SPARC, an extracellular matrix glycoprotein containing the follistatin module, is expressed by astrocytes in synaptic enriched regions of the adult brain

BRAIN RESEARCH ELSEVIER

Brain Research 676 (1995) 69-79

Research report

SPARC, an extracellular matrix glycoprotein containing the follistatin module, is expressed by astrocytes in synaptic enriched regions of the adult brain Duane B. Mendis a, Luc Malaval b, Ian R. Brown a,* a Departmen,~ of Zoology, University of Toronto, Scarborough Campus, West Hill, Ont., MIC 1A4, Canada b INSERM, U403, Edouard Herriot Hospital, Lyon, France

Accepted 27 December 1994

Abstract

Although extracellular matrix (ECM) glycoproteins play important roles in neural development, their levels are generally believed to decrease in the adult brain. Immunohistochemical analysis indicates that the anti-adhesive ECM glycoprotein SPARC/osteonectin, which contains a follistatin 'module,' is expressed in the adult rabbit nervous system. In the cerebellum, SPARC is present in Bergmann glia, with a strong signal along their radial fibres. SPARC, while enriched in membrane fractions, is not a transmemb~rane protein. In the hippocampus, colocalization of SPARC is observed in cells which express the astrocytic marker GFAP. The expression of SPARC by a subset of astrocytes, particularly in synaptic enriched areas, suggests a continuing role for the ECM in the adult brain. Keywords: Bergmann glia; Immunohistochemistry; Western blotting; Astroglia

1. Introduction

SPARC (Secreted Protein Acidic and Rich in Cysteine) is a well-characterized component of the extracellular matrix (ECM), originally isolated from developing bone and referred to as osteonectin, but later shown to be expressed by a wide range of tissues and culture systems [23]. When exposed to exogenous SPARC, endothelial ceils grown in tissue culture undergo dramatic changes, such as adopting a rounded morphology, arrest of cell migration, and inhibition of cell cycle progression [10,13,22]. Furthermore, in the presence of SPARC, the synthesis of secreted molecules such as fibronectin, thrombospondin, and plasminogen activator inhibitor (PAl-l) has been shown to be altered [21]. These results suggest that SPARC could be involved in developmental events such as cell migration and tissue reorganization. Concerning neural development, however, previous reports have shown low levels of SPARC in the embryonic and neonatal brain [29,35]

* Corresponding author. Fax: (1) (416) 287-7642. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSD! 0006-8993(95)00101-8

unlike other ECM molecules such as laminin, fibronectin, and thrombospondin [30,36,40]. We have noted significant levels of SPARC m R N A in the adult brain by Northern blotting during the analysis of SC1, a neural protein that shows partial sequence homology to SPARC [20]. Recently we examined the expression of SPARC m R N A during postnatal neural development by in situ hybridization, and demonstrated that the m R N A is present at low levels at birth [27]. However, levels of SPARC m R N A increase in caudal regions of the adult brain such as the cerebellum and brainstem as postnatal development proceeds. Thus, SPARC appears to be an ECM component which is expressed both in the developing and adult nervous system. Although the expression of many ECM molecules such as laminin, fibronectin, and thrombospondin decrease as neural development proceeds, a small number of ECM components including SPARC have been identified in the adult brain [16-19,28]. Recent observations on the homology between a region of SPARC and the follistatin domain or 'module' have heightened our interest in establishing the

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pattern of SPARC expression in the mammalian nervous system. The follistatin module is present in SPARC, the related proteins SC1 and QR1, as well as agrin and follistatin [6,12,14,20,23,33]. This module has been suggested to play a role in neural differentiation by accumulating, protecting and modulating the activity of growth factors [11,33]. Follistatin has been implicated in the inhibition of activin and promotion of neuralizing activity in early embryogenesis [14,15]. The present investigation looks at the cellular and subcellular distribution of SPARC protein in the adult rabbit brain utilizing two anti-SPARC monoclonal antibodies [25]. Highest levels of expression were seen in the cerebellum, followed by brain stem, with other brain regions showing approximately equal amounts to that observed in heart. Subcellular fractionation indicates that SPARC is enriched in membrane-containing fractions, but is not a transmembrane protein. SPARC is expressed by Bergmann glial cells and their radial fibres in the adult cerebellum and also at the time of granule neuron migration during development. Double immunohistochemistry demonstrates that SPARC is expressed in astrocytes in the hippocampus.

to yield a P2 pellet which contains light and synaptic membranes. The supernatant ($2) fraction was then centrifuged at 105,000 × gay for 90 min to give a P3 fraction, which contain microsomes. A total of 20/~g of the homogenate, P1, $2, P2, and P3 fractions were then electrophoresed and analyzed by Western blot analysis with the anti-SPARC ON6 antibody as described above.

2.3. Phase separation of SPARC in 1% Triton X-114 For the separation of membrane components, 100 /zg of the P2 cerebellar fraction was suspended in a final concentration of 10 mM Tris buffer, pH 7.4, containing 150 mM NaC1 and 1% Triton X-114 at a final protein concentration of 1 mg/ml at 4°C for 30 min. The suspension was layered over 250 ~1 of 6% sucrose, incubated at 30°C for 3 min, followed by centrifugation at 325 x gay for 3 min at room temperature. The lower detergent and upper aqueous phases were recovered, and processed for Western blot analysis.

2.4. Immunohistochemistry 2. Materials and methods

2.1. Western blotting Tissue homogenates were prepared from adult rabbit brain regions and heart. Aliquots of total protein (100 /zg) were separated on a linear 10% polyacrylamide gel according to Mendis et al. [27]. The proteins were then transferred onto nitrocellulose membranes and processed for Western analysis. The blot was blocked for 1 h at room temperature in 2% purified BSA in TBST (10 mM Tris, 0.25 M NaCI, 0.3% Tween 20, pH 7.4) with 0.02% sodium azide and then incubated with the anti-SPARC monoclonal antibodies ON6 (1:2,000 dilution of 1.7 mg/ml stock) or ON9 (1:1,000 dilution of 2.0 mg/ml stock) for 16 h [25]. Anti-GFAP (Boehringer Mannheim) or anti-actin (Amersham) monoclonal antibodies were incubated at 1:10,000 for 2 h. The blots were rinsed 4 X 10 min in TBST, and incubated for 2 h at room temperature with a peroxidase-labelled polyclonal anti-mouse IgG diluted 1:5,000 in TBST and then washed 4 X 10 min in TBST. The presence of immunoreactive bands was visualized by use of ECL Western blotting detection reagents (Amersham, RPN 2106).

2.2. Isolation of subcellular fractions Total cerebellar homogenates were centrifuged at 800 x gay for 5 min to yield a P1 nuclear pellet. The supernatant was centrifuged at 11,000 x gav for 15 min

Adult or 4-day-old rabbits were perfused with 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS), pH 7.4. Brain tissue was removed and placed in 4% paraformaldehyde overnight at 4°C and then allowed to equilibrate in 25% sucrose in PBS, mounted in OCT embedding compound, and then cut at 25 t~m using a cryostat. For peroxidase immunohistochemistry with the ON6 or ON9 antibodies, sections were allowed to air dry for 2 h and then incubated for 20 min at room temperature in buffer 1 (PBS, 0.2% Triton X-100, 0.1% BSA). Sections were then incubated 14-16 h at room temperature in primary antibody (1:200 in buffer 1), followed by processing with the Vectastain Elite ABC kit (Vector labs) using diaminobenzidine (DAB) as the substrate. For double immunohistochemistry, sections were incubated in absolute methanol at -20°C for 3 min, followed by detection of the GFAP antibody (1:100) or non-specific antibody (Mouse IgG3; Organon Teknika, Cat. #50333/36351) diluted 1:100 in the same manner as mentioned above. After staining with the peroxidase method, sections were washed with tap water for 10 min, followed by incubation with buffer 1 for 30 min. Sections were incubated with either ON6 or nonspecific antibody as mentioned above and visualized with an anti-mouse alkaline phosphatase conjugated enzyme (Boehringer Mannheim), using NBT and BCIP (Biorad) as substrates. Sections were then dehydrated and mounted with permount. For detection of microglia, sections were immersed in 3% hydrogen peroxide for 3 min followed by an

D.B. Mendis et al. ~Brain Research 676 (1995) 69-79

incubation in buffer 1 for 30 min. This was followed by incubation with peroxidase labelled lectin (Sigma, Cat # L-5391) diluted to 2 0 / z g / m l in buffer 1 for 2 h and then processed using DAB as a substrate and mounted as above.

3. Results

3.1. Distribution of SPARC protein in regions of the adult brain Total protein isolated from adult brain regions and heart was separated on polyacrylamide gels for Western blot analysis (Fig. 1). Immunoreactive bands were

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visualized using the ON6 (panel A) and ON9 (panel B) anti-SPARC monoclonal antibodies [25]. Both antibodies detected a doublet of 40/43 kDa in the cerebellum (lane 1) while the 43 kDa component predominated in the cerebral hemispheres (lane 2), hippocampus (lane 3), brainstem (lane 4) and heart (lane 5). The strongest SPARC signal was observed in the cerebellum. Since the astrocytic marker G F A P is used in this study for immunohistochemistry to determine if SPARC is expressed by astrocytes, a parallel blot was incubated with an anti-GFAP antibody (panel C). G F A P was detected in all brain regions (lanes 1 to 4) but not in heart (lane 5). This blot was then stripped and re-in-

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Acfin 12345 Fig. 1. Western blot analysis of SPARC protein in regions of the adult rabbit brain. Aliquots of total protein (100/~g) isolated from various brain regions and heart were separated on polyacrylamide gels and immunoreactive bands visualized using the anti-SPARC monoclonal antibodies ON6 (panel A) and ON9 (panel B), or an anti-GFAP monoclonal antibody (panel C). The scale on the left indicates the position of molecular weight markers (kDa). The GFAP blot in panel C was stripped and re-incubated with an anti-actin antibody (shown in lower portion of panel). Lane 1--cerebellum; 2 --cerebral hemispheres; 3--hippocampus; 4--brain stem; 5--heart.

Fig. 2. Distribution of SPARC in membranous fractions of the cerebellum. Panel A: cerebellar homogenates were separated into nuclear, total membranous, and microsomal fractions. Aliquots of 20 /.~g of protein from each fraction was loaded per lane and processed for Western blot analysis using the ON6 anti-SPARC antibody. The scale on the left indicates the position of molecular weight markers (kDa). Lane 1--total cerebellar homogenate, 2--nuclear fraction (P1), 3--supernatant fraction ($2), 4--total membranous fraction (P2), 5--microsomes (P3). Panel B: phase partitioning of SPARC in 1% Triton X-114. The cerebellar P2 total membranous fraction (40 /zg) shown in lane 1 was separated into a detergent extract phase (lane 2) and an aqueous phase (lane 3) and processed for Western blot analysis as above.

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cubated with an anti-actin antibody to verify that equal amounts of protein were loaded per lane (shown in the lower portion of panel C).

3.2. Association of SPARC with membranous fractions of the cerebellum Subcellular fractionation was performed in order to determine where SPARC protein is localized in the rabbit brain. Cerebellar homogenates were separated into nuclear, total membranous, and microsomal fractions and analyzed by Western blotting with the ON6 anti-SPARC antibody (Fig. 2A). SPARC was enriched in the total membranous (P2) and microsome (P3) fractions (lanes 4 and 5) compared to the total homogenate, nuclear (P1), and supernatant fractions (lanes 1 to 3). In Fig. 2B, phase partitioning of the cerebellar P2 total membranous fraction (lane 1) was used to determine if SPARC is an integral membrane protein. SPARC immunoreactivity was not detected in the detergent extract phase (lane 2) and was observed in the aqueous phase which would contain the detergent insoluble membrane fraction (lane 3), suggesting that SPARC, while associated with neural membranes, is not an integral membrane protein. Extracellular matrix components in the adult brain have previously been reported to be associated with detergent-insoluble membrane-containing fractions [16,17].

3.3. Localization of SPARC protein in cellular layers of the adult cerebellum As shown in Fig. 1 by Western blotting, the brain region which exhibited the highest level of SPARC protein was the cerebellum. In Fig. 3, immunohistochemical studies were undertaken to determine which cerebellar cells in the adult brain express SPARC. As shown in panel A, a striking pattern of SPARC immunoreactivity was observed in Bergmann glial fibres in the molecular layer (ML) which show a parallel orientation. SPARC was also detected in (i) astrocytic Bergmann glial cell bodies in the Purkinje cellular layer (PCL) which are better resolved at higher magnification in panel B (labelled as BG) and ii) in scattered cells in the deep white matter (WM) and granule cell layer (GCL) which may be astrocytes (labelled as A in panel B). Expression was not detected in neuronal cells, for example Purkinje neurons (PC) in panel B. As shown in panel C, SPARC immunoreactivity is seen to extend along Bergmann glial fibres (BGF) up to glial end feet (EF) that impinge on the pial surface (P) of the cerebellum.

3.4. Expression of SPARC in the developing cerebellum As shown in Fig. 3, high levels of SPARC were observed in Bergmann glial fibres in the adult rabbit

Fig. 3. Immunohistochemical localization of SPARC protein in the adult cerebellum. Sagittal sections (25/~m) of the rabbit cerebellum were incubated with a 1:200 dilution of the ON9 anti-SPARC monoclonal antibody. Signal was visualized using peroxidase. Bars in A = 100 /zm, B and C = 50 /.~m. A, astrocyte; BG, Bergmann glial cells; BGF, Bergmann glial fibres; EF, glial end feet; GCL, granule cell layer; ML, molecular layer; P, pial surface; PC, Purkinje neuron; PCL, Purkinje cellular layer; WM, deep white matter.

cerebellum. We next analyzed whether SPARC is present along these fibres in the developing cerebellum at postnatal day 4 (PD4) during granule cell migration. Nissl staining of the PD4 cerebellar layers in Fig. 4A indicates the presence of granule cells that migrate

D.B. Mendis et al. / Brain Research 676 (1995) 69-79

from the external granule cell layer (EGL) to the internal granule cell layer (GCL). As shown in Fig. 4B, immunohistochemistry with an anti-GFAP antibody identifies Bergmann glial fibres (denoted by arrowheads) which extend from the Purkinje cellular layer (PCL) through the molecular layer (ML) and EGL, terminating at the pial surface (P). A similar pattern is observed for SPARC (denoted by arrowheads in Panel C, and also at higher magnification in panel D). The presence of SPARC along Bergmann glial fibres at this developmental stage suggests a role for this molecule in granule cell migration. While a fibre-like staining pattern is noted for SPARC in the molecular layer (Fig. 4C), the protein appears to be associated primarily with cell bodies, not cell processes (denoted by arrows) in astrocytes of the white matter (WM). 3.5. Localization o f S P A R C protein to astrocytes in the adult hippocampus

Immunohistochemistry indicated that SPARC was not detected in pyramidal neurons of the CA1 region

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of the adult hippocampus, however expression was noted in cells scattered throughout the molecular layer (ML) (Fig. 5A). Given the expression of SPARC in astrocytic Bergmann glial cells which was noted in the cerebellum (Fig. 3), we decided to explore whether the SPARC-positive cells in the molecular layer of the hippocampus were astrocytes. This involved the use of G F A P as an astrocyte-specific marker. G F A P signal was apparent in cellular processes while the SPARC protein was localized to the cell body in astrocytes of the hippocampal molecular layer (ML). As shown in Fig. 5B and at higher magnification in panel C, double immunohistochemistry revealed that a large proportion of G F A P positive cells (identified by brown stained cell processes after the peroxidase treatment - - see arrowheads) are also SPARC positive (identified by dark blue staining in cell bodies after the alkaline phosphatase treatment - - see arrows). The following controls were also included: panel D - - prior peroxidase staining with a non-specific antibody did not interfere with the subsequent detection of SPARC by the alkaline phosphatase treatment which produces a blue cel-

Fig. 4. SPARC expression in the developingcerebellum. Sagittal sections of the developing cerebellum at postnatal day 4 (PD4) were stained with Cresyl violet (panel A) for cellular detail, or subjected to immunohistochemistrywith either anti-GFAP antibody (panel B), or the anti-SPARC ON6 antibody (panels C and D). Bars in A,B,C = 100/zm, D = 50/zm. EGL, external granule layer; GCL, granule cell layer; ML, molecular layer; P, pial surface; PCL, Purkinje cellular layer; WM, deep white matter; arrows, astrocytes; arrowhead, Bergmann glial fibres.

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lular stain; Panel E - - cross-reactivity was not observed (as indicated by lack of blue stain) when GFAP was detected by the peroxidase treatment, followed by incubation with a non-specific antibody and the alkaline phosphatase detection. This is seen at higher magnification in panel F as both cell processes (arrowhead) and the cell body (arrow) stained brown with no detectable blue staining.

3.6. Expression of SPARC in other regions of the adult brain As can be seen in Fig. 6A, SPARC positive cells in the cerebral cortex exhibilted an astrocytic appearance (A), while neurons (N) were immunonegative. A SPARC signal was also observed in the glial limitans (GL) and in cells associated with blood vessels (BV). This pattern of SPARC expression did not correspond to microglia cells (panel B), which make up approximately 20% of all glial cells in the CNS. Double immunohistochemistry with a lectin microglial marker and the anti-SPARC antibodies indicated that SPARC expression was not detecl:ed in this type of glial cell (data not shown). In agreement with our previous mRNA study [28], SPARC protein is expressed within brain stem nuclei such as the inferior olive (IO) (panel C). At higher magnification (panel D) it is apparent that SPARC protein is expressed by glial cells (G) and not by neurons (N) in this brain region. SPARC expression within the non-myelinated globus palladus (GP) demonstrates a fibre-like pattern radiating into the corpus callosum (CC). This pattern may reflect the presence of an extracellular matrix surrounding striatal and subthalamic projections that are known to form compact bundles around pallidal neurons [32].

4. Discussion

The extracellular matrb: (ECM) is believed to maintain tissue integrity and structure as well as to modulate intercellular interactions. The ECM is often characterized as an electron dense region surrounding cells which consists of fibrils of collagen and proteoglycan proteins, associated with a wide range of other extracellular glycoproteins [1]. In contrast, the developing and adult brain lacks collagen and well-defined mot-

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phological ECM structures such as the basal lamina, with the exception of the barrier layers of the pial surface and surrounding blood vessels [38]. In the developing brain, a growing number of ECM components have been identified [43]. The pattern of expression of these proteins, combined with their effect on cellular interactions in vitro, has suggested that the ECM plays important functions in directing cell migration, apoptosis, differentiation, lineage, and cell division [43]. Given that the expression of structural ECM components such as laminin, fibronectin, and thrombospondin decreases following developmental events and that collagens are lacking in the mature CNS [38], it might be concluded that there is no ECM in the adult brain. Recently, however, a small number of ECM components, most notably proteoglycans, have been shown to be expressed in the adult CNS [1619,24]. These molecules have been localized to insoluble membrane-containing subcellular fractions, but are not transmembrane. Little is known of their functional properties. The role of an ECM in the adult brain might be to stabilize synaptic structure by interaction with various cell adhesion molecules and inhibit further neurite outgrowth [17]. In addition, the adult brain ECM may help to regulate the extracellular environment through binding of calcium, as well as diffusible proteins such as growth factors and cytokines [17]. SPARC is a well-characterized modulator of cellmatrix interactions which is found in a wide range of mineralized and non-mineralized tissues [23]. Previous studies have reported low levels of SPARC protein and mRNA in the embryonic nervous system [3,29,35], however we have demonstrated significant levels of SPARC mRNA during the postnatal neural development with maximal expression in caudal regions of the adult brain, such as the cerebellum and brainstem [28]. Our interest in establishing the pattern of SPARC expression in the mammalian nervous system has intensified following recent observations on the homology between a region of SPARC and the follistatin domain or 'module' [14,23,33]. This module has been suggested to play a role in neural differentiation by accumulating, protecting, and modulating the activity of growth factors [11,33]. The follistatin module is present in SPARC and the related proteins SC1 and QR1, as well as agrin and follistatin [12,20,23,28]. In the present report, we have utilized two anti-SPARC monoclonal antibodies,

Fig. 5. Double immunohistochemistry of SPARC and GFAP localization in the hippocampus. Coronal sections of the adult forebrain were reacted with the anti-SPARC ON6 antibody and detected with peroxidase (panel A) or using double immunohistochemistry to determine if SPARC is expressed in GFAP positive astrocytes (panels B and C). For double immunohistochemistry, binding of the first antibody (anti-GFAP) was detected with peroxidase (brown) followed by the detection of the second antibody (anti-SPARC) with alkaline phosphatase (blue). The following controls were included: panel D: non-specific antibody detected with peroxidase (brown) followed by anti-SPARC ON6 antibody detected with alkaline phosphatase (blue); Panels E and F: anti-GFAP antibody detected with peroxidase (brown) followed by non-specific antibody detected with alkaline phosphatase (blue). Bars in A,B,D,E = 100/~m, C and F = 50 gm. CA1, pyramidal neurons of the hippocampus; ML, molecular layer; arrow, cell body; arrowheads, cell processes.

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Fig. 6. Localization of SPARC protein in various adult brain regions. Coronal sections were reacted with the ON9 anti-SPARC antibody. Panel A: SPARC staining in the cerebral cortex, Panel B: localization of microglia in cerebral cortex utilizing lectin-peroxidase histochemistry, Panels C and D: SPARC localization in the inferior olivary nuclei of the brain stem, and Panel E: SPARC immunoreactivity within the giobus paUidus. A, astrocytes; BV, blood vessels; CC, corpus callosum; G, glial cells; GL, glial limitans; GP, globus pallidus; IO, inferior olive; N, neuron. Bars in A and B = 75 pm, C and E = 200 ftm, and D = 40/~m.

D.B. Mendis et al. /Brain Research 676 (1995) 69-79

ON6 and ON9 [25], to determine the cellular and subcellular distribution of SPARC protein in the adult rabbit brain. The ON6 and ON9 antibodies indicate that the highest levels of SPARC protein are found in the cerebellum and brainstem while lower levels are found in the hippocampus and cerebral hemispheres. Although enriched in membrane fractions, SPARC is not a transmembrane protein as it is insoluble in Triton X-114. This agrees with pr~wious reports which suggest that SPARC is associated with membranes through binding to a cell surface receptor or by the interaction with other ECM molecules 1142].Insolubility in non-ionic detergents appears to be a general property of other adult brain ECM molecules such as the proteoglycans Cat-301 and T1 antigen [17,18]. This finding has led investigators to propose that there is an insoluble matrix in the adult brain [17]. Our results suggest that SPARC may be associated with an insoluble matrix. The highest level of SPARC protein in the brain is observed in the cerebellum where Bergmann glial cells and their processes show intense staining, with relatively little signal in the granule cell and white matter layers. The related SC1 glycoprotein is also associated with Bergmann glial fibres in the developing and adult cerebellum, although differences in the expression pattern within the cerebellum are apparent [27,28]. For example, SC1 is expressed by neurons of the deep cerebellar nuclei and SPARC is not. Bergmann glia are a specialized form of astrocytes, which like Mueller cells of the retina, do not ~transform into astrocytes or ependymal cells, but remain as radial gila as development proceeds [4,8,39]. Radial glia do not transform in amphibians, birds, or reptiles, where they are believed to function in a similar manner to astrocytes by surrounding and stabilizing synaptic contacts through perinodal processes [41]. The molecular layer of the cerebellum is a synapse-rich region of the brain, for example, a single Purkinje neuron may form as many as 105 dendritic contacts. The presence of SPARC along cellular processes of Bergmann gila may reflect a role in plasticity since the molecule has been reported to exhibit anti-adhesive properties [22,37]. Interestingly, the related SC1 protein is also present along Bergmann glial fibres in the adult cerebellum [27]. Bergmann glial fibres in the developing cerebellum serve as a well-defined migratory pathway for granule neurons. The movement of cells would require both adhesive and anti-adhesiw~. interactions between the cell and the substrate. Thrombospondin, which has been shown to form a complex with SPARC [9] is expressed by migratory granule neurons [30]. Thrombospondin antibodies have been shown to inhibit migration of cerebellar granule neurons from the external to the internal granule cell layer [31]. SPARC, which has been shown to have anti-adhesive properties [22,37],

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is associated with Bergrnann glial fibres and may interact with thrombospondin or other ECM molecules on granule neurons in order to facilitate the migration of these cells during cerebellar development. It is of interest that astrocytes in the deep white matter of the developing cerebellum, which are not involved in neuronal migration, show little evidence of SPARC in their cellular processes. The use of double-immunohistochemistry has clearly demonstrated that SPARC is expressed by astrocytes in other synaptic enriched regions of the brain such as in the molecular layer of the hippocampus. This finding agrees with the previously reported secretion of SPARC by astrocytes in cell culture [26,44]. The present observations describing the expression of SPARC by astrocytes and Bergmann glia, as well as our previous SPARC mRNA study [28] differ with the recent report of SPARC mRNA in neurons of the rat locus coeruleus [5]. It is possible that the bovine probe used in that rat mRNA study hybridized to related sequences such as SC1 [20,27]. SC1 protein and mRNA are expressed by a range of neurons throughout the brain including the locus coeruleus (Mendis et al., manuscript in progress). The high level of SPARC mRNA previously observed in brain stem nuclei [28] is shown in the present report to reflect expression by glial cells and not neurons in the inferior olive. Our immunoreactivity data are consistent with Golgi impregnation studies and immunohistochemistry with anti-proteoglycan antibodies which have reported perineuronal nets of ECM covering neuronal soma and dendrites of the superior olive and cerebral cortex [2,7]. Likewise, the fibre-like pattern of SPARC immunoreactivity radiating into the corpus callosum may reflect an ECM associated with striatal and subthalamic projections that are known to form compact bundles surrounding pallidal neurons [32]. In summary, our results indicate that SPARC protein is expressed in regions of the mammalian brain which are enriched in synapses such as the molecular layer of the cerebellum, and the hippocampus with lower levels evident in fibre tract regions. Synapses within these regions are plastic and undergo rearrangements. SPARC may be involved in these processes through the modulation of growth factors since SPARC contains a follistatin domain capable of binding growth factors [14,33] and has been shown to inhibit receptor binding of the PDGF AB and BB dimers [34]. SPARC may influence the morphology of mature neural cells and their cellular processes by modulating adhesive and anti-adhesive interactions [22,37]. The ECM is known to play an important role in neural development [38,43]. Our identification of SPARC in the adult brain suggests that the extracellular matrix may continue to play a dynamic role in the mature nervous system. As

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more adult brain ECM molecules are identified, their interactions with SPARC will need to be examined as well as the general role of the ECM with respect to synaptic plasticity and neuronal transmission.

Acknowledgements We thank P. Manzerra for supplying some of the rabbit brain tissue sections and homogenates and J. Foster for the perfusion of the PD4 rabbit brains. We are grateful to Dr. M. Ringuette for his gift of the non-specific antibody, and to S. Rush for critical proof-reading of this manuscript. The anti-bovine SPARC monoclonal antibodies ON6 and ON9 were produced in the laboratories of CIS-Biointernational, Marcoule, France. This work was funded by a grant to I.R.B. from the Natural Sciences and Engineering Research Council (NSERC) Canada.

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