M. Andriske

Influence of Different Promoters on the Expression Pattern of Mutated Human α-Synuclein in Transgenic Mice

Two missense mutations (A53T and A30P) in the gene encoding the presynaptic protein α-synuclein (asyn) are associated with rare, dominantly inherited forms of Parkinson’s disease (PD) and its accumulation in Lewy bodies and Lewy neurites. As an initial step in investigating the role of asyn in the pathogenesis of PD, we have generated C57BL/6 transgenic mice overexpressing the doubly mutated human asyn under the control of three different promoters; the chicken β-actin (chβactin), the mouse tyrosine hydroxylase 9.6 kb (msTH) and the mouse prion protein (msprp). In this study we compared the regional and cellular expression pattern of the transgenic protein in the brain and peripheral organs of various transgenic mouse lines. Western blot analysis and immunohistochemistry consistently showed that all three promoters successfully drive the expression of the transgene. The msprp promoter was found to give the highest level of transgene expression. All promoters directed the expression into the brain and specific neuron types. However, the promoters differed with respect to (i) the expression pattern in peripheral organs, (ii) the number and (iii) the regional distribution of expressing cells in the brain. Furthermore, remarkable line-to-line variation of expression patterns was observed in mouse lines carrying the same construct. Future studies will analyze how the variations in transgene expression affect the pathogenesis in the animals.

Expression of MUPP1 protein in mouse brain

Localizing cell surface receptors to specific subcellular sites can be crucial for proper functioning. PDZ proteins apparently play central roles in such protein localizations. 5-HT(2C) receptors have previously been shown to interact with MUPP1, a multi PDZ domain protein, in heterologous systems and in rat choroid plexus. We now report the generation and characterization of two independent MUPP1 antisera, which recognise distinct areas of the mouse brain in agreement with previous in-situ hybridization studies. Our results indicate that MUPP1 immunoreactivity co-localizes with 5-HT(2A) or 5-HT(2C) receptor expression in all regions of the mouse brain, including the choroid plexus where 5-HT(2C) receptors are highly enriched.

A controlled aquatic ecological life support system (CAELSS) for combined production of fish and higher plant biomass suitable for integration into a lunar or planetary base.

Based on the construction principle of the already operative Closed Equilibrated Biological Aquatic System (C.E.B.A.S.) the concept of an aquaculture system for combined production of animal and plant biomass was developed. It consists of a tank for intensive fish culture which is equipped with a feeding lock representing also a trap for biomass removal followed by a water recycling system. This is an optimized version of the original C.E.B.A.S. filters adapted to higher water pollutions. It operates in a fully biological mode and is able to convert the high ammonia ion concentrations excreted by the fish gills into nitrite ions. The second biomass production site is a higher plant cultivator with an internal fiber optics light distributor which may utilize of solar energy. The selected water plant is a tropical rootless duckweed of the genus Wolffia which possesses a high capacity in nitrate elimination and is terrestrially cultured as a vegetable for human nutrition in Southeast Asia. It is produced in an improved suspension culture which allows the removal of excess biomass by tangential centrifugation. The plant cultivator is able to supply the whole system with oxygen for respiration and eliminates vice versa the carbon dioxide exhaled by the fish via photosynthesis. A gas exchanger may be used for emergency purposes or to deliver excess oxygen into the environment and may be implemented into the air regeneration system of a closed environment of higher order. The plant biomass is fed into a biomass processor which delivers condensed fresh and dried biomass as pellets. The recovered water is fed back into the aquaculture loop. The fresh plants can be used for human nutrition immediately or can be stored after sterilization in an adequate packing. The dried Wolffia pellets are collected and brought into the fish tank by an automated feeder. In parallel the water from the plant cultivator is driven back to the animal tank by a pump. The special feature of the system described is, however, the used fish species. It is the herbivorous teleost Ctenopharyngodon idellus (Chinese Grass Carp) which can be raised solely with plant biomass. In this case, moreover, it can be useful for the bioregeneration of plant biomass inedible for humans which can be used easily as additional food for the fishes thus resulting in an intensivation of animal protein production. The resupply of removed fish biomass has to be guaranteed by a separate hatchery.

Animal protein production modules in biological life support systems: novel combined aquaculture techniques based on the Closed Equilibrated Biological Aquatic System (C.E.B.A.S.).

Based on the experiences made with the Closed Equilibrated Biological Aquatic System (C.E.B.A.S.) which was primarily developed for long-term and multi-generation experiments with aquatic animals and plants in a space station highly effective fresh water recycling modules were elaborated utilizing a combination of ammonia oxidizing bacteria filters and higher plants. These exhibit a high effectivity to eliminate phosphate and anorganic nitrogen compounds and are, in addition, able to contribute to the oxygen supply of the aquatic animals. The C.E.B.A.S. filter system is able to keep a closed artificial aquatic ecosystem containing teleost fishes and water snails biologically stable for several month and to eliminate waste products deriving from degraded dead fishes without a decrease of the oxygen concentration down to less than 3.5 mg/l at 25 degrees C. More advanced C.E.B.A.S. filter systems, the BIOCURE filters, were also developed for utilization in semiintensive and intensive aquaculture systems for fishes. In fact such combined animal-plant aquaculture systems represent highly effective productions sites for human food if proper plant and fish species are selected. The present papers elucidates ways to novel aquaculture systems in which herbivorous fishes are raised by feeding them with plant biomass produced in the BIOCURE filters and presents the scheme of a modification which utilizes a plant species suitable also for human nutrition. Special attention is paid to the benefits of closed aquaculture system modules which may be integrated into bioregenerative life support systems of a higher complexity for, e.g., lunar or planetary bases including some psychological aspects of the introduction of animal protein production into plant-based life support systems. Moreover, the basic reproductive biological problems of aquatic animal breeding under reduced gravity are explained leading to a disposition of essential research programs in this context.

The C.E.B.A.S.-Minimodule: Behaviour of an artificial aquatic ecological system during spaceflight

The C.E.B.A.S.-Minimodule, a closed aquatic ecosystem integrated into a middeck locker and consisting of a Zoological (animal tanks), a Botanical (plant bioreactor), a Microbial (bacteria filter) and an Electronic Component (data acquisition/control system) was flown on the STS-89 spaceshuttle mission in January 1998 for 9 days. Preflight the plant bioreactor was loaded with 53 g of Ceratophyllum demersum (coontail) and the animal tanks with 4 adult pregnant females of the fish, Xiphophorus helleri (sword-tails), 200 juveniles of the same species less than 1 week of age, 38 large and 30 juvenile Biomphalaria glabrata water snails. The filter compartment was filled with 200 g of lava grain inoculated with laboratory strains of ammonia-oxidizing bacteria. A ground reference was undertaken with the same biological setup with a delay of 4 d. After an adaptation period of 5 d the system was closed and integrated into the spaceshuttle one day before launch. Video recordings of the animals were automatically taken for 10 minutes in 2-hour periods; the tapes were changed daily by the astronauts. The chemical and physical data for the aquatic system were within the expected range and were closely comparable in comparison to the ground reference. After 9 d under space conditions, the plant biomass increased to 117 g. The plants were all found in very good condition. All 4 adult female fish were retrieved in a good physiological condition. The juvenile fishes had a survival rate of about 33%. Almost 97% of the snails had survived and produced more than 250 neonates and 40 spawning packs. All samples were distributed according to a defined schedule and satisfied all scientific needs of the involved 12 principal investigators. This was the first successful spaceflight of an artificial aquatic ecosystem containing vertebrates, invertebrates, higher plants and microorganisms self-sustained by its inhabitants only. C.E.B.A.S. in a modified form and biological setup is a promising candidate for the early space station utilization as a first midterm experiment.

Effect of salmon gonadotropic hormone on sex steroids in male rainbow trout: plasma levels and testicular secretion in vitro

SummaryMale rainbow trout were treated with salmon gonadotropic hormone (GTH) at different stages of the circannual reproductive cycle; spawning fish were also treated with an antiserum against salmon GTH. Injection of GTH led to a several-fold increase of plasma sex steroid levels during spermatogenesis and in the spawning season but was without effect at early stages of testicular development. GTH neutralization during the spawning season was followed by a several-fold decrease of plasma sex steroid levels. During spermatogenesis and in the spawning season, both treatment regimes resulted in an increased sensitivity of testicular explants in response to a subsequent stimulation of steroid secretion in vitro. This up-regulatory response may facilitate and maintain the high sex steroid plasma levels observed during the spawning season. It may also be necessary to allow for concomitant peak values of plasma GTH and sex steroids in the spawning season, a situation difficult to understand within the negative feedback concept. The adaptive capacities of the testicular steroidogenic system indicate that it is not only an effector site for GTH but also an active part of the endocrine system controling reproduction.

Differential expression of Cathepsin S and X in the spinal cord of a rat neuropathic pain model

BackgroundAmple evidence suggests a substantial contribution of cellular and molecular changes in the spinal cord to the induction and persistence of chronic neuropathic pain conditions. While for a long time, proteases were mainly considered as protein degrading enzymes, they are now receiving growing interest as signalling molecules in the pain pathology. In the present study we focused on two cathepsins, CATS and CATX, and studied their spatiotemporal expression and activity during the development and progression of neuropathic pain in the CNS of the rat 5th lumbar spinal nerve transection model (L5T).ResultsImmediately after the lesion, both cathepsins, CATS and CATX, were upregulated in the spinal cord. Moreover, we succeeded in measuring the activity of CATX, which was substantially increased after L5T. The differential expression of these proteins exhibited the same spatial distribution and temporal progression in the spinal cord, progressing up to the medulla oblongata in the late phase of chronic pain. The cellular distribution of CATS and CATX was, however, considerably different.ConclusionThe cellular distribution and the spatio-temporal development of the altered expression of CATS and CATX suggest that these proteins are important players in the spinal mechanisms involved in chronic pain induction and maintenance.

Hypoxia facilitates neurogenic dural plasma protein extravasation in mice: a novel animal model for migraine pathophysiology.

Migraine animal models generally mimic the onset of attacks and acute treatment processes. A guinea pig model used the application of meta-chlorophenylpiperazine (mCPP) to trigger immediate dural plasma protein extravasation (PPE) mediated by 5-HT2B receptors. This model has predictive value for antimigraine drugs but cannot explain the delayed onset of efficacy of 5-HT2B receptor antagonists when clinically used for migraine prophylaxis. We found that mCPP failed to induce dural PPE in mice. Considering the role 5-HT2B receptors play in hypoxia-induced pulmonary vessel muscularization, we were encouraged to keep mice under hypoxic conditions and tested whether this treatment will render them susceptible to mCPP-induced dural PPE. Following four-week of hypoxia, PPE, associated with increased transendothelial transport, was induced by mCPP. The effect was blocked by sumatriptan. Chronic application of 5-HT2B receptor or nitric oxide synthase blockers during hypoxia prevented the development of susceptibility. Here we present a migraine model that distinguishes between a migraine-like state (hypoxic mice) and normal, normoxic mice and mimics processes that are related to chronic activation of 5-HT2B receptors under hypoxia. It seems striking, that chronic endogenous activation of 5-HT2B receptors is crucial for the sensitization since 5-HT2B receptor antagonists have strong, albeit delayed migraine prophylactic efficacy.

Investigation of 5-HT2B receptor induced dural plasma protein extravasation in a mouse migraine model

Migraine attacks originate in the meninges which are densely innervated by trigeminal nerve fibers. Stimulated endothelial cells of dural blood vessels secrete nitric oxide, where neuropeptides are released from trigeminal nerve fibers. This, in turn, leads to meningeal Plasma Protein Extravasation (PPE), serving as an established indicator for migraine attacks in animal models. In our mouse migraine model, we sensitized mice against the 5-HT2B receptor agonist meta-chlorphenylpiperazine (mCPP). Mice kept under hypoxic conditions for four weeks displayed significantly elevated PPEs in the dura mater upon mCPP injection. Tissue accumulation of the tracer Evans Blue was measured to quantify the extent of the PPE. In this study, several histological tracers were employed to identify the part of the vasculature where PPE occurs and the cellular mechanism associated with it. Injections of BSA-FITC (FITC-linked bovine serum albumin) verified the mCPP induced PPE in the dura mater. After leaving the blood vessels, the tracer was incorporated into perivascular, CD68-positive macrophages. With electron microscopic studies using the tracer HRP (horse radish peroxidase) we demonstrated that the PPE is associated with increased transcytotic transport. After 5-HT2B receptor activation, HRP escapes from capillaries and venules of hypoxic mice via an increased transcytotic transport in the endothelium. The hypoxic treatment itself increases transcytosis in arterioles, which may be indicative of a proinflammatory state of the endothelium. HRP was also detectable in intercellular clefts, but was always retained at tight junctions In summary, we demonstrated that in mice hypoxic treatment induces dural PPE via increased transcytotis at arterioles and that this is elevated to capillaries and venules after mCPP-injection.