Neuroinflammation in the pathogenesis of axonal Charcot-Marie-Tooth disease caused by lack of GDAP1.

Mutations in the GDAP1 mitochondrial outer membrane gene cause Charcot-Marie-Tooth (CMT) neuropathy. Reduction or absence of GDAP1 has been associated with abnormal changes in the mitochondrial morphology and dynamics, oxidative stress and changes in calcium homeostasis. Neuroinflammation has been described in rodent models of genetic demyelinating CMT neuropathies but not in CMT primarily associated with axonopathy. Inflammatory processes have also been related to mitochondrial changes and oxidative stress in central neurodegenerative disorders. Here we investigated the presence of neuroinflammation in the axonal neuropathy of the Gdap1-/- mice. We showed by transcriptome profile of spinal cord and the in vivo detection of activated phagocytes that the absence of GDAP1 is associated with upregulation of inflammatory pathways. We observed reactive gliosis in spinal cord with increase of the astroglia markers GFAP and S100B, and the microglia marker IBA1. Additionally, we found significant increase of inflammatory mediators such as TNF-α and pERK, and C1qa and C1qb proteins of the complement system. Importantly, we observed an increased expression of CD206 and CD86 as M2 and M1 microglia and macrophage response markers, respectively, in Gdap1-/- mice. These inflammatory changes were also associated with abnormal molecular changes in synapses. In summary, we demonstrate that inflammation in spinal cord and sciatic nerve, but not in brain and cerebellum, is part of the pathophysiology of axonal GDAP1-related CMT.

Gene expression network analysis reveals new transcriptional regulators as novel factors in human ischemic cardiomyopathy.

BACKGROUND: Ischemic cardiomyopathy (ICM) is characterized by transcriptomic changes that alter cellular processes leading to decreased cardiac output. Because the molecular network of ICM is largely unknown, the aim of this study was to characterize the role of new transcriptional regulators in the molecular mechanisms underlying the responses to ischemia. METHODS: Myocardial tissue explants from ICM patients and control (CNT) subjects were analyzed by RNA-Sequencing (RNA-Seq) and quantitative Real-Time PCR. RESULTS: Enrichment analysis of the ICM transcriptomic profile allowed the characterization of novel master regulators. We found that the expression of the transcriptional regulators SP100 (-1.5-fold, p < 0.05), CITED2 (-3.8-fold, p < 0.05), CEBPD (-4.9-fold, p < 0.05) and BCL3 (-3.3-fold, p < 0.05) were lower in ICM than in CNT. To gain insights into the molecular network defined by the transcription factors, we identified CEBPD, BCL3, and HIF1A target genes in the RNA-Seq datasets. We further characterized the biological processes of the target genes by gene ontology annotation. Our results suggest that CEBPD-inducible genes with roles in the inhibition of apoptosis are downregulated and that BCL3-repressible genes are involved in the regulation of cellular metabolism in ICM. Moreover, our results suggest that CITED2 downregulation causes increased expression of HIF1A target genes. Functional analysis of HIF1A target genes revealed that hypoxic and stress response genes are activated in ICM. Finally, we found a significant correlation between the mRNA levels of BCL3 and the mRNA levels of both CEBPD (r = 0.73, p < 0.001) and CITED2 (r = 0.56, p < 0.05). Interestingly, CITED2 mRNA levels are directly related to ejection fraction (EF) (r = 0.54, p < 0.05). CONCLUSIONS: Our data indicate that changes in the expression of SP100, CITED2, CEBPD, and BCL3 affect their transcription regulatory networks, which subsequently alter a number of biological processes in ICM patients. The relationship between CITED2 mRNA levels and EF emphasizes the importance of this transcription factor in ICM. Moreover, our findings identify new mechanisms used to interpret gene expression changes in ICM and provide valuable resources for further investigation of the molecular basis of human cardiac ischemic response.

RNA-sequencing analysis reveals new alterations in cardiomyocyte cytoskeletal genes in patients with heart failure.

Changes in cardiomyocyte cytoskeletal components, a crucial scaffold of cellular structure, have been found in heart failure (HF); however, the altered cytoskeletal network remains to be elucidated. This study investigated a new map of cytoskeleton-linked alterations that further explain the cardiomyocyte morphology and contraction disruption in HF. RNA-Sequencing (RNA-Seq) analysis was performed in 29 human LV tissue samples from ischemic cardiomyopathy (ICM; n=13) and dilated cardiomyopathy (DCM, n=10) patients undergoing cardiac transplantation and six healthy donors (control, CNT) and up to 16 ICM, 13 DCM and 7 CNT tissue samples for qRT-PCR. Gene Ontology analysis of RNA-Seq data demonstrated that cytoskeletal processes are altered in HF. We identified 60 differentially expressed cytoskeleton-related genes in ICM and 58 genes in DCM comparing with CNT, hierarchical clustering determined that shared cytoskeletal genes have a similar behavior in both pathologies. We further investigated MYLK4, RHOU, and ANKRD1 cytoskeletal components. qRT-PCR analysis revealed that MYLK4 was downregulated (-2.2-fold; P<0.05) and ANKRD1 was upregulated (2.3-fold; P<0.01) in ICM patients vs CNT. RHOU mRNA levels showed a statistical trend to decrease (-2.9-fold). In DCM vs CNT, MYLK4 (-4.0-fold; P<0.05) and RHOU (-3.9-fold; P<0.05) were downregulated and ANKRD1 (2.5-fold; P<0.05) was upregulated. Accordingly, MYLK4 and ANKRD1 protein levels were decreased and increased, respectively, in both diseases. Furthermore, ANKRD1 and RHOU mRNA levels were related with LV function (P<0.05). In summary, we have found a new map of changes in the ICM and DCM cardiomyocyte cytoskeleton. ANKRD1 and RHOU mRNA levels were related with LV function which emphasizes their relevance in HF. These new cytoskeletal changes may be responsible for altered contraction and cell architecture disruption in HF patients. Moreover, these results improve our knowledge on the role of cytoskeleton in functional and structural alterations in HF.

miRNA signature and Dicer requirement during human endometrial stromal decidualization in vitro.

Decidualization is a morphological and biochemical transformation of endometrial stromal fibroblast into differentiated decidual cells, which is critical for embryo implantation and pregnancy establishment. The complex regulatory networks have been elucidated at both the transcriptome and the proteome levels, however very little is known about the post-transcriptional regulation of this process. miRNAs regulate multiple physiological pathways and their de-regulation is associated with human disorders including gynaecological conditions such as endometriosis and preeclampsia. In this study we profile the miRNAs expression throughout human endometrial stromal (hESCs) decidualization and analyze the requirement of the miRNA biogenesis enzyme Dicer during this process. A total of 26 miRNAs were upregulated and 17 miRNAs downregulated in decidualized hESCs compared to non-decidualized hESCs. Three miRNAs families, miR-181, miR-183 and miR-200, are down-regulated during the decidualization process. Using miRNAs target prediction algorithms we have identified the potential targets and pathways regulated by these miRNAs. The knockdown of Dicer has a minor effect on hESCs during in vitro decidualization. We have analyzed a battery of decidualization markers such as cell morphology, Prolactin, IGFBP-1, MPIF-1 and TIMP-3 secretion as well as HOXA10, COX2, SP1, C/EBPß and FOXO1 expression in decidualized hESCs with decreased Dicer function. We found decreased levels of HOXA10 and altered intracellular organization of actin filaments in Dicer knockdown decidualized hESCs compared to control. Our results provide the miRNA signature of hESC during the decidualization process in vitro. We also provide the first functional characterization of Dicer during human endometrial decidualization although surprisingly we found that Dicer plays a minor role regulating this process suggesting that alternative biogenesis miRNAs pathways must be involved in human endometrial decidualization.

Inhibition of histone deacetylase activity in human endometrial stromal cells promotes extracellular matrix remodelling and limits embryo invasion.

Invasion of the trophoblast into the maternal decidua is regulated by both the trophoectoderm and the endometrial stroma, and entails the action of tissue remodeling enzymes. Trophoblast invasion requires the action of metalloproteinases (MMPs) to degrade extracellular matrix (ECM) proteins and in turn, decidual cells express tissue inhibitors of MMPs (TIMPs). The balance between these promoting and restraining factors is a key event for the successful outcome of pregnancy. Gene expression is post-transcriptionally regulated by histone deacetylases (HDACs) that unpacks condensed chromatin activating gene expression. In this study we analyze the effect of histone acetylation on the expression of tissue remodeling enzymes and activity of human endometrial stromal cells (hESCs) related to trophoblast invasion control. Treatment of hESCs with the HDAC inhibitor trichostatin A (TSA) increased the expression of TIMP-1 and TIMP-3 while decreased MMP-2, MMP-9 and uPA and have an inhibitory effect on trophoblast invasion. Moreover, histone acetylation is detected at the promoters of TIMP-1 and TIMP-3 genes in TSA-treated. In addition, in an in vitro decidualized hESCs model, the increase of TIMP-1 and TIMP-3 expression is associated with histone acetylation at the promoters of these genes. Our results demonstrate that histone acetylation disrupt the balance of ECM modulators provoking a restrain of trophoblast invasion. These findings are important as an epigenetic mechanism that can be used to control trophoblast invasion.

A novel model of human implantation: 3D endometrium-like culture system to study attachment of human trophoblast (Jar) cell spheroids.

There is an urgent need to develop optimized experimental models to examine human implantation. These studies aimed to (i) establish a human endometrium-like three-dimensional (3D) culture system, and (ii) examine the attachment of trophoblast-like Jar spheroids to the culture. In the present work, 3D endometrial cultures were constructed with fibrin-agarose as matrix scaffold, and using epithelial and stromal cells from both human primary cultures and established cell lines. An attachment assay between trophoblast cells and the 3D culture was developed. Epithelial cells (cytokeratin(+)) concentrated on top of the matrix forming a monolayer, and stromal cells (vimentin(+)) resided within the matrix, resembling the normal endometrial structure. The capability of primary epithelial cells to form glands spontaneously was observed. Human trophoblast cells (Jar cells) were hCG(+) by immunostaining, allowed to form spheroids, and confirmed to secrete hCG into the medium. Time-dependent experiments demonstrated a high rate of attachment of Jar spheroids to the epithelium, and adhesion was strongly related to the various cell types present in the 3D culture. An architecturally and functionally competent 3D endometrial culture system was established, that coupled with Jar spheroids mimicking trophoblast cells, provides a unique in vitro model for the study of certain aspects of human implantation.