Identification and characterization of neuroblasts in the subventricular zone and rostral migratory stream of the adult human brain

It is of great interest to identify new neurons in the adult human brain, but the persistence of neurogenesis in the subventricular zone (SVZ) and the existence of the rostral migratory stream (RMS)-like pathway in the adult human forebrain remain highly controversial. In the present study, we have described the general configuration of the RMS in adult monkey, fetal human and adult human brains. We provide evidence that neuroblasts exist continuously in the anterior ventral SVZ and RMS of the adult human brain. The neuroblasts appear singly or in pairs without forming chains; they exhibit migratory morphologies and co-express the immature neuronal markers doublecortin, polysialylated neural cell adhesion molecule and βIII-tubulin. Few of these neuroblasts appear to be actively proliferating in the anterior ventral SVZ but none in the RMS, indicating that neuroblasts distributed along the RMS are most likely derived from the ventral SVZ. Interestingly, no neuroblasts are found in the adult human olfactory bulb. Taken together, our data suggest that the SVZ maintains the ability to produce neuroblasts in the adult human brain.     

Thrombospondin-1 binds to ApoER2 and VLDL receptor and functions in postnatal neuronal migration

Apolipoprotein E receptor 2 (ApoER2), very low-density lipoprotein receptor (VLDLR), and Dab1 are the main components of the Reelin signalling cascade. Reelin is the sole ligand defined so far in signalling through this pathway. Postnatal migration of neuronal precursors from the subventricular zone (SVZ) to the olfactory bulb (OB), however, depends on ApoER2 and Dab1, but functions independently of Reelin. Here, we show that thrombospondin-1 (THBS-1) is a novel physiological ligand for ApoER2 and VLDLR. THBS-1 is present in the SVZ and along the entire rostral migratory stream (RMS). It binds to ApoER2 and VLDLR and induces phosphorylation of Dab1. In contrast to Reelin, it does not induce Dab1 degradation or Akt phosphorylation, but stabilizes neuronal precursor chains derived from subventricular explants. Lack of THBS-1 results in anatomical abnormalities of the RMS and leads to a reduction of postnatal neuronal precursors entering the OB.     

Changes in cell migration and survival in the olfactory bulb of the pcd/pcd mouse

Postnatally, the Purkinje cell degeneration mutant mice lose the main projecting neurons of the main olfactory bulb (OB): mitral cells (MC). In adult animals, progenitor cells from the rostral migratory stream (RMS) differentiate into bulbar interneurons that modulate MC activity. In the present work, we studied changes in proliferation, tangential migration, radial migration patterns, and the survival of these newly generated neurons in this neurodegeneration animal model. The animals were injected with bromodeoxyuridine 2 weeks or 2 months before killing in order to label neuroblast incorporation into the OB and to analyze the survival of these cells after differentiation, respectively. Both the organization and cellular composition of the RMS and the differentiation of the newly generated neurons in the OB were studied using specific markers of glial cells, neuroblasts, and mature neurons. No changes were observed in the cell proliferation rate nor in their tangential migration through the RMS, indicating that migrating neuroblasts are only weakly responsive to the alteration in their target region, the OB. However, the absence of MC does elicit differences in the final destination of the newly generated interneurons. Moreover, the loss of MC also produces changes in the survival of the newly generated interneurons, in accordance with the dramatic decrease in the number of synaptic targets available.     

Changes in cytosolic CA2+ are not involved in DDT-induced loss of gap junctional communication in WB-F344 cells

Recent studies have demonstrated that the insecticide DDT is a tumor promoting agent. Similar to many other tumor promoting agents, DDT has been shown to inhibit gap junctional intercellular communication (GJIC) between cells in culture, and it has been suggested that DDT-induced loss of communication between adjacent cells may depend on changes in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i). In the present study, the role of[Ca²⁺]_i in DDT-induced loss of GJIC was investigated in WB-F344 rat liver cells using the scrape-loading/dye transfer assay (SLDT) and the Ca²⁺ fourescent indicator, furà-2. Our results show that DDT at non-cytotoxic concentrations caused a reversible loss of GJIC. Inhibition of GJIC was not associated with detectable increases in [Ca²⁺]_i, and was not prevented by loading cells with the intracellular Ca²⁺ chelator, BAPTA. In addition, the hydroquinone, tBuBHQ, which caused a 2+3 fold sustained increase in [Ca²⁺]_i, did not inhibit GJIC. Conversely, when untreated cells were loaded with increasing BAPTA concentrations, GJIC were lost. These results indicate that increases in [Ca²⁺]_i are not responsible for DDT-induced loss of communication and that, in general an increase in [Ca²⁺]_i, within physiological levels is not sufficient to abolish GJIC. However, Ca²⁺-dependent processes that are active at normal resting [Ca²⁺ _i appear to be required for the maintenance of GJIC.Abbreviations [Ca²⁺] cytosolic free Ca²⁺ concentration - GJIC gap junctional intercellular communication - SLDT scrape-loading/dye transfer assay - DDT 1,1,1-trichloro-2,2-di-(4-chlorophenyl)ethane - tBuBHQ 2,5-di(tert-butyl)-1,4-benzohydroquinone - LDH lactate dehydrogenase - ER endoplasmic reticulum - Fura-2 1-[2-(5carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxyl]-2-(2amino-5-methylphenoxy)-ethane-N,N,N,N-tetraacetic acid - BAPTA bis-(o-aminophenoxy)-ethane-N,N,N,N-tetraaceticacid - Fura-2/AM and BAPTA/AM are the cell permeant acetoxymethyl ester forms of fura-2 and BAPTA, respectively     

Permeability and structural studies of heart cell gap junctions under normal and altered ionic conditions

Summary The permeability and ultrastructure of communicating junctions of cultured neonatal rat ventricular cells are examined under control conditions and during treatments which raise intracellular Ca²⁺. Lucifer Yellow (487 mol wt) is used to examine junctional permeability. Under normal ionic conditions dye transfer from an injected muscle cell to neighboring muscle cells occurs rapidly (in less than 6 sec) while transfer to neighboring fibroblasts occurs more slowly. Application of monensin, which results in a partial contracture with superimposed asynchrony, or A23187, which results in a partial contracture, do not inhibit the transfer of dye between the muscle cells. A23187 did result in junctional blockade between muscle cells and fibroblasts. Freeze-fractured gap junctions from control and monensin-treated cells exhibit no distinguishable differences. Center-to-center spacing was not significantly different, 9.0 nm±1.4sd versus 9.2 nm±1.3sd, respectively; and particle diameters were virtually unchanged, 8.69 nm±0.9sd versus 8.61 nm±1.07sd, respectively. These results suggest that concentrations of intracellular Ca²⁺ sufficient to support a partial contracture and asynchronous contractile activity do not result in a block of intercellular junctions in cultured myocardial cells. These results are discussed in terms of intracellular Ca²⁺-buffering and junctional sensitivity to Ca²⁺.     

Selective localization of G protein γ5 subunit in the subventricular zone of the lateral ventricle and rostral migratory stream of the adult rat brain

G proteins play important roles in transmembrane signal transduction, and various isoforms of each subunit, alpha, beta and gamma, are highly expressed in the brain. The Ggamma5 subunit is a minor isoform in the adult brain, but we have previously shown it to be highly expressed in the proliferative region of the ventricular zone in the rat embryonic brain. We show here that Ggamma5 is also selectively localized in a proliferative region in the adult rat brain, including the subventricular zone of the lateral ventricle and rostral migratory stream. The Galphai2 subunit colocalized with Ggamma5 in these regions, the two subunits being present in neuronal precursors and ependymal cells but not in proliferating astrocytes. In addition, intense staining of Ggamma5 was seen in axons of the olfactory neurons, which are known to regenerate. These results suggest specific roles for Ggamma5 in precursor cells during neurogenesis so that this isoform might be a useful biological marker.     

Jagged1 signals in the postnatal subventricular zone are required for neural stem cell self-renewal

Neural stem cells (NSCs) in the postnatal mammalian brain self-renew and are a source of neurons and glia. To date, little is known about the molecular and cellular mechanisms regulating the maintenance and differentiation of these multipotent progenitors. We show that Jagged1 is required by mitotic cells in the subventricular zone (SVZ) and stimulates self-renewal of multipotent epidermal growth factor-dependent NSCs. Jagged1-expressing cells line the adult SVZ and are juxtaposed to Notch1-expressing cells, some of which are putative NSCs. In vitro, endogenous Jagged1 acts through Notch1 to promote NSC maintenance and multipotency. In vivo, reducing Jagged1/Notch1 signaling decreases the number of proliferating cells in the SVZ. In addition, soluble Jagged1 promotes self-renewal and neurogenic potential of multipotent neural progenitors in vitro. Our findings suggest a central role for Jagged1 in the NSC niche in the SVZ for maintaining a population of NSCs in the postnatal brain.     

Gap junctions assemble in the presence of cytoskeletal inhibitors, but enhanced assembly requires microtubules

The role of cytoskeletal elements in gap junction (GJ) assembly has been studied using Novikoff hepatoma cells treated with cytochalasin B (CB) to disrupt actin filaments or with colchicine or nocodazole to disrupt microtubules. After 60 min of cell reaggregation, freeze-fracture was used to evaluate quantitatively the "initiation," "maturation," and "growth" phases of GJ assembly. The development of junctional permeability to fluorescent dyes was also analyzed. The only effects of CB on the structure or permeability of the developing junctions involved an elongation of GJ aggregates and a small decrease in formation plaque areas. Colchicine (but not the inactive form, lumicolchicine) prevented the enhancement of GJ growth by cholesterol, but its effect on basal growth was equivocal. Nocodazole inhibited the growth of GJ, even under basal conditions, without an effect on initiation. Nocodazole also blocked the forskolin-enhanced increase in the growth of GJs and, in living MDCK cells, reduced the movement of transport intermediates containing green fluorescent protein-tagged connexin43. Thus, neither actin filaments nor microtubules appear to restrict GJ assembly by anchoring intramembrane GJ proteins, nor are they absolutely required for functional GJs to form. However, microtubules are necessary for enhanced GJ growth and likely for facilitating connexin trafficking under basal conditions.     

Longterm quiescent cells in the aged human subventricular neurogenic system specifically express GFAP‐δ

A main neurogenic niche in the adult human brain is the subventricular zone (SVZ). Recent data suggest that the progenitors that are born in the human SVZ migrate via the rostral migratory stream (RMS) towards the olfactory bulb (OB), similar to what has been observed in other mammals. A subpopulation of astrocytes in the SVZ specifically expresses an assembly‐compromised isoform of the intermediate filament protein glial fibrillary acidic protein (GFAP‐δ). To further define the phenotype of these GFAP‐δ expressing cells and to determine whether these cells are present throughout the human subventricular neurogenic system, we analysed SVZ, RMS and OB sections of 14 aged brain donors (ages 74‐93). GFAP‐δ was expressed in the SVZ along the ventricle, in the RMS and in the OB. The GFAP‐δ cells in the SVZ co‐expressed the neural stem cell (NSC) marker nestin and the cell proliferation markers proliferating cell nuclear antigen (PCNA) and Mcm2. Furthermore, BrdU retention was found in GFAP‐δ positive cells in the SVZ. In the RMS, GFAP‐δ was expressed in the glial net surrounding the neuroblasts. In the OB, GFAP‐δ positive cells co‐expressed PCNA. We also showed that GFAP‐δ cells are present in neurosphere cultures that were derived from SVZ precursors, isolated postmortem from four brain donors (ages 63‐91). Taken together, our findings show that GFAP‐δ is expressed in an astrocytic subpopulation in the SVZ, the RMS and the OB. Importantly, we provide the first evidence that GFAP‐δ is specifically expressed in longterm quiescent cells in the human SVZ, which are reminiscent of NSCs.     

Dye coupling and connexin expression by cortical radial glia in the early postnatal subventricular zone

In this study, we have analyzed the specific contribution of the cortical radial glia (RG) for gap junctional communication (GJC) within the postnatal subventricular zone (SVZ). To specifically target RG as source of dye‐coupling in situ, we have developed a new technique that involves direct cell loading through the processes that reach the pial surface, with a mix of gap junction permeant (Lucifer yellow, LY) and nonpermeant (rhodamine‐conjugated dextran 3 KDa, RD) fluorochromes, the latter used as a marker for direct loaded cells. Tissue sections were analyzed for identification of directly loaded (LY+RD+) and coupled cells (LY+RD–) in the SVZ. Directly loaded cells were restricted to the region underlying the pial loading surface area. Coupled cells were distributed in a bistratified manner, along the outer dorsal surface of the SVZ and aligning the ventricle, leaving the SVZ core relatively free. Blocking GJC prior to pial loading greatly reduced dye coupling. Phenotypic analysis indicated that coupling by RG excludes neuroblasts and is mostly restricted to cells of glial lineage. Notwithstanding, no corresponding restriction to specific cell phenotype was found for two connexin isotypes, Cx43 and Cx45, in the postnatal SVZ. The extensive homocellular cell coupling by RG suggests an important role in the regulation of neurogenesis and functional compartmentalization of the postnatal SVZ. © 2012 Wiley Periodicals, Inc. Develop Neurobiol 2012     

应用FRAP技术分析小鼠嵌合体和正常胚胎发育中细胞间隙连接介导通讯

应用激光扫描共聚焦显微镜的光漂白恢复(fluorescence redistribution after photobleaching,FRAP)技术分析小鼠嵌合体胚胎和正常胚胎的卵裂球之间细胞间隙连接介导通讯(gap junctional inter-cellular communica-tion,GJIC),结果发现:8-细胞期嵌合体胚胎的光漂白恢复率(24.3%)明显低于正常胚胎(64.2%),提示GJIC的降低可能是影响嵌合体胚胎发育率降低的因素之一;囊胚的光漂白恢复率也较低(22.7%),提示随着细胞分化,GJIC的水平有所降低。     

Maintaining Connexin43 Gap Junctional Communication in v-Src Cells Does Not Alter Growth Properties Associated with the Transformed Phenotype

Loss of connexin expression and/or gap junctional communication (GJC) has been correlated with increased rates of cell growth in tumor cells compared to their normal communication-competent counterparts. Conversely, reduced rates of cell growth have been observed in tumor cells that are induced to express exogenous connexins and re-establish GJC. It is not clear how this putative growth-suppressive effect of the connexin proteins is mediated and some data has suggested that this function may be independent of GJC. In mammalian cells that express v-Src, connexin43 (Cx43) is phosphorylated on Tyr247 and Tyr265 and this results in a dramatic disruption of GJC. Cells that express a Cx43 mutant with phenylalanine mutations at these tyrosine sites form functional gap junctions that, unlike junctions formed by wild type Cx43, remain functional in cells that co-express v-Src. These cells still appear transformed; however, it is not known whether their ability to maintain GJC prevents the loss of growth restraints that confine “normal” cells, such as the inability to grow in an anchorage-independent manner or to form foci. In these studies, we have examined some of the growth properties of cells with Cx43 gap junctions that remain communication-competent in the presence of the co-expressed v-Src oncoprotein.     

Intercellular communication via gap junctions affected by mechanical load in the bovine annulus fibrosus

Cells in the intervertebral disc, as in other connective tissues including tendon, ligament and bone, form interconnected cellular networks that are linked via functional gap junctions. These cellular networks may be necessary to affect a coordinated response to mechanical and environmental stimuli. Using confocal microscopy with fluorescence recovery after photobleaching methods, we explored the in situ strain environment of the outer annulus of an intact bovine disc and the effect of high-level flexion on gap junction signalling. The in situ strain environment in the extracellular matrix of the outer annulus under high flexion load was observed to be non-uniform with the extensive cellular processes remaining crimped sometimes at flexion angles greater than 25°. A significant transient disruption of intercellular communication via functional gap junctions was measured after 10 and 20 min under high flexion load. This study illustrates that in healthy annulus fibrosus tissue, high mechanical loads can impede the functioning of the gap junctions. Future studies will explore more complex loading conditions to determine whether losses in intercellular communication can be permanent and whether gap junctions in aged and degenerated tissues become more susceptible to load. The current research suggests that cellular structures such as gap junctions and intercellular networks, as well as other cell–cell and cell–matrix interconnections, need to be considered in computational models in order to fully understand how macroscale mechanical signals are transmitted across scales to the microscale and ultimately into a cellular biosynthetic response in collagenous tissues.     

Contact stimulation of prostate cancer cell migration: the role of gap junctional coupling and migration stimulated by heterotypic cell-to-cell contacts in determination of the metastatic phenotype of Dunning rat prostate cancer cells

BACKGROUND INFORMATION: Motile activity of tumour cells is regarded as a critical factor determining their metastatic potential. We have shown previously that contrary to the majority of normal cells, homotypic contacts between some tumour cells, among them low metastatic (AT-2) and highly metastatic (MAT-LyLu) rat prostate cancer cells, increase the speed of their movements. The aim of the present study was to determine the effect of heterotypic cell-to-cell contacts on the migration of rat prostate cancer cells differing in metastatic potential, and to correlate it with the intensity of homo- and heterologous gap junctional coupling. RESULTS: MAT-LyLu and AT-2 cells moving on the surface of fibroblasts displayed significantly greater cell displacement than those moving on plastic substrata. This effect correlated with the polarization (contact guidance) and increased speed of cell movements. However, in contrast with the migration on plastic substrata, where MAT-LyLu cells displayed considerably higher motility than AT-2 cells, no differences between both cell lines were observed on the surface of fibroblasts. On the other hand, in contrast with AT-2, Mat-LyLu cells displayed extensive homologous coupling mediated by connexin43 and were able to couple with normal fibroblasts. CONCLUSION: Heterotypic contacts between migrating prostatic cancer cells and normal fibroblasts can strongly stimulate their migration during invasion; however, this effect does not correlate with the gap junctional coupling between cancer cells and normal fibroblasts.     

Increased gap junctional intercellular communication is directly related to the anti-tumor effect of all-trans-retinoic acid plus tamoxifen in a human mammary cancer cell line

Additive effects against tumor cells might be achieved by combining anti-neoplastic agents directed against one or more altered mechanisms in cancer. We investigated the participation of gap junctional intercellular communication (GJIC), which is commonly dysfunctional in tumor cells as a possible mediating mechanism of the effect of all-trans-retinoic acid (RA) and tamoxifen (Tx) in MCF-7 human breast cancer cell lines. The combination of RA + Tx stimulated GJIC in approximately 53 +/- 3% of MCF-7 cells as early as after 6 h of treatment remaining communicated through 144 h of culture. The GJIC enhancement occurred along with immunolocalization of Cx26 and 43 at the membrane of contacting cells and correlated with higher protein levels. Cx40 immunoreactive plaques were detected at cell-to-cell contacts during 48 h of RA + Tx treatment that did not involve higher protein expression, to the contrary, a downregulation occurred after 72 h of treatment. Cell proliferation inhibition upon RA + Tx exposure was observed with optimal effects at 96-120 h of culture with an accumulation of cells primarily in G2/M and G0/G1 cell cycle boundaries. An enhancement of the pre-existing E-cadherin levels was observed after drug exposure along with a downregulation of Bcl-2 and C-myc protein levels and a reduction of telomerase activity, suggesting partial tumor phenotype reversion. Blockage of the RA + Tx-induced GJIC with 18-beta-glycyrrhetinic acid (beta-Gly) prevented in 34% the inhibition of MCF-7 proliferation and the E-cadherin increment in 30% at 96 h of culture. GJIC blockage did not alter the downregulation of Bcl-2, c-Myc, or telomerase activity induced by RA + Tx. Our results showed the participation of GJIC as a mediator mechanism of the combined action of RA and Tx in MCF-7 cells. The chemopreventive modulation of GJIC might represent an approachable alternative for the improvement of cancer therapy.     

Preendocytotic alterations in cumulus cell gap junctions precede meiotic resumption in the rat cumulus-oocyte complex

Cumulus cells in the mammalian ovary are normally connected to each other and to their enclosed oocyte by an extensive network of gap junctions (GJs). We have shown that the loss of cumulus cell GJs is correlated temporally with meiotic resumption in the intact preovulatory rat follicle (Larsen et al., 1986). Here we describe morphological changes in GJ particle packing patterns (PPPs) that occur prior to GJ loss and meiotic resumption in hormonally stimulated rat cumulus-oocyte complexes (COCs). In the PMSG-primed rat, 89% of the cumulus cell GJ area detected by freeze-fracture electron microscopy consists of tightly packed junctional particles: 4% exhibit loose PPPs of randomly dispersed particles; and 7% contain a mixture of both tight and loose PPPs. One to 2 hr after stimulation with hCG, the area of GJs containing tight PPPs drops by 50%-60%, while junctions exhibiting loosely organized and mixed patterns increase concomitantly. These shifts in PPPs are accompanied by the appearance of unusual particle-free areas of puckered or ruffled nonjunctional membrane at the GJ periphery. Cumulus cell GJs from isolated COCs incubated in FSH-containing medium demonstrate a similar shift in PPPs prior to meiotic resumption. The appearance of fusing areas of particle-free nonjunctional membrane at the GJ periphery in vitro is correlated with GJ loss and is not seen in COCs treated with dihydrocytochalasin B to inhibit endocytotic removal of cumulus GJs. The structural and temporal nature of these morphological observations supports the hypothesis that interruption of junctional communication plays a role in meiotic maturation of the preovulatory oocyte.     

Different pathways are involved in the early development of the transient oral apparatus in anuran tadpoles (Anura: Leiuperidae)

The oral apparatus of anuran tadpoles is a unique structure composed of soft and keratinized parts surrounding the mouth. Among the many variations, a common oral apparatus involves a dorsal gap in the marginal papillae, keratinized jaw sheaths, and two upper and three lower rows of labial teeth. In Leiuperidae, besides this generalized morphology, four configurations are distinguished by the arrangement of the lower marginal papillae and the number of lower tooth rows. Study of the early oral ontogeny in 12 species representing these five configurations shows variations in the development of the lower marginal papillae and the third lower labial tooth row. Similar configurations may result from similar pathways (e.g. Physalaemus cuvieri group and Pseudopaludicola falcipes) or different pathways (e.g. generalized oral discs of Pleurodema and Physalaemus). Different oral configurations may result from overlapping trajectories ending at different stages (e.g. Physalaemus riograndensis and Ph. biligonigerus) or different trajectories (e.g. Ph. henselii and Ph. gracilis). Further studies are needed to interpret the role that heterochrony has played in evolutionary change within this family. The unsuspected variation occurring in this transient structure highlights its evolutionary potential and might be insightful in studies of anuran phylogenies that are largely based on adult characters. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 104 , 330–345.