Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies.

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

RhoA inhibits neural differentiation in murine stem cells through multiple mechanisms.

Spontaneous neural differentiation of embryonic stem cells is induced by Noggin-mediated inhibition of bone morphogenetic protein 4 (BMP4) signaling. RhoA is a guanosine triphosphatase (GTPase) that regulates cytoskeletal dynamics and gene expression, both of which control stem cell fate. We found that disruption of Syx, a gene encoding a RhoA-specific guanine nucleotide exchange factor, accelerated retinoic acid-induced neural differentiation in murine embryonic stem cells aggregated into embryoid bodies. Cells from Syx(+/+) and Syx(-/-) embryoid bodies had different abundances of proteins implicated in stem cell pluripotency. The differentiation-promoting proteins Noggin and RARγ (a retinoic acid receptor) were more abundant in cells of Syx(-/-) embryoid bodies, whereas the differentiation-suppressing proteins SIRT1 (a protein deacetylase) and the phosphorylated form of SMAD1 (the active form of this transcription factor) were more abundant in cells of Syx(+/+) embryoid bodies. These differences were blocked by the overexpression of constitutively active RhoA, indicating that the abundance of these proteins was maintained, at least in part, by RhoA activity. The peripheral stress fibers in cells from Syx(-/-) embryoid bodies were thinner than those in Syx(+/+) cells. Furthermore, less Noggin and fewer vesicles containing Rab3d, a GTPase that mediates Noggin trafficking, were detected in cells from Syx(-/-) embryoid bodies, which could result from increased Noggin exocytosis. These results suggested that, in addition to inhibiting Noggin transcription, RhoA activity in wild-type murine embryonic stem cells also prevented neural differentiation by limiting Noggin secretion.

VEGF and Angiopoietin-1 exert opposing effects on cell junctions by regulating the Rho GEF Syx.

Vascular endothelial growth factor (VEGF) and Ang1 (Angiopoietin-1) have opposing effects on vascular permeability, but the molecular basis of these effects is not fully known. We report in this paper that VEGF and Ang1 regulate endothelial cell (EC) junctions by determining the localization of the RhoA-specific guanine nucleotide exchange factor Syx. Syx was recruited to junctions by members of the Crumbs polarity complex and promoted junction integrity by activating Diaphanous. VEGF caused translocation of Syx from cell junctions, promoting junction disassembly, whereas Ang1 maintained Syx at the junctions, inducing junction stabilization. The VEGF-induced translocation of Syx from EC junctions was caused by PKD1 (protein kinase D1)-mediated phosphorylation of Syx at Ser(806), which reduced Syx association to its junctional anchors. In support of the pivotal role of Syx in regulating EC junctions, syx(-/-) mice had defective junctions, resulting in vascular leakiness, edema, and impaired heart function.

Rab13-dependent trafficking of RhoA is required for directional migration and angiogenesis.

Angiogenesis requires concomitant remodeling of cell junctions and migration, as exemplified by recent observations of extensive endothelial cell movement along growing blood vessels. We report that a protein complex that regulates cell junctions is required for VEGF-driven directional migration and for angiogenesis in vivo. The complex consists of RhoA and Syx, a RhoA guanine exchange factor cross-linked by the Crumbs polarity protein Mupp1 to angiomotin, a phosphatidylinositol-binding protein. The Syx-associated complex translocates to the leading edge of migrating cells by membrane trafficking that requires the tight junction recycling GTPase Rab13. In turn, Rab13 associates with Grb2, targeting Syx and RhoA to Tyr(1175)-phosphorylated VEGFR2 at the leading edge. Rab13 knockdown in zebrafish impeded sprouting of intersegmental vessels and diminished the directionality of their tip cells. These results indicate that endothelial cell mobility in sprouting vessels is facilitated by shuttling the same protein complex from disassembling junctions to the leading edges of cells.

Membrane traffic as a coordinator of cell migration and junction remodeling.

The change in the overall shape of developing organs is a consequence of the cumulative movement, reshaping, and proliferation of the individual mural cells that make up the walls of these organs. Recent observations suggest that the shape and the position of endothelial cells (ECs) in growing blood vessels are highly dynamic, implying that these cells remodel their junctions extensively and do not preserve their initial relative positions. In order to determine the mechanisms that confer the dynamic behavior of mural ECs, we tracked the trafficking of a cell junction protein complex that consists of the RhoA-specific guanine exchange factor (GEF) Syx, the scaffold protein Mupp1, and the phospholipid binding protein Amot.1 We found that RhoA co-trafficked with this complex on the same endocytic vesicles, and that its cellular activity pattern was determined by Rab13-dependent trafficking. The vesicles were targeted by a Rab13-associated protein complex to Tyr(1175)-phosphorylated VEGFR2 at the leading edge of ECs migrating under a VEGF gradient. These results indicate that the dynamic behavior of ECs in sprouting vessels is conferred by using the same protein complex for the regulation of both cell junctions and cell motility. Together with previous studies that demonstrated regulation of Rac signaling by Rab5-dependent trafficking,(2) it appears now that membrane traffic is tightly coupled to the regulation of Rho GTPases, and, consequently, to the regulation of the actin cytoskeleton, cell junctions, and cell migration.

Beta4 tubulin identifies a primitive cell source for oligodendrocytes in the mammalian brain.

We have identified a novel population of cells in the subventricular zone (SVZ) of the mammalian brain that expresses beta4 tubulin (betaT4) and has properties of primitive neuroectodermal cells. betaT4 cells are scattered throughout the SVZ of the lateral ventricles in adult human brain and are significantly increased in the SVZs bordering demyelinated white matter in multiple sclerosis brains. In human fetal brain, betaT4 cell densities peak during the latter stages of gliogenesis, which occurs in the SVZ of the lateral ventricles. betaT4 cells represent <2% of the cells present in neurospheres generated from postnatal rat brain but >95% of cells in neurospheres treated with the anti-mitotic agent Ara C. betaT4 cells produce oligodendrocytes, neurons, and astrocytes in vitro. We compared the myelinating potential of betaT4-positive cells with A2B5-positive oligodendrocyte progenitor cells after transplantation (25,000 cells) into postnatal day 3 (P3) myelin-deficient rat brains. At P20, the progeny of betaT4 cells myelinated up to 4 mm of the external capsule, which significantly exceeded that of transplanted A2B5-positive progenitor cells. Such extensive and rapid mature CNS cell generation by a relatively small number of transplanted cells provides in vivo support for the therapeutic potential of betaT4 cells. We propose that betaT4 cells are an endogenous cell source that can be recruited to promote neural repair in the adult telencephalon.

Relationship between blood pressure and Alzheimer's disease in Linxian County, China.

To investigate the relationship between hypertension and Alzheimer's disease(AD) and the change of Alzheimer's patients' blood pressure(BP) before and after the onset of AD, we conducted this epidemiological study. Subjects for this study were individuals who participated in a large scale, randomized controlled trial of nutritional intervention from 1984 to 1991. Participants were initially screened for dementia using Chinese Mini-Mental State Examination (CMMS) and Activities of Daily Living (ADL). Positive subjects were subsequently administered a detailed neuropsychological and neurobehavioral examination. The diagnosis of AD was made by a consensus conference of psychiatrists using Diagnostic And Statistical Manual Of Mental Disorders-Fourth Edition(DSM-IV) criteria. 16488 subjects were examined and 301 were diagnosed as AD. We compared the prevalence of AD in different populations that were stratified with 1984's systolic or diastolic blood pressure(those four stratifications being high blood pressure, borderline blood pressure, normal, low blood pressure), and compared the change of blood pressure of 301 AD patients between 1984 and 1999-2000, which is before and after the onset of AD respectively. Multiple Logistic Regression (1:1 nested case-control study) was used to assess if hypertension is an independent risk factor for AD, and Trend test was used to assess the relationship between blood pressure and AD. Here we demonstrate that there was a significant difference in AD prevalence among different populations stratified by systolic or diastolic blood pressure (P < 0.01). The prevalence is highest in hypertension group, and lowest in hypotension group. Multiple Logistic Regression identified high blood pressure as a risk factor for AD (OR = 1.97, 95%CI:1.09-3.54, P = 0.02). Trend test showed that there is a significant dose-response relationship between blood pressure and AD (P < 0.0002). For hypertensive AD patients, there was no significant difference in systolic blood pressure(SBP) before and after the onset of AD, but diastolic blood pressure(DBP) decreased dramatically after the onset of AD (P < 0.01); however, the result also showed that DBP decrease occurred in the non-demented group. Based on this, we think the DBP decrease is not related to AD. We further investigated whether BP values differed crossed-sectionally between the AD-patients and non-demented individuals. We found that regardless of SBP or DBP, the BP values of the AD group were all significantly higher than that of non-demented. In summary, these data suggest there is a strong relationship between hypertension and AD; however, the mechanism remains to be studied.

[The association between angiotensin I converting enzyme gene polymorphism and Chinese late onset Alzheimer disease].

OBJECTIVE: To investigate the relationship between angiotensin I converting enzyme gene insertion/deletion polymorphism and Alzheimer disease (AD), as well as the effect of hypertension on the relationship. METHODS: This case-control study, included 96 AD patients meeting the DSM-IV diagnosis, and 96 subjects as controls coming from the same area and in the same environmental condition. Using the polymerase chain reaction (PCR) amplified the DNA segments, and the PCR products were identified by 2% agarose gel and visualized by ethidium bromide staining. RESULTS: There was significant difference between AD patients and controls in ACE genotypes and alleles distribution, as well as between AD patients with high blood pressure and controls with high blood pressure. But between normotensive AD patients and normotensive controls, there was no significant difference in ACE genotypes distribution (P>0.05). CONCLUSION: ACE genotypes associated with the risk of AD, but II genotype as risk genetic factor only restricted in subjects with high blood pressure.