SEMA6A drives GnRH neuron-dependent puberty onset by tuning median eminence vascular permeability.

Innervation of the hypothalamic median eminence by Gonadotropin-Releasing Hormone (GnRH) neurons is vital to ensure puberty onset and successful reproduction. However, the molecular and cellular mechanisms underlying median eminence development and pubertal timing are incompletely understood. Here we show that Semaphorin-6A is strongly expressed by median eminence-resident oligodendrocytes positioned adjacent to GnRH neuron projections and fenestrated capillaries, and that Semaphorin-6A is required for GnRH neuron innervation and puberty onset. In vitro and in vivo experiments reveal an unexpected function for Semaphorin-6A, via its receptor Plexin-A2, in the control of median eminence vascular permeability to maintain neuroendocrine homeostasis. To support the significance of these findings in humans, we identify patients with delayed puberty carrying a novel pathogenic variant of SEMA6A. In all, our data reveal a role for Semaphorin-6A in regulating GnRH neuron patterning by tuning the median eminence vascular barrier and thereby controlling puberty onset.

Generation of a Triadin KnockOut Syndrome Zebrafish Model.

Different forms of sudden cardiac death have been described, including a recently identified form of genetic arrhythmogenic disorder, named "Triadin KnockOut Syndrome" (TKOS). TKOS is associated with recessive mutations in the TRDN gene, encoding for TRIADIN, but the pathogenic mechanism underlying the malignant phenotype has yet to be completely defined. Moreover, patients with TKOS are often refractory to conventional treatment, substantiating the need to identify new therapeutic strategies in order to prevent or treat cardiac events. The zebrafish (Danio rerio) heart is highly comparable to the human heart in terms of functions, signal pathways and ion channels, representing a good model to study cardiac disorders. In this work, we generated the first zebrafish model for trdn loss-of-function, by means of trdn morpholino injections, and characterized its phenotype. Although we did not observe any gross cardiac morphological defect between trdn loss-of-function embryos and controls, we found altered cardiac rhythm that was recovered by the administration of arrhythmic drugs. Our model will provide a suitable platform to study the effect of TRDN mutations and to perform drug screening to identify new pharmacological strategies for patients carrying TRDN mutations.

Targeting HDAC8 to ameliorate skeletal muscle differentiation in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) causes progressive skeletal muscle degeneration and currently there are few therapeutic options. The identification of new drug targets and their validation in model systems of DMD could be a promising approach to make progress in finding new treatments for this lethal disease. Histone deacetylases (HDACs) play key roles in myogenesis and the therapeutic approach targeting HDACs in DMD is in an advanced phase of clinical trial. Here, we show that the expression of HDAC8, one of the members of the HDAC family, is increased in DMD patients and dystrophic zebrafish. The selective inhibition of HDAC8 with the PCI-34051 inhibitor rescues skeletal muscle defects, similarly to the treatment with the pan-HDAC inhibitor Givinostat. Through acetylation profile of zebrafish with HDAC8 dysregulation, we identified new HDAC8 targets involved in cytoskeleton organization such as tubulin that, when acetylated, is a marker of stable microtubules. Our work provides evidence of HDAC8 overexpression in DMD patients and zebrafish and supports its specific inhibition as a new valuable therapeutic approach in the treatment of this pathology.

The Genome-Wide Impact of Nipblb Loss-of-Function on Zebrafish Gene Expression.

Transcriptional changes normally occur during development but also underlie differences between healthy and pathological conditions. Transcription factors or chromatin modifiers are involved in orchestrating gene activity, such as the cohesin genes and their regulator NIPBL. In our previous studies, using a zebrafish model for nipblb knockdown, we described the effect of nipblb loss-of-function in specific contexts, such as central nervous system development and hematopoiesis. However, the genome-wide transcriptional impact of nipblb loss-of-function in zebrafish embryos at diverse developmental stages remains under investigation. By RNA-seq analyses in zebrafish embryos at 24 h post-fertilization, we examined genome-wide effects of nipblb knockdown on transcriptional programs. Differential gene expression analysis revealed that nipblb loss-of-function has an impact on gene expression at 24 h post fertilization, mainly resulting in gene inactivation. A similar transcriptional effect has also been reported in other organisms, supporting the use of zebrafish as a model to understand the role of Nipbl in gene regulation during early vertebrate development. Moreover, we unraveled a connection between nipblb-dependent differential expression and gene expression patterns of hematological cell populations and AML subtypes, enforcing our previous evidence on the involvement of NIPBL-related transcriptional dysregulation in hematological malignancies.

HDAC8: A Promising Therapeutic Target for Acute Myeloid Leukemia.

Histone deacetylase 8 (HDAC8), a class I HDAC that modifies non-histone proteins such as p53, is highly expressed in different hematological neoplasms including a subtype of acute myeloid leukemia (AML) bearing inversion of chromosome 16 [inv(16)]. To investigate HDAC8 contribution to hematopoietic stem cell maintenance and myeloid leukemic transformation, we generated a zebrafish model with Hdac8 overexpression and observed an increase in hematopoietic stem/progenitor cells, a phenotype that could be reverted using a specific HDAC8 inhibitor, PCI-34051 (PCI). In addition, we demonstrated that AML cell lines respond differently to PCI treatment: HDAC8 inhibition elicits cytotoxic effect with cell cycle arrest followed by apoptosis in THP-1 cells, and cytostatic effect in HL60 cells that lack p53. A combination of cytarabine, a standard anti-AML chemotherapeutic, with PCI resulted in a synergistic effect in all the cell lines tested. We, then, searched for a mechanism behind cell cycle arrest caused by HDAC8 inhibition in the absence of functional p53 and demonstrated an involvement of the canonical WNT signaling in zebrafish and in cell lines. Together, we provide the evidence for the role of HDAC8 in hematopoietic stem cell differentiation in zebrafish and AML cell lines, suggesting HDAC8 inhibition as a therapeutic target in hematological malignancies. Accordingly, we demonstrated the utility of a highly specific HDAC8 inhibition as a therapeutic strategy in combination with standard chemotherapy.

Enigmatic Ladies of the Rings: How Cohesin Dysfunction Affects Myeloid Neoplasms Insurgence.

The genes of the cohesin complex exert different functions, ranging from the adhesion of sister chromatids during the cell cycle, DNA repair, gene expression and chromatin architecture remodeling. In recent years, the improvement of DNA sequencing technologies allows the identification of cohesin mutations in different tumors such as acute myeloid leukemia (AML), acute megakaryoblastic leukemia (AMKL), and myelodysplastic syndromes (MDS). However, the role of cohesin dysfunction in cancer insurgence remains elusive. In this regard, cells harboring cohesin mutations do not show any increase in aneuploidy that might explain their oncogenic activity, nor cohesin mutations are sufficient to induce myeloid neoplasms as they have to co-occur with other causative mutations such as NPM1, FLT3-ITD, and DNMT3A. Several works, also using animal models for cohesin haploinsufficiency, correlate cohesin activity with dysregulated expression of genes involved in myeloid development and differentiation. These evidences support the involvement of cohesin mutations in myeloid neoplasms.

HDAC8 regulates canonical Wnt pathway to promote differentiation in skeletal muscles.

Histone deacetylase 8 (HDAC8) is a class 1 histone deacetylase and a member of the cohesin complex. HDAC8 is expressed in smooth muscles, but its expression in skeletal muscle has not been described. We have shown for the first time that HDAC8 is expressed in human and zebrafish skeletal muscles. Using RD/12 and RD/18 rhabdomyosarcoma cells with low and high differentiation potency, respectively, we highlighted a specific correlation with HDAC8 expression and an advanced stage of muscle differentiation. We inhibited HDAC8 activity through a specific PCI-34051 inhibitor in murine C2C12 myoblasts and zebrafish embryos, and we observed skeletal muscles differentiation impairment. We also found a positive regulation of the canonical Wnt signaling by HDAC8 that might explain muscle differentiation defects. These findings suggest a novel mechanism through which HDAC8 expression, in a specific time window of skeletal muscle development, positively regulates canonical Wnt pathway that is necessary for muscle differentiation.

Modeling Cornelia de Lange syndrome in vitro and in vivo reveals a role for cohesin complex in neuronal survival and differentiation.

Cornelia de Lange syndrome (CdLS), which is reported to affect ∼1 in 10 000 to 30 000 newborns, is a multisystem organ developmental disorder with relatively mild to severe effects. Among others, intellectual disability represents an important feature of this condition. CdLS can result from mutations in at least five genes: nipped-B-like protein, structural maintenance of chromosomes 1A, structural maintenance of chromosomes 3, RAD21 cohesin complex component and histone deacetylase 8 (HDAC8). It is believed that mutations in these genes cause CdLS by impairing the function of the cohesin complex (to which all the aforementioned genes contribute to the structure or function), disrupting gene regulation during critical stages of early development. Since intellectual disorder might result from alterations in neural development, in this work, we studied the role of Hdac8 gene in mouse neural stem cells (NSCs) and in vertebrate (Danio rerio) brain development by knockdown and chemical inhibition experiments. Underlying features of Hdac8 deficiency is an increased cell death in the developing neural tissues, either in mouse NSCs or in zebrafish embryos.

Multiple Layers of CDK5R1 Regulation in Alzheimer's Disease Implicate Long Non-Coding RNAs.

Cyclin-dependent kinase 5 regulatory subunit 1 (CDK5R1) gene encodes for p35, the main activator of Cyclin-dependent kinase 5 (CDK5). The active p35/CDK5 complex is involved in numerous aspects of brain development and function, and its deregulation is closely associated to Alzheimer's disease (AD) onset and progression. We recently showed that miR-15/107 family can negatively regulate CDK5R1 expression modifying mRNA stability. Interestingly, miRNAs belonging to miR-15/107 family are downregulated in AD brain while CDK5R1 is upregulated. Long non-coding RNAs (lncRNAs) are emerging as master regulators of gene expression, including miRNAs, and their dysregulation has been implicated in the pathogenesis of AD. Here, we evaluated the existence of an additional layer of CDK5R1 expression regulation provided by lncRNAs. In particular, we focused on three lncRNAs potentially regulating CDK5R1 expression levels, based on existing data: NEAT1, HOTAIR, and MALAT1. We demonstrated that NEAT1 and HOTAIR negatively regulate CDK5R1 mRNA levels, while MALAT1 has a positive effect. We also showed that all three lncRNAs positively control miR-15/107 family of miRNAs. Moreover, we evaluated the expression of NEAT1, HOTAIR, and MALAT1 in AD and control brain tissues. Interestingly, NEAT1 displayed increased expression levels in temporal cortex and hippocampus of AD patients. Interestingly, we observed a strong positive correlation between CDK5R1 and NEAT1 expression levels in brain tissues, suggesting a possible neuroprotective role of NEAT1 in AD to compensate for increased CDK5R1 levels. Overall, our work provides evidence of another level of CDK5R1 expression regulation mediated by lncRNAs and points to NEAT1 as a biomarker, as well as a potential pharmacological target for AD therapy.