The role of the actin-binding proteins cofilin1 and INF2 on mitochondrial dynamics and cellular resilience

Neurological diseases, such as stroke, Alzheimer’s disease and related dementias are among the most prevalent disorders leading to disability and death worldwide. Many cell death pathways, including apoptosis, necrosis and necroptosis, oxytosis or ferroptosis, relevant for these pathologies, converge at the level of neuronal demise through oxidative stress. In the last decades, many efforts were accomplished to identify underlying pathophysiological mechanisms leading to neuronal demise and subsequent deficits in brain function. However, it still remains obscure which molecular mechanisms contribute to these pathologies and how they are interconnected. The role of mitochondria and respective dynamics dependent on the actin-binding proteins cofilin1 and INF2 contributing to mitochondrial regulation and to neuronal demise are illuminated in the present work. In particular, cofilin1 loss-of-function studies in MEF cells, demonstrated that absence of this actin-binding protein indirectly contributes to mitochondrial fission via DRP1 activation. Mitochondrial dynamics is especially substantial for the generation as well as delivery of ATP to cellular areas with high energy demand; and fission events are frequently associated with impaired mitochondrial function as a prerequisite for cell death. In the case of cofilin1 knockdown, however, mitochondrial fragmentation, was not associated with any mitochondrial impairment, substantiated by an identical bioenergetic profile of cofilin1-/- cells and control cells, unaltered ATP levels and a preserved mitochondrial integrity as assessed by TMRE measurements. Additionally, cofilin1 knockout was linked to increased basal mitochondrial Ca2+ level through elevated MCU expression, putatively contributing to mitochondrial fission. The role of cofilin1 in cell death paradigms induced by erastin or glutamate was negligible in MEF cells, and deletion of the protein had no relevant effect on cellular resilience. In neuronal cells, however, cofilin1 was identified, for the first time, as a redox sensor in oxidative stress-induced cell death pathways, namely oxytosis and ferroptosis, thereby linking detrimental cellular ROS accumulation to mitochondrial demise through this actin-regulating protein. In particular, cofilin1 deletion in neuronal HT22 cells exerted substantial beneficial effects on mitochondrial resilience, assessed by quantification of mitochondrial ROS production, mitochondrial membrane potential or bioluminescent-based measurement of ATP levels. Intriguingly, HT22 cells deficient for cofilin1 exhibited a profound glycolytic shift as a response to erastin or glutamate toxicity to meet their energy demand, whereas control cells were metabolically inactive. Surprisingly, interfering with another actin-binding protein, namely INF2, exerted similar effects on cellular resistance of neuronal HT22 cells comparable to cofilin1 knockdown. Accordingly, mitochondrial parameters were significantly preserved after oxytosis and ferroptosis induction resulting in enhanced cellular survival. Recent findings from this study and by others, suggesting that actin dynamics is directly linked to the regulation of mitochondrial fusion and fission, guided this project towards uncovering the potential of INF2 to impact mitochondrial morphology. This study unraveled an indirect role for INF2 on the regulation of mitochondrial fission by affecting actin dynamics and DRP1 activity in neuronal cells. Notably, cofilin1 was confirmed being as a key player under pathophysiological conditions induced by glutamate in primary cortical neurons, as cofilin1 deficient cells were substantially protected against the induced excitotoxicity. Mitochondrial respiration was significantly preserved in cofilin1-/- primary neurons under excitotoxic conditions, thereby maintaining cellular survival. Additionally, decrease of cofilin-actin rod formation in cofilin1-/- deficient neurons might also contribute to the observed protective effects. The present data on isolated mitochondria treated with the recombinant cofilin1 protein provide a further link to toxicity-related mitochondrial impairment by cofilin1 itself. Direct effects of cofilin1 were demonstrated by assessing the mitochondrial membrane potential, mitochondrial ROS accumulation and mitochondrial respiration. Interestingly, the detrimental impact of cofilin1 on mitochondria is dependent on oxidation of crucial cysteine residues at position 139 and 147, as mutations of these cysteine residues to serine abolished the noxious character of cofilin1. Overall, the present findings reveal, for the first time, that the actin-binding proteins cofilin1 and INF2 play a crucial role in paradigms of oxidative stress and that inhibition of these proteins results in protective effects in neuronal cells that were particularly attributed to the preserved mitochondrial integrity and function. Thus, interfering with the pathological activation of actin-binding proteins, such as cofilin1 or INF2 may offer an effective therapeutic strategy in neurodegenerative diseases

Studies on the role of protein secretion systems in Pseudomonas fluorescens biofilm formation, and development of a tunable gene expression system for such analyses

Plant growth-promoting rhizobacteria (PGPRs), such as Pseudomonas fluorescens, support plants via different mechanisms such as biocontrol (suppressing pathogenic organisms), phytostimulation (modulation of plant-hormone levels) or biofertilization (by improving nutrient supply) and thus are important for growth yields. Therefore, it is important to understand interactions of the bacteria and plant roots. Many PGPRs are able to form a biofilm on plant roots, so they live in an adherent cell community on the root surface. Different bacterial surface structures (e.g. pili or fimbriae) and secreted proteins (e.g. adhesins or enzymes) are involved in biofilm formation or plant growth promotion. Therefore, the first part of this study investigated the influence of selected secreted proteins or protein secretion systems on biofilm formation of P. fluorescens. Genes encoding translocated substrates or secretion system components were deleted to abolish their function. Two genes encoding type 1 secretion system (T1SS) components (lapA, encoding an adhesin and aprE, encoding a membrane fusion protein) were identified, whose deletion lead to weaker biofilm formation. Further analyses of the Apr T1SS revealed that its substrate AprA (an extracellular protease) was not directly involved in biofilm formation. The deletion of aprE most likely resulted in mistargeting AprA into the periplasm, which could influence biofilm formation. In the second part of the study, a new tightly controlled sugar-independent expression system was established for P. fluorescens A506. The gene PflA506_4486 was predicted to encode a putative anthranilic acid inducible regulator for the expression of an adjacent antABC operon for anthranilic acid degradation to catechol. PflA506_4486 and the putative promoter region for antABC expression were cloned into a shuttle vector system for Escherichia coli and Pseudomonas. gfp was put under control of this anthranilate-inducible promoter (PantA) system as reporter for the examination of promoter activity. In this study it was shown that the anthranilate-inducible promoter system is tightly controlled by inducer concentration and can be tuned to expression levels suitable for physiological analyses. Compared with a rhamnose-inducible promoter system, the PantA-system was advantageous in P. fluorescens A506. Moreover, this new system is working in P. fluorescens as well as in Pseudomonas putida and Pseudomonas aeruginosa, which are the most intensively studies pseudomonads

Ansätze für eine muttergebundene Aufzucht von Milchschaflämmern in der Praxis

Ziel der Studie war die Erhebung von Erfahrungen aus der Praxis mit muttergebundener Aufzucht bei Milchschafen. Es wurden 11 Leitfadengestützte Experteninterviews geführt. Die Betriebe unterschieden sich in ihren Aufzuchtmethoden vor allem durch Beginn des Melkens (Spanne 1. - 56. Tag nach der Geburt), Anzahl Melkungen am Tag (1 oder 2), Dauer des Kontakts zwischen Mutterschafen und Lämmern (ganz- oder halbtags), sowie der Dauer der Säugeperiode (45 - 183 Tage), bei z.T. sehr unterschiedlichen Kombinationen. Die Betriebsleiter waren trotz der reduzierten Milchmenge zufrieden mit ihren Aufzuchtmethoden

Der Einfluss der akustischen Stimulation des Slow-Wave-Sleeps auf die Konsolidierung prozeduraler Gedächtnisinhalte bei gesunden Kindern und dazu im Vergleich bei Kindern mit einer Aufmerksamkeitsdefizit-/Hyperaktivitäts-Störung (ADHS)

Schlaf fördert die Konsolidierung hippokampus-abhängiger deklarativer Gedächtnisinhalte bei Erwachsenen und in besonderem Maße bei Kindern. Für den entwicklungsbedingten Charakter der schlafassoziierten Gedächtniskonsolidierung wird der bei Kindern reifungsbedingt erhöhte Slow Oscillation-Aktivität (SO; <1Hz) im Slow-Wave-Sleep (SWS) verantwortlich gemacht. Diese nierderfrequente, hochamplitudige EEG-Aktivität steht ursächlich mit der Festigung hippokampus-abhängiger Gedächtnisinhalte im Schlaf in Verbindung. Im Gegensatz zu Erwachsenen profitieren gesunde Kinder jedoch nicht bei der Konsolidierung prozeduraler Gedächtnisinhalte. Prozedurale Gedächtnisinhalte besitzen neben rein motorischen Komponenten auch hippokampus-abhängige deklarative Anteile. Beide Aspekte können während der Konsolidierung jedoch hemmend miteinander interagieren. Somit wird vermutet, dass bei gesunden Kindern aufgrund des vermehrten SWS bevorzugt hippokampus-abhängig Aspekte prozeduraler Gedächtnisinhalte zulasten rein motorischer Anteile konsolidiert werden, welches sich in Form einer reduzierten schlafassoziierten Gedächtniskonsolidierung äußert. Kinder mit einer ADHS zeigen im Vergleich zu Gesunden Defizite in der schlafassoziierten Konsolidierung deklarativer Gedächtnisinhalte, jedoch Vorteile bei prozeduralen Gedächtnisleistungen. Als Ursache werden hierfür dysfunktionale SO während des SWS vermutet. Mit Hilfe der transkraniellen Gleichstromstimulation (tDCS) wurde bereits gezeigt, dass eine Induktion von SO bei Kindern mit einer ADHS deklarative Gedächtnisleistungen gesteigert und gleichzeitig prozedurale Gedächtnisleistungen reduziert. Da die tDCS bislang nicht an gesunden Kindern durchgeführt werden konnte, soll nun mittels akustischer Stimulation die SO-Aktivität bei Kindern mit und ohne ADHS während des SWS erhöht werden. Es wird dabei erwartet, dass sich infolge der akustischen Stimulation bei Kindern mit einer ADHS die zuvor beobachteten Vorteile bei der Konsolidierung prozeduraler Gedächtnisinhalte reduzieren, bzw. gesunde Kindern deutlich schlechtere Konsolidierungsleistungen im Vergleich zu einer nicht-Stimulationsbedingung aufweisen. Um diese Fragen beantworten zu können, wird eine doppelt verblindete Studie durchgeführt, an der sowohl gesunde Kinder als Kontrollen als auch Kinder mit einer ADHS als Patienten teilnehmen. In beiden Gruppen sollen jeweils 16 Kinder im Alter von 9-12 Jahren untersucht werden. Alle Kinder verbringen drei Nächte im Schlaflabor. Bei allen Nächten wird ein Polysomnogramm abgeleitet. In allen drei Nächten werden zusätzlich mithilfe eines weiteren EEG-Systems SO in Echtzeit detektiert. Die erste Nacht im Schlaflabor soll als Eingewöhnungsnacht dienen, Nacht 2 und Nacht 3 sind Experimentalnächte. In einer der beiden Experimentalnächte werden die detektierten SO mithilfe akustischer Signale (50ms, 55 dB rosa Rauschen) für 3,5h verstärkt. In der anderen Experimentalnacht werden SO zwar detektiert, jedoch nicht verstärkt. Am Abend der beiden Experimentalnächte erlernen die Kinder eine prozedurale Aufgabe in Form einer Serial-Reaction-Time-Task. Am Morgen darauf erfolgt der Abruf

A tunable anthranilate-inducible gene expression system for Pseudomonas species

Pseudomonads are among the most common bacteria in soils, limnic ecosystems, and human, animal, or plant host environments, including intensively studied species such as Pseudomonas aeruginosa, P. putida, or P. fluorescens. Various gene expression systems are established for some species, but there is still a need for a simple system that is suitable for a wide range of pseudomonads and that can be used for physiological applications, i.e., with a tuning capacity at lower expression levels. Here, we report the establishment of the anthranilate-dependent PantA promoter for tunable gene expression in pseudomonads. During studies on P. fluorescens, we constructed an anthranilate-inducible AntR/PantA-based expression system, named pUCP20-ANT, and used GFP as reporter to analyze gene expression. This system was compared with the rhamnose-inducible RhaSR/PrhaB-based expression system in an otherwise identical vector background. While the rhamnose-inducible system did not respond to lower inducer concentrations and always reached high levels over time when induced, expression levels of the pUCP20-ANT system could be adjusted to a range of distinct lower or higher levels by variation of anthranilate concentrations in the medium. Importantly, the anthranilate-inducible expression system worked also in strains of P. aeruginosa and P. putida and therefore will be most likely useful for physiological and biotechnological purposes in a wide range of pseudomonads

The relation between memory and decision-making in Multiple Sclerosis patients

Background. Impairments in long-term and working memory are widespread in Multiple Sclerosis (MS), setting on in early disease stages. These memory impairments may limit patients’ ability to take informed and competent medical decisions, too. In healthy populations, memory abilities predict decision quality across a wide range of tasks. These studies suggest that higher working memory capacity supports decisions in cognitively taxing tasks, whereas better semantic memory facilitates decisions in tasks requiring knowledge retrieval. In individuals with MS, previous studies have linked less accurate decisions to memory deficits and reduced executive functioning, too. However, these studies focussed on decisions under risk and did not broadly assess decision making skills. We aimed to fill this gap in a cross-sectional study. Methods. Hundred thirty-seven participants with MS were recruited during their stay in an MS specialized rehabilitation centre. In a first test session, participants completed a standardized test battery for working memory and semantic memory, the inventory for memory diagnostics. In a second test session, participants filled out the Adult Decision Making Competence battery (A-DMC). This version of the A-DMC measured decision making competence on five subscales: Resistance to Framing Effects, Under/Overconfidence, Applying Decision Rules, Consistency in Risk Perception, and Resistance to Sunk Cost Effects. In addition, participants were screened for depression and cognitive fatigue. Results. Working memory was impaired in most participants, whereas semantic memory was not impaired. To understand which memory abilities underlie distinct components of decision making in people with MS, we used structural equation modelling. Replicating previous findings in a healthy sample, working memory capacity was associated with the ability to recall semantic knowledge. Participants with lower working memory capacity were less resistant to framing effects and adhered to decision rules less. In contrast, participants with worse semantic memory assessed their own knowledge less accurately, perceived risks less consistently, and made more errors in applying decision rules. Cognitive fatigue and depression unlikely explain these relationships. Conclusions. Taken together, our study suggests that the memory problems, frequently reported in MS patients, may reach out to higher-order cognitive functions, such as decision making skills. Supporting shared decision-making and patient autonomy within MS thus requires to take memory impairments into account and to match the information provided to the patient’s memory abilities

Lebenswelten gehörloser MigrantInnen

In Hinblick auf die Situation von Gehörlosen mit Migrationshintergrund stellte sich in der vorliegenden Arbeit die Frage, welche Bedeutung die gehörlose Identität und das Zugehörigkeitsgefühl zur Gehörlosengemeinschaft und der Kontakt zu anderen Gehörlosen einnimmt und wie sich das Leben in mehreren Welten – der „gehörlosen Welt“ und der „hörenden Welt“ sowohl im Herkunfts- als auch im Aufnahmeland und über nationale Grenzen hinweg – gestaltet bzw. wie gehörlose MigrantInnen diese verschiedenen Lebenswelten gestalten. Dabei liegt das Hauptaugenmerk auf den individuellen Erfahrungen und Sichtweisen der InterviewpartnerInnen, die mittels lebensgeschichtlicher Interviews erfasst wurden. Aufbauend auf deren Analyse werden die verschiedenen Aspekte der hörenden und der gehörlosen Lebenswelten dargestellt und im Sinne der Grounded Theory mit entsprechender Literatur in Zusammenhang gebracht. Als weitere methodische Herangehensweise diente die der Kultur- und Sozialanthropologie zugrunde liegende Methode der teilnehmenden Beobachtung, durch die sich der Zugang – neben dem Erlernen der Österreichischen Gebärdensprache – zum Feld erst eröffnete und den gesamten Forschungsprozess hindurch begleitete. Als theorieorientierte Einleitung wird auf migrationsgeschichtliche Aspekte, auf Gehörlosigkeit und auf die Bedeutung von Sprache und Kultur für die Identitätskonstruktionen gehörloser MigrantInnen im Sinne hybrider Identitäten und Kulturen eingegangen. Der Darstellung vergangener und teils immer noch bestehender Vorstellungen und Vorurteile Hörender gegenüber Gehörlosen folgt ein Einblick in den Gehörlosendiskurs, der aufzeigt, dass es innerhalb der Gehörlosengemeinschaften zu einem wieder erstarkenden Bewusstsein der eigenen kulturellen Besonderheiten und der damit verbundenen Stärkung der gehörlosen Identität kommt. Damit verbunden werden Fragen der Eigen- und Fremdbezeichnung Gehörloser und die Zugehörigkeit zur Gemeinschaft thematisiert. Wesentlich ist es, aufzuzeigen, dass es sich um aktive AkteurInnen im Migrationsprozess und im Prozess der Vergemeinschaftung handelt. Gehörlose (MigrantInnen) sollen nicht als defizitär und als von der hörenden Mehrheitsgesellschaft unterdrückte Opfer dargestellt werden; vielmehr sollen ihre Strategien und Handlungsspielräume aufgezeigt werden. Aus den Erzählungen der InterviewpartnerInnen geht hervor, dass ein Leben als gehörlose/r MigrantIn in einer hörenden Mehrheitsgesellschaft im Aufnahmeland neben den Kommunikationsschwierigkeiten eine immerwährende Konfrontation mit einer ihnen gegenüber distanzierten, unterdrückenden und oft auch fremdenfeindlich gestimmten Umwelt mit sich bringt. Die Interviewantworten zeigen, dass dies auf der einen Seite zu einer verstärkten Distanzierung gegenüber der hörenden Welt und zu einem Rückzug in die gehörlose Welt führen kann. Auf der anderen Seite kann das Eingebundensein in die Gehörlosengemeinschaft, in der es möglich ist, die Kultur und Sprache frei zu leben und die somit das Selbstbewusstsein der gehörlosen Identität fördert, dazu führen, dass es zu einem verstärkten Interesse am Kontakt zu Hörenden beiträgt, um eben diese Vorurteile zu beseitigen und für ein positives Bild Gehörloser in der Gesellschaft zu sorgen

Beyond society's desire for a slowed‐down temporal experience: Toward a nomological network of individuals' need‐for deceleration

This study expands on past deceleration and slow consumption research by introducing and validating a measure of need-for-deceleration, an individual's motivational ability to engage in activities aimed at slowing down the perceived fast passage of time. Following initial scale development, two studies establish construct validity by placing need-for-deceleration into a nomological network. Results indicate that the measure correlated with, but was distinct from, variables involving negative affective states, such as state anxiety and neuroticism. Need-for-deceleration scores were not related to materialism, but negatively correlated with self-efficacy, life satisfaction, work-life balance, and conscientiousness. Correlations were positive with need-for-uniqueness, future time orientation, and susceptibility to normative influence. Need-for-deceleration was also associated with regulatory focus (positively with prevention, and negatively with promotion focus). To explore criterion validity, a third study establishes associations between need-for-deceleration and consumer lifestyle variables. Developing and validating the scale can help researching and managing products relating to the consumption of time, wellness, mindfulness, and simplicity

Gap Junction Dependent Cell Communication Is Modulated During Transdifferentiation of Mesenchymal Stem/Stromal Cells Towards Neuron-Like Cells

In vitro transdifferentiation of patient-derived mesenchymal stem/stromal cells (MSCs) into neurons is of special interest for treatment of neurodegenerative diseases. Although there are encouraging studies, little is known about physiological modulations during this transdifferentiation process. Here, we focus on the analysis of gap junction dependent cell-cell communication and the expression pattern of gap junction-building connexins during small molecule-induced neuronal transdifferentiation of human bone marrow-derived MSCs. During this process, the MSC markers CD73, CD90, CD105, and CD166 were downregulated while the neuronal marker Tuj1 was upregulated. Moreover, the differentiation protocol used in the present study changed the cellular morphology and physiology. The MSCs evolved from a fibroblastoid morphology towards a neuronal shape with round cell bodies and neurite-like processes. Moreover, depolarization evoked action potentials in the transdifferentiated cells. MSCs expressed mRNAs encoding Cx43 and Cx45 as well as trace levels of Cx26, Cx37- and Cx40 and allowed transfer of microinjected Lucifer yellow. The differentiation protocol increased levels of Cx26 (mRNA and protein) and decreased Cx43 (mRNA and protein) while reducing the dye transfer. Cx36 mRNA was nearly undetectable in all cells regardless of treatment. Treatment of the cells with the gap junction coupling inhibitor carbenoxolone (CBX) only modestly altered connexin mRNA levels and had little effect on neuronal differentiation. Our study indicates that the small molecule-based differentiation protocol generates immature neuron-like cells from MSCs. This might be potentially interesting for elucidating physiological modifications and mechanisms in MSCs during the initial steps of differentiation towards a neuronal lineage