Signal-regulated proteins and fibroblast growth factor receptors: comparative immunolocalization in rat retina

ELSEVIER

Neuroscience Letters 214 (1996) 135-138

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Signal-regulated proteins and fibroblast growth factor receptors: comparative immunolocalization in rat retina P.R. Blanquet*, L. J o n e t INSERM U450, D~veloppement, Vieillissement et Pathologie de la r~tine, 29 rue Wilhem, 75016 Paris, France Received 12 June 1996; revised version received 5 July 1996; accepted 12 July 1996

Abstract We compared the immunolocalization of fibroblast growth factor (FGF)-R1 and FGF-R2 with that of several intracellular signalling proteins in rat neural retina. Only the serine/threonine extracellular signal-related kinases (ERK) and lipocortin/annexin 6, a major calcium-binding protein, .appeared to be co-localized with FGF-R1 and FGF-R2 in all subfields of the neural retina. In particular, ERK appeared to be present in perikarya of ganglion cells and synaptic layers as did these receptors. Possible implications of these results with regard to neuroprot~tive role of FGF are discussed.

Keywords: Neural retina; lmmunostaining; Signalling proteins; Fibroblast growth factor receptor; Rat

Fibroblast growth factor (FGF) is involved in controlling neuronal survival and plasticity in the nervous system and may play an important role in the brain response to injury and neurodegenerative insults [6,17]. In addition, FGF receptors (FGF-Rs) are widely expressed in the central nervous system (CNS) [9,15]. Surprisingly, however, exceedingly little attention has been paid to signal transduction mechanisms that are activated by FGF/receptor interactions in the CNS. Although the disposition of anatomical layers in the neural retina is clear-cut, even less is known regarding the FGF signalling pathways in this tissue. Recently, several proteins known to be involved in FGF signalling cascades [2] were found to be present in some regions of the brain. These proteins include: (a) phospholipase C-7 (PLC-7), a regulator of phosphatidyl inositol metabolism [21]; (b) extracellular signal-related kinases (ERK), a family of serine/threonine kinases that play a central position :in the cellular phosphorylation network [10]; (c) Raf, a serine/threonine kinase that is one of activators of ERK [22]; (d) a phosphotyrosine phosphatase designed as Syp/SH-PTP2, that may also be involved in activation of the ERK pathway [11]; and (c) lipocortins/ * Corresponding author. Urtit~ de Recherches de Physiopharmacologie du Syst~me Nerveux, INSERM U161, 2 rue d'A16sia, 75014 Paris, France. Tel.: +33 45893662; fax: +33 45881304.

annexins 1, 2 and 6 (lipo 1, 2, 6), a family of structurally related proteins that appear to be capable of controlling both calcium buffering systems and activity of phospholipase A2 [1,2,20]. As a first step towards better understanding of FGF transduction mechanisms in the CNS, we compared the regional distribution of these potential regulators with that of FGF-R1 and FGF-R2 in adult rat neural retina. The localization of receptors was investigated using two rabbit antisera specific for flg/FGF-R1 and bek/FGF-R2, designated as anti-fig 1A and antibek 1A. Anti-fig 1A and anti-bek 1A, prepared against sequences in the carboxy termini of the two receptors [13], were kindly provided by Dr. Craig Dionne (Rorer Biotechnology, Inc., USA). We also used a rabbit antiserum named anti-FGF-R, which was raised to the extracellular region of FGF-R2 as described elsewhere [15]. To examine the localization of signalling proteins, we performed immunostaining with several monoclonal antibodies of demonstrated reactivity for rat tissues: anti-PLC-7 (Tranduction Lab., Lexington, USA); anti-ERK (ERK1 + 2) (Zymed Lab., Inc. San Francisco); anti-Syp / SH-FI'Ir2 (Transduction Lab.); and antineurofilaments 68 (anti-NF68; Sigma Chemical Co., St Louis, MO). In addition, we used the following polyclonal antibodies: anti-Raf-1 (Santa Cruz Biotechno., Inc., CA); anti-lipo 1, anti-lipo 2 and anti-lipo 6, prepared using

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human lymphocyte lipocortins by Drs Rothhut and F. Russo-Marie (U332 INSERM, ICGM, Paris). These antibodies were shown also to be broadly reactive with rat tissues. In order to obtain accurately the immunolocalization of FGF-Rs and signalling proteins, different conditions of tissue fixation and development of the secondary antibody were tested as indicated elsewhere [16]. Finally, the immunohistochemical studies were essentially performed according to protocols described previously [15]. Briefly, retinas were sectioned on a Bright cryostat, washed, and fixed for 30 min with 4% paraformaldehyde. Sections were then saturated for 15 min with 1% normal goat serum and 1% bovine serum albumin and incubated for 1 h at 25 ° with one of polyclonal antibodies (1/100 dilution) or with one of monoclonal antibodies (1/400 dilution). For control incubations, anti-PLC-3,, anti-ERK and anti-Syp/SH-PTP2 were replaced with ascites fluid from a non-immunoglobulin-secreting mouse tumor or were preadsorbed with an excess of corresponding available control antigens. Polyclonal anti-flglA, anti-beklA and antiRaf-1 were also pre-adsorbed with available control peptides. Polyclonal anti-FGF-R and anti-lipol,2,6 were replaced with control preimmune sera. After washing, sections were incubated for 1 h at 25°C with one of secondary antibodies (1/100 dilution), rinsed again and coverslipped. For double-staining experiments, each of polyclonal antireceptor antibodies were incubated with one of monoclonal antibodies. Also, the polyclonal anti-Raf-1 antibody was incubated with the monoclonal anti-ERK antibody. Polyclonal antibodies and monoclonal antibodies were localized using secondary fluorescein isothiocyanate (FITC)-conjugated sheep anti-rabbit IgG (1/100 dilution; Biosys) and secondary Rhodamine-conjugated goat antimouse IgG (1/100 dilution; Cooper Biomedica). As shown in Fig. la,c,d, similar fluorescence patterns were observed with anti-FGF-R, anti-fig 1A and anti-bek 1A. Interestingly, a regular distribution of stained patches was seen near the inner limiting membrane. From the location of the optic fiber layer recognized by anti-NF68 (Fig. lj,k), these bright dots corresponded to the ganglion cell bodies. In fact, this immunoreactivity appeared to be restricted to perikarya of ganglion cells (Fig. l j). In addition, an intense staining was seen in the laminae corresponding to inner and outer plexiform layers, which are the synaptic layers, as well as the segments of photoreceptors and the pigmented epithelium (Fig. 1a,c,d). The use of antibodies to signalling proteins led to several distinct staining patterns. Staining with anti-lipo 1 and anti-Iipo 2 was absent or barely detectable (Fig. li). Anti-PLC-), only significantly labelled the choroidcapilaris region (Fig. lb), whereas anti-Syp/SH-PTP2 decorated both the segments of photoreceptors and the pigmented epithelium (Fig. le). In contrast, there was a general overlap in the distribution of fluorescent layers obtained with anti-ERK and anti-receptor antibodies (Fig. l a,c,d,f). In particular, as shown at higher magnification in Fig. 11, reactivity of

ERK1 and ERK2 appeared to occur in the perikarya of ganglion cells as did reactivity of FGF-R1 and FGF-R2. Likewise, lipo 6 immunostaining could be found in all subfields of the retinal tissue (Fig. lh). In this case, however, the fluorescence picture was not similar to that seen after using anti-ERK, since a widespread staining was found in areas from the inner plexiform layer to the inner limiting membrane. On the other hand, despite the reported role of Raf-1 in ERK activation by FGF [2], the innermost region of the retina was almost completely devoid of Raf-1 immunostaining (Fig. lg,m). Reactivity of Raf-1 was only intense in the segments of photoreceptors and the pigmented epithelium. Our results clearly show that FGF-R1 and FGF-R2 have similar distribution patterns in rat retina. However, we cannot exclude the possibility that other FGF-Rs could also be detected. Anti-FGF-R, indeed, is potentially capable of recognizing all four mammalian FGF-Rs, since it was raised to the full-length extracellular region of FGFR2 some domains of which are conserved amongst the other FGF-Rs [2,15]. Comparison of reactivity sites of signal-regulated proteins with those of FGF-R1 and FGF-R2 suggests that FGF may potentially activate some of signalling proteins documented here within each retinal region. It follows that different regions in retina might exhibit different anatomic circuitry and/or biochemical mechanisms induced by FGF/receptor interactions. In this regard, it is interesting that only staining of ERK1 and ERK2 occurred coincidentally in all areas of the neural retina where FGF-R1 and FGF-R2 were labelled. Particularly noteworthy also was the widespread distribution of lipo 6 staining in the neural regions where other calciumbinding proteins co-exist [1]. The fact that FGF-R1 and FGF-R2 were detected in the ganglion cell perikarya and the inner plexiform layer which contains ganglion dentritic processes is consistent with a number of reports. For example, studies reported that FGF is anterogradely transported from the dentritic processes of ganglion cells to the superior colliculus in rat brain, thus suggesting that receptors on ganglion dentrites mediate the uptake of FGF and are transported to the vicinity of ganglion cell bodies before beginning their journey away from the retina [5]. Since internalization of FGF molecules in neurons was shown to generate fragments that could yet trigger receptor activity [19], this raises the possibility that receptor-bound FGF may act as an anterograde trophic complex in ganglion cells. There is now accumulating evidence to suggest that the FGF signalling network may be a dual regulation system in which both signalling cascades activated by surface membraneassociated receptors and internalized receptor/FGF complexes are involved [2,8]. Therefore, internalized FGF-Rs might provide an additive new mechanism for activation of some signal-regulated proteins such as ERK in neural retina. The molecular link by which FGF-R1 and FGF-R2 could lead to activation of ERK in neural retina is not

P~R. Blanquet, L. Jonet / Neuroscience Letters 214 (1996) 135-138

137

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INL..~ ONL.-~

Fig. 1. Distribution of signalling proteins and FGF receptors in rat retina detected by indirect immunofluorescence. Retina consists of two distinct regions: the non-neuronal retinal pigmented epithelium (RPE) above the choroidcapilaris (CC); and the neural retina bordered by the inner limiting membrane. Neural retina aligns into thre,~ highly ordered cellular layers: the outer nuclear layer (ONL) above the photoreceptor segments (PRS); the inner nuclear layer (INL); and the ganglion cell layer (GCL). In between the cellular layers, processes of retinal neurons, called the inner plexiform layer (IPL) and the outer plexiform layer (OPL), are assembled to make synaptic contacts. Paraformaldehyde-fixed sections were incubated with one of antisera designated as anti-fig 1A (e), anti-bek 1A (d), anti-Raf-1 (g), anti-lipo 6 (h) and anti-lipo 1 (i) or one of monoclonal antibodies named anti-SH-PTP2 (e) and anti-ERK (f). Some sections were also double-labelled with anti-FGF-R antiserum (a) and monoclonal anti-PLC-3, antibody (b). with anti-FGF-R antiserum (j) and monoclonal anti-NF68 antilxpdy (k), or with monoclonal anti-ERK antibody (I) and anti-Raf-1 antiserum (m). These antibodies were localized using either F1TC-conjugated sheep, anti-rabbit IgG (a,c,d,g,h,i,j,m) or Rhodamine-conjugated goat anti-mouse IgG (b,e,f,k,1). Note that only anti-FGF-R, antifig 1A, anti-bek 1A, anti-ERK and anti-lipo 6 decorated all subfields of the neural retina (a,c,d,f,h). Note also that reactivity of ERK appeared to occur in the perikarya of ganglion cel2s (arrows, f, 1) as did reactivity of FGF receptors (arrow, j), just beneath the NF68-positive staining corresponding to the optic fiber layer (arrow, k). The retinal pigmented epithelium and the photoreceptor layer have become separated due to disruption of the tissue (a,b,c,d,f,g). Scale bars: 69/zm (a-k); 51 #m (1,m). clear, s i n c e i m m u n o r e a c t i v e Raf-1 w a s n o t significantly d e t e c t e d in t h e g a n g l i o n cell l a y e r a n d t h e p l e x i f o r m layers. C o n s i s t e n t l y , h o w e v e r , it h a s b e e n s h o w n that acti-

v a t i o n o f E R K a p p e a r s to b e d e p e n d e n t o n R a f - B b u t n o t o n Raf-1 in rat b r a i n [22]. P r e s u m a b l y , t h e r e f o r e , o n e o f t h e c a n d i d a t e E R K a c t i v a t o r s in rat r e t i n a m i g h t b e B - R a f .

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Studies have established that F G F strongly protects cultured rat n e u r o n s from glutamate toxicity [6] and rat neural retina from ischemic injury [17]. Various experiments also showed that E R K is activated in neurons after transient ischemia as well as glutamate receptor activation and calcium influx [3,7,12]. In addition, lipo 6 in rat brain was found to interact with calspectin [20], a protein whose c a l c i u m - d e p e n d e n t cleavage appears to be i n v o l v e d in the regulation of glutamate receptor b i n d i n g and n e u r o n a l degenerescence [14]. It is therefore tempting to speculate that E R K 1 , E R K 2 and lipo 6 might function as 'cross-talk' monitors in fully differentiated neural retina, ensuring that F G F receptor-induced signals act as neuroprotective events in response to excitotoxic assaults of calcium. Such an interpretation needs to be taken, obviously, with caution as E R K can be also potentially activated by other growth factor signalling pathways ~ retina [2,18]. H o w ever, this interpretation is strengthened b y the fact that only ganglion and Mtiller cells were stainable with antibodies specific for trkA [4] and trkB (preliminary data not shown), two neurotrophin receptors thought to be capable of i n d u c i n g E R K activation in n e u r o n s [18]. These restricted staining patterns, indeed, compared to those of F G F - R s and ERK, tend to support the hypothesis that E R K might be m a i n l y associated with the signalling transduction of F G F - R s in rat retina. The use of subsets of cultured retinal cells should help to verify these assertions. W e wish to thank Dr. C. D i o n n e for his contribution in providing antisera against F G F - R s and Drs. B. Rothhut and F. Russo-Marie for supplying antisera against lipocortins/annexins. W e are also indebted to Dr. J.C. J e a n n y for his advice o n i m m u n o c h e m i s t r y and to H. Co~t for the photographs. [1] Baimbridge, K.G., Celio, M.R. and Rogers, J.H., Calcium-binding proteins in the nervous system, Trends Neurosci., 15 (1996) 303308. [2] Blanquet, P.R., FGF signalling: a mechanism on the way of being deciphered, M&I. Sci., 12 (1996) 303-312. [3] Campos-Gonzalez, R. and Kindy, M.S., Tyrosine phosphorylation of microtubule-associated protein kinase after transient ischemia in the gerbil brain, J. Neurochem., 59 (1992) 1955-1958. [4] Carmignoto, G., Comelli, M.C., Candeo, P., Cavicchioli, L., Yan, Q., Merighi, A. and Maffei, L., Expression of NGF receptor and NGF receptor mRNA in the developing and adult rat retina, Exp. Neurol., 111 (1991) 302-311. [5] Ferguson, A., Schweitzer, J.B. and Johnson, Jr., E.M., Basic fibreblast growth factor: receptor-mediated internalization, metabolism, and anterograde axonal transport in retinal ganglion cells, J. Neurosci., 10 (1990) 2176-2189. [6] Femandez-Sanchez, M.T. and Novelli, A., Basic fibroblast growth factor protects cerebellar neurons in primary culture from NMDA

and non-NMDA receptor mediated neurotoxicity, FEBS Lett., 335 (1993) 124-131. [7] Fiore, R.S., Murphy, T.H., Sanghera, J.S., Pelech, S.L. and Baraban, J.M., Activation of p42 mitogen-activated protein kinase by glutamate receptor stimulation in rat primary cortical cultures, J. Neurochem., 61 (1993) 1626-1633. [8] Johnston, C.L., Cox, H.C., Gomm, J.J. and Coombes, R.C., Fibreblast growth factor receptors (FGFRs) localize in different cellular compartments, J. Biol. Chem., 270 (1995) 30643-30650. [9] Matsuo, A., Tooyama, I., Isobe, S., Oomura, Y., Akiguchi, I., Hanai, K., Kimura, J. and Kimura, H., Immunohistochemical localization in the rat brain of an epitote corresponding to the fibroblast growth factor receptor-I, Neuroscience, 60 (1994) 49-66. [10] Ortiz, J., Harris, H.W., Guitart, X., Terwilliger, R.Z., Haycock, J.W. and Nestler, E.J., ExtraceUular signal-regulated protein kinases (ERKs) and ERK kinase (MEK) in brain regional distribution and regulation by chronic morphine, J. Neurosci., 15 (1995) 1285-1297. [11] Reeves, S.A., Ueki, K., Sinha, B., Difiglia, M. and Louis, D.N., Regional expression and subcellular localization of the tyrosinspecific phosphatase in the adult human nervous system, Neuroscience, 71 (1996) 1037-1042. [12] Rosen, L.B., Ginty, D.D., Weber, M.J. and Greenberg, M.E., Membrane depolarization and calcium influx stimulate MEK and MAP kinase via activation of Ras, Neuron, 12 (1994) 1207-1221. [13] Ruta, M., Burgess, W., Givol, D., Epstein, J., Neiger, N., Kapiow, J., Crumley, G., Dionne, C., Jaye, M. and Schlessinger, J., Receptor for acidic fibroblast growth factor is related to the tyrosine kinase encoded by the fms-like gene (FLG), Prec. Natl. Acad. Sci. USA, 86 (1989) 8722-8726. [14] Siman, R., Baudry, M. and Lynch, G., Regulation of glutamate receptor binding by the cytoskeletal protein fodrin, Nature, 313 (1985) 225-228. [15] Torriglia, A. and Blanquet, P.R., Immunological evidence for a fibroblast growth factor receptor in adult retinal optic fiber and synaptic layers, Neuroscience, 60 (1994) 969-981. [16] Torriglia, A., Jeanny, J.-C. and Blanquet, P.R., Immunohistochemical analysis of fibroblast growth factor receptor in bovine retina, Neurosci. Lett., 172 (1994) 125-128. [17] Unoki, K. and La Vail, M.M., Protection of the rat retina from ischemic injury by brain-derived neurolxophic factor, ciliary neurotrophic factor, and basic fibroblast growth-factor, Invest. Ophthalmol. Vis. Sci., 35 (1994) 907-912. [18] Van der Geer, P. and Hunter, T., Receptor protein-tyrosine kinases and their signal transduction pathways, Annu. Rev. Cell Biol., 10 (1994) 251-337. [19] Walicke, P,A. and Baird, A., Internalization and processing of basic fibroblast growth factor by neurons and astrocytes, J. Neurosci., 11 (1991) 2249-2258. [20] Watanabe, T., Inui, M., Chen, B.-Y., Iga, M. and Sobue, K., Annexin 6-binding proteins in brain, J. Biol. Chem., 269 (1994) 17656-17662. [21] Williams, E.J., Furness, J., Walsh, F.S. and Doherty, P., Characterisation of the second messenger pathway underlying neurite outgrowth stimulated by FGF, Development, 120 (1994) 1685-1693. [22] Yamamori, B., Kuroda, S., Shimizu, K., Fukui, K., Ohtsuka, T. and Takai, Y., Purification of a Ras-dependent mitogen-activated protein kinase kinase kinase from bovine brain cytosol and its identification as a complex of B-Raf and 14-3-3 proteins, J. Biol. Chem., 270 (1995) 11723-11726.