No abstract

Despite extensive efforts on probing the mechanism of Alzheimer’s disease (AD) and enormous investments into AD drug development, the lack of effective disease-modifying therapeutics and the complexity of the AD pathogenesis process suggest a great need for further insights into alternative AD drug targets. Herein, we focus on the chiral effects of truncated amyloid beta (Aβ) and offer further structural and molecular evidence for epitope region-specific, chirality-regulated Aβ fragment self-assembly and its potential impact on receptor-recognition. A multidimensional ion mobility-mass spectrometry (IM-MS) analytical platform and in-solution kinetics analysis reveal the comprehensive structural and molecular basis for differential Aβ fragment chiral chemistry, including the differential and cooperative roles of chiral Aβ N-terminal and C-terminal fragments in receptor recognition. Our method is applicable to many other systems and the results may shed light on the potential development of novel AD therapeutic strategies based on targeting the D-isomerized Aβ, rather than natural L-Aβ.

Neuropeptides are one of the most diverse classes of signaling molecules and have attracted great interest over the years owing to their roles in regulation of a wide range of physiological processes. However, there are unique challenges associated with neuropeptide studies stemming from the highly variable molecular sizes of the peptides, low concentrations, high degree of structural diversity and large number of isoforms. As a result, much effort has been focused on developing new techniques for studying neuropeptides, as well as novel applications directed towards learning more about these endogenous peptides. The areas of importance for neuropeptide studies include structure, localization within tissues, interaction with their receptors, including ion channels, and physiological function. Here, we discuss these aspects and the associated techniques, focusing on technologies that have demonstrated potential in advancing the field in recent years. Most identification and structural information has been gained by mass spectrometry, either alone or with confirmations from other techniques, such as nuclear magnetic resonance spectroscopy and other spectroscopic tools. While mass spectrometry and bioinformatic tools have proven to be the most powerful for large-scale analyses, they still rely heavily on complementary methods for confirmation. Localization within tissues, for example, can be probed by mass spectrometry imaging, immunohistochemistry and radioimmunoassays. Functional information has been gained primarily from behavioral studies coupled with tissue-specific assays, electrophysiology, mass spectrometry and optogenetic tools. Concerning the receptors for neuropeptides, the discovery of ion channels that are directly gated by neuropeptides opens up the possibility of developing a new generation of tools for neuroscience, which could be used to monitor neuropeptide release or to specifically change the membrane potential of neurons. It is expected that future neuropeptide research will involve the integration of complementary bioanalytical technologies and functional assays.

Matrix-assisted laser desorption/ionization (MALDI)-MS imaging has been utilized to image a variety of biomolecules, including neuropeptides. Washing a tissue section is an effective way to eliminate interfering background and improve detection of low concentration target analyte molecules; however, many previous methods have not been tested for neuropeptide analysis via MALDI-MS imaging. Using crustaceans as a neurological model organism, we developed a new, simple washing procedure and applied this method to characterize neuropeptide changes due to hypoxia stress. With a 10 s 50:50 EtOH:H 2 O wash, neuropeptide coverage was improved by 1.15-fold, while normalized signal intensities were increased by 5.28fold. Specifically, hypoxia and hypercapnia stress conditions were investigated due to their environmental relevance to marine invertebrates. Many neuropeptides, including RFamides, pyrokinin, and cardioactive peptides, showed distinct up-and down-regulation for specific neuropeptide isoforms. Since crustacean neuropeptides are homologous to those found in humans, results from these studies can be applied to understand potential roles of neuropeptides involved in medical hypoxia and hypercapnia.

The decrease of pH level in the water affects animals living in aquatic habitat, such as crustaceans. The molecular mechanisms enabling these animals to survive this environmental stress remain unknown. To understand the modulatory function of neuropeptides in crustaceans when encountering drops in pH level, we developed and implemented a multifaceted mass spectrometric platform to investigate the global neuropeptide changes in response to water acidification in the Atlantic blue crab, Callinectes sapidus. Neural tissues were collected at different incubation periods to monitor dynamic changes of neuropeptides under different stress conditions occurring in the animal. Neuropeptide families were found to exhibit distinct expression patterns in different tissues and even each isoform had its specific response to the stress. Circulating fluid in the crabs (hemolymph) was also analyzed after 2-hour exposure to acidification condition, and together with results from tissue analysis, enabled the discovery of neuropeptides participating in the stress accommodation process as putative hormones. Two novel peptide sequences were detected in the hemolymph that appeared to be involved in the stress-related regulation in the crabs. The data have been deposited to the ProteomeXchange with identifier PXD013536.