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One of the key molecular events underlying the pathogenesis of Parkinson's disease (PD) is the aberrant misfolding and aggregation of the α-synuclein (αS) protein into higher-order oligomers that play a key role in neuronal dysfunction and degeneration. A wealth of experimental data supports the hypothesis that the neurotoxicity of αS oligomers is intrinsically linked with their ability to interact with, and disrupt, biological membranes; especially those membranes having negatively-charged surfaces and/or lipid packing defects. Consequences of αS–lipid interaction include increased membrane tension, permeation by pore formation, membrane lysis and/or leakage due to the extraction of lipids from the bilayer. Moreover, we assert that the interaction of αS with a liquid-disordering phospholipid uniquely enriched in mitochondrial membranes, namely cardiolipin (1,3-diphosphatidyl- sn -glycerol, CL), helps target the αS oligomeric complexes intracellularly to mitochondria. Binding mediated by CL may thus represent an important pathomechanism by which cytosolic αS could physically associate with mitochondrial membranes and disrupt their integrity. Impaired mitochondrial function culminates in a cellular bioenergetic crisis and apoptotic death. To conclude, we advocate the accelerated discovery of new drugs targeting this pathway in order to restore mitochondrial function in PD. [ABSTRACT FROM AUTHOR]

Pathogenic generation of amyloid β-peptide (Aβ) by sequential cleavage of β-amyloid precursor protein (APP) by β- and γ-secretases is widely believed to causally underlie Alzheimer disease (AD). β-Secretase initially cleaves APP thereby generating a membrane-bound APP C-terminal fragment, from which γ-secretase subsequently liberates 37-43-amino acid long Aβ species. Although the latter cleavages are intramembranous and although lipid alterations have been implicated in AD, little is known of how the γ-secretase-mediated release of the various Aβ species, in particular that of the pathogenic longer variants Aβ42 and Aβ43, is affected by the lipid environment. Using a cell-free system, we have directly and systematically investigated the activity of γ-secretase reconstituted in defined model membranes of different thicknesses. We found that bilayer thickness is a critical parameter affecting both total activity as well as cleavage specificity of γ-secretase. Whereas the generation of the pathogenic Aβ42/43 species was markedly attenuated in thick membranes, that of the major and rather benign Aβ40 species was enhanced. Moreover, the increased production of Aβ42/43 by familial AD mutants of presenilin 1, the catalytic subunit of γ-secretase, could be substantially lowered in thick membranes. Our data demonstrate an effective modulation of γ-secretase activity by membrane thickness, which may provide an approach to lower the generation of the pathogenic Aβ42/43 species. [ABSTRACT FROM AUTHOR]

Anti-amyloidogenic processing of the amyloid precursor protein APP by α-secretase prevents formation of the amyloid-β peptide, which accumulates in senile plaques of Alzheimer disease patients. α-Secretase belongs to the family of a disintegrin and metalloproteases (ADAMs), and ADAM10 is the primary candidate for this anti-amyloidogenic activity. We recently demonstrated that ADAM10 translation is repressed by its 5′-UTR and that in particular the first half of ADAM10 5′-UTR is responsible for translational repression. Here, we asked whether specific sequence motifs exist in the ADAM10 5′-UTR that are able to form complex secondary structures and thus potentially inhibit ADAM10 translation. Using circular dichroism spectroscopy, we demonstrate that a G-rich region between nucleotides 66 and 94 of the ADAM10 5′-UTR forms a highly stable, intramolecular, parallel G-quadruplex secondary structure under physiological conditions. Mutation of guanines in this sequence abrogates the formation of the G-quadruplex structure. Although the G-quadruplex structure efficiently inhibits translation of a luciferase reporter in in vitro translation assays and in living cells, inhibition of G-quadruplex formation fails to do so. Moreover, expression of ADAM10 was similarly repressed by the G-quadruplex. Mutation of the G-quadruplex motif results in a significant increase of ADAM10 levels and consequently APPsα secretion. Thus, we identified a critical RNA secondary structure within the 5′-UTR, which contributes to the translational repression of ADAM10. [ABSTRACT FROM AUTHOR]

Abstract: Opposing views exist as to how unesterified fatty acids (FA) enter and leave cells. It is commonly believed that for short- and medium-chain FA free diffusion suffices whereas it is questioned whether proteins are required to facilitate transport of long-chain fatty acid (LCFA). Furthermore, it is unclear whether these proteins facilitate binding to the plasma membrane, trans-membrane movement, dissociation into the cytosol and/or transport in the cytosol. In this mini-review we approach the controversy from a different point of view by focusing on the membrane permeability constant (P) of FA with different chain length. We compare experimentally derived values of the P of short and medium-chain FA with values of apparent permeability coefficients for LCFA calculated from their dissociation rate constant (k off), flip-flop rate constant (k flip) and partition coefficient (K p) in phospholipid bilayers. It was found that Overton''s rule is valid as long as k flip≪k off. With increasing chain length, the permeability increases according to increasing K p and reaches a maximum for LCFA with chain length of 18 carbons or longer. For fast flip-flop (e.g. k flip=15s−1), the apparent permeability constant for palmitic acid is very high (P app=1.61cm/s). Even for a slow flip-flop rate constant (e.g. k flip=0.3s−1), the permeability constant of LCFA is still several orders of magnitude larger than the P of water and other small non-electrolytes. Since polyunsaturated FA have basically the same physico-chemical properties as LCFA, they have similar membrane permeabilities. The implications for theories involving proteins to facilitate uptake of FA are discussed. [Copyright &y& Elsevier]

The intracellular deposition of fibrillar aggregates of α-synuclein is a characteristic feature of Parkinson disease. Alternatively, as a result of its unusual conformational plasticity, α-synuclein may fold into an amphipathic helix upon contact with a lipid-water interface. Using spin label ESR and fluorescence spectroscopy, we show here that α-synuclein affects the lipid packing in small unilamellar vesicles. The ESR hyperfine splittings of spin-labeled phospholipid probes revealed that a-synuclein induces chain ordering at carbon 14 of the acyl chains below the chain melting phase transition temperature but not in the liquid crystalline state of electroneutral vesicle membranes. Binding of α-synuclein leads to an increase in the temperature and cooperativity of the phase transition according to the fluorescence anisotropy of the hydrophohic polyene 1,6-diphenylhexatriene and of the fluorescence emission maxima of the amphiphilic probe 6-dodecanoyl-2-dimethylaminonaphthalene. Binding parameters were obtained from the fluorescence anisotropy measurements in combination with our previous determinations by titration calorimetry (Nuscher, B., Kamp, F., Mehnert, T., Odoy, S., Haass, C., Kahle, P. J., and Beyer, K. (2004) J. Biol. Chem. 279, 21966-21975). We also show that a-synuclein interacts with vesicle membranes containing sphingomyelin and cholesterol. We propose that the protein is capable of annealing defects in curved vesicle membranes, which may prevent synaptic vesicles from premature fusion. [ABSTRACT FROM AUTHOR]

A number of neurodegenerative disorders, including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, are characterized by the intracellular deposition of fibrillar aggregates that contain a high proportion of α-synuclein (αS). The interaction with the membrane-water interface strongly modulates folding and aggregation of the protein. The present study investigates the lipid binding and the coil-helix transition of αS, using titration calorimetry, differential scanning calorimetry, and circular dichroism spectroscopy. Titration of the protein with small unilamellar vesicles composed of zwitterionic phospholipids below the chain melting temperature of the lipids yielded exceptionally large exothermic heat values. The sigmoidal titration curves were evaluated in terms of a simple model that assumes saturable binding sites at the vesicle surface. The cumulative heat release and the ellipticity were linearly correlated as a result of simultaneous binding and helix folding. There was no heat release and folding of αS in the presence of large unilamellar vesicles, indicating that a small radius of curvature is necessary for the αS-membrane interaction. The heat release and the negative heat capacity of the proteinvesicle interaction could not be attributed to the coilhelix transition of the protein alone. We speculate that binding and helix folding of αS depends on the presence of defect structures in the membrane-water interface, which in turn results in lipid ordering in the highly curved vesicular membranes. This will be discussed with regard to a possible role of the protein for the stabilization of synaptic vesicle membranes. [ABSTRACT FROM AUTHOR]

Studies the rapid flip-flop of oleic acid across the plasma membrane. Binding of oleic acid to the plasma membrane by measuring the fluorescent fatty acid-binding protein; Measurement of changes in internal hydrogen-ion concentration caused by flip-flop and metabolism; Evaluation of the metabolic end products of oleic acid.

[Display omitted] • A validated sample preparation protocol for simultaneous GC–MS analysis of different steroid classes is presented. • The method allows untargeted screening as well as targeted analysis and quantification in brain and liver tissue and cultured cells. • Endogenous steroid concentrations of mouse brain, mouse liver and of cultured cells are reported. Steroids play an important role in cell regulation and homeostasis. Many diseases like Alzheimer's disease or Smith-Lemli-Opitz syndrome are known to be associated with deviations in the steroid profile. Most published methods only allow the analysis of small subgroups of steroids and cannot give an overview of the total steroid profile. We developed and validated a method that allows the analysis of free neutral steroids, including intermediates of cholesterol biosynthesis, free oxysterols, C 19 and C 21 steroids, free steroid acids, including bile acids, and sterol sulfates using gas chromatography-mass spectrometry. Samples were analyzed in scan mode for screening purposes and in dynamic multiple reaction monitoring mode for highly sensitive quantitative analysis. The method was validated for mouse brain and liver tissue and consists of sample homogenization, lipid extraction, steroid group separation, deconjugation, derivatization and gas chromatography-mass spectrometry analysis. We applied the method on brain and liver samples of mice (10 months and 3 weeks old) and cultured N2a cells and report the endogenous concentrations of 29 physiological steroids. [ABSTRACT FROM AUTHOR]

Imbalances in the amounts of amyloid-β peptides (Aβ) generated by the membrane proteases β- and γ-secretase are considered as a trigger of Alzheimer's disease (AD). Cell-free studies of γ-secretase have shown that increasing membrane thickness modulates Aβ generation but it has remained unclear if these effects are translatable to cells. Here we show that the very long-chain fatty acid erucic acid (EA) triggers acyl chain remodeling in AD cell models, resulting in substantial lipidome alterations which included increased esterification of EA in membrane lipids. Membrane remodeling enhanced γ-secretase processivity, resulting in the increased production of the potentially beneficial Aβ37 and/or Aβ38 species in multiple cell lines. Unexpectedly, we found that the membrane remodeling stimulated total Aβ secretion by cells expressing WT γ-secretase but lowered it for cells expressing an aggressive familial AD mutant γ-secretase. We conclude that EA-mediated modulation of membrane composition is accompanied by complex lipid homeostatic changes that can impact amyloidogenic processing in different ways and elicit distinct γ-secretase responses, providing critical implications for lipid-based AD treatment strategies. [ABSTRACT FROM AUTHOR]

GGGGCC (G4C2) repeat expansion in the C9orf72 gene has been shown to cause frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Dipeptide repeat proteins produced through repeat-associated non-AUG (RAN) translation are recognized as potential drivers for neurodegeneration. Therefore, selective inhibition of RAN translation could be a therapeutic avenue to treat these neurodegenerative diseases. It was previously known that the porphyrin TMPyP4 binds to G4C2 repeat RNA. However, the consequences of this interaction have not been well characterized. Here, we confirmed that TMPyP4 inhibits C9orf72 G4C2 repeat translation in cellular and in in vitro translation systems. An artificial insertion of an AUG codon failed to cancel the translation inhibition, suggesting that TMPyP4 acts downstream of non-AUG translation initiation. Polysome profiling assays also revealed polysome retention on G4C2 repeat RNA, along with inhibition of translation, indicating that elongating ribosomes stall on G4C2 repeat RNA. Urea-resistant interaction between G4C2 repeat RNA and TMPyP4 likely contributes to this ribosome stalling and thus to selective inhibition of RAN translation. Taken together, our data reveal a novel mode of action of TMPyP4 as an inhibitor of G4C2 repeat translation elongation. [ABSTRACT FROM AUTHOR]

Abstract: Cumulative evidence now suggests that the abnormal aggregation of the protein α-synuclein (αS) is a critical factor in triggering neurodegeneration in Parkinson''s disease (PD). In particular, a fundamental pathogenetic mechanism appears to involve targeting of neuronal membranes by soluble oligomeric intermediates of αS, leading to their disruption or permeabilisation. Therefore, a model assay was developed in which fluorophore-loaded unilamellar vesicles were permeabilised by soluble oligomers, the latter formed by aggregation of human recombinant αS protein. The αS oligomers induced an impairment of membrane integrity similar to that of the pore-forming bacterial peptide gramicidin. The lipid vesicle permeabilisation assay was then utilised to screen 11 natural polyphenolic compounds, 8 synthetic N′-benzylidene-benzohydrazide compounds and black tea extract for protection against membrane damage by wild-type and mutant (A30P, A53T) synuclein aggregates. A select group of potent inhibitory compounds included apigenin, baicalein, morin, nordihydroguaiaretic acid, and black tea extract. Structure–activity analysis further suggests that a 5,7-dihydroxy-chromen-4-one moiety appears to be favourable for the inhibition reaction. In conclusion, we have identified a group of polyphenols that can effectively hinder membrane damage by αS aggregates. These may serve as a viable source of lead compounds for the development and design of novel therapeutic agents in PD. [Copyright &y& Elsevier]

The biophysical properties and biological functions of membranes are highly dependent on lipid composition. Supplementing cellular membranes with very long chain fatty acids (vlcFAs) is notoriously difficult given the extreme insolubility of vlcFAs in aqueous solution. Herein, we report a solvent-free, photochemical approach to enrich target membranes with vlcFA. To prevent aggregation of vlcFA, we created light-sensitive micelles composed exclusively of poly-ethylene-glycol-nervonic acid amphiphiles (NA-PEG), which spontaneously disassemble in the presence of lipid bilayers. Once embedded within a membrane, UV light is used to cleave off PEG, leaving free nervonic acid (NA, i.e. FA24:1) in the target membrane. When applied to living cells, free NA was processed by the cell to generate various species of membrane and other lipids with incorporated vlcFAs. In this way, we were able to alter the membrane lipid composition of cellular membranes and modulate the enzymatic activity of γ-secretase, an intramembrane protease whose dysfunction has been implicated in the onset and progression of Alzheimer's disease. Unlabelled Image • Delivery of very long chain fatty acids to cell membranes • Light induced cleavage of fatty acid-PEG conjugates results in membrane modulation. • The enzymatic activity of γ-secretase is modulated by modifying the cell membrane. • The dysfunction of an intramembrane protease has been implicated in the onset and progression of Alzheimer disease. [ABSTRACT FROM AUTHOR]

• Oligomeric tau compromised the membrane integrity of isolated mitochondria. • Tau nanopores exhibited multi-level ion conductances in mito-mimetic bilayers. • Tau oligomers preferentially targeted membranes rich in cardiolipin. • NAO, a cardiolipin-specific dye, protected against damage by tau oligomers. Misfolding and aggregate formation by the tau protein has been closely related with neurotoxicity in a large group of human neurodegenerative disorders, which includes Alzheimer's disease. Here, we investigate the membrane-active properties of tau oligomers on mitochondrial membranes, using minimalist in vitro model systems. Thus, exposure of isolated mitochondria to oligomeric tau evoked a disruption of mitochondrial membrane integrity, as evidenced by a combination of organelle swelling, efflux of cytochrome c and loss of the mitochondrial membrane potential. Tau-induced mitochondrial dysfunction occurred independently of the mitochondrial permeability transition (mPT) pore complex. Notably, mitochondria were rescued by pre-incubation with 10-N-nonyl acridine orange (NAO), a molecule that specifically binds cardiolipin (CL), the signature phospholipid of mitochondrial membranes. Additionally, NAO prevented direct binding of tau oligomers to isolated mitochondria. At the same time, tau proteins exhibited high affinity to CL-enriched membranes, whilst permeabilisation of lipid vesicles also strongly correlated with CL content. Intriguingly, using single-channel electrophysiology, we could demonstrate the formation of non-selective ion-conducting tau nanopores exhibiting multilevel conductances in mito-mimetic bilayers. Taken together, the data presented here advances a scenario in which toxic cytosolic entities of tau protein would target mitochondrial organelles by associating with their CL-rich membrane domains, leading to membrane poration and compromised mitochondrial structural integrity. [ABSTRACT FROM AUTHOR]