Synergistic interactions between Alzheimer's Aß40 and Aß42 on the surface of primary neurons revealed by single molecule microscopy.

Two amyloid-ß peptides (Aß40 and Aß42) feature prominently in the extracellular brain deposits associated with Alzheimer's disease. While Aß40 is the prevalent form in the cerebrospinal fluid, the fraction of Aß42 increases in the amyloid deposits over the course of disease development. The low in vivo concentration (pM-nM) and metastable nature of Aß oligomers have made identification of their size, composition, cellular binding sites and mechanism of action challenging and elusive. Furthermore, recent studies have suggested that synergistic effects between Aß40 and Aß42 alter both the formation and stability of various peptide oligomers as well as their cytotoxicity. These studies often utilized Aß oligomers that were prepared in solution and at µM peptide concentrations. The current work was performed using physiological Aß concentrations and single-molecule microscopy to follow peptide binding and association on primary cultured neurons. When the cells were exposed to a 1:1 mixture of nM Aß40:Aß42, significantly larger membrane-bound oligomers developed compared to those formed from either peptide alone. Fluorescence resonance energy transfer experiments at the single molecule level reveal that these larger oligomers contained both Aß40 and Aß42, but that the growth of these oligomers was predominantly by addition of Aß42. Both pure peptides form very few oligomers larger than dimers, but either membrane bound Aß40/42 complex, or Aß40, bind Aß42 to form increasingly larger oligomers. These findings may explain how Aß42-dominant oligomers, suspected of being more cytotoxic, develop on the neuronal membrane under physiological conditions.

Single-molecule imaging reveals aß42:aß40 ratio-dependent oligomer growth on neuronal processes.

Soluble oligomers of the amyloid-ß peptide have been implicated as proximal neurotoxins in Alzheimer's disease. However, the identity of the neurotoxic aggregate(s) and the mechanisms by which these species induce neuronal dysfunction remain uncertain. Physiologically relevant experimentation is hindered by the low endogenous concentrations of the peptide, the metastability of Aß oligomers, and the wide range of observed interactions between Aß and biological membranes. Single-molecule microscopy represents one avenue for overcoming these challenges. Using this technique, we find that Aß binds to primary rat hippocampal neurons at physiological concentrations. Although amyloid-ß(1-40) as well as amyloid-ß(1-42) initially form larger oligomers on neurites than on glass slides, a 1:1 mix of the two peptides result in smaller neurite-bound oligomers than those detected on-slide or for either peptide alone. With 1 nM peptide in solution, Aß40 oligomers do not grow over the course of 48 h, Aß42 oligomers grow slightly, and oligomers of a 1:1 mix grow substantially. Evidently, small Aß oligomers are capable of binding to neurons at physiological concentrations and grow at rates dependent on local Aß42:Aß40 ratios. These results are intriguing in light of the increased Aß42:Aß40 ratios shown to correlate with familial Alzheimer's disease mutations.

Plasma 3-nitrotyrosine and outcome in neonates with severe bronchopulmonary dysplasia after inhaled nitric oxide.

Plasma protein levels of 3-nitrotyrosine and 3-chlorotyrosine were measured by LC-MS/MS at 0 and 72 h after the initiation of inhaled nitric oxide (INO) at 20 ppm in 22 prematurely born infants with clinically documented bronchopulmonary dysplasia. Infants were classified at the time of hospital discharge as either "off mechanical ventilation," "on mechanical ventilation," or "expired/organ failure." These outcomes were tested for association with changes in plasma levels of 3-nitrotyrosine and 3-chlorotyrosine and selected clinical risk factors. Infants whose 3-nitrotyrosine levels decreased over the 72 h period were more likely to wean off of mechanical ventilation (p =.03). There was no significant association between changes in 3-chlorotyrosne levels and outcome. After controlling for other variables, an odds ratio of 8.3 (95% CI: 1.3-54.4) for improved outcomes was observed if the 3-nitrotyrosine levels decreased. These data suggest that nitrative and oxidative stress may be related to the severity of lung disease and, consequentially, the overall outcome in this select group of infants with severe bronchopulmonary dysplasia.