Opposing functions for retromer and Rab11 in extracellular vesicle traffic at presynaptic terminals

Neuronal extracellular vesicles (EVs) play important roles in intercellular communication and pathogenic protein propagation in neurological disease. However, it remains unclear how cargoes are selectively packaged into neuronal EVs. Here, we show that loss of the endosomal retromer complex leads to accumulation of EV cargoes including amyloid precursor protein (APP), synaptotagmin-4 (Syt4), and neuroglian (Nrg) at Drosophila motor neuron presynaptic terminals, resulting in increased release of these cargoes in EVs. By systematically exploring known retromer-dependent trafficking mechanisms, we show that EV regulation is separable from several previously identified roles of neuronal retromer. Conversely, mutations in rab11 and rab4, regulators of endosome-plasma membrane recycling, cause reduced EV cargo levels, and rab11 suppresses cargo accumulation in retromer mutants. Thus, EV traffic reflects a balance between Rab4/Rab11 recycling and retromer-dependent removal from EV precursor compartments. Our data shed light on previous studies implicating Rab11 and retromer in competing pathways in Alzheimer’s disease, and suggest that misregulated EV traffic may be an underlying defect.     

Molecular evidence that the deadliest sea snake Enhydrina schistosa (Elapidae: Hydrophiinae) consists of two convergent species

We present a striking case of phenotypic convergence within the speciose and taxonomically unstable Hydrophis group of viviparous sea snakes. Enhydrina schistosa, the ‘beaked sea snake’, is abundant in coastal and inshore habitats throughout the Asian and Australian regions, where it is responsible for the large majority of recorded deaths and injuries from sea snake bites. Analyses of five independent mitochondrial and nuclear loci for populations spanning Australia, Indonesia and Sri Lanka indicate that this ‘species’ actually consists of two distinct lineages in Asia and Australia that are not closest relatives. As a result, Australian “E. schistosa” are elevated to species status and provisionally referred to Enhydrina zweifeli. Convergence in the characteristic ‘beaked’ morphology of these species is probably associated with the wide gape required to accommodate their spiny prey. Our findings have important implications for snake bite management in light of the medical importance of beaked sea snakes and the fact that the only sea snake anti-venom available is raised against Malaysian E. schistosa.     

The physical chemistry of cytoplasm and its influence on cell function: an update

From the point of view of intermolecular interactions, the cytoplasmic space is more like a crowded party in a house full of furniture than a game of tag in an empty field. Understanding the physical chemical properties of cytoplasm is thus of key importance for understanding cellular function. This article attempts to provide an entrée into the current literature on this subject and offers some general guidelines for thinking about intracellular biochemistry.     

Chronic antioxidant therapy reduces oxidative stress in a mouse model of Alzheimer's disease

Oxidative modifications are a hallmark of oxidative imbalance in the brains of individuals with Alzheimer's, Parkinson's and prion diseases and their respective animal models. While the causes of oxidative stress are relatively well-documented, the effects of chronically reducing oxidative stress on cognition, pathology and biochemistry require further clarification. To address this, young and aged control and amyloid-β protein precursor-over-expressing mice were fed a diet with added R-alpha lipoic acid for 10 months to determine the effect of chronic antioxidant administration on the cognition and neuropathology and biochemistry of the brain. Both wild type and transgenic mice treated with R-alpha lipoic acid displayed significant reductions in markers of oxidative modifications. On the other hand, R-alpha lipoic acid had little effect on Y-maze performance throughout the study and did not decrease end-point amyloid-β load. These results suggest that, despite the clear role of oxidative stress in mediating amyloid pathology and cognitive decline in ageing and AβPP-transgenic mice, long-term antioxidant therapy, at levels within tolerable nutritional guidelines and which reduce oxidative modifications, have limited benefit.     

Interrogating cAMP-dependent Kinase Signaling in Jurkat T Cells via a Protein Kinase A Targeted Immune-precipitation Phosphoproteomics Approach

In the past decade, mass-spectrometry-based methods have emerged for the quantitative profiling of dynamic changes in protein phosphorylation, allowing the behavior of thousands of phosphorylation sites to be monitored in a single experiment. However, when one is interested in specific signaling pathways, such shotgun methodologies are not ideal because they lack selectivity and are not cost and time efficient with respect to instrument and data analysis time.Here we evaluate and explore a peptide-centric antibody generated to selectively enrich peptides containing the cAMP-dependent protein kinase (PKA) consensus motif. This targeted phosphoproteomic strategy is used to profile temporal quantitative changes of potential PKA substrates in Jurkat T lymphocytes upon prostaglandin E₂ (PGE₂) stimulation, which increases intracellular cAMP, activating PKA. Our method combines ultra-high-specificity motif-based immunoaffinity purification with cost-efficient stable isotope dimethyl labeling. We identified 655 phosphopeptides, of which 642 (i.e. 98%) contained the consensus motif [R/K][R/K/X]X[pS/pT]. When our data were compared with a large-scale Jurkat T-lymphocyte phosphoproteomics dataset containing more than 10,500 phosphosites, a minimal overlap of 0.2% was observed. This stresses the need for such targeted analyses when the interest is in a particular kinase.Our data provide a resource of likely substrates of PKA, and potentially some substrates of closely related kinases. Network analysis revealed that about half of the observed substrates have been implicated in cAMP-induced signaling. Still, the other half of the here-identified substrates have been less well characterized, representing a valuable resource for future research.The identification and quantification of protein phosphorylation under system perturbations is an integral part of systems biology (1, 2). The combination of phosphopeptide enrichment (3–6), stable isotope labeling, and high-resolution mass spectrometry (MS) methods (7–9) has become the method of choice for the identification of novel phosphorylation sites and for the quantitation of temporal dynamics within signaling networks (10, 11), allowing the behavior of thousands of phosphorylation sites to be studied in a single experiment (10, 12, 13). Nowadays, one of the most commonly adopted high-throughput phosphoproteomics strategies utilizes two consecutive separation steps: (i) an initial fractionation to reduce the sample complexity, and (ii) a phosphopeptide-specific affinity purification. Such techniques include strong cation exchange fractionation under acidic conditions (3), followed by a chelation-based method with the use of metal ions (i.e. immobilized metal ion affinity chromatography (4), metal oxide affinity chromatography (10, 14), or Ti⁴⁺ immobilized metal ion affinity chromatography (6)). Alternatives to strong cation exchange for the first sample fractionation step have also been reported, including the use of electrostatic repulsion liquid chromatography (15, 16), which is well suited for the identification of multiply phosphorylated peptides, or hydrophilic interaction chromatography (17).Although the number of detected phosphorylated peptides is nowadays impressive, these kinds of methodologies are still inclined to identify/quantify the more abundant phosphoproteins present in a sample. For example, phosphotyrosine peptides are underrepresented because of their relatively lower abundance.In order to analyze key signaling events that may occur on less abundant phosphoproteins, more targeted approaches, focused on a specific pathway or a specific post-translational modification, are thus still essential. Studies examining post-translational modifications are often based on immunoaffinity purification at the protein or peptide level using dedicated antibodies. Recent examples include the selective enrichment of acetylated lysines (18) and phosphorylated tyrosines (19, 20). More recently, the first specific methods targeting serine/threonine phosphorylation motifs using immune-affinity assays have emerged (21, 22). The advantages of targeted approaches are their potentially higher sensitivity and more specific throughput with, as a consequence, relatively faster and easier data interpretation, which make them attractive for many systems biology applications.Immunoaffinity enrichment can be applied at both the protein and the peptide level, and both have been explored to study protein tyrosine phosphorylation (23). The first one results mainly in information on total protein phosphorylation levels. The detection of the actual phosphoresidue might be hampered by the high content of unmodified peptides derived from the immune-purified phosphoprotein and its binding partners. Immunoprecipitation at the peptide level (20, 24, 25), in contrast, leads to improved phosphosite characterization, with the identification of hundreds of sites, albeit with the loss (generally) of information regarding total protein expression.To profile the dynamic regulation of phosphorylation events via mass spectrometry, stable isotope labeling is often implemented, either with the use of amino acids in cell culture (10) or via chemical peptide labeling of the proteolytic digests (26, 27). To identify low-abundant signaling events, phosphoprotein/phosphopeptide immunoprecipitation is typically performed on several milligrams of material because of the substoichiometric abundance of post-translational modifications. This may hamper the use of expensive isotope-labeling reagents such as iTRAQ or tandem mass tag reagents, given the large amount of chemicals needed. Boersema et al. (28) introduced an alternative sensitive and accurate triplex labeling approach using inexpensive reagents (i.e. formaldehyde) that is much less limited in terms of the sample type or amount. We combined this latter stable-isotope dimethyl labeling approach (27–29) with highly specific antibodies raised against a set of cAMP-dependent protein kinase (PKA) phosphorylated substrates as based on the current literature (11, 30–34). It is generally accepted that PKA phosphorylates sites with the reasonably stringent consensus motif [R/K][R/K/X]X[pS/pT]. It should be noted that this consensus motif resembles somewhat the motifs of other AGC kinases (e.g. Akt, PKG, PKC).The basicity of the PKA motifs may hamper their analysis via MS-based proteomics, especially when trypsin is used as a protease, as the peptides may become too small to be sequenced. The use of trypsin is also unfavorable in the approach presented here when attempting to immunoprecipitate peptides bearing the PKA motif. Therefore, we decided to use Lys-C in order to keep the (dominant (RRX[pS/pT])) phosphorylated motif intact. To enhance identification, we applied decision-tree MS/MS technology (9), which makes use of HCD and ETD for more efficient fragmentation, higher mass accuracy in tandem MS mode, and less background noise (35).We applied this method to screen the response of Jurkat T cells to prostaglandin E₂ (PGE₂) treatment. PGE₂ is a potent inflammatory mediator that plays an important role in several immune-regulatory actions (36). It is produced by many different cell types, including tumor cells, where carcinogenesis is associated with chronic inflammatory responses (37). PGE₂ signaling in T cells is initiated by its binding to the G protein–coupled receptors EP1, -2, -3, and -4. Signaling pathways that are initiated by PGE₂ are for the most part under control of the second messenger cyclic adenosine monophosphate (cAMP),¹ which is generated from ATP by adenylyl cyclase when PGE₂ binds to EP2 or EP4 receptors. One of the primary targets of cAMP is PKA—cAMP binding releases the catalytic subunit activating the kinase. In the current study, we efficiently enriched close to 650 phosphopeptides containing the [R/K][R/K/X]X[pS/pT] consensus motif. Almost all these sites were absent in a recently reported comprehensive phosphoproteomics dataset of Jurkat T cells (12), compiled using shotgun strong cation exchange–immobilized metal ion affinity chromatography analysis and containing ∼10,500 phosphorylation sites, illustrative of the complementarity and selectivity of our approach. The qualitative and quantitative data presented here provide a wide-ranging and credible resource of likely PKA substrates. Network analysis confirmed several established cAMP-dependent signaling nodes in our dataset, although most identified potential PKA substrates are “novel” (i.e. not previously reported and/or linked to PKA). Therefore, the dataset presented here can be considered as a comprehensive and reliable resource for future research into cAMP-related signaling.     

Live fluorescence and transmission-through-dye microscopic study of actinomycin D-induced apoptosis and apoptotic volume decrease

The effect of actinomycin D on HeLa cells was studied by live fluorescence and transmission-through-dye microscopy—a recently developed technique that permits volume measurements in live cells. In particular, it is well suited for the observation and quantification of the apoptotic volume decrease (AVD), which is widely viewed as an essential feature of apoptosis. The main results from our study are as follows. (1) Apoptosis caused in HeLa cells by actinomycin D proceeds in two morphologically distinct stages: the early stage is characterized by extensive blebbing, and the late stage by a more compact shape. The loss of mitochondrial membrane potential occurs at about the same time as blebbing, and chromatin condensation follows 30–90 min later. Caspase-3 and 7 become activated during the late stage. (2) Because blebbing occurs before activation of caspase-3, it has to be initiated by a different mechanism. Although blebbing is one of the earliest observable changes, it can be selectively inhibited without affecting other apoptotic reactions. (3) The majority of cells experience a temporary volume increase after the appearance of blebs. Eventually, AVD takes over and the cells shrink by approximately 40 % of their initial volume; the volume loss becomes noticeable at the end of the blebbing phase and continues through the late stage. Sometimes, at the end of long incubations, shrinkage gives way to swelling, possibly indicating secondary necrosis. (4) Both early and late apoptosis are accompanied by intracellular accumulation of Na⁺, while low-sodium medium prevents apoptosis. Except for a partial protective effect of quinine, all of the tested blockers of Na⁺, K⁺ and Cl^? channels failed to prevent apoptosis or AVD.     

Diagnostics method for the rapid quantitative detection and identification of low-level contamination of high-purity water with pathogenic bacteria

High-purity water (HPW) can be contaminated with pathogenic microorganisms, which may result in human infection. Current culture-based techniques for the detection of microorganisms from HPW can be slow and laborious. The aim of this study was to develop a rapid method for the quantitative detection and identification of pathogenic bacteria causing low-level contamination of HPW. A novel internally controlled multiplex real-time PCR diagnostics assay was designed and optimized to specifically detect and identify Pseudomonas aeruginosa and the Burkholderia genus. Sterile HPW, spiked with a bacterial load ranging from 10 to 10³ cfu/100 ml, was filtered and the bacterial cells were removed from the filters by sonication. Total genomic DNA was then purified from these bacteria and subjected to testing with the developed novel multiplex real-time PCR diagnostics assay. The specific P. aeruginosa and Burkholderia genus assays have an analytical sensitivity of 3.5 genome equivalents (GE) and 3.7 GE, respectively. This analysis demonstrated that it was possible to detect a spiked bacterial load of 1.06 × 10² cfu/100 ml for P. aeruginosa and 2.66 × 10² cfu/100 ml for B. cepacia from a 200-ml filtered HPW sample. The rapid diagnostics method described can reliably detect, identify, and quantify low-level contamination of HPW with P. aeruginosa and the Burkholderia genus in <4 h. We propose that this rapid diagnostics method could be applied to the pharmaceutical and clinical sectors to assure the safety and quality of HPW, medical devices, and patient-care equipment.