Understanding the Adult Mammalian Heart at Single-Cell RNA-Seq Resolution.

During the last decade, extensive efforts have been made to comprehend cardiac cell genetic and functional diversity. Such knowledge allows for the definition of the cardiac cellular interactome as a reasonable strategy to increase our understanding of the normal and pathologic heart. Previous experimental approaches including cell lineage tracing, flow cytometry, and bulk RNA-Seq have often tackled the analysis of cardiac cell diversity as based on the assumption that cell types can be identified by the expression of a single gene. More recently, however, the emergence of single-cell RNA-Seq technology has led us to explore the diversity of individual cells, enabling the cardiovascular research community to redefine cardiac cell subpopulations and identify relevant ones, and even novel cell types, through their cell-specific transcriptomic signatures in an unbiased manner. These findings are changing our understanding of cell composition and in consequence the identification of potential therapeutic targets for different cardiac diseases. In this review, we provide an overview of the continuously changing cardiac cellular landscape, traveling from the pre-single-cell RNA-Seq times to the single cell-RNA-Seq revolution, and discuss the utilities and limitations of this technology.

Single-Cell RNA Sequencing Analysis Reveals a Crucial Role for CTHRC1 (Collagen Triple Helix Repeat Containing 1) Cardiac Fibroblasts After Myocardial Infarction.

BACKGROUND: Cardiac fibroblasts (CFs) have a central role in the ventricular remodeling process associated with different types of fibrosis. Recent studies have shown that fibroblasts do not respond homogeneously to heart injury. Because of the limited set of bona fide fibroblast markers, a proper characterization of fibroblast population heterogeneity in response to cardiac damage is lacking. The purpose of this study was to define CF heterogeneity during ventricular remodeling and the underlying mechanisms that regulate CF function. METHODS: Collagen1α1-GFP (green fluorescent protein)-positive CFs were characterized after myocardial infarction (MI) by single-cell and bulk RNA sequencing, assay for transposase-accessible chromatin sequencing, and functional assays. Swine and patient samples were studied using bulk RNA sequencing. RESULTS: We identified and characterized a unique CF subpopulation that emerges after MI in mice. These activated fibroblasts exhibit a clear profibrotic signature, express high levels of Cthrc1 (collagen triple helix repeat containing 1), and localize into the scar. Noncanonical transforming growth factor-ß signaling and different transcription factors including SOX9 are important regulators mediating their response to cardiac injury. Absence of CTHRC1 results in pronounced lethality attributable to ventricular rupture. A population of CFs with a similar transcriptome was identified in a swine model of MI and in heart tissue from patients with MI and dilated cardiomyopathy. CONCLUSIONS: We report CF heterogeneity and their dynamics during the course of MI and redefine the CFs that respond to cardiac injury and participate in myocardial remodeling. Our study identifies CTHRC1 as a novel regulator of the healing scar process and a target for future translational studies.

An early dysregulation of FAK and MEK/ERK signaling pathways precedes the ß-amyloid deposition in the olfactory bulb of APP/PS1 mouse model of Alzheimer's disease.

UNLABELLED: Olfactory dysfunction is an early event of Alzheimer's disease (AD). However, the mechanisms associated to AD neurodegeneration in olfactory areas are unknown. Here we used double-transgenic amyloid precursor protein/presenilin 1 (APPswe/PS1dE9) mice and label-free quantitative proteomics to analyze early pathological effects on the olfactory bulb (OB) during AD progression. Prior to ß-amyloid plaque formation, 9 modulated proteins were detected on 3-month-old APP/PS1 mice while 16 differential expressed proteins were detected at 6months, when ß-amyloid plaques appear, indicating a moderate imbalance in cytoskeletal rearrangement, and synaptic plasticity in APP/PS1 OBs. Moreover, ß-amyloid induced an inactivation of focal adhesion kinase (FAK) together with a transient activation of MEK1/2, leading to inactivation of ERK1/2 in 6-months APP/PS1 OBs. In contrast, the analysis of human OBs revealed a late activation of FAK in advanced AD stages, whereas ERK1/2 activation was enhanced across AD staging respect to controls. This survival potential was accompanied by the inhibition of the proapototic factor BAD in the OB across AD phenotypes. Our data contribute to a better understanding of the early molecular mechanisms that are modulated in AD neurodegeneration, highlighting significant differences in the regulation of survival pathways between APP/PS1 mice and sporadic human AD. SIGNIFICANCE: Loss of smell is involved in early stages of Alzheimer's disease (AD), usually preceding classic disease symptoms. However, the mechanisms governing this dysfunction are still poorly understood, losing its potential as a useful tool for clinical diagnosis. Our study characterizes potential AD-associated molecular changes in APP/PS1 mice olfactory bulb (OB) using MS-quantitative proteomics, revealing early cytoskeletal disruption and synaptic plasticity impairment. Moreover, an opposite pattern was found when comparing the activation status of specific survival pathways between APP/PS1 OBs and OBs derived from sAD subjects with different neuropathological grading. Our data reflect, in part, the progressive effect of APP overproduction and Aß accumulation on the OB proteome during AD progression.

Drafting the proteome landscape of myeloid-derived suppressor cells.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that are defined by their myeloid origin, immature state, and ability to potently suppress T-cell responses. They regulate immune responses and the population significantly increases in the tumor microenvironment of patients with glioma and other malignant tumors. For their study, MDSCs are usually isolated from the spleen or directly of tumors from a large number of tumor-bearing mice although promising ex vivo differentiated MDSC production systems have been recently developed. During the last years, proteomics has emerged as a powerful approach to analyze MDSCs proteomes using shotgun-based mass spectrometry (MS), providing functional information about cellular homeostasis and metabolic state at a global level. Here, we will revise recent proteome profiling studies performed in MDSCs from different origins. Moreover, we will perform an integrative functional analysis of the protein compilation derived from these large-scale proteomic studies in order to obtain a comprehensive view of MDSCs biology. Finally, we will also discuss the potential application of high-throughput proteomic approaches to study global proteome dynamics and post-translational modifications (PTMs) during the differentiation process of MDSCs that will greatly boost the identification of novel MDSC-specific therapeutic targets to apply in cancer immunotherapy.

Olfactory bulb proteome dynamics during the progression of sporadic Alzheimer's disease: identification of common and distinct olfactory targets across Alzheimer-related co-pathologies.

Olfactory dysfunction is present in up to 90% of Alzheimer's disease (AD) patients. Although deposition of hyperphosphorylated tau and ß-amyloid substrates are present in olfactory areas, the molecular mechanisms associated with decreased smell function are not completely understood. We have applied mass spectrometry-based quantitative proteomics to probe additional molecular disturbances in postmortem olfactory bulbs (OB) dissected from AD cases respect to neurologically intact controls (n=20, mean age 82.1 years). Relative proteome abundance measurements have revealed protein interaction networks progressively disturbed across AD stages suggesting an early imbalance in splicing factors, subsequent interrupted cycling of neurotransmitters, alteration in toxic and protective mechanisms of ß-amyloid, and finally, a mitochondrial dysfunction together with disturbance in neuron-neuron adhesion. We also present novel molecular findings in the OB in an autopsy cohort composed by Lewy body disease (LBD), frontotemporal lobar degeneration (FTLD), mixed dementia, and progressive supranuclear palsy (PSP) cases (n = 41, mean age 79.7 years). Olfactory mediators deregulated during the progression of AD such as Visinin-like protein 1, RUFY3 protein, and Copine 6 were also differentially modulated in the OB in LBD, FTLD, and mixed dementia. Only Dipeptidyl aminopeptidase-like protein 6 showed a specific down-regulation in AD. However, no differences were observed in the olfactory expression of this protein panel in PSP subjects. This study demonstrates an olfactory progressive proteome modulation in AD, unveiling cross-disease similarities and differences especially for specific proteins involved in dendritic and axonic distributions that occur in the OB during the neurodegenerative process.

Toward defining the anatomo-proteomic puzzle of the human brain: An integrative analysis.

The human brain is exceedingly complex, constituted by billions of neurons and trillions of synaptic connections that, in turn, define ∼900 neuroanatomical subdivisions in the adult brain (Hawrylycz et al. An anatomically comprehensive atlas of the human brain transcriptome. Nature 2012, 489, 391-399). The human brain transcriptome has revealed specific regional transcriptional signatures that are regulated in a spatiotemporal manner, increasing the complexity of the structural and molecular organization of this organ (Kang et al. Spatio-temporal transcriptome of the human brain. Nature 2011, 478, 483-489). During the last decade, neuroproteomics has emerged as a powerful approach to profile neural proteomes using shotgun-based MS, providing complementary information about protein content and function at a global level. Here, we revise recent proteome profiling studies performed in human brain, with special emphasis on proteome mapping of anatomical macrostructures, specific subcellular compartments, and cerebrospinal fluid. Moreover, we have performed an integrative functional analysis of the protein compilation derived from these large-scale human brain proteomic studies in order to obtain a comprehensive view of human brain biology. Finally, we also discuss the potential contribution of our meta-analysis to the Chromosome-centric Human Proteome Project initiative.