Antidepressant treatment effects on hippocampal protein turnover: Molecular and spatial insights from mass spectrometry

A major challenge in managing depression is that antidepressant drugs take a long time to exert their therapeutic effects. For the development of faster-acting treatments, it is crucial to get an improved understanding of the molecular mechanisms underlying antidepressant mode of action. Here, we used a novel mass spectrometry-based workflow to investigate how antidepressant treatment affects brain protein turnover at single-cell and subcellular resolution. We combined stable isotope metabolic labeling, quantitative Tandem Mass Spectrometry (qTMS) and Multi-isotope Imaging Mass Spectrometry (MIMS) to simultaneously quantify and image protein synthesis and turnover in hippocampi of mice treated with the antidepressant paroxetine. We identified changes in turnover of individual hippocampal proteins that reveal altered metabolism-mitochondrial processes and report on subregion-specific turnover patterns upon paroxetine treatment. This workflow can be used to investigate brain protein turnover changes in vivo upon pharmacological interventions at a resolution and precision that has not been possible with other methods to date. Our results reveal acute paroxetine effects on brain protein turnover and shed light on antidepressant mode of action.     

Stable Isotope Metabolic Labeling with a Novel 15N-Enriched Bacteria Diet for Improved Proteomic Analyses of Mouse Models for Psychopathologies

The identification of differentially regulated proteins in animal models of psychiatric diseases is essential for a comprehensive analysis of associated psychopathological processes. Mass spectrometry is the most relevant method for analyzing differences in protein expression of tissue and body fluid proteomes. However, standardization of sample handling and sample-to-sample variability are problematic. Stable isotope metabolic labeling of a proteome represents the gold standard for quantitative mass spectrometry analysis. The simultaneous processing of a mixture of labeled and unlabeled samples allows a sensitive and accurate comparative analysis between the respective proteomes. Here, we describe a cost-effective feeding protocol based on a newly developed ¹⁵N bacteria diet based on Ralstonia eutropha protein, which was applied to a mouse model for trait anxiety. Tissue from ¹⁵N-labeled vs. ¹⁴N-unlabeled mice was examined by mass spectrometry and differences in the expression of glyoxalase-1 (GLO1) and histidine triad nucleotide binding protein 2 (Hint2) proteins were correlated with the animals'' psychopathological behaviors for methodological validation and proof of concept, respectively. Additionally, phenotyping unraveled an antidepressant-like effect of the incorporation of the stable isotope ¹⁵N into the proteome of highly anxious mice. This novel phenomenon is of considerable relevance to the metabolic labeling method and could provide an opportunity for the discovery of candidate proteins involved in depression-like behavior. The newly developed ¹⁵N bacteria diet provides researchers a novel tool to discover disease-relevant protein expression differences in mouse models using quantitative mass spectrometry.     

A MS data search method for improved 15N‐labeled protein identification

Quantitative proteomics using stable isotope labeling strategies combined with MS is an important tool for biomarker discovery. Methods involving stable isotope metabolic labeling result in optimal quantitative accuracy, since they allow the immediate combination of two or more samples. Unfortunately, stable isotope incorporation rates in metabolic labeling experiments using mammalian organisms usually do not reach 100%. As a consequence, protein identifications in ¹⁵N database searches have poor success rates. We report on a strategy that significantly improves the number of ¹⁵N‐labeled protein identifications and results in a more comprehensive and accurate relative peptide quantification workflow.     

A Method of Drusen Measurement Based on the Geometry of Fundus Reflectance

Background  

The hallmarks of age-related macular degeneration, the leading cause of blindness in the developed world, are the subretinal deposits known as drusen. Drusen identification and measurement play a key role in clinical studies of this disease. Current manual methods of drusen measurement are laborious and subjective. Our purpose was to expedite clinical research with an accurate, reliable digital method.     

Isolation of a ribonucleoprotein complex involved in mRNA localization in Drosophila oocytes

Localization of bicoid (bcd) mRNA to the anterior and oskar (osk) mRNA to the posterior of the Drosophila oocyte is critical for embryonic patterning. Previous genetic studies implicated exuperantia (exu) in bcd mRNA localization, but its role in this process is not understood. We have biochemically isolated Exu and show that it is part of a large RNase-sensitive complex that contains at least seven other proteins. One of these proteins was identified as the cold shock domain RNA-binding protein Ypsilon Schachtel (Yps), which we show binds directly to Exu and colocalizes with Exu in both the oocyte and nurse cells of the Drosophila egg chamber. Surprisingly, the Exu-Yps complex contains osk mRNA. This biochemical result led us to reexamine the role of Exu in the localization of osk mRNA. We discovered that exu-null mutants are defective in osk mRNA localization in both nurse cells and the oocyte. Furthermore, both Exu/Yps particles and osk mRNA follow a similar temporal pattern of localization in which they transiently accumulate at the oocyte anterior and subsequently localize to the posterior pole. We propose that Exu is a core component of a large protein complex involved in localizing mRNAs both within nurse cells and the developing oocyte.