Autophagy is a fundamental cellular quality-control mechanism that maintains cellular homeostasis by degrading damaged organelles, protein aggregates, and other intracellular debris. During this process, material destined for degradation is enclosed within a double-membrane vesicle, the autophagosome, which subsequently fuses with a lysosome to form an autolysosome. Lysosomal enzymes then degrade the cargo into its basic components, allowing their recycling by the cell.

Our research focuses on IST1, a key component of the ESCRT machinery involved in membrane remodeling. Recently a patient with biallelic loss-of-function variants in IST1 was identified who presents with severe neurodevelopmental disease characterized by profound cognitive impairment and motor dysfunction. Patient-derived fibroblasts exhibit a striking reduction in lysosome abundance, suggesting that defective lysosomal homeostasis may impair autophagy and contribute to disease pathogenesis.

In this project, we will investigate how IST1 deficiency affects the autophagy-lysosome pathway. We will combine immunoblotting and immunofluorescence microscopy with dynamic autophagic flux assays using a tandem fluorescent reporter that distinguishes autophagosomes from autolysosomes. These complementary approaches will allow us to pinpoint where the autophagic pathway is disrupted.

Finally, we will evaluate whether pharmacological stimulation of autophagy can restore cellular function, providing initial evidence for potential therapeutic strategies.

This project offers the opportunity to gain hands-on experience with state-of-the-art cell biology techniques while addressing a clinically relevant question in rare neurodevelopmental disease.

Principal Investigator:
Prof. Dr. Christian Hübner (Institute of Human Genetics)

Autophagy plays a key role in tumorigenesis and in the development and progression of neurodegenerative diseases. Although the inhibition of autophagy is important for cancer treatment, research into neurodegenerative diseases is investigating whether activating autophagy could promote the breakdown of toxic protein aggregates.

The tumor suppressor Nit1 (Nitrilase1) exists in different isoforms, the main ones of which are localised in the cytoplasm and in mitochondria, and the nucleus. Nit1 is downregulated in several cancer types, such as oesophageal and colon cancer and reduced expression has been linked to poor prognosis. Nit1 belongs to the tenth branch of the nitrilase superfamily and is classified as a Rosetta Stone protein and tumor suppressor together with its partner Fhit (fragile histidine triad). Additionally, Nit1 acts as a metabolite repair enzyme, hydrolysing dGSH (deaminated glutathione). Studies with colon cancer cell lines have observed a Nit1-dependent regulation of SMAD/TGFb-signalling. TGF-β frequently activates autophagy. Interestingly, clinical case reports describe patients with bi-allelic Nit1 variants as suffering from brain small vessel diseases, which is characterized by movement disorders, massively dilated perivascular spaces and intracerebral haemorrhage.

In previous studies, we identified both Fhit and Nit1 as b-catenin interaction partners, which repress canonical Wnt/b-catenin signalling in an additive manner (1-3). The Wnt/b-catenin signalling pathway plays a key role in the development of colon cancer, but also numerous neurological and neurodegenerative disorders. It is well known that the Wnt-signalling pathway and autophagy influence each other. In preliminary experiments we observed alterations in Nit1 expression levels in response to Wnt activation or autophagy modulation. These findings will analysed in detail to uncover the regulation of Nit1 in autophagy.

This project aims to answer the following questions:

1. Is Nit1 regulated by autophagy induction or inhibition?

2. Is there an interaction of important autophagy key players with Nit1 and how can it be modulated?

3. Vice versa: Is autophagy (mitophagy) affected by Nit1 knock-down or overexpression?

References:
1. Weiske J, Albring KF, Huber O. The tumor suppressor Fhit acts as a repressor of b-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/b-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.

Principal Investigator:
Prof. Dr. Otmar Huber (Department of Biochemistry II)

• understand, if and how the interaction between adipogenic / fibrogenic TME and NSCLC cells influences autophagy in tumor cells and non-tumor cells within the TME.
• understand, if and how the co-culture induced modulation of autophagy has influence on tumor biological behavior in NSCLC.
• understand, how the interaction of tumor cells with adipocytes / fibroblasts influences the autophagic, adipogenic and fibrotic pathways.
• understand, if in vitro results can be confirmed in primary lung cancer samples

• Establishing fibroblast cell line 3T3-L1-differentiated adipocytes. Co-culture of adipocytes/tumor cells and fibroblasts/tumor cells w/wo nutrient starvation
• Expression analysis of autophagy markers (p62, beclin-1, LC3I/II, etc.), adipogenic proteins (FABPs, CD36, ACSLs, FASN etc.), and ECM proteins (fibronectin, integrins, tenascin, MMPs, etc.) by RT-qPCR and Western blotting
• Performing cell-based functional assays (MTT, migration/invasion, seahorse, mitochondrial staining)
• Analysis of the markers identified from in vitro co-culture models selectively by using immunohistochemistry and multiplexing immunofluorescence in primary lung cancer samples

Ma Y, Nenkov M, Berndt A, Abubrig M, Schmidt M, Sandhaus T, Huber O, Clement JH, Lang SM, Chen Y, Gaßler N. The Diagnostic Value of ACSL1, ACSL4, and ACSL5 and the Clinical Potential of an ACSL Inhibitor in Non-Small-Cell Lung Cancer. Cancers (Basel). 2024 Mar 16;16(6):1170. doi: 10.3390/cancers16061170.

Ma Y, Nenkov M, Schröder DC, Abubrig M, Gassler N, Chen Y. Fibulin 2 Is Hypermethylated and Suppresses Tumor Cell Proliferation through Inhibition of Cell Adhesion and Extracellular Matrix Genes in Non-Small Cell Lung Cancer. Int J Mol Sci. 2021 Oct 31;22(21):11834. doi: 10.3390/ijms222111834.

Chen Y, Schnitzler K, Ma Y, Nenkov M, Theis B, Petersen I. The clinical influence of autophagy-associated proteins on human lung cancer. Disease Markers 2018; 2018:8314963. doi: 10.1155/2018/8314963.

PD Dr. Y. Chen, Prof. Dr. N. Gaßler (Section Pathology of the Institute of Forensic Medicine)

Diabetic nephropathy is a major complication of diabetes mellitus characterized by complex molecular mechanisms such as oxidative stress, inflammation, and disturbances in cellular metabolism. Autophagy, as a cellular defense mechanism, plays a potentially protective role under metabolic stress, with the mTOR signaling pathway playing a crucial regulatory role. The aim of this project is to investigate the effects of hyperglycemic conditions on autophagy in HEK cells and to analyze its interaction with the mTOR pathway. Through pharmacological modulation of mTOR (e.g., with rapamycin), we aim to determine whether and to what extent autophagy is activated or inhibited, and whether this contributes to protection against cellular damage. Changes in autophagy and mTOR activities will be assessed using Western blot analyses, fluorescence microscopy, and proteomics. The findings could form the basis for new therapeutic strategies for diabetic nephropathy by identifying the regulation of autophagy-mTOR cascades as a potential target. This project thus contributes to a better understanding of the molecular mechanisms underlying kidney damage in diabetes.

This project aims to investigate the role of autophagy in HEK (human embryonic kidney) cells under hyperglycemic conditions, with a particular focus on its interaction with the mTOR signaling pathway. The goal is to determine whether and how the mTOR pathway influences autophagic activity and whether this modulation has a potentially nephroprotective effect.

• How do hyperglycemic conditions alter autophagy in HEK cells?
• What role does the mTOR signaling pathway play in regulating autophagy in this context?
• Can pharmacological modulation of mTOR (e.g., with rapamycin as an inhibitor or mTOR activators) influence the autophagic process and protect cell function?

• Short term (up to 6 months): Establishment of cell cultures, optimization of hyperglycemic conditions and treatment protocols
• Medium term (up to 9 months): Conducting autophagy and mTOR analyses, statistical evaluation
• Long term (up to 12 months): Evaluation and interpretation of the data
• Long term (up to 18 months): Summary, publication, and presentation

1. Jung et al. (2010). mTOR regulation of autophagy. Cell. (doi: 10.1016/j.febslet.2010.01.017)
2. Yang et al. (2017). Autophagy in diabetic kidney disease. Cell Mol Life Sci. (doi: 10.1007/s00018-017-2639-1)
3. Tanaka et al. (2012). Autophagy as a therapeutic target in diabetic nephropathy. Exp Diabetes Res. (doi: 10.1155/2012/628978)

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

Cisplatin is an effective chemotherapeutic agent whose nephrotoxic side effects limit its use in therapy. Autophagy is a cellular defense mechanism against stress that is regulated by the mTOR signaling pathway. Previous studies suggest a complex, time-dependent effect of cisplatin on autophagy, ranging from activation to inhibition. These seemingly contradictory results indicate a high degree of complexity in mechanistic and experimental factors. To understand the molecular mechanisms underlying these processes, comprehensive analyses at the protein level are necessary.

This project provides a solid foundation for identifying fundamental cellular and molecular response patterns involved in cisplatin-induced kidney damage and establishes an analytical basis for further in-depth follow-up experiments.

Within one year, the fundamental effects of cisplatin on autophagy and the mTOR pathway in HEK cells will be investigated, specifically:

• Characterization of the autophagic response at various time points.
• Analysis of mTOR activation processes.
• Initial comprehensive proteomic analyses using LC-MS/MS to identify signaling cascades and protein dynamics.

As part of the one-year research plan, optimal experimental conditions and standardization protocols for protein extraction and Western blot analyses will first be established. This will be followed by experimental investigations of autophagy and mTOR phosphorylation using Western blot and fluorescence microscopy, supplemented by an initial proteomic analysis via LC-MS/MS to identify relevant signaling pathways and protein dynamics. Data analysis will involve integrating the results related to autophagic processes and mTOR activation to identify potential target proteins and signaling cascades.

1. Hu X, Ma Z, Wen L, Li S, Dong Z. Autophagy in cisplatin-induced nephrotoxicity during cancer therapy. Cancers (Basel). November 10, 2021;13(22):5618. doi: 10.3390/cancers13225618.
2. Wilmes A, et al. Mechanism of cisplatin toxicity in the proximal tubule, revealed by the integration of transcriptomics, proteomics, metabolomics, and biokinetics. Toxicol In Vitro. Dec. 25, 2015;30(1 Pt A) :117–27. doi: 10.1016/j.tiv.2014.10.006.
3. Jung CH, Ro SH, Cao J, Otto NM, Kim DH. mTOR regulation of autophagy. FEBS Lett. Apr. 2, 2010;584(7):1287–95. doi: 10.1016/j.febslet.2010.01.017.

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

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:
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)

Our group has established a robust and highly reproducible rat liver NMP model using organs obtained after cardiac death (DCD) with and without human erythrocytes as oxygen carriers. Using this model, we demonstrated that donor age, warm ischemia time, and perfusion strategy critically determine graft performance and are associated with impaired metabolic function. Preliminary analyses further revealed the accumulation of p62, along with altered LC3 expression, in livers subjected to prolonged warm ischemia, suggesting alterations in autophagy.

We aim to determine whether autophagy-associated alterations are linked to impaired mitochondrial quality control, increased apoptosis, and reduced graft recovery during NMP following warm ischemia.

Building on our preliminary findings, the results of this project will establish a mechanistic basis for future studies investigating whether the pharmacological modulation of autophagy during non-ischemic machine perfusion (NMP) can enhance the quality control of mitochondria and improve the functional recovery of ischaemic donor livers.

1. Chen H, Dirsch O, Albadry M, Paz AH, Dahmen U. Normothermic ex vivo liver machine perfusion in mouse. J Vis Exp. 2023 (199):e65363.
2. Chen X. Ischemia-reperfusion injury in a rat model of normothermic oxygenated machine perfusion and liver transplantation.Dissertation. Friedrich Schiller University Jena; 2024.
3. Chen H, Li F, Ismael MA, Chen X, Nocke F, Cantore M, et al. Challenges in establishing a normothermic oxygenated machine perfusion system using livers from mice after cardiac death. J Investigative Surgery. Under revision.
4. Klionsky DJ, Abdel-Aziz AK, Abdelfatah S, et al. Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition). Autophagy. 2021;17(1):1–382. https://doi.org/10.1080/15548627.2020.1797280
5. Mao B, Yuan W, Wu F, et al. Autophagy in hepatic ischemia-reperfusion injury. Cell Death Discovery. 2023;9:115. https://doi.org/10.1038/s41420-023-01387-0

Transplantationschirurgie)

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)