Projects 2nd Funding Period

Nathalie Albert, Louisa von Baumgarten
Temporospatial changes of TSPO expression in glioblastoma in response to therapy and during disease progression – a multimodal TSPO PET, amino acid PET and MRI study

TSPO is overexpressed in primary brain tumors and has been shown to correlate with tumor malignancy and a poor prognosis. Our previous work performed in the first funding period has shown that the PET signal of gliomas in TSPO PET and amino acid PET ([18F]FET) are different and also differ from contrast enhancement in MRI. Furthermore, PET signals may change in response to therapy and during disease progression and its impact on the clinical outcome and individual prognosis of glioblastoma patients has not been assessed so far.

Therefore, this project aims to visualize temporal and spatial changes of TSPO expression by means of a single arm observational study using longitudinal multimodal TSPO / [18F]FET PET and ceMRI for patients with glioblastoma, and to correlate imaging findings with changes on the cellular and molecular level as well with the clinical outcome.

In work package 1 patients with newly diagnosed brain tumors suspicious for glioblastoma are prospectively enrolled in an observational study. All data retrieved from clinical workup, clinical-follow-up, imaging and neuropathological analysis are collected. Tumor specimen retrieved from tumor resection or stereotactic biopsies are spatially correlated with PET and MRI data, which enables a precise comparison of imaging information with histopathological findings and pathway analyses for project A2/3. This collaboration with project A2/A3 allows in-depth analyses of changes of the tumor microenvironment, cellular contributions to the TSPO signal (myeloid cells vs. tumor cells) and molecular alterations in a unique longitudinal study cohort with tissue samples obtained with TSPO PET image guidance at primary diagnosis and at relapse.

Work package 2 focusses on advanced image analysis of the multimodal imaging data and its correlation with clinical and neuropathological data obtained in work package 1. The analyses include pharmacokinetic modelling, machine learning and deep learning approaches to perform non-invasive tissue classification, distinguish therapy responders from non-responders at an early time point and to predict the time and localization of tumor recurrence and ultimately, to link imaging features with overall survival.

Markus Zweckstetter
Nuclear magnetic resonance (NMR) spectroscopy of human TSPO

The three-dimensional structure of mouse TSPO (mTSPO) was previously determined by my lab using solution-state NMR spectroscopy. In addition, we studied the influence of a clinically relevant polymorphism in mTSPO, which results in the substitution of alanine by threonine at position 147 and decreases the affinities of many radioligands and therapeutics, on the mTSPO structure. We also found out that mTSPO is more flexible when no high-affinity radioligand is bound to the protein. A further focus of our studies was the analysis of the interaction of mTSPO with cholesterol using solid-state NMR spectroscopy. The three-dimensional structure of human TSPO is, however, unknown. In this project, we thus propose to study the structure of human TSPO (hTSPO) using solution- and solid-state NMR spectroscopy. To this end, we will first – using a model protein – perform tests on how to optimize the NMR process of resonance assignment and structure determination of alpha-helical membrane proteins. Subsequently, we will apply this methodology to hTSPO in the absence and presence of high-affinity ligands that are studied within the research unit (such as XBD173). This work will address the question if hTSPO folds – in the absence of hTSPO-specific ligands – into a stable conformation when it is solubilized in either detergents or lipids with distinct properties. Subsequent steps will be the determination of the structure of hTSPO, analysis of the molecular details of its interaction with selected high-affinity ligands and the influence of the A147T polymorphism on these interactions.

TSPO ligands in the treatment of depression: proof-of-concept and underlying mechanisms of action.
Benzodiazepines are fast acting anxiolytics which primarily target synaptic GABAA receptors. However, common side effects are sedation, tolerance development, and abuse liability. TSPO ligands promote endogenous neurosteroidogenesis which modulate extrasynaptic GABAA receptors. Meanwhile, exogenous neurosteroids have been shown to exert rapid antidepressant effects in postpartum depression and major depressive disorder (Rupprecht et al. 2022, 2026; Riebel et al. 2025). To delineate putative differences between bezodiazepines and TSPO ligands, in the first funding period we perfomed a clinical study in 36 healthy male volunteers to compare the effects of the TSPO ligand etifoxine with the benzodiazepine alprazolam regarding anxiolytic efficacy, modulation of GABergic excitability, modulation of neuronal networks, and microbiome composition. In the second funding period we conducted a placebo controlled clinical proof of concept study in patients with major depression to evaluate whether the TSPO ligand etifoxine exerts rapid antidepressant effects similar to exogenous neurosteroids such as zuranolone (Rupprecht et al. 2022, 2026). Moreover, we investigated the impact of etifoxine on cognition and depression related neuronal networks (Brunner et al. 2024).
In the first funding period we showed that alprazolam had more pronounced GABAergic effects than etifoxine in a double pulse TMS paradigm and produced more sedation (Riebel et al. 2023). Moreover, sedation induced by alprazolam disconnects low-level resting state functional networks but simultaneously increases their within-network connectivity (Wein et al. 2024). Moreover, we showed additional effects of alprazolam-associated sedation using our inhouse developed fusion searchlight (FuSL) framework, which integrates complementary information from multiple resting-state fMRI metrics, thereby enhancing the accuracy of pharmacological treatment prediction from rs-fMRI data (Wein et al. 2025). Treatment with etifoxine reduced the abundance of a few bacterial species, which are currently seen as beneficial components of a healthy intestinal microbiome (Manook et al. 2023), whereas absolute plasma neurosteroid levels were not affected (Riebel et al. 2025). In a task-based paradigm, alprazolam reduced subjective nervousness more prominently than etifoxine, while no deprsignificant effect of either medication could be detected by means of fMRI (Riebel et al. in preparation).
In the second funding period we conducted a clinical proof of concept study in 50 patients with major depressive disorder to assess whether adjunct treatment with the TSPO ligand etifoxine may fasten the clinical response to antidepressant treatment via enhancement of endogenous neurosteroidogenesis (Brunner et al. 2024). Moreover, a putative impact on cognition is inverstigated by a cognitive test battery. Finally, the investigation of depression related neuronal networks will provide insight into the mechanisms of action of putative antidepressant effects of TSPO ligands. The recruitment for the study is completed now. The respective data analyses are under way.