N-Adamantyl Phthalimidine: A New Thalidomide-like Drug That Lacks Cereblon Binding and Mitigates Neuronal and Synaptic Loss, Neuroinflammation, and Behavioral Deficits in Traumatic Brain Injury and LPS Challenge.

Neuroinflammation contributes to delayed secondary cell death following traumatic brain injury (TBI), has the potential to chronically exacerbate the initial insult, and represents a therapeutic target that has largely failed to translate into human efficacy. Thalidomide-like drugs have effectively mitigated neuroinflammation across cellular and animal models of TBI and neurodegeneration but are complicated by adverse actions in humans. We hence developed N-adamantyl phthalimidine (NAP) as a new thalidomide-like drug to mitigate inflammation without binding to cereblon, a key target associated with the antiproliferative, antiangiogenic, and teratogenic actions seen in this drug class. We utilized a phenotypic drug discovery approach that employed multiple cellular and animal models and ultimately examined immunohistochemical, biochemical, and behavioral measures following controlled cortical impact (CCI) TBI in mice. NAP mitigated LPS-induced inflammation across cellular and rodent models and reduced oligomeric α-synuclein and amyloid-ß mediated inflammation. Following CCI TBI, NAP mitigated neuronal and synaptic loss, neuroinflammation, and behavioral deficits, and is unencumbered by cereblon binding, a key protein underpinning the teratogenic and adverse actions of thalidomide-like drugs in humans. In summary, NAP represents a new class of thalidomide-like drugs with anti-inflammatory actions for promising efficacy in the treatment of TBI and potentially longer-term neurodegenerative disorders.

The transcription factor PITX1 drives astrocyte differentiation by regulating the SOX9 gene.

Astrocytes perform multiple essential functions in the developing and mature brain, including regulation of synapse formation, control of neurotransmitter release and uptake, and maintenance of extracellular ion balance. As a result, astrocytes have been implicated in the progression of neurodegenerative disorders such as Alzheimer's disease, Huntington's disease, and Parkinson's disease. Despite these critical functions, the study of human astrocytes can be difficult because standard differentiation protocols are time-consuming and technically challenging, but a differentiation protocol recently developed in our laboratory enables the efficient derivation of astrocytes from human embryonic stem cells. We used this protocol along with microarrays, luciferase assays, electrophoretic mobility shift assays, and ChIP assays to explore the genes involved in astrocyte differentiation. We demonstrate that paired-like homeodomain transcription factor 1 (PITX1) is critical for astrocyte differentiation. PITX1 overexpression induced early differentiation of astrocytes, and its knockdown blocked astrocyte differentiation. PITX1 overexpression also increased and PITX1 knockdown decreased expression of sex-determining region Y box 9 (SOX9), known initiator of gliogenesis, during early astrocyte differentiation. Moreover, we determined that PITX1 activates the SOX9 promoter through a unique binding motif. Taken together, these findings indicate that PITX1 drives astrocyte differentiation by sustaining activation of the SOX9 promoter.

Nurr1 performs its anti-inflammatory function by regulating RasGRP1 expression in neuro-inflammation.

Nurr1, a transcription factor belonging to the orphan nuclear receptor, has an essential role in the generation and maintenance of dopaminergic neurons and is important in the pathogenesis of Parkinson' disease (PD). In addition, Nurr1 has a non-neuronal function, and it is especially well known that Nurr1 has an anti-inflammatory function in the Parkinson's disease model. However, the molecular mechanisms of Nurr1 have not been elucidated. In this study, we describe a novel mechanism of Nurr1 function. To provide new insights into the molecular mechanisms of Nurr1 in the inflammatory response, we performed Chromatin immunoprecipitation sequencing (ChIP-Seq) on LPS-induced inflammation in BV2 cells and finally identified the RasGRP1 gene as a novel target of Nurr1. Here, we show that Nurr1 directly binds to the RasGRP1 intron to regulate its expression. Moreover, we also identified that RasGRP1 regulates the Ras-Raf-MEK-ERK signaling cascade in LPS-induced inflammation signaling. Finally, we conclude that RasGRP1 is a novel regulator of Nurr1's mediated inflammation signaling.

Biophysiochemical properties of endothelial cells cultured on bio-inspired collagen films.

BACKGROUND: In this study, we investigated the effect of the extracellular matrix on endothelial dysfunction by careful observation of human umbilical vein endothelial cells (HUVECs) cultured on denatured collagen film. RESULTS: HUVECs on denatured collagen film showed relatively high surface roughness compared with normal HUVECs. The expression levels of MMP-1, MMP-2 and CD146 increased in the ECs on denatured collagen film. In addition, we examined the accumulation of fluorescent beads on HUVEC layers subjected to circulatory flow. The number of accumulated fluorescent beads increased on the disorganized HUVEC layers. CONCLUSIONS: The proposed in vitro study using bio-inspired collagen films could potentially be used in the size- and ligand-based design of drugs to treat endothelial dysfunction caused by circulatory vascular diseases.

Phosphoinositides differentially regulate protrudin localization through the FYVE domain.

Protrudin is a FYVE (Fab 1, YOTB, Vac 1, and EEA1) domain-containing protein involved in transport of neuronal cargoes and implicated in the onset of hereditary spastic paraplegia. Our image-based screening of the lipid binding domain library revealed novel plasma membrane localization of the FYVE domain of protrudin unlike canonical FYVE domains that are localized to early endosomes. The membrane binding study by surface plasmon resonance analysis showed that this FYVE domain preferentially binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)), phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P(2)), and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) unlike canonical FYVE domains that specifically bind phosphatidylinositol 3-phosphate (PtdIns(3)P). Furthermore, we found that these phosphoinositides (PtdInsP) differentially regulate shuttling of protrudin between endosomes and plasma membrane via its FYVE domain. Protrudin mutants with reduced PtdInsP-binding affinity failed to promote neurite outgrowth in primary cultured hippocampal neurons. These results suggest that novel PtdInsP selectivity of the protrudin-FYVE domain is critical for its cellular localization and its role in neurite outgrowth.

Vitronectin promotes oligodendrocyte differentiation during neurogenesis of human embryonic stem cells.

We demonstrate enhanced differentiation of oligodendrocytes during neurogenesis of human embryonic stem cells (hESCs) using an extracellular matrix protein, vitronectin (VN). We show that VN is expressed in the ventral part of the developing human spinal cord. Combined treatment of retinoic acid, sonic hedgehog, and noggin in the presence of VN allows hESCs to differentiate into O4-positive oligodendrocytes. Particularly, VN profoundly promotes the derivation of oligodendrocyte progenitors that proliferate and differentiate into oligodendrocytes in response to mitogenic and survival factors. These results support the beneficial effect of VN on oligodendrocytic differentiation of hESCs.

Brain cancer stem-like cell genesis from p53-deficient mouse astrocytes by oncogenic Ras.

Here, we show that H-ras(V12) causes the p53-knockout mouse astrocytes (p53-/- astrocytes) to be transformed into brain cancer stem-like cells. H-ras(V12) triggers the p53-/- astrocytes to express a Nestin and a Cd133, which are expressed in normal and cancer neural stem cells. H-ras(V12) also induces the formation of a single cell-derived neurosphere under neural stem cell culture conditions. Furthermore, H-ras(V12)-overexpressing p53-/- astrocytes (p53-/-ast-H-ras(V12)) possess an in vitro self-renewal capacity, and are aberrantly differentiated into Tuj1-positve neurons both in vitro and in vivo. Amongst a variety of Ras-mediated canonical signaling pathways, we demonstrated that the MEK/ERK signaling pathway is responsible for neurosphere formation in p53-deficient astrocytes, whereas the PI3K/AKT signaling pathway is involved in oncogenic transformation in these cells. These findings suggest that the activation of Ras signaling pathways promotes the generation of brain cancer stem-like cells from p53-deficient mouse astrocytes by changing cell fate and transforming cell properties.

Comparative analysis of the developmental competence of three human embryonic stem cell lines in vitro.

One of the goals of stem cell technology is to control the differentiation of human embryonic stem cells (hESCs), thereby generating large numbers of specific cell types for many applications including cell replacement therapy. Although individual hESC lines resemble each other in expressing pluripotency markers and telomerase activity, it is not clear whether they are equivalent in their developmental potential in vitro. We compared the developmental competence of three hESC lines (HSF6, Miz-hES4, and Miz-hES6). All three generated the three embryonic germ layers, extraembryonic tissues, and primordial germ cells during embryoid body (EB) formation. However, HSF6 and Miz-hES6 readily formed neuroectoderm, whereas Miz-hES4 differentiated preferentially into mesoderm and endoderm. Upon terminal differentiation, HSF6 and Miz-hES6 produced mainly neuronal cells whereas Miz-hES4 mainly formed mesendodermal derivatives, including endothelial cells, leukocyte progenitors, hepatocytes, and pancreatic cells. Our observations suggest that independently-derived hESCs may differ in their developmental potential.

Neurogenic effect of vascular endothelial growth factor during germ layer formation of human embryonic stem cells.

Vascular endothelial growth factor (VEGF), a potent mitogen for vascular endothelial cells, has been suggested as a modulator that is involved in neurogenesis as well as angiogenesis. Here, we directly examined the effect of VEGF on neuroectodermal differentiation using human embryonic stem cells (hESCs). VEGF treatment upregulated the expression of neuroectodermal genes (Sox1 and Nestin) during germ layer formation in embryoid bodies (EBs) and efficiently increased the number of neural rosettes expressing both Pax6 and Nestin. The neural progenitors generated from VEGF-treated EBs further differentiated into cells that showed a similar pattern of gene expression observed in the development of dopaminergic neurons upon terminal differentiation. These results support the neurogenic effect of VEGF on hESC differentiation.

Nonylphenol and octylphenol-induced apoptosis in human embryonic stem cells is related to Fas-Fas ligand pathway.

Human embryonic stem (hES) cells have been proposed as a source of various cell types for cell replacement therapy. Besides their potential in therapeutic uses, ES cells also have other potential applications, such as in drug discovery and in vitro screening assays of various toxicants. Nonylphenol (NP) and octylphenol (OP) are common environmental contaminants, known to disrupt the reproductive and endocrine system. However, little is known about their toxicological effects on early embryonic development in humans. In this study, we used undifferentiated hES cells and the neural progenitor cells derived from them to investigate the potential toxicity of NP and OP. Our results show that the cytotoxic effects of NP and OP involve DNA fragmentation, the major characteristic of apoptosis. The NP- and OP-induced apoptosis was concomitant with the increased activity of Caspase-8 and -3. Moreover, both Fas and Fas ligand (FasL) protein expressions were markedly increased in the NP- or OP-exposed hES cells. These results suggest that NP and OP are able to trigger apoptosis in hES cells via a pathway dependent on caspase activation and Fas-FasL interaction. In particular, hES cell-derived neural progenitor cells had a higher sensitivity to the toxicants than undifferentiated hES cells, thereby suggesting that the toxic stress response may differ depending on the developmental stage. These findings offer new perspectives for understanding the fundamental mechanisms in chemical-induced apoptosis in hES cells.

ERK activation by thymosin-beta-4 (TB4) overexpression induces paclitaxel-resistance.

The development of paclitaxel-resistance in tumors is one of the most significant obstacles to successful therapy. Thymosin-beta-4 (TB4) has been known as actin-sequestering protein and functions in tumor metastasis. Here, we overexpressed TB4 in HeLa cells (TB4-HeLa) and examined the effect of TB4 in paclitaxel-induced cell death. TB4-HeLa cells showed a higher growth rate and a lower percentage of basal apoptosis than HeLa cells. TB4-HeLa cells were more resistant to paclitaxel-induced cell death than HeLa cells. TB4 transcript expression with paclitaxel treatment was dose-dependently increased in HeLa cells but that was not in TB4-HeLa cells. Small interfering RNA (siRNA) of TB4 inhibited HeLa cell growth and enhanced paclitaxel-induced cell death. Basal ERK phosphorylation was elevated and basal p38 kinase phosphorylation was reduced in paclitaxel non-treated TB4-HeLa cells. When treated with paclitaxel, cell death and resistance-induction were independent of ERK and p38 kinase activation. Paclitaxel-resistance of TB4-HeLa cells was overcome by the inhibition of basal ERK activity with PD98059 pre-treatment. The inhibition of basal p38 kinase activity with SB203580 pre-treatment attenuated the paclitaxel-induced HeLa cell death. In conclusion, TB4 induced paclitaxel-resistance through the elevation of basal level of ERK phosphorylation. Therefore, TB4 could be a novel target to regulate paclitaxel-resistance.