Neurovaskuläre Veränderungen nach fokaler zerebraler Ischämie in Mäusen mit Alzheimer-artigen Merkmalen

Alzheimer-Demenz als häufigste Demenzform wird vorrangig als eigenständige neurodegenerative Erkrankung gesehen, teilt jedoch mit dem akuten Schlaganfall einige Risikofaktoren. Das Alter scheint dabei der zentrale Faktor zu sein, der die Überlappung beider Erkrankungen erklärt, da sowohl kardiovaskuläre Risikofaktoren wie arterielle Hypertonie, Hypercholesterinämie und Diabetes als auch die Häufigkeit der Alzheimerschen Erkrankung und der zerebralen Ischämie mit steigender Lebenszeit zunehmen. Das steigende Interesse an der genauen Aufklärung der Pathomechanismen beider Erkrankungen, kann mit dem demografischen Wandel begründet werden, aus dem die erhebliche absolute Zunahme beider Erkrankungen resultiert und ein enormer volkswirtschaftlicher Kostenaufwand erwächst. \\Experimentelle Studien sowie klinische Beobachtungen lassen vermuten, dass es einen kausalen Zusammenhang zwischen der Alzheimerschen Erkrankung und dem Schlaganfall gibt. Es wurde gezeigt, dass Patienten mit einer zerebralen Ischämie in der Anamnese ein erhöhtes Risiko haben an der Alzheimerschen Demenz zu erkranken. Zudem wurde nachgewiesen, dass chronische Hypertonie und experimentelle Schlaganfall-Induktion Alzheimer-artige Veränderungen verursachen, wie z.B. höhere Konzentration von APP und Tau-Phosphorylierung. Gleichsinnig dazu haben Alzheimer-Patienten zum einen ein erhöhtes Risiko für ein ischämisches Ereignis, zum anderen ist das Auftreten eines Schlaganfalls bei Alzheimer-Patienten mit einer schlechteren Prognose vergesellschaftet. Es wurde postuliert, dass $\beta$-Amyloid und Ischämie bei gemeinsamem Auftreten schlechtere Konsequenzen hervorrufen, als wenn diese Faktoren einzeln vorkommen. \\Im Tiermodell konnte gezeigt werden, dass ischämische Ereignisse, insbesondere mildere Formen, in Anwesenheit von hohen Konzentrationen an $\beta$-Amyloid größere Infarktvolumina, eine fulminantere Entzündungsreaktion und einen schwereren Gedächtnisverlust verursachen im Vergleich zum alleinigen Auftreten von Ischämie oder $\beta$-Amyloid. Die offensichtliche wechselseitige Beeinflussung der pathologischen Veränderungen erschwert die Konzipierung erfolgreicher Behandlungsstrategien. Daher wandten sich verschiedene Forschungsgruppen ab von den Angriffspunkten an typischen Merkmalen der Alzheimerschen Erkrankung (wie $\beta$-Amyloid, Tau-Hyperphosphorylierung und cholinerge Dysfunktion) hin zu einem komplexeren Denkansatz mit Berücksichtigung von Neuronen in Assoziation mit Gefäßstrukturen sowie Astro- und Mikroglia, zusammengefasst als neurovaskuläre Einheit (NVU). Das Verstehen der komplexen neurovaskulären Pathologie im Alzheimer-Ischämie-Kontext sollte zur Entwicklung neuerer, spezifischer Behandlungsstrategien führen. \\Die vorliegende Studie fokussierte dabei auf cholinerge, Cholinazetyltransferase (ChAT)-immunpositive, und katecholaminerge, Tyrosinhydroxylase (TH)-immunpositive, Nervenfasern in Assoziation mit endothelialen Gefäßstrukturen, um die NVU im Alzheimer-Gehirn mit begleitendem Schlaganfall zu untersuchen. Ein Schwerpunkt lag dabei auf der Analyse der ChAT als dem Enzym, dessen Aktivitätsverlust in Zusammenhang mit der altersabhängigen Degeneration von cholinergen Neuronen im basalen Vorderhirn steht. Die Degeneration des cholinergen Systems ist nachweislich involviert in die Progression der Alzheimerschen Erkrankung sowie mitverantwortlich für einen kognitiven Funktionsverlust, bedingt durch Aβ-Vorkommen und Tau-Phosphorylierung. \\Für die vorliegende Studie wurde in Wildtyp-Mäusen und triple-transgenen (3xTg) Mäusen verschiedener Altersgruppen (3 und 12 Monate) eine unilaterale fokale zerebrale Ischämie induziert. Die 3xTg-Mäuse vereinigen zwei menschliche Transgene (Amyloid-Präkursorprotein und Tau) und sind homozygot für das Knock-in-Konstrukt Präsenilin. Zusätzlich wurden drei 2 Jahre alte 3xTg-Mäuse untersucht um altersabhängige Alzheimer-artige Merkmale nachzuweisen. \\Histochemische Analysen dienten zum Vergleich der neuronalen und vaskulären Komponenten der NVU sowie der Alzheimer-spezifischen Merkmale. Fluoreszenzmarkierungen bestätigten die Existenz Alzheimer-typischer Charakteristika wie $\beta$-Amyloid-Vorkommen und Phospho-Tau zusammen mit glialen Reaktionen und morphologisch verändertem Endothel, das mit \textit{Solanum tuberosum} Lektin (STL, Kartoffellektin) dargestellt wurde. Neben deutlichen morphologischen Veränderungen der Gefäßstrukturen kam es 24 Stunden nach Ischämie-Induktion zu einem drastischen Rückgang der Immunreaktivität für TH und ChAT als neuronalen Komponenten der NVU, vor allem im ischämie-betroffenen Striatum aber auch in ischämiefernen Arealen wie der ischämischen Grenzzone und dem ipsilateralen Neokortex. Aufgrund von Korrelationsanalysen, die eine gleichsinnige Degeneration des cholinergen Systems und des Endothels gezeigt haben, kann man von gleichzeitigen Veränderungen multipler Zelltypen innerhalb der NVU im Ischämie-betroffenen Gehirn mit Alzheimer-Phänotyp ausgehen. Ausgeprägter erschien der beschriebene Effekt auf cholinerge Komponenten jüngerer Mäusen zuzutreffen. Die vorgelegten Ergebnisse unterstreichen das komplexe Zusammenspiel der verschiedenen Zelltypen innerhalb der NVU im Alzheimer-Gehirn mit ischämischen Veränderungen, einschließlich der Veränderungen des cholinergen Systems in Verbindung mit vaskulären Pathologien. Daher sollten neue Behandlungsstrategien neben bereits etablierten Ansätzen gegen neuronale Degeneration und Tau-Phosphorylierung auch auf zellulärer Ebene eingreifen, z.B. durch Stabilisierung der Integrität von endothelialen und glialen Elementen, eingreifen. Derartige Strategien könnten die Krankheitsentwicklung verzögern und das Krankheitsbild mildern

Preventing Conflicts in Sharing Communities as a Means of Promoting Sustainability

The sharing economy is a new promising trend with many positive outcomes on society and the environment, as it provides potential for sustainable solutions due to the reduction of resource consumption and less waste. However, research and practice show that sharing comes with its own share of problems. People often act selfishly, and in worst-case scenarios try to take advantage of others without contributing to the shared good. To achieve the higher goal of sustainability, it is important that conflicts in the sharing economy are prevented, and a setting is achieved that allows people to easily behave in a cooperative and sustainable way. The present research examines which conflicts emerge in sharing communities (study 1) and community gardens in particular (study 2), and whether regulation can prevent conflicts in large groups. Two exploratory studies were conducted. First, a qualitative study with consumers and non-consumers of the sharing economy revealed that regulatory systems are perceived as important for preventing the exploitation of other community members, but also that cooperation should not be enforced with strict controls and punishment. Rather, problems should be discussed in a democratic group setting, rules and goals should be set up together, and trust should be built. Second, a questionnaire study with community gardeners in Austria confirmed these results, and showed that trust is related to less conflict in community gardens, while harsh forms of regulation are related to a potential for greater conflict. Additionally, the results indicate that soft forms of regulation are related to fewer relationship and task conflicts, better conflict resolutions, a high sense of community, and greater trust in the community. We then discuss how these findings can be used to regulate sharing economy activities and give limitations and directions for future studies

The Influence of Regulation on Trust and Risk Preference in Sharing Communities

Sharing within communities has gained popularity in recent years. However, taking part in a community also comes with a certain amount of risk. This perceived amount of risk can be contained by regulations within a community as well as by potential participants’ trust in the community and the other members. We argue for a relation between regulation and the willingness to take the risk of joining a sharing community with trust as a mediator. Thereby, we distinguish between two kinds of regulation (soft and harsh regulation) and two kinds of trust (implicit and reason-based trust) on two different levels (vertical and horizontal trust). In one laboratory and one online experiment with 432 participants overall, we found that the compound of high soft and low harsh regulation increases participants’ willingness to take the risk of participation and that the effect of soft regulation is mediated mainly by vertical and horizontal reason-based trust. Based on our results, we encourage sharing communities to count on soft regulation in order to increase potential members’ trust in the community and therefore take the risk to participate

An Intracellular Nanotrap Redirects Proteins and Organelles in Live Bacteria

Owing to their small size and enhanced stability, nanobodies derived from camelids have previously been used for the construction of intracellular "nanotraps," which enable redirection and manipulation of green fluorescent protein (GFP)-tagged targets within living plant and animal cells. By taking advantage of intracellular compartmentalization in the magnetic bacterium Magnetospirillum gryphiswaldense, we demonstrate that proteins and even entire organelles can be retargeted also within prokaryotic cells by versatile nanotrap technology. Expression of multivalent GFP-binding nanobodies on magnetosomes ectopically recruited the chemotaxis protein CheW(1)-GFP from polar chemoreceptor clusters to the midcell, resulting in a gradual knockdown of aerotaxis. Conversely, entire magnetosome chains could be redirected from the midcell and tethered to one of the cell poles. Similar approaches could potentially be used for building synthetic cellular structures and targeted protein knockdowns in other bacteria. IMPORTANCE Intrabodies are commonly used in eukaryotic systems for intracellular analysis and manipulation of proteins within distinct subcellular compartments. In particular, so-called nanobodies have great potential for synthetic biology approaches because they can be expressed easily in heterologous hosts and actively interact with intracellular targets, for instance, by the construction of intracellular "nanotraps" in living animal and plant cells. Although prokaryotic cells also exhibit a considerable degree of intracellular organization, there are few tools available equivalent to the well-established methods used in eukaryotes. Here, we demonstrate the ectopic retargeting and depletion of polar membrane proteins and entire organelles to distinct compartments in a magnetotactic bacterium, resulting in a gradual knockdown of magneto-aerotaxis. This intracellular nanotrap approach has the potential to be applied in other bacteria for building synthetic cellular structures, manipulating protein function, and creating gradual targeted knockdowns. Our findings provide a proof of principle for the universal use of fluorescently tagged proteins as targets for nanotraps to fulfill these tasks

Enhancement of the efficiency of non-viral gene delivery by application of pulsed magnetic field

New approaches to increase the efficiency of non-viral gene delivery are still required. Here we report a simple approach that enhances gene delivery using permanent and pulsating magnetic fields. DNA plasmids and novel DNA fragments (PCR products) containing sequence encoding for green fluorescent protein were coupled to polyethylenimine coated superparamagnetic nanoparticles (SPIONs). The complexes were added to cells that were subsequently exposed to permanent and pulsating magnetic fields. Presence of these magnetic fields significantly increased the transfection efficiency 40 times more than in cells not exposed to the magnetic field. The transfection efficiency was highest when the nanoparticles were sedimented on the permanent magnet before the application of the pulsating field, both for small (50 nm) and large (200-250 nm) nanoparticles. The highly efficient gene transfer already within 5 min shows that this technique is a powerful tool for future in vivo studies, where rapid gene delivery is required before systemic clearance or filtration of the gene vectors occur

Enhancement of the efficiency of non-viral gene delivery by application of pulsed magnetic field

New approaches to increase the efficiency of non-viral gene delivery are still required. Here we report a simple approach that enhances gene delivery using permanent and pulsating magnetic fields. DNA plasmids and novel DNA fragments (PCR products) containing sequence encoding for green fluorescent protein were coupled to polyethylenimine coated superparamagnetic nanoparticles (SPIONs). The complexes were added to cells that were subsequently exposed to permanent and pulsating magnetic fields. Presence of these magnetic fields significantly increased the transfection efficiency 40 times more than in cells not exposed to the magnetic field. The transfection efficiency was highest when the nanoparticles were sedimented on the permanent magnet before the application of the pulsating field, both for small (50 nm) and large (200–250 nm) nanoparticles. The highly efficient gene transfer already within 5 min shows that this technique is a powerful tool for future in vivo studies, where rapid gene delivery is required before systemic clearance or filtration of the gene vectors occurs

PINK1-Interacting Proteins: Proteomic Analysis of Overexpressed PINK1

Recent publications suggest that the Parkinson's disease- (PD-) related PINK1/Parkin pathway promotes elimination of dysfunctional mitochondria by autophagy. We used tandem affinity purification (TAP), SDS-PAGE, and mass spectrometry as a first step towards identification of possible substrates for PINK1. The cellular abundance of selected identified interactors was investigated by Western blotting. Furthermore, one candidate gene was sequenced in 46 patients with atypical PD. In addition to two known binding partners (HSP90, CDC37), 12 proteins were identified using the TAP assay; four of which are mitochondrially localized (GRP75, HSP60, LRPPRC, and TUFM). Western blot analysis showed no differences in cellular abundance of these proteins comparing PINK1 mutant and control fibroblasts. When sequencing LRPPRC, four exonic synonymous changes and 20 polymorphisms in noncoding regions were detected. Our study provides a list of putative PINK1 binding partners, confirming previously described interactions, but also introducing novel mitochondrial proteins as potential components of the PINK1/Parkin mitophagy pathway

Potential Repercussions of Offshore Wind Energy Development in the Northeast United States for the Atlantic Surfclam Survey and Population Assessment

The Atlantic surfclam Spisula solidissima fishery, which spans the U.S. Northeast continental shelf, is among the most exposed to offshore wind energy development impacts because of the overlap of fishing grounds with wind energy lease areas, the hydraulic dredges used by the fishing vessels, and the location of vessel home ports relative to the fishing grounds. The Atlantic surfclam federal assessment survey is conducted using a commercial fishing vessel in locations that overlap with the offshore wind energy development. Once wind energy turbines, cables, and scour protection are installed, survey operations within wind energy lease areas may be curtailed or eliminated due to limits on vessel access, safety requirements, and assessment survey protocols. The impact of excluding the federal assessment survey from wind energy lease areas was investigated using a spatially explicit, agent-based modeling framework that integrates Atlantic surfclam stock biology, fishery captain and fleet behavior, and federal assessment survey and management decisions. Simulations were designed to compare assessment estimates of spawning stock biomass (SSB) and fishing mortality (F) for scenarios that excluded the survey from (1) wind energy lease areas or (2) wind energy lease areas and potential wind energy lease areas (“call areas”). For the most restricted scenario, the simulated stock assessment estimated 17% lower SSB relative to an unrestricted survey, placing it below the SSB target. The simulated F increased by 7% but was still less than the accepted F threshold. Changes in biological reference points were driven by the inability to access the Atlantic surfclam biomass within the wind energy lease areas. Deviations in reference points reflected the proportion of the population excluded from the survey. Excluding the Atlantic surfclam assessment surveys from the regions designated for offshore wind development can alter long-term stock assessments by increasing uncertainty in metrics that are used to set fishing quotas

The Atlantic Surfclam Fishery and Offshore Wind Energy Development: 1. Model Development and Verification

Competing pressures imposed by climate-related warming and offshore development have created a need for quantitative approaches that anticipate fisheries responses to these challenges. This study used a spatially explicit, ecological-economic agent-based model integrating dynamics associated with Atlantic surfclam stock biology, decision-making behavior of fishing vessel captains, and fishing fleet behavior to simulate stock biomass, and fishing vessel catch, effort and landings. Simulations were implemented using contemporary Atlantic surfclam stock distributions and characteristics of the surfclam fishing fleet. Simulated distribution of fishable surfclam biomass was determined by a spatially varying mortality rate, fishing by the fleet was controlled by captain decisions based on previous knowledge, information sharing, and the ability to search and find fishing grounds. Quantitative and qualitative evaluation of simulation results showed that this modeling approach sufficiently represents Atlantic surfclam fishery dynamics. A fishing simulation showed that the captain\u27s decision-making and stock knowledge, and the distribution of fishing grounds relative to home ports controlled the landed catch. The approach used herein serves as the basis for future studies examining response of the Atlantic surfclam fishery to a nexus of simultaneous, complex natural and anthropogenic pressures, and provides a framework for similar models for other resources facing similar pressures

The Atlantic surfclam fishery and offshore wind energy development: 1. Model development and verification

Competing pressures imposed by climate-related warming and offshore development have created a need for quantitative approaches that anticipate fisheries responses to these challenges. This study used a spatially explicit, ecological-economic agent-based model integrating dynamics associated with Atlantic surfclam stock biology, decision-making behavior of fishing vessel captains, and fishing fleet behavior to simulate stock biomass, and fishing vessel catch, effort and landings. Simulations were implemented using contemporary Atlantic surfclam stock distributions and characteristics of the surfclam fishing fleet. Simulated distribution of fishable surfclam biomass was determined by a spatially varying mortality rate, fishing by the fleet was controlled by captain decisions based on previous knowledge, information sharing, and the ability to search and find fishing grounds. Quantitative and qualitative evaluation of simulation results showed that this modeling approach sufficiently represents Atlantic surfclam fishery dynamics. A fishing simulation showed that the captain’s decision-making and stock knowledge, and the distribution of fishing grounds relative to home ports controlled the landed catch. The approach used herein serves as the basis for future studies examining response of the Atlantic surfclam fishery to a nexus of simultaneous, complex natural and anthropogenic pressures, and provides a framework for similar models for other resources facing similar pressures