Ole Steen Jørgensen

Brain specific synaptosomal membrane proteins demonstrated by crossed immunoelectrophoresis

CROSSHI immunoelectrophoresis has been introduced as a method for the investigation of complex antigen mixtures because of its quantitative properties and because of its excellent resolution power (LAURELL, 1965; CLARKE & FREEMAN, 1968; BOCK et al., 1971). We are currently using crossed immunoelectrophoresis together with other immunochemical methods for the study of antigens in extracts of brain subcellular particles (BOCK et al., 1974). In this study we report the demonstration of the brain specificity of five different antigens from rat brain synaptosomal plasma membranes (SPM) by means of crossed-line immunoelectrophoresis with absorption in situ of antibodies.

DETERMINATION OF BRAIN‐SPECIFIC ANTIGENS IN SHORT TERM CULTIVATED RAT ASTROGLIAL CELLS AND IN RAT SYNAPTOSOMES

—The brain‐specific antigens 14·3·2, GFA, A5, F3, D1, D2, D3 and C1 were quantitated in a short‐term astroglial cell culture taken as a model of glial cells, and in synaptosomes, synaptosomal membranes and synaptic vesicles as neuronal material. Furthermore, the antigens were quantitated in newborn rat brain, as this served as the starting material for the cell culture. The membrane antigens C1, D1, D2 and D3 were absent from the cultured astroglia, indicating a neuronal origin for these antigens. C1 was enriched 3‐fold in synaptosomes and synaptosomal membranes and more than 10‐fold in synaptic vesicles indicating that this antigen might be a marker protein for nerve endings. The name Synaptin is introduced for this antigen. Conversely, the data on the antigens D1, D2 and D3 indicated that these antigens were not restricted to the synaptosomes although they were of neuronal origin. Trace amounts of the cathodal part of the heterogeneous cytoplasmic antigen 14·3·2 were present in the cell culture, possibly originating from a few contaminating neurons. The cytoplasmic antigens A5 and F3 were found both in the astroglial culture and in the synaptosomal fraction. F3, however, was found in low concentration in the synaptosomes and 3‐fold enriched in newborn rat brain compared to rat brain from 35‐day‐old rats or to 21‐day‐old brain cell cultures. It was therefore regarded as a brain specific fetal antigen. The antigen GFA was highly enriched in the astroglial culture compared to whole brain and only trace amounts were found in the synaptosomal fraction supporting the astroglial origin of this antigen.

Induction by Hydrocortisone of Glutamine Synthetase in Mouse Primary Astrocyte Cultures

Glutaminc synthetase activity was investigated in developing primary astroglial cultures established from newborn mouse cerebral hemispheres. Between the 2nd and 4th week of culture there was little change in activity under our standard culturing conditions; however, when hydrocortiwne (10 μM) was added to the cultures for 48 h, the enzyme activity increased two‐ to fourfold, depending upon the age of the culture, with maximum response in 2‐week‐old cultures. The addition of dibutyryl cyclic AMP (dRcAMP) to the culture medium caused morphological differentiation of the astroglial cells but eliminated the response of the cells to hydrocortisone. Culturing in elevated serum levels, which delays morphological differentiation and inhibits astroglial cytodifferentiation after exposure to dBcAMP, shifted the time of maximal response to hydrocortisone from 2 to 3 weeks and prevented the abolishment of glutamine synthetase induction by dBcAMP. The induction of glutamine synthetase by hydrocortisone was prevented by actinomycin D (0.5 μg/ml), indicating its dependence upon RNA and protein synthesis. The present work thus confirms reports in the literature that hydrocortisone induces glutamine synthetase in neural tissues, but differs from the findings of Moscona and co‐workers in the chick retina that intact tissues are required for the induction to occur.

The nervous system specific protein D2 is involved in adhesion among neurites from cultured rat ganglia

The rat nervous system specific protein D2 is an integral membrane protein present on all bodies and neurites of cultured neurons from brain fetuses [l-3]. In adult brain, D2 is found mainly on the outside of synaptic membranes [4-61. It has been hypothesized that this protein may be involved in the recognition mechanism during the early stage of synaptogenesis 15451. Here we have examined the effect of anti-D2 antibodies on the formation of neurite fascicles from cultured rat sympathetic ganglia. The results indicated that D2 may be involved in neurite-neurite interaction, a property which has been reported for the cell adhesion molecule (CAM) isolated from chick embryo neural tissue [7,8]. This property and similarities in molecular weight, membrane distribution and localization raise the possibility that D2 and CAM may be evolutionarily related proteins. This hypothesis is strengthened by the finding, that anti-CAM antibodies crossreact with D2 protein.

Erythropoietin in patients with aneurysmal subarachnoid haemorrhage: a double blind randomised clinical trial

SummaryBackground. Erythropoietin (EPO) is neuroprotective in experimental models of stroke and subarachnoid haemorrhage (SAH) and possibly in patients with thromboembolic stroke. We studied the efficacy and safety of EPO in patients with SAH. Methods. A larger scale clinical trial was planned but preliminarily terminated because of a lower than expected inclusion rate. However, 73 patients were randomised to treatment with EPO (500 IU/kg/day for three days) or placebo. The primary endpoint was Glasgow Outcome Score at six months. We further studied surrogate measures of secondary ischaemia, i.e. transcranial Doppler (TCD) flow velocity, symptomatic vasospasm, cerebral metabolism (microdialysis) and jugular venous oximetry, biochemical markers of brain damage (S-100β and neuron specific enolase) and blood–brain barrier integrity. Findings. The limited sample size precluded our primary hypotheses being verified and refuted. However, data from this study are important for any other study of SAH and as much raw data as possible are presented and can be included in future meta analyses. On admission the proportion of patients in a poor condition was higher in the EPO group compared with the placebo group but the difference was statistically insignificant. In the EPO-treated patients the CSF concentration of EPO increased 600-fold. Except for a higher extracelullar concentration of glycerol in the EPO group probably caused by the poorer clinical condition of these patients, there were no statistically significant group differences in the primary or secondary outcome measures. EPO was well tolerated. Conclusions. Beneficial effects of EPO in patients with SAH cannot be excluded or concluded on the basis of this study and larger scale trials are warranted.

Synaptic Plasma Mèmbrane Antigen D2 Measured in Human Cerebrospinal Fluid by Rocket-Line Immunoelectrophoresis. Determination in Psychiatric and Neurological Patients

The concentration of an antigen partially identical to the rat brain synaptic membrane antigen D2, was measured in human cerebrospinal fluid. The method used was rocket‐line immunoelectrophoresis in agarose gel containing the detergent Triton X‐100, and line‐forming extract of rat brain membranes. No significant differences were found between the controls and patients with multiple sclerosis, inflammatory‐degenerative neurological diseases, atrophic brain diseases, acute cerebro‐vascular disease, schizophrenia, or affective psychosis.

Neural cell adhesion molecule (NCAM) as a quantitative marker in synaptic remodeling

The neural cell adhesion molecule (NCAM) participates in adhesion and neuritic outgrowth during nervous system development. In the adult brain, NCAM is considered to be involved in neuronal sprouting and synaptic remodeling. the NCAM concentration of brain tissue has proved to be a useful marker of these processes, especially when viewed in comparison with the concentration of a marker of mature synapses, e.g. D3-protein (SNAP-25) or synaptophysin. The present review focusses on studies of adult brain in which NCAM concentration estimates and NCAM/D3 ratios have been used to evaluate the rate of synaptic remodeling in brain damage and degenerative diseases.

Nervous System‐Specific Proteins in Developing Rat Cerebral Cells in Culture

Abstract: The nervous system‐specific proteins: synaptin, D1, D2, D3, glial fibrillary acidic protein (GFA) and 14‐3‐2, were quantified in dissociated cerebral cells from the foetal rat brain at various times of growth in culture. By approximately 1 week in culture, the neuronal membrane markers synaptin, D1, D2, and D3 could all be demonstrated. A maximum concentration of 10–20% for synaptin, D1, and D3 and 160% for D2, in comparison with the levels in adult forebrain, was attained during the 2nd week in vitro. The astroglial gliofilament marker GFA increased continuously, reaching by 38 days of cultivation an 18‐fold higher level than the concentration in adult forebrain. The neuronal cytoplasm marker 14‐3‐2 could be demonstrated in trace amounts, and only after more than 1 week in vitro. Neuronal cell bodies and processes stained by indirect immunofluorescence using an anti‐D2 serum were strongly fluorescent after 1 week in vitro. Immunofluorescence staining for GFA revealed a cytoplasmatic filamentous network in perinuclear areas and processes of, presumably, astroblasts.

Synaptic membrane protein D2 in the cerebrospinal fluid of manic-melancholic patients.

The synaptic membrane protein D2 was measured in the cerebrospinal fluid (CSF) of manic‐melancholic patients. The concentration of D2 increased with the age of the patients until about 35 years of age. No difference was found between the D2‐concentration in CSF from a control group compared with different manic‐melancholic subgroups. The D2‐concentration in CSF collected from the patients during depression or mania was compared with CSF collected from the same patients when their moods were normalized. In the case of the depressed patients, we found that the D2‐concentration increased slightly when the mood was normalized.

BRAIN-SPECIFIC ANTIGENS IN THE QUAKING MOUSE DURING ONTOGENY

Abstract— By means of crossed Immunoelectrophoresis the concentrations of 7 brain‐specific antigens have been investigated during the ontogenic development of normal and Quaking mice. Two proteins, the glial fibrillary acidic protein and the brain‐specific membrane protein D5 were found to be strongly increased in mutant brains. The synaptosomal antigen synaptin (Cl), the 14‐3‐2 protein of neuronal cytoplasm, and the neuronal membrane antigens D1, D2 and D3 were all present at normal levels in mutant brains.