14
Neuroscience Letters, 112 (1990) 14-18 Elsevier Scientific Publishers Ireland Ltd.
NSL 06783
Analysis of a neuronal antigen (Hu) expression in the developing rat brain detected by autoantibodies from patients with paraneoplastic encephalomyelitis F r a n c e s c G r a u s I a n d Isidre F e r r e r 2 tDepartment of Neurology, Hospital Clinic i Provincial, Barcelona and :Neuropathology Unit, Department of Pathology, Hospital 'Prineipes Espana', Hospitalet del Llobregat, Barcelona (Spain)
(Received 6 November 1989; Revised version received 14 December 1989; Accepted 19 December 1989) Key words." Autoantibody; Paraneoplastic disease; Neuronal differentiation; lmmunohistochemistry
Anti-Hu is an autoantibody that recognizes an antigen (Hu) highly restricted to neuronal nuclei. In the developing rat brain all neurons and the germinal cell layer were anti-Hu positive. Ependyma and choroid plexus were positive only in the early stages of development. The strongest expression of Hu was seen in the most mature neurons. The transitory nature of Cajal-Retzius and subplate neurons was confirmed with the anti-Hu staining. Although the Hu is also expressed by neural cells other than neurons, the strongest staining of mature neurons could indicate that Hu plays a role in the process of neuronal differentiation. Patients with p a r a n e o p l a s t i c e n c e p h a l o m y e l i t i s a s s o c i a t e d with small cell lung cancer h a r b o r an a u t o a n t i b o d y in their serum a n d C S F called a n t i - H u [1,6]. By i m m u n o h i s t o c h e m i s t r y , a n t i - H u stains the nuclei o f n e u r o n s a n d small cell lung cancer b u t not the nuclei o f o t h e r n o r m a l tissues o r o t h e r t u m o r s [3, 6]. The antigen identified is a set o f basic p r o t e i n s o f 38-40 k D a o f m o l e c u l a r weight [6]. The function o f these p r o t e i n s in the b i o l o g y o f n e u r o n s is u n k n o w n . In the present study we a n a l y z e d the r e l a t i o n s h i p between the expression o f the H u antigen a n d the n o r m a l differentiation o f n e u r o n s a n d its possible presence in o t h e r cell types in the d e v e l o p i n g rat brain. P r e g n a n t W i s t a r rats f r o m o u r own c o l o n y were h o u s e d in i n d i v i d u a l cages a n d kept u n d e r a u t o m a t i c a l l y c o n t r o l l e d h u m i d i t y , t e m p e r a t u r e a n d 12 h l i g h t , l a r k cycles. A t birth, p u p s were restricted to 8-10 p e r litter so each p u p could have access to a similar a m o u n t o f milk. R a t s o f either sex at fetal stages E l 8 a n d E20, a n d postnatal d a y s P0 (newborn), P1, P3, P5, P7, P10, P I 5 a n d P60 were used in the study. T w o a n i m a l s were e x a m i n e d at every d e v e l o p m e n t a l stage. Biotinylated I g G (gift o f Dr. Posner) a n d two sera from 3 p a t i e n t s with high titer o f the a n t i - H u a n t i b o d y were used in the study. All patients h a d p a r a n e o p l a s t i c enceCorrespondence: F. Graus, Department of Neurology, Hospital Clinic i Provincial, Villarroel 170, 08036 Barcelona, Spain.
0304-3940/90/$ 03.50 {'3 1990 Elsevier Scientific Publishers Ireland Ltd.
15 phalomyelitis and small cell lung cancer. The detailed clinical and immunological features of these patients are described elsewhere [1]. Biotinylated normal human IgG and serum of two normal subjects were used as controls. Rats were killed under deep diethyl ether anesthesia, the brains were fixed with 5% formalin at 4°C for 6 h and kept in 30% sucrose in phosphate-buffered saline (PBS) overnight at 4°C. Brain sections were embedded in OCT compound (Milles Scientific, U.S.A.), snap-frozen in isopentane chilled by liquid nitrogen, and stored at -70°C. Sections were cut at 7 pm on a cryostat, washed in PBS and sequentially incubated with 10% normal goat serum for 20 min, patient's serum (1:1000) overnight at 4°C, 0.3% hydrogen peroxide for 20 min, biotylinated goat anti-human IgG (Vector Labs, U.S.A.) for 30 min, and vectastain-avidin-biotin complex (Vector Labs, U.S.A.) for 30 min. The substrate staining was developed with 0.05% diaminobenzidine with 0.01% hydrogen peroxide. Some sections were lightly counterstained with hematoxylin. When the biotylinated human IgG was used, sections were incubated with 10% normal human serum for 20 min, 0.3% hydrogen peroxide for 20 min, the biotylinated human IgG (1:160) overnight at 4°C followed by the ABC for 30 min. The reaction was developed as described above. All dilutions were done in PBS. In some experiments serial dilutions of the anti-Hu antibody were used in consecutive brain sections to evaluate intensity differences of the immunoreactivity among different cerebral areas. The same results were obtained with the 3 sources of the anti-Hu antibody. However, the biotylinated anti-Hu was superior with a stronger staining. In the adult rat brain, the nucleus and cytoplasm of all neurons were positive. Staining of the cytoplasm was more intense than that seen in our previous studies of adult human brain [6]. This diffence may be explained by the use in this study of a different fixative and a much more sensitive immunohistochemical technique. At lower dilutions, some glial nuclei were positive but the nuclei of ependyma, choroid plexus and non-neural cells were negative. Sections studied at E 18 and E20 included the whole head and all cells other than those neural in origin were negative. The anti-Hu stained all neurons of the brainstem, diencephalon, and neuroblasts of the primordium hippocampi and cortical plate. Moreover, a positive reaction was observed in the periventricular germinal layer, ependyma and choroid plexus when low dilutions of the antibodies were used (Fig. 1A,B). Radial glial fibers were negative. The staining was stronger in the most mature neurons as suggested by the observation that neurons of the trigeminal ganglia remained positive at dilutions that gave no staining in the cortical plate. During postnatal development, the intensity of the immunoreaction decreased in the ependyma and choroid plexus that became negative at P15. Cells in the germinal layer were positive even at P10 but the staining was weaker than in the cortex, and in the sections counterstained with hematoxylin some of the cell nuclei were negative. In contrast, neurons became more clearly stained and the cytoarchitectonics of the different regions were clearly delineated with the antibody(Fig. 1C). In the cerebellum, Purkinje cells were strongly positive at P0. In the following days, the inner region of the external granular layer was more positive than the outer part of this layer
16
Fig. 1. Reactivity of the anti-Hu antibody in the cerebral cortex of the developingrat (without counterstaining). A-C: parietal cortex, A, E20; B, P0; C, P5. M, molecular layer; CP, cortical plate; SP, subcortical plate (subplate); G, germinal layer; I-VI, cortical layers. D-F: occipital cortex. D, P 1; E, P5; F, P7. CajalRetzius and globular cells of the molecular layer at different postnatal ages. A, C, bar = I00 /zm; B, bar = 200/zm; D-F, bar = 50/zm.
suggesting a progressive expression of the Hu antigen at the time of cell migration. The staining of the internal granular layer was weaker than that of the Purkinje cells throughout all the stages of development (Fig. 2). A similar difference in the intensity of the staining was observed in the granule cells of the fascia dentata that appeared weaker than the projective and local-circuit neurons of the hippocampus. In the neocortex at E20 and P0 the strongest staining was present in neurons of layer VIb and Cajal-Retzius (CR) cells in layer I. During the first week of postnatal life, neurons of layers Via and V and, later those of layer III were strongly stained by the antibody, whereas neurons of layers II and IV exhibited a less intense immunoreactivity throughout these developmental stages probably related to their smaller size (Fig.
17
Fig. 2. Reactivityof the anti-Hu antibody in the cerebellumof the developingrat (without counterstaining). A, P0; B, P5; C, P7. Bar= 100/zm. EGL, external granular layer; IGL, internal granular layer; P, Purkinje cell layer; F, Fastigial nucleus. 1A-C). In accordance with this interpretation neurons of layer II in the piriform and entorhinal cortices, which are larger than those of layer II in the neocortex, had a stronger staining. The neuronal staining by the anti-Hu was helpful to analyze the transitory nature of subplate neurons and CR cells. Subplate neurons were clearly positive during the first week of postnatal life but the immunoreactivity was markedly reduced at P15. This feature agrees with the observation that subplate neurons probably degenerate and die once migrating neuroblasts reach their position in the cerebral cortex and definitive connections are established [4, 9]. The anti-Hu staining clearly identified the morphology of CR cells from E20 to P5. In the following days
18 C R cells were rarely seen and globular cells appeared in the molecular layer (Fig. 1D-F). However, at P15 neither C R n o r globular cells were seen in the molecular layer. M o s t probably, typical C R cells disappear [2] or are transformed into large globular cells during the second week o f postnatal life [5]. W h e t h e r this globular morphology precedes degeneration and death or represents an intermediate stage o f C R cell transformation into n o n - p y r a m i d a l neurons o f the molecular layer is presently u n k n o w n [8]. O u r findings confirm that anti-Hu immunoreactivity is highly restricted to neural cells. In the developing rat brain the H u antigen is expressed in m a n y cells o f the germinal cell layer, even at stages where most o f the cells are committed to glial rather than neuronal differentiation. This observation suggests that the presence o f the H u antigen does not always mean that cells will develop into mature neurons and agrees with the finding o f the H u antigen in the small cell lung cancer [3, 6], a t u m o r o f presumable neuroendocrine origin. The strongest staining in well differentiated neurons and the loss o f expression o f the H u antigen in e p e n d y m a choroid plexus cells that were initially positive in the fetal and early postnatal days o f life suggest the H u antigen m a y be important, along with other non-histone c h r o m o s o m a l proteins [7], in the process o f neuronal differentiation. We wish to acknowledge the expert technical assistance o f Ms. M.J. Iranzo. We thank Dr. J.B. Posner o f Memorial Sloan-Kettering Cancer Center for providing the biotylinated a n t i - H u antibody. Supported in part by G r a n t FISS-89/0058 from the F o n d o de Investigaciones Sanitarias de la Seguridad Social, Spain. 1 Anderson, N.E., Rosenblum, M.K., Graus, F., Wiley, R.G. and Posner, J.B., Autoantibodies in paraneoplastic syndromes associated with small-cell lung cancer, Neurology, 38 (1988) 1391- 1398. 2 Bradford, R., Parnavelas, J.G. and Lieberman, A.R., Neurons in layer I of the developing occipital cortex of the rat, J. Comp. Neurol., 176 (1977) 121 132. 3 Budde-Steffen, C., Anderson, N.E., Rosenblum, M.K. and Posner, J.B., Expression of an antigen in small cell lung carcinoma lines detected by antibodies from patients with paraneoplastic dorsal root ganglionopathy, Cancer Res., 48 (1988) 430~34. 4 Chun, J.J.M., Nakamura, M.J., Shatz, C.J., Transient cells of the developing mammalian telencephalon are peptide immunoreactive neurons, Nature (Lond.), 325 (I 987) 617~-620. 5 Edmunds, S.M. and Parnavelas, J.G., Retzius-Cajal cells: an ultrastructural study in the developing visual cortex of the rat, J. Neurocytol., 11 (1982) 427-446. 6 Graus, F., Elkon, K.B., Cordon-Cardo, C. and Posner, J.B., Sensory neuropathy and small cell lung cancer. Antineuronal antibody that also reacts with the tumor, Am. J. Med., 80 (1986) 45-52. 7 Heizmann, C.W., Arnold, E.M. and Kuenzle, C.C., Fluctuations of non-histone chromosomal proteins in differentiating brain cortex and cerebellar neurons, J. Biol. Chem., 255 (1980) 11504-11511. 8 Marin-Padilla, M., Neurons of layer I. A developmental analysis. In A. Peters and E.G. Jones (Eds.), Cerebral Cortex, Vot 1, Cellular Components of the Cerebral Cortex, Plenum, New York, 1985, pp. 447-478. 9 Shatz, C.J., Chun, J.J.M. and Luskin, M.B., The role of the subplate in the development of the mammalian telencephalon. In A. Peters and E.G. Jones (Eds.), Cerebral Cortex, Vol. 7, Developmental and Maturation of the Cerebral Cortex, Plenum, New York, 1988, pp. 35-38.
Neuroscience Letters, 112 (1990) 14-18 Elsevier Scientific Publishers Ireland Ltd.
NSL 06783
Analysis of a neuronal antigen (Hu) expression in the developing rat brain detected by autoantibodies from patients with paraneoplastic encephalomyelitis F r a n c e s c G r a u s I a n d Isidre F e r r e r 2 tDepartment of Neurology, Hospital Clinic i Provincial, Barcelona and :Neuropathology Unit, Department of Pathology, Hospital 'Prineipes Espana', Hospitalet del Llobregat, Barcelona (Spain)
(Received 6 November 1989; Revised version received 14 December 1989; Accepted 19 December 1989) Key words." Autoantibody; Paraneoplastic disease; Neuronal differentiation; lmmunohistochemistry
Anti-Hu is an autoantibody that recognizes an antigen (Hu) highly restricted to neuronal nuclei. In the developing rat brain all neurons and the germinal cell layer were anti-Hu positive. Ependyma and choroid plexus were positive only in the early stages of development. The strongest expression of Hu was seen in the most mature neurons. The transitory nature of Cajal-Retzius and subplate neurons was confirmed with the anti-Hu staining. Although the Hu is also expressed by neural cells other than neurons, the strongest staining of mature neurons could indicate that Hu plays a role in the process of neuronal differentiation. Patients with p a r a n e o p l a s t i c e n c e p h a l o m y e l i t i s a s s o c i a t e d with small cell lung cancer h a r b o r an a u t o a n t i b o d y in their serum a n d C S F called a n t i - H u [1,6]. By i m m u n o h i s t o c h e m i s t r y , a n t i - H u stains the nuclei o f n e u r o n s a n d small cell lung cancer b u t not the nuclei o f o t h e r n o r m a l tissues o r o t h e r t u m o r s [3, 6]. The antigen identified is a set o f basic p r o t e i n s o f 38-40 k D a o f m o l e c u l a r weight [6]. The function o f these p r o t e i n s in the b i o l o g y o f n e u r o n s is u n k n o w n . In the present study we a n a l y z e d the r e l a t i o n s h i p between the expression o f the H u antigen a n d the n o r m a l differentiation o f n e u r o n s a n d its possible presence in o t h e r cell types in the d e v e l o p i n g rat brain. P r e g n a n t W i s t a r rats f r o m o u r own c o l o n y were h o u s e d in i n d i v i d u a l cages a n d kept u n d e r a u t o m a t i c a l l y c o n t r o l l e d h u m i d i t y , t e m p e r a t u r e a n d 12 h l i g h t , l a r k cycles. A t birth, p u p s were restricted to 8-10 p e r litter so each p u p could have access to a similar a m o u n t o f milk. R a t s o f either sex at fetal stages E l 8 a n d E20, a n d postnatal d a y s P0 (newborn), P1, P3, P5, P7, P10, P I 5 a n d P60 were used in the study. T w o a n i m a l s were e x a m i n e d at every d e v e l o p m e n t a l stage. Biotinylated I g G (gift o f Dr. Posner) a n d two sera from 3 p a t i e n t s with high titer o f the a n t i - H u a n t i b o d y were used in the study. All patients h a d p a r a n e o p l a s t i c enceCorrespondence: F. Graus, Department of Neurology, Hospital Clinic i Provincial, Villarroel 170, 08036 Barcelona, Spain.
0304-3940/90/$ 03.50 {'3 1990 Elsevier Scientific Publishers Ireland Ltd.
15 phalomyelitis and small cell lung cancer. The detailed clinical and immunological features of these patients are described elsewhere [1]. Biotinylated normal human IgG and serum of two normal subjects were used as controls. Rats were killed under deep diethyl ether anesthesia, the brains were fixed with 5% formalin at 4°C for 6 h and kept in 30% sucrose in phosphate-buffered saline (PBS) overnight at 4°C. Brain sections were embedded in OCT compound (Milles Scientific, U.S.A.), snap-frozen in isopentane chilled by liquid nitrogen, and stored at -70°C. Sections were cut at 7 pm on a cryostat, washed in PBS and sequentially incubated with 10% normal goat serum for 20 min, patient's serum (1:1000) overnight at 4°C, 0.3% hydrogen peroxide for 20 min, biotylinated goat anti-human IgG (Vector Labs, U.S.A.) for 30 min, and vectastain-avidin-biotin complex (Vector Labs, U.S.A.) for 30 min. The substrate staining was developed with 0.05% diaminobenzidine with 0.01% hydrogen peroxide. Some sections were lightly counterstained with hematoxylin. When the biotylinated human IgG was used, sections were incubated with 10% normal human serum for 20 min, 0.3% hydrogen peroxide for 20 min, the biotylinated human IgG (1:160) overnight at 4°C followed by the ABC for 30 min. The reaction was developed as described above. All dilutions were done in PBS. In some experiments serial dilutions of the anti-Hu antibody were used in consecutive brain sections to evaluate intensity differences of the immunoreactivity among different cerebral areas. The same results were obtained with the 3 sources of the anti-Hu antibody. However, the biotylinated anti-Hu was superior with a stronger staining. In the adult rat brain, the nucleus and cytoplasm of all neurons were positive. Staining of the cytoplasm was more intense than that seen in our previous studies of adult human brain [6]. This diffence may be explained by the use in this study of a different fixative and a much more sensitive immunohistochemical technique. At lower dilutions, some glial nuclei were positive but the nuclei of ependyma, choroid plexus and non-neural cells were negative. Sections studied at E 18 and E20 included the whole head and all cells other than those neural in origin were negative. The anti-Hu stained all neurons of the brainstem, diencephalon, and neuroblasts of the primordium hippocampi and cortical plate. Moreover, a positive reaction was observed in the periventricular germinal layer, ependyma and choroid plexus when low dilutions of the antibodies were used (Fig. 1A,B). Radial glial fibers were negative. The staining was stronger in the most mature neurons as suggested by the observation that neurons of the trigeminal ganglia remained positive at dilutions that gave no staining in the cortical plate. During postnatal development, the intensity of the immunoreaction decreased in the ependyma and choroid plexus that became negative at P15. Cells in the germinal layer were positive even at P10 but the staining was weaker than in the cortex, and in the sections counterstained with hematoxylin some of the cell nuclei were negative. In contrast, neurons became more clearly stained and the cytoarchitectonics of the different regions were clearly delineated with the antibody(Fig. 1C). In the cerebellum, Purkinje cells were strongly positive at P0. In the following days, the inner region of the external granular layer was more positive than the outer part of this layer
16
Fig. 1. Reactivity of the anti-Hu antibody in the cerebral cortex of the developingrat (without counterstaining). A-C: parietal cortex, A, E20; B, P0; C, P5. M, molecular layer; CP, cortical plate; SP, subcortical plate (subplate); G, germinal layer; I-VI, cortical layers. D-F: occipital cortex. D, P 1; E, P5; F, P7. CajalRetzius and globular cells of the molecular layer at different postnatal ages. A, C, bar = I00 /zm; B, bar = 200/zm; D-F, bar = 50/zm.
suggesting a progressive expression of the Hu antigen at the time of cell migration. The staining of the internal granular layer was weaker than that of the Purkinje cells throughout all the stages of development (Fig. 2). A similar difference in the intensity of the staining was observed in the granule cells of the fascia dentata that appeared weaker than the projective and local-circuit neurons of the hippocampus. In the neocortex at E20 and P0 the strongest staining was present in neurons of layer VIb and Cajal-Retzius (CR) cells in layer I. During the first week of postnatal life, neurons of layers Via and V and, later those of layer III were strongly stained by the antibody, whereas neurons of layers II and IV exhibited a less intense immunoreactivity throughout these developmental stages probably related to their smaller size (Fig.
17
Fig. 2. Reactivityof the anti-Hu antibody in the cerebellumof the developingrat (without counterstaining). A, P0; B, P5; C, P7. Bar= 100/zm. EGL, external granular layer; IGL, internal granular layer; P, Purkinje cell layer; F, Fastigial nucleus. 1A-C). In accordance with this interpretation neurons of layer II in the piriform and entorhinal cortices, which are larger than those of layer II in the neocortex, had a stronger staining. The neuronal staining by the anti-Hu was helpful to analyze the transitory nature of subplate neurons and CR cells. Subplate neurons were clearly positive during the first week of postnatal life but the immunoreactivity was markedly reduced at P15. This feature agrees with the observation that subplate neurons probably degenerate and die once migrating neuroblasts reach their position in the cerebral cortex and definitive connections are established [4, 9]. The anti-Hu staining clearly identified the morphology of CR cells from E20 to P5. In the following days
18 C R cells were rarely seen and globular cells appeared in the molecular layer (Fig. 1D-F). However, at P15 neither C R n o r globular cells were seen in the molecular layer. M o s t probably, typical C R cells disappear [2] or are transformed into large globular cells during the second week o f postnatal life [5]. W h e t h e r this globular morphology precedes degeneration and death or represents an intermediate stage o f C R cell transformation into n o n - p y r a m i d a l neurons o f the molecular layer is presently u n k n o w n [8]. O u r findings confirm that anti-Hu immunoreactivity is highly restricted to neural cells. In the developing rat brain the H u antigen is expressed in m a n y cells o f the germinal cell layer, even at stages where most o f the cells are committed to glial rather than neuronal differentiation. This observation suggests that the presence o f the H u antigen does not always mean that cells will develop into mature neurons and agrees with the finding o f the H u antigen in the small cell lung cancer [3, 6], a t u m o r o f presumable neuroendocrine origin. The strongest staining in well differentiated neurons and the loss o f expression o f the H u antigen in e p e n d y m a choroid plexus cells that were initially positive in the fetal and early postnatal days o f life suggest the H u antigen m a y be important, along with other non-histone c h r o m o s o m a l proteins [7], in the process o f neuronal differentiation. We wish to acknowledge the expert technical assistance o f Ms. M.J. Iranzo. We thank Dr. J.B. Posner o f Memorial Sloan-Kettering Cancer Center for providing the biotylinated a n t i - H u antibody. Supported in part by G r a n t FISS-89/0058 from the F o n d o de Investigaciones Sanitarias de la Seguridad Social, Spain. 1 Anderson, N.E., Rosenblum, M.K., Graus, F., Wiley, R.G. and Posner, J.B., Autoantibodies in paraneoplastic syndromes associated with small-cell lung cancer, Neurology, 38 (1988) 1391- 1398. 2 Bradford, R., Parnavelas, J.G. and Lieberman, A.R., Neurons in layer I of the developing occipital cortex of the rat, J. Comp. Neurol., 176 (1977) 121 132. 3 Budde-Steffen, C., Anderson, N.E., Rosenblum, M.K. and Posner, J.B., Expression of an antigen in small cell lung carcinoma lines detected by antibodies from patients with paraneoplastic dorsal root ganglionopathy, Cancer Res., 48 (1988) 430~34. 4 Chun, J.J.M., Nakamura, M.J., Shatz, C.J., Transient cells of the developing mammalian telencephalon are peptide immunoreactive neurons, Nature (Lond.), 325 (I 987) 617~-620. 5 Edmunds, S.M. and Parnavelas, J.G., Retzius-Cajal cells: an ultrastructural study in the developing visual cortex of the rat, J. Neurocytol., 11 (1982) 427-446. 6 Graus, F., Elkon, K.B., Cordon-Cardo, C. and Posner, J.B., Sensory neuropathy and small cell lung cancer. Antineuronal antibody that also reacts with the tumor, Am. J. Med., 80 (1986) 45-52. 7 Heizmann, C.W., Arnold, E.M. and Kuenzle, C.C., Fluctuations of non-histone chromosomal proteins in differentiating brain cortex and cerebellar neurons, J. Biol. Chem., 255 (1980) 11504-11511. 8 Marin-Padilla, M., Neurons of layer I. A developmental analysis. In A. Peters and E.G. Jones (Eds.), Cerebral Cortex, Vot 1, Cellular Components of the Cerebral Cortex, Plenum, New York, 1985, pp. 447-478. 9 Shatz, C.J., Chun, J.J.M. and Luskin, M.B., The role of the subplate in the development of the mammalian telencephalon. In A. Peters and E.G. Jones (Eds.), Cerebral Cortex, Vol. 7, Developmental and Maturation of the Cerebral Cortex, Plenum, New York, 1988, pp. 35-38.
