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)

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 Trauma, Hand and Reconstructive Surgery, Experimental Trauma Surgery)

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)

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)

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)

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)

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)

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)

Background and previous work:
Respiratory pathogens, including viruses and bacteria, are causing severe acute lung infections accompanied with long-term alterations. Over the last few years, the role of autophagy has been recognized as a vital cellular process in infectious diseases. Mitophagy is a specialized form of autophagy, the cellular process responsible for degrading and recycling cellular components. Specifically, mitophagy refers to the selective degradation of mitochondria by autophagy. The activation or suppression of mitophagy can be triggered by pathogenic microorganisms infecting cells. Our own data indicates that viruses lead via entry mechanism to premature, cellular senescence. Additionally, inflammatory processes mainly induced by alveolar macrophages can influence the homeostasis of lung alveoli, influencing autophagy.

Specific aims:
In this study, we aim to investigate (1) the influence of Influenza A Virus and Staphylococcus aureus on mitophagy in alveolar macrophages, (2) the impact of alveolar macrophages on lung fibroblasts and (3) the effect of activation/inhibition on mitophagy in alveolar macrophages on the production of extracellular matrix proteins of lung fibroblasts.

Working programme:
Work Package 1 - Cultivation of alveolar macrophages and viral/bacterial infection.
Aim: To identify mitophagy pathways in alveolar macrophages w/wo infection.
Methods:
- Infection with Influenza A-Virus (H1N1 Virus, H1N1/PR8), Staphylococcus aureus (USA300)
- cultivation of human monocyte derived alveolar like macrophages
- virological and bacteriological standard measurements (Plaque-Assay, colony-forming units, viral qRT-PCR, immunofluorescence, inflammation, ELISA)
- mitophagy pathway analysis and investigation of the inflammasome

Work Package 2 - Investigation of alveolar macrophages from patients with fibrosis.
Aim: To investigate fibrosis-related alterations of mitophagy during infection.
Methods:
- Isolation of alveolar macrophages from bronchoalveolar lavage of patients with fibrosis (positive ethic vote obtained)
- viral/bacterial infection of alveolar macrophages and healthy volunteers (commercially obtained)
- analysis of pathogen load, and inflammation (protein based, e.g. ELISA)
- analysis of mitophagy (e.g. RNA-Sequencing)

Work Package 3 - Influence of alveolar macrophages on lung fibroblasts.
Aim: To understand the impact of alveolar macrophages on the establishment of lung fibrosis.
Methods:
- Cocultivation of alveolar-like macrophages and human primary lung fibroblast
- viral/bacterial infection of alveolar-like macrophages and subsequent cocultivation
- analysis of fibrosis markers (alpha-sma, vimentin etc), extra cellular matrix proteins (ECM) and parameters of cellular senescence (p16, p21, MMP3, LB1)
- immunofluorescence of cocultivation with focus on ECM

Principal Investigator:
PD Dr. Stefanie Deinhardt-Emmer (Institute of Medical Microbiology)

Background and previous work:
Autophagy is a process to maintain homeostasis of cells by degrading dysfunctional cell organelles and long-lived macromolecules. Thi process is initiated by formation of a double membrane vacuole named autophagosome that engulfes parts of the cytoplasm. After fusing with a lysosome the inner membrane and the autophagolysosome´s content are degraded and reused. Autophagy has cytoprotective effects on cells under stressful conditions. There is strong evidence that Toll-like receptors crosstalk with autophagy pathways e.g. TLR4.
The group of Experimental Nephrology has a system TLR-expressing cells with a luciferase-based reporter system to follow TLR-activation in living cells. The primary objective of this project is to develop a novel approach to investigate the interplay between autophagy and TLR4 receptor signaling using the LC3B biosensor. This biosensor has been shown to monitor the priming of LC3B by ATG4B, a key event in autophagy initiation.

Specific aims are:
1. To investigate the effect of TLR4 activation on autophagy induction and LC3B priming using the LC3B biosensor.
2. To examine the role of autophagy in regulating TLR4-mediated inflammatory responses.
3. To identify key molecular players involved in the crosstalk between autophagy and TLR4 signaling.

Working programme:
1) Molecular cloning of autophagy biosensors and different reporter genes
2) Transient and stable Lentiviral-based transfection of human cell lines with TLRs and autophagy biosensor LC3B.
3) Stimulation of cell lines with TLR ligands:Cells will be stimulated with LPS, a TLR4 agonist,
and monitoring effects on the LC3B biosensor.
4) Characterization of cellular response via RNAseq

Principal Investigator:
Prof. Dr. Ralf Mrowka (Department of Internal Medicine III, Experimental Nephrology)

Background and previous work:
Functioning autophagy is a prerequisite for the maintenance of complex endothelial functions such as barrier function and angiogenesis (Spengler 2020, Cells 9(3), 687). Previous work by the group has demonstrated that stressors that promote endothelial dysfunction, such as elevated levels of saturated fatty acids or dicarbonyls, can inhibit autophagy. Our data show that treatment of cells with pathophysiological concentrations of palmitate led to saturation of membrane phospholipids and significant changes in endoplasmic reticulum (ER) and mitochondrial membrane morphology. This was accompanied by an inhibition of bulk autophagy, probably due to an inhibition of autophagosome-lysosome fusion. These data may partly explain the development of endothelial dysfunction in conditions with high levels of saturated fatty acids, such as diabetes.

Specific aims:
In this project we aim to investigate the effect of palmitate stress on selective autophagy, i.e. on the lysosomal clearance of portions of mitochondria (mitophagy) and ER (ER-phagy) in human endothelial cells.

Working programme:
The experiments will be performed in primary endothelial cells isolated from human umbilical veins and exposed to palmitate in a dose- and time-dependent manner. Selective autophagy will be monitored using non-selective (PK-hLC3B) or organelle-specific autophagy reporters such as mito-Keima and mito-SRAI for mitophagy or pCW57-CMV-ssRFP-GFP-KDEL and mCherry-GFP-FAM134B for ER-phagy. These tandem fluorescence-based reporters will be introduced into endothelial cells via a lentiviral approach. As they contain pH-sensitive fluorophores, the fluorescence will change upon fusion of autophagosomes with lysosomes, allowing the evaluation of the final step of autophagy, mitophagy or ER-phagy. Depending on the results, we will investigate the role of autophagy receptors in palmitate-induced changes in mitophagy or ER-phagy. The evaluation of organelle-selective autophagy will be accompanied by studies of organelle function. For example, mitochondrial permeability will be measured by FACS using fluorescent probes and ER stress will be monitored by qRT-PCR or immunoblotting techniques.

Principal Investigator:
Dr. Katrin Spengler, Prof. Dr. Regine Heller (Institute for Molecular Cell Biology)

Background and previous work:
The autophagic response to viral infection varies depending on the cell type and the infecting virus. Autophagy can fight viral infections, but viruses have also developed mechanisms to evade intracellular degradation by autophagy or to use the process of autophagy for their replication. Our previous work has shown that activation of AMP-activated protein kinase (AMPK), a metabolic sensor and regulator, inhibits herpes simplex virus type 1 (HSV-1) replication in endothelial cells (Doshi et al., Microbiol Spectr. 2023 Sep 13:e0041723). AMPK is known to stimulate autophagy by phosphorylating two autophagy initiators, i.e. ULK1 and Beclin-1. Preliminary data have shown that ULK1 and Beclin-1 protect endothelial cells against HSV-1 infection and replication. In addition, we found that the phosphorylation state of Beclin-1 is altered upon HSV-1 infection suggesting that HSV-1 targets Beclin-1-dependent pathways, including autophagy.

Specific aims:
In this project, we will investigate the role of ULK1 and Beclin-1 in HSV-1 replication in endothelial cells. In particular, we will determine if and how the virus affects the ULK1-Beclin-1 pathway and whether the protective role of ULK1 and Beclin-1 is related to the induction of autophagy or to non-autophagic functions of both proteins.

Working programme:
The experiments will be performed in primary endothelial cells isolated from human umbilical veins and infected with the HSV-1 KOS strain, a laboratory strain, provided by the Institute of Medical Microbiology, Section for Experimental Virology, Jena University Hospital. Virus replication in endothelial cells is measured by TCID50 titrations to quantify the amount of virus required to produce a cytopathic effect in reporter cells. To further characterize the effect of HSV-1 infection on ULK1 and Beclin-1, the phosphorylation state of both proteins will be characterized after protein enrichment using a phosphoproteomic approach. Potential upstream kinases will be identified using pharmacological inhibitors or RNAi technology. Downstream pathways will be assessed by Western blot analysis. Autophagy will be monitored using LC3B staining to detect autophagosome formation and tandem fluorescence-based reporters to describe autophagosome-lysosome fusion.
Co-localisation of autophagosome markers and viral proteins will be performed to determine if and how HSV-1 associates with autophagic vesicles. To monitor non-autophagic functions of ULK1 and Beclin-1, the inflammatory state of cells will be compared in wild type and ULK1- or Beclin-1-depleted cells. The expression of cytokines and inflammatory markers will be measured by FACS.

Principal Investigator:
Dr. Heena Doshi, Prof. Dr. Regine Heller (Institute for Molecular Cell Biology)

Background and previous work:
The pathogenesis of chronic myeloid leukemia is driven by the expression of BCR::ABL1 - a constitutively active tyrosine kinase. CML is well treatable using tyrosine kinase inhibitors (TKIs), allowing most patients to reach a near-normal life expectancy. However, life-long treatment is associated with a multitude of toxicity-related issues, which has led to several studies exploring TKI discontinuation and the probability of long-term therapy-free remission (TFR). Unfortunately, at least half of the patients attempting TKI discontinuation experience relapse. However, it is known that relapse is driven by TKI-resistant CML stem cells.
A key survival factor of CML stem cells is the anti-apoptotic protein BCL-2, which also inhibits autophagy by interacting with the autophagy regulator Beclin-1. The binding of BCL-2 to Beclin-1 inhibits the formation of the PI3K complex, thereby suppressing autophagy induction. As a BCL-2 antagonist, venetoclax affects both apoptosis and autophagy by perturbing the Bcl-2/Beclin-1 complex. Thereby venetoclax could enable the elimination of CML cells, including quiescent stem cells, to improve TFR. Previous analyses have shown that targeted dual blockade of BCL-2 and BCR::ABL1 results in a synergistic enhancement of apoptosis of CML stem cells in vivo (Carter et al. SciTranslMed 2016) and elicits a stronger effect on the viability of CD34+ CML cells compared to healthy cells in vitro.
The VARIANT clinical trial assesses the reduction of CML stem cells in the bone marrow of patients who receive venetoclax after TKI discontinuation. This offers the possibility to investigate autophagy upon combination and sequential TKI and venetoclax treatment, not only in CML cell line models, but also in primary patient material.

Specific aims:
1. Analyses of effects of combined TKI and venetoclux application (simultaneously and sequentially) on TKI-sensitive and -resistant K562 CML cell lines, in terms of viability and expression of key markers of autophagy (Beclin-1, LC3, p62).
2. Combined TKI and venetoclux incubation of CD34+ stem cells of CML patients and healthy donors, to assess viability and expression of key markers of autophagy.

Working program:
Month 1 to 2
- Familiarize with laboratory routine and methods.
- Optimize cell culture conditions for K562 cells, including appropriate concentrations of TKI and venetoclax
- Practice thawing and culturing of primary cells.
- Validate Western Blot antibodies and primers used for quantitative real-time PCR.

Month 3 to 7
- Treat TKI-sensitive and -resistant K562 cell lines with combined TKI and venetoclax and establish growth curves, using real-time cell viability monitoring (RealTimeGlo by Promega).
- Harvest cells at various time points for Western blotting to analyze protein levels of lipidated LC3-II which is recruited to autophagosomal membranes, p62 as an indicator of autophagic flux, and Beclin-1.
- Simultaneously, collect samples for qRT-PCR to measure mRNA expression of LC3, p62, and Beclin-1.
- Data analysis and interpretation of results from cell line experiments.

Month 8 to 11
- Plan and refine experimental conditions for analyses of CD34+ primary cells based on cell line data.
- Considering limited proliferation capacity and availability, carry out analyses analogue to cell line studies, where possible.
- Analyze differences in viability and autophagy marker expression between cell lines and patient samples.
- Identify potential correlations between treatment response and autophagy dysregulation.

Month 12
- Finalize thesis

Principal Investigator:
Prof. Dr. Thomas Ernst, Dr. Jenny Rinke (Department of Internal Medicine II, Division of Hematology and Oncology)

Background and previous work:
Liver failure is a critical complication of sepsis and severe infections due to the central role of the liver in the overall immunometabolic homeostasis. The liver responds to systemic infection with a robust reprogramming of its metabolic and secretory landscape, a process known as the phase I response, to help fight infection. One poorly understood part of this response is the role of autophagy. The liver reacts to pharmacological and metabolic stress with particularly high levels of autophagy; indeed, autophagy was first described in rodent liver in the 1960s. However, the significance and consequences of hepatic autophagy are not well understood because published evidence is conflicting as to whether autophagy contributes to the Phase I response or whether it plays a protective or destructive, pro-apoptotic role in stressed hepatocytes. Indeed, even less is known in the case of inflammation, as most studies have focused on alcoholic, pharmacological or metabolic responses to stress.
We have previously investigated the role and mode of action of calorie restriction as a priming cue for hepatocyte protection and induction of disease tolerance in inflammation. We observed that calorie restriction induced autophagy in hepatocytes, but this form of autophagy was unconventional as it was uncoupled from mTORC1 activity and did not correlate with cell viability. Thus, while autophagy induction is well documented in stressed hepatocytes, its role and regulation remain enigmatic.

Specific aims:
We aim to study the regulation and role of hepatocyte autophagy in the context of inflammatory stress in pre-clinical, animal-free liver models, including primary hepatocytes and liver organoids.

Working programme:
Using primary hepatocytes and genetically engineered liver organoids, we will study the regulation and consequences of hepatocyte autophagy and identify potential targets for pharmacological manipulation of this critical process. Hepatocyte/organoids will first be characterised with respect to their metabolic properties and ability to induce a phase I switch in response to inflammation. In a second step, hepatocytes/organoids will be exposed to inflammatory (cytokine/PAMP mixture), metabolic (calorie restriction/hyperglucemia) or pharmacological (paracetamol) stress, followed by assessment of autophagy using various alternative readouts, including microscopy-based analysis of autophagic flux using Cyto.ID© or mCherry-GFP-LC3B-based fluorescent biosensors. The autophagy readout will be compared with parallel measurements of cell viability (PI/Hoechst staining), ER stress (Western blot) and protein synthesis (metabolic methionine labelling). Finally, we will modulate autophagy using established inhibitors (chloroquine, leupeptin) or activators (rapamycin, trehalose) and examine the outcome for stress-induced cell death or cell damage. Looking ahead, the results of these experiments will be validated in a follow-up study using human patient/donor liver samples, possibly from human autopsies. In summary, we expect to gain a comprehensive view of how stress-induced autophagy modulates the adaptive responses of hepatocytes, which may provide new approaches to enhance the resilience of the liver during severe infections or other conditions of liver failure.

Principal Investigator:
Prof. Dr. Ignacio Rubio (Department for Anesthesiology & Intensive Care Medicine, Work Group Host Response to Sepsis)

Background and previous work:
Autophagy is a cellular bulk degradation system for long-lived proteins, damaged organelles and aggregated proteins. It acts as quality control and helps to maintain cellular homeostasis. This prevents various diseases, such as neuronal and muscle degeneration (Anding et al., 2017 Dev. Cell). During autophagy, double-membrane autophago- somes surround cytoplas- mic materials and fuse with lysosomes to degrade their contents. Yet, the molecular compo- nents enabling these steps are far from clear.
Recently, syndapin I (PACSIN1) was identified as autophagy regulator using CRISPR/Cas9- mediated gene ablation in HeLa cells. In line with syndapins playing some role in autophagy, C. elegans mutants deficient for the only syndapin family member in worms (sdpn-1) showed an impaired protein clearance in muscles and movement deficits that may suggest accelerations of age-dependent impairment of locomotion due to defective aggregation clearance (Oe et al., 2022 PLoS Genetics).
In mammals, syndapin I is the nervous system-enriched member of the syndapin family of membrane-shapers (Qualmann et al., 1999 Mol. Biol. Cell). Syndapin I KO causes defects in synaptic transmission and post-synaptic plasticity correlating with seizures and schizophrenia- like symptoms in mice (Koch et al., 2011 EMBO J, Koch et al., 2022 Cereb. Cortex). The muscle-enriched isoform is syndapin III. Syndapin III KO leads to impaired caveolar invagina- tions, as demonstrated by 3D-electron microscopy, expanded variations in muscle fiber dia- meters and muscle defects after physical exercise manifesting in internally positioned nuclei and signs of inflammation and necrosis (Seemann et al., 2017 eLIFE). These defects are remi- niscent of caveolinopathy symptoms. Interestingly, a recent case report showed that biallelic PACSIN3 gene truncation variants caused clinical symptoms in humans. Patients showed early fatigue, muscle pain and exercise intolerance, as well as elevated creatin kinase levels in the circulation – a clinical parameter also known from caveolinopathies. Histopathological examinations of muscle biopsies showed increased fiber diameter variations, atropic fibers and internally positioned nuclei. Yet, at least at the light microscopical level, no obvious defects in anti-caveolin3 immunostaining patterns were observed. However, the authors report the occasional occurrence of cytoplasmic bodies with proliferations of tubes and tubular aggregate-like stacks of unknown nature (Distelmaier et al., 2024 Brain).

Specific aims:
We propose that these structures may be accumulated autophagosomes and will study the effects of syndapin deficiency on autophagy by targeted gene knock-out in mice using light microscopy and electron microscopy on cultured neurons and muscle samples as well as cultured cardiomyocytes and fibroblasts from WT and KO animals.

Working programme:
With the syndapin I and syndapin III KO mice we generated, we are in the unique position to do so. We will furthermore compare the observed defects with those in human patients. We will also evaluate which autophagy processes are impaired (aggrephagy, lysophagy, mito- phagy) and test whether this applies to both nutrient-rich and stress conditions.
Together, these efforts will highlight the mechanisms of autophagy contributing to cellular homeostasis – a key requisite for post-mitotic cells, such as neurons and muscles, which need to maintain their integrity life-long to ensure functionality and healthy aging.

Principal Investigator:
Prof. Dr. Britta Qualmann, PD Dr. Michael Kessels (Institute of Biochemistry I)

Background and previous work:
Patients with acute myeloid leukemia (AML) still have a very poor prognosis and survival, largely influenced by underlying molecular aberrations as well as the development of therapy resistances. The impact of autophagy has been extensively studied in AML, demonstrating an important mechanism for cell survival and resistance to therapeutic stress. For example, synergistic effects of classical cytotoxic chemotherapy and autophagy inhibitors have been shown. With an increasing molecular understanding of AML, targeted therapies such as Menin inhibitors are currently in focus. These show promising results in the presence of NPM1 mutations or a KMT2A rearrangement (both can find in approximately 30% of AML patients), but also lead to early development of resistances and thus reduced efficacy. Recently, the research group published results on potential synergistic effects by combining Menin inhibition with Tamibarotene, an all-trans retinoic acid agonist, in NPM1- and KMT2A-mutated cell lines as well as patient cells, providing a possible new approach to improve effectiveness of Menin inhibition.
It is known that NPM1 mutations induce autophagy through the accumulation of PML in the cytosol and so can induce survival of AML cells through subsequent AKT activation. In the case of a KMT2A mutation, which is associated with particularly poor overall survival, the protein LAMP5, acting as an autophagy repressor, prevents the selective autophagic degradation of the fusion protein and supports leukemic transformation. Here, preclinical use of autophagy inducers showed improved degradation of the KMT2A fusion protein. Therefore, the presumed relevance of autophagy modulation in these cells suggests a rationale for combining it with Menin inhibitors in AML cells depending on the presence of NPM1 mutation or a KMT2A rearrangement.

Specific aims:
1. Testing of various autophagy modulators in established FLT3-, KMT2A-, and NPM1-mutated AML cell lines.
2. Investigation of the influence of Menin inhibition on autophagy in AML cells.
3. Enhancement of the efficacy of Menin inhibitors through combination with autophagy modulators

Working programme:
Due to the previous work of the research group on the topic, the cell lines (MV4-11, MOLM13, and OCI-AML3) as well as the experimental setup (viability analyses, apoptosis assays, flow cytometry, Western blot, microscopy) are already well established. Initially, various autophagy inhibitors will be tested for efficacy in the mentioned cell models using apoptosis and viability assays. Subsequently, the influence of Menin inhibition on the regulation of autophagy markers already established in the group (e.g., LC3B) will be examined using Western blot. Following this, combination experiments will investigate potential synergistic effects in terms of apoptosis induction. Furthermore, potential effects on primary AML patient samples from a biobank of the research group can be evaluated.

Principal Investigator:
Prof. Dr. Sebastian Scholl, Dr. Maximilian Fleischmann (Department of Internal Medicine II)

Background and previous work:
MAPK organizer protein 1 (MORG1), also known as WDR83, is a multifunctional scaffold protein of the HIF and MAPK/ERK pathways involved in the polarization of epithelial cells (Vomastek et al. Proc Natl Acad Sci U S A. 2004;101(18) 6981-6; Hopfer et al. J Biol Chem. 2006;281(13):8645-55; Hayase et al. J Cell Biol. 2013;200(5):635-50). We have found that a reduction in MORG1 expression (heterozygous MORG1 knockout (KO)) attenuates diabetes-induced renal damage (glomerulosclerosis and tubulointerstitial fibrosis) in mouse models of type 2 diabetes mellitus (Loeffler et al. Nephrol Dial Transplant 2017;32: 2017-2034). We assume that this is mainly due to MORG1's role in the HIF signaling pathway and thus the stabilization of the protective HIF2 upon MORG1 reduction. Interestingly, in old heterozygous MORG1-KO animals we see a loss of renoprotectivity or even an enhancement of renal fibrosis compared to old wild-type animals. A possible explanation for these unpublished findings could be a newly described function of MORG1 in the mTORC1 signaling pathway: MORG1 reportedly primes basal autophagy by inhibiting lysosomal recruitment of mTORC1 (Abudu et al. Molecular Cell 2024;84:1-18). We speculate that sustained MORG1 reduction results in an amplification of aging-induced autophagy deficiency, hence compromising cellular integrity. Furthermore, we observe sex-dependent MORG1 regulation in aging kidneys that is associated with known sex differences in age-induced renal damage, for which there is also no conclusive mechanistic explanation yet. Transforming growth factor beta 1, which is induced in the ageing kidney and is thought to be responsible for profibrotic changes, has also been shown to induce autophagy/mTORC1 (Ding et al. Semin Nephrol 2014;34(1):62-71). The signaling pathway that TGF-ß1 activates for this function is via p38, a MAPK such as Erk1/2, for which MORG1 acts as a scaffold protein.

Specific aims:
One aim of this project is to investigate in animal models whether A) age-induced autophagy deficiency is influenced by the MORG1 genotype and B) whether there are sex differences in this respect. The analyses will differentiate between autophagy in podocytes and autophagy in renal tubular cells, since both cell types are affected by age-induced damage and are characterized by a high autophagy rate under physiological conditions.

A second major aim is to investigate whether MORG1 plays a role in the dual function of TGF-ß1 as an inducer of collagen synthesis (via the classical Smad2/3 pathway) and as an inducer of autophagy and collagen degradation (via the alternative p38 pathway).

Working programme:
Work package 1: Renal tissue sections from wild type and heterozygous MORG1-KO mice of different ages and both sexes will be analyzed by OPAL-based multiplex immunofluorescence for the number of different autophagy particles, autophagy-associated protein expressions and signaling pathways in podocytes and tubule cells (LAMP1, LC3, Beclin1, Atg13 in combination with podocyte marker Synaptopodin and WT1 as well as the proximal tubule marker GGT1 and Villin).
Work package 2: TGF-ß1 stimulation experiments in combination with MORG1-siRNA with one podocyte and two proximal tubule cell lines (insulin-dependent TKPTS and insulin-independent MTC) will be used to analyze TGF-ß1 signaling pathways (classical and alternative), mTORC signalling and autophagic flux as a function of MORG1 expression by Western blot and fluorescence microscopy.

Principal Investigator:
PD Dr. Ivonne Löffler, Prof. Dr. Gunter Wolf, (Department of Internal Medicine III)