Experimental proteome analysis was combined with a genome-wide prediction screen to characterize the protein content of the thylakoid lumen of Arabidopsis chloroplasts. Soluble thylakoid proteins were separated by two-dimensional electrophoresis and identified by mass spectrometry. The identities of 81 proteins were established, and N termini were sequenced to validate localization prediction. Gene annotation of the identified proteins was corrected by experimental data, and an interesting case of alternative splicing was discovered. Expression of a surprising number of paralogs was detected. Expression of five isomerases of different classes suggests strong (un)folding activity in the thylakoid lumen. These isomerases possibly are connected to a network of peripheral and lumenal proteins involved in antioxidative response, including peroxiredoxins, m-type thioredoxins, and a lumenal ascorbate peroxidase. Characteristics of the experimentally identified lumenal proteins and their orthologs were used for a genome-wide prediction of the lumenal proteome. Lumenal proteins with a typical twin-arginine translocation motif were predicted with good accuracy and sensitivity and included additional isomerases and proteases. Thus, prime functions of the lumenal proteome include assistance in the folding and proteolysis of thylakoid proteins as well as protection against oxidative stress. Many of the predicted lumenal proteins must be present at concentrations at least 10,000-fold lower than proteins of the photosynthetic apparatus.

The haplochromine cichlids of Lake Victoria constitute a classical example of explosive speciation. Extensive intra- and interspecific variation in male nuptial coloration and female mating preferences, in the absence of postzygotic isolation between species, has inspired the hypothesis that sexual selection has been a driving force in the origin of this species flock. This hypothesis rests on the premise that the phenotypic traits that underlie behavioural reproductive isolation between sister species diverged under sexual selection within a species. We test this premise in a Lake Victoria cichlid, by using laboratory experiments and field observations. We report that a male colour trait, which has previously been shown to be important for behavioural reproductive isolation between this species and a close relative, is under directional sexual selection by female mate choice within this species. This is consistent with the hypothesis that female choice has driven the divergence in male coloration between the two species. We also find that male territoriality is vital for male reproductive success and that multiple mating by females is common.

BackgroundDespite the clear physical association between activated astrocytes and amyloid-β (Aβ) plaques, the importance of astrocytes and their therapeutic potential in Alzheimer’s disease remain elusive. Soluble Aβ aggregates, such as protofibrils, have been suggested to be responsible for the widespread neuronal cell death in Alzheimer’s disease, but the mechanisms behind this remain unclear. Moreover, ineffective degradation is of great interest when it comes to the development and progression of neurodegeneration. Based on our previous results that astrocytes are extremely slow in degrading phagocytosed material, we hypothesized that astrocytes may be an important player in these processes. Hence, the aim of this study was to clarify the role of astrocytes in clearance, spreading and neuronal toxicity of Aβ.ResultsTo examine the role of astrocytes in Aβ pathology, we added Aβ protofibrils to a co-culture system of primary neurons and glia. Our data demonstrates that astrocytes rapidly engulf large amounts of Aβ protofibrils, but then store, rather than degrade the ingested material. The incomplete digestion results in a high intracellular load of toxic, partly N-terminally truncated Aβ and severe lysosomal dysfunction. Moreover, secretion of microvesicles containing N-terminally truncated Aβ, induce apoptosis of cortical neurons.ConclusionsTaken together, our results suggest that astrocytes play a central role in the progression of Alzheimer’s disease, by accumulating and spreading toxic Aβ species.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0098-z) contains supplementary material, which is available to authorized users.

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Amyloid-β (Aβ) immunotherapy for Alzheimer's disease (AD) has good preclinical support from transgenic mouse models and clinical data suggesting that a long-term treatment effect is possible. Soluble Aβ protofibrils have been shown to exhibit neurotoxicity in vitro and in vivo, and constitute an attractive target for immunotherapy. Here, we demonstrate that the humanized antibody BAN2401 and its murine version mAb158 exhibit a strong binding preference for Aβ protofibrils over Aβ monomers. Further, we confirm the presence of the target by showing that both antibodies efficiently immunoprecipitate soluble Aβ aggregates in human AD brain extracts. mAb158 reached the brain and reduced the brain protofibril levels by 42% in an exposure-dependent manner both after long-term and short-term treatment in tg-ArcSwe mice. Notably, a 53% reduction of protofibrils/oligomers in cerebrospinal fluid (CSF) that correlated with reduced brain protofibril levels was observed after long-term treatment, suggesting that CSF protofibrils/oligomers could be used as a potential biomarker. No change in native monomeric Aβ42 could be observed in brain TBS extracts after mAb158-treatment in tg-ArcSwe mice. By confirming the specific ability of mAb158 to selectively bind and reduce soluble Aβ protofibrils, with minimal binding to Aβ monomers, we provide further support in favor of its position as an attractive new candidate for AD immunotherapy. BAN2401 has undergone full phase 1 development, and available data indicate a favorable safety profile in AD patients.

We have used a novel polyelectrolyte multilayer (PEM) coating consisting of the polyelectrolytes collagen and hyaluronic acid. The build-up by the layer-by-layer deposition technique is outlined by ex situ and in situ ellipsometric measurements. When collagen was added, the thickness of the PEM was increased, and the refractive index was decreased. Corresponding but opposite effects were noted when hyaluronic acid was added. These changes are considered to be explained by a diffusion mechanism. It was also found that the PEM layers were unstable at physiological pH. However, by cross-linking using N-(3-di-methylaminopropyl)-N'-ethylcarbodiimide together with N-hydroxysuccinimide, a stable PEM layer resulted. These tissue friendly PEM layers are expected to have a great impact in the design of artificial extracellular matrixes. Also, the insertion of fluorescence labels demonstrates the potential for incorporation of other functionalities.

Oligomeric assemblies of amyloid‐β (Aβ) are suggested to be central in the pathogenesis of Alzheimer’s disease because levels of soluble Aβ correlate much better with the extent of cognitive dysfunctions than do senile plaque counts. Moreover, such Aβ species have been shown to be neurotoxic, to interfere with learned behavior and to inhibit the maintenance of hippocampal long‐term potentiation. The tg‐ArcSwe model (i.e. transgenic mice with the Arctic and Swedish Alzheimer mutations) expresses elevated levels of Aβ protofibrils in the brain, making tg‐ArcSwe a highly suitable model for investigating the pathogenic role of these Aβ assemblies. In the present study, we estimated Aβ protofibril levels in the brain and cerebrospinal fluid of tg‐ArcSwe mice, and also assessed their role with respect to cognitive functions. Protofibril levels, specifically measured with a sandwich ELISA, were found to be elevated in young tg‐ArcSwe mice compared to several transgenic models lacking the Arctic mutation. In aged tg‐ArcSwe mice with considerable plaque deposition, Aβ protofibrils were approximately 50% higher than in younger mice, whereas levels of total Aβ were exponentially increased. Young tg‐ArcSwe mice showed deficits in spatial learning, and individual performances in the Morris water maze were correlated inversely with levels of Aβ protofibrils, but not with total Aβ levels. We conclude that Aβ protofibrils accumulate in an age‐dependent manner in tg‐ArcSwe mice, although to a far lesser extent than total Aβ. Our findings suggest that increased levels of Aβ protofibrils could result in spatial learning impairment.

BackgroundAmyloid-β (Aβ) immunotherapy is one of the most promising disease-modifying strategies for Alzheimer’s disease (AD). Despite recent progress targeting aggregated forms of Aβ, low antibody brain penetrance remains a challenge. In the present study, we used transferrin receptor (TfR)-mediated transcytosis to facilitate brain uptake of our previously developed Aβ protofibril-selective mAb158, with the aim of increasing the efficacy of immunotherapy directed toward soluble Aβ protofibrils.MethodsAβ protein precursor (AβPP)-transgenic mice (tg-ArcSwe) were given a single dose of mAb158, modified for TfR-mediated transcytosis (RmAb158-scFv8D3), in comparison with an equimolar dose or a tenfold higher dose of unmodified recombinant mAb158 (RmAb158). Soluble Aβ protofibrils and total Aβ in the brain were measured by enzyme-linked immunosorbent assay (ELISA). Brain distribution of radiolabeled antibodies was visualized by positron emission tomography (PET) and ex vivo autoradiography.ResultsELISA analysis of Tris-buffered saline brain extracts demonstrated a 40% reduction of soluble Aβ protofibrils in both RmAb158-scFv8D3- and high-dose RmAb158-treated mice, whereas there was no Aβ protofibril reduction in mice treated with a low dose of RmAb158. Further, ex vivo autoradiography and PET imaging revealed different brain distribution patterns of RmAb158-scFv8D3 and RmAb158, suggesting that these antibodies may affect Aβ levels by different mechanisms.ConclusionsWith a combination of biochemical and imaging analyses, this study demonstrates that antibodies engineered to be transported across the blood-brain barrier can be used to increase the efficacy of Aβ immunotherapy. This strategy may allow for decreased antibody doses and thereby reduced side effects and treatment costs.