Reducing Reactive Astrogliosis in vitro using Altered Gravity

Kurzfassung

In the current study, astrocytes reacted to 2g hypergravity, as well as to simulated microgravity by clinorotation with similar defects in spreading, leading to reduced cell size. This reaction to the changes in their gravitational environment indicates their ability to “sense” gravity in a so far unknown manner. Indeed, astrocytes are known to be mechanosensitive and as an integral part of the blood-brain barrier can react to changes in blood pressure and brain perfusion. Candidate mechanosensitive proteins in astrocytes are the transient receptor cation channel TRPV4 and Connexin43 (Turovsky, Braga et al. 2020) and the mechano-gated ion channel Piezo1 (Shaopeng Chi 2022). It would therefore be interesting to repeat the hypergravity- and simulated microgravity experiments with inhibitors against or knock-out cell lines of these mechanosensitive channels. This would further bring to light how mammalian cells, and astrocytes in particular, sense gravitational cues in their environment. Viewing astrocytic cellular responses to hypergravity, the observed spreading defects as well as the decreased migration should be examined with the same assays but under increased hypergravity of 3g and more to find out if there is a dose-response curve with regards to the severity of the changes of cell morphology and behavior under hypergravity. When exposing cells to increased hypergravity regimen, viability needs again to be controlled for. To find out if the 95 decrease in cell spreading and cell area is going hand in hand with a decrease in cell volume, or if the measured spreading reduction is due to the cells becoming more spherical, but keeping their volume constant, astrocytes should be analyzed by cytosolic, or membrane staining and imaged in a confocal microscope using z-stacking. By this, a 3D model of each cell can be generated to calculate the cell volume. These volumes can then be compared between normal and hypergravity to see if the cell volume is reduced by increased gravitational loading. It is still not clear if the observed reduction in astrocyte migration under hypergravity is solely caused by changes in cytoskeletal dynamics or if other cellular pathways which influence migration behavior play a part in this phenotype. To this end, more thorough biochemical and genetic analyses should be performed, taking into account other proteins which are implicated in astrocyte migration such as MMP-2 and MMP-13 (Verslegers, Lemmens et al. 2013) and other Rho-associated proteins (Hodge and Ridley 2016). RhoA for example is implicated in actin stress fiber and focal adhesion formation (Pellegrin and Mellor 2007), and a more than 1.5 times increase in RhoA protein expression was detected after one day of hypergravity exposure (see 2.7). More thorough analyses using protein mass spectrometry or RNA sequencing of astrocytes exposed to altered gravity conditions can be performed to yield more in-depth results of the cells’ transcriptome and protein content. Indeed, using the experiment payload Cellfix on the sounding rocket MAPHEUS 13, primary murine astrocytes were exposed to six minutes of real microgravity and fixed to be retrieved for transcriptomic analysis. These samples will be compared to 1g ground controls and hypergravity-exposed cells with regards to the already identified proteins but also to find newly regulated targets. Cellular migration should also be investigated not only in hypergravity, but also in (simulated) microgravity. Astrocyte reactivity is a complex and not yet thoroughly defined mechanism which could be defined by many more markers than investigated in this study. Studied by many researchers, novel markers are constantly found which will help to identify and further understand various reactivity phenotypes. Newly found targets can also be investigated in altered gravity exposed cells to learn more about astrocyte reactivity in hyper- and microgravity. A reactive phenotype in astrocytes could be chemically evoked by addition of e.g., pro-inflammatory cytokines such as IL-1β and TNF, or by the addition of medium from activated glia (Retamal, Froger et al. 2007, Lagos-Cabre, Alvarez et al. 2017). Live-cell epifluorescence microscopy employing LifeAct-GFP astrocytes showed alterations of the actin cytoskeleton under hypergravity in a qualitative analysis. These experiments could also be repeated under increased hypergravity loading of 3g and more to see if the observed changes in actin dynamics increase in severity. In this regard, projects have been initiated to develop software solutions for artificial intelligence-supported recognition of cells and intracellular filaments such as actin, microtubules, and intermediate filaments. For accurate 96 machine learning algorithms, large training datasets with the features that shall be recognized (e.g., intracellular fibers) labeled must first be generated. This in itself of course is a high workload and has to be evaluated as a trade-off. Additionally, it is not yet clear how cell-type specific such a model will be. To have scientific merit, a potential automatic program for recognition of intracellular features needs to output discrete and objective data, where the “black box” paradigm of machine learning algorithms could prove problematic. With regards to these challenges, more classical approaches using mechanisms such as thresholding and watershed segmentation are also being evaluated. A functioning, and ideally universal program will ultimately help to not only qualitatively examine microscopy images, but also objectively quantify changes in cytoskeletal morphology after altered gravity exposure not only in epifluorescence but also in confocal and STED microscopy images. Live-cell microscopy on the Hyperscope is logically confined to hypergravity exposure, but a prototype clinostat microscope is being developed at the DLR which will enable live-cell microscopy also in simulated microgravity. Live-cell microscopy can be enhanced with further channels or fluorophores by employing live-staining solutions for other cytoskeletal elements, such as SiR-tubulin, Pkmito, or by using other transgenic mouse models to visualize GFAP or other intermediate filaments. Live-cell microscopy in real microgravity will in the future be possible on the ISS by means of the FLUMIAS instrument which is being developed by the DLR. LifeAct-GFP astrocytes are part of a planned project (Live Assessment of Astrocytic Reactivity Adaptations under Space Conditions – LAARA) and shall be imaged in microgravity and increments up to 1g with added tubulin and calcium live stains to elucidate astrocyte adaptation to real microgravity and eventual changes in morphology, behavior, and reactivity. By imaging the cells while they are exposed to different centrifugation speeds between 0g and 1g, the thresholds of reaction to gravity will also be investigated and can be directly compared to the hypergravity and simulated microgravity data in this thesis. Ground-based studies using simulated microgravity and mechanical unloading with clinostats started in this work (see 2.8) are planned to be continued in more detail. A clinostat that can hold Ibidi 3.5 cm dishes (see 4.2.3.4, 4.2.3.5) is in construction, so that migration assays in simulated microgravity can be performed to compare the astrocyte migration speed under various gravity conditions. The employed methods of hypergravity exposure in the MuSIC for fluorescence and STED microscopy and live-cell microscopy under hypergravity of scratch assays and cell spreading or intracellular actin dynamics have been newly developed for this project and can be translated to many other cell types or model systems to enrich the field of research under altered gravity conditions. 97 Altogether, the findings of this work pave the way for new insights into cellular reactions to hypergravity and open up new avenues of research for the treatment of astrogliosis and resulting impairments of the CNS. The underlying pathways responsible for the observed hypergravity phenotypes could, when characterized in detail, be exploited to alter astrocyte reactivity in vivo and thus lead to new treatments for neurological disease or injury. For these aims, other model systems such as 3D cell culture, brain organoids, ex vivo techniques such as acute brain slices and in vivo mouse models need to be employed to move closer to the level of the whole organism. The timed attenuation but not complete ablation of astrocyte reactivity through hypergravity exposure may possibly be established as a non-invasive means of reducing the negative effects of astrogliosis and enhance neuronal regeneration. This approach can be combined with classical drug-based therapies to selectively diminish the adverse effects of glial scarring in vivo but retain the positive properties of reactive astrocytes and astrogliosis.