Expression of the neurosecretory process in pc12 cells is governed by rest

The neurosecretory process is acquired during differentiation and can be lost en block by differentiated cells. To investigate the role of REST/NRSF, a transcription repressor, in the maintenance of the process we studied two PC12 clones, one wt and one defective, expressing low and high levels of endogenous RE-1 silencing transcription (factor) (REST), respectively. Stable transfection of constructs demonstrated that REST represses 10 genes coding for proteins of neurosecretory vesicles and their exocytosis, eight including and two lacking the REST-binding sequence, RE-1. Of these genes, those of chromogranins were strongly repressed by fewfold increases of REST, those of VAMP2 and syntaxin1a required much higher levels. Moreover, in wt cells transfected with an active construct the dense-core vesicles, still competent for regulated exocytosis, were much smaller, with lighter cores; in defective cells, the dominant-negative construct induced the rescue of many vesicle/exocytosis genes but not of those of chromogranins. Small dense-core vesicles, exocytized upon stimulation, were rescued when the construct-transfected defective cells were transfected also with chromograninA or treated with trichostatinA, a blocker of histone deacetylases. Our results identify REST, working by direct and indirect mechanisms, as the factor governing the maintenance of the neurosecretory process and the properties of dense-core vesicles in PC12 cells

The Rest Repression of the Neurosecretory Phenotype Is Negatively Modulated by BHC80, a Protein of the BRAF/HDAC Complex

Expression of neurosecretion by nerve cells requires the levels of the transcription repressor element-1 silencing transcription factor (REST) to be very low. However, when high-REST clones of PC12 cells, defective of neurosecretion, were fused to other high-REST, non-neurosecretory cells, some neurosecretion was recovered. To clarify the mechanism of this recovery, we fused defective PC12 cells with human lymphocytes. A cytogenetic analysis revealed all hybrid clones that recovered neurosecretion to contain a fragment of chromosome 11 including the gene encoding BHC80, a protein of one of the complexes that mediate REST repression. In these clones, REST levels were as high as in defective PC12, whereas BHC80, localized in the nucleus, was 4- to 5-fold higher. Transient transfection of defective PC12 with various amounts of BHC80 cDNA induced (1) in defective PC12, the reexpression of only neurosecretion mRNAs; (2) in defective PC12 cotransfected with the REST negative construct DNA-binding domain (to attenuate gene repression), the recovery of a weak, but complete neurosecretory phenotype, including dense-core granules and their regulated exocytosis. Chromatin immunoprecipitation and immunodepletion analyses revealed the extensive BHC80 association with REST at the genes of two neurosecretion proteins, chromograninB and SNAP25, however only in the low-REST PC12, whereas in high-REST defective PC12 no association was appreciable. In defective PC12 transfected with BHC80 some association was reestablished. Therefore, the recovery of neurosecretion observed after fusion/transfection of defective PC12 depends on the reciprocal level of BHC80 and REST, with BHC80 working as a negative modulator of REST repression. This role appears of possible cell physiological and pathological importance

The Rest Repression of the Neurosecretory Phenotype Is Negatively Modulated by BHC80, a Protein of the BRAF/HDAC Complex

Expression of neurosecretion by nerve cells requires the levels of the transcription repressor element-1 silencing transcription factor (REST) to be very low. However, when high-REST clones of PC12 cells, defective of neurosecretion, were fused to other high-REST, non-neurosecretory cells, some neurosecretion was recovered. To clarify the mechanism of this recovery, we fused defective PC12 cells with human lymphocytes. A cytogenetic analysis revealed all hybrid clones that recovered neurosecretion to contain a fragment of chromosome 11 including the gene encoding BHC80, a protein of one of the complexes that mediate REST repression. In these clones, REST levels were as high as in defective PC12, whereas BHC80, localized in the nucleus, was 4- to 5-fold higher. Transient transfection of defective PC12 with various amounts of BHC80 cDNA induced (1) in defective PC12, the reexpression of only neurosecretion mRNAs; (2) in defective PC12 cotransfected with the REST negative construct DNA-binding domain (to attenuate gene repression), the recovery of a weak, but complete neurosecretory phenotype, including dense-core granules and their regulated exocytosis. Chromatin immunoprecipitation and immunodepletion analyses revealed the extensive BHC80 association with REST at the genes of two neurosecretion proteins, chromograninB and SNAP25, however only in the low-REST PC12, whereas in high-REST defective PC12 no association was appreciable. In defective PC12 transfected with BHC80 some association was reestablished. Therefore, the recovery of neurosecretion observed after fusion/transfection of defective PC12 depends on the reciprocal level of BHC80 and REST, with BHC80 working as a negative modulator of REST repression. This role appears of possible cell physiological and pathological importance

ZFP423, a transcription factor implicated in Joubert Syndrome and Cerebellar Vermis Hypoplasia, orchestrates the pace and mode of cerebellar neurogenesis

Neurogenesis is a tightly regulated process, both in the embryonic and in the adult brain. Its success depends on the ability of a germinative epithelium to establish the appropriate balance between maintaining an undifferentiated progenitor pool and giving birth to sequential generations of neurons and glia. The Zfp423 gene encodes a 30 Zn-finger transcription factor (TF) which interacts with the SMAD1- SMAD4 complex (BMP signaling), Notch intracellular domain, retinoic acid receptors and Collier/Olf-1/EBF TFs. This gene has been previosly implicated in cerebellar development. Mutations in the human ortholog ZNF423 have been identified in patients carrying cerebellar vermis hypoplasia (CVH) or Joubert Syndrome (JS), and/ or exhibiting other signs of ciliopathy outside the central nervous system. We have been analyzing two mouse mutant lines carrying allelic in-frame deletions of Zfp423. One of them lacks Zn-finger domains 9-20 (Δ9-20), implicated in BMP and Notch signal transduction, while the other lacks a C-terminal domain (Δ28-30). Both mutants exhibit cerebellar malformations and severe ataxia. However, our results indicate that the two protein domains play sharply distinct roles in the context of cerebellar neurogenesis. In Zfp423Δ9-20/Δ9-20 mutants, GABAergic Purkinje cell (PC) neurogenesis is impaired and the PC progenitor pool in the ventricular zone is precociously depleted. Conversely, Zfp423Δ28-30/Δ28-30 mutants display a selective impairment in the development of glutamatergic cerebellar neurons

Heparin Protects Human Neural Progenitor Cells from Zika Virus-Induced Cell Death While Preserving Their Differentiation into Mature Neuroglial Cells

Zika virus (ZIKV) is an arbovirus member of the Flaviviridae family that causes severe congenital brain anomalies in infected fetuses. The key target cells of ZIKV infection, human neural progenitor cells (hNPCs), are highly permissive to infection that causes the inhibition of cell proliferation and induces cell death. We have previously shown that pharmaceutical-grade heparin inhibits virus-induced cell death with negligible effects on in vitro virus replication in ZIKV-infected hNPCs at the "high" multiplicity of infection (MOI) of 1. Here, we show that heparin inhibits formation of ZIKV-induced intracellular vacuoles, a signature of paraptosis, and inhibits necrosis and apoptosis of hNPCs grown as neurospheres (NS). To test whether heparin preserved the differentiation of ZIKV-infected hNPCs into neuroglial cells, hNPCs were infected at the MOI of 0.001. In this experimental condition, heparin inhibited ZIKV replication by ca. 2 log10, mostly interfering with virion attachment, while maintaining its protective effect against ZIKV-induced cytopathicity. Heparin preserved differentiation into neuroglial cells of hNPCs that were obtained from either human-induced pluripotent stem cells (hiPSC) or by fetal tissue. Quite surprisingly, multiple additions of heparin to hNPCs enabled prolonged virus replication while preventing virus-induced cytopathicity. Collectively, these results highlight the potential neuroprotective effect of heparin that could serve as a lead compound to develop novel agents for preventing the damage of ZIKV infection on the developing brain. IMPORTANCE ZIKV is a neurotropic virus that invades neural progenitor cells (NPCs), causing inhibition of their proliferation and maturation into neurons and glial cells. We have shown previously that heparin, an anticoagulant also used widely during pregnancy, prevents ZIKV-induced cell death with negligible inhibition of virus replication. Here, we demonstrate that heparin also exerts antiviral activity against ZIKV replication using a much lower infectious inoculum. Moreover, heparin interferes with different modalities of virus-induced cell death. Finally, heparin-induced prevention of virus-induced NPC death allows their differentiation into neuroglial cells despite the intracellular accumulation of virions. These results highlight the potential use of heparin, or pharmacological agents derived from it, in pregnant women to prevent the devastating effects of ZIKV infection on the developing brain of their fetuses

REST/NRSF governs the expression of dense-core vesicle gliosecretion in astrocytes

The REST/NRSF transcriptional repressor prevents cultured astrocytes from forming DCVs, and its variable expression in human brain cortex astrocytes may account for their functional heterogeneity

Neuronal models of TDP-43 proteinopathy display reduced axonal translation, increased oxidative stress, and defective exocytosis

Amyotrophic lateral sclerosis (ALS) is a progressive, lethal neurodegenerative disease mostly affecting people around 50–60 years of age. TDP-43, an RNA-binding protein involved in pre-mRNA splicing and controlling mRNA stability and translation, forms neuronal cytoplasmic inclusions in an overwhelming majority of ALS patients, a phenomenon referred to as TDP-43 proteinopathy. These cytoplasmic aggregates disrupt mRNA transport and localization. The axon, like dendrites, is a site of mRNA translation, permitting the local synthesis of selected proteins. This is especially relevant in upper and lower motor neurons, whose axon spans long distances, likely accentuating their susceptibility to ALS-related noxae. In this work we have generated and characterized two cellular models, consisting of virtually pure populations of primary mouse cortical neurons expressing a human TDP-43 fusion protein, wt or carrying an ALS mutation. Both forms facilitate cytoplasmic aggregate formation, unlike the corresponding native proteins, giving rise to bona fide primary culture models of TDP-43 proteinopathy. Neurons expressing TDP-43 fusion proteins exhibit a global impairment in axonal protein synthesis, an increase in oxidative stress, and defects in presynaptic function and electrical activity. These changes correlate with deregulation of axonal levels of polysome-engaged mRNAs playing relevant roles in the same processes. Our data support the emerging notion that deregulation of mRNA metabolism and of axonal mRNA transport may trigger the dying-back neuropathy that initiates motor neuron degeneration in ALS

Secretome of in vitro cultured human embryos contains extracellular vesicles that are uptaken by the maternal side

Communication between embryo and maternal endometrium occurs during a specific time frame in which implantation is possible. Here we demonstrate for the first time that conditioned media from non-manipulated human embryos cultured in vitro for 3 days or up to the blastocyst stage contain extracellular vesicles (EVs) with a diameter of 50 to 200 nm and bearing the traditional microvesicle and exosome marker proteins CD63, CD9 and ALIX. The embryonic origin of these EVs has been confirmed by the presence of stemness gene transcripts and their enrichment in the non-classical HLA-G protein. NANOG and POU5F1 transcripts were shown to be contained in vesicles deriving from embryos at different stages of development. In line with a higher detection rate of the HLA-G protein in blastocysts compared to cleavage stage embryos, a significantly higher amount of HLA-G was found in vesicles accumulated in spent media from day 3 to day 5 of development compared to those isolated from the earlier stage. Uptake of dye-labeled embryo-derived EVs by human primary endometrial epithelial and stromal cells was also demonstrated with a fluorescence intensity signal significantly higher for cells treated with vesicles derived from blastocysts. Based on these findings, EV exchange may be suggested as an emerging way of communication at the maternal-fetal interface

Enlargeosome, an Exocytic Vesicle Resistant to Nonionic Detergents, Undergoes Endocytosis via a Nonacidic Route

Enlargeosomes, a new type of widely expressed cytoplasmic vesicles, undergo tetanus toxin-insensitive exocytosis in response to cytosolic Ca(2+) concentration ([Ca(2+)](i)) rises. Cell biology of enlargeosomes is still largely unknown. By combining immunocytochemistry (marker desmoyokin-Ahnak, d/A) to capacitance electrophysiology in the enlargeosome-rich, neurosecretion-defective clone PC12-27, we show that 1) the two responses, cell surface enlargement and d/A surface appearance, occur with similar kinetics and in the same low micromolar [Ca(2+)](i) range, no matter whether induced by photolysis of the caged Ca(2+) compound o-nitrophenyl EGTA or by the Ca(2+) ionophore ionomycin. Thus, enlargeosomes seem to account, at least in large part, for the exocytic processes triggered by the two stimulations. 2. The enlargeosome membranes are resistant to nonionic detergents but distinct from other resistant membranes, rich in caveolin, Thy1, and/or flotillin1. 3. Cell cholesterol depletion, which affects many membrane fusions, neither disrupts enlargeosomes nor affects their regulated exocytosis. 4. The postexocytic cell surface decline is [Ca(2+)](i) dependent. 5. Exocytized d/A-rich membranes are endocytized and trafficked along an intracellular pathway by nonacidic organelles, distinct from classical endosomes and lysosomes. Our data define specific aspects of enlargeosomes and suggest their participation, in addition to cell differentiation and repair, for which evidence already exists, to other physiological and pathological processes