RNA sequencing and proteomics approaches reveal novel deficits in the cortex of Mecp2-deficient mice, a model for Rett syndrome.

BACKGROUND: Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by mutations in the transcriptional regulator MeCP2. Much of our understanding of MeCP2 function is derived from transcriptomic studies with the general assumption that alterations in the transcriptome correlate with proteomic changes. Advances in mass spectrometry-based proteomics have facilitated recent interest in the examination of global protein expression to better understand the biology between transcriptional and translational regulation. METHODS: We therefore performed the first comprehensive transcriptome-proteome comparison in a RTT mouse model to elucidate RTT pathophysiology, identify potential therapeutic targets, and further our understanding of MeCP2 function. The whole cortex of wild-type and symptomatic RTT male littermates (n = 4 per genotype) were analyzed using RNA-sequencing and data-independent acquisition liquid chromatography tandem mass spectrometry. Ingenuity® Pathway Analysis was used to identify significantly affected pathways in the transcriptomic and proteomic data sets. RESULTS: Our results indicate these two "omics" data sets supplement one another. In addition to confirming previous works regarding mRNA expression in Mecp2-deficient animals, the current study identified hundreds of novel protein targets. Several selected protein targets were validated by Western blot analysis. These data indicate RNA metabolism, proteostasis, monoamine metabolism, and cholesterol synthesis are disrupted in the RTT proteome. Hits common to both data sets indicate disrupted cellular metabolism, calcium signaling, protein stability, DNA binding, and cytoskeletal cell structure. Finally, in addition to confirming disrupted pathways and identifying novel hits in neuronal structure and synaptic transmission, our data indicate aberrant myelination, inflammation, and vascular disruption. Intriguingly, there is no evidence of reactive gliosis, but instead, gene, protein, and pathway analysis suggest astrocytic maturation and morphological deficits. CONCLUSIONS: This comparative omics analysis supports previous works indicating widespread CNS dysfunction and may serve as a valuable resource for those interested in cellular dysfunction in RTT.

A rapid MALDI-TOF mass spectrometry workflow for Drosophila melanogaster differential neuropeptidomics.

BACKGROUND: Neuropeptides are a diverse category of signaling molecules in the nervous system regulating a variety of processes including food intake, social behavior, circadian rhythms, learning, and memory. Both the identification and functional characterization of specific neuropeptides are ongoing fields of research. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of nervous tissues from a variety of organisms allows direct detection and identification of neuropeptides. Here, we demonstrate an analysis workflow that allows for the detection of differences in specific neuropeptides amongst a variety of neuropeptides being simultaneously measured. For sample preparation, we describe a straight-forward and rapid (minutes) method where individual adult Drosophila melanogaster brains are analyzed. Using a MATLAB-based data analysis workflow, also compatible with MALDI-TOF mass spectra obtained from other sample preparations and instrumentation, we demonstrate how changes in neuropeptides levels can be detected with this method. RESULTS: Over fifty isotopically resolved ion signals in the peptide mass range are reproducibly observed across experiments. MALDI-TOF MS profile spectra were used to statistically identify distinct relative differences in organ-wide endogenous levels of detected neuropeptides between biological conditions. In particular, three distinct levels of a particular neuropeptide, pigment dispersing factor, were detected by comparing groups of preprocessed spectra obtained from individual brains across three different D. melanogaster strains, each of which express different amounts of this neuropeptide. Using the same sample preparation, MALDI-TOF/TOF tandem mass spectrometry confirmed that at least 14 ion signals observed across experiments are indeed neuropeptides. Among the identified neuropeptides were three products of the neuropeptide-like precursor 1 gene previously not identified in the literature. CONCLUSIONS: Using MALDI-TOF MS and preprocessing/statistical analysis, changes in relative levels of a particular neuropeptide in D. melanogaster tissue can be statistically detected amongst a variety of neuropeptides. While the data analysis methods should be compatible with other sample preparations, the presented sample preparation method was sufficient to identify previously unconfirmed D. melanogaster neuropeptides.

Amyotrophic lateral sclerosis-linked FUS/TLS alters stress granule assembly and dynamics.

BACKGROUND: Amyotrophic lateral sclerosis (ALS)-linked fused in sarcoma/translocated in liposarcoma (FUS/TLS or FUS) is concentrated within cytoplasmic stress granules under conditions of induced stress. Since only the mutants, but not the endogenous wild-type FUS, are associated with stress granules under most of the stress conditions reported to date, the relationship between FUS and stress granules represents a mutant-specific phenotype and thus may be of significance in mutant-induced pathogenesis. While the association of mutant-FUS with stress granules is well established, the effect of the mutant protein on stress granules has not been examined. Here we investigated the effect of mutant-FUS on stress granule formation and dynamics under conditions of oxidative stress. RESULTS: We found that expression of mutant-FUS delays the assembly of stress granules. However, once stress granules containing mutant-FUS are formed, they are more dynamic, larger and more abundant compared to stress granules lacking FUS. Once stress is removed, stress granules disassemble more rapidly in cells expressing mutant-FUS. These effects directly correlate with the degree of mutant-FUS cytoplasmic localization, which is induced by mutations in the nuclear localization signal of the protein. We also determine that the RGG domains within FUS play a key role in its association to stress granules. While there has been speculation that arginine methylation within these RGG domains modulates the incorporation of FUS into stress granules, our results demonstrate that this post-translational modification is not involved. CONCLUSIONS: Our results indicate that mutant-FUS alters the dynamic properties of stress granules, which is consistent with a gain-of-toxic mechanism for mutant-FUS in stress granule assembly and cellular stress response.

Recent advances in single-cell MALDI mass spectrometry imaging and potential clinical impact.

Single-cell analysis is gaining popularity in the field of mass spectrometry as a method for analyzing protein and peptide content in cells. The spatial resolution of MALDI mass spectrometry (MS) imaging is by a large extent limited by the laser focal diameter and the displacement of analytes during matrix deposition. Owing to recent advancements in both laser optics and matrix deposition methods, spatial resolution on the order of a single eukaryotic cell is now achievable by MALDI MS imaging. Provided adequate instrument sensitivity, a lateral resolution of approximately 10 µm is currently attainable with commercial instruments. As a result of these advances, MALDI MS imaging is poised to become a transformative clinical technology. In this article, the crucial steps needed to obtain single-cell resolution are discussed, as well as potential applications to disease research.

Strategies for stabilizing superoxide dismutase (SOD1), the protein destabilized in the most common form of familial amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a disorder characterized by the death of both upper and lower motor neurons and by 3- to 5-yr median survival postdiagnosis. The only US Food and Drug Administration-approved drug for the treatment of ALS, Riluzole, has at best, moderate effect on patient survival and quality of life; therefore innovative approaches are needed to combat neurodegenerative disease. Some familial forms of ALS (fALS) have been linked to mutations in the Cu/Zn superoxide dismutase (SOD1). The dominant inheritance of mutant SOD1 and lack of symptoms in knockout mice suggest a "gain of toxic function" as opposed to a loss of function. A prevailing hypothesis for the mechanism of the toxicity of fALS-SOD1 variants, or the gain of toxic function, involves dimer destabilization and dissociation as an early step in SOD1 aggregation. Therefore, stabilizing the SOD1 dimer, thus preventing aggregation, is a potential therapeutic strategy. Here, we report a strategy in which we chemically cross-link the SOD1 dimer using two adjacent cysteine residues on each respective monomer (Cys111). Stabilization, measured as an increase in melting temperature, of ∼20 °C and ∼45 °C was observed for two mutants, G93A and G85R, respectively. This stabilization is the largest for SOD1, and to the best of our knowledge, for any disease-related protein. In addition, chemical cross-linking conferred activity upon G85R, an otherwise inactive mutant. These results demonstrate that targeting these cysteine residues is an important new strategy for development of ALS therapies.