Differential effects of familial Alzheimer’s disease-causing mutations on amyloid precursor protein (APP) trafficking, proteolytic conversion, and synaptogenic activity

The amyloid precursor protein (APP) is a key player in Alzheimer`s disease (AD) and the precursor of the Aβ peptide, which is generated by consecutive cleavages of β- and γ-secretases. Familial Alzheimer’s disease (FAD) describes a hereditary subgroup of AD that represents a low percentage of AD cases with an early onset of the disease. Different APP FAD mutations are thought to have qualitatively different effects on its proteolytic conversion. However, few studies have explored the pathogenic and putative physiological differences in more detail. Here, we compared different FAD mutations, located at the β- (Swedish), α- (Flemish, Arctic, Iowa) or γ-secretase (Iberian) cleavage sites. We examined heterologous expression of APP WT and FAD mutants in non-neuronal cells and their impact on presynaptic differentiation in contacting axons of co-cultured neurons. To decipher the underlying molecular mechanism, we tested the subcellular localization, the endocytosis rate and the proteolytic processing in detail by immunoprecipitation–mass spectrometry. Interestingly, we found that only the Iberian mutation showed altered synaptogenic function. Furthermore, the APP Iowa mutant shows significantly decreased α-secretase processing which is in line with our results that APP carrying the Iowa mutation was significantly increased in early endosomes. However, most interestingly, immunoprecipitation–mass spectrometry analysis revealed that the amino acid substitutions of APP FAD mutants have a decisive impact on their processing reflected in altered Aβ profiles. Importantly, N-terminally truncated Aβ peptides starting at position 5 were detected preferentially for APP Flemish, Arctic, and Iowa mutants containing amino acid substitutions around the α-secretase cleavage site. The strongest change in the ratio of Aβ40/Aβ42 was observed for the Iberian mutation while APP Swedish showed a substantial increase in Aβ1–17 peptides. Together, our data indicate that familial AD mutations located at the α-, β-, and γ-secretase cleavage sites show considerable differences in the underlying pathogenic mechanisms

Hygiene inspections on passenger ships in Europe - an overview

<p>Abstract</p> <p>Background</p> <p>Hygiene inspections on passenger ships are important for the prevention of communicable diseases. The European Union (EU) countries conduct hygiene inspections on passenger ships in order to ensure that appropriate measures have been taken to eliminate potential sources of contamination which could lead to the spread of communicable diseases. This study was implemented within the framework of the EU SHIPSAN project and it investigates the legislation applied and practices of hygiene inspections of passenger ships in the EU Member States (MS) and European Free Trade Association countries.</p> <p>Methods</p> <p>Two questionnaires were composed and disseminated to 28 countries. A total of 92 questionnaires were completed by competent authorities responsible for hygiene inspections (n = 48) and the creation of legislation (n = 44); response rates were 96%, and 75.9%, respectively.</p> <p>Results</p> <p>Out of the 48 responding authorities responsible for hygiene inspections, a routine programme was used by 19 (39.6%) of these to conduct inspections of ships on national voyages and by 26 (54.2%) for ships on international voyages. Standardised inspection forms are used by 59.1% of the authorities. A scoring inspection system is applied by five (11.6%) of the 43 responding authorities. Environmental sampling is conducted by 84.1% of the authorities (37 out of 44). The inspection results are collected and analysed by 54.5% (24 out of 44) of the authorities, while 9 authorities (20.5%) declared that they publish the results. Inspections are conducted during outbreak investigations by 75% and 70.8% of the authorities, on ships on national and international voyages, respectively. A total of 31 (64.6%) and 39 (81.3%) authorities conducted inspections during complaint investigations on ships on international and on national voyages, respectively. Port-to-port communication between the national port authorities was reported by 35.4% (17 out of 48) of the responding authorities and 20.8% (10 out of 48) of the port authorities of other countries.</p> <p>Conclusion</p> <p>This study revealed a diversity of approaches and practices in the conduct of inspections, differences in the qualifications/knowledge/experience of inspectors, the legislation applied during inspections, and the lack of communication and training among many EU countries. An integrated European inspection programme involving competent expert inspectors in each EU Member States and special training for ship hygiene delivered to crew members and inspectors would help to minimize the risk of communicable diseases. Common inspection tools at a European level for hygiene inspection practices and port-to-port communication are needed.</p

Microarray analysis of Foxa2 mutant mouse embryos reveals novel gene expression and inductive roles for the gastrula organizer and its derivatives

<p>Abstract</p> <p>Background</p> <p>The Spemann/Mangold organizer is a transient tissue critical for patterning the gastrula stage vertebrate embryo and formation of the three germ layers. Despite its important role during development, there are still relatively few genes with specific expression in the organizer and its derivatives. Foxa2 is a forkhead transcription factor that is absolutely required for formation of the mammalian equivalent of the organizer, the node, the axial mesoderm and the definitive endoderm (DE). However, the targets of Foxa2 during embryogenesis, and the molecular impact of organizer loss on the gastrula embryo, have not been well defined.</p> <p>Results</p> <p>To identify genes specific to the Spemann/Mangold organizer, we performed a microarray-based screen that compared wild-type and <it>Foxa2 </it>mutant embryos at late gastrulation stage (E7.5). We could detect genes that were consistently down-regulated in replicate pools of mutant embryos versus wild-type, and these included a number of known node and DE markers. We selected 314 genes without previously published data at E7.5 and screened for expression by whole mount <it>in situ </it>hybridization. We identified 10 novel expression patterns in the node and 5 in the definitive endoderm. We also found significant reduction of markers expressed in secondary tissues that require interaction with the organizer and its derivatives, such as cardiac mesoderm, vasculature, primitive streak, and anterior neuroectoderm.</p> <p>Conclusion</p> <p>The genes identified in this screen represent novel Spemann/Mangold organizer genes as well as potential Foxa2 targets. Further investigation will be needed to define these genes as novel developmental regulatory factors involved in organizer formation and function. We have placed these genes in a Foxa2-dependent genetic regulatory network and we hypothesize how Foxa2 may regulate a molecular program of Spemann/Mangold organizer development. We have also shown how early loss of the organizer and its inductive properties in an otherwise normal embryo, impacts on the molecular profile of surrounding tissues.</p

Decreasing the expression of PICALM reduces endocytosis and the activity of β-secretase: Implications for Alzheimer's disease

© 2016 The Author(s). Background: Polymorphisms in the gene for phosphatidylinositol binding clathrin assembly protein (PICALM), an endocytic-related protein, are associated with a small, increased risk of developing Alzheimer's disease (AD), strongly suggesting that changes in endocytosis are involved in the aetiology of the disease. We have investigated the involvement of PICALM in the processing of amyloid precursor protein (APP) to understand how PICALM could be linked to the development of AD. We used siRNA to deplete levels of PICALM, its isoforms and clathrin heavy chain in the human brain-derived H4 neuroglioma cell line that expresses endogenous levels of APP. We then used Western blotting, ELISA and immunohistochemistry to detect intra- and extracellular protein levels of endocytic-related proteins, APP and APP metabolites including β-amyloid (Aβ). Levels of functional endocytosis were quantified using ALEXA 488-conjugated transferrin and flow cytometry as a marker of clathrin-mediated endocytosis (CME). Results: Following depletion of all the isoforms of PICALM by siRNA in H4 cells, levels of intracellular APP, intracellular β-C-terminal fragment (β-CTF) and secreted sAPPβ (APP fragments produced by β-secretase cleavage) were significantly reduced but Aβ40 was not affected. Functional endocytosis was significantly reduced after both PICALM and clathrin depletion, highlighting the importance of PICALM in this process. However, depletion of clathrin did not affect APP but did reduce β-CTF levels. PICALM depletion altered the intracellular distribution of clathrin while clathrin reduction affected the subcellular pattern of PICALM labelling. Both PICALM and clathrin depletion reduced the expression of BACE1 mRNA and PICALM siRNA reduced protein levels. Individual depletion of PICALM isoforms 1 and 2 did not affect APP levels while clathrin depletion had a differential effect on the isoforms, increasing isoform 1 while decreasing isoform 2 expression. Conclusions: The depletion of PICALM in brain-derived cells has significant effects on the processing of APP, probably by reducing CME. In particular, it affects the production of β-CTF which is increasingly considered to be an important mediator in AD independent of Aβ. Thus a decrease in PICALM expression in the brain could be beneficial to slow or prevent the development of AD

The extraordinary evolutionary history of the reticuloendotheliosis viruses

The reticuloendotheliosis viruses (REVs) comprise several closely related amphotropic retroviruses isolated from birds. These viruses exhibit several highly unusual characteristics that have not so far been adequately explained, including their extremely close relationship to mammalian retroviruses, and their presence as endogenous sequences within the genomes of certain large DNA viruses. We present evidence for an iatrogenic origin of REVs that accounts for these phenomena. Firstly, we identify endogenous retroviral fossils in mammalian genomes that share a unique recombinant structure with REVs—unequivocally demonstrating that REVs derive directly from mammalian retroviruses. Secondly, through sequencing of archived REV isolates, we confirm that contaminated Plasmodium lophurae stocks have been the source of multiple REV outbreaks in experimentally infected birds. Finally, we show that both phylogenetic and historical evidence support a scenario wherein REVs originated as mammalian retroviruses that were accidentally introduced into avian hosts in the late 1930s, during experimental studies of P. lophurae, and subsequently integrated into the fowlpox virus (FWPV) and gallid herpesvirus type 2 (GHV-2) genomes, generating recombinant DNA viruses that now circulate in wild birds and poultry. Our findings provide a novel perspective on the origin and evolution of REV, and indicate that horizontal gene transfer between virus families can expand the impact of iatrogenic transmission events

Reviewing progress: 7 Year Trends in Characteristics of Adults and Children Enrolled at HIV Care and Treatment Clinics in the United Republic of Tanzania.

To evaluate the on-going scale-up of HIV programs, we assessed trends in patient characteristics at enrolment and ART initiation over 7 years of implementation. Data were from Optimal Models, a prospective open cohort study of HIV-infected (HIV+) adults (>=15 years) and children (<15 years) enrolled from January 2005 to December 2011 at 44 HIV clinics in 3 regions of mainland Tanzania (Kagera, Kigoma, Pwani) and Zanzibar. Comparative statistics for trends in characteristics of patients enrolled in 2005--2007, 2008--2009 and 2010--2011 were examined. Overall 62,801 HIV+ patients were enrolled: 58,102(92.5%) adults, (66.5% female); 4,699(7.5%) children.Among adults, pregnant women enrolment increased: 6.8%, 2005--2007; 12.1%, 2008--2009; 17.2%, 2010--2011; as did entry into care from prevention of mother-to-child HIV transmission (PMTCT) programs: 6.6%, 2005--2007; 9.5%, 2008--2009; 12.6%, 2010--2011. WHO stage IV at enrolment declined: 27.1%, 2005--2007; 20.2%, 2008--2009; 11.1% 2010--2011. Of the 42.5% and 29.5% with CD4+ data at enrolment and ART initiation respectively, median CD4+ count increased: 210cells/muL, 2005--2007; 262cells/muL, 2008--2009; 266cells/muL 2010--2011; but median CD4+ at ART initiation did not change (148cells/muL overall). Stavudine initiation declined: 84.9%, 2005--2007; 43.1%, 2008--2009; 19.7%, 2010--2011.Among children, median age (years) at enrolment decreased from 6.1(IQR:2.7-10.0) in 2005--2007 to 4.8(IQR:1.9-8.6) in 2008--2009, and 4.1(IQR:1.5-8.1) in 2010--2011 and children <24 months increased from 18.5% to 26.1% and 31.5% respectively. Entry from PMTCT was 7.0%, 2005--2007; 10.7%, 2008--2009; 15.0%, 2010--2011. WHO stage IV at enrolment declined from 22.9%, 2005--2007, to 18.3%, 2008--2009 to 13.9%, 2010--2011. Proportion initiating stavudine was 39.8% 2005--2007; 39.5%, 2008--2009; 26.1%, 2010--2011. Median age at ART initiation also declined significantly. Over time, the proportion of pregnant women and of adults and children enrolled from PMTCT programs increased. There was a decline in adults and children with advanced HIV disease at enrolment and initiation of stavudine. Pediatric age at enrolment and ART initiation declined. Results suggest HIV program maturation from an emergency response

Intracellular Trafficking and Synaptic Function of APL-1 in Caenorhabditis elegans

Background: Alzheimer’s disease (AD) is a neurodegenerative disorder primarily characterized by the deposition of b-amyloid plaques in the brain. Plaques are composed of the amyloid-b peptide derived from cleavage of the amyloid precursor protein (APP). Mutations in APP lead to the development of Familial Alzheimer’s Disease (FAD), however, the normal function of this protein has proven elusive. The organism Caenorhabditis elegans is an attractive model as the amyloid precursor-like protein (APL-1) is the single ortholog of APP, and loss of apl-1 leads to a severe molting defect and early larval lethality. Methodology/Principal Findings: We report here that lethality and molting can be rescued by full length APL-1, C-terminal mutations as well as a C-terminal truncation, suggesting that the extracellular region of the protein is essential for viability. RNAi knock-down of apl-1 followed by drug testing on the acetylcholinesterase inhibitor aldicarb showed that loss of apl-1 leads to aldicarb hypersensitivity, indicating a defect in synaptic function. The aldicarb hypersensitivity can be rescued by full length APL-1 in a dose dependent fashion. At the cellular level, kinesins UNC-104/KIF-1A and UNC-116/kinesin-1 are positive regulators of APL-1 expression in the neurons. Knock-down of the small GTPase rab-5 also leads to a dramatic decrease in the amount of apl-1 expression in neurons, suggesting that trafficking from the plasma membrane to the early endosome is important for apl-1 function. Loss of function of a different small GTPase, UNC-108, on the contrary, leads t

Substrate docking to γ-secretase allows access of γ-secretase modulators to an allosteric site

γ-Secretase generates the peptides of Alzheimer's disease, Aβ40 and Aβ42, by cleaving the amyloid precursor protein within its transmembrane domain. γ-Secretase also cleaves numerous other substrates, raising concerns about γ-secretase inhibitor off-target effects. Another important class of drugs, γ-secretase modulators, alter the cleavage site of γ-secretase on amyloid precursor protein, changing the Aβ42/Aβ40 ratio, and are thus a promising therapeutic approach for Alzheimer's disease. However, the target for γ-secretase modulators is uncertain, with some data suggesting that they function on γ-secretase, whereas others support their binding to the amyloid precursor. In this paper we address this controversy by using a fluorescence resonance energy transfer-based assay to examine whether γ-secretase modulators alter Presenilin-1/γ-secretase conformation in intact cells in the absence of its natural substrates such as amyloid precursor protein and Notch. We report that the γ-secretase allosteric site is located within the γ-secretase complex, but substrate docking is needed for γ-secretase modulators to access this site

Epidemic infectious gastrointestinal illness aboard U.S. Navy ships deployed to the Middle East during peacetime operations – 2000–2001

BACKGROUND: Infectious gastrointestinal illness (IGI) outbreaks have been reported in U.S. Navy ships and could potentially have an adverse mission impact. Studies to date have been anecdotal. METHODS: We conducted a retrospective analysis of weekly reported disease and non-battle injury health data collected in 2000 – 2001 from 44 U.S. Navy ships while sailing in the 5(th )Fleet (Persian Gulf and nearby seas). RESULTS: During this period, 11 possible IGI outbreaks were identified. Overall, we found 3.3 outbreaks per 100 ship-weeks, a mean outbreak duration of 4.4 weeks, and a mean cumulative ship population attack rate of 3.6%. Morbidity, represented by days lost due to personnel being placed on sick-in-quarters status, was higher during outbreak weeks compared to non-outbreak weeks (p = 0.002). No clear seasonal distribution was identified. CONCLUSION: Explosive outbreaks due to viruses and bacteria with the potential of incapacitating large proportions of the crew raise serious concerns of mission impact and military readiness

Real-Time Imaging and Quantification of Amyloid-β Peptide Aggregates by Novel Quantum-Dot Nanoprobes

Background: Protein aggregation plays a major role in the pathogenesis of neurodegenerative disorders, such as Alzheimer’s disease. However, direct real-time imaging of protein aggregation, including oligomerization and fibrillization, has never been achieved. Here we demonstrate the preparation of fluorescent semiconductor nanocrystal (quantum dot; QD)-labeled amyloid-b peptide (QDAb) and its advanced applications. Methodology/Principal Findings: The QDAb construct retained Ab oligomer-forming ability, and the sizes of these oligomers could be estimated from the relative fluorescence intensities of the imaged spots. Both QDAb coaggregation with intact Ab42 and insertion into fibrils were detected by fluorescence microscopy. The coaggregation process was observed by real-time 3D imaging using slit-scanning confocal microscopy, which showed a typical sigmoid curve with 1.5 h in the lag-time and 12 h until saturation. Inhibition of coaggregation using an anti-Ab antibody can be observed as 3D images on a microscopic scale. Microglia ingested monomeric QDAb more significantly than oligomeric QDAb, and the ingested QDAb was mainly accumulated in the lysosome. Conclusions/Significance: These data demonstrate that QDAb is a novel nanoprobe for studying Ab oligomerization an