Transient expression of neurofilament-like (RT97) immunoreactivity in cerebellar granule cells

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Developmental Brain Research, 28 (1986) 282-280 Elsevier

BRD 60160

Transient expression of neurofilament-like (RT97) immunoreactivity in cerebellar granule cells M.A. CAMBRAY-DEAKIN and R.D. BURGOYNE The Physiological Laboratory, University of Liverpool, Liverpool ( U. K. ) (Accepted April 15th, 1986) Key words: neurofilament - - cerebellar granule cell - - transient expression - - RT97 - - immunochemistry

The expression of the 200-kDa neurofilament subunit in developing rat cerebellar granule cells in vivo and in vitro was examined by immunocytochemistry and immunoblotting. Granule cells in vitro were found to express neurofilament-like immunoreactivity. In cryostat sections, neurofilament immunoreactivity was evident between P6 and P13 as a band of staining in the molecular layer indicative of staining of parallel fibres. No labelling of parallel fibres was seen at P18 or in adult animals.

Neurofilaments (NF's) are a class of intermediate filaments localised to some but not all neurons in the central and peripheral nervous systems 15,2°. Notably, in the rat cerebellum NF's are found in Golgi, stellate and basket cells but are not generally considered to be present in Purkinje cells or granule cells 2°. Mammalian NF's are commonly taken to consist of 3 protein subunits of high (200 kDa; HNF), medium (150 kDa; MNF) and low (70 kDa; LNF) mol. wts. with a central core of LNF, with M N F and H N F side projections 19. The exact role(s) of NF's is (are) unclear. NF's have been suggested to provide a structural lattice for axons 12 and to be involved with (i) axonal transport 9, (ii) tubulin polymerisation is, (iii) partitioning of the neuronal cytoskeleton 24. The distribution of NF subunits has to some extent been studied in the mature and developing nervous system by immunocytochemistry 3'4'9'13'16'19'22'23and NF's have not been reported to occur in adult cerebellar granule cells. The possibility that developing granule cells express NF's has not been examined. However, transient expression of N F subunits has been observed in other cell types, for example, Purkinje cells which do not show marked NF immunoreactivity in the hen are labelled by NF antibodies between embryonic days 12 and 20 (ref 3). Further-

more, granule cell axons (parallel fibres) are known to express proteins transiently during development e,g. tyrosylated a-tubulin 6'7, M A P 1 (x) (ref. 5), M A P 3 (ref. 2), Con A binding glycoproteins 2s and a cell surface determinant 21. Moreover, whilst parallel fibres in the adult cerebellum are not labelled with NF antibodies 22, presumed granule cells in vitro from newborn rats do express NF immtmoreaetivit~2~, Thus granule cells may express NF's at some stage(s) of their development. To investigate this poss~ilit-y we have examined the appearance in the developing rat cerebellum of immunoreactivity to RT97, a monoclonal antibody which binds predominantly to the H N F 25. Cryostat sections (10 /~m) were p r e p a r e d from freshly frozen, unfixed rat cerebella from postnatal days 6, 10, 13 and 18 (P6-18) and adult animals. After sectioning, the tissue was fixed in 4% formaldehyde in phosphate-buffered saline (PBS) for 30 min. The sections were then washed in PBS followed by incubation in 0.1% Triton X-100, 0.3% bovine serum albumin (BSA) in PBS (PBT) for 30 rain. R e sections were then incubated overnight at 10 °C with RT97 (ref. 25; ascites fluid, a gift from Dr. B. Anderton) or non-immune mouse serum (10 /~g/ml, 1:500 in PBT). The sections were then washed in PBS

Correspondence: M.A. Cambray-Deakin, The Physiological LaboratOry, University of Liverpool, P.O. Box 147, Brownlow Hill, Liverpool, L69 3BX, U.K. 0165-3806/86/$03.50© 1986 Elsevier Science Publishers B.V. (Biomedical Division)

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Fig. 1. a-e: cryostat sections of developing rat cerebellum labelled with RT97. a: P6 (×150). b: P10 (><180). c: P13 (×150). d: P18 (× 160). e: adult (× 150). egl, external germinal layer; m, molecular layer; p, Purkinje cell layer; g, granular layer; w, white matter. Dotted line indicates the line of the pial surface, f: RT97-1abelled cerebellar neuron culture at 11 DIV ()<410) showing stained neuronal processes and cell bodies (large filled arrowhead) and labelled nuclei of underlying flat cells (small unfilled arrowhead).

284 and incubated with sheep anti-mouse-biotin complex (1:100 in PBT, Amersham) for 1 h at room temperature, washed in PBS and incubated with streptavidin-horseradish peroxidase complex (1:150 in PBT, Amersham) for 30 min at room temperature. After washing, the bound peroxidase was visualised with diaminobenzidine. Cerebellar homogenates for immunoblotting were prepared from frozen rat cerebella by homogenisation in ice-cold 5 mM Tris-HCl, pH 8.0 containing 2 mM E G T A and 0.1 mM PMSF, methanol precipitation and resuspension in dissociation buffer (1.25% SDS, 2 mM E D T A , 10% sucrose, 125 mM Tris-HCl, 1% 2-mercaptoethanol, pH 6.8). Duplicate aliquots of the homogenates were run on a 7.5% SDS-polyacrylamide gel 14 and the separated proteins transferred to nitrocellulose paper by transverse electrophoresis. The transferred proteins were then visualised using Ponceau S stain and, after removal of the stain, were incubated with 0.3% BSA, 0.2% Triton X-100 in PBS for 5 h. The duplicate samples were then incubated with either RT97 or non-immune mouse serum (2.5pg/ml, 1:2000) for 1 h at room temperature and then overnight at 10 °C. The immunoblots were developed using the biotin-streptavidin system as before (sheep anti-mouse-biotin 1:200, streptavidin-horseradish peroxidase, 1:300). Cerebellar cell cultures were prepared from P6 rat cerebella and maintained as described previously 8,u in 96 well trays at a density of 4 × 104 cells per well. At 11 days in vitro (DIV) the cultures were fixed with 4% formaldehyde (30 min) and incubated with RT97 or non-immune mouse serum (10/~g/ml or 1:500) for 2 h, the bound antibodies being visualised by the biotin-streptavidin system as for cryostat sections. When the RT97-1abelled cryostat sections were examined (Fig. l a - e ) it was evident that in addition to staining of the developing white matter, there was a band of immunoreactivity in the molecular layer of P6-P13 rats which was not present in P18 or adult animals. In P6 rats (Fig. la) where the molecular layer is small and the Purkinje cell layer is as yet not clearly delineated, immunoreactivity appeared to extend into the developing Purkinje cell layer. However, in P10 and P13 animals (Fig. lb,c) RT97 labelling was restricted to the molecular layer, where it formed a distinct dense band of staining, and the white matter. In some sections of P13 rats RT97 immunoreactivity

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Fig. 2. RT97 immunoblot of rat cerebellar homogenates. The tracks are (from left to right) P6, P10, P13, P18 and adult rat homogenates. Arrowhead indicates an apparent mol. wt. of 200 kDa. A duplicate immunoblot incubated with non-immune mouse serum was unstained.

appeared to be less intense at the inner (older) parts of the molecular layer although this was rare. The pattern of labelling seen in P6, P10 and P13 rats is consistent with the expression of RT97 immunoreactivity in the growing, immature parallel fibres. By P18, however (Fig. ld), the homogenous staining of the molecular layer was replaced by a staining pattern more typical of the adult cerebellum (Fig. le); that is, a marked labelling of the basket cell fibres surrounding the base of the Purkinje cells and a labelling of fibres (probably derived from Golgi and basket cells) running tangentially and perpendicularly to the surface of the folium. The labelling of the fibres in the molecular layer was confined to the lower third of the layer in P18 rats but in the adult stained fibres were visible running to just below the pial membrane. The immunoblotting studies (Fig. 2) clearly showed that RT97 predominantly labelled a band of 200 kDa at all ages examined indicating that HNF is expressed early in development. Except for P6 there was no evidence for the presence of any cross-reaction of the RT97 antiserum with proteins of mol. wt. greater than ca. 200 kDa although there is some faint labelling of bands of lower mol. wt. and the gel front which may be due to the presence of NF breakdown products 4. The cross-reacting material with a mol. wt. greater than 200 kDa was not detected later than P6 despite the persistence of parallel fibre staining until P13.

285 In cerebellar granule cell cultures (Fig. lf) the processes and p e r i k a r y a of the granule cells were labelled with RT97 after 11 D I V (also at 2 D I V , data not shown) as are the nuclei of the flat cells ( p r o b a b l y astrocytes) also present in the cultures. The staining of histone proteins in cell nuclei 26 p r e s u m a b l y accounts for the a p p a r e n t labelling of the p e r i k a r y a of granule cells in vitro. F u r t h e r evidence for the presence of H N F in the developing molecular layer is noticeable in the w o r k of Leclerc et a1.16 using a different monoclonal antibody to the H N F , which labelled the m o l e c u l a r layer of P 9 - P 1 4 cerebellum, although this was not comm e n t e d on by the authors. Since the e p i t o p e recognised by RT97 is present on polypeptides o t h e r than H N F such as histone proteins 26 and rhodopsin a it is possible that the RT97 labelling in parallel fibres is due to an alternative p o l y p e p t i d e . H o w e v e r , the observation that a n o t h e r a n t i - H N F m o n o c l o n a l labels parallel fibres 16 argues that it is H N F that is transiently expressed. Conclusive evidence will require studies using o t h e r a n t i - H N F antibodies. It should also be n o t e d that the loss of RT97 labelling m a y be due to the loss of the p h o s p h o r y l a t e d e p i t o p e rather than the H N F subunit. The transient presence of H N F in the developing molecular layer suggests that H N F m a y play a role in the outgrowth of i m m a t u r e parallel fibres. With regard to the possible molecular mechanisms by which

H N F might influence such neurite formation, H N F has been r e p o r t e d to p r o m o t e tubulin polymerisation TMand it is evident that two o t h e r microtubule-associated proteins, M A P 1 (x) (ref. 5) and M A P 3 (ref. 2) are also transiently expressed in parallel fibres at about the same time as H N F . In particular M A P 3 is enriched in N F containing axons in adult brain and has been suggested to act in cross-linking microtubules and NF's. In addition, the developing parallel fibres express tyrosylated a - t u b u l i n 6 over the same p e r i o d as H N F , M A P 1 (x) and M A P 3. It is t e m p t i n g therefore to speculate that all these cytoskeletal components are intimately involved in the complex processes of neurite growth in granule cells. It is noteworthy that in PC12 cells, neurite outgrowth stimulated by nerve growth factor is also associated with an increased expression of N F ' s 17 and microtubule-associated proteins 1° and increased microtubule assembly. H o w e v e r , it is unlikely that H N F expression is a prerequisite for neurite outgrowth from all neurons as in some brain regions (i.e. b r a i n s t e m and optic nerve) H N F expression occurs late in develo p m e n t 13'19'23. I n d e e d , the early a p p e a r a n c e of H N F

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in the cerebellum is in contrast to its late a p p e a r a n c e in these other brain regions. This work was s u p p o r t e d by a project grant from the M R C . W e thank Mrs. K.-M. N o r m a n for technical assistance.

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