Distinct functions for anterograde and retrograde sorting of SORLA in amyloidogenic processes in the brain

SORLA is a neuronal sorting receptor implicated both in sporadic and familial forms of AD. SORLA reduces the amyloidogenic burden by two mechanisms, either by rerouting internalized APP molecules from endosomes to the trans-Golgi network (TGN) to prevent proteolytic processing or by directing newly produced Aβ to lysosomes for catabolism. Studies in cell lines suggested that the interaction of SORLA with cytosolic adaptors retromer and GGA is required for receptor sorting to and from the TGN. However, the relevance of anterograde or retrograde trafficking for SORLA activity in vivo remained largely unexplored. Here, we generated mouse models expressing SORLA variants lacking binding sites for GGA or retromer to query this concept in the brain. Disruption of retromer binding resulted in a retrograde-sorting defect with accumulation of SORLA in endosomes and depletion from the TGN, and in an overall enhanced APP processing. In contrast, disruption of the GGA interaction did not impact APP processing but caused increased brain Aβ levels, a mechanism attributed to a defect in anterograde lysosomal targeting of Aβ. Our findings substantiated the significance of adaptor-mediated sorting for SORLA activities in vivo, and they uncovered that anterograde and retrograde sorting paths may serve discrete receptor functions in amyloidogenic processes

Mutation of Tyrosine Sites in the Human Alpha-Synuclein Gene Induces Neurotoxicity in Transgenic Mice with Soluble Alpha-Synuclein Oligomer Formation

Overexpression of α-synuclein with tyrosine mutated to phenylalanine at position 125 leads to a severe phenotype with motor impairment and neuropathology in Drosophila. Here, we hypothesized that tyrosine mutations would similarly lead to impaired motor performance with neuropathology in a rodent model. In transgenic mice (ASO), tyrosines at positions 125, 133, and 136 in human α-synuclein were mutated to phenylalanine and cloned into a Thy1.2 expression vector, which was used to create transgenic mouse lines on a mixed genetic background TgN(Thy-1-SNCA-YF)4Emfu (YF). The YF mice had a decreased lifespan and displayed a dramatic motor phenotype with paralysis of both hind- and forelegs. Post-translational modification of α-synuclein due to phosphorylation of serine 129 is often seen in inclusions in the brains of patients with α-synucleinopathies. We observed a slight but significant increase in phosphorylation of serine 129 in the cytosol in YF mice compared to age-matched human α-synuclein transgenic mice (ASO). Conversely, significantly decreased phosphorylation of serine 129 was seen in synaptosomes of YF mice that also contained higher amounts of soluble oligomers. YF mice deposited full-length α-synuclein aggregates in neurons widespread in the CNS with the main occurrence in the forebrain structures of the cerebral cortex, the basal ganglia, and limbic structures. Full-length α-synuclein labeling was also prominent in many nuclear regions of the brain stem, deep cerebellar nuclei, and cerebellar cortex. The study shows that the substitution of tyrosines to phenylalanine in α-synuclein at positions 125, 133, and 136 leads to severe toxicity in vivo. An insignificant change upon tyrosine substitution suggests that the phosphorylation of serine 129 is not the cause of the toxicity

The effect of heterogeneity on invasion in spatial epidemics: from theory to experimental evidence in a model system

Heterogeneity in host populations is an important factor affecting the ability of a pathogen to invade, yet the quantitative investigation of its effects on epidemic spread is still an open problem. In this paper, we test recent theoretical results, which extend the established “percolation paradigm” to the spread of a pathogen in discrete heterogeneous host populations. In particular, we test the hypothesis that the probability of epidemic invasion decreases when host heterogeneity is increased. We use replicated experimental microcosms, in which the ubiquitous pathogenic fungus Rhizoctonia solani grows through a population of discrete nutrient sites on a lattice, with nutrient sites representing hosts. The degree of host heterogeneity within different populations is adjusted by changing the proportion and the nutrient concentration of nutrient sites. The experimental data are analysed via Bayesian inference methods, estimating pathogen transmission parameters for each individual population. We find a significant, negative correlation between heterogeneity and the probability of pathogen invasion, thereby validating the theory. The value of the correlation is also in remarkably good agreement with the theoretical predictions. We briefly discuss how our results can be exploited in the design and implementation of disease control strategies

Isolation of cancer stem cells by selection for miR-302 expressing cells

Background Cancer stem cells are believed to be a major reason for long-term therapy failure because they are multi-drug resistant and able to rest mitotically inactive in the hypoxic center of tumors. Due to their variable number and their often low proliferation rate, cancer stem cells are difficult to purify in decent quantities and to grow in cell culture systems, where they are easily outcompeted by faster growing more ‘differentiated’, i.e., less stem cell-like tumor cells. Methods Here we present a proof of principle study based on the idea to select cancer stem cells by means of the expression of a stem cell-specific gene. A selectable egfp-neo coding sequence was inserted in the last exon of the non-coding murine miR-302 host gene. As a stem cell specific regulatory element, 2.1 kb of the genomic region immediately upstream of the miR-302 host gene transcription start site was used. Stable transgenic CJ7 embryonic stem cells were used to induce teratomas. Results After three weeks, tumors were removed for analysis and primary cultures were established. Stem cell-like cells were selected from these culture based on G418 selection. When the selection was removed, stem cell morphology and miR-302 expression were rapidly lost, indicating that it was not the original ES cells that had been isolated. Conclusions We show the possibility to use drug resistance expressed from a regulatory sequence of a stem cell-specific marker, to isolate and propagate cancer stem cells that otherwise might be hidden in the majority of tumor cells

Targeted inactivation of the Septin2 and Septin9 genes in myelinating Schwann cells of mice

The formation of axon-enwrapping myelin sheaths by oligodendrocytes in the central nervous system (CNS) involves the assembly of a scaffolding septin filament comprised of the subunits SEPTIN2, SEPTIN4, SEPTIN7 and SEPTIN8. Conversely, in the peripheral nervous system (PNS) myelin is synthesized by a different cell type termed Schwann cells, and it remained unknown if septins also assemble as a multimer in PNS myelin. According to prior proteome analysis, PNS myelin comprises the subunits SEPTIN2, SEPTIN7, SEPTIN8, SEPTIN9 and SEPTIN11, which localize to the paranodal and abaxonal myelin sub-compartments. Here we use the Cre/loxP-system to delete the Septin9-gene specifically in Schwann cells, causing a markedly reduced abundance of SEPTIN9 in sciatic nerves, implying that Schwann cells are the main cell type expressing SEPTIN9 in the nerve. However, Septin9-deficiency in Schwann cells did not affect the abundance or localization of other septin subunits. In contrast, when deleting the Septin2-gene in Schwann cells the abundance of all relevant septin subunits was markedly reduced, including SEPTIN9. Notably, we did not find evidence that deleting Septin2 or Septin9 in Schwann cells impairs myelin biogenesis, nerve conduction velocity or motor/sensory capabilities, at least at the assessed timepoints. Our data thus show that SEPTIN2 but not SEPTIN9 is required for the formation or stabilization of a septin multimer in PNS myelin in vivo; however, its functional relevance remains to be established

In Situ Detection of Active Edge Sites in Single-Layer MoS2_2 Catalysts

MoS2 nanoparticles are proven catalysts for processes such as hydrodesulphurization and hydrogen evolution, but unravelling their atomic-scale structure under catalytic working conditions has remained significantly challenging. Ambient pressure X-ray Photoelectron Spectroscopy (AP-XPS) allows us to follow in-situ the formation of the catalytically relevant MoS2 edge sites in their active state. The XPS fingerprint is described by independent contributions to the Mo3d core level spectrum whose relative intensity is sensitive to the thermodynamic conditions. Density Functional Theory (DFT) is used to model the triangular MoS2 particles on Au(111) and identify the particular sulphidation state of the edge sites. A consistent picture emerges in which the core level shifts for the edge Mo atoms evolve counter-intuitively towards higher binding energies when the active edges are reduced. The shift is explained by a surprising alteration in the metallic character of the edge sites, which is a distinct spectroscopic signature of the MoS2 edges under working conditions

Evaluation of yield, yield stability, and yield–protein relationship in 17 commercial faba bean cultivars

Abstract Faba bean is a legume crop with high protein content and considerable potential for wider cultivation in cool climates. However, it has a reputation for having unstable yield with large interannual variability, mostly attributed to yearly variation in rainfall. In this study, 17 commercial cultivars of faba bean were evaluated for seed yield, yield stability and the relationship between seed yield and protein content at four locations in Denmark and Finland during 2016?2018. We found that location and year effects accounted for 89% of the total seed yield variation. Cultivar ? environment (GxE) interactions were small (2.4%) and did not cause reranking of cultivars across environments. Yield stability contributed little to the mean yield of the cultivars, as high-yielding cultivars consistently outperformed the lower yielding genotypes, even under the most adverse conditions. Similarly, GxE effects on protein content were limited, and we found an overall negative correlation of ?0.61 between seed yield and protein content for the cultivars and environments studied. These data may be helpful for selecting cultivars for field use or for use in breeding programmes, considering that future faba bean pricing could depend on both protein quantity and concentration.Peer reviewe

Pulse-shaped two-photon excitation of a fluorescent base analogue approaches single-molecule sensitivity

Fluorescent nucleobase analogues (FBAs) have many desirable features in comparison to extrinsic fluorescent labels, but they are yet to find application in ultrasensitive detection. Many of the disadvantages of FBAs arise from their short excitation wavelengths (often in the ultraviolet), making two-photon excitation a potentially attractive approach. Pentacyclic adenine (pA) is a recently developed FBA that has an exceptionally high two-photon brightness. We have studied the two-photon-excited fluorescence properties of pA and how they are affected by incorporation in DNA. We find that pA is more photostable under two-photon excitation than via resonant absorption. When incorporated in an oligonucleotide, pA has a high two-photon cross section and emission quantum yield, varying with sequence context, resulting in the highest reported brightness for such a probe. The use of a two-photon microscope with ultrafast excitation and pulse shaping has allowed the detection of pA-containing oligonucleotides in solution with a limit of detection of ∼5 molecules, demonstrating that practical single-molecule detection of FBAs is now within reach