Postnatal neurogenesis in hippocampal slice cultures: early in vitro labeling of neural precursor cells leads to efficient neuronal production

Neurogenesis continues throughout life in the hippocampus. To study postnatal neurogenesis in vitro, hippocampal slices from rats on postnatal day 5 (P5) were cultured on a porous membrane for 14 or 21 days. In the initial experiments, precursor cells were labeled with bromodeoxyuridine (BrdU) after 7 days in culture because hippocampal slices are generally used in experiments after 1-2 weeks in culture. Fourteen days after labeling, however, only about 10% of BrdU-labeled cells expressed neuronal markers, although in living rats, about 80% of cells labeled with BrdU on P5 had become neurons by P19. Next, rats were injected with BrdU 30 min before culture, after which hippocampal slices were cultured for 14 days to examine the capacity of in vivo-labeled neural precursors to differentiate into neurons in vitro. In this case, more than two-thirds of BrdU-labeled cells expressed neuronal markers, such as Hu, NeuN, and PSA-NCAM. Furthermore, precursor cells underwent early in vitro labeling by incubation with BrdU or a modified retrovirus vector carrying EGFP for 30 min from the beginning of the culture. This procedure resulted in a similar high rate of neuronal differentiation and normal development into granule cells. In addition, time-lapse imaging with retrovirus-EGFP revealed migration of neural precursors from the hilus to the granule cell layer. These results indicate that in vivo- and early in vitro-labeled cultures are readily available ex vivo models for studying postnatal neurogenesis and suggest that the capacity of neural precursors to differentiate into neurons is reduced during the culture period.     

The cytotoxic action of lymphokine activated killer cells upon the human glioma cell line U251 is stimulated by bispecific monoclonal antibody (MoAb) constructs

The ability of IL-2 stimulated mononuclear cells to kill the human glioblastoma cell line U251 has been investigated. Highest cytotoxic activity was generated in low cell density cultures incubated for 15 days with 250–1000 U/ml IL-2. Sub-optimal killing was noted, with cells only exposed to IL-2 for three days. Under the latter conditions, bispecific monoclonal antibodies (MoAbs) of either anti-CD3 or anti-CD16 and an anti-NCAM MoAb stimulated LAK cell activity markedly. Anti-CD16 conjugates were found more effective than anti-CD3 and (Fab)₂ constructs more efficacious than those made with whole Ig molecules. Maximal stimulation of LAK cell activity was noted with bispecific MoAbs. Little effect was observed with either single or mixtures of monomeric MoAbs. Furthermore, no effect of bispecific MoAbs was observed when target cells lacked expression of NCAM. These results could be of clinical importance as it is not always feasible to screen LAK cells for optimal activity before administration to patients. Whilst bispecific MoAbs have no effect on optimally stimulated LAK cells, they are not inhibitory and can stimulate killing under sub-optimal IL-2 stimulation.     

Papillary glioneuronal tumour: clinicopathological and biochemical study of one case with 7-year follow up

Among the mixed glioneuronal tumours, a new variant called papillary glioneuronal tumour has recently been delineated. A case occurring in a 23-year-old man is reported. The tumour was cystic with a mural nodule enhanced by gadolinium injection. It was located within the left parieto-occipital lobe. Surgical excision showed a greyish friable tumour with cystic areas. Histopathological examination revealed a pseudopapillary component comprising a single layer of regular cells, arranged around hyalinised vessels. These cells were immunoreactive with anti-glial fibrillary acidic protein and HNK1 antibodies. A neurocytoma-like component coexisted with round blind cells and focal fibrillary rosettes. These cells were immunostained by anti-neuron-specific enolase and anti-synaptophysin antibodies. Neither mitoses nor ganglioid cells were seen. HNK1, the three isoforms of NCAM, and the L1 adhesion molecule were detected by Western blot analysis. Ultrastructural study showed three different types of cells. The first contained gliofilaments, the second showed long processes with true synapses, and the third was poorly differentiated. However, all had identical nuclei and contained dense bodies. These findings suggest a common origin for the tumour cells derived from a bipotential neuroglial progenitor. As for other mature mixed neuroglial tumours, the prognosis is good. Our patient is free of disease 7 years after complete surgical treatment. Received: 17 May 1999 / Revised, accepted: 21 July 1999     

慢性醋酸铅染毒对小鼠皮层神经细胞粘附分子mRNA表达的观察

目的观察慢性醋酸铅染毒对小鼠记忆功能的影响,探讨神经细胞粘附分子(NCAM)与学习记忆的关系。方法小鼠通过饮水饲以不同浓度醋酸6周后,测定水迷宫、突触传递长时程增强(LTP)及大脑皮层NCAM mRNA表达量。结果随着染毒铅浓度的增加,小鼠血清、大脑皮层铅浓度明显升高,水迷宫实验的潜伏期明显延长、错误次数显著增加,在脑海马CAL区的LTP受到的抑制程度加大,PS振幅平均增长率降低,与正常对照组比较,差异均有显著性(P<0.05)。36、9、mmol.L-1醋酸铅组大脑皮层NCAM mRNA表达量比正常对照组分别低25.6%、56.6%和80.8%(P<0.05);9 mmol.L-1醋酸铅组比3 mmol.L-1醋酸铅组低74.2%,比6 mmol.L-1醋酸铅组低55.7%,差异均有显著性(P<0.05)。结论铅的摄入可降低小鼠大脑皮层NCAM mRNA表达水平,对学习记忆能力有抑制作用;NCAM mRNA表达量随铅浓度升高而下降。     

Clinicopathological correlates of plasma cell CD56 (NCAM) expression in multiple myeloma

The aim of this prospective, long-term study was to define the flow cytometric characteristics of plasma cell CD56 expression as well as determine the clinical characteristics of 204 multiple myeloma (MM) patients and 26 plasma cell leukemia (PCL) patients with regard to CD56 expression. CD56 expression intensity was determined by measurement of antigen molecules on the cell defined as Antibodies Bound per Cell (ABC) and calculation of Relative Fluorescence Intensity (RFI). CD56 expression was found in 66% of MM and 54% of PCL cases. The RFI values for individual MM patients ranged from 7.6 to 27.4 while ABC values on MM plasma cells from 2255 to 58469. There was a correlation between the proportion of all bone marrow CD38⁺⁺/CD138⁺ cells with CD56 expression and ABC and RFI indices. With regard to CD56 expression positive patients, the CD56^- MM patients presented lower frequency of osteolysis (p = 0.01). The median survival was 48 months in CD56⁺ patients and 43 months (p = 0.84) in CD56^- cases. In conclusion, CD56 expression carries no distinct adverse prognosis and the lack of CD56 expression does not define a unique clinicopathological or prognostic entity in MM. A remarkable heterogeneity of CD56 expression intensity in CD56⁺ patients imposes the necessity of determining CD56 expression intensity in candidates to antibody-based therapy.     

Specificity of peripheral nerve regeneration: interactions at the axon level

Peripheral nerves injuries result in paralysis, anesthesia and lack of autonomic control of the affected body areas. After injury, axons distal to the lesion are disconnected from the neuronal body and degenerate, leading to denervation of the peripheral organs. Wallerian degeneration creates a microenvironment distal to the injury site that supports axonal regrowth, while the neuron body changes in phenotype to promote axonal regeneration. The significance of axonal regeneration is to replace the degenerated distal nerve segment, and achieve reinnervation of target organs and restitution of their functions. However, axonal regeneration does not always allows for adequate functional recovery, so that after a peripheral nerve injury, patients do not recover normal motor control and fine sensibility. The lack of specificity of nerve regeneration, in terms of motor and sensory axons regrowth, pathfinding and target reinnervation, is one the main shortcomings for recovery. Key factors for successful axonal regeneration include the intrinsic changes that neurons suffer to switch their transmitter state to a pro-regenerative state and the environment that the axons find distal to the lesion site. The molecular mechanisms implicated in axonal regeneration and pathfinding after injury are complex, and take into account the cross-talk between axons and glial cells, neurotrophic factors, extracellular matrix molecules and their receptors. The aim of this review is to look at those interactions, trying to understand if some of these molecular factors are specific for motor and sensory neuron growth, and provide the basic knowledge for potential strategies to enhance and guide axonal regeneration and reinnervation of adequate target organs.     

Patterns of immunoreactivity specific for gustducin and for NCAM differ in developing rat circumvallate papillae and their taste buds

α-Gustducin and neural cell adhesion molecule (NCAM) are molecules previously found to be expressed in different cell types of mammalian taste buds. We examined the expression of α-gustducin and NCAM during the morphogenesis of circumvallate papillae and the formation of their taste buds by immunofluorescence staining and laser-scanning microscopy of semi-ultrathin sections of fetal and juvenile rat tongues. Images obtained by confocal laser scanning microscopy in transmission mode were also examined to provide outlines of histology and cell morphology. Morphogenesis of circumvallate papillae had already started on embryonic day 13 (E13) and was evident as the formation of placode. By contrast, taste buds in the circumvallate papillae started to appear between postnatal day 0 (P0) and P7. Although no cells with immunoreactivity specific for α-gustducin were detected in fetuses from E13 to E19, cells with NCAM-specific immunoreactivity were clearly apparent in the entire epithelium of the circumvallate papillary placode, the rudiment of each circumvallate papilla and the developing circumvallate papilla itself from E13 to E19. However, postnatally, both α-gustducin and NCAM became concentrated within taste cells as the formation of taste buds advanced. After P14, neither NCAM nor α-gustducin was detectable in the epithelium around the taste buds. In conclusion, α-gustducin appeared in the cytoplasm of taste cells during their formation after birth, while NCAM appeared in the epithelium of the circumvallate papilla-forming area. However, these two markers of taste cells were similarly distributed within mature taste cells.     

Increased sensitivity of human lung adenocarcinoma cells to cisplatin associated with downregulated contactin-1

Contactin-1 (CNTN-1), a glycosyl phosphatidylinositol anchor neural cell adhesion molecule (ACAM), is thought to function not only in nervous system development but also in the invasion and metastasis of several tumours. To investigate whether CNTN-1 is involved in multidrug resistance (MDR) in lung adenocarcinoma, CNTN-1 expression was compared between MDR human lung adenocarcinoma A549/cisplatin (A549/DDP) cells and its progenitor A549 cells. The comparison showed that CNTN-1 expression in A549/DDP cells was significantly higher than in A549 cells both at the mRNA level and the protein level. In order to confirm the physiological function of the abnormal expression, lentivirus-mediated short hairpin RNA (shRNA) was used to silence CNTN-1. Cell cytotoxicity assay and cell apoptosis assay revealed that silencing CNTN-1 both in A549 cells and in A549/DDP cells not only rendered cells more sensitive to cisplatin than the negative control, but also increased the cisplatin-induced apoptosis. Metastasis and invasion assays demonstrated that CNTN-1 knockdown reduced metastasis and invasion but did not affect A549 or A549/DDP cell proliferation. To investigate whether the abnormal expression of CNTN-1 is associated with characteristics of patients with non-small cell lung cancer (NSCLC), immunohistochemistry was used to detect CNTN-1 expression in 143 tissue samples from NSCLC patients and the results showed that the degree of CNTN-1 expression positively correlated with lymphatic invasion in patients with lung adenocarcinoma who received adjuvant cisplatin- or carboplatin-based treatment after surgery. Thus, we concluded that CNTN-1 is closely related with MDR of lung adenocarcinoma. Additionally, CNTN-1 is a novel marker to predict chemotherapeutic efficacy of patients with lung adenocarcinoma, especially with regard to cisplatin- or carboplatin-based regimens.     

Insight into the structural mechanism of the bi-modal action of an NCAM mimetic,the C3 peptide

C3, a synthetic peptide binding to the Ig1 module of the neural cell adhesion molecule (NCAM) has previously been identified and shown to inhibit NCAM homophilic binding and NCAM-mediated activation of the fibroblast growth factor (FGF) receptor (FGFR). However, C3 can also stimulate signalling on its own in a way similar to NCAM. Here we show that in the absence of NCAM, C3 can bind and activate FGFR, whereas in the presence of NCAM, C3 inhibits the NCAM-stimulated FGFR activation without activating FGFR on its own. Several competing models of FGFR activation by NCAM have been previously proposed. In one of them, the FGFR Ig2–Ig3 modules are involved in binding to NCAM, whereas in another – the FGFR “acid box” region mediates the interaction. The bi-modal effect of C3 can be explained in the context of the former model and is not consistent with the latter, thus providing evidence in support of the former model.     

Causal evidence for the involvement of the neural cell adhesion molecule,NCAM, in chronic stress‐induced cognitive impairments

In rodents, chronic stress induces long‐lasting structural and functional alterations in the hippocampus, as well as learning and memory impairments. The neural cell adhesion molecule (NCAM) was previously hypothesized to be a key molecule in mediating the effects of stress due to its role in neuronal remodeling and since chronic stress diminishes hippocampal NCAM expression in rats. However, since most of the evidence for these effects is correlative or circumstantial, we tested the performance of conditional NCAM‐deficient mice in the water maze task to obtain causal evidence for the role of NCAM. We first validated that exposure to chronic unpredictable stress decreased hippocampal NCAM expression in C57BL/6 wild‐type mice, inducing deficits in reversal learning and mild deficits in spatial learning. Similar deficits in water maze performance were found in conditional NCAM‐deficient mice that could not be attributed to increased anxiety or enhanced corticosterone responses. Importantly, the performance of both the conditional NCAM‐deficient mice and chronically stressed wild‐type mice in the water maze was improved by post‐training injection of the NCAM mimetic peptide, FGLs. Thus, these findings support the functional involvement of NCAM in chronic stress‐induced alterations and highlight this molecule as a potential target to treat stress‐related cognitive disturbances. © 2009 Wiley‐Liss, Inc.