Distinct immunoreactivity to 110 kDa laminin-binding protein in adult and lesioned rat forebrain

Brain Research, 555 (1991) 305-312 Elsevier ADONIS 0006899391168515

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BRES 16851

Distinct immunoreactivity to 110 kDa laminin-binding protein in adult and lesioned rat forebrain Mathias Jucker 1, Hynda K. Kleinman 2, Christine F. Hthmann 3, J. Mark Ordy 4 and Donald K. Ingram 1 1Laboratory of Cellular and Molecular Biology, The Nathan W. Shock Laboratories, Gerontology Research Center, National Institute on Aging, NIH, Baltimore, MD 21224 (U.S.A.), ZLaboratory of Developmental Biology and Anomalies, National Institute of Dental Research, NIH, Bethesda, MD 20892 (U.S.A.), 3Department of Psychiatry, The Johns Hopkins Medical School, Baltimore, MD 21205 (U.S.A.) and 4CNS Research Lab., Fisons Corporation, Rochester, NY 14624 (U.S.A.) (Accepted 12 March 1991) Key words: Laminin-binding protein; Synthetic laminin-derived peptide PA22-2; Neurotrophic factor; Immunocytochemistry; Stab wound; Ischemic lesion; Reactive astrocyte

A phosphorylated, ~- 110 kDa laminin-binding protein (110 kDa LBP) from mouse brain has been previously identified. This protein recognizes a neurite-outgrowth promoting 19-amino acid synthetic peptide (PA22-2) derived from the laminin A chain. In the present study, an antibody against the 110 kDa LBP was used to localize immunoreactivity in the normal adult rat brain and also following a stab wound and ischemic lesion. Immunorcactive cells were found in layers II/III and V of the cerebral cortex and within apical dendrites of pyramidal neurons. Specific immunoreactivity was also found in the stratum lucidum in the CA3 region of the hippocampus which exhibited densely stained mossy fibers and terminals. Mechanical and ischemic lesions induced intense immunolabeling of reactive glial cells around the lesion site. The distinct and anatomically restricted localization of the immunostain in adult and lesioned rat brain suggests that 110 kDa LBP-like molecules might have an important function in forebrain structures and may be involved in the response to CNS injury.

INTRODUCTION Laminin is a large basement membrane glycoprotein with many biological activities 14,2°,33. It is a potent p r o m o t e r of neurite outgrowth for a variety of cells, including forebrain neurons in vitro and in vivo 1"12"19'42. In contrast to the developing CNS where laminin is expressed transiently, laminin was originally believed to be limited to the basement membrane surrounding capillaries in the adult CNS and to reactive glial cells after lesioning the brain 7A6,24,3°. Recent studies using monoclonal and affinity purified laminin antibody, however, have reported an intraneuronal laminin-like immunoreactivity in the adult CNS 8,31,4°. A 19-amino acid synthetic peptide (PA22-2) derived from the laminin A chain has been identified as a neurite-promoting site 28,32. In vitro, PA22-2 stimulates neurite outgrowth in cerebellar granule cells 2a and fetal septal cells 12. Using affinity chromatography, a M r ~- 110 k D a phosphorylated laminin-binding protein (110 kDa

LBP) from newborn mouse brain was recently identified 1~. This binding protein can also be purified on a PA22-2 affinity column and has been characterized as a non-integrin, cell surface protein 15. Several other non-integrin and integrin lamininbinding proteins/receptors have been described and can be found on the surface of various neuronal cells in culture 5'14'24'27'33'34'38. In the PNS, interactions between Schwann cell-derived laminin and integrin receptors have been demonstrated during development and in nerve regeneration 24'aS'a8. Nothing is known about laminin receptors in the adult CNS or in response to CNS injury. In the present study using immunohistochemical techniques, we demonstrate a distinct and anatomically restricted distribution of 110 k D a LBP-like immunoreactivity in the normal adult and lesioned rat forebrain. The findings suggest a function of 110 kDa LBP-like molecules in normal adult rat brain and in response to CNS injury.

Correspondence: D.K. Ingram, Gerontology Research Center, NIA, NIH, Francis Scott Key Medical Center, 4940 Eastern Avenue, Baltimore, MD 21224, U.S.A. Fax: (301) 550-1704.

306 MATERIALS AND METHODS

Antibody Antiserum against purified M~ ~ 110 kDa LBP was prepared in rabbits as described in Kleinman et ai. 15. The immunogen has recently been isolated from a detergent extract of newborn mouse brain and identified to bind 19-amino acid laminin A chain-derived peptide PA22-2 (laminin A chain residues 2091-2108). Brain tissue and lesion procedures Adult male 3-7 month old Fischer-344 rats (Harlan SpragueDawley Inc., Indianapolis, IN) were used. Some of the rats received a mechanical lesion 3, 8, or 14 days prior to sacrifice. Using a Hamilton microsyringe (300 gm diameter), 1 gl saline was injected over 1 rain unilaterally into the striatum (AP = 0.5 from Bregma, L = 3, DV --- 5 from skull) and hippocampus (AP = -3.8, L = 3, DV = 3.5). In addition, 3-month old male Sprague-Dawley rats subjected to 15 rain of transient ischemia by means of 4-vessel occlusion were used21. The rats were run in a complex maze task 11 and sacrificed 2 months after ischemic treatment. For immunocytochemistry, rats were sacrificed by pentobarbital overdose and transcardially perfused with 50 ml 0.1 M PBS, pH 7.4, followed by 300 ml filtrated 10% formalin in 0.1 M PB, pH 7.4, at room temperature. Subsequently, brains were removed, post-fixed overnight in the same fixative at 4 °C, and placed in 30% sucrose in PBS for 2 days at 4 °C. Brains were frozen in 2-methylbutane and stored at -70 °C until sectioning at 10--40/~mon a freezing sliding microtome. For immunoblotting, normal rats and stab wound lesioned rats (8 days prior to sacrifice) were perfused with 50 ml 0.1 M PBS, pH 7.4, at 4 °C. Brains were removed, separated in control and lesioned forebrain hemispheres, frozen in 2-methylbutane, and stored at -70 °C until further processing. lmmunocytochemistry Free-floating tissue sections were washed with TBS-NaCI (0.05 M Tris-buffer containing 1.5% NaCI, pH 7.4). Sections were incubated for 10 min in 0.3% Triton X-100 in TBS-NaCI, followed by 30 min in 5% goat serum in TBS-NaCI at room temperature. Thereafter, sections were reacted for 2 days at 4 °C with the primary antibody diluted 1:500 to 1:8000 in TBS-NaCi containing 2% goat serum and 0.3% Triton X-100. Subsequently, brain tissue was treated for 1.5 h at 4 °C with biotinylated goat anti-rabbit IgG (1:200, Vectastain Elite ABC Kit, Vector Laboratories, Burlingame, CA) in 1% goat serum in TBS-NaCI, followed by 1.5 h with the ABC complex (1:100, Vectastain Elite ABC Kit) in TBS-NaCI. Washes between treatments were done with TBS-NaC1 at 4 °C. The immunoreaction was visualized by treating sections with TBS-NaC! containing 0.05% 3,3"-diaminobenzidine-HCi4 (Fluka), 0.03% H202, and 0.06% NiCI2 for 3-7 rain at room temperature. Finally, the tissue was washed again, mounted on gelatine/chrome alum-coated slides, dehydrated, cleared and coverslipped. Specific and distinct immunoreactivity was observed with dilutions 1:2000 up to 1:8000. Control sections were processed identically, but the primary antiserum was (i) either omitted from the staining procedure or (ii) replaced by primary antiserum preabsorbed with HPLC-purified immunogen, or (iii) replaced by serum from non-immunized rabbits. All controls were negative. However, high concentrations of normal rabbit serum (up to 1:2000) frequently resulted in a clearly different, but appreciable cellular immunoreactivity. Western immunoblot Normal and lesioned rat forebrain tissue was Dounce homogenized at 4 °C in 0.01 M PB, pH 7.4, containing 0.15 M NaCI, 10 mM EDTA, 1 mM NEM, and 0.15% Triton X-100. Homogenates were centrifuged at 10,000 g for 15 min at 4 °C. A Lowry protein assay was performed on the supernatant fraction and 300 #g protein was loaded per lane. Finally, 5-7.5% SDS-polyacrylamide gels under reducing conditions were run and transferred to nitrocellulose

by the Western blot method 37. The antiserum to 110 kDa LBP was used at a dilution of 1:50.

RESULTS

110 kDa LBP-like immunoreactivity in the normal adult rat brain Coronal brain sections were i m m u n o s t a i n e d with antibody to the 110 k D a LBP (Fig. 1A). I m m u n o s t a i n i n g was observed throughout the neocortex including frontoparietal, cingulate, and occipital cortex confirming preliminary findings 15. The reaction product was confined to perikarya in layers II/III, V and to a lesser extent to Via (Fig. 1D,E). Pyramidal cells with their main apical and proximal basal dendritic processes were clearly labeled (Fig. 1B). Apical dendrites were i m m u n o s t a i n e d from the soma up to the molecular layer (Fig. 1E). In addition, densely labeled horizontal fibers were observed in layers II/III. The reaction product appeared to be localized to processes and possibly to n e u r o n a l surfaces but was also present in the cytoplasm of perikarya. Nuclei always appeared free of immunoreactivity (Fig. 1B). U n d e r high magnification m a n y thin i m m u n o l a b e l e d processes could be detected throughout the neocortex. Similar small processes could be seen in the corpus callosum, fimbriafornix, other white matter, as well as traversing the striatum, findings that indicated some axonal staining was also present. Intense immunoreactivity in hippocampus was observed in the CA3 region and in the hilus of the dentate gyrus (Fig. 2A). Confined to the stratum lucidum, the reaction product stained mossy fibers and terminals (Fig. 2 C - E ) . Pyramidal cells and granule cells of the dentate gyrus lacked immunoreactivity. Weakly stained glial cells could be observed frequently in the hippocampus (Fig. 2B) but not in the neocortex or striatum. All immunoreactivity disappeared when the primary antibody was (a) omitted, (b) replaced by normal rabbit serum (Fig. 1C), or (c) when the antibody was preabsorbed with purified i m m u n o g e n (Fig. 3C). I10 kDa LBP-like immunoreactivity in response to mechanical and ischemic lesions Rats were subjected to a unilateral needle injury in forebrain 3, 8, or 14 days prior to sacrifice to study possible expression of 110 k D a LBP-like molecules in response to injury. Immunostaining with a n t i - l l 0 kDa LBP showed an intense glial immunoreactivity around the lesion tract (Fig. 3A). Darkly stained astrocytes (Fig. 3B) could be recognized by their stellate-shaped cell morphology2'23. Neuronal processes within a narrow zone about the lesion were also intensely labeled. Needle injury-induced immunoreactivity in astroglia was most intense in the

307

Fig. 1. Immunoreactivity to 110 kDa LBP in the normal adult rat forebrain. Coronal brain sections were immunoreacted by the ABC-peroxidase technique. A: hemisection through the parietal cortex and dorsal hippocampus revealing intense immunoreactivity in the more superficial cortical layers and in the CA3 region of the hippocampus. D, E: immunolabeled pyramidal cells with their apical and proximal basal dendrites in cortical layers II/III and V. Many more pyramidal cells in layer V could be immunolabeled when sections were cut at 15/~m (E) compared to 40/~m (D). C: no immunostaining was observed with normal rabbit antiserum 1:6000. B: high magnification of 110 kDa LBP-positive pyramidal neurons in layers II/III. Antibody dilutions are 1:4000-1:6000. Sections are 40/zm ( A - D ) and 15/zm (E). Calibration bars are 250 /zm (E) and 50/zm (B).

308 fronto-parietal cortex where injury was greatest but was also clearly present in the underlying hippocampus which frequently had weakly stained glia and in the striatum which normally presented no cellular staining. In all sites,

immunoreactivity was localized to the site of the injury. No reactive astrocytes could be observed in the unlesioned hemisections. The 4-vessel occlusion (4-VO) model of ischemic

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Fig. 2. Immunoreactivity to 110 kDa LBP in the normal adult rat dorsal hippocampus. A: immunoreactivity was primarily associated with the CA3 region of the hippocampus and the hilus (H) of the dentate gyrus. C: higher magnification demonstrates that the immunoreactivity is confined to mossy fibers (mf) and terminals (stratum lucidum). CA3 pyramidal cells (p) and granule cells of the dentate gyrus (g) are free of immunostaining. D: note the close vicinity of immunostained mossy fiber terminals and CA3 pyramidal cells. E: in the rostral part of the hippocampus, long mossy fibers could be labeled (co: corpus callosum). B: weakly stained gial cells were frequently observed in the hippocampus. The framed area in A is shown in higher magnification in B. Antibody dilutions are 1:4000. Sections are 40/~m (A,B,D) and 15/zm (C,E). Calibration bars are 500/tin (A), 250/~m (C,E), and 50/zm (B,D).

309

Fig. 3. Immunoreactivity to 110 kDa LBP in response to a needle injury in the adult rat brain. A mechanical lesion was appfied by inserting a micropipette in the rat brain and injecting 1/~1 saline. A: immunostained reactive astrocytes could be observed around the wound side in the fronto-parietal cortex 8 days after surgery. B: reactive astrocytes in higher magnification. C: note the absence of immunoreactive astrocytes when antiserum was preabsorbed with 0.75 mg/ml purified 110 kDa LBP. Arrows in (A) and (C) indicate needle tract. Antiserum was used at 1:4000-1:6000. Sections are 40/~m. Calibration bars are 250 pm (A,C) and 50/~m (B).

Fig. 4. Thionin Nissl stain and immunoreactivity to 110 kDa LBP in the dorsal hippocampus after ischemic lesion. By using the 4-vessel occlusion (4-VO) technique, rats were subjected to transient cerebral ischemia. A- thionin Nissl staining revealed degeneration of CA1 pyramidal cells (p) in the dorsal hippocampus 8 weeks after surgery. B: sham-operated control. Note the shrinkage of the CA1 region in response to the ischemic lesion shown in A compared to B. C: adjacent section to A immunoreacted with anti-ll0 kDa LBP reveals an anatomically restricted immunopositive reactive gliosis in the region where cell degeneration occurred. D: higher magnification of framed box in C showing immunostained reactive astrocytes in CA1 for the 4-VO rat. E- only weakly immunostained glial cells could be observed in the sham-operated control. Antibody dilutions are 1:4000. Sections are 40/zm. Calibration bars are 500/~m (B) and 50/~m (E).

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Fig. 5. Immunoblot of normal and lesioned adult rat brain with anti-110 kDa LBE Unilateral mechanical lesioned rat forebrain was separated in unlesioned control (lane 1) and lesioned (lane 2) hemisphere. Brain tissue was homogenized in the presence of Triton and centrifuged. A Lowry protein assay was performed on the supernatant fraction. Polyacrylamide-SDS gels were run under reducing conditions, transferred to nitrocellulose, and stained with polyclonai anti-110 kDa LBP (1:50). The reactive bands at ~ 110 kDa (arrow) correspond to the mobility of the 110 kDa immunogen isolated from newborn mouse brain 1~. A similar band pattern was observed in control (lane 1) and lesioned brain tissue (lane 2), suggesting that the reactive glial-derived protein recognized by anti-ll0 kDa LBP is also expressed in normal adult rat tissue. damage was used to study involvement of 110 kDa LBP-like molecules in the ischemic lesion response in the adult rat CNS. This experimental surgical technique produces transient ischemia and causes a loss of selectively vulnerable neurons in the brain 21. It has been used as a model for certain neurological diseases. As demonstrated with thionin Nissl stain 8 weeks after 4 - V O , the ischemic lesion caused extensive degeneration of CA1 pyramidal cells bilaterally in the dorsal hippocampus compared to normal brain (Fig. 4A,B). As seen after needle injury, intense immunolabeled reactive glia could be observed. Reactive astrocytes were found anatomically restricted to the hippocampal region where cell loss occurred (Fig. 4C). Both a markedly increased staining density and an increased size and number of darkly immunostained astrocytes as well as processes per cell could be observed compared to control (Fig. 4D,E). Preliminary analysis showed that the severity of the ischemic lesion as determined by Nissl stain was reflected by the extent of the 110 kDa LBP-like immunoreactive gliosis (data not shown). Immunolabeled reactive astrocytes were not observed in sham-operated rats (Fig. 4E).

Western immunoblot Immunoblotting of a detergent extract of adult and lesioned forebrain was used to verify 110 kDa LBP antibody specificity. No differences were detected be-

tween normal adult brain tissue and tissue from the unlesioned control hemisphere of rats which received a stab wound 8 days prior to sacrifice. Immunoblot results revealed a band at ~ 110 kDa (Fig. 5, lane 1) suggesting that the antibody recognizes a protein in adult rat brain similar in molecular size to the immunogen extracted from mouse brain 15. The additional slightly lower molecular weight band is frequently found in brain tissue (ref. 15 and H.K. Kleinman, unpublished observation) and may be due to non-specific binding and/or breakdown of the protein. No additional band could be detected in lesioned brain tissue (lane 2) compared to controls. The 110 kDa band appears to be more intense in the lesioned compared to the control side. DISCUSSION Using avidin-biotin peroxidase immunohistochemical techniques, our results demonstrate that antibody to 110 kDa LBP strongly immunostains distinct cell populations and fibers in the normal adult rat brain. Cellular immunoreactivity was found in cortical pyramidal neurons in layers II/III and V with their apical dendrites clearly labeled. Furthermore, the reaction product was specifically associated with mossy fibers and terminals in the stratum lucidum of the hippocampus. The exact location of the immunoreactivity requires further clarification using thinner sections and high resolution microscopy. This approach is in progress since the same staining pattern can also be observed in vibratome sections in the absence of Triton (results not shown). In two types of forebrain injury, needle injury and ischemia, our results further demonstrate an intense 110 kDa LBP-like immunostaining of reactive astrocytes. It is unclear at this point whether the 110 kDa LBP immunogen used in this study and isolated from newborn mouse brain is identical with previously described laminin receptors of similar size isolated from membrane fractions of NG108-15 neuroblastoma x glioma cells 4'13'29. Many common properties were noted 15. Antibodies against the 110 kDa laminin receptor isolated from NG108-15 cells have been found to block neurite outgrowth induced by a PA22-2 containing domain of laminin 13. Since the mouse brain-derived 110 kDa LBP used in this study was isolated on a PA22-2 affinity column, it is likely that the neuroblastoma x gliomaderived protein is identical or related to the brain-derived 110 kDa LBP. Furthermore, studying migration of neural crest cells in chick embryo, Pomeranz et al. 22 reported similar immunoreactivity to 110 kDa LBP and to 'cranin', a 120 kDa protein derived from NG108-15 neuroblastoma × glioma cells29. Antiserum to cranin does indeed immunostain certain processes in embryonic rat brain.

311 However, no immunoreactivity in adult rat brain was reported 29. In contrast to the developing CNS where laminin is transiently expressed, laminin and other extracellular matrix components, such as fibronectin, collagen, and proteoglycans, were generally believed to be absent from the neuropil in the adult brain 3'24. However, recent studies challenged this view. Intraneuronal laminin-like molecules in the adult brain have been reported 8'31'4° and proteoglycans were found to be associated with some neurons in the mature CNS 41. The function of intraneuronal laminin-like molecules is unknown but deserves special consideration since the present findings suggest that in certain neuronal populations, e.g. cortical pyramidal cells, both laminin-like and 110 kDa LBP-like immunoreactivity can be demonstrated. Reactive gliosis in response to mechanical and ischemic injuries can be seen by increased glial fibrillary acidic protein (GFAP) staining 9,36. Reactive glial cells at the site of injury may have an important role in CNS regeneration. They might exert beneficial effects on successful regeneration or inhibit axonal regeneration 23. The present immunohistochemical findings suggest that astrocytes express 110 kDa LBP-like molecules in response to injury. This reactivity is intense and anatomically specific. Since immunoblotting of stab wound lesioned tissue showed no additional band compared to normal brain tissue, it is likely that the molecules expressed by reactive glia are identical to the 110 kDa LBP-like protein expressed in neurons in normal brain. The same conclusion can be drawn from preliminary immunoblot experiments in which normal and ischemic hippocampal tissue were compared. Of particular interest in this context are reports that astrocytes also express laminin molecules in response to injury TM. The function of such astroglia-derived laminin in mediating an injury response in the CNS is unclear but was suggested to REFERENCES 1 Baron-Van Evercooren, A., Kleinman, H.K., Ohno, S., Marangos, P., Schwartz, J.P. and Dubois-Dalcq, M.E., Nerve growth factor, laminin, and fibronectin promote neurite growth in human fetal sensory ganglia cultures, J. Neurosci. Res., 8 (1982) 179-193. 2 Bignami, A, and Dahl, D., Astrocyte-specificprotein and radial glia in the cerebral cortex of newborn rat, Nature, 252 (1974) 55-56. 3 Carbonetto, S., Evans, D. and Cochard, P., Nerve fiber growth

in culture on tissue substrata from central and peripheral nervous system, J. Neurosci., 7 (1987) 610-620. 4 Douville, P.J., Harvey, W.J. and Carbonetto, S., Isolation and partial characterization of high affinity laminin receptors in neural cells, J. Biol. Chem., 263 (1988) 14964-14969. 5 Edgar, D., Neuronal laminin receptors, Trends Neurosci., 12 (1989) 248-251. 6 Ehrig, K., Leivo, I., Argraves, W.S., Ruoslathi, E. and Engvali,

contribute to neovascularization after injury 7. Since reactive astrocytes might produce both laminin and a laminin binding protein in response to CNS injury, a role for laminin molecules in the injury response is likely, In cultured CNS neurons from the septal-basal forebrain, an increased laminin response after axotomy was found and might be related to the expression or modification of a laminin receptor 26. In the developing mammalian CNS, the transiently expressed laminin produced by astrocytes appears to be an important factor for axon elongation ls. In culture, however, glial-derived laminin appears to be a variant form of EHS tumor-derived basement membrane laminin lacking the 400 kDa A chain 17'39. Other variant forms of laminin have also been described and form a family of proteins 6'1°'2°'25. It is unknown if the PA22-2 amino acid sequence of the laminin A chain recognized by the 110 kDa LBP is present in reactive astroglial-derived laminin. In summary, the anatomically specific cellular localization of 110 kDa LBP-like immunoreactivity in the normal adult rat forebrain and the intensive immunolabeling of reactive astrocytes suggest a function of 110 kDa LBP-like molecules in the adult brain and in response to CNS injury. The astroglia-derived antigen does not appear to be a glia-specific protein but is also expressed in neurons in the normal brain. Further studies and characterization of 110 kDa LBP are necessary to understand the structure, interactions, and function of this laminin-binding protein which is likely involved in CNS neuronal-glial communication.

Acknowledgements. We thank E. Williams and C. Adler (GRC) for the excellent help with the photography, and E. Mesco, A. Passaniti (GRC), E Cannon (NIDR), N. Muth (Essex Community College, Baltimore) and E Bialobok (Fisions Corp.) for their experimental support. The GRC is fully accredited by the American Association for the Accreditation of Laboratory Animal Care.

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