l o u r ~ olf N.worhunirtry. 1978. Vol. 30. pp. 7-14. Pergamon Press Printed in Great Britain.

SHORT REVIEW

NERVOUS SYSTEM SPECIFIC PROTEINS ELISABETH BOCK The Protein Laboratory, University of Copenhagen, Sigurdsgade 34, DK-2200 Copenhagen N, Denmark

IN mE nervous system specific functions are con- procedures have also been described by UYEMUIUet & LEWNE(1971). The small nected to the various cell types. Nerve cell function al. (1971)and DANNIES includes the conduction of action potentials, synaptic molecule fMW 21.OOO-24.OOO)is of low immunoeenetransmission and the establishment of specific connee city. LEVINE& MOORE(1965)first made an antiserum tions Astrocytes have uptake mechanisms for neuro- by coupling the antigen to methylated bovine serum transmitters, and oligodendrocytes produce the albumin before immunization. The protein has been closely packed plasma membranes of myelin. These quantified by means of a complement fixation assay specific functions are to a large extent mediated by (MOOREet a[., 19683, quantitative immunoelectroproteins, soluble or membrane bound, which are phoresis (JACQUE et al., 19741 and a radioimmunoasunique to the nervous system. For the proteinchemi- say (UOZOMI& RYAN,1973). Sl00 was found to be MI characterization of neural cells and cell com- at least 10,CKKLfold more concentrated in brain than ponents several approaches can be used to choose in other tissues and closely cross-reactive forms were those proteins which probably are involved in specific found in all vertebrate species examined including functions One approach is to select proteins related mammals, birds, fish and reptiles (MOORE, 1972). to certain structures, which are important in the oper- Sl00 has been demonstrated both in the central and ation of the nervous system, such as the proteins of the periferal nervous system, and generally the level microtubules or myelin. Another is to make use of in white matter is higher than in grey; particularly high affinity markers such as r-neurotoxins, which high levels were found in cerebellum (MOORE,1972). specifically bind to the nicotinic acetylcholine recep The protein has been localized in both glial and neurtor. Determination of enzymes involved in specific onal cells. Glial localization was first demonstrated functions such as metabolic pathways of neurotrans- by HYDEN& MCEWEN(1966)by immunofluorescence mitters have also proven very useful. Finally, nervous staining and by microdissection of single neurons and system specific proteins, detected by immunochemical gha which were subsequently assayed for S-100 contechniques represent a group of markers of various tent. These authors also demonstrated S-100in neurcells and cell structures The study of proteins which OMI nuclei. Glial localization was suggested by two judged by immunochemical methods are specific to degeneration studies (CICERO et al., 19706; PEREZ et the nervous system, is based on the assumption that al., 1970)in which S-100 levels rose during degenera protein, which is not required by any other tissue, ation of the optic nerve and retrograde degeneration has an important function probably specific to the of the dorsal thalamus However, during Wallerian nervous system. Many of these proteins have been degeneration of the tibia1 nerve the S-100 level fell demonstrated in a wide range of species. The conser- (PEREZ & MOORE,1968). As a possible explanation vation during evolution further supports this assump for these contradictory findings it was suggested that tion. In the following, a series of nervous system S-100 was localized in the Schwann cells in the perispecific proteins found in normal mammalian brain feral nerve, but its continued synthesis there required will be described Proteins, which are soluble or at an intact axon. Determination of S-100in bulk-preleast partly soluble at neutral pH in conventional pared neurons and glial cells from rat indicated a 1976) Using buffer systems were the first to be isolated, and they glial localization (BOCK& HAMBERGER, the peroxidase-labelled antibody technique LUDWIN are therefore the best characterized. et al. (1976)and MBLLER et a[. (1977)found S100 $100 in astrocytes, both in the nucleus and the cytoplasm. This protein was first isolated by MOORE(1965)and It was also demonstrated in oligodendroglia, whereas MOORE& MCGREGOR (1965) It was named S-100 no staining in any structure of the neurons was because of its solubility in saturated ammonium sul- observed in rat brain. MOOREet al. (1977) studied phate at pH 7,a property which together with a very the localization in rat and chick by immunofluoresacidic isoelectric pH has been employed in the purifi- cence. In rat S-100 was restricted to astrocytes and cation procedure (MOORE,1965, 3972). Purification oligodendrocytes. In chick S-100 was detected in 7

8

ELISABETTI BCKK

several groups of neurons early in development, l O could only be detected whereas at later stages SO in oligodendrocytes and myelinated fibres In several reports S-100 has been localized to the nucleus ( M I C H Eet~ al., 1974) and specifically the nuclei of nerve cells (HYDEN & MCEWEN,1966; HANSSON et al., 1975). SlOO has also been demonstrated in isolated synaptosomes by DONATOet al. (1975) in two forms: soluble and membrane bound. These authors demonstrated a binding of SlOO to a receptor on the synaptic membranes. The binding was specific, saturable, reversible, and Caz+,time and temperature dependent. By the peroxidase-labelled antibody technique HAGLIDet al. (1974) demonstrated on the ultrastructural level S-100 on the postsynaptic density. It has been proposed that the protein is transported along the axon (MIANIet al. 1972). Although the possible presence of S-100 in the Schwann cells calls for a confirmation of an intraaxonal localization of the protein. Developmental studies in chick have shown that S l0 0 synthesis occur relatively late in the development compared to histological maturity, the SlOO level rising some time after cell division has ceased (CICERO et a/., 197b). BENDAet al. (1968) described the adaption to culture of cells derived from chemically induced rat glial tumors. A clonal cell line, the C-6 cell line, produced $100. P ~ F F EetRal. (1970) demonstrated the importance of homologous C-6 cell contact for Sl00 acet al. (1973) demonstrated, cumulation. HERSCHMAN that the absolute rate of synthesis of S-100 protein increased by a factor at least five to six, when C-6 cells entered the stationary phase, and they concluded, that the observed increase of S-100in stationary phase was due to a density dependent, contact mediated induction of synthesis. The degradation rate of S l 0 0 in stationary phase C-6 cell cultures was a p proximately.the same as the rate observed for total protein, and HERSCHMAN et al. (1973) concluded that this result was in agreement with the in uivo degrada& MOORE(1970), who tion data obtained by CICERO found a half life in the order of 12-15 days. In contrast, MCEWEN& HYDEN(1966) found in rat brain a half life of several hours, while total protein had a half life of several days. The differences may be due to different experimental approaches giving different degrees of contamination during isolation of $100. The methodology employed in these studies for determination of half life, i.e. labelling the proteins with a radioactive precursor followed by isolation of the spedic protein at various times after labelling, has however been shown to be of limited value (POOLE, 1971).The presence of SlOO has been demonstrated in several other cell lines derived from the & WECHSLER (1972) demonnervous system. PFEIFFER strated SlOO in a tumor Schwann cell line, RN-2, which also was able to synthesize myelin basic proet al. (1974) chemically induced tein. SCHUBERT tumors of neuronal and glial origin and adapted the resultant cells to clonal cell culture. SlOO was demon-

strated in both putative dial and putative neuronal clonal cell lines. However, not all glial cell lines contained S100. Many chemical studies have been performed giving clues as to what to examine in the search for a possible function of S-100. The molecular weight is approx 21,000-24,000(MOORE, 1965). It is composed of three subunits of about 7000 daltons which cannot be identical, since there is a single tryptophan residue per 21.000 daltons (DANNIES& LEMNE,1971). There is a growing body of evidence that SlOO is not a single homogenous protein species, but rather a heterogenous mixture of polypeptide chains possessing similar physicochemical properties (UYEMURA et al., 1971). In the presence of calcium ions S-100 has been shown to undergo a conformational change which exposes hydrophobic parts of the molecule and allows strong binding to artificial membranes, and facilitates the membrane transport of monovalent cations (CALIS %NO & BANGHAM, 1971 ; CALISSANO, 1973; CALISSANO et al., 1974). The 8-10 calcium binding sites have been shown to be of two species, and the high-affinity sites were inhibited and cooperative in the presence of K'. At physiological concentrations of K + and Na' these effects of Caz+occurred with calcium concentrations also in the physiological range. In the search for a function of SO l O these studies seen to provide promising leads. On the other hand the studies indicating that S l 0 0 is an integral constituent of the chromatin acidic proteins suggest that it may be involved in genomic regulation within neural tissue. 14-3-2 Using the same general method of preparation as described for the S l 0 0 protein (MOORE,1965) it was also possible to isolate the 14-3-2 protein and prepare antibodies against it (MOORE& PEREZ, 1968). Antigen-a isolated by BENNETT& EDELMAN (1968) and the nerve specific rat protein (NSP-R) isolated by MARANGOS et al. (1975a) have been shown to be antigenically similar to 14-3-2 (BENNETT,1974; MARANGOS et al., 19756). Suitable isolation procedures for 14-3-2 have been described by several authors (BENNETT & EDELMAN, 1968; MOORE, 1972; MARANGOS et a/., 1975a; G R A et~al., 1977). The protein.has been quantified by means of a complement fixation assay (MOOREet a/., 1968), quantitative immunoelectrophoresis (BOCK et ol., 1975) and a radioimmunoassay (MARANGOS et al., 19756; REVOLTELLA et al., 1976). 14-3-2 is specific to the nervous system, and brain was shown by MOORE& PEREZ (1968) to contain 100-200 times more 14-3-2 than any other organ tested. The protein was found in cross-reacting forms in all mammalian species and in birds. The content of 14-3-2 was higher in grey matter than in white and it was present in peripheral nerve. 14-3-2 has been localized to the neuronal cytoplasm by degeneret al., 19706). ation studies (PEREZet al.. 1970; OCERO Microdissection of single neurons with subsequent quantitative measurement of the protein demon-

Nervous system specific proteins strated the presence of 14-3-2 in the cell bodies, and also in the surrounding neuropil (MOORE,1975). RCKU.et al. (1976) localized 14-3-2 to neurons using the immunoperoxidase technique. Glial cells were never stained. However, the authors noted that not all neurons were stained. CIMMO et al. (1977) studied the localization during development by the immunofluorescence technique. Three classes of neurons were distinguished: a class of neurons that had 14-3-2 early in development and continuing throughout the life of the animal; a class of neurons where the protein only was present in a limited period during develop ment (e.g. Purkinje cells); and a class of neurons which never contained 14-3-2 (e.g. cortical motor neurons). Developmental studies have shown that 14-3-2 synthesis occurs relatively late in the process of development of the nervous system (CICEROet al., 197Oa). Axonal transport of 14-3-2 appeared to be a relatively slow process with little or no rapid component (MARANGOS et al., 1975~).BENNETT& EDELMAN (1968) noted a heterogeneity in electrophoretic mobility of 14-3-2, when analysing brain extracts by immunoelectrophoresis, revealed as a double arc precipitate. They isolated the anodally migrating component which showed a tendency to aggregate. By sedimentation velocity and equilibrium data 14-3-2 was found to be a dimer of two subunits of molecular weight of 39,000, although the protein by SDS polyacrylamide gel electrophoresis produced a band of an apparent molecular weight of 48,OOO (MARANGOS et al., 19750). SCHUBERT et al. (1974) chemically induced tumors of neuronal and glial origin and adapted the resultant cells to clonal cell culture. 14-3-2 was demonstrated in both glial and neuronal cell lines All the five neuronal clonal cell lines established by SCHUBERT and coworkers contained 14-3-2, however, of two clones (S20 and CIA) derived from the C1300 mouse neuroblastoma only CIA contained measurable amounts of 14-3-2. Neither 14-3-2 nor S-100 could be demonstrated in'a clonal myoblast line (L6)or a fibroblast line (3T3). HERSCHMAN & LERNm (1973) were unable to demonstrate 14-3-2 in two C1300 clones (N18 and NB41), whereas the human neuroblastoma line IMR-32 reacted strongly with antisera to human 14-3-2. In contrast to the results obtained for Sl00 protein in C-6 cells, 143-2 seemed to accumulate in growing IMR-32 cells Similar results were obtained by Aucusn-Tmo et al. (1973) indicating that 14-3-2 production in neuroblastoma cells is independent of cell division and cell contact. 14-3-2 has also been demonstrated in the tumor Schwann cell line RN-2 (Bum et al., 1976). The data on Sl00 and 14-3-2 indicate that demonstration of nervous system specific proteins in a clonal cell line may be used as a proof of a neuroectodermal origin, but it is not possible to distinguish between neuronal or dial origin of a cell line by means of immunochemical markers BOCK & DISSMG (1975) demonstrated enolase

9

activity connected to 14-3-2 immunoprecipitate. They also showed that the two electrophoretically different forms of the protein were immunochemically partially identical. Furthermore, they were the first to demonstrate the presence of brain specific isoenzymes of enolase. MARANGOS et al. (1976) showed coincident chromatographic elution of 14-3-2 and two of the three brain enolase isoenzymes. Furthermore, they demonstrated enolase activity in preparations of 14-3-2 from several species The isoenzymes apparently unique to the brain have recently been shown to be two dimers. yy and ay, made of the brain specific y subunit and the Q subunit of wide tissue distribution. yy corresponded to the 14-3-2 protein as originally described (called 14-3-2 anodal component by BOCK & DISING, 19753, and ay (by BCCK & DISSING called 14-3-2 cathodal component) cross-reacted with the yy enolase (BOCK et a/.. 1977~).An antiserum against 14-3-2 and an antiserum against the y subunit of enolase were identical in their effect on activity of enolase isoenzymes. The question is whether enolase activity can be attributed to the 14-3-2 protein or whether the enzyme and the brain-specific protein are firmly attached to each other. If the latter is true, the attachment must be strong, since the data on isolated brain enolase and 143-2 accord well as regards heterogeneity, molecular weight and amount (WOOD, 1964). More research has to be carried out to clarify this matter. At present, this is the first brain-specific protein to which an enzyme activity has been attributed. The conversion of 2-phospho-glycerate to phosphoenol pyruvate is common to virtually all living cells Since it has not been possible to detect any major kinetic differences between the several isoenzymes of enolase, specific functional differences for the neuronal form must be sought outside the active site. Gliul fibrillary acidic protein (GFA)

GFA was first isolated by ENG et 01. (1971) from tissues rich in fibrous astrocytes obtained from human brains with pathological conditions leading to fibrous ghosis, e.g. multiple sclerosis plaques. The protein can also be isolated from normal brain tissue by a procedure described by DAHL& BIGNAMI(1975). GFA isolated from multiple sclerosis plaques was very immunogenic, and antiserum could easily be prepared (UYEDAet al., 1972). Immunogenecity seemed to vary depending on the preparative procedure (DAHL 8c BIGNAMI, 19760). The protein has been determined by quantitative immunoelectrophoresis (JACQUE et al., 1974) and an immunoradiometric assay (ENGet al., 1976). UYEDA et al. (1972) demonstrated the brain specificity of GFA. N o GFA could be found in peripheral nerve or other organs. The level in white matter was higher than in grey matter. GFA could be demonstrated in a wide range of vertebrates including birds and fish @AHL & BIGNAMI,1973). By immunofluorescence technique BIoNAMI et al. (1972) found a selective staining of fibrous astrocytes and

10

ELISABETH BOCK

their processes. This was confirmed by the peroxi- subunits. The characteristics of GFA and the neurodase-labelled antibody technique (LUDW et al., filament protein differed very much from those of 1976; M ~ L L EetRa[., 1977). These authors also noted actin and of tubulin and, therefore, encourages the a somewhat weaker staining of protoplasmic a s t r e consideration of intermediate filaments as a separate cytes. During development immunofluorescence first class of organelles on a biochemical as well as a morappeared in the period when bundles of glial filaments phological basis. Based on these considerations a probecame visible with electron microscope (BIGNAMI & tein immunuchemically related to GFA has recently DAHL,1973). Determination of GFA in bulk-prepared been demonstrated in human fibroblasts in culture neurons and glial cells indicated a glial localization by means of immunoperoxidase staining of the cul(BOCK & HAMBERGER, 1976). In a quantitative study ture and by absorption of the specific GFA antiserum on brain specific proteins during ontogeny in mice by fibroblast extracts (BOCK et al., 1977b). Concerning the function of GFA, DAHL & BIGNAM et al., 1976) a peak of the specific concen(JACQUE tration of GFA relative to total protein content was (1973) hypothesized that one function of a fibrous found between days 10 and 14 postnatally, corre- astrocyte was to provide support for myelinated censponding to the outburst of astroglial differentiation tral nervous system axons, which are highly suscep ai the time of myelination. ANTANITUSet al. (1975) tible to tearing in trauma, and which are not suridentified astrocytes by immunoperoxydase staining rounded, as in peripheral nerves, by the basal laminae for GFA in primary tissue culture explants of human of the Schwann cells and by. the collagen fibrils of fetal forebrain (gestational ages 12-20 weeks). BOCK the endoneurium. The astroglial fibre, of which GFA et al. (1975) determined GFA, 14-3-2, D1, D2, D3. presumably is a major constituent, m a y provide this and synaptin in a primary culture of hemispheres support. from newborn rats. The cells were cultivated for 21 days in falcon flasks. At the time of harvesting no a2 glycoprotein WARECKA& BAUER (1967) demonstrated a soluble neuronal cells could be identified and the cells were supposed to be astroglial cells, contaminated with a L Y ~ glycoprotein specific to the nervous system. The few percent of ependyma cells. The supposedly neur- protein could be isolated by immuno-affinity chromaonal membrane proteins D1, D2, D3, and synaptin tography (WARECKA et al., 1972). a2 Glycoprotein was were all absent from the culture. The anodal com- mainly present in the white matter of the hemispheres, ponent of 14-3-2 was also absent, whereas 14-3-2 the spinal chord and the optic nerve. Traces were cathodal component was present in an amount corre- also demonstrated in grey matter (putamen, n. causponding to loo/, of adult whole brain homogenate, datus), whereas the protein was absent in peripheral expressed relatively to total protein. The level of GFA nerves. In bulk-prepared neurons only trace amounts et al., 1972; VOGEL, expressed relatively to total protein was more than were demonstrated (WARECKA 40 times higher in the cultured cells than in whole 1972). These data indicated a glial localization of the adult brain. Immunofluoremce staining for GFA of protein. The presence of r 2 glycoprotein in various cells from the rat C-6 glioma cell line was positive brain tumors has been studied. A marked difference in only an occasional cell in monolayer or suspension was found between astrocytoma and glioblastoma cell cultures, whereas positive staining was obtained multiforme: a2 glycoprotein was always present in the 1975). in most cells maintained for several days on sponge former and absent from the latter (WARECKA, foam matrices (BISSELet al., 1974). In extracts of The major protein component obtained by the imhuman glioblastomas GFA was found 2 4 times munoaffinity chromatography was PAS positive et al., 1974). Binding to Concanavalin enriched compared to brain homogenate (DITTMA" (BRUNGRABER et al., 1977), and MORIet a/ (1975) have demonstrated A was employed in the purification by BRUNGRABER high levels of GFA in both CSF and cyst fluid of et al. (1975). The function of a2 glycoprotein is unknown. tumors from patients with glioblastomas. By SDS polyacrylamide gel electrophoresis GFA seemed to be composed of 1-7 peptides in the range GP-350 A brain specific sialoglycoprotein, GP-350 was isoof 54,00(1-40,500 daltons; the pattern varied depending on the preparation procedure (DAHL, 1976). The lated from the soluble fraction of calf brain by VAN glial filament, 8-10nm in diameter, is morphologi- NIEW AMERONGENet al. (1972). Regional distribucally similar to the neurofilament and the inter- tion studies showed the presence of GP-350 in all mediate filament found in a variety of cells and tissues areas studied; in relatively large amounts in the (SHELANSKI et al., 1976). Recent comparative studies regions rich in ganglia such as caudate nucleus, cereof GFA and neurofilament protein (YEN et al., 1976), bellar grey matter, and in relatively small amounts and the isolation of GFA-like protein from peripheral in the regions poor in ganglia such as corpus callonerve where glial fibres are absent (DAHL & BIGNAMI, sum, cerebral white and cerebral grey matter (VAN 1976b) indicated similarity of the protein subunit of NIEW AMERONGEN& ROUKEMA,1973). Subcellular glial and nerve filaments. These findings also sug- distribution studies revealed GP-350 in the soluble gested the possibility that cytofilaments in other non- cell fraction and in the synaptosomal membrane fracneural cells might be composed of similar protein tion (VAN NIEUWAMERONGLT& ROUKEMA,1973,

Nervous system specific proteins 1974). By immunofluorescence GP-350 was localized in neuronal structures (VAN NIEUWAMERONGENet al., 1974b). Several areas were studied and positive staining was seen iv Purkinje cells, pyramidal cells and stellate cells. No fluorescence was visible in glial cells or gliomas In sciatic nerve fluorescence was present in long parallel fibres. By measurement of incorporation of ~-[~H]glucosamineinto GP-350, a maximal incorporation into soluble and membranebound GP-350 was found after 2 h and 3 h respectively. The half life was estimated to be 19 h for soluble GP-350 and 18 h for membrane-bound GP-350 (VAN NIEUWAMERONGENet al., 1974a). The apparent molecular weight determined by SDS polyacrylamide gel electrophoresis was found to be 11,600 and the isoelectric pH was determined to be about 2 (VAN 1973). The soluble NIEUWAMERONGEN& ROUKEMA, and membrane-bound GP-350 seemed to be identical immunochemically and with regard to aminoacid composition and carbohydrate composition (VAN NIEUWAMERONGEN & ROUKEMA, 1974). The function of GP-350 is unknown.

I1

philic protein in charge-shift electrophoresis (BOCK, NORRILD & BHAKDI, in preparation). The function of synaptin is unknown, but the topographic localization in combination with a carbohydrate moiety on the protein makes it a plausible hypothesis that it may be involved in the exocytotic process of synaptic vesicles during synaptic transmission. D1

This brain specific membrane protein was identified by J0RGENsEN & BOCK (1974) by means of an antiserum raised against rat synaptosomal plasma membranes. By quantitative immunoelectrophoresis it was found to be enriched four times in synaptosomal plasma membranes compared to whole brain homo1975; BOCK et a/., 1975). genate (BOCK & J~RGENSEN, It has also been demonstrated in axonal preparations (Jargensen, personal communication) No D1 could be demonstrated in primary cultures of rat astroglial cell (BOCK et al., 1975). D1 could be demonstrated in all regions studied in the CNS of rats (BOCK& BRRSTRUP, in preparation). In an ontogenic study on mice by JACQUE et al. (1976) the level of D1, expressed Synaptin relatively to the total protein content, rose steadily By immunization with rat brain synaptic vesicles, from day 12 to day 40 where a plateau was reached. BOCKet al. (1974) rased antiserum against a vesicle An immunochemically partially identical form of D1 membrane protein, synaptin (initially designated Cl). was present at days, 1, 5, and 8. The topographic This protein was spedic to the nervous system and localization in the synaptosomal membrane was inpresent in several mammalian species including vestigated by an immunoabsorption technique 1976). The antigenic determinants were mouse, ox, and man. By quantitative immunoelectro- J~RGENSEN, phoresis it was shown to be enriched in the synaptic located mainly if not totally on the outside of the vesicle fraction more than ten times compared to the synaptosomal plasma membrane. By SDS polyacryllevel in whole brain homogenates (BOCK & J~RGEN- amide gel electrophoresis D1 has been shown to be composed of two polypeptide chains: D1-1 of molecuSEN, 1975; BOCK et al., 1975). It was also demonstrated on synaptosomal plasma membranes, enriched lar weight 50,000, and D1-2 of 116,000 (J0RGENSEN. three times compared to whole brain homogenate. 1977~).By charge-shift electrophoresis D1 behaved as Conversely, synaptin was absent from primary cul- an amphiphilic protein (J~RGENSEN,1977b). The functures of rat astrocytes (BOCK et al., 1975). The regional tion of D1 is unknown. distribution of synaptin in CNS of rats has been determined (BOCK & BRASTRUP, in preparation) and 0 2 the protein was present in all areas studied. In cereThis brain specific membrane protein was identified bellum, pons, and medulla oblongata relatively low in a similar manner as the D1 protein, using an anti& levels were demonstrated. No correlation was found synaptosomal membrane antiserum (J~RGENSEN to known and putative neurotransmitters, neurotrans- BOCK, 1974). By quantitative immunoelectrophoresis mitter synthesizing enzymes, or neurotransmitter it was found to be enriched in synaptosomal plasma receptors. In an ontogenic study on mice by J A C ~ U E membranes three times compared to whole brain et al. (1976) the level of synaptin expressed relative homogenate. An immunochemically partially identito total protein content, was shown to rise from day cal form of D2 has been demonstrated in human 8 to day 40 postnatally; at d a y 40 a plateau was cerebrospinal fluid (J0RGENSEN & BOCK, 1975). NO reached At days 1 and 5 an immunochemically par- D2 could be demonstrated in primary cultures of rat tially identical form of synaptin was present. By astroglial cells (BOCK et al., 1975). D2 could be means of an immunoabsorption technique synaptin demonstrated in all areas studied in the C N S of rats has been demonstrated on the outside of the vesicle (WK & BRRSTRUP,'in preparation). JACQUE et al., membrane and of the membrane of the chromaffine 1976, found that the level of D2 in mice expressed granule and on the inside of the synaptosomal plasma relatively to total protein content, decreased with age membrane (BOCK& HELLE,1977). The protein con- just after the brain growth spurt, steady adult level tains a carbohydrate moiety, which reacts with reached at day 40 being only 50% of the level at day various lectins, it has an apparent molecular weight 12 The level of D2 thus rather paralleled the rate of 45,000 determined by SDS polyacrylamide gel elec- of synapse formation than the amount of formed trophoresis, a pl of 4.2, and it behaves like an amphi- synapses Topographically it has been localized on

42

ELISABETH BOCK

the outside of the synaptosomal plasma membrane 1976). By SDS polyacrylamide gel electrophoresis D2 was shown to be a polypeptide of a molecular weight of 139,OOO (JBRGENSEN, 1977a). D2 behaved like an amphiphilic protein in charge-shift electrophoresis (JBRGENSEN, 1977b). The decrease in concentration during developmcnt in connection with thc topographic localization made JQRGENSEN (1976) hypothesize that D2 was involved in intercellular recognition processes during synaptogenesis. (JQRGENSEN,

03

This brain specific membrane protein was demonstrated in the same investigations which led to the demonstration of two previously mentioned membrane proteins, D1 and D2 (JQRGENSEN & BOCK, 1974). By quantitative immunoelectrophoresis it was shown to be enriched in synaptosomal plasma membranes four times compared to whole brain homogenate (BOCK & J ~ R G M S E N , 1975: BOCK et a!., 1975). Conversely, D3 was absent from primary cultures of rat astrocytes (BOCK et al., 1975). D3 has been demonstrated in all areas of rat CNS studied (BOCK & BRESTRUP, 1977). JACQUE et al. (1976) found that the level of D3 in mice, expressed relative to total protein, rose during ontogeny from day 12 to day 40 postnatally. At day 1, 5, and 8 an immunochemically partially identical form of D3 was present. Topographically it was localized on the inside of the synaptosoma1 plasma membrane (J~RGENsEN, 1976). In SDS polyacrylamide gel electrophoresis the protein gave a rather complex pattern consisting of at least three polypeptides of molecular weights between 50,ooO and 14,000 (JQRGENSEN, 1977a). By charge-shift electrophoresis D3 was shown to be an amphiphilic protein (JBRGMSEN, 1977b). The function of D3 is unknown.

P-400 P-400 is a protein with an apparent molecular weight of 400,000 MALL^ et al., 1975, 1976). It is membrane bound, present in normal cerebellum, absent in cerebral cortex, and lost as a consequence of both the nervous and the staggerer mutations. Purified Purkinje cell somas from rat contained significant amounts of the protein. P-400 has been studied in homogenates of molecular layer, granular layer, and white matter from cerebellu'm and was found exclusively in the molecular layer, also if the molecular layer was freed from Purkinje cell somas, indicating that the protein is present in the dendritic arborization of the Purkinje cell (MIKOSHIBA et al., 1977). The function of P-400 is unknown. Nerwus system antigen-1 (NS-1)

N S I is a brain specific membrane protein (or proteins) defined by means of antiserum raised against a chemically induced glioblastoma in mice (SCHACHNER,1974). By the cytotoxicity test the protein was demonstrated on the cell surface. It was found in brains of other mammals. The content of

NS-1 was higher in white than in grey matter and it was also present in peripheral nerve. Myelin deficient mutant mice had low amounts of the antigen@). (1974) suggest that These findings made SCHACHNER it was located exclusively on glial membranes. Other brain specific surface proteins located both on neurons and on glial cells have been described recently using this approach, see review by FIELDS (1976). Concluding remarks Several mammalian nervous system specific proteins have been described in the last decade. The proteins are defined as antigens, which by means of their corresponding antibodies only can be demonstrated in the nervous system. Depending on the sensitivity of the immunochemical assay employed this means that the protein is present in brain extracts in at least 25-50 times higher concentration than in extracts of other tested tissues. Investigations on these proteins have been based on the assumption that they have important functions probably specific to the nervous system. However, it has turned out to be an extremely difficult task to determine these functions and presently the only protein, to which a function has been attributed, is the neuronal cytoplasmic protein 14-3-2, which probably is a brain specific isoenzyme of enolase, an enzyme activity common to virtually all living cells. Another purpose for investigauons on these proteins has been to establish immunochemically defined markers of neural cells and cell structures. The present methods for localization of proteins have here been of varying value. Clonal cell lines seem to be less valid models of their in vivo counterparts than primary cultures, bulk prepared cells or subcellular fractions Localization by means of labelled antibodies at light microscopical or ultrastructural level seems to be the method of choice. Recent reports indicate that the same protein m a y vary in localization during development and that the localization of a protein may differ from one species to another. This has a number of implications with regard to gene expression during differentiation and maturation, and it stresses the necessity of further localization studies of nearly all nervous system specific proteins, before valid conclusions can be made, when these proteins are employed as immunochemical markers. Finally, it should be mentioned that the newly developed sensitive assays for measurement in blood and cerebrospinal fluid may provide new tools for diagnosis and monitoring of neurological diseases. Also, further immunohistochemical studies of pathological nervous tissue are awaited with interest, since correlation between morphology and biochemistry m a y introduce new perspectives in neuropathology. Acknowledgement-The financial * support of Warwara Larsens Fond is gratefully acknowledged. REFERENCES

D.'s., C H B. ~ H. & Brain Res. 89, 363-367.

AWNITUS

LAWAM

L. w. (1975)

Nervous system specific proteins

13

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