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Brain Research, 518 (19911) 324--328 Elsevier

BRES 24110

GAP-43 in adult visual cortex Helen McIntosh* and David Parkinson Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, MO 63110 (U.S.A.)

(Accepted 6 February 1990) Key words: GAP-43; Visual cortex; Purification; Adult; Distribution

GAP-43 was purified from cat brain by a rapid isolation procedure and was used to raise highly specific polyclonal antibodies in rabbits. Immunoblots of proteins from adult cat, monkey and human visual cortex as well as bovine cortex also showed specific staining of a single protein that was present in both soluble and membrane fractions. Immunocytochemistryof both cat and human adult visual cortex showed that GAP-43 has a laminar distribution. The growth-associated protein, GAP-43, has been linked with neuronal development and regeneration 18 as well as synaptic plasticity 3. Although its function is unknown, it may be involved in neurotransmitter release 6 and/or calmodulin-mediated events 1. It is one of a few proteins whose change in phosphorylation state is linked to the duration of long-term potentiation in the hippocampus 12. GAP-43 m R N A levels have been reported as lowest in primary sensory areas and highest in associative areas 2"5'x4. Similarly, in the primate visual system, endogenous phosphorylation of GAP-43 was lowest in the striate cortex, where visual input enters the cortex, and was highest in regions associated with memory storage 13. GAP-43 expression may be involved in the plasticity of cat visual cortex, both during the peak of the critical period, which extends from 4-6 weeks postnatally H, and in defining the duration of the critical period. In order to investigate these ideas, we have purified GAP-43 from cat brain and raised specific antibodies. These antibodies have been used to investigate the GAP-43 in adult visual cortex in preparation for developmental studies. GAP-43 was purified from a membrane fraction of neonatal cat brain. The initial steps involving alkaline pH extraction were as described 16'1°. In a departure from these methods, the proteins that remained soluble in the presence of ammonium sulfate at 50% saturation were extracted by passing this high salt solution over a small column (1-2-ml bed volume) of C4-bonded reversephase silica. The adsorption of GAP-43 was probably enhanced by the high salt concentration. This step also served to de-salt and concentrate the GAP-43 simulta-

neously. The column was washed with 0.1% TFA and then GAP-43 was eluted with 50% acetonitrile. Further purification was by reverse-phase HPLC (Fig. 1). This rapid procedure yields about 20/~g of GAP-43 per gram of brain in 2-3 days. Purified cat GAP-43 was used to raise antisera in rabbits by standard procedures. Gel electrophoresis and western blotting were performed as described m. The reactivity of antiserum CG-1 was investigated with membrane and soluble fractions prepared from cerebral cortex of cow, and from visual cortex of cat, human and macaque monkey. Protein from each soluble fraction was concentrated by first precipitating with chloroform/ methanol 2° and then redissolving in SDS sample buffer for SDS-PAGE (Fig. 2B). A single band of about 48 kDa was stained by CG-1 in both fractions of all four species. The intensity of staining suggested that the GAP-43 content of the membrane fractions was about two-fold that of the soluble fractions from the same species. The mobilities of soluble and membrane GAP-43 were similar in each species, but there were small differences in mobilities between species (i.e. monkey = human > cat = bovine). CG-1 also stained a single band on immunoblots of rat cerebral cortex, but at a higher mobility than seen for these other species, (data not shown), confirming previous observations a°. Amino acid sequence data predicts slight differences in molecular weight and hydrophilic character for GAP-43 from different species 8" 9,15,19. Thus, the small differences in relative mobilities may reflect the small differences in molecular weight or hydrophilic character as well as other factors such as

* Present address: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 4566 Scott Avenue, St. Louis, MO 63110, U.S.A. Correspondence: D. Parkinson, Department of Cell Biologyand Physiology,Washington University School of Medicine, 4566 Scott Avenue, St Louis, MO 63110, U.S.A. 0006-8993/90/$03.50 © 199(I Elsevier Science Publishers B.V. (Biomedical Division)

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Fig. 1. A: HPLC purification of GAP-43. The GAP-43-enriched extract from 3 g of kitten forebrain was dissolved in I ml 0.1% TFA and applied to a reversed phase column (Vydac C4, 25 x 0.46 cm, 10 gm). Sample was eluted with a linear gradient from 5 to 70% acetonitrile in 0.1% TFA. The GAP-43 peak (arrow) eluted at 24 min (45% acetonitrile). The vertical bar represents an absorbance of 0.1 at 214 nm. B: electrophoretic analysis of HPLC fractions from GAP-43 enriched extract. Sequential 1-min fractions eluted in an acetonitrile gradient were analyzed for GAP-43 content. Lane A, pure cat GAP-43; lanes B-O, fractions 22-35 from the HPLC. Lanes D and E contain cat GAP-43.

p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n s . T h e s e results s h o w that

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Fig. 2. GAP-43 antiserum cross-reacts with a single protein from membrane and soluble fractions of adult visual cortex. Proteins were separated by PAGE, transferred to nitrocellulose and then reacted with antiserum CG-1 at 1:1000. Bound antibodies were visualized with a biotinylated secondary antibody and avidin-biotinylated horseradish peroxidase complex. Lane 1, pure cat GAP-43; even-numbered lanes, membrane proteins; odd numbered lanes, soluble proteins from: monkey (2,3), cat (4,5), human (6,7), cow (8,9). A: stained with Coomassie blue, B: immunoblot of gel duplicating lanes 2-9 of gel A. Fifteen gg of membrane or cytoplasmic protein were applied per lane.

326

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Fig. 3. Immunostaining of area 17 is laminar. Coronal sections from adult visual cortex were stained with CG-1 antiserum (A and C) or preimmune serum (B and D), both diluted 1:2000. Bound antibodies were visualized as described in the legend to Fig. 2. Bar = l mm. A, B: cat visual cortex. C, D: human visual cortex. G A P - 4 3 by immunoassay gives values of about 0.5 mg p e r gram m e m b r a n e protein in adult cat visual cortex ( M c l n t o s h et al., submitted). M u c h of the work on G A P - 4 3 has focussed on the m e m b r a n e - a s s o c i a t e d form 3, yet our data suggest that significant amounts are also present in the soluble fraction. The samples in Fig. 2 were o b t a i n e d after

homogenization in 5 m M E D T A and 1 m M P M S F and centrifugation at 100,000 g for 60 min. Similar results were o b t a i n e d after centrifugation at 109,000 g for 90 rain (unpublished data). In this case pepstatin and leupeptin (1 /~g/ml each) were included in the homogenization buffer along with P M S F and E D T A . Thus, the presence of protease inhibitors during h o m o g e n i z a t i o n and the use

327 Of a centrifugation protocol which is known to precipitate microsomes from tissue homogenates 4"7 left a protein with the immunoreactivity and electrophoretic mobility of GAP-43 in the soluble phase. Therefore, the soluble GAP-43 is unlikely to be an artifact of proteolysis or centrifugation. The significance of GAP-43 in the soluble fraction is unclear. GAP-43 solubilized from membranes binds calmodulin 1, so it is possible that the GAP-43 in the soluble fraction also has this property and so may be involved in modulation of calmodulin-dependent events. Alternatively, soluble GAP-43 may be part of a pool awaiting the fatty acylation that is thought to be responsible for its m e m b r a n e association 17. The distribution of GAP-43 in visual cortex was determined by immunocytochemical labelling of sections from adult cat and human primary visual cortex with GAP-43 antiserum (Fig. 3 A - D ) . Tissue from both sources showed positive staining with primary antiserum, but essentially no staining with preimmune serum. Tissue staining was laminar in both the cat and human primary visual cortex. In both species the staining in area 17 was most intense in layers I and V/VI, intermediate in layers II/III, and least in layer IV. In both cat and human, the low level staining in layer IV was restricted to area 17. The intensity of staining and the contrast between layers was greater in the human visual cortex (Fig. 3C) than in the cat visual cortex (Fig. 3A). There was no evidence of columnar staining with GAP-43 antibodies in these or other sections from cat and human visual cortex. The absence of detectable GAP-43 in layer IV of the

immunostained section of cat and human visual cortex (Fig. 3C) is particularly interesting since that is the layer that receives most of the visual input from the lateral geniculate nucleus. This observation raises the possibility that the critical period is defined by factors controlling the expression of GAP-43 in layer IV cells and in the geniculocortical afferents that project to them. Our preliminary immunocytochemical studies show that this may be the case because GAP-43 staining is seen in all layers of kitten visual cortex. The presence of GAP-43 in superficial and deep layers of adult primary visual cortex leaves open the possibility of continued plasticity in these regions. In summary, we have described a rapid and efficient procedure for the purification of GAP-43 from cat brain. Polyclonal antiserum raised against this protein crossreacts with a single protein in visual cortex from monkey and human as well as in bovine cerebral cortex. GAP-43 is present in significant amounts in both soluble and membrane-associated fractions from adult cortex of all of these species. GAP-43 distribution is laminar in the adult visual cortex, with layer IV showing the lowest concentration. This data will provide the basis for studies to determine the changes in GAP-43 during development of visual cortex.

1 Alexander, K.A., Wakim, B.A., Doyle, G.S., Walsh, K.A. and Storm, D.R., Identification and characterization of the calmodulin-binding domain of neuromodulin, a neurospecific calmodulin-binding protein, J. Biol. Chem., 263 (1988) 7544-7549. 2 Benowitz, L.I., Apostolides, P.J., Perrone-Bizzozero, N., Finkelstein, S.P. and Zwiers, H., Anatomical distribution of the growth-associated protein GAP-43/B-50 in the adult rat brain, J. Neurosci., 8 (1988) 339-352. 3 Benowitz, L.I. and Routtenberg A., A membrane phosphoprotein associated with neural development, axonal regeneration, phospholipid metabolism, and synaptic plasticity, Trends in Neurosci., 10 (1987) 527-532. 4 De Robertis, E., Arnaiz, G.R.D.L., Alberici, M., Butcher, R.W. and Sutherland, E.W., Subcellular distribution of adenyl cyclase and cyclic phosphodiesterase in rat brain cortex, J. Biol. Chem., 242 (1967) 3487-3493. 5 De la Monte, S.M., Federoff, H.J., Ng, S.-C., Grabezyk, E. and Fishman, M.C., GAP-43 gene expression during development: persistence in a distinctive set of neurons in the mature central nervous system, Dev. Brain Res., 46 (1989) 161-168. 6 Dekker, L.V., De Graan, P.N.E., Versteeg, D.H.G., Oestreicher, A.B. and Gispen, W.H., Phosphorylation of B-50 (GAP43) is correlated with neurotransmitter release in rat hippocampal slices, J. Neurochem., 52 (1989) 24-30. 7 Gray, E.G. and Whittaker, V.P., The isolation of nerve endings from brain: an electron-microscopic study of cell fragments derived by homogenization and centrifugation, J. Anat., 96 (1962) 79-96. 8 Karns, L.R., Ng, S.-C., Freeman, J.A. and Fishman, M.C.,

Cloning of complementary DNA for GAP-43, a neuronal growth-related protein, Science, 236 (1987) 597-600. 9 Kosik, K.S., Orecchio, L.D., Bruns, G.A.P., Benowitz, L.I., MacDonald, G.P., Cox, D.R. and Neve, R.L., Human GAP-43: its deduced amino acid sequence and chromosomal localization in mouse and human, Neuron, 1 (1988) 127-132. 10 Mclntosh, H., Parkinson, D., Meiri, K., Daw, N. and Willard, M., A GAP-43-1ikeprotein in cat visual cortex, Visual Neurosci., 2 (1989) 583-591. 11 Mitchell, D.M., and Timney, B., Postnatal development of function in the mammalian visual system. In J.M. Brookhart, V.B. Mountcastle, I. Darian-Smith and S.R. Geiger, (Eds.),

The authors wish to thank Dr. Nigel Daw for his support and his helpful suggestions throughout this work. We would also like to thank Dr. Karina Meiri for help and advice on the initial HPLC purification of GAP-43. This work was supported by NIH Program Project Grant PO1 NS15070 and NEI Training Grant EY 07057.

Handbook of Physiology, Section 1. The nervous system, Vol. IlL Sensory Processes, Part 1, Physiological Society, Bethesda, MD,

1984, pp. 507-555. 12 Nelson, R.B., Linden, D.J., and Routtenberg, A., Phosphoproteins localized to presynaptic terminal linked to persistence of long-term potentiation (LTP): quantitative analysis of two dimensional gels, Brain Research, 497 (1989) 30-42. 13 Nelson, R.B., Reideman, D.P., O'Neill, J.B., Mishkin, M. and Routtenberg, A., Gradients of protein Kinase C substrate phosphorylation in primate visual system peak in visual memory storage areas, Brain Research, 416 (1987) 387-392. 14 Neve, R.L, Finch, E.A., Bird, E.D. and Benowitz, L.I., Growth-associated protein GAP-43 is expressed selectively in associative regions of the adult human brain, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 3638-3642. 15 Ng, S.-C., de la Monte, S.M., Conboy, G.L., Karns, L.R. and Fishman, M.C., Cloning of human GAP-43: Growth association

328 and ischemic resurgence, Neuron, 1 (1988) 133-139. 16 Oestreicher, A.B., Van Duin, M., Zwiers, H. and Gispen, W.H., Cross-reaction of anti-rat B-50: characterization and isolation of a 'B-50 phosphoprotein' from bovine brain, J. Neurochem., 43 (1984) 935-943. 17 Skene, J.H.P. and Virag, I., Posttranslational membrane attachment and dynamic fatty acylation of a neuronal growth cone protein, GAP-43, J, Cell Biol., 108 (1989) 613-624. 18 Skene, J.H,P. and Willard, M.B., Changes in axonally transported proteins during axon regeneration in toad retinal ganglion

cells, and axonally transported proteins associated with growth in rabbit central and peripheral nervous systems, J. Cell Biol., 89 (1981) 86-103. 19 Wakim, B.T., Alexander, K.A., Masure, H.R., Cimler, B.M., Storm, D.R. and Walsh K.A., Amino acid sequence of P-57, a neurospecific calmodulin-binding protein, Biochemistry, 26 (1987) 7466-7470. 20 Wessel, D. and Flugge, U.I., A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids, Anal. Biochem., 138 (1984) 141-143

GAP-43 in adult visual cortex.

GAP-43 was purified from cat brain by a rapid isolation procedure and was used to raise highly specific polyclonal antibodies in rabbits. Immunoblots ...
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