Sox9 Inhibits Cochlear Hair Cell Fate by Upregulating Hey1 and HeyL Antagonists of Atoh1.

It is widely accepted that cell fate determination in the cochlea is tightly controlled by different transcription factors (TFs) that remain to be fully defined. Here, we show that Sox9, initially expressed in the entire sensory epithelium of the cochlea, progressively disappears from differentiating hair cells (HCs) and is finally restricted to supporting cells (SCs). By performing ex vivo electroporation of E13.5-E14.5 cochleae, we demonstrate that maintenance of Sox9 expression in the progenitors committed to HC fate blocks their differentiation, even if co-expressed with Atoh1, a transcription factor necessary and sufficient to form HC. Sox9 inhibits Atoh1 transcriptional activity by upregulating Hey1 and HeyL antagonists, and genetic ablation of these genes induces extra HCs along the cochlea. Although Sox9 suppression from sensory progenitors ex vivo leads to a modest increase in the number of HCs, it is not sufficient in vivo to induce supernumerary HC production in an inducible Sox9 knockout model. Taken together, these data show that Sox9 is downregulated from nascent HCs to allow the unfolding of their differentiation program. This may be critical for future strategies to promote fully mature HC formation in regeneration approaches.

Epigenetic profiles of elevated cell free circulating H3.1 nucleosomes as potential biomarkers for non-Hodgkin lymphoma.

During cell death, nucleosomes, the basic structural unit of chromatin, are released into the blood stream and elevated levels have been found in the plasma of patients with solid cancers. In this study, we demonstrate an increase in cell free circulating H3.1-nucleosomes levels in plasma samples from patients with hematological malignancy, non-Hodgkin lymphoma (NHL), relative to healthy donors. As histone post-translational modifications (PTMs) of circulating nucleosomes are described as potential biomarkers of various solid cancers, we investigated the epigenetic profile of nucleosomes from NHL patients following nucleosome enrichment (Nu.Q® capture) combined with mass spectrometry. Eight histones PTMs, including the acetylation of histone H3 at lysine 9, 14 and 18 as well as the methylation state of histone H3 at lysine 9, 27 and 36, were identified at a higher level in the plasma of NHL patients compared to healthy donors. These results were confirmed in a larger clinical cohort by immunoassay. Subsequently, the temporal profile of these histone PTMs in NHL patients undergoing treatment course highlighted the potential use of these new biomarkers to monitor treatment response and/or disease progression. Our results substantiate that levels of H3.1-nucleosomes are particularly elevated in NHL patients and may be a useful diagnostic tool. Moreover, our work emphasizes the crucial roles of the epigenetic marks present on circulating nucleosomes to detect and monitor tumor progression and/or treatment response of non-Hodgkin Lymphoma.

Serial profiling of cell-free DNA and nucleosome histone modifications in cell cultures.

Recent advances in basic research have unveiled several strategies for improving the sensitivity and specificity of cell-free DNA (cfDNA) based assays, which is a prerequisite for broadening its clinical use. Included among these strategies is leveraging knowledge of both the biogenesis and physico-chemical properties of cfDNA towards the identification of better disease-defining features and optimization of methods. While good progress has been made on this front, much of cfDNA biology remains uncharted. Here, we correlated serial measurements of cfDNA size, concentration and nucleosome histone modifications with various cellular parameters, including cell growth rate, viability, apoptosis, necrosis, and cell cycle phase in three different cell lines. Collectively, the picture emerged that temporal changes in cfDNA levels are rather irregular and not the result of constitutive release from live cells. Instead, changes in cfDNA levels correlated with intermittent cell death events, wherein apoptosis contributed more to cfDNA release in non-cancer cells and necrosis more in cancer cells. Interestingly, the presence of a ~ 3 kbp cfDNA population, which is often deemed to originate from accidental cell lysis or active release, was found to originate from necrosis. High-resolution analysis of this cfDNA population revealed an underlying DNA laddering pattern consisting of several oligo-nucleosomes, identical to those generated by apoptosis. This suggests that necrosis may contribute significantly to the pool of mono-nucleosomal cfDNA fragments that are generally interrogated for cancer mutational profiling. Furthermore, since active steps are often taken to exclude longer oligo-nucleosomes from clinical biospecimens and subsequent assays this raises the question of whether important pathological information is lost.

A novel proteomics approach to epigenetic profiling of circulating nucleosomes.

Alteration of epigenetic modifications plays an important role in human cancer. Notably, the dysregulation of histone post-translational modifications (PTMs) has been associated with several cancers including colorectal cancer (CRC). However, the signature of histone PTMs on circulating nucleosomes is still not well described. We have developed a fast and robust enrichment method to isolate circulating nucleosomes from plasma for further downstream proteomic analysis. This method enabled us to quantify the global alterations of histone PTMs from 9 CRC patients and 9 healthy donors. Among 54 histone proteoforms identified and quantified in plasma samples, 13 histone PTMs were distinctive in CRC. Notably, methylation of histone H3K9 and H3K27, acetylation of histone H3 and citrullination of histone H2A1R3 were upregulated in plasma of CRC patients. A comparative analysis of paired samples identified 3 common histone PTMs in plasma and tumor tissue including the methylation and acetylation state of lysine 27 of histone H3. Moreover, we highlight for the first time that histone H2A1R3 citrulline is a modification upregulated in CRC patients. This new method presented herein allows the detection and quantification of histone variants and histone PTMs from circulating nucleosomes in plasma samples and could be used for biomarker discovery of cancer.

Anxiety-like features and spatial memory problems as a consequence of hippocampal SV2A expression.

The Synaptic Vesicle Protein 2A (SV2A) is a transmembrane protein whose presence is reduced both in animal models and in patients with chronic epilepsy. Besides its implication in the epileptic process, the behavioural consequences of the changes in its expression remain unclear. The purpose of our research is to better understand the possible role(s) of this protein through the phenotype of cKO (Grik4 Cre+/-, SV2A lox/lox) mice, male and female, which present a specific decrease of SV2A expression levels in the hippocampal glutamatergic neurons but without any epileptic seizures. In this study, we compare the cKO mice with cHZ (Grik4 Cre+/-, SV2A lox/+) and WT (Grik4 Cre+/+, SV2A lox/lox) mice through a battery of tests, used to evaluate different features: the anxiety-related features (Elevated Plus Maze), the locomotor activity (Activity Chambers), the contextual fear-related memory (Contextual Fear Conditioning), and the spatial memory (Barnes Maze). Our results showed statistically significant differences in the habituation to a new environment, an increase in the anxiety levels and spatial memory deficit in the cHZ and cKO groups, compared to the WT group. No statistically significant differences due to the genotype appeared in the spontaneous locomotor activity or the fear-linked memory. However, sexual differences were observed in this last feature. These results highlight not only an important role of the SV2A protein in the cognitive and anxiety problems typically encountered in epileptic patients, but also a possible role in the symptomatology of other neurodegenerative diseases, such as the Alzheimer's disease.

Aurora A plays a dual role in migration and survival of human glioblastoma cells according to the CXCL12 concentration.

Primary glioblastoma is the most frequent human brain tumor in adults and is generally fatal due to tumor recurrence. We previously demonstrated that glioblastoma-initiating cells invade the subventricular zones and promote their radio-resistance in response to the local release of the CXCL12 chemokine. In this work, we show that the mitotic Aurora A kinase (AurA) is activated through the CXCL12-CXCR4 pathway in an ERK1/2-dependent manner. Moreover, the CXCL12-ERK1/2 signaling induces the expression of Ajuba, the main cofactor of AurA, which allows the auto-phosphorylation of AurA.We show that AurA contributes to glioblastoma cell survival, radio-resistance, self-renewal, and proliferation regardless of the exogenous stimulation with CXCL12. On the other hand, AurA triggers the CXCL12-mediated migration of glioblastoma cells in vitro as well as the invasion of the subventricular zone in xenograft experiments. Moreover, AurA regulates cytoskeletal proteins (i.e., Actin and Vimentin) and favors the pro-migratory activity of the Rho-GTPase CDC42 in response to CXCL12. Altogether, these results show that AurA, a well-known kinase of the mitotic machinery, may play alternative roles in human glioblastoma according to the CXCL12 concentration.

The miR-183/ItgA3 axis is a key regulator of prosensory area during early inner ear development.

MicroRNAs are important regulators of gene expression and are involved in cellular processes such as proliferation or differentiation, particularly during development of numerous organs including the inner ear. However, it remains unknown if miRNAs are required during the earliest stages of otocyst and cochlear duct development. Here, we report that a conditional loss of Dicer expression in the otocyst impairs the early development of the inner ear as a result of the accumulation of DNA damage that trigger p53-mediated apoptosis. Moreover, cochlear progenitors in the prosensory domain do not exit the cell cycle. Our unbiased approach identified ItgA3 as a target of miR-183, which are both enriched in the otic vesicle. We observed that the repression of integrin alpha 3 by miR-183 controls cell proliferation in the developing cochlea. Collectively, our results reveal that Dicer and miRNAs play essential roles in the regulation of early inner ear development.

Puzzling Out Synaptic Vesicle 2 Family Members Functions.

Synaptic vesicle proteins 2 (SV2) were discovered in the early 80s, but the clear demonstration that SV2A is the target of efficacious anti-epileptic drugs from the racetam family stimulated efforts to improve understanding of its role in the brain. Many functions have been suggested for SV2 proteins including ions or neurotransmitters transport or priming of SVs. Moreover, several recent studies highlighted the link between SV2 and different neuronal disorders such as epilepsy, Schizophrenia (SCZ), Alzheimer's or Parkinson's disease. In this review article, we will summarize our present knowledge on SV2A function(s) and its potential role(s) in the pathophysiology of various brain disorders.

Development and Validation of a New Mouse Model to Investigate the Role of SV2A in Epilepsy.

SV2A is a glycoprotein present in the membranes of most synaptic vesicles. Although it has been highly conserved throughout evolution, its physiological role remains largely unknown. Nevertheless, Levetiracetam, a very effective anti-epileptic drug, has been recently demonstrated to bind to SV2A. At present, our understanding of the normal function of SV2A and its possible involvement in diseases like epilepsy is limited. With this study, we sought to develop a relevant model enabling analysis of SV2A's role in the occurrence or progression of epilepsy. For this purpose, we generated a floxed SV2A mouse model with conditional alleles carrying LoxP sites around exon 3 by means of a gene-targeting strategy. The SV2A lox/lox mouse line is indistinguishable from wild-type mice. When the recombination was observed in all cells, a model of mice with both SV2A alleles floxed around exon 3 recapitulated the phenotype of SV2A KO mice, including seizures. However, the specific invalidation of SV2A in the CA3 hippocampal region was not followed by epileptic seizures or decrease in the epileptic threshold on pentylenetetrazol (PTZ) test. These results demonstrate that the floxed SV2A mouse line has been successfully established. This transgenic mouse model will be useful for investigating SV2A functions related to cell types and developmental stages.

MicroRNA-124 Regulates Cell Specification in the Cochlea through Modulation of Sfrp4/5.

The organ of Corti, the auditory organ of the mammalian inner ear, contains sensory hair cells and supporting cells that arise from a common sensory progenitor. The molecular bases allowing the specification of these progenitors remain elusive. In the present study, by combining microarray analyses with conditional deletion of Dicer in the developing inner ear, we identified that miR-124 controls cell fate in the developing organ of Corti. By targeting secreted frizzled-related protein 4 (Sfrp4) and Sfrp5, two inhibitors of the Wnt pathway, we showed that miR-124 controls the ß-catenin-dependent and also the PCP-related non-canonical Wnt pathways that contribute to HC differentiation and polarization in the organ of Corti. Thus, our work emphasizes the importance of miR-124 as an epigenetic safeguard that fine-tunes the expression of genes critical for cell patterning during cochlear differentiation.