Dorte Linnemann

NCAM-antibodies modulate induction of long-term potentiation in rat hippocampal CA1.

The neural cell adhesion molecule (NCAM) probably plays a role in neural plasticity in the adult vertebrate brain. We here present evidence that NCAM may be involved in long-term potentiation (LTP) in the CA1-region of rat hippocampal slices. It is shown that local application of antibodies against NCAM inhibits subsequent LTP-induction. Thus NCAM may be directly involved in the initial phase of LTP-induction. These results have important implications for the possible involvement of NCAM in learning and memory.

Cell adhesion molecules in neural development.

Adhesive mechanisms have been implicated in several morphogenetic processes during development. In the last decade several molecules mediating specific adhesion between cells and between cells and their environment have been characterized. Possible roles in morphogenesis have been established for some of these molecules. In the brain, four cell-cell adhesion molecules have been characterized: NCAM, L1, the myelin-associated glycoprotein and N-cadherin. Furthermore, a cell-substrate adhesion molecule, cytotactin, and its proteoglycan ligand have been described. We here review the data on structure, localization, developmental regulation and function of these molecules in brain.

Characterization of soluble forms of NCAM.

Neural cell adhesion molecule (NCAM) has been described as a family of membrane glycoproteins. However, soluble NCAM immunoreactivity has long been recognized. We here show that soluble NCAM is composed of two quantitatively major polypeptides of M r 180 000 and 115 000 and two minor components of M r 160 000 and 145 000. Soluble NCAM was immunochemically identical to membrane NCAM, was polysialylated and carried the HNK‐1 epitope. It only constituted 0.8% of total NCAM in newborn rat brain. Soluble NCAM appeared in neuronal cell culture medium 15–30 min after the start of synthesis preceding accumulation of membrane‐associated NCAM on the cell surface. This indicates that soluble NCAM contains a secreted component.

Regional Changes in Expression of NCAM, GFAP, and S100 in Aging Rat Brain

In aging brain degenerative processes occur. However, the aging brain still have regenerative capacity although diminished compared to young rats. The neural cell adhesion molecule (NCAM) may be involved in neuroplasticity during regenerative events. In this study, the polypeptide composition and amount of NCAM was determined in regions of brain from young, mature and old rats. During adult life, the amount of NCAM decreased in several brain regions whereas in aged rats, NCAM was enhanced in all brain regions examined. The amount of the glial fibrillary acidic protein (GFAP) increased during aging in all brain regions reflecting general gliosis in the aged rat brain. The amount of the neuro- and gliotrophic protein S100 increased from young adult to mature age in all brain regions investigated followed by a decrease during old age. Aged rats were tested in a Morris water maze and a group of rats (20%) with learning impairment was defined. However, no differences in amount of NCAM, GFAP, or S100 were observed between aged rats with and without spatial learning impairment.

Heterogeneity of Soluble Neural Cell Adhesion Molecule

Abstract: Soluble neural cell adhesion molecule (NCAM) from rat brain neuronal cell culture media consists predominantly of a polypeptide of Mr∼ 115,000. Minor amounts of a polypeptide of Mr∼ 180,000 and two inconsistently appearing components of Mr 160,000 and 145,000 are also observed. The Mr 115,000 component is derived from the neuronal membrane NCAM components NCAM‐A of Mr 190,000, NCAM‐B of Mr 140,000, or both. Thus, as a part of the catabolism of membrane NCAM‐A plus ‐B, a minor fraction is posttranslationally cleaved and recovered in the media as discernible soluble NCAM polypeptides. The half‐life of membrane NCAM‐A plus ‐B is <24 h. Astrocyte culture media contains a predominant soluble NCAM component of Mr 120,000 derived from membrane‐associated NCAM‐C. A close comparison of deglycosylated soluble NCAM from astrocyte and neuronal cultures showed a small but consistent difference in Mr, a result suggesting that different NCAM polypeptides are released from the membrane of neurons and astrocytes. In contrast to the Mr 115,000‐120,000 NCAM polypeptides, the Mr 180,000 polypeptide from neuronal culture media does not seem to be derived from membrane‐attached NCAM and may therefore represent a secreted NCAM isoform

Developmental Study of the Cell Adhesion Molecule L1

Cell adhesion molecules are involved in several morphogenetic events during development. A spatiotemporal regulation of the expression of the neural cell adhesion molecule NCAM has previously been demonstrated. In this study, we describe developmental changes in the rat brain in the expression of another neural cell adhesion molecule, L1. The amount of L1 varies with age showing a peak value in early post-natal life. L1 is synthesized as a polypeptide with a relative molecular weight (Mr) of 200,000 in explant cultures of rat forebrain from embryonic day 17 to post-natal day 15, but L1 synthesis decreases relative to total protein synthesis during the same period. L1 sulphation does not appear to change developmentally whereas the degree of phosphorylation seems to decrease. At the plasma membrane L1 is present as three polypeptides with Mr 200,000, 140,000 and 80,000. The latter is phosphorylated like the 200,000 Mr polypeptide. Furthermore, L1 exists in a soluble form which consists of 170,000, 140,000 and 80,000 Mr polypeptides. During development a slight decrease in Mr of the 200,000 and 140,000 Mr polypeptides is observed, possibly due to changes in glycosylation. To conclude, L1 expression is temporally regulated and its function may thus, like NCAM, be developmentally modified by some of these changes.

Tissue MicroRNAs as Predictors of Outcome in Patients with Metastatic Colorectal Cancer Treated with First Line Capecitabine and Oxaliplatin with or without Bevacizumab

Purpose We tested the hypothesis that expression of microRNAs (miRNAs) in cancer tissue can predict effectiveness of bevacizumab added to capecitabine and oxaliplatin (CAPEOX) in patients with metastatic colorectal cancer (mCRC). Experimental Design Patients with mCRC treated with first line CAPEOX and bevacizumab (CAPEOXBEV): screening (n = 212) and validation (n = 121) cohorts, or CAPEOX alone: control cohort (n = 127), were identified retrospectively and archival primary tumor samples were collected. Expression of 754 miRNAs was analyzed in the screening cohort using polymerase chain reaction (PCR) arrays and expression levels were related to time to disease progression (TTP) and overall survival (OS). Significant miRNAs from the screening study were analyzed in all three cohorts using custom PCR arrays. In situ hybridization (ISH) was done for selected miRNAs. Results In the screening study, 26 miRNAs were significantly correlated with outcome in multivariate analyses. Twenty-two miRNAs were selected for further study. Higher miR-664-3p expression and lower miR-455-5p expression were predictive of improved outcome in the CAPEOXBEV cohorts and showed a significant interaction with bevacizumab effectiveness. The effects were strongest for OS. Both miRNAs showed high expression in stromal cells. Higher expression of miR-196b-5p and miR-592 predicted improved outcome regardless of bevacizumab treatment, with similar effect estimates in all three cohorts. Conclusions We have identified potentially predictive miRNAs for bevacizumab effectiveness and additional miRNAs that could be related to chemotherapy effectiveness or prognosis in patients with mCRC. Our findings need further validation in large cohorts, preferably from completed randomized trials.

Expression of NCAM mRNA and polypeptides in aging rat brain

In aging brain, degenerative as well as compensatory regenerative processes are believed to occur. The neural cell adhesion molecule NCAM is involved in developmental and regenerative processes in the brain. However, the role of NCAM in aging brain has not been characterized. In this study, the expression of NCAM mRNAs and polypeptides was investigated in aging rat brain. The 7.4 and 6.7 kb NCAM mRNAs were selectively downregulated during postnatal development, and the 5.2 and 2.9 kb NCAM mRNAs were upregulated. However, from postnatal day 40 to old age no change in NCAM mRNA classes was observed. The fraction of NCAM mRNA containing the VASE exon increased postnatally but remained stable during adult life. VASE, which is believed to modulate the binding capacity, seemed to be relatively more abundant in the 7.4 and 6.7 kb NCAM mRNAs, encoding transmembrane NCAM forms, than in the 5.2 and 2.9 kb NCAM mRNAs, coding for glycosyl phosphatidylinositol (GPI) linked NCAM. Conversely, insertion of exons a and AAG between exons 12 and 13, a region containing two fibronectin type III repeats, seemed to be more pronounced in 5.2 and 2.9 kb NCAM mRNAs than in the 7.4 and 6.7 kb mRNAs. During postnatal development an increase in the fraction of 6.7 kb NCAM mRNA containing the exons a and AAG was observed. However, during aging the fraction of NCAM mRNAs containing this exon combination seemed constant. At the protein level, NCAM‐A was downregulated both during development and aging. No changes were observed during aging in the composition of soluble NCAM forms in the brain, cerebrospinal fluid or blood plasma. The amount of NCAM in rat brain decreased during postnatal development, but remained at a constant level from postnatal day 40 to old age.

A developmental study of the biosynthesis of the neural cell adhesion molecule

The neural cell adhesion molecule (N-CAM) is a glycoprotein found in neurons, glial cells and muscle cells. In this report we describe developmental changes in biosynthesis of N-CAM polypeptides in rat forebrain explant cultures. N-CAM was synthesized as the following polypeptides: HMr (Mr between 250,000 and 350,000), A (200,000 Mr), B (135,000 Mr) and C (115,000 Mr). The biosynthetic pattern of N-CAM polypeptides changed during development: the biosynthesis of HMr and C increased relative to A and B. N-CAM biosynthesis decreased 100-fold from embryonic day 17 to postnatal day 25; N-CAM turnover decreased 350-fold during the same period. N-CAM polysialylation and sulfatation decreased markedly with age, whereas phosphorylation seemed to be constant during development. Only polypeptides A and B were phosphorylated, whereas A, B and C were sulfated. A was more sulfated and phosphorylated than B. It is concluded that the above described modulations of N-CAM may be of importance in the developmental regulation of cell-cell adhesion.

Tumour-infiltrating lymphocyte scores effectively stratify outcomes over and above p16 post chemo-radiotherapy in anal cancer

Background:The majority (90%) of anal cancers are human papillomavirus (HPV)-driven, identified using immunochemistry for p16. Compared with HPV− patients, those with HPV+ disease generally show improved survival, although relapse rates around 25% indicate a need for further stratification of this group.Methods:Using two cohorts of anal cancer, previously characterised for p16, we assessed the prognostic value of tumour-infiltrating lymphocytes (TILs).Results:Tumour-infiltrating lymphocyte scores were used to stratify p16+ cases, where tumours with absent/low levels of TIL had a relapse-free rate of 63%, as opposed to 92% with high levels of TIL (log rank P=0.006).Conclusions:Assessment of TIL adds to p16 status in the prognosis of anal cancer following chemo-radiotherapy and provides evidence of the clinical importance of the immune response.