Docosahexaenoic Acid-Derived Neuroprotectin D1 Induces Neuronal Survival via Secretase- and PPARγ-Mediated Mechanisms in Alzheimer's Disease Models

Neuroprotectin D1 (NPD1) is a stereoselective mediator derived from the omega-3 essential fatty acid docosahexaenoic acid (DHA) with potent inflammatory resolving and neuroprotective bioactivity. NPD1 reduces Aβ42 peptide release from aging human brain cells and is severely depleted in Alzheimer's disease (AD) brain. Here we further characterize the mechanism of NPD1's neurogenic actions using 3xTg-AD mouse models and human neuronal-glial (HNG) cells in primary culture, either challenged with Aβ42 oligomeric peptide, or transfected with beta amyloid precursor protein (βAPP)sw (Swedish double mutation APP695sw, K595N-M596L). We also show that NPD1 downregulates Aβ42-triggered expression of the pro-inflammatory enzyme cyclooxygenase-2 (COX-2) and of B-94 (a TNF-α-inducible pro-inflammatory element) and apoptosis in HNG cells. Moreover, NPD1 suppresses Aβ42 peptide shedding by down-regulating β-secretase-1 (BACE1) while activating the α-secretase ADAM10 and up-regulating sAPPα, thus shifting the cleavage of βAPP holoenzyme from an amyloidogenic into the non-amyloidogenic pathway. Use of the thiazolidinedione peroxisome proliferator-activated receptor gamma (PPARγ) agonist rosiglitazone, the irreversible PPARγ antagonist GW9662, and overexpressing PPARγ suggests that the NPD1-mediated down-regulation of BACE1 and Aβ42 peptide release is PPARγ-dependent. In conclusion, NPD1 bioactivity potently down regulates inflammatory signaling, amyloidogenic APP cleavage and apoptosis, underscoring the potential of this lipid mediator to rescue human brain cells in early stages of neurodegenerations

Alzheimer disease models and human neuropathology: similarities and differences

Animal models aim to replicate the symptoms, the lesions or the cause(s) of Alzheimer disease. Numerous mouse transgenic lines have now succeeded in partially reproducing its lesions: the extracellular deposits of Aβ peptide and the intracellular accumulation of tau protein. Mutated human APP transgenes result in the deposition of Aβ peptide, similar but not identical to the Aβ peptide of human senile plaque. Amyloid angiopathy is common. Besides the deposition of Aβ, axon dystrophy and alteration of dendrites have been observed. All of the mutations cause an increase in Aβ 42 levels, except for the Arctic mutation, which alters the Aβ sequence itself. Overexpressing wild-type APP alone (as in the murine models of human trisomy 21) causes no Aβ deposition in most mouse lines. Doubly (APP × mutated PS1) transgenic mice develop the lesions earlier. Transgenic mice in which BACE1 has been knocked out or overexpressed have been produced, as well as lines with altered expression of neprilysin, the main degrading enzyme of Aβ. The APP transgenic mice have raised new questions concerning the mechanisms of neuronal loss, the accumulation of Aβ in the cell body of the neurons, inflammation and gliosis, and the dendritic alterations. They have allowed some insight to be gained into the kinetics of the changes. The connection between the symptoms, the lesions and the increase in Aβ oligomers has been found to be difficult to unravel. Neurofibrillary tangles are only found in mouse lines that overexpress mutated tau or human tau on a murine tau −/− background. A triply transgenic model (mutated APP, PS1 and tau) recapitulates the alterations seen in AD but its physiological relevance may be discussed. A number of modulators of Aβ or of tau accumulation have been tested. A transgenic model may be analyzed at three levels at least (symptoms, lesions, cause of the disease), and a reading key is proposed to summarize this analysis

Determination of internal volume and volume distribution of lipid vesicles from dynamic light scattering data

The total internal volume, enclosed by the spherical, unilamellar membranes of a lipid vesicle dispersion, is calculated from the vesicle's size distribution according to the relationships derived here. The size distribution of phosphatidylcholine vesicles is determined from dynamic light scattering measurements. Mean vesicle radii of about 100 nm and rather symmetric distributions with standard deviations of about 25 nm are characteristic of vesicles being prepared by dialysis of mixed octyle glucoside/egg yolk phosphatidylcholine micelles. A total internal volume of about 6 ml per mmol of phospholipid has been determined from the dynamic light scattering data. This is in good agreement with the results obtained from determinations of the amount of 5(6)-carboxyfluorescein trapped by the vesicles employing absorption- and fluorescence spectroscopy, belonging to the hitherto commonly used methods. In addition, the new procedure is applied to determine the density distribution of the volume enclosed by the vesicles of a given radius as well as the number density distribution of vesicles of a given internal volume, required for a detailed interpretation of flux measurements. The latter distribution with standard deviations around 3 · 106 nm3 is rather unsymmetric with respect to the corresponding mean value of the internal volume enclosed by a single vesicle, which is here typically about 4 · 106 nm3

Iodinated photoreactive vasopressin antagonists: labelling of hepatic vasopressin receptor subunits

To identify and characterize V1 vasopressin receptors, photoreactive antagonists of the glycogenolytic and vasoconstrictor activity of vasopressin have been synthesized. The following analogues with 3‐mercapto‐ 3,3‐cyclopentamethylene‐propionic acid (Mca) and N‐methylalanine (MeAla) in position 1 and 7 of vasopressin (VP) were effective V1 antagonists: [Mca1,D‐Tyr2,MeAla7,Lys8]VP (l), [Mca1,MeAla7,Arg8,Lys9]VP (2), [Mca1,MeAla7,Arg8,d‐Lys9]VP (3). Introduction of the photoreactive 4‐azidophenylamidino group into the side‐ chain of Lyss8 in analogue 1 or into Lys9 in analogues 2 and 3 increased the potency (for analogue 1 a tenfold increase in the antiglycogenolytic effect and a fivefold increase in the antivasopressor effect) and binding affinity for the rat hepatic V1 receptor. Mono‐iodination at Tyr2 with 125I resulted in photoreactive antagonists of high specific radioactivity, which had roughly the same binding affinity as vasopressin for the rat hepatic V1 receptor (Kd= 0.9—1.8 nM). In photoaffinity labelling experiments with purified rat liver membranes, containing 2–3 pmol V1 receptor/ mg protein, the analogues labelled specifically two proteins with the relative molecular masses (Mr) of 30000 and 38 000. These results and the results of a recent study using 3H‐labelled photoreactive vasopressin agonists [Boer, R. and Fahrenholz, F. (1985) J. Bid. Chem. 260, 15051 ‐ 150541 provide evidence that both vasopressin agonists and antagonists can interact with the same two subunits of the heterodimeric hepatic V1 receptor. Furthermore the radioiodinated photoreactive V1 antagonists should be helpful to identify V1 receptor proteins in membranes of other cell types

Asymmetric reconstitution of the erythrocyte anion transport system in vesicles of different curvature: implications for the shape of the band 3 protein

The anion transport protein of the human erythrocyte membrane, band 3, was solubilized and purified in solutions of the non-ionic detergent nonaethylene glycol lauryl ether and then reconstituted in spherical egg phosphatidylcholine bilayers as described earlier (U. Scheuring, K. Kollewe, W. Haase, and D. Schubert, J. Membrane Biol. 90, 123-135 (1986)). The resulting paucilamellar proteoliposomes of average diameter 70 nm were transformed into smaller vesicles by French press treatment and fractionated according to size by gel filtration. The smallest protein-containing liposomes obtained had diameters around 32 nm; still smaller vesicles were free of protein. All proteoliposome samples studied showed a rapid sulfate efflux which was sensitive to specific inhibitors of band 3-mediated anion exchange. In addition, the orientation of the transport protein in the vesicle membranes was found to be "right-side-out" in all samples. This suggests that the orientation of the protein in the vesicle membranes is dictated by the shape of the protein's intramembrane domain and that this domain has the form of a truncated cone or pyramid

Conformation of [8‐arginine]vasopressin and V₁ antagonists in dimethyl sulfoxide solution derived from two‐dimensional NMR spectroscopy and molecular dynamics simulation

Structural and dynamic properties of [8‐arginine]vasopressin and a class of highly potent vasopressin V1 antagonists which contain 3‐mercepto‐3, 3‐cyclopentamethylene propionic acid (Mca) in position 1 of the vasopressin sequence have been determined. On the basis of two‐dimensional NMR experiments in dimethyl sulfoxide solution, interproton distances were derived according to which model conformations were built and refined using molecular dynamics simulations. The antagonistic property was found to be related to an inversed chirality of the disulfide bridge. In all investigated molecules, characteristic β‐turn structure elements were found for the backbone conformation of the endocyclic residues Tyr2–Asn5. For this portion of the peptide sequence, various conformational equilibria were detected which matched different time scales. For [Arg8]vasopressin, averaged NMR parameters were obtained which could be explained by rapid interconversion between different β‐turn geometries, whereas multiple slowly exchanging conformations were observed for the V1 antagonists. V1 antagonists containing sarcosine in position 7 exhibited multiple spectral patterns for the exocyclic part attributed tocis/trans isomerization. The X‐ray structure of deamino‐oxytocin [Wood, S. P., Tickle, I. J., Treharne, A. M., Pitts, J. E., Mascarenhas, Y., Li, J. Y., Husain, J., Cooper, S., Blundell, T. L., Hruby, V. J., Buku, A., Fischman, A. J. & Wyssbrod, H. R. (1986) Science 232, 633–636] was found to represent one sample of the conformational space covered by the multiple conformations found for [Mca1, Arg8]vasopressin

Arginine-vasopressin analogues with high antidiuretic/vasopressor selectivity. Synthesis, biological activity, and receptor binding affinity of arginine-vasopressin analogues with substitutions in positions 1, 2, 4, 7, and 8

In a search for more selective agonists of arginine-vasopressin (AVP), 10 analogues of [Sar7]- and [MeLa7]AVP with additional substitutions in positions 1 (beta-mercaptopropionic acid), 2 (phenylalanine), 4 (valine), or 8 (D-arginine) were synthesized and tested for antidiuretic and vasopressor activities. All analogues are characterized by a relatively high antidiuretic activity and by a sharp decrease in pressor activity. Their antidiuretic/vasopressor (A/P) selectivities were 2-3 orders higher (except for peptides 2 and 3) than that of the parent hormone. The additivity of the effects of changes in positions 1, 2, 4, and 8 combined with the sarcosine or N-methylalanine substitutions in position 7 on the biological activity is observed. Binding affinities of AVP analogues to plasma membranes from bovine kidney inner medulla and from rat liver containing specific vasopressin receptors were also determined. Generally, these analogues retained high binding affinities to renal vasopressin receptors, and on the other hand they are characterized by a large decrease in binding affinities to hepatic vasopressin receptors, which share characteristics with vasopressor receptors