Neuronal Systems Potentially Involved in Autism Spectrum Disorder

Pfn2 is a brain specific actin binding protein involved in actin dynamics. The Pfn2 knock-out mouse model shows an autistic-like phenotype with all of the features commonly reported in autism patients such as social and communicational impairment, repetitive behavior and epileptic seizures. The role(s) and function(s) of Pfn2 in this striking phenotype are not understood completely. A first step in understanding these roles is investigating the expression pattern of Pfn2 in the brain to shed light on the different neuronal systems that could potentially contribute to the many aspects of the autistic-like and epileptic phenotypes. A Profilin2-GFP fusion protein knock-in mouse model was used in this thesis to investigate the expression of Pfn2 in neuronal cell subtypes that could possibly contribute to the autistic-like phenotype. Pfn2 has been found expressed in glutamatergic, dopaminergic, serotonergic, adrenergic and cholinergic neurons, both pre- and post-synaptically, while GABAergic neurons showed a complex subtype specific expression pattern dependent on the respective brain area. Pfn2 was missing from all glial cell subtypes. Neural stem cells expressed Pfn2, but stem cell-derived progenitor cells were devoid of Pfn2, but it is re-expressed upon their differentiation into post-mitotic neurons. The P2-GFP fusion protein is expressed in the knock-in mouse model at reduced levels (app. half) compared to the wt in heterozygous animals. P2-GFP also showed a minor impairment in poly-Lproline binding efficiency (app. 75% of Pfn2), but no changes in G-actin binding efficiency. Nevertheless, P2-GFP was able to bind to nearly all ligands that Pfn2 could interact with including a novel interaction partner, cortactin. The P2-GFP KI mouse model is therefore validated to study Pfn2 trafficking and function in vivo. Finally, comparative analysis of wt and Pfn2 KO primary hippocampal neuron cultures as a model system revealed that Pfn2 also has a role during neuronal development. Pfn2 KO neurons showed an accelerated neuronal development, reaching each developmental stage faster than wt cells, possibly due to altered actin dynamics. The P2-GFP KI model was then employed to produce mature hippocampal neuron cultures and the trafficking of Pfn2 following different stimulation protocols was studied. Upon application of various stimulants, Pfn2 mostly re-localized into post-synaptic terminals, as previously described, but also into pre-synaptic terminals and, to a lesser extent, into axons. The view of Pfn2 and its function possibly involved in actin-dependent mechanisms during neuronal development as well as during induced synaptic activity requires further studies, but a complex role of Pfn2 in these processes has been discovered with the help of the P2-GFP mouse model as a valuable tool

Differential effects of hypergravity on immune dysfunctions induced by simulated microgravity

Microgravity (μg) is among the major stressors in space causing immune cell dysregulations. These are frequently expressed as increased pro-inflammatory states of monocytes and reduced activation capacities in T cells. Hypergravity (as artificial gravity) has shown to have beneficial effects on the musculoskeletal and cardiovascular system both as a countermeasure option for μg-related deconditioning and as gravitational therapy on Earth. Since the impact of hypergravity on immune cells is sparsely explored, we investigated if an application of mild mechanical loading of 2.8 g is able to avoid or treat μg-mediated immune dysregulations. For this, T cell and monocyte activation states and cytokine pattern were first analyzed after whole blood antigen incubation in simulated μg (s-μg) by using the principle of fast clinorotation or in hypergravity. Subsequent hypergravity countermeasure approaches were run at three different sequences: one preconditioning setting, where 2.8 g was applied before s-μg exposure and two therapeutic approaches in which 2.8 g was set either intermediately or at the end of s-μg. In single g-grade exposure experiments, monocyte pro-inflammatory state was enhanced in s-μg and reduced in hypergravity, whereas T cells displayed reduced activation when antigen incubation was performed in s-μg. Hypergravity application in all three sequences did not alleviate the increased pro-inflammatory potential of monocytes. However, in T cells the preconditioning approach restored antigen-induced CD69 expression and IFNy secretion to 1 g control values and beyond. This in vitro study demonstrates a proof of concept that mild hypergravity is a gravitational preconditioning option to avoid adaptive immune cell dysfunctions induced by (s-)μg and that it may act as a booster of immune cell function

Hypergravity attenuates Reactivity in Primary Murine Astrocytes

Neuronal activity is the key modulator of nearly every aspect of behavior, affecting cognition, learning and memory as well as motion. Alterations or even disruptions of the transmission of synaptic signals are the main cause of many neurological disorders. Lesions to nervous tissues are associated with phenotypic changes mediated by astrocytes becoming reactive. Reactive astrocytes form the basis of astrogliosis and glial scar formation. Astrocyte reactivity is often targeted to inhibit axon dystrophy and thus promote neuronal regeneration. Here, we use increased gravitational (mechanical) loading induced by hypergravity to identify a potential method to modify key features of astrocyte reactivity. We exposed primary murine astrocytes as a model system closely resembling the reactivity phenotype in vivo on custom-built centrifuges for cultivation as well as for livecell imaging under hypergravity conditions in a physiological range (2g and 10g). This resulted in significant changes to astrocyte morphology, behavior and reactivity phenotypes, with the ultimate goal being to enhance neuronal regeneration for novel therapeutic approaches

Streamlining Culture Conditions for the Neuroblastoma Cell Line SH-SY5Y: A Prerequisite for Functional Studies

The neuroblastoma cell line SH-SY5Y has been a well-established and very popular in vitro model in neuroscience for decades, especially focusing on neurodevelopmental disorders, such as Parkinson’s disease. The ability of this cell type to differentiate compared with other models in neurobiology makes it one of the few suitable models without having to rely on a primary culture of neuronal cells. Over the years, various, partly contradictory, methods of cultivation have been reported. This study is intended to provide a comprehensive guide to the in vitro cultivation of undifferentiated SH-SY5Y cells. For this purpose, the morphology of the cell line and the differentiation of the individual subtypes are described, and instructions for cell culture practice and long-term cryoconservation are provided. We describe the key growth characteristics of this cell line, including proliferation and confluency data, optimal initial seeding cell numbers, and a comparison of different culture media and cell viability during cultivation. Furthermore, applying an optimized protocol in a long-term cultivation over 60 days, we show that cumulative population doubling (CPD) is constant over time and does not decrease with incremental passage, enabling stable cultivation, for example, for recurrent differentiation to achieve the highest possible reproducibility in subsequent analyses. Therefore, we provide a solid guidance for future research that employs the neuroblastoma cell line SH-SY5

Hypergravity attenuates Reactivity in Primary Murine Astrocytes

Neuronal activity is the key modulator of nearly every aspect of behavior, affecting cognition, learning, and memory as well as motion. Hence, disturbances of the transmission of synaptic signals are the main cause of many neurological disorders. Lesions to nervous tissues are associated with phenotypic changes mediated by astrocytes becoming reactive. Reactive astrocytes form the basis of astrogliosis and glial scar formation. Astrocyte reactivity is often targeted to inhibit axon dystrophy and thus promote neuronal regeneration. Here, we aim to understand the impact of gravitational loading induced by hypergravity to potentially modify key features of astrocyte reactivity. We exposed primary murine astrocytes as a model system closely resembling the in vivo reactivity phenotype on custom-built centrifuges for cultivation as well as for live-cell imaging under hypergravity conditions in a physiological range (2g and 10g). We revealed spreading rates, migration velocities, and stellation to be diminished under 2g hypergravity. In contrast, proliferation and apoptosis rates were not affected. In particular, hypergravity attenuated reactivity induction. We observed cytoskeletal remodeling of actin filaments and microtubules under hypergravity. Hence, the reorganization of these key elements of cell structure demonstrates that fundamental mechanisms on shape and mobility of astrocytes are affected due to altered gravity conditions. In future experiments, potential target molecules for pharmacological interventions that attenuate astrocytic reactivity will be investigated. The ultimate goal is to enhance neuronal regeneration for novel therapeutic approache

Microelectrode Array Electrophysiological Recording of Neuronal Network Activity during a Short-Term Microgravity Phase

During spaceflight, humans are subjected to a variety of environmental factors which deviate from Earth conditions. Especially the lack of gravity poses a big challenge to the human body and has been identified as a major trigger of many detrimental effects observed in returning astronauts but also in participants of spaceflight-analog studies. Structural alterations within the brain as well as declines in cognitive performance have been reported, which has brought the topic of brain health under microgravity into the focus of space research. However, the physiological mechanisms underlying these observations remain elusive. Every aspect of human cognition, behavior and psychomotor function is processed by the brain based on electro-chemical signals of billions of neurons, which relay information via neuronal networks throughout the body. Alterations in neuronal activity are the main cause of a variety of mental disorders and changed neuronal transmission may also lead to diminished human performance in space. Thus, understanding the functioning of these fundamental processes under the influence of altered gravity conditions on a cellular level is of high importance for any manned space mission. Previous electrophysiological experiments using patch clamp have shown that propagation velocity of action potentials (APs) is dependent on gravity. With this project, we aim to advance the electrophysiological approach from a single-cell level to a complex network level by employing Microelectrode array (MEA) technology. MEAs feature the advantage of real-time electrophysiological recording of a complex and mature neuronal network in vitro, without the need for invasive patch clamp insertion into cells. Using a custom-built pressure chamber, we were able to integrate and conduct our experiment on the ZARM Drop Tower platform, exposing the entire system to 4.7 s of high-quality microgravity (10-6 to 10-5 x g0). With this setup we were able to evaluate the functional activity patterns of iPSC-derived neuronal networks subjected to microgravity, while keeping them under controlled and stable temperature and pressure conditions. Activity data was acquired constantly - immediately before the drop, during the free-fall (microgravity) phase and during a subsequent post-drop recording phase. For neuronal activity analysis the action potential frequency in each experiment phase was calculated for the single electrodes. We found that during the 4.7 s lasting microgravity phase the mean action potential frequency across the neuronal networks was significantly elevated. Additionally, electrical activity readapted back to baseline level within 10 minutes of post-drop recordings. Our preliminary data shows that real-time, electrophysiological recording of neuronal network activity based on MEA technology is possible under altered gravity conditions and that differences in activity can be detected already in very short time frames in the second range. Furthermore, the observation that microgravity has an effect on the electrophysiological activity of neuronal networks is in line with previously published findings in single neurons and poses further questions with regards to astronaut brain health on manned space missions. The MEA payload system was approved for autonomous recording of redundant cellular electrophysiological data in the Drop Tower. It will be applied on other microgravity platforms such as sounding rockets and parabolic flights and thus increased experimental time. Apart from neurons, various other electrically active cellular systems such as myocytes or myotubes could be examined using this hardware

UNRAVELING ASTROCYTE BEHAVIOUR IN THE SPACE BRAIN: RADIATION RESPONSE OF PRIMARY ASTROCYTES

Exposure to ionizing radiation as part of space radiation, is a major limiting factor for crewed space exploration. Astronauts will encounter different types of space radiation, which may cause cognitive damage causing detrimental effects on learning and attention, elevated anxiety and depression. Due to its limited regenerative potential, especially the central nervous system (CNS) is very vulnerable towards radiation-induced damage. Astrocytes, the most abundant glial cells of the CNS, have different crucial functions in the CNS, e.g. maintaining normal brain function. In this work, the response of astrocytes towards low linear energy transfer (LET) X-rays and high-LET carbon ions was compared to unravel possible specific effects of space-relevant high-LET radiation. [...

In Prostate Cancer Cells Cytokines are early Responders to Gravitational Changes occurring in Parabolic Flights

The high mortality in men with metastatic prostate cancer (PC) establishes the need for diagnostic optimization by new biomarkers. Mindful of the effect of real microgravity on metabolic pathways of carcinogenesis, we attended a parabolic flight (PF) mission to perform an experiment with the PC cell line PC-3, and submitted the resulting RNA to next generation sequencing (NGS) and quantitative real-time PCR (qPCR). After the first parabola, alterations of the F-actin cytoskeleton-like stress fibers and pseudopodia are visible. Moreover, numerous significant transcriptional changes are evident. We were able to identify a network of relevant PC cytokines and chemokines showing differential expression due to gravitational changes, particularly during the early flight phases. Together with differentially expressed regulatory lncRNAs and micro RNAs, we present a portfolio of 298 potential biomarkers. Via qPCR we identified IL6 and PIK3CB to be sensitive to vibration effects and hypergravity, respectively. Per NGS we detected five upregulated cytokines (CCL2, CXCL1, IL6, CXCL2, CCL20), one zink finger protein (TNFAIP3) and one glycoprotein (ICAM1) related to c-REL signaling and thus relevant for carcinogenesis as well as inflammatory aspects. We found regulated miR-221 and the co-localized lncRNA MIR222HG induced by PF maneuvers. miR-221 is related to the PC-3 growth rate and MIR222HG is a known risk factor for glioma susceptibility. These findings in real microgravity may further improve our understanding of PC and contribute to the development of new diagnostic tools