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 Neurology)

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)