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:
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:
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:
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)