Open Access

Olga Tschesnokowa

• Subject of this thesis was the investigation of the actin-interacting and glucocorticoid-sensitive Protein DRR1 (or Fam107a) and its role in promoting stress resilience in the murine hippocampus. We proposed the hypothesis that DRR1 through its actin-binding properties specifically modulates neuronal actin dynamics and promotes resilience through synaptic plasticity leading to subsequently improvement of cognitive performance and social behavior. The accompanied AMPA-receptor transport could create an efficient way regulating neural function and complex behavior during stress episodes. By utilizing fluorescent immunohistochemistry, we showed basal expression of DRR1 primarily in the murine cerebellum and hippocampal CA3 and CA1 area. Co-staining with different cell marker proteins showed DRR1 expression in neurons, microglia and especially in astrocytic end-feet, which create contact to the brain vasculature. To test whether DRR1 and AMPA receptor function correlate to modulate stress-associated consequences, primary hippocampal neuron cultures were transduced with adeno-associated virus (AAV) for overexpression or suppression of the protein. Western Blot analysis showed a positive correlation between the AMPA-receptor subunit GluR2 and DRR1 amounts. Further the application of the proximity ligation assay (PLA) in untreated neural cultures indicated interaction between DRR1 and the AMPA receptor subunit GluR2. To address whether DRR1 even affects AMPAR trafficking we performed the “newly inserted assay” after AAV-treatment of primary hippocampal neuron cultures. Suppression of DRR1 revealed less newly inserted GluR2 subunits as compared to controls. Inconclusive were the results upon DRR1 overexpression, however they point to no changes. In the second part we correlated behavioral phenotypes originating from in vivo overexpression and suppression of DRR1 in the murine hippocampus with potential alterations in neuronal morphology. Therefore, in vitro analysis was performed utilizing AAV transduced primary hippocampal cultures overexpressing or suppressing DRR1. Synchronously the viral vector included a green fluorescent protein (GFP) being expressed throughout the complete neural cell. GFP staining was used to verify successful transfection and for reconstruction of dendritic arbors and dendritic stretches for spine classification. DRR1 suppression showed reduced total spine numbers especially evoked by reduced numbers of immature spine classes – namely long thin spines and filopodia. Whereas mature mushroom spines and stubby spines were unaffected. By overexpressing DRR1, tendencies inclined against higher total dendritic lengths, branch points and increased dendritic arbors in comparison to controls. In regard of spines, total numbers were unaffected. However, mature mushroom spines were significantly declined in numbers, but compensated by increased numbers of immature long thin spines and filopodia. Chronic social defeat stress (CSDS) is widely used in mouse models to study the effects of stress and resilience. We exposed C57Bl/6J mice expressing GFP under the Thy1 promoter CSDS and categorized them into resilient (R+/-), susceptible (R-/-) and non-learning (R+/+) mice following a modified social interaction test (MSIT). We found alterations in CA1 spine compositions with resilient animals resembling the untreated phenotype. Stress susceptible and non-learning animals displayed reduced numbers in stubby spines with simultaneous increases in mature mushroom spines. In addition, we could detect a tendency towards more immature spines in susceptible animals and non-learners, mirroring our in vitro results. Finally, we present a different investigative approach in this thesis. Sequenced acute stress was previously found to compromise cognition including spine loss. We aimed to investigate the implication of acute stress on DRR1 levels and its occurrence in diverse cell types of the brain. We subjected one group of C57Bl/6J mice to acute stress and injected another group with the artificial glucocorticoid DEX. Six hours post stress, animals were perfused and brains were subsequently immunobiologically analyzed. We found DRR1 protein levels elevated in the hippocampus of stressed and DEX-treated animals compared to controls. Interestingly, DRR1 seemed was especially elevated in endothelial cells. This coincides with our investigations finding DRR1 present in astrocytic end-feet under basal conditions and might claim a participation of DRR1 in the blood-brain-barrier integrity. Our results show DRR1 as actin-interacting and glucocorticoid-sensitive gene affecting structural plasticity of hippocampal spines. Moreover, DRR1 directly interacts with AMPA glutamate receptors and presumably is involved in AMPA trafficking to the postsynaptic membrane. In addition, this study could demonstrate that DRR1 is expressed by other cell types of the brain. Of special interest is DRR1’s occurrence in astrocytic end-feet and endothelial cells suggesting a role as integrator of cell-cell communication and to this end also acting as modifier of stress-induced consequences at the neurovascular unit. In vivo data of chronically stressed mice displayed no phenotypic differences in hippocampal pyramidal neurons of resilient animals as compared to unstressed mice. Morphological alterations of spine structures were particularly visible in stress susceptible and non-learning animals. Integrating our findings with existing behavioral data, we can conclude that DRR1 plays a role in stress resilience whereby it needs to be expressed in a tightly managed homeostatic equilibrium.
• Vielzählige epidemiologische Langzeitstudien weisen darauf hin, dass Stress und dessen neurobiologische Korrelate bei der Entstehung und dem Verlauf von psychiatrischen Erkrankungen mitwirken. Insbesondere soziale Belastungsfaktoren in frühkindlichen Entwicklungsphasen begünstigen die Entstehung stressbedingter psychischer Störungen, wie beispielsweise Depression. Auch Umwelt-Traumata und variierende Lebensumstände in adulten Jahren erhöhen den Krankheitsdruck. Bei vielen Patienten evoziert Stress Krankheitsperioden aufgrund von genetischer Prädisposition, der sogenannten individuellen Vulnerabilität. Allerdings ist das Wissen über die individuelle Stressanfälligkeit noch unvollständig geklärt. Vor allem neurobiologische Mechanismen, die stressvolle Ereignisse in stressbedingte psychiatrische Mechanismen manifestieren, sind unzureichend erforscht. Dies führt dazu, dass entsprechende Erkrankungen erst spät erkannt und therapiert werden. Akuter Stress versetzt den Organismus in Alarmbereitschaft („Kampf-oder-Flucht-Reaktion“), indem das autonome Nervensystem aktiviert wird, um die Stresssituation zu bewältigen. Die dabei freigesetzten Neurotransmitter und Hormone lösen (über-) lebensnotwendige Anpassungen in Organen aus. Dadurch werden Energiereserven über die Gluconeogenese speziell zu wichtigen körperlichen Instanzen wie Muskeln oder dem Gehirn mobilisiert. Gleichzeitig setzt sich die zelluläre und humorale Immunabwehr herab. Im zentralen Nervensystem aktiviert Stress die Hypothalamus-Hypophysen-Nebennierenrinden-Achse (HPAAchse), wodurch die Ausschüttung des „Stresshormons“ Cortisol reguliert wird. Cortisol wiederum besitzt einen Feedback-Mechanismus zum zentralen Nervensystem, um die Stressreaktion nach der akuten Belastungssituation zu stoppen und den Grundzustand wieder herzustellen. Mineral- und Glucocorticoid-Rezeptoren stellen bei der Rückkopplung der HPAAchse, die wichtigsten Regulationselemente dar. Hält Stress über einen längeren Zeitraum an, wird von chronischem Stress gesprochen. Hierbei bleibt eine vollständige Rückregulation der HPA-Achse aus, was zu maladaptiven Anpassungen führt. Neben Belastungen des Herz-Kreislaufsystems, wird der Stoffwechsel gestört und die Immunabwehr beeinträchtigt. Daneben befördern Lern- und Gedächtnisstörungen neuropsychiatrische Erkrankungen. Die Erforschung von Resilienz-Mechanismen, stellt einen neuen wissenschaftlichen Ansatz dar, um präventiv gegen die Entstehung von Depressionen vorzugehen. In diesem Kontext, wird Resilienz als schnelle Erholung oder Aufrechterhaltung mentaler Gesundheit nach Perioden schwerer psychologischer oder körperlicher Belastung definiert. Interessanterweise weisen verschiedene Individuen unterschiedliche Stressresilienz auf. Heutzutage werden Tiermodelle verwendet, um die zugrundeliegenden neurobiologischen Mechanismen von Stressresilienz genauer zu erforschen. Chronische soziale Ablehnung fungiert dabei als Tiermodell erster Wahl, um Stress resiliente und Stress suszeptible Mäuse zu erzeugen. Unzählige Studien an Nagern zeigten, dass soziales Verhalten mit Stress stark verknüpft ist und die Freisetzung von Glucocorticoiden die Glutamat-Neurotransmission im Präfrontalen Kortex und im Hippocampus beeinflusst, wodurch die kognitive Leistung beeinträchtigt wird. Die dabei einhergehenden Veränderungen in der neuronalen Erregbarkeit stellen einen Kernbefund stressassoziierter mentaler Störungen dar.