Monoacylated Cellular Prion Proteins Reduce Amyloid-beta-Induced Activation of Cytoplasmic Phospholipase A2 and Synapse Damage

Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by the accumulation of amyloid-β (Aβ) and the loss of synapses. Aggregation of the cellular prion protein (PrPC) by Aβ oligomers induced synapse damage in cultured neurons. PrPC is attached to membranes via a glycosylphosphatidylinositol (GPI) anchor, the composition of which affects protein targeting and cell signaling. Monoacylated PrPC incorporated into neurons bound “natural Aβ”, sequestering Aβ outside lipid rafts and preventing its accumulation at synapses. The presence of monoacylated PrPC reduced the Aβ-induced activation of cytoplasmic phospholipase A2 (cPLA2) and Aβ-induced synapse damage. This protective effect was stimulus specific, as treated neurons remained sensitive to α-synuclein, a protein associated with synapse damage in Parkinson’s disease. In synaptosomes, the aggregation of PrPC by Aβ oligomers triggered the formation of a signaling complex containing the cPLA2.a process, disrupted by monoacylated PrPC. We propose that monoacylated PrPC acts as a molecular sponge, binding Aβ oligomers at the neuronal perikarya without activating cPLA2 or triggering synapse damage

Preventing Alzheimer's Disease

http://dx.doi.org/10.1126/science.122854

Recent pace of change in human impact on the world's ocean.

Humans interact with the oceans in diverse and profound ways. The scope, magnitude, footprint and ultimate cumulative impacts of human activities can threaten ocean ecosystems and have changed over time, resulting in new challenges and threats to marine ecosystems. A fundamental gap in understanding how humanity is affecting the oceans is our limited knowledge about the pace of change in cumulative impact on ocean ecosystems from expanding human activities - and the patterns, locations and drivers of most significant change. To help address this, we combined high resolution, annual data on the intensity of 14 human stressors and their impact on 21 marine ecosystems over 11 years (2003-2013) to assess pace of change in cumulative impacts on global oceans, where and how much that pace differs across the ocean, and which stressors and their impacts contribute most to those changes. We found that most of the ocean (59%) is experiencing significantly increasing cumulative impact, in particular due to climate change but also from fishing, land-based pollution and shipping. Nearly all countries saw increases in cumulative impacts in their coastal waters, as did all ecosystems, with coral reefs, seagrasses and mangroves at most risk. Mitigation of stressors most contributing to increases in overall cumulative impacts is urgently needed to sustain healthy oceans

A New Method for Quantitative Immunoblotting of Endogenous α-Synuclein

β-Sheet-rich aggregates of α-synuclein (αSyn) are the hallmark neuropathology of Parkinson’s disease and related synucleinopathies, whereas the principal native structure of αSyn in healthy cells - unfolded monomer or α-helically folded oligomer - is under debate. Our recent crosslinking analysis of αSyn in intact cells showed that a large portion of endogenous αSyn can be trapped as oligomers, most notably as apparent tetramers. One challenge in such studies is accurately quantifying αSyn Western blot signals among samples, as crosslinked αSyn trends toward increased immunoreactivity. Here, we analyzed this phenomenon in detail and found that treatment with the reducible amine-reactive crosslinker DSP strongly increased αSyn immunoreactivity even after cleavage with the reducing agent β-mercaptoethanol. The effect was observed with all αSyn antibodies tested and in all sample types from human brain homogenates to untransfected neuroblastoma cells, permitting easy detection of endogenous αSyn in the latter, which had long been considered impossible. Coomassie staining of blots before and after several hours of washing revealed complete retention of αSyn after DSP/β-mercaptoethanol treatment, in contrast to a marked loss of αSyn without this treatment. The treatment also enhanced immunodetection of the homologs β- and γ-synuclein and of histones, another group of small, lysine-rich proteins. We conclude that by neutralizing positive charges and increasing protein hydrophobicity, amine crosslinker treatment promotes adhesion of αSyn to blotting membranes. These data help explain the recent report of fixing αSyn blots with paraformaldehyde after transfer, which we find produces similar but weaker effects. DSP/β-mercaptoethanol treatment of Western blots should be particularly useful to quantify low-abundance αSyn forms such as extracellular and post-translationally modified αSyn and splice variants

N-Alpha-Acetylation of α-Synuclein Increases Its Helical Folding Propensity, GM1 Binding Specificity and Resistance to Aggregation

A switch in the conformational properties of α-synuclein (αS) is hypothesized to be a key step in the pathogenic mechanism of Parkinson’s disease (PD). Whereas the beta-sheet-rich state of αS has long been associated with its pathological aggregation in PD, a partially alpha-helical state was found to be related to physiological lipid binding; this suggests a potential role of the alpha-helical state in controlling synaptic vesicle cycling and resistance to β-sheet rich aggregation. N-terminal acetylation is the predominant post-translational modification of mammalian αS. Using circular dichroism, isothermal titration calorimetry, and fluorescence spectroscopy, we have analyzed the effects of N-terminal acetylation on the propensity of recombinant human αS to form the two conformational states in interaction with lipid membranes. Small unilamellar vesicles of negatively charged lipids served as model membranes. Consistent with previous NMR studies using phosphatidylserine, we found that membrane-induced α-helical folding was enhanced by N-terminal acetylation and that greater exothermic heat could be measured upon vesicle binding of the modified protein. Interestingly, the folding and lipid binding enhancements with phosphatidylserine in vitro were weak when compared to that of αS with GM1, a lipid enriched in presynaptic membranes. The resultant increase in helical folding propensity of N-acetylated αS enhanced its resistance to aggregation. Our findings demonstrate the significance of the extreme N-terminus for folding nucleation, for relative GM1 specificity of αS-membrane interaction, and for a protective function of N-terminal-acetylation against αS aggregation mediated by GM1

Proteomic Profiling of γ-Secretase Substrates and Mapping of Substrate Requirements

The presenilin/γ-secretase complex, an unusual intramembrane aspartyl protease, plays an essential role in cellular signaling and membrane protein turnover. Its ability to liberate numerous intracellular signaling proteins from the membrane and also mediate the secretion of amyloid-β protein (Aβ) has made modulation of γ-secretase activity a therapeutic goal for cancer and Alzheimer disease. Although the proteolysis of the prototypical substrates Notch and β-amyloid precursor protein (APP) has been intensely studied, the full spectrum of substrates and the determinants that make a transmembrane protein a substrate remain unclear. Using an unbiased approach to substrate identification, we surveyed the proteome of a human cell line for targets of γ-secretase and found a relatively small population of new substrates, all of which are type I transmembrane proteins but have diverse biological roles. By comparing these substrates to type I proteins not regulated by γ-secretase, we determined that besides a short ectodomain, γ-secretase requires permissive transmembrane and cytoplasmic domains to bind and cleave its substrates. In addition, we provide evidence for at least two mechanisms that can target a substrate for γ cleavage: one in which a substrate with a short ectodomain is directly cleaved independent of sheddase association, and a second where a substrate requires ectodomain shedding to instruct subsequent γ-secretase processing. These findings expand our understanding of the mechanisms of substrate selection as well as the diverse cellular processes to which γ-secretase contributes

A Coalescent Sampler Successfully Detects Biologically Meaningful Population Structure Overlooked by F‐Statistics

Assessing the geographic structure of populations has relied heavily on Sewell Wright\u27s F‐statistics and their numerous analogues for many decades. However, it is well appreciated that, due to their nonlinear relationship with gene flow, F statistics frequently fail to reject the null model of panmixia in species with relatively high levels of gene flow and large population sizes. Coalescent genealogy samplers instead allow a model‐selection approach to the characterization of population structure, thereby providing the opportunity for stronger inference. Here, we validate the use of coalescent samplers in a high gene flow context using simulations of a stepping‐stone model. In an example case study, we then re‐analyze genetic datasets from 41 marine species sampled from throughout the Hawaiian archipelago using coalescent model selection. Due to the archipelago\u27s linear nature, it is expected that most species will conform to some sort of stepping‐stone model (leading to an expected pattern of isolation by distance), but F‐statistics have only supported this inference in ~10% of these datasets. Our simulation analysis shows that a coalescent sampler can make a correct inference of stepping‐stone gene flow in nearly 100% of cases where gene flow is ≤100 migrants per generation (equivalent to FST = 0.002), while F‐statistics had mixed results. Our re‐analysis of empirical datasets found that nearly 70% of datasets with an unambiguous result fit a stepping‐stone model with varying population sizes and rates of gene flow, although 37% of datasets yielded ambiguous results. Together, our results demonstrate that coalescent samplers hold great promise for detecting weak but meaningful population structure, and defining appropriate management units

Hydrophilic loop 1 of Presenilin-1 and the APP GxxxG transmembrane motif regulate γ-secretase function in generating Alzheimer-causing Aβ peptides

γ-Secretase is responsible for the proteolysis of amyloid precursor protein (APP) into amyloid-beta (Aβ) peptides, which are centrally implicated in the pathogenesis of Alzheimer’s disease (AD). The biochemical mechanism of how processing by γ-secretase is regulated, especially as regards the interaction between enzyme and substrate, remains largely unknown. Here, mutagenesis reveals that the hydrophilic loop-1 (HL-1) of presenilin-1 (PS1) is critical for both γ-secretase step-wise cleavages (processivity) and its allosteric modulation by heterocyclic γ-modulatory compounds. Systematic mutagenesis of HL-1, including all of its familial AD mutations and additional engineered variants, and quantification of the resultant Aβ products show that HL-1 is necessary for proper sequential γ-secretase processivity. We identify Y106, L113, and Y115 in HL-1 as key targets for heterocyclic γ-secretase modulators (GSMs) to stimulate processing of pathogenic Aβ peptides. Further, we confirm that the GxxxG domain in the APP transmembrane region functions as a critical substrate motif for γ-secretase processivity: a G29A substitution in APP-C99 mimics the beneficial effects of GSMs. Together, these findings provide a molecular basis for the structural regulation of γ-processivity by enzyme and substrate, facilitating the rational design of new GSMs that lower AD-initiating amyloidogenic Aβ peptides