Miguel Weil

IKAP/Elp1 involvement in cytoskeleton regulation and implication for familial dysautonomia

Deficiency in the IKAP/Elp1 protein leads to the recessive sensory autosomal congenital neuropathy which is called familial dysautonomia (FD). This protein was originally identified as a role player in transcriptional elongation being a subunit of the RNAPII transcriptional Elongator multi-protein complex. Subsequently, IKAP/Elp1 was shown to play various functions in the cytoplasm. Here, we describe experiments performed with IKAP/Elp1 downregulated cell lines and FD-derived cells and tissues. Immunostaining of the cytoskeleton component α-tubulin in IKAP/Elp1 downregulated cells revealed disorganization of the microtubules (MTs) that was reflected in aberrant cell shape and process formation. In contrast to a recent report on the decrease in α-tubulin acetylation in IKAP/Elp1 downregulated cells, we were unable to observe any effect of IKAP/Elp1 deficiency on α-tubulin acetylation in the FD cerebrum and in a variety of IKAP/Elp1 downregulated cell lines. To explore possible candidates involved in the observed aberrations in MTs, we focused on superior cervical ganglion-10 protein (SCG10), also called STMN2, which is known to be an MT destabilizing protein. We have found that SCG10 is upregulated in the IKAP/Elp1-deficient FD cerebrum, FD fibroblasts and in IKAP/Elp1 downregulated neuroblastoma cell line. To better understand the effect of IKAP/Elp1 deficiency on SCG10 expression, we investigated the possible involvement of RE-1-silencing transcription factor (REST), a known repressor of the SCG10 gene. Indeed, REST was downregulated in the IKAP/Elp1-deficient FD cerebrum and IKAP/Elp1 downregulated neuroblastoma cell line. These results could shed light on a possible link between IKAP/Elp1 deficiency and cytoskeleton destabilization.

Nitric oxide is involved in establishing the balance between cell cycle progression and cell death in the developing neural tube.

Endogenous nitric oxide (NO) has recently been shown to affect cell cycle progression in the neural tube (NT) of the chick embryo. High NO levels trigger entry into S phase basally, while low NO levels facilitate mitosis apically. Here, we further explore the involvement of NO in determining cell numbers in the chick NT. In addition to the effect of short-term (6 h) NOS inhibition, we have observed a concomitant decrease in programmed cell death (PCD). Paradoxically, long-term (12 h) NOS inhibition caused an increase in PCD to compensate for the high proliferation rate under these conditions. Long-term treatment with a NO donor caused a decrease in S phase and increased PCD. The effects produced by the NO donor could be alleviated by folic acid that facilitated entry into S phase and prevented PCD. The effects produced by NOS inhibition (12 h) could be overcome by an embryo extract, used as a source of extracellular survival factors that enhanced proliferation and prevented PCD. Taken together, these data demonstrate that changing endogenous NO levels affect the balance between cell proliferation and PCD in NT of the developing chick embryo.

Evidence that nitric oxide regulates cell-cycle progression in the developing chick neuroepithelium

In all developing epithelia, the nuclei continually migrate between the apical and basal sides of the cell during the cell cycle, with S phase occurring basally and mitosis occurring apically. The purpose and mechanism of this nuclear migration are unknown. Here, we show that nitric oxide (NO) is endogenously produced in the developing chicken neural tube, where it apparently regulates cell‐cycle progression. We provide evidence that high NO levels promote entry into S phase basally, whereas low levels of NO facilitate entry into mitosis apically.

Serum free cultured bone marrow mesenchymal stem cells as a platform to characterize the effects of specific molecules.

Human mesenchymal stem cells (hMSC) are easily isolated from the bone marrow by adherence to plastic surfaces. These cells show self-renewal capacity and multipotency. A unique feature of hMSC is their capacity to survive without serum. Under this condition hMSC neither proliferate nor differentiate but maintain their biological properties unaffected. Therefore, this should be a perfect platform to study the biological effects of defined molecules on these human stem cells. We show that hMSC treated for five days with retinoic acid (RA) in the absence of serum undergo several transcriptional changes causing an inhibition of ERK related pathways. We found that RA induces the loss of hMSC properties such as differentiation potential to either osteoblasts or adipocytes. We also found that RA inhibits cell cycle progression in the presence of proliferating signals such as epidermal growth factor (EGF) combined with basic fibroblast growth factor (bFGF). In the same manner, RA showed to cause a reduction in cell adhesion and cell migration. In contrast to these results, the addition of EGF+bFGF to serum free cultures was enough to upregulate ERK activity and induce hMSC proliferation and cell migration. Furthermore, the addition of these factors to differentiation specific media instead of serum was enough to induce either osteogenesis or adipogenesis. Altogether, our results show that hMSCs ability to survive without serum enables the identification of signaling factors and pathways that are involved in their stem cell biological characteristics without possible serum interferences.

Thymic involution, a co‐morbidity factor in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating disease, characterized by extremely rapid loss of motor neurons. Our studies over the last decade have established CD4+ T cells as important players in central nervous system maintenance and repair. Those results, together with recent findings that CD4+ T cells play a protective role in mouse models of ALS, led us to the current hypothesis that in ALS, a rapid T‐cell malfunction may develop in parallel to the motor neuron dysfunction. Here, we tested this hypothesis by assessing thymic function, which serves as a measure of peripheral T‐cell availability, in an animal model of ALS (mSOD1 [superoxide dismutase] mice; G93A) and in human patients. We found a significant reduction in thymic progenitor‐cell content, and abnormal thymic histology in 3–4‐month‐old mSOD1 mice. In ALS patients, we found a decline in thymic output, manifested in the reduction in blood levels of T‐cell receptor rearrangement excision circles, a non‐invasive measure of thymic function, and demonstrated a restricted T‐cell repertoire. The morbidity of the peripheral immune cells was also manifested in the increase of pro‐apoptotic BAX/BCXL2 expression ratio in peripheral blood mononuclear cells (PBMCs) of these patients. In addition, gene expression screening in the same PBMCs, revealed in the ALS patients a reduction in key genes known to be associated with T‐cell activity, including: CD80, CD86, IFNG and IL18. In light of the reported beneficial role of T cells in animal models of ALS, the present observation of thymic dysfunction, both in human patients and in an animal model, might be a co‐pathological factor in ALS, regardless of the disease aetiology. These findings may lead to the development of novel therapeutic approaches directed at overcoming the thymic defect and T‐cell deficiency.

Structural profiling and biological performance of phospholipid-hyaluronan functionalized single-walled carbon nanotubes

In spite of significant insolubility and toxicity, carbon nanotubes (CNTs) erupt into the biomedical research, and create an increasing interest in the field of nanomedicine. Single-walled CNTs (SWCNTs) are highly hydrophobic and have been shown to be toxic while systemically administrated. Thus, SWCNTs have to be functionalized to render water solubility and biocompatibility. Herein, we introduce a method for functionalizing SWCNT using phospholipids (PL) conjugated to hyaluronan (HA), a hydrophilic glycosaminoglycan, with known receptors on many types of cancer and immune cells. This functionalization allowed for CNT solubilization, endowed the particles with stealth properties evading the immune system, and reduced immune and mitochondrial toxicity both in vitro and in vivo. The CNT-PL-HA internalized into macrophages and showed low cytotoxicity. In addition, CNT-PL-HA did not induce an inflammatory response in macrophages as evidenced by the cytokine profiling and the use of image-based high-content analysis approach in contrast to non-modified CNTs. In addition, systemic administration of CNT-PL-HA into healthy C57BL/6 mice did not alter the total number of leukocytes nor increased liver enzyme release as opposed to CNTs. Taken together, these results suggest an immune protective mechanism by the PL-HA coating that could provide future therapeutic benefit.

Folic acid rescues nitric oxide-induced neural tube closure defects

The importance of folic acid (FA) in the prevention of neural tube defects (NTD) involving failure of neural tube (NT) closure in the developing embryo is well established. Various forms of NTD, especially spina bifida and anencephaly, can be prevented by supplementing the diet with FA in the periconceptional period. How FA acts to prevent NTD is still unknown. Nitric oxide (NO) had been shown to be able to induce NTD in 10.5-day rat embryos, and biochemical studies showed that NO inhibits methionine synthase (MS), by interfering with the transfer of the methyl group from the methyl donor 5-methyl-tetrahydrofolate (5mTHF) to homocysteine. We have shown that endogenously produced NO in the neuroepithelium, at the time of NT closure, plays a role in cell cycle progression and the regulation of cell numbers in the developing NT. We also found that high NO levels increase the demand for folates. Here, we describe experiments that examine the direct effect of FA and NO produced by the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) on the process of NT closure in the chick embryo ex ovo. The data here demonstrate that exposure to NO produced by SNAP results in an open NT by inhibition of the MS reaction, thus interfering with the flow of one carbon unit through the folate pathway, possibly by vitamin B12 poisoning. These effects of NO can be effectively alleviated by FA or vitamin B12. Thus, the findings described here may explain why FA supplementation during pregnancy prevents NT closure defects. The experimental system used here is focused on a defined 6 h window between the eight and 12 somite stage in chick embryo development. This is the time when NT closure takes place by a process that involves programmed cell death. Eight somite-stage embryos were explanted as previously described, and cultured for 6 h individually in DMEM conditioned agarose dishes, in the presence of the NO donor SNAP (2 mM) with or without 50 mg/ml FA. The effects of these treatments on normal NT closure in whole mount and serial NT transverse sections of 12 somite stage embryos are shown in Figure 1a. The NO levels produced by SNAP treatment in our experimental system clearly interfere with NT closure. While FA alone had no effect on NT closure, its addition to the SNAP-treated embryos alleviated the SNAP effect by a complete restoration of NT closure. To further verify that NO is indeed the factor responsible for the NTD, experiments were performed using 4mg/ml hemoglobin to quench NO. This hemoglobin treatment in fact counteracted the SNAP effect on NT closure, resulting in normal NT closure rates (2273% open NT transverse sections, Po0.001 significance by two-way ANOVA). The observation that SNAP produced NO inhibits NT closure in the chick embryo and that this inhibition is counteracted by FA raised the possibility that NO interferes with one carbon unit metabolism. This conclusion is corroborated by a recent report suggesting that NO inhibits the MS reaction, thereby blocking the flow of one carbon units through the folate pathway. Our results and this report prompted us to examine the possibility that NO induction of NTD is caused by inhibition of the MS reaction. To this end, eight somite stage embryos were treated with 2 mM SNAP in the presence or absence of FA. For the last 4 h of incubation, 5-[C]m-THF was added to the embryo culture. At the end of the experiment (12 somite stage), NT closure was assessed in whole mount preparations and the embryos were processed for assaying the MS reaction by measuring the incorporation of labelled methyl groups into TCA-precipitable material. The correlation between MS activity and NT closure is demonstrated in Figure 1b. High MS activity was detected in 12 somite embryos with closed NT morphology. In this category were most of the untreated embryos and those treated with a combination of SNAP and FA. Decreased MS activity was detected in embryos treated with SNAP alone, in which open NT morphology was observed. These results suggest that normal NT closure requires normal MS activity. One carbon unit flow associated with MS activity can be used therefore as a biochemical marker of normal NT closure. To further characterize the inhibitory effects of NO on the MS reaction and its dependence on FA availability, we assayed MS activity in dose response experiments with FA and SNAP in pooled embryo cultures. A clear concentrationdependent effect of SNAP on MS activity was observed, and that FA at a concentration of 50 mg/ml completely restored normal MS activity (Figure 1c). Since NO is known to interact with cobalamin (vitamin B12), 4 a known cofactor in the methyl transfer reaction, we argued that the effect of NO on the methyl group transfer reaction may be a result of sequestration of this cofactor by NO. To test this possibility, we added 50 mg/ml of vitamin B12 to the MS assay system, and found that, at this concentration, vitamin B12 significantly restores MS activity in embryos exposed to 2 mM SNAP (Figure 1c). It is therefore tempting to speculate that the molecular mechanism by which NO inhibits the carbon flow through the folate pathway involves cobalamin poisoning by NO. This Cell Death and Differentiation (2004) 11, 361–363 & 2004 Nature Publishing Group All rights reserved 1350-9047/04

Assessing cellular toxicities in fibroblasts upon exposure to lipid-based nanoparticles: a high content analysis approach

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Enriched Population of PNS Neurons Derived from Human Embryonic Stem Cells as a Platform for Studying Peripheral Neuropathies

Lipid-based nanoparticles (LNPs) are widely used for the delivery of drugs and nucleic acids. Although most of them are considered safe, there is confusing evidence in the literature regarding their potential cellular toxicities. Moreover, little is known about the recovery process cells undergo after a cytotoxic insult. We have previously studied the systemic effects of common LNPs with different surface charge (cationic, anionic, neutral) and revealed that positively charged LNPs ((+)LNPs) activate pro-inflammatory cytokines and induce interferon response by acting as an agonist of Toll-like receptor 4 on immune cells. In this study, we focused on the response of human fibroblasts exposed to LNPs and their cellular recovery process. To this end, we used image-based high content analysis (HCA). Using this strategy, we were able to show simultaneously, in several intracellular parameters, that fibroblasts can recover from the cytotoxic effects of (+)LNPs. The use of HCA opens new avenues in understanding cellular response and nanotoxicity and may become a valuable tool for screening safe materials for drug delivery and tissue engineering.

Neural tube closure depends on nitric oxide synthase activity

Background The absence of a suitable cellular model is a major obstacle for the study of peripheral neuropathies. Human embryonic stem cells hold the potential to be differentiated into peripheral neurons which makes them a suitable candidate for this purpose. However, so far the potential of hESC to differentiate into derivatives of the peripheral nervous system (PNS) was not investigated enough and in particular, the few trials conducted resulted in low yields of PNS neurons. Here we describe a novel hESC differentiation method to produce enriched populations of PNS mature neurons. By plating 8 weeks hESC derived neural progenitors (hESC-NPs) on laminin for two weeks in a defined medium, we demonstrate that over 70% of the resulting neurons express PNS markers and 30% of these cells are sensory neurons. Methods/Findings Our method shows that the hNPs express neuronal crest lineage markers in a temporal manner, and by plating 8 weeks hESC-NPs into laminin coated dishes these hNPs were promoted to differentiate and give rise to homogeneous PNS neuronal populations, expressing several PNS lineage-specific markers. Importantly, these cultures produced functional neurons with electrophysiological activities typical of mature neurons. Moreover, supporting this physiological capacity implantation of 8 weeks old hESC-NPs into the neural tube of chick embryos also produced human neurons expressing specific PNS markers in vivo in just a few days. Having the enriched PNS differentiation system in hand, we show for the first time in human PNS neurons the expression of IKAP/hELP1 protein, where a splicing mutation on the gene encoding this protein causes the peripheral neuropathy Familial Dysautonomia. Conclusions/Significance We conclude that this differentiation system to produce high numbers of human PNS neurons will be useful for studying PNS related neuropathies and for developing future drug screening applications for these diseases.