genes Mkx and Tnmd and changes in ECM-related genes (Col1 and 3, MMP3) and IL6 synthesis after 7 days of loading (Pentzold et al. 2022, 10.1186/s13036-022-00283-y ), indicating dedifferentiation is taking place. These results suggest that tissue reorganisation occurs in the tendon that is subject to overload and/or inflammation. However, the role of autophagy in this context is still unclear and will be investigated in this project.
Specific aims:
This study will investigate the role of autophagy under the influence of overloading and Il1ß treatment on BATs. 1) In particular, the effect of mechanical stress on key markers of autophagy such as Map1LC3B, Ulk1 and Atg12 and main players of the mTor signalling pathway will be investigated by gene expression analysis and immunohistochemistry. 2) In addition, the extent to which the presence of IL1ß, a pro-inflammatory cytokine, promotes autophagy under mechanical stress will be analysed.
Working programme:
Bioarteficial tendons (BATs) will be generated using murine C3H10T1/2 cells cultured in collagen I gel at 4% (physiological) and 8% (overload) loading conditions using the FlexCell-System. Inflammation will be imitated by the administration of Il-1ß. The effect on the BATs should be investigated under physiological conditions and mechanical overload, with and without inflammatory stimulation. In addition, the effect of autophagy should be demonstrated by its inhibition.
Methods and analyses:
- Culturing of BATs in FlexCell-System under physiological and overloading conditions for 7d with and without Il-1β stimulation
Inhibition trial: 3-Methyladenine (PI3K-Inhibitor); MRT 67307 (Ulk1 inhibitor)
- Gene expression analyses (qPCR, array) will performed with regard to the most important genes for autophagy (Atg5, Atg7,
Atg12, Atg10, Bcl2, Becn1, Map1LC3B, Ulk1 a.o.) and mTor signalling pathway (Raptor, Rictor, Akt1, Pi3k a.o.) as well as tenocyte
specific marker and markers of ECM.
- Histology (cell count, cell morphology) and immunofluorescence (e.g. LC3B, Beclin1, mTorC1, tenocyte markers), evaluation by
microscopy (evaluation of relative positive-stained cells, total cell count)
- ELISA for IL-1β and IL-6 secretion (cell culture supernatant) and LC3B (cell lysate)
- Western blotting analyses (LC3B, mTorC1)
Principal Investigator:
Dr. Diana Freitag (Department of Neurology)
Studying the role of autophagy in a congenital disorder of glycosylation
Background and previous work:
We previously reported that a defect of GMPPA causes a rare congenital disorder of glycosylation (CDGs). We further showed that GMPPA controls the levels of the sugar donor GDP-mannose and thus promotes the incorporation of GDP-mannose into glycan structures.
Specific aims:
We found that several proteins involved in autophagy are regulated in tissues of GMPPA deficient mice. Therefore, we here aim to study whether autophagy is involved in the pathogenesis of the disorder.
Working programme:
We recently obtained fibroblasts from a patient with compound heterozygosity for GMPPA loss-of-function variants. We will use these cells to assess whether we can recapitulate our findings obtained for fibroblasts obtained from Gmppa knockout mice such as alterations of the Golgi apparatus. We will also test whether we find similar alterations in proteins involved in autophagy. To judge whether autophagy is impaired, we will transfect cells with different autophagy reporters such as LC3-GFP-RFP. Because GFP is quenched in acidic compartments, autolyosomes will be labelled red but not green, while autophagosomes will be label red and green. This will allow us to quantify autophagosomes and autolysosomes at steady state and upon starvation or pharmacological induction of autophagy. If time allows, we will also study autophagy in iPSC derived neurons.
Selected readings:
Mutations in GMPPA cause a glycosylation disorder characterized by intellectual disability and autonomic dysfunction. Koehler K, et al., Am J Hum Genet. 2013 Oct 3;93(4):727-34
GMPPA defects cause a neuromuscular disorder with α-dystroglycan hyperglycosylation. Franzka P, et al., J Clin Invest. 2021, 131(9):e139076
Principal Investigator:
Dr. Patricia Franzka, Prof. Dr. Christian Hübner (Institute of Human Genetics)
Autophagy and hereditary spastic paraplegia associated with defects in the adaptor protein complex 5 (AP5)
Background and previous work:
Previously, we could show that the AP5-related neurodegenerative disorders SPG11, SPG15 and SPG48 are characterized by defective autophagy with accumulation of undegraded intracellular material. We further showed that this defect is associated with altered phosphoinositide signaling due to the upregulation and mislocalization of PI4K2A, a kinase generating PI4P.
Specific aims:
We will assess whether the knockout or knock-down of PI4K2A can rescue the autophagy defect observed in SPG11, SPG15 and SPG48.
Working programme:
We will perform immunostainings to assess the effects of the knockout of PI4K2A on different subcellular compartments such as the Golgi apparatus, early and late endosomes as well as lysosomes. Next we will study the possible consequences for the degradative pathway. To this end, we will transfect cells with different autophagy reporters such as LC3-GFP-RFP and study autophagy flux at steady state and upon starvation or pharmacological induction of autophagy. Because GFP is quenched in the acidic compartment of autolysosomes, acidic compartments such as autolyosomes will be labelled red, while autophagosomes will be labeled both red and green. These studies will be flanked by semiquantitative Western blot analyses for different markers of the degradative pathway. Finally, we will address whether defective autophagy in SPG11, SPG15 and SPG48 can be rescued by inhibiting or knocking down PI42A using siRNAs. If effective, we will potentially also assess the consequences of PI4K2A knock-down in existing Spg11-knockout mice.
Selected reading:
Mouse models for hereditary spastic paraplegia uncover a role of PI4K2A in autophagic lysosome reformation.
Khundadze M, Ribaudo F, Hussain A, Stahlberg H, Brocke-Ahmadinejad N, Franzka P, Varga RE, Zarkovic M, Pungsrinont T, Kokal M, Ganley IG, Beetz C, Sylvester M, Hübner CA.
Autophagy. 2021 Nov;17(11):3690-3706.
Principal Investigator:
Dr. Mukhran Khundadze, Prof. Dr. Christian Hübner (Institute of Human Genetics)
Studying the role of the ER-resident membrane shaping protein Reticulon-2 in autophagy
Background and previous work:
RTN-2 encodes an ER-resident membrane shaping protein, which is associated with neurodegeneration. Members of the Reticulon family of proteins have been found in the interactome of FAM134B, a receptor for the specific degradation of ER-fragments via autophagy, also called ER-phagy, which is also linked to neurodegeneration.
Specific aims:
Because of its interaction with proteins involved in ER-phagy and similar phenotypes of patients with RTN-2 loss of function, we here propose to assess whether RTN-2 is involved in autophagy as well.
Working programme:
We recently obtained fibroblasts from a patient homozygous for a RTN-2 loss-of-function variant, who suffers from autosomal recessive hereditary spastic paraplegia. We will immortalize these cells by introducing the large SV40 T-cell antigen. We will use this cell line to assess both bulk autophagy and ER-phagy. We will use a set of markers for different intracellular compartments such as ER, mitochondria and the Golgi apparatus to compare the basic cellular morphology. Moreover, we will pharmacologically induce ER-stress and the consequences for cell viability. To judge whether autophagy is imparied, we will transfect cells with different autophagy reporters such as LC3-GFP-RFP and quantify autophagosomes and autolyosomes at steady state and upon starvation of pharmacological induction of autophagy. Because GFP is quenched in the acidic compartment o such as autolyosomes, these will be labelled red but not green. Depending on the results obtained, we will confirm our findings in primary neuros obtained from available KO mice or iPSC-derived human neurons.
Selected reading:
Heteromeric clusters of ubiquitinated ER-shaping proteins drive ER-phagy.
Foronda H, Fu Y, Covarrubias-Pinto A, Bocker HT, González A, Seemann E, Franzka P, Bock A, Bhaskara RM, Liebmann L, Hoffmann ME, Katona I, Koch N, Weis J, Kurth I, Gleeson JG, Reggiori F, Hummer G, Kessels MM, Qualmann B, Mari M, Dikić I, Hübner CA.
Nature. 2023 Jun;618(7964):402-410
Principal Investigator:
Prof. Dr. Christian Hübner (Institute of Human Genetics)
The role of a Nit1-FoxO3a axis in the regulation of autophagy
Background and previous work:
Autophagy is an intracellular process frequently upregulated in response to different forms of cellular stress such as nutrient deprivation, DNA damage and hypoxia as well as metabolic and oxidative stress. Autophagy needs to be tightly regulated to maintain cell and tissue homeostasis.
The nitrilase superfamily member Nit1 (branch 10) was classified as a Rosetta Stone protein acting as a tumor suppressor together with its partner Fhit (fragile histidine triad). In addition, Nit1 has a metabolite repair activity as a dGSH (deaminated glutathione) hydrolase. In previous work, we identified both Fhit and Nit1 as interaction partners of ß-catenin which additively repress canonical Wnt/ß-catenin signaling (1-3). Mechanistically, this appears to depend on a disruption of the ß-catenin/TCF transcription complex or the prevention of ß-catenin/TCF complex formation. In consequence, this may contribute to the observed translocation of ß-catenin from TCF to FoxO3a transcription factor in response to oxidative stress (4,5). In this context, our preparatory work revealed that Nit1 also binds to FoxO3a thereby regulating FoxO3a transcriptional activity. Since FoxO3a was shown to regulate genes involved in autophagy initiation, autophagosome formation and elongation we postulate (5), that Nit1 is involved in regulation of autophagy. Thus, the aim of our project is to confirm this hypothesis and to decipher the role of a Nit1/FoxO3a axis in autophagy.
Specific aims:
This project aims to answer the following questions:
1. How is Nit1 expression modulated during autophagy?
2. Is autophagy affected by Nit1 knock-down or overexpression?
3. How does the Nit1/FoxO3a axis modulate the crosstalk between autophagy and apoptosis?
Working programme:
Ad 1) Autophagy will be induced by nutrient starvation, rapamycin treatment or by activation of AMPK and expression of mitochondrial and cytosolic isoforms of Nit1 will be analyzed by qRT-PCR. Nit1 protein levels will be studied by Western blotting. Potential changes in localization of Nit1 will be investigated by immunofluorescence microscopy.
Ad 2) Vice versa, we want to address the question how Nit1 affects the autophagy program. In this context, the effect of a knock-down and of overexpression of Nit1 (mitochondrial and cytosolic variant as well as enzymatic-dead variants) on autophagy will be examined by analyzing autophagic marker proteins such as LC3 and Ulk-1 variants, Beclin-1 and p62 by Western blotting and in immunofluorescence microscopy.
Ad 3) Autophagy is of certain importance for cell survival and under certain conditions competes with apoptosis. Nit1 also is known to promote apoptosis. This crosstalk will be tested by analyzing the expression of apoptotic markers such as cleavage of caspases and PARP as well as M30 CytoDeath staining. In addition, FoxO3a levels will be modulated by siRNA or FoxO3a overexpression to investigate epistatic effects.
References:
1. Weiske J, Albring KF, Huber O. The tumor suppressor Fhit acts as a repressor of ß-catenin transcriptional activity. Proc Natl Acad Sci USA (2007) 104(51):20344-20349.
2. Mittag S, Valenta T, Weiske J, Bloch L, Klingel S, Gradl D, Wetzel F, Chen Y, Petersen I, Basler K, Huber O. A novel role for the tumour suppressor Nitrilase1 modulating the Wnt/ß-catenin signalling pathway. Cell Discovery (2016) 2:15039.
3. Mittag S, Wetzel F, Müller SY, Huber O. The Rosetta Stone hypothesis-based interaction of the tumor suppressor proteins Nit1 and Fhit. Cells (2023) 12:353.
4. Essers MAG, de Vries-Smits LMM, Barker N, Polderman PE, Burgering BMT, Korswagen HC. Science (2005) 308:1181-1184.
5. Rodrguez-Colman MJ, Dansen TB, Burgering BMT. FOXO transcription factors as mediators of stress adaptation. Nat Rev Mol Cell Biol (2024) 25:46-64.
6. Cheng Z. The FoxO-autophagy axis in health and disease. Trends Endocrinol Metab (2019) 30:658-671.
Principal Investigator:
Prof. Dr. Otmar Huber (Institute of Biochemistry II)
Analysing AUTOphagy to determine individual RESIlience to STress (AUTORESIST)
Genome-wide and proteome-wide association studies have highlighted autophagy-related pathways in various brain disorders, suggesting a role in conditions like Schizophrenia, Bipolar Disorder, Autism Spectrum Disorder, Major Depression, and Alzheimer’s disease. However, a comprehensive analysis of autophagy's role in stress-related mental disorders and investigation into intervention strategies remains unexplored and an investigation of autophagy-inducing strategies have not been conducted to date.
Functional autophagy, crucial for cellular health, is best understood through "autophagic flux," representing its degradation activity and hallmark of this cellular process. While animal models allow dynamic assessment through genetic modification, measuring autophagic flux in humans remains challenging despite advances. The overarching goal of AutoResist is to provide causal evidence that autophagy is centrally implicated in mental health and to deliver solutions to monitor autophagy/autophagic flux in humans and battle mental health problems through specific biomarkers, pharmacological and non-pharmacological intervention strategies and innovative technological solutions. To this end, the Opel and Gassen labs will join forces and combine their particular expertise in molecular, cellular, metabolic, physiological and clinical approaches. Specifically, to gain a more detailed understanding, we will target the following objectives: Current methods, focusing on indirect markers, lack comprehensive evaluation of autophagic flux under inducing conditions. Our project aims to (i) establish a quantifiable method for assessing autophagic flux in humans, (ii) investigate its relationship with mental health symptoms and underlying neural circuits and (iii) test its modulation via a fasting intervention.
To this end, we will examine cause-effect mechanisms by identifying relevant metabolites and proteins (in plasma and circulating leukocytes). We will employ a recently established assay to assess autophagic flux in peripheral blood. Venous blood will be treated ex vivo with an autophagy modulator and subsequently analyzed. This process enables the determination of autophagic flux, and in combination with targeted proteomic analysis, allows for the molecular assessment of affected substrates such as organelles or proteins. After incubation with the Autophagosome-Lysosome fusion blocker Chloroquine, PBMCs are isolated and directly processed for immunoblotting against a set of markers for autophagic flux and regulation, including the autophagy core proteins LC3A/B-I, LC3A/B-II, Atg3, Atg5, Atg7, Atg12-Atg5, Atg16L1, Beclin-1 and Ambra1, the adapter proteins and cargo receptors for selective autophagy SQSTM1 and NBR1 as well as the nutrient sensor and master regulator of autophagy mTOR. A major effort using untargeted metabolomics of human plasma and PBMCs will be performed to identify an autophagy-related signature in metabolomic alteration. Using the outlined autophagy essay, we will investigate the relationship between autophagy flux and stress related disorders based on a longitudinal investigation in n= 50 depressive patients undergoing inpatient treatment for depression at Jena University Hospital, and n= 25 healthy controls. We will collect blood samples for autophagy essays, MRI neuroimaging (rsfMRI, sMRI, DTI) and mental health assessments at baseline prior to start of treatment, and again at follow-up approximately 8 weeks later at treatment cessation. This design will allow us to disentangle the relationship between autophagy flux and both specific symptom clusters, treatment response over time as well as the interaction of autophagy, changes in neural circuits and mental health. Lastly, we will conduct a fasting intervention in n=20 MDD participants to probe if the well known authophagy inducing effect of fasting correlates with mental health improvement and restoration of abberrant neural signalling major depression.
Principal Investigator:
Prof. Nils Opel (Department of Psychiatry, University Clinic Jena)
Dr. Nils Gassen (Department of Psychiatry, University Clinic Bonn)
Determining autophagosome-mediated antigen presentation in dendritic cell subpopulations
Background and previous work:
The Dudziak laboratory is focusing on understanding the development and function of dendritic cells in mouse and man. Dendritic cells (DCs) are key cells in the induction of primary immune responses. DCs are sentinels of the immune system, recognizing invading pathogens such as viruses or bacteria. Pathogenic material is taken up and processed into small peptides. The Dudziak group has been able to show that the dendritic cell subtype cDC1 (CD8+ DCs) is superior in presenting antigens on MHC-class-I to CD8+ cytotoxic T cells by a mechanism called cross-presentation, while the dendritic cell subtype cDC2 (CD8-) excels in presenting antigens on MHC-class-II to CD4+ T cells (Dudziak et al., Science 2007, doi: 10.1126/science.1136080; Neubert et al., JI, 2014, doi: 10.4049/jimmunol.1300975; Lehmann et al., JEM, 2017, doi: 10.1084/jem.20160951). We recently found that the cDC2 subset can be further subdivided into Clec12A+ and Clec12A- DC2 (Amon et al., Cell Reports, 2024, doi: 10.1016/j.celrep.2024.113949) and that the small Clec12A+ DC2 subset is capable of inducing CD8+ cytotoxic T cell responses, when both viral or bacterial stimuli were used for DC-activation (unpublished). We further found that Clec12A- DCs only present antigen to CD8+ T cells, when the DCs were activated with bacterial but not viral stimuli. Importantly, a very recent study suggests that autophagy is involved in a TAP-1 independent presentation of antigens on MHC-class-I molecules indicating an alternative activation pathway of CD8+ cytotoxic T cell responses (Sengupta et al., JI, 2024; doi: 10.4049/jimmunol.2200446). This data indicate that autophagic processes occur in antigen presentation allowing for CD4+ as well as CD8+ T cell activation. Understanding how the process of autophagy is involved in antigen processing and presentation in primary DCs in vivo has not been addressed, yet.
Specific aims:
We here hypothesize that autophagy is a select process in the cDC2 compartment under specific inflammatory/disease conditions allowing for a TAP-1 independent antigen presentation on MHC-class-I. To prove our hypothesis, we will
1. Perform a characterization of autophagy protein components in DC subsets in steady state and inflammatory conditions
2. Perform DC-T cell antigen-presentation analyses in activating and steady state conditions in absence and presence of TAP-1 as well as autophagy inhibitors
Working program:
3.1 Perform a characterization of autophagy protein components in DC subsets in steady state and inflammatory conditions
In the first aim we want to better understand, which DC subset is expressing molecules needed in the process of autophagy. Thus, we will analyze the expression of the involved molecules including ULK1 kinase complex (P200, ATG13 and ATG101), class III phosphatidylinositol 3-kinase (PIK3C3) complex (composed of BECN1, AMBRA1, ATG14L, VPS15 and VPS3), PtdIns3, WIPI proteins, ATG5–ATG12–ATG16L1 (E3) complex, LC3, ATG4 protease, E1 (ATG7), E2 (ATG3) and E3 components, phosphatidylethanolamine (PE), LIR-containing autophagy receptors (AR; such as p62), RAB proteins, SNARE proteins, HOPS complex, as well as other molecules in autophagy processes (e.g. p62/SQSTM1). We will perform multicolor flow cytometry, as well as protein analysis using protein simple system. Existing transcriptome data will be checked for expression of autophagy related molecules. A high-throughput multicolor microscopy (HCI) system (currently applied, Prof. Huebner) will allow for direct assessment of localization as well as colocalization of proteins involved in autophagy within the DC subsets. As controls, common markers of the endosomal/lysosomal compartment will be costained (e.g. Rab5, Rab7, EEA). All experiments will be performed using DCs in steady state as well as under inflammatory conditions (we will start with our established conditions using TLR ligand activation e.g. TLR3 (poly-IC), TLR4 (LPS), TLR7/8 (R848), TLR9 (CpG). We will extend these analyses and use described autophagy activators such as mTOR dependent activator Rapamycin or mTOR independent reagents such as Lithium chloride. We expect to identify autophagic pathways in the DC subsets and their regulation under inflammatory conditions.
3.2 Perform DC-T cell antigen-presentation analyses in activating and steady state conditions in absence and presence of autophagy inhibitors
In the next step and in case it is time, we will perform in vivo antigen targeting experiments combined with in vitro T cell proliferation and T cell activation experiments. Here, we will make use of antigen targeting antibodies in the presence or absence of inflammatory stimuli (TLR3, TLR4, TLR7/8, TLR9 stimulus, or identified mTOR dependent or mTOR independent reagents) (please see Dudziak et al., Science 2007, doi: 10.1126/science.1136080; Lehmann et al., JEM, 2017, doi: 10.1084/jem.20160951). After 12h, DCs will be sorted from lymph nodes or spleen, respectively, into Clec12A+ and Clec12A- cDC2, cDC1, pDCs, as well as DC3 (see Amon et al., Cell Reports, 2024; Heger et al., PNAS, 2023). DCs will be co-cultured with CFSE labeled transgenic T cells carrying a T cell receptor specific for ovalbumin antigen when presented on MHC-class-I (OT-I mice, CD8+ T cells recognizing OVA257-264), or on MHC-class-II (OT-II mice, CD4+ T cells recognizing OVA323-339). On day 3, T cells will be analyzed for their proliferation. In a second part of the experiments, T cell activation will be controlled after 6, 12, 24, or 72 hrs. After establishing the conditions and after full hands-on-lab training, experiments for understanding autophagy processes in antigen presentation will be performed. We will use various genetic mouse models, in which antigen processing is influenced (e.g. TAP-1-/-, ATG7-/-, ATG16l1-/-, GABARAP-/- mice. In parallel, autophagy inhibition experiments will be performed. Here, we will directly FACS-sort DCs from lymph nodes and spleens of TAP-1-/- mice and add autophagy inhibitors while in parallel DCs will be loaded with ovalbumin antigen or antigen targeting antibodies and providing co-stimulation of DCs using TLR ligands or mTOR dependent/independent reagents.
Principal Investigator:
Prof. Dr. Diana Dudziak (Institute of Immunology)